U.S. patent application number 15/034490 was filed with the patent office on 2016-09-29 for compositions and methods for selecting a treatment for b-cell neoplasias.
This patent application is currently assigned to THE BROAD INSTITUTE, INC.. The applicant listed for this patent is THE BRIGHAM AND WOMEN'S HOSPITAL, INC., THE BROAD INSTITUTE, INC.. Invention is credited to STEVEN A. CARR, BENJAMIN LEVINE EBERT, EMMA FINK, JAN KRONKE, NAMRATA D. UDESHI.
Application Number | 20160282354 15/034490 |
Document ID | / |
Family ID | 53180373 |
Filed Date | 2016-09-29 |
United States Patent
Application |
20160282354 |
Kind Code |
A1 |
EBERT; BENJAMIN LEVINE ; et
al. |
September 29, 2016 |
COMPOSITIONS AND METHODS FOR SELECTING A TREATMENT FOR B-CELL
NEOPLASIAS
Abstract
The present invention features compositions and methods for
identifying a subject having a B cell neoplasia or related
condition responsive to treatment with lenalidomide and
lenalidoinide-related compounds.
Inventors: |
EBERT; BENJAMIN LEVINE;
(BOSTON, MA) ; CARR; STEVEN A.; (CAMBRIDGE,
MA) ; KRONKE; JAN; (BOSTON, MA) ; UDESHI;
NAMRATA D.; (CAMBRIDGE, MA) ; FINK; EMMA;
(BOSTON, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
THE BROAD INSTITUTE, INC.
THE BRIGHAM AND WOMEN'S HOSPITAL, INC. |
Cambridge
Boston |
MA
MA |
US
US |
|
|
Assignee: |
THE BROAD INSTITUTE, INC.
CAMBRIDGE
MA
THE BRIGHAM & WOMEN'S HOSPITAL, INC.
BOSTON
MA
|
Family ID: |
53180373 |
Appl. No.: |
15/034490 |
Filed: |
November 7, 2014 |
PCT Filed: |
November 7, 2014 |
PCT NO: |
PCT/US14/64629 |
371 Date: |
May 4, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61902066 |
Nov 8, 2013 |
|
|
|
61915439 |
Dec 12, 2013 |
|
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01N 33/94 20130101;
G01N 33/5011 20130101; C12Q 1/6886 20130101; C12Q 2600/106
20130101; A01K 2267/03 20130101; C12Q 2600/136 20130101; A01K
67/0278 20130101; G01N 33/57496 20130101; G01N 33/5088 20130101;
A01K 2227/105 20130101; A01K 2217/072 20130101; C12Q 1/6876
20130101; G01N 2333/47 20130101; C12Q 2600/158 20130101; C12Q
2600/156 20130101 |
International
Class: |
G01N 33/574 20060101
G01N033/574; G01N 33/94 20060101 G01N033/94; G01N 33/50 20060101
G01N033/50 |
Goverment Interests
STATEMENT OF RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED
RESEARCH
[0002] This work was supported by the following grants from the
National Institutes of Health, Grant Nos: R01HL082945 and P01
CA108631. The government has certain rights in the invention.
Claims
1. A method of characterizing the lenalidomide- or lenalidomide
analog sensitivity of a subject having a neoplasia characterized by
increased IKZF1 or IKZF3 polypeptide expression, the method
comprising (a) contacting a cell derived from the neoplasia with
lenalidomide or a lenalidomide analog; and (b) detecting the level
of an IKZF1 or IKZF3 polypeptide or polypeptide ubiquitination in
the cell relative to the level in an untreated control cell,
wherein detection of a decrease in IKZF1 or IKZF3 polypeptide level
identifies the neoplasia as sensitive to lenalidomide or a
lenalidomide analog and the absence of a decrease in IKZF1 or IKZF3
polypeptide level or polypeptide ubiquitination identifies the
neoplasia as lenalidomide- or lenalidomide analog resistant.
2. The method of claim 1, wherein the lenalidomide analog is
thalidomide or pomalidomide.
3. The method of claim 1, wherein the neoplasia is a B or T cell
neoplasia.
4. The method of claim 3, wherein the B or T cell neoplasia is
mantle cell lymphoma, chronic lymphocytic leukemia, multiple
myeloma, or B cell lymphoma.
5. The method of claim 1, wherein the decrease in IKZF1 or IKZF3
polypeptide level is by at least about 20% or is undetectable by
Western blot.
6. The method of claim 1, wherein IKZF1 or IKZF3 polypeptide level
is detected by immunoassay, radioimmunoassay, immunohistochemistry,
FACS analysis, or quantitative fluorescent microscopy.
7-9. (canceled)
10. The method of claim 1, the method further comprising detecting
binding of CRBN to an IKZF1 or IKZF3 polypeptide in a biological
sample of the subject relative to the level present in a reference,
wherein detection of binding is indicative of lenalidomide- or
lenalidomide analog sensitivity and a reduction in binding is
indicative of lenalidomide- or lenalidomide analog resistance.
11. The method of claim 1, the method further comprising detecting
the sequence of an IKZF1 or IKZF3 polypeptide or polynucleotide in
a biological sample obtained from the subject relative to a IKZF1
or IKZF3 reference sequence, wherein detection of a mutation in the
IKZF1 or IKZF3 polypeptide or polynucleotide sequence is indicative
of lenalidomide resistance and failure to detect a mutation is
indicative of lenalidomide sensitivity.
12-19. (canceled)
20. A method of reducing the proliferation of a cell characterized
by increased IKZF1 or IKZF3 polypeptide expression, the method
comprising contacting the cell with lenalidomide or a lenalidomide
analog and an inhibitory nucleic acid molecule that reduces the
expression or activity of an IKZF1 or IKZF3 polypeptide or casein
kinase 1.
21. The method of claim 20, wherein the inhibitory nucleic acid
molecule is an antisense nucleic acid molecule, shRNA, siRNA
molecule, or Crispr.
22-23. (canceled)
24. The method of claim 20, wherein the inhibitory nucleic acid
molecule is an antisense nucleic acid molecule, shRNA, siRNA
molecule, or CRISPRi.
25. (canceled)
26. A recombinant murine cell, transgenic mouse, or knock-in mouse
comprising a polynucleotide encoding a human CRBN polypeptide
27. (canceled)
28. The recombinant murine cell of claim 26, wherein the CRBN
polypeptide comprises at least one substitution selected from the
group consisting of S369C, V380E, and I391V.
29-33. (canceled)
34. A method for assessing teratogenicity of lenalidomide or an
analog thereof, the method comprising contacting the mouse of claim
26 with lenalidomide or an analog thereof, and assessing
teratogenicity in pups produced by said mouse.
35. (canceled)
36. A method of assessing lenalidomide sensitivity in the murine
cell, transgenic mouse, or knock-in mouse of claim 26, the method
comprising contacting the murine cell, transgenic mouse, or
knock-in mouse of claim 26 with lenalidomide or an analog thereof,
and assessing lenalidomide sensitivity.
37-38. (canceled)
39. A derivative of lenalidomide of formula 1: ##STR00003##
40. A method of screening for agents that bind lenalidomide, the
method comprising contacting the derivative of claim 39 with the
agent and detecting binding to said derivative.
41. A method of screening for agents that increase lenalidomide
binding to CRBN, the method comprising contacting the derivative of
claim 39 and CRBN with an agent, and detecting increased binding of
CRBN to the derivative.
42. The method of claim 40, wherein binding to CRBN is assayed by
detecting the affinity of binding, by detecting ubiquination of
IKZF1 or IKZF3, by detecting degradation of IKZF1 or IKZF3.
43. A method of screening for agents that activate ubiquitin
ligase, the method comprising contacting a ubiquitin ligase with
the agent in the presence of IKZF1 or IKZF3, and detecting
ubiquitin ligase activation.
44-48. (canceled)
49. A method of identifying an agent that treats a myelodysplastic
syndrome, the method comprising contacting the cell with the agent
and detecting a decrease in casein kinase 1A1 polypeptide level in
the cell relative to the level present in a reference, thereby
identifying the agent as treating myelodysplastic syndrome.
50. (canceled)
51. A method of characterizing the lenalidomide or lenalidomide
analog sensitivity of a subject having myelodysplastic syndrome,
the method comprising (a) contacting a biological sample of the
subject with lenalidomide or a lenalidomide analog; and (b)
detecting altered casein kinase 1A1 polypeptide ubiquitination
relative to the level present in a reference.
52. (canceled)
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of and priority to U.S.
Provisional Application Ser. No. 61/902,066, filed Nov. 8, 2013,
and U.S. Provisional Application Ser. No. 61/915,439, filed Dec.
12, 2013, the contents of which are incorporated herein by
reference.
BACKGROUND OF THE INVENTION
[0003] B lymphocytes are an important cellular component of the
adaptive immune system. When normal B-cell development goes awry,
B-cell neoplasia can result. B cell neoplasms include multiple
myeloma, mantle cell lymphoma and chronic lymphocytic leukemia.
Multiple myeloma is a malignant plasma cell disorder and is the
second most common hematologic malignancy in the United States,
with about 20 000 patients diagnosed annually. Most patients
diagnosed with multiple myeloma survive for only 2-3 years. In
contrast, patients with mantle cell lymphoma may survive between 5
and 7 years. However, for most multiple myeloma patients, the
disease eventually progresses or returns, and over time treatment
resistance often develops. Chronic lymphocytic leukemia (CLL) is
the most common form of adult leukemia. In the U.S. alone, about
15,000 patients will be diagnosed with CLL in 2013, and almost
5,000 deaths from CLL will occur. MDS is diagnosed in more than
15,000 new patients per year, and deletions of chromosome 5q are
the most common cytogenetic abnormality. As with virtually all
cancers, prognosis is improved by the early identification of
disease and initiation of an appropriate therapeutic regimen.
Similarly, it is important to detect treatment resistance to a
particular agent early, so that alternate forms of therapy can be
provided.
SUMMARY OF THE INVENTION
[0004] As described below, the present invention features
compositions and methods for identifying a subject having a B cell
neoplasia or myelodysplastic syndrome/acute myeloid leukemia
responsive to treatment with lenalidomide and lenalidomide-related
compounds.
[0005] In one aspect, the invention provides a method of
characterizing the lenalidomide- or lenalidomide analog sensitivity
of a subject having a neoplasia characterized by increased IKZF1 or
IKZF3 polypeptide expression, the method involving contacting a
cell derived from the neoplasia with lenalidomide or a lenalidomide
analog; and detecting the level of an IKZF1 or IKZF3 polypeptide in
the cell relative to the level in an untreated control cell, where
detection of a decrease in IKZF1 or IKZF3 polypeptide level
identifies the neoplasia as sensitive to lenalidomide or a
lenalidomide analog and the absence of a decrease in IKZF1 or IKZF3
polypeptide level identifies the neoplasia as lenalidomide- or
lenalidomide analog resistant.
[0006] In another aspect, the invention provides a method of
characterizing the lenalidomide, thalidomide, or pomalidomide
sensitivity of a subject having a B cell neoplasia, the method
involving detecting increased IKZF1 or IKZF3 polypeptide level in a
biological sample of the subject relative to the level present in a
reference, where an increased level of IKZF1 or IKZF3 polypeptide
is indicative of lenalidomide, thalidomide, or pomalidomide
sensitivity.
[0007] In yet another aspect, the invention provides a method of
characterizing the lenalidomide or lenalidomide analog sensitivity
of a subject having a neoplasia or related condition, the method
involving contacting a biological sample of the subject with
lenalidomide or a lenalidomide analog; and detecting increased or
decreased IKZF1 or IKZF3 polypeptide ubiquitination relative to the
level present in a reference. In one embodiment, the method is
carried out in the presence of a proteasome inhibitor.
[0008] In still another aspect, the invention provides a method of
characterizing the lenalidomide- or lenalidomide analog sensitivity
of a subject having a neoplasia or related condition, the method
involving detecting binding of CRBN to an IKZF1 or IKZF3
polypeptide in a biological sample of the subject relative to the
level present in a reference, where detection of binding is
indicative of lenalidomide- or lenalidomide analog sensitivity and
a reduction in binding is indicative of lenalidomide- or
lenalidomide analog resistance.
[0009] In another aspect, the invention provides a method of
characterizing lenalidomide sensitivity in a subject having a B
cell neoplasia or related condition, the method involving detecting
the sequence of an IKZF1 or IKZF3 polypeptide or polynucleotide in
a biological sample obtained from the subject relative to a IKZF1
or IKZF3 reference sequence, where detection of a mutation in the
IKZF1 or IKZF3 polypeptide or polynucleotide sequence is indicative
of lenalidomide resistance and failure to detect a mutation is
indicative of lenalidomide sensitivity.
[0010] In another aspect, the invention provides a method of
monitoring lenalidomide sensitivity in a subject having a B cell
neoplasia or related condition, the method involving detecting the
sequence of an IKZF1 or IKZF3 polypeptide or polynucleotide in a
biological sample obtained from the subject relative to a IKZF1 or
IKZF3 reference sequence, where detection of a mutation in the
IKZF1 or IKZF3 polypeptide or polynucleotide sequence is indicative
of lenalidomide resistance and failure to detect a mutation is
indicative of lenalidomide sensitivity.
[0011] In still another aspect, the invention provides a method of
reducing the proliferation of a cell characterized by increased
IKZF1 or IKZF3 polypeptide expression, the method involving
contacting the cell with an inhibitory nucleic acid molecule that
reduces the expression of activity of an IKZF1 or IKZF3
polypeptide. In one embodiment, the inhibitory nucleic acid
molecule is an antisense nucleic acid molecule, shRNA, siRNA
molecule, or Crispr. In another embodiment, the inhibitory nucleic
acid molecule is expressed from a viral vector.
[0012] In yet another aspect, the invention provides a method of
reducing the proliferation of a cell, the method comprising
contacting the cell with an inhibitory nucleic acid molecule that
decreases the expression of casein kinase 1 and lenalidomide or a
lenalidomide analog. In one embodiment, the inhibitory nucleic acid
molecule is an antisense nucleic acid molecule, shRNA, siRNA
molecule, or Crispr. In another embodiment, the inhibitory nucleic
acid molecule is expressed from a viral vector.
[0013] In another aspect, the invention provides a recombinant
murine cell containing a polynucleotide encoding a human CRBN
polypeptide. In one embodiment, the cell comprises an expression
vector encoding the CRBN polypeptide. In another embodiment, the
expression vector is a viral vector.
[0014] In still another aspect, the invention provides a
recombinant murine cell containing a polynucleotide encoding a
mutant CRBN polypeptide. In one embodiment, the mutant CRBN
polypeptide comprises at least one substitution selected from the
group consisting of S369C, V380E, and I391V. In another embodiment,
the cell comprises an expression vector encoding the CRBN
polypeptide. In still another embodiment, the expression vector is
a viral vector.
[0015] In another aspect, the invention provides a transgenic mouse
or knock-in mouse containing a polynucleotide encoding a human CRBN
polypeptide. In one embodiment, the mouse is pregnant.
[0016] In another aspect, the invention provides a transgenic mouse
or knock-in mouse containing a polynucleotide encoding a mutant
CRBN polypeptide. In one embodiment, the mouse is pregnant.
[0017] In another aspect, the invention provides a method for
assessing teratogenicity of lenalidomide or an analog thereof, the
method involving contacting the pregnant mouse of claim 27 with
lenalidomide or an analog thereof, and assessing teratogenicity in
the murine pups produced by the pregnant mouse. In one embodiment,
teratogenicity is assessed prenatally or post natally.
[0018] In another aspect, the invention provides a method of
assessing lenalidomide sensitivity in the transgenic mouse of claim
26, the method involving contacting a mouse of claim 26 with
lenalidomide or an analog thereof, and assessing lenalidomide
sensitivity. In one embodiment, lenalidomide sensitivity is
assessed by assaying IKZF1 or IKZF3 levels or ubiquitination, by
assessing CRBN binding, by assaying for an alteration in the immune
system, or by assaying neoplastic cell proliferation. In another
embodiment, the immune system is assayed by analyzing B cell and T
cell function
In another aspect, the invention provides a derivative of
lenalidomide of formula 1:
##STR00001##
In another aspect, the invention provides a method of screening for
agents that bind lenalidomide, the method involving contacting the
derivative of the above aspect with the agent and detecting binding
to the derivative.
[0019] In yet another aspect, the invention provides a method of
screening for agents that increase lenalidomide binding to CRBN,
the method involving contacting the derivative of a previous aspect
and CRBN with an agent, and detecting increased binding of CRBN to
the derivative. In one embodiment, binding to CRBN is assayed by
detecting the affinity of binding, by detecting ubiquination of
IKZF1 or IKZF3, by detecting degradation of IKZF1 or IKZF3.
[0020] In another aspect, the invention provides a method of
screening for agents that activate ubiquitin ligase, the method
involving contacting a ubiquitin ligase with the agent in the
presence of IKZF1 or IKZF3, and detecting ubiquitin ligase
activation. In one embodiment, the screen is carried out in a cell.
In another embodiment, global protein ubiquitination and
alterations in global protein levels are assayed. In yet another
embodiment, the method involves detecting increased IKZF1 or IKZF3
ubiquitination, increased IKZF1 or IKZF3 degradation, or increased
IKZF1 or IKZF3 binding to CRBN. In another embodiment, the
ubiquitin ligase is present in a normal or neoplastic cell. In
another embodiment, the method involves detecting a decrease in
neoplastic cell proliferation.
[0021] In another aspect, the invention provides a method of
identifying an agent that treats a myelodysplastic syndrome, the
method involving contacting the cell with the agent and detecting a
decrease in casein kinase 1A1 polypeptide level in the cell
relative to the level present in a reference, thereby identifying
the agent as treating myelodysplastic syndrome. In one embodiment,
the a cell containing a 5q deletion.
[0022] In another aspect, the invention provides a method of
characterizing the lenalidomide or lenalidomide analog sensitivity
of a subject having myelodysplastic syndrome, the method involving
contacting a biological sample of the subject with lenalidomide or
a lenalidomide analog; and detecting altered casein kinase 1A1
polypeptide ubiquitination relative to the level present in a
reference. In one embodiment, the method is carried out in the
presence of a proteasome inhibitor.
[0023] In various embodiments of the above aspects or any other
aspect of the invention delineated herein, the lenalidomide analog
is thalidomide or pomalidomide. In other embodiments of the above
aspects or any other aspect of the invention delineated herein, the
neoplasia is a B or T cell neoplasia. In other embodiments, the B
or T cell neoplasia is mantle cell lymphoma, chronic lymphocytic
leukemia, multiple myeloma, or B cell lymphoma. In other
embodiments, the decrease in IKZF1 or IKZF3 polypeptide level is by
at least about 20% or is undetectable by Western blot. In other
embodiments, IKZF1 or IKZF3 polypeptide level is detected by
immunoassay, radioimmunoassay, immunohistochemistry, FACS analysis,
or quantitative fluorescent microscopy. In other embodiments, the B
cell neoplasia is mantle cell lymphoma, chronic lymphocytic
leukemia, multiple myeloma cell, or B cell lymphoma. In another
embodiment, the mutation is in amino acids 160-180 of IKZF3. In
other embodiments, the mutation reduces IKZF3 binding to CRBN or
reduces IKZF3 degradation in response to lenalidomide. In other
embodiments, the mutation is at amino acid position 147, 150, 161,
or 162. In other embodiments, the mutation is Q147H, Q1501-1,
L161R, or L162R. In other embodiments, a mutation in an IKZF1 or
IKZF3 polypeptide is detected by assaying antibody binding to amino
acids 160-180. In another embodiment, a mutation is an IKZF1 or
IKZF3 polynucleotide is detected by sequencing or
hybridization.
[0024] Other features and advantages of the invention will be
apparent from the detailed description, and from the claims.
DEFINITIONS
[0025] Unless defined otherwise, all technical and scientific terms
used herein have the meaning commonly understood by a person
skilled in the art to which this invention belongs. The following
references provide one of skill with a general definition of many
of the terms used in this invention: Singleton et al., Dictionary
of Microbiology and Molecular Biology (2nd ed. 1994); The Cambridge
Dictionary of Science and Technology (Walker ed., 1988); The
Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer
Verlag (1991); and Hale & Marham, The Harper Collins Dictionary
of Biology (1991). As used herein, the following terms have the
meanings ascribed to them below, unless specified otherwise.
[0026] By "IKZF1 polypeptide" is meant a polypeptide having at
least about 85% amino acid sequence identity to a sequence provided
at NCBI Accession No. AAH18349, NP_006051, NP_001207694, or a
fragment thereof and having DNA binding or transcriptional
regulatory activity.
[0027] For IKZF1 Isoform 1, the degron is from 130-270. For IKZF1
Isoform 2, the degron is from amino acid 136-180/236-249. Both
isoforms are responsive to lenalidomide. Exemplary amino acid
sequences for the two isoforms are provided below:
TABLE-US-00001 IKZF1 isoform 2 NCBI Reference No. NP_001207694 1
mdadegqdms qvsgkesppv sdtpdegdep mpipedlstt sggqqssksd rvvasnvkve
61 tqsdeengra cemngeecae dlrmldasge kmngshrdqg ssalsgvggi
rlpngklkcd 121 icgiicigpn vlmvhkrsht gerpfqcnqc gasftqkgnl
lrhiklhsge kpfkchlcny 181 acrrrdaltg hlrthsvike etnhsemaed
lckigsersl vldrlasnva krkssmpqkf 241 lgdkglsdtp ydssasyeke
nemmkshvmd qainnainyl gaeslrplvq tppggsevvp 301 vispmyqlhk
plaegtprsn hsaqdsaven llllskaklv psereaspsn scqdstdtes 361
nneeqrsgli yltnhiapha rnglslkeeh raydllraas ensqdalrvv stsgeqmkvy
421 kcehcrvlfl dhvmytihmg chgfrdptec nmcgyhsqdr yefsshitrg ehrfhms
IKZF1 isoform 1 NCBI Reference No. NP_006051 1 mdadegqdms
qvsgkesppv sdtpdegdep mpipedlstt sggqqssksd rvvasnvkve 61
tqsdeengra cemngeecae dlrmldasge kmngshrdqg ssalsgvggi rlpngklkcd
121 icgiicigpn vlmvhkrsht gerpfqenqc gasftqkgnl lrhiklhsge
kpfkchlcny 181 acrrrdaltg hlrthsvgkp hkcgycgrsy kqrssleehk
erchnylesm glpgtlypvi 241 keetnhsema edlckigser slvldrlasn
vakrkssmpq kflgdkglsd tpydssasye 301 kenemmkshv mdqainnain
ylgaeslrpl vqtppggsev vpvispmyql hkplaegtpr 361 snhsaqdsav
enllllskak lvpsereasp snscqdstdt esnneeqrsg liyltnhiap 421
harnglslke ehraydllra asensqdalr vvstsgeqmk vykcehcrvl fldhvmytih
481 mgchgfrdpf ecnmcgyhsq dryefsshit rgehrfhms
[0028] By "IKZF1 polynucleotide" is meant a polynucleotide encoding
an IKZF1 polypeptide. An exemplary IKZF1 polynucleotide is provided
at NM_006060.4 and reproduced below:
TABLE-US-00002 1 ggcagcagag gaaccttttg gaggaggaag aggacacaga
ggccctgtag ccaggcacca 61 agatccctcc caggtggctg ggtctgaggg
gaactccgag cagccctagg tcctcaaagt 121 ctggatttgt gtggaaaagg
cagctctcac ttggccttgg cgaggcctcg gttggttgat 181 aacctgagga
ccatggatgc tgatgagggt caagacatgt cccaagtttc agggaaggaa 241
agcccccctg taagcgatac tccagatgag ggcgatgagc ccatgccgat ccccgaggac
301 ctctccacca cctcgggagg acagcaaagc tccaagagtg acagagtcgt
ggccagtaat 361 gttaaagtag agactcagag tgatgaagag aatgggcgtg
cctgtgaaat gaatggggaa 421 gaatgtgcgg aggatttacg aatgcttgat
gcctcgggag agaaaatgaa tggctcccac 481 agggaccaag gcagctcggc
tttgtcggga gttggaggca ttcgacctcc taacggaaaa 541 ctaaagtgtg
atatctgtgg gatcatttgc atcgggccca atgtgctcat ggttcacaaa 601
agaagccaca ctggagaacg gcccttccag tgcaatcagt gcggggcctc attcacccag
661 aagggcaacc tgctccggca catcaagctg cattccgggg agaagccctt
caaatgccac 721 ctctgcaact acgcctgccg ccggagggac gccctcactg
gccacctgag gacgcactcc 781 gtcattaaag aagaaactaa tcacagtgaa
atggcagaag acctgtgcaa gataggatca 841 gagagatctc tcgtgctgga
cagactagca agtaacgtcg ccaaacgtaa gagctctatg 901 cctcagaaat
ttcttgggga caagggcctg tccgacacgc cctacgacag cagcgccagc 961
tacgagaagg agaacgaaat gatgaagtcc cacgtgatgg accaagccat caacaacgcc
1021 atcaactacc tgggggccga gtccctgcgc ccgctggtgc agacgccccc
gggcggttcc 1081 gaggtggtcc cggtcatcag cccgatgtac cagctgcaca
agccgctcgc ggagggcacc 1141 ccgcgctcca accactcggc ccaggacagc
gccgtggaga acctgctgct gctctccaag 1201 gccaagttgg tgccctcgga
gcgcgaggcg tccccgagca acagctgcca agactccacg 1261 gacaccgaga
gcaacaacga ggagcagcgc agcggtctca tctacctgac caaccacatc 1321
gccccgcacg cgcgcaacgg gctgtcgctc aaggaggagc accgcgccta cgacctgctg
1381 cgcgccgcct ccgagaactc gcaggacgcg ctccgcgtgg tcagcaccag
cggggagcag 1441 atgaaggtgt acaagtgcga acactgccgg gtgctcttcc
tggatcacgt catgtacacc 1501 atccacatgg gctgccacgg cttccgtgat
ccttttgagt gcaacatgtg cggctaccac 1561 agccaggacc ggtacgagtt
ctcgtcgcac ataacgcgag gggagcaccg cttccacatg 1621 agctaaagcc
ctcccgcgcc cccaccccag accccgagcc accccaggaa aagcacaagg 1681
actgccgcct tctcgctccc gccagcagca tagactggac tggaccagac aatgttgtgt
1741 ttggatttgt aactgttttt tgttttttgt ttgagttggt tgattggggt
ttgagttggt 1801 tttgaaaaga tttttatttt tagaggcagg gctgcattgg
gagcatccag aactgctacc 1861 ttcctagatg tttccccaga ccgctggctg
agattccctc acctgtcgct tcctagaatc 1921 cccttctcca aacgattagt
ctaaattttc agagagaaat agataaaaca cgccacagcc 1981 tgggaaggag
cgtgctctac cctgtgctaa gcacggggtt cgcgcaccag gtgtcttttt 2041
ccagtcccca gaagcagaga gcacagcccc tgctgtgtgg gtctgcaggt gagcagacag
2101 gacaggtgtg ccgccaccca agtgccaaga cacagcaggg ccaacaacct
gtgcccaggc 2161 cagcttcgag ctacatgcat ctagggcgga gaggctgcac
ttgtgagaga aaatactatt 2221 tcaagtcata ttctgcgtag gaaaatgaat
tggttgggga aagtcgtgtc tgtcagactg 2281 ccctgggtgg agggagacgc
cgggctagag cctttgggat cgtcctggat tcactggctt 2341 tgcggaggct
gctcagatgg cctgagcctc ccgaggcttg ctgccccgta ggaggagact 2401
gtcttcccgt gggcatatct ggggagccct gttccccgct ttttcactcc cataccttta
2461 atggccccca aaatctgtca ctacaattta aacaccagtc ccgaaatttg
gatcttcttt 2521 ctttttgaat ctctcaaacg gcaacattcc tcagaaacca
aagctttatt tcaaatctct 2581 tccttccctg gctggttcca tctagtacca
gaggcctctt ttcctgaaga aatccaatcc 2641 tagccctcat tttaattatg
tacatctgtt tgtagccaca agcctgaatt tctcagtgtt 2701 ggtaagtttc
tttacctacc ctcactatat attattctcg ttttaaaacc cataaaggag 2761
tgatttagaa cagtcattaa ttttcaactc aatgaaatat gtgaagccca gcatctctgt
2821 tgctaacaca cagagctcac ctgtttgaaa ccaagctttc aaacatgttg
aagctcttta 2881 ctgtaaaggc aagccagcat gtgtgtccac acatacatag
gatggctggc tctgcacctg 2941 taggatattg gaatgcacag ggcaattgag
ggactgagcc agaccttcgg agagtaatgc 3001 caccagatcc cctaggaaag
aggaggcaaa tggcactgca ggtgagaacc ccgcccatcc 3061 gtgctatgac
atggaggcac tgaagcccga ggaaggtgtg tggagattct aatcccaaca 3121
agcaagggtc tccttcaaga ttaatgctat caatcattaa ggtcattact ctcaaccacc
3181 taggcaatga agaatatacc atttcaaata tttacagtac ttgtcttcac
caacactgtc 3241 ccaaggtgaa atgaagcaac agagaggaaa ttgtacataa
gtacctcagc atttaatcca 3301 aacaggggtt cttagtctca gcactatgac
attttgggct gactacttat ttgttaggca 3361 ggagctctcc tgtgcattgt
aggataatta gcagtatccc tggtggctac ccaatagacg 3421 ccagtagcac
cccgaattga caacccaaac tctccagaca tcaccaactg tcccctgcga 3481
ggagaaatca ctcctggggg agaaccactg acccaaatga attctaaacc aatcaaatgt
3541 ctgggaagcc ctccaagaaa aaaaaaaaaa aa
[0029] By "IKZF3 polypeptide" is meant a protein having at least
about 85% amino acid sequence identity to NCBI Accession No.
NP_036613.2 (UnitPro Identifier No. Q9UKT9-1) or a fragment thereof
and having DNA binding or transcriptional regulatory activity. An
exemplary amino acid sequence of IKZF3 is provided below.
TABLE-US-00003 10 20 30 40 50 60 MEDIQTNAEL KSTQEQSVPA ESAAVLNDYS
LTKSHEMENV DSGEGPANED EDIGDDSMKV 70 80 90 100 110 120 KDEYSERDEN
VLKSEPMGNA EEPEIPYSYS REYNEYENIK LERHVVSFDS SRPTSGKMNC 130 140 150
160 170 180 DVCGLSCISF NVLMVHKRSH TGERPFQCNQ CGASFTQKGN LLRHIKLHTG
EKPFKCHLCN 190 200 210 220 230 240 YACQRRDALT GHLRTHSVEK PYKCEFCGRS
YKQRSSLEEH KERCRTFLQS TDPGDTASAE 250 260 270 280 290 300 ARHIKAEMGS
ERALVLDRLA SNVAKRKSSM PQKFIGEKRH CFDVNYNSSY MYEKESELIQ 310 320 330
340 350 360 TRMMDQAINN AISYLGAEAL RPLVQTPPAP TSEMVPVISS MYPIALTRAE
MSNGAPQELE 370 380 390 400 410 420 KKSIHLPEKS VPSERGLSPN NSGHDSTDTD
SNHEERQNHI YQQNHMVLSR ARNGMPLLKE 430 440 450 460 470 480 VPRSYELLKP
PPICPRDSVK VINKEGEVMD VYRCDHCRVL FLDYVMFTIH MGCHGFRDPF 490 500
ECNMCGYRSH DRYEFSSHIA RGEHRALLK
[0030] By "IKZF3 polynucleotide is meant a nucleic acid sequence
encoding an IKZF3 polypeptide. An exemplary polynucleotide sequence
is provided at NCBI Accession No. NM_012481, which is reproduced
below:
TABLE-US-00004 1 gcaggagcac gtggagaggc cgagtagcca cagcggcagc
tccagcccgg cccggcagcg 61 acatggaaga tatacaaaca aatgcggaac
tgaaaagcac tcaggagcag tctgtgcccg 121 cagaaagtgc agcggttttg
aatgactaca gtttaaccaa atctcatgaa atggaaaatg 181 tggacagtgg
agaaggccca gccaatgaag atgaagacat aggagatgat tcaatgaaag 241
tgaaagatga atacagtgaa agagatgaga atgttttaaa gtcagaaccc atgggaaatg
301 cagaagagcc tgaaatccct tacagctatt caagagaata taatgaatat
gaaaacatta 361 agttggagag acatgttgtc tcattcgata gtagcaggcc
aaccagtgga aagatgaact 421 gcgatgtgtg tggattatcc tgcatcagct
tcaatgtctt aatggttcat aagcgaagcc 481 atactggtga acgcccattc
cagtgtaatc agtgtggggc atcttttact cagaaaggta 541 acctcctccg
ccacattaaa ctgcacacag gggaaaaacc ttttaagtgt cacctctgca 601
actatgcatg ccaaagaaga gatgcgctca cggggcatct taggacacat tctgtggaga
661 aaccctacaa atgtgagttt tgtggaagga gttacaagca gagaagttcc
cttgaggagc 721 acaaggagcg ctgccgtaca tttcttcaga gcactgaccc
aggggacact gcaagtgcgg 781 aggcaagaca catcaaagca gagatgggaa
gtgaaagagc tctcgtactg gacagattag 841 caagcaatgt ggcaaaacga
aaaagctcaa tgcctcagaa attcattggt gagaagcgcc 901 actgctttga
tgtcaactat aattcaagtt acatgtatga gaaagagagt gagctcatac 961
agacccgcat gatggaccaa gccatcaata acgccatcag ctatcttggc gccgaagccc
1021 tgcgcccctt ggtccagaca ccgcctgctc ccacctcgga gatggttcca
gttatcagca 1081 gcatgtatcc catagccctc acccgggctg agatgtcaaa
cggtgcccct caagagctgg 1141 aaaagaaaag catccacctt ccagagaaga
gcgtgccttc tgagagaggc ctctctccca 1201 acaatagtgg ccacgactcc
acggacactg acagcaacca tgaagaacgc cagaatcaca 1261 tctatcagca
aaatcacatg gtcctgtctc gggcccgcaa tgggatgcca cttctgaagg 1321
aggttccccg ctcttacgaa ctcctcaagc ccccgcccat ctgcccaaga gactccgtca
1381 aagtgatcaa caaggaaggg gaggtgatgg atgtgtatcg gtgtgaccac
tgccgcgtcc 1441 tcttcctgga ctatgtgatg ttcacgattc acatgggctg
ccacggcttc cgtgaccctt 1501 tcgagtgtaa catgtgtgga tatcgaagcc
atgatcggta tgagttctcg tctcacatag 1551 ccagaggaga acacagagcc
ctgctgaagt gaatatctgg tctcagggat tgctcctatg 1621 tattcagcat
cgtttctaaa aaccaatgac ctcgcctaac agattgctct caaaacatac 1681
tcagttccaa acttcttttc ataccatttt tagctgtgtt cacaggggta gccagggaaa
1741 cactgtcttc cttcagaaat tattcgcagg tctagcatat tattactttt
gtgaaacctt 1801 tgttttccca tcagggactt gaattttatg gaatttaaaa
gccaaaaagg tatttggtca 1861 ttatcttcta cagcagtgga atgagtggtc
ccggagatgt gctatatgaa acattctttc 1921 tgagatatat caaccacacg
tggaaaagcc tttcagtcat acatgcaaat ccacaaagag 1981 gaagagctga
ccagctgacc ttgctgggaa gcctcaccct tctgcccttc acaggctgaa 2041
gggttaagat ctaatctccc taatctaaat gacagtctaa gagtaagtaa aagaacagcc
2101 ataaaataag tatctgttac gagtaactga agaccccatt ctccaagcat
cagatccatt 2161 tcctatcaca acatttttaa aaaatgtcat ctgatggcac
ttctgcttct gtcctttacc 2221 ttcccatctc cagtgaaaag ctgagctgct
ttgggctaaa ccagttgtct atagaagaaa 2281 atctatgcca gaagaactca
tggttttaaa tatagaccat catcgaaact ccagaaattt 2341 atccactgtg
gatgatgaca tcgctttcct ttggtcaagg ttggcagagc aagggtataa 2401
agggggaaat tgtttggcag caccaacaga aaacaaacaa acaaaaaaca gctacctaaa
2461 acttcttgaa agagttcatg gagaattggt gatacagacc caaagcaaat
ttgccaatga 2521 tattttccac aaaaaaagtc caaaaagtat ggctcagcct
ccccctcccc acaggagagg 2581 aattggagat agatggcatg tgtgtttaga
tcggagttga gctccggaat ggggtgagga 2641 gggacacctc tattgagagg
ttctccttga tcaggcaggc ttcggccctt tttttcccat 2701 ttaaatggaa
ctgctgtatt ccatgaaaat tcctgaaagt ctgatcacgg ttctgcagat 2761
gtataagtca tccttgtcac tcataatatg tacatactat caggaggagt gctgttatca
2821 tggtaaaatt agcactggaa taggaggtca caaaatgctg gctaattagc
tatgtgactt 2881 tgagaaatcg tttaactttt tttttttttt tttttttgag
acaggatctc actctgttgc 2941 ccaggctgga gtgcagtggt gcaatcatgg
ctcagtgcag cctcgacctc cccaggctca 3001 ggtgatcctc ccacctcagc
ctcttgagta ctgggacaac aagtgcacac caccatgtct 3061 ggctacattt
tgttcttttt gtagagatag gggtctcact atgttgccca tgctggtctt 3121
gaactcctgg gctcaagcaa tcagcccgcc tcagccccct aaagtgctgg gattacaggt
3181 gtgagccacc acacccagcc ttatttaact cttaaaactc agtttccggc
caggctcggt 3241 ggctcacacc tgtaatccca acactttggg aagccgaggc
aggcgcatca tttgaggtca 3301 ggagttcgag accagcctga cccacatggt
gaaaccctgt ctctactaaa aatacaaaaa 3361 ttagctgggc agtagtggca
catgcctgta atcccagcta ctccggaggc tgaggcagaa 3421 aaatcgctta
agcctgggag gttgaggttg cggtgagtgg agatcacact actgcactcc 3481
agtctgggcg acagagtgag accctgtctc aaacaaaaca aaacaaaaac aaacaaacaa
3541 aaacaaaaaa aactcagttt cctcatccat aaaataggaa ttagatttca
atgttctctt 3601 aggtcccttc tagctttaat tcatatgtga ttatgcagta
accacaaggt attttttaaa 3661 cctcctaatg tatggatatt aagcagaaga
gtatttatat gaatacatgt ttcacattcc 3721 tttggtatga aaatggtgtg
ttaagttttt cctttaacca ctgagttgtg aatgtgaaga 3781 aggtggtgga
gaggaacaaa aaacagaaag gtattttgat cttgccacaa agcatacaca 3841
caaattggca catgcagctg tttgccaaag ccttcttttt ttttttactt tttaagaaat
3901 tatgttaggg aaaataaatt ctgcttccag ggacaacttc atggagccta
tttacaaatt 3961 aagagtcagc ttaatttgta acatttctac cagagccaag
aatcccaaat tcctggtaga 4021 ttagtgtttt atttctaagg ggcttatgca
ttcggctcca actcaactcg tctatgtgct 4081 gccagtaatt aaaatgttcc
acctcagact gcacaaatgg cttatccttc tttgtggcat 4141 ggcgtctgtc
tcaggaaaaa aggttttatg aaattccatg gcaacagtcc caacatgttt 4201
gagacttcag ctaaaggaat ggatgtattt tggtgtgtag tcttcagtat atcactgtat
4261 ttccgtaata ctagactcca agctatgcca gattgcttat tccctttgtg
aaagaggagt 4321 tgctcattac gttcttgaaa tatcgcacat cctgttggtt
cttcaaggga caagagaaag 4381 agaattLgga agcagggatt agtagaagag
aaaacgaggg aaaggaagcc tttccaccag 4441 attagtgttc aagtctttgc
agaggagacc aacttttttt gttttctttt gttttgagac 4501 agtctctcgc
tctgctgccc aggctggagt gcagtggcgc gatctcggct cacggcaacc 4561
tccgcctccc gggttcaagc aattctcctg cctcagcctc ccaagtagct gggattacag
4621 gtgctcacca ccaagcccgg ctaattcttg tatttttagt agagacaagg
tttcaccatg 4681 ttggccaggc cagtctcaaa ctcctgacct caggtgatct
gcccgccttg gcctcccaca 4741 gtgctgggat tacaggcatg agctaccgca
cccagcctga gaccaccttt tgcatctcaa 4801 gattgtgaaa ccaaggccca
ttccaccagc ctggggactc tttttataga tatgatcctc 4861 ctttttcctg
tgactaatga atttgctgca tgatttctat tcttctgagg ttagttttct 4921
gagtaaggtg accactcaca aaggcacttt ctttgtggca ttctgagcct agattggggc
4981 ccatcaattc cagaaaaaat ttatgtgtgg aaactctgca tcctcaagtc
ttgaagttga 5041 accagatatg cagtggttac catcacacag ataaacgctg
ccttctgtac atacccctta 5101 tgctgtacta attaacaaac cccttgccag
ggctggggag gtgagggtga aggagaatct 5161 tagcagaagg gcagagtcag
gacttgcatc tgccactgct gggcactgaa gccctggagc 5221 agcttcagat
agtacctgta ctttctcatg cagactccct ctgaacaaga gccttgtagg 5281
cccctctcct tcatttccca ccagcctctt atcaggcggg ctttccacca tacacccagg
5341 aggccacggt ctgaggaaca accaaaccca tgcaaagggc cgggcgcgat
agctcacgcc 5401 tgtaatgcca gcactttggg aggctggggc aggcagatca
cctgaggttg ggagttcgag 5161 acctgcctga ccaacatgga gaaaccccca
tctctactaa aaatacaaaa ttagccgggc 5521 gtgatggcac atgcctgtaa
tcccagctac tcaggaggct gaggcaggag aatcgcttga 5581 acccgggagg
cggaggttgc ggtgagccga gatggcacca ctgcactcca gcctcggcaa 5641
caagagcgaa actctgtcta aaacaaaaac aaacaaacaa acaaaaaaac ccaggcaaag
5701 tttcuttgca gccaaggtga cagaactggg ctgagggtgg aaaagaaaca
gaaccagtgc 5761 tccaggtgtt ttttaatttt ttaatttatt tttatttttt
ttgtatatgt atatatatgt 5821 atgtatattt tagaggacca gggtctcact
atgttgccta ggccagactc aaactcctgt 5881 gctcaagcaa tcctgcctca
gcctcccaag tagctgggat tacaggcatg cacaaacaat 5941 gcccagctct
ccaaatgttt tctgtcacta cctgaagtgt tgcatcggta cttcctacgg 6001
aaagaaaact aaatagaagt gtctctcccg tgagccccca ccactaccac cagaaaaaaa
6061 aaagagagaa aatgaactca tcagtcttta gtttcctcaa gttattctcc
caaaaagaca 6121 ttcgccttgg cacagataag ccagctaatc ttatgcttta
tgacccactg tgagctgttc 6181 ctgacacagc ttctgacttt gtcagtgaca
aaatttctca ccttttaaat gcagtgctta 6241 acattttgtt aggcccatac
tcaaaatcgg ccagatataa aatgacctca gattttgatc 6301 tcctaggctc
aaacaatcct cctacctcag cctcccaagt agctgggact ataggcacac 6361
caccatgcac agctaatttt ttttgtattt ttctgcagag atggcgtttc gccatactgc
6421 ccaggctagt ctcaaaatcc tgggctcaag caatctgccc acctcagcct
cccaaagtgc 6481 tggaactaca ggcaagagcc actgcgccca gccacaacct
cagatttctt tggcaaacag 6541 aaatgtttaa aaacacaaaa ttttgctcag
gtgaaacact gtgttactat caaatctcac 6601 atccacataa agtttttctt
ttcggctttg tttcgtgagg aacagacaga acaaagtttt 6661 tccaggtagc
atctgtatca ctattattct cctatttcct gtaccacccc cacctcccca 6721
agccctactg aatgtgaggt ttagaatgtt ttaaggaggg tcaggtgcgg tggctcacgc
6781 ctgtaatccc agcactttgg gaggccaagg cgggcggatc acctgagttt
gggagttcga 6841 gaccagcctg accaacatgg agaaaccctg tctctactaa
aaatacaaaa ttagccaggc 6901 gtggtggcac atgcctgtaa tcccagctac
ttaggaggct gaggcaggag aatcgcttga 6961 acccaggagg aggaggttgt
ggtgagccga gatcgtgcca ttgcactcca gcctgggtga 7021 cagagtgaga
ctccatctcg aaaaaaaaaa tacaaaaatt agctgggtgt ggtggtgcac 7081
acctgtaatc ccagctactc gggaggctga cgcaggagaa ttgcttgaac ctgggaggtg
7141 gaggttgcag tgagccgaga tcgcgccatt gcaatccagc ctggacaaca
gagtgagact 7201 ccatctcaaa aaaaaaaaaa aaaagaatgt tttaaggaaa
aaaatagtac tgttacatat 7261 aatcccaggt gataagacca caatggaaat
gtttaagtcc tcactttaaa gagtacccca 7321 ctgagaagag gtatgttgga
ctctagcaga gatttggaaa ctctgggaca ctcaagatgt 7381 gaaagagcct
ggctatctga ggactcaaag agtcagcatc gggacttgtg agctcaagaa 7441
gagaaaaggg agtggtgaaa ctttgtccta aaagttagca ccaggaacag
aagaaaaaaa
7501 cccgatatat agtgatacct catcttttag agaatgggaa gctatttttg
tgttcacaca 7561 gaaagtatag ttcaaaaaac ctctatatcc agagttcaga
caaggagaat gatttgagat 7621 ataagtgccg atgaaggagg tcaattttga
tctgaaacca gcagctggac ctgggccacc 7681 tcaggaaaag gactctgttc
tccaaggcag cacgactgaa tggttctgag aataagccag 7741 ggttcaggac
tcctgaccct ttaggaccat ggactcagaa gagcctgaag gacaattgtg 7801
ggctttaaac ttctgagagc ttgtaaagta acacaagact gtgcctctcc cttgccccag
7861 ctgtagatag tctttgcccc accattgtta tgaagataca cagggttttg
cagtttgaat 7921 aaattggata caagtttcct cttttttttt ttctttttga
gacaaagtct cgctctgttt 7981 ccccaggctg agtgcagtgg cacaatcaag
gcttacttgc cgcctcaacc tcctgggctc 8041 aagcaacgag ccatcctccc
gtcttagcct cccaactagc tgagactaca ggcgtgggtc 8101 accacaccca
gctaattttt gtactttttg tagagacagg gtctcaccat gttgcccagg 8161
ctggtcctga actcctgggc tcaagtaatc tgcccacctc agcctcccaa agtgttgggg
8221 ttacaggcgt gaggcaccgc ggctggcctg agtttcttct taatactgta
tcacaattgt 8281 gggctgtctt atgtgttgat atcgattgag ctatttgaaa
taggaatgtt aatgggtgta 8341 ttaaattttt gtaaggatat aacaatatct
accttccaag gatgttgtga ggttttccat 8401 gattttgtat atgagctaat
gttacctttg aggggtggtg tgcattatgt tggatgattg 8461 taaattttca
gtggaaaatg taccgtgtcc taaatttaaa gacatgaaaa atatcccaag 8521
atcatactag atcataatag caattccttt acaaatgaat tatggaggta actgatctct
8581 aacagtttcc ttcatgttgt tttaatgcac aagggcagag gatctgctga
cccttggaac 8641 cagcgtgagc taaccacgtg ctatagacac ttcatgttgt
cgcacccagg gaagtcaaag 8701 cgctttgctc cctcactgtc tgtgagtcct
cagccattag taccccaccc cccgctgctc 8761 caaaacttga gttatttcaa
atgtttctca ctgttcatct ctccactgac cccactccag 8821 aaagcctgga
gagagtccca agatgccacc caccttcccc aatccctcgc cacagatctg 8881
tgtctatctc acactctgta agtgccgctt tgcttcttcc tctcttgaaa agactgagaa
8941 cacacatttt aacatgttag gaaaatgggg cagcctaaaa aatgactgat
cccaccgcca 9001 gtgactcatg tatactccag gctagcagac aaggcccttt
ttggtgggcc tgcttccgtg 9061 ggttcacaga aaccaaatta ctgtgggttg
caaagaatta gcaggtcatt tacaaagcag 9121 acatcccttc acccagactg
tggttttgca tgctcaggtt ctcagtctat gagctttggt 9181 gcaggatcat
tttggctact ggaaaaacca tagcttattt taaatttctg gttgccaaag 9241
ccaccacacg tgtggtctgt ggatgaccat tgtctgcaga atgacgagga aggaacagaa
9301 tgtggtttgg ggctcagggt ggccttccca ctgggaggga aggcgggagg
gagccottgc 9361 cctgggtttt gacacagcct gtgctcacag cctctcctct
catctgcatt tctcagaaat 9421 gccctccctg cccagtggtg actttccctc
gtcactccta tggagttcta cctggagccc 9481 agccatgtgt ggaactgtga
agtttactcc tctgtaaaga tggtttaaag aaagtcagct 9541 tctgaaatgt
aacaatgcta acccttgctg gaaccctgta agaaatagcc ctgctgatag 9601
ttttctaggt ttatcatgtt tgatttttac actgaaaaat aaaaaaatcc tggtatgttt
9661 gaaattaaaa aaaaaaaaaa aaaaaa
[0031] By "human CRBN polypeptide" is meant an amino acid sequence
or fragment thereof having at least 85% amino acid sequence
identity to NCBI Accession No. AAH67811.1 or NP_01166953.1 and
having IKZF3 binding activity. Exemplary CRBN polypeptide sequences
are provided below:
TABLE-US-00005 AAF167811.1 1 magegdqqda ahnmgnhlpl lpeseeedem
evedqdskea kkpniinfdt slptshtylg 61 admeefhgrt lhdddscqvi
pvlpqvmmil ipgqtlplql fhpqevsmvr nliqkdrtfa 121 vlaysnvqer
eaqfgttaei yayreeqdfg ieivkvkaig rqrfkvlelr tqsdgiqqak 181
vqilpecvlp stmsavqles lnkcqifpsk pvsredqcsy kwwqkyqrrk fhcanltswp
241 rwlyslydae tlmdrikkql rewdenlkdd slpsnpidfs yrvaaclpid
dvlriqllki 301 gsaiqrlrce ldimnkctsl cckqcqetei ttkneifsls
lcgpmaayvn phgyvhetlt 361 vykacnlnli grpstehswf pgyawtvaqc
kicashigwk ftatkkdmsp qkfwgltrsa 421 llptipdted eispdkvilc l
NP_001166953.1 1 magegdqqda ahnmgnhlpl lpeseeedem evedqdskea
kkpniinfdt slptshtylg 61 admeefhgrt lhdddscqvi pvlpqvmmil
ipgqtlplql fhpqevsmvr nliqkdrtfa 121 vlaysnvqer eaqfgttaei
yayreeqdfg ieivkvkaig rqrfkvlelr tqsdgiqqak 181 vgilpecvlp
stmsavqles lnkcqifpsk pvsredqcsy kwwqkyqkrk fhcanltswp 241
rwlyslydae tlmdrikkql rewdenlkdd slpsnpidfs yrvaaclpid dvlriqllki
301 gsaiqrlrce ldimnkctsl cckqcqetei ttkneifsls lcgpmaayvn
phgyvhetlt 361 vykacnlnli grpstehswf pgyawtvaqc kicashigwk
ftatkkdmsp gkfwgltrsa 421 llptipdted eispdkvilc l
[0032] By "human CRBN polynucleotide" is meant a nucleic acid
molecule encoding a CRBN polypeptide. An exemplary CRBN
polynucleotide sequence is provided at NCBI Accession No. BC067811,
which is reproduced below:
TABLE-US-00006 1 gcgtgtaaac agacatggcc ggcgaaggag atcagcagga
cgctgcgcac aacatgggca 61 accacctgcc gctcctgcct gagagtgagg
aagaagatga aatggaagtt gaagaccagg 121 atagtaaaga agccaaaaaa
ccaaacatca taaattttga caccagtctg ccgacatcac 181 atacatacct
aggtgctgat atggaagaat ttcatggcag gactttgcac gatgacgaca 241
gctgtcaggt gattccagtt cttccacaag tgatgatgat cctgattccc ggacagacat
301 tacctcttca gctttttcac cctcaagaag tcagtatggt gcggaattta
attcagaaag 361 atagaacctt tgctgttctt gcatacagca atgtacagga
aagggaagca cagtttggaa 421 caacagcaga gatatatgcc tatcgagaag
aacaggattt tggaattgag atagtgaaag 481 tgaaagcaat tggaagacaa
aggttcaaag tccttgagct aagaacacag tcagatggaa 541 tccagcaagc
taaagtgcaa attcttcccg aatgtgtgtt gccttcaacc atgtctgcag 601
ttcaattaga atccctcaat aagtgccaga tatttccttc aaaacctgtc tcaagagaag
661 accaatgttc atataaatgg tggcagaaat accagaggag aaagtttcat
tgtgcaaatc 721 taacttcatg gcctcgctgg ctgtattcct tatatgatgc
tgagacctta atggacagaa 781 tcaagaaaca gctacgtgaa tgggatgaaa
atctaaaaga tgattctctt ccttcaaatc 841 caatagattt ttcttacaga
gtagctgctt gtcttcctat tgatgatgta ttgagaattc 901 agctccttaa
aattggcagt gctatccagc gacttcgctg tgaattagac attatgaata 961
aatgtacttc cctctgctgt aaacaatgtc aagaaacaga aataacaacc aaaaatgaaa
1021 tattcagttt atccttatgt gggccgatgg cagcttatgt gaatcctcat
ggatatgtgc 1081 atgagacact tactgtgtat aaggcttgca acttgaatct
gataggccgg ccttctacag 1141 aacacagctg gtttcctggg tatgcctgga
ctgttgccca gtgtaagatc tgtgcaagcc 1201 atattggatg gaagtttacg
gccaccaaaa aagacatgtc acctcaaaaa ttttggggct 1261 taacgcgatc
tgctctgttg cccacgatcc cagacactga agatgaaata agtccagaca 1321
aagtaatact ttgcttgtaa acagatgtga tagagataaa gttagttatc taacaaattg
1381 gttatattct aagatctgct ttggaaatta ttgcctctga tacataccta
agtaaacata 1441 acattaatac ctaagtaaac ataacattac ttggagggtt
gcagtttcta agtgaaactg 1501 tatttgaaac ttttaagtat actttaggaa
acaagcatga acggcagtct agaataccag 1561 aaacatctac ttgggtagct
tggtgccatt atcctgtgga atctgatatg tctggtagcg 1621 tgtcattgat
gggacatgaa gacatctttg gaaatgatga gattatttcc tgtgttaaaa 1681
aaaaaaaaaa aatcttaaat tcctacaatg tgaaactgaa actaataatt tgatcctgat
1741 gtatgggaca gcgtatctgt accagtgctc taaataacaa aagctagggt
gacaagtaca 1801 tgttcctttt ggaaagaagc aaggcaatgt atattaatta
ttctaaaagg gctttgttcc 1861 tttccatttt ctttaacttc tctgagatac
tgatttgtaa attttgaaaa ttagttaaaa 1921 tatgcagttt tttgagccca
cgaatagttg tcatttcctt tatgtgcctg ttagtaaaaa 1981 gtagtattgt
gtatttgctc agtatctgaa ctataagccc atttatactg ttccatacaa 2041
aagctatttt tcaaaaatta atttgaacca aaactactac tatagggaaa agatgccaaa
2101 acatgtcccc tcacccaggc taaacttgat actgtattat tttgttcaat
gtaaattgaa 2161 gaaaatctgt aagtaagtaa accttaagtg tgaaactaaa
aaaaaaaaaa aaa
[0033] By "murine CRBN polypeptide" is meant an amino acid sequence
or fragment thereof having at least 85% amino acid sequence
identity to NCBI Accession No. BC086488.1 or NP_67424 and having
IKZF3 binding activity. Exemplary CRBN polypeptide sequence is
provided below:
TABLE-US-00007 BC086488.1 1 mgnhlpllpd sededdeiem evedqdskea
rkpniinfdt slptshtylg admeefhgrt 61 lhdddscqvi pvlpevlmil
ipgqtlplql shpqevsmvr nliqkdrtfa vlaysnvqer 121 eaqfgttaei
yayreeqefg ievvkvkaig rqrfkvlelr tqsdgiqqak vqilpecvlp 181
stmsavqves lnkcqvfpsk piswedqysc kwwqkyqkrk fhcanltswp rwlyslydae
241 tlmdrikkql rewdenlkdd slpenpidfs yrvaaclpid dvlriqllki
gsaiqrlrce 301 ldimnkctsl cckqcqetei ttkneifsls lcgpmaayvn
phgyvhetlt vykasnlnli 361 grpstvhswf pgyawtiaqc kicashigwk
ftatkkdmsp qkfwgltrsa llptipeted 421 eispdkvilc l NP_067424 1
magegdqqda ahnmgnhlpl lpadsededd eiemevedqd skearkpnii nfdtslptsh
61 tylgadmeef hgrtlhddds cqvipvlpev lmilipgqtl plqlshpqev
smvrnliqkd 121 rtfavlaysn vqereaqfgt taeiyayree qefgievvkv
kaigrqrfkv lelrtqsdgi 181 qqakvqilpe cvlpstmsav qleslnkcqv
fpskpiswed qysckwwqky qkrkfhcanl 241 tswprwlysl ydaetlmdri
kkqlrewden lkddslpenp idfsyrvaac lpiddvlriq 301 llkigsaiqr
lrceldimnk ctslcckqcq eteittknei fslslcgpma ayvnphgyvh 361
etltvykasn lnligrpstv hswfpgyawt iaqckicash igwkftatkk dmspqkfwgl
421 trsallptip etedeispdk vilcl
[0034] By "murine CRBN polynucleotide" is meant a nucleic acid
molecule encoding a murine CRBN polypeptide. An exemplary murine
CRBN polynucleotide sequence is provided at NCBI Accession No.
NM_021449 or NM_175357, which are reproduced below:
TABLE-US-00008 NM_021449 1 tttcccaggc tcctttgcgg gtaaacagac
atggccggcg agggagatca gcaggacgct 61 gcgcacaaca tgggaaacca
cctgccgctt ctgcctgaca gtgaagatga agatgatgaa 121 attgaaatgg
aagttgaaga ccaagatagt aaagaagcca gaaaaccgaa tatcataaac 181
tttgacacca gtctgccaac ctcacataca tacctgggag ctgatatgga ggagttccac
241 gggagaactt tgcatgacga cgacagctgc caggtgatcc cagtccttcc
tgaggtgctg 301 atgatcctga ttcctgggca gacactccca ctgcaqctct
ctcacccaca ggaagtcagc 361 atggtgcgga acttaatcca gaaagacagg
acctttgcag tccttgcata cagtaatgtg 421 caagaaaggg aagcacagtt
tgggacaaca gcagagatct atgcctatcg agaagagcag 481 gagtttggaa
ttgaagtagt gaaagtgaaa gcaattggaa ggcagcggtt caaggtcctc 541
gaacttcgaa cacagtcaga tggaatccag caagctaaag tgcagatttt gccagagtgt
601 gtgttgccgt caaccatgtc tgcagtgcag ttagaatcac tcaataagtg
ccaggtattt 661 ccttcaaaac ccatctcctg ggaagaccag tattcatgta
aatggtggca gaaataccag 721 aagagaaagt ttcactgtgc aaatctaaca
tcatggcctc gctggctgta ttcattatat 781 gatgctgaaa cattaatgga
tagaattaag aaacagctac gtgaatgqqa tgaaaatctc 841 aaagatgatt
ctcttcctga aaatccaata gacttttctt acagagtagc tgcttgtctt 901
cctattgatg atgtattgag aattcagctc cttaaaatcg gcagtgctat tcaacggctt
961 cgctgtgaat tggacatcat gaacaaatgt acttcccttt gctgtaaaca
atgtcaagaa 1021 acagaaataa cgacaaagaa tgaaatattt agtttatcct
tatgtggtcc aatggcagca 1081 tatgtgaatc ctcatggata tgtacatgag
acactgactg tgtataaagc gtccaacctg 1141 aatctgatag gccggccttc
tacagtgcac agctggtttc ccgggtatgc atggaccatt 1201 gcccagtgca
agatctgtgc aaqccatatt ggatggaaat ttacagccac aaaaaaagac 1261
atgtcacctc aaaaattttg gggcttaact cgctctgctc tgttacccac aattccagag
1321 actgaagatg aaataagtcc agacaaagta atactttgtt tataagtgca
cctgtaggag 1381 tgacttcctg acagatattt cctcaagtca gatctgccca
gtcatcactg cctctgatat 1441 atgtgtatag tgggttacag catttgccta
ccaagttcaa gagcatattt agggaatgag 1501 aaagcagtat aaaacataag
gctgggttcc aaaatacttg ctttttagta gcttggtgcc 1561 atggattatc
ctgttgagtc tatgtcatga caggatagga aaacacagtt gaaataatgg 1621
gaatggccat ggaacaggat aggggcacca ctgctctaaa tgatgaagct ctaaatgatg
1681 aatgctccag aaactgggtt ggtaagcaca agatagaggc aaggcagtgt
aattttaaaa 1741 ggactttgct cctttcaatt ttccttagct tgtctgagat
actgacctgt acattttgaa 1801 catattaaag agtaactaag tattctgagc
agaaatagca gcatttggtg tagttgcact 1861 tttgatttga tgagcctgtg
atgtgctaga tccctttaac taatgtatat gtccattttg 1921 cattttattt
gcaaatataa gtgaacagta tatatttcta ggattatacc atttaggaaa 1981
caggtttaca taaacataaa tatccaaatc tattctattt ggctgaatta tgtcaaagta
2041 atcaagtaga atactgaaaa gtgtaagtac gtaataaaat gcaactcaag
aataggctgc 2101 tccttaatgt cattttttca aaagttctac ttgtqtttca
ttcaagctgc tgtgatggag 2161 tggggaatta tgcctttact gctgcagtat
aatctqatga tccatggact gtttaccatt 2221 actttcagat aggactgttt
aaaggaatct tacacaatat agcagctttg atgtcactcc 2281 atctgtgcag
atgacaacag cagaaactcc atagtttaaa atccaggtat ttactgacct 2341
gggtgaagta gattttgaca cgccctttta tagcacatca ccttatttga cttcaagaaa
2401 attcaaaatc caaaagctgc tgtttacttg tacagtacac agatatctat
gagcagctat 2461 gcagtaagta actatgtaag ctatcagaaa gctaagccat
atccatctaa cttgtaaaat 2521 aaacaacgtg ttcactatct gtggcacctg
atataaaggc aagagtctca gcacaagccc 2581 tcctgttatt cctgcaactt
tctgaaatca gaacaatcct gttataaata gatgctacta 2641 tggactcatt
caggaaacca ctaagaaaac atagettcec ttcaacagtt actacatttt 2701
aagatcaaca gcactgctcc acaagcattg ggaaattcag gaggtagact tgagcttagt
2761 ttttctacct acactcatgc tggttctggg gtctcagtaa cacaggaggg
gagaacacca 2821 gccttaccaa gacttcccct gtttcataca gggctcatct
ttaggtcttc tttatgtaac 2881 ttagtagttc atctttttcc atccggtaac
cacttttctt ccactgttca cgcaactgct 2941 gtagcagggc cccaatttcc
ttccctgaag aaatacccac tttcctgatg tcatgtccac 3001 tcacagggaa
cggcgggaca gaccactgct gcatctcttt gagaagacca tgctctcctt 3061
ggtacttcag cagctcacaa acacgggcag ttgcatctgg ttccctagac taaatgacac
3121 agttttatca catactaaac actcacaatt tcattctact atttaaatac
ttacatcaaa 3181 totacagtgt gaaaaagtta cttttctcta gttagtgaga
actattttct gctcagacct 3241 aatacatact tacgtctatc acaaagtctt
ggtatggttt caatggttct gaactatctg 3301 ttgctttaat caagtctttc
ctgtttttaa ctataaataa acccaggttt ttctcctctt 3361 ttgaaatttt
caatctcaag tccaattttg tgacatcatc ttgtactttg aataaagaag 3421
ccaaaagagt cattggtttt ggtgaaaagc cttcaacatt tttactgact ttgttaaatt
3481 cttctaaatt tgcattagca ggtaaacctg taaacaaaag agaaaagtca
tttttttctt 3541 aactacaaaa ccctcactca cctcttaaac tatgcagatt
tttaagaatg tgtagtgttc 3601 tttctccact gcttattatc agcccattcg
tcactccctt aactcctaga agaaatctat 3661 catgttcctg tttcctgtag
cagcatgtct tgtgaagctc aggagctgtg atcatatcag 3721 gtaccagcat
atgccttctc agtcatgatc ctgtctgcac acattcccta ctcagcaatt 3781
gtatgttctt gtaaaacagt caaagttact gtctaaaata tactggctat agttattaat
3841 ttcctttcta tatattaagt gttttgtgaa agagcttatt atacattaac
ttattgcttc 3901 atcctcctct ctatgaagta gcttttattt tgaacccttt
gtgattataa accaacccaa 3961 cctgcaaaac cagtaagctt catcaaattc
aggtgttctc tctgaactat tctttaccaa 4021 taaataaact atttccatct
ttaatcccaa aaaaaaaaaa aaaa NM_175357 1 tttcccaggc tcctttgcgg
gtaaacagac atggccggcg agggagatca gcaggacgct 61 gcgcacaaca
tgggaaacca cctgccgctt ctgcctgcag acagtgaaga tgaagatgat 121
gaaattgaaa tggaagttga agaccaagat agtaaagaag ccagaaaacc gaatatcata
181 aactttgaca ccagtctgcc aacctcacat acatacctgg gagctgatat
ggaggagttc 241 cacgggagaa ctttgcatga cgacgacagc tgccaggtga
tcccagtcct tcctgaggtg 301 ctgatgatcc tgattcctgg gcagacactc
ccactgcagc tctctcaccc acaggaagtc 361 agcatggtgc ggaacttaat
ccagaaagac aggacctttg cagtccttgc atacagtaat 421 gtgcaagaaa
gggaagcaca gtttgggaca acagcagaga tctatgccta tcgagaagag 481
caggagtttg gaattgaagt agtgaaagtg aaagcaattg gaaggcagcg gttcaaggtc
541 ctcgaacttc gaacacagtc agatggaatc cagcaagcta aagtgcagat
tttgccagag 601 tgtgtgttgc cgtcaaccat gtctgcagtg cagttagaat
cactcaataa gtgccaggta 661 tttccttcaa aacccatctc ctgggaagac
cagtattcat gtaaatggtg gcagaaatac 721 cagaagagaa agtttcactg
tgcaaatcta acatcatggc ctcgctggct gtattcatta 781 tatgatgctg
aaacattaat ggatagaatt aagaaacagc tacgtgaatg ggatgaaaat 841
ctcaaagatg attctcttcc tgaaaatcca atagactttt cttacagagt agctgcttgt
901 cttcctattg atgatgtatt gagaattcag ctccttaaaa tcggcagtgc
tattcaacgg 961 cttcgctgtg aattggacat catgaacaaa tgtacttccc
tttgctgtaa acaatgtcaa 1021 gaaacagaaa taacgacaaa gaatgaaata
tttagtttat ccttatgtgg tccaatggca 1081 gcatatgtga atcctcatgg
atatgtacat gagacactga ctgtgtataa agcgtccaac 1141 ctgaatctga
taggccggcc ttctacagtg cacagctggt ttcccgggta tgcatggacc 1201
attgcccagt gcaagatctg tgcaagccat attggatgga aatttacagc cacaaaaaaa
1261 gacatgtcac ctcaaaaatt ttggggctta actcgctctg ctctgttacc
cacaattcca 1321 gagactgaag atgaaataag tccagacaaa gtaatacttt
gtttataagt gcacctgtag 1381 gagtgacttc ctgacagata tttcctcaag
tcagatctgc ccagtcatca ctgcctctga 1441 tatatgtgta tagtgggtta
cagcatttgc ctaccaagtt caagagcata tttagggaat 1501 gagaaagcag
tataaaacat aaggctgggt tccaaaatac ttgcttttta gtagcttggt 1561
gccatggatt atcctgttga gtctatgtca tgacaggata ggaaaacaca gttgaaataa
1621 tgggaatggc catggaacag gataggggca ccactgctct aaatgatgaa
gctctaaatg 1681 atgaatgctc cagaaactgg gttggtaagc acaagataga
ggcaaggcag tgtaatttta 1741 aaaggacttt gctcctttca attttcctta
gcttgtctga gatactgacc tgtacatttt 1801 gaacatatta aagagtaact
aagtattctg agcagaaata gcagcatttg gtgtagttgc 1861 acttttgatt
tgacgagcct gtgatgtgct agatcccttt aactaatgta tatgtccatt 1921
ttgcatttta tttgcaaata taagtgaaca gtatatattt ctaggattat accatttagg
1981 aaacaggttt acataaacat aaatatccaa atctattcta tttggctgaa
ttatgtcaaa 2041 gtaatcaagt agaatactga aaagtgtaag tacgtaataa
aatgcaactc aagaataggc 2101 tgctccttaa tgtcattttt tcaaaagttc
tacttgtgtt tcattcaagc tgctgtgatg 2161 gagtggggaa ttatgccttt
actgctgcag tataatctga tgatccatgg actgtttacc 2221 attactttca
gataggactg tttaaaggaa tcttacacaa tatagcagct ttgatgtcac 2281
tccatctgtg cagatgacaa cagcagaaac tccatagttt aaaatccagg tatttactga
2341 cctgggtgaa gtagattttg acacgccctt ttatagcaca tcaccttatt
tgacttcaag 2401 aaaattcaaa atccaaaagc tgctgtttac ttgtacagta
cacagatatc tatgagcagc 2461 tatgcagtaa gtaactatgt aagctatcag
aaagctaagc catatccatc taacttgtaa 2521 aataaacaat gtgttcacta
tctgtggcac ctqatataaa ggcaagagtc tcagcacaag 2581 ccctcctgtt
attcctgcaa ctttctgaaa tcagaacaat cctgttataa atagatgcta 2641
ctatggactc attcaggaaa ccactaagaa aacatagttt ctcttcaaca gttactacat
2701 tttaagatca acagcactgc tccacaagca ttgggaaatt caggaggtag
acttgagctt 2761 agtttttcta cctacactca tgctggtttt ggggtctcag
taacacagga ggggagaaca 2821 ccagccttac caagacttcc cctgtttcat
acagggctca tctttaggtc ttctttatgt 2881 aacttagtag ttcatctttt
tccatccggt aaccactttt cttccactgt tcacgcaact 2941 gctgtagcag
ggccccaatt tccttccctg aagaaatacc cactttcctg atgtcatgtc 3001
cactcacagg gaacggcggg acagaccact gctgcatctc tttgagaaga ccatgctctc
3061 cttggtactt cagcagctca caaacacggg cagttgcatc tggttcccta
gactaaatga 3121 cacagtttta tcacatacta aacactcaca atttcattct
actatttaaa tacttacatc 3181 aaatctacag tgtgaaaaag ttacttttct
ctagttagtg agaactattt tctgctcaga 3241 cctaatacat acttacgtct
atcacaaagt cttggtatgg tttcaatggt tctgaactat 3301 ctgttgcttt
aatcaagtct ttcctgtttt taactataaa taaacccagg tttttctcct
3361 cttttgaaat tttcaatctc aagtccaatt ttgtgacatc atcttgtact
ttgaataaag 3421 aagccaaaag agtcattggt tttggtgaaa agccttcaac
atttttactg actttgttaa 3481 attcttctaa atttgcatta gcaggtaaac
ctgtaaacaa aagagaaaag tcattttttt 3541 cttaactaca aaaccctcac
tcacctctta aactatgcag atttttaaga atgtgtagtg 3601 ttctttctcc
actgcttatt atcagcccat tcgtcactcc cttaactcct agaagaaatc 3661
tatcatgttc ctgtttcctg tagcagcatg tcttgtgaag ctcaggagct gtgatcatat
3721 caggtaccag catatgcctt ctcagtcatg atcctgtctg cacacattcc
ctactcagca 3781 attgtatgtt cttgtaaaac agtcaaagtt actgtctaaa
atatactggc tatagttatt 3841 aatttccttt ctatatatta agtgttttgt
gaaagagctt attatacatt aacttattgc 3901 ttcatcctcc tctctatgaa
gtagctttta ttttgaaccc tttgtgatta taaaccaacc 3961 caacctgcaa
aaccagtaag cttcatcaaa ttcaggtgtt ctctctgaac tattctttac 4021
caataaataa actatttcca tctttaatcc caaaaaaaaa aaaaaaa
[0035] By "casein kinase 1A1 polypeptide" is meant a protein having
at least about 85% or greater identity to Unit Pro Accession No.
P48729-1 or P48729-2 (having a phosphor serine at position 156) and
having kinase activity.
TABLE-US-00009 10 20 30 40 50 60 MASSSGSKAE FIVGGKYKLV RKIGSGSFGD
IYLAINITNG EEVAVKLESQ KARHPQLLYE 70 80 90 100 110 120 SKLYKILQGG
VGIPHIRWYG QEKDYNVLVM DLLGPSLEDL FNFCSRRFTM KTVLMLADQM 130 140 150
160 170 180 ISRIEYVHTK NFIHRDIKPD NFLMGIGRHC NKLFLIDFGL AKKYRDNRTR
QHIRYREDKN 190 200 210 220 230 240 LTGTARYASI NAHLGIEQSR RDDMESLGYV
LMYFNRTSLP WQGLKAATKK QKYEKISEKK 250 260 270 280 290 300 MSTPVEVLCK
GFPAEFAMYL NYCRGLRFEE APDYMYLRQL FRILFRTLNH QYDYTFDWTM 310 320 330
LKQKAAQQAA SSSGQGQQAQ TPTGKQTDKT KSNMKGF
[0036] By "casein kinase 1A1 polynucleotide" is meant a
polynucleotide encoding a casein kinase 1A1 polypeptide.
[0037] By "B cell neoplasia" is meant any neoplasia arising from a
B-cell progenitor or other cell of B cell lineage. In particular
embodiments, a B cell neoplasia arises from a cell type undergoing
B cell differentiation. In other embodiments, a B cell neoplasia
includes plasma cells.
[0038] By "mutant CRBN" is meant any mutation of murine CRBN to
include at least one of S369C, V380E, I391V, or any other
substitution, deletion or addition of the murine CRBN that confers
lenalidomide sensitivity to CSNK1A1.
[0039] By "myeloid malignancy" is meant a condition associated with
a defect in the proliferation of a hematopoietic cell.
Myelodysplastic syndrome with deletion of 5q.
[0040] By "agent" is meant any small molecule chemical compound,
antibody, nucleic acid molecule, or polypeptide, or fragments
thereof.
[0041] By "ameliorate" is meant decrease, suppress, attenuate,
diminish, arrest, or stabilize the development or progression of a
disease.
[0042] By "alteration" is meant a change. In one embodiment, an
alteration characterized in accordance with the methods of the
invention is a change in the sequence of a polypeptide or
polynucleotide. In another embodiment, an alteration characterized
in accordance with the methods of the invention is an increase or
decrease in the level, biological activity, or post-transcriptional
modification of a polypeptide (e.g., IKZF1, IKZF3) as detected by
standard art known methods such as those described herein. As used
herein, an alteration includes 10%, 25%, 50%, 75%, 85%, 95% or
greater increase or decrease in level or biological activity.
[0043] By "lenalidomide sensitivity" is meant that at least one
symptom of a pathological condition is ameliorated by treatment
with lenalidomide or a lenalidomide analog.
[0044] By "lenalidomide resistant" is meant that a neoplastic cell
has acquired an alteration that allows it to escape an
anti-neoplastic effect of lenalidomide. Exemplary anti-neoplastic
effects include, but are not limited to, any effect that reduces
proliferation, reduces survival, and/or increases cell death (e.g.,
increases apoptosis).
[0045] By "analog" is meant a molecule that is not identical, but
has analogous functional or structural features. Lenalidomide
analogs include, but are not limited to, thalidomide or
pomalidomide. By "biological sample" is meant any liquid, cell, or
tissue obtained from a subject.
[0046] In this disclosure, "comprises," "comprising," "containing"
and "having" and the like can have the meaning ascribed to them in
U.S. patent law and can mean "includes," "including," and the like;
"consisting essentially of" or "consists essentially" likewise has
the meaning ascribed in U.S. patent law and the term is open-ended,
allowing for the presence of more than that which is recited so
long as basic or novel characteristics of that which is recited is
not changed by the presence of more than that which is recited, but
excludes prior art embodiments.
[0047] "Detect" refers to identifying the presence, absence or
amount of the analyte to be detected.
[0048] By "detectable label" is meant a composition that when
linked to a molecule of interest renders the latter detectable, via
spectroscopic, photochemical, biochemical, immunochemical, or
chemical means. For example, useful labels include radioactive
isotopes, magnetic beads, metallic beads, colloidal particles,
fluorescent dyes, electron-dense reagents, enzymes (for example, as
commonly used in an ELISA), biotin, digoxigenin, or haptens.
[0049] By "effective amount" is meant the amount of an agent
required to ameliorate the symptoms of a disease relative to an
untreated patient. The effective amount of active compound(s) used
to practice the present invention for therapeutic treatment of a
disease varies depending upon the manner of administration, the
age, body weight, and general health of the subject. Ultimately,
the attending physician or veterinarian will decide the appropriate
amount and dosage regimen. Such amount is referred to as an
"effective" amount.
[0050] By "fragment" is meant a portion of a polypeptide or nucleic
acid molecule. This portion contains, preferably, at least 10%,
20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the entire length of
the reference nucleic acid molecule or polypeptide. A fragment may
contain 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100, 200, 300, 400,
500, 600, 700, 800, 900, or 1000 nucleotides or amino acids.
[0051] The invention provides a number of targets that are useful
for the development of highly specific drugs to treat or a disorder
characterized by the methods delineated herein. In addition, the
methods of the invention provide a facile means to identify
therapies that are safe for use in subjects. In addition, the
methods of the invention provide a route for analyzing virtually
any number of compounds for effects on a disease described herein
with high-volume throughput, high sensitivity, and low
complexity.
[0052] By "inhibitory nucleic acid" is meant a double-stranded RNA,
siRNA, shRNA, or antisense RNA, or a portion thereof, or a mimetic
thereof, that when administered to a mammalian cell results in a
decrease (e.g., by 10%, 25%, 50%, 75%, or even 90-100%) in the
expression of a target gene. Typically, a nucleic acid inhibitor
comprises at least a portion of a target nucleic acid molecule, or
an ortholog thereof, or comprises at least a portion of the
complementary strand of a target nucleic acid molecule. For
example, an inhibitory nucleic acid molecule comprises at least a
portion of any or all of the nucleic acids delineated herein.
[0053] The terms "isolated," "purified," or "biologically pure"
refer to material that is free to varying degrees from components
which normally accompany it as found in its native state. "Isolate"
denotes a degree of separation from original source or
surroundings. "Purify" denotes a degree of separation that is
higher than isolation. A "purified" or "biologically pure" protein
is sufficiently free of other materials such that any impurities do
not materially affect the biological properties of the protein or
cause other adverse consequences. That is, a nucleic acid or
peptide of this invention is purified if it is substantially free
of cellular material, viral material, or culture medium when
produced by recombinant DNA techniques, or chemical precursors or
other chemicals when chemically synthesized. Purity and homogeneity
are typically determined using analytical chemistry techniques, for
example, polyacrylamide gel electrophoresis or high performance
liquid chromatography. The term "purified" can denote that a
nucleic acid or protein gives rise to essentially one band in an
electrophoretic gel. For a protein that can be subjected to
modifications, for example, phosphorylation or glycosylation,
different modifications may give rise to different isolated
proteins, which can be separately purified.
[0054] By "isolated polynucleotide" is meant a nucleic acid (e.g.,
a DNA) that is free of the genes which, in the naturally-occurring
genome of the organism from which the nucleic acid molecule of the
invention is derived, flank the gene. The term therefore includes,
for example, a recombinant DNA that is incorporated into a vector;
into an autonomously replicating plasmid or virus; or into the
genomic DNA of a prokaryote or eukaryote; or that exists as a
separate molecule (for example, a cDNA or a genomic or cDNA
fragment produced by PCR or restriction endonuclease digestion)
independent of other sequences. In addition, the term includes an
RNA molecule that is transcribed from a DNA molecule, as well as a
recombinant DNA that is part of a hybrid gene encoding additional
polypeptide sequence.
[0055] By an "isolated polypeptide" is meant a polypeptide of the
invention that has been separated from components that naturally
accompany it. Typically, the polypeptide is isolated when it is at
least 60%, by weight, free from the proteins and
naturally-occurring organic molecules with which it is naturally
associated. Preferably, the preparation is at least 75%, more
preferably at least 90%, and most preferably at least 99%, by
weight, a polypeptide of the invention. An isolated polypeptide of
the invention may be obtained, for example, by extraction from a
natural source, by expression of a recombinant nucleic acid
encoding such a polypeptide; or by chemically synthesizing the
protein. Purity can be measured by any appropriate method, for
example, column chromatography, polyacrylamide gel electrophoresis,
or by HPLC analysis.
[0056] By "marker" or "biomarker" is meant any protein or
polynucleotide having an alteration in expression level or activity
that is associated with a disease or disorder.
[0057] As used herein, "obtaining" as in "obtaining an agent"
includes synthesizing, purchasing, or otherwise acquiring the
agent.
[0058] By "reference" is meant a standard or control condition.
[0059] A "reference sequence" is a defined sequence used as a basis
for sequence comparison. A reference sequence may be a subset of or
the entirety of a specified sequence; for example, a segment of a
full-length cDNA or gene sequence, or the complete cDNA or gene
sequence. For polypeptides, the length of the reference polypeptide
sequence will generally be at least about 16 amino acids,
preferably at least about 20 amino acids, more preferably at least
about 25 amino acids, and even more preferably about 35 amino
acids, about 50 amino acids, or about 100 amino acids. For nucleic
acids, the length of the reference nucleic acid sequence will
generally be at least about 50 nucleotides, preferably at least
about 60 nucleotides, more preferably at least about 75
nucleotides, and even more preferably about 100 nucleotides or
about 300 nucleotides or any integer thereabout or
therebetween.
[0060] Nucleic acid molecules useful in the methods of the
invention include any nucleic acid molecule that encodes a
polypeptide of the invention or a fragment thereof. Such nucleic
acid molecules need not be 100% identical with an endogenous
nucleic acid sequence, but will typically exhibit substantial
identity. Polynucleotides having "substantial identity" to an
endogenous sequence are typically capable of hybridizing with at
least one strand of a double-stranded nucleic acid molecule.
Nucleic acid molecules useful in the methods of the invention
include any nucleic acid molecule that encodes a polypeptide of the
invention or a fragment thereof. Such nucleic acid molecules need
not be 100% identical with an endogenous nucleic acid sequence, but
will typically exhibit substantial identity. Polynucleotides having
"substantial identity" to an endogenous sequence are typically
capable of hybridizing with at least one strand of a
double-stranded nucleic acid molecule. By "hybridize" is meant pair
to form a double-stranded molecule between complementary
polynucleotide sequences (e.g., a gene described herein), or
portions thereof, under various conditions of stringency. (See,
e.g., Wahl, G. M. and S. L. Berger (1987) Methods Enzymol. 152:399;
Kimmel, A. R. (1987) Methods Enzymol. 152:507).
[0061] For example, stringent salt concentration will ordinarily be
less than about 750 mM NaCl and 75 mM trisodium citrate, preferably
less than about 500 mM NaCl and 50 mM trisodium citrate, and more
preferably less than about 250 mM NaCl and 25 mM trisodium citrate.
Low stringency hybridization can be obtained in the absence of
organic solvent, e.g., formamide, while high stringency
hybridization can be obtained in the presence of at least about 35%
formamide, and more preferably at least about 50% formamide.
Stringent temperature conditions will ordinarily include
temperatures of at least about 30.degree. C., more preferably of at
least about 37.degree. C., and most preferably of at least about
42.degree. C. Varying additional parameters, such as hybridization
time, the concentration of detergent, e.g., sodium dodecyl sulfate
(SDS), and the inclusion or exclusion of carrier DNA, are well
known to those skilled in the art. Various levels of stringency are
accomplished by combining these various conditions as needed. In a
preferred: embodiment, hybridization will occur at 30.degree. C. in
750 mM NaCl, 75 mM trisodium citrate, and 1% SDS. In a more
preferred embodiment, hybridization will occur at 37.degree. C. in
500 mM NaCl, 50 mM trisodium citrate, 1% SDS, 35% formamide, and
100.mu.g/ml denatured salmon sperm DNA (ssDNA). In a most preferred
embodiment, hybridization will occur at 42.degree. C. in 250 mM
NaCl, 25 mM trisodium citrate, 1% SDS, 50% formamide, and 200
.mu.g/ml ssDNA. Useful variations on these conditions will be
readily apparent to those skilled in the art.
[0062] For most applications, washing steps that follow
hybridization will also vary in stringency. Wash stringency
conditions can be defined by salt concentration and by temperature.
As above, wash stringency can be increased by decreasing salt
concentration or by increasing temperature. For example, stringent
salt concentration for the wash steps will preferably be less than
about 30 mM NaCl and 3 mM trisodium citrate, and most preferably
less than about 15 mM NaCl and 1.5 mM trisodium citrate. Stringent
temperature conditions for the wash steps will ordinarily include a
temperature of at least about 25.degree. C., more preferably of at
least about 42.degree. C., and even more preferably of at least
about 68.degree. C. In a preferred embodiment, wash steps will
occur at 25.degree. C. in 30 mM NaCl, 3 mM trisodium citrate, and
0.1% SDS. In a more preferred embodiment, wash steps will occur at
42 C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. In a
more preferred embodiment, wash steps will occur at 68.degree. C.
in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. Additional
variations on these conditions will be readily apparent to those
skilled in the art. Hybridization techniques are well known to
those skilled in the art and are described, for example, in Benton
and Davis (Science 196:180, 1977); Grunstein and Hogness (Proc.
Natl. Acad. Sci., USA 72:3961, 1975); Ausubel et al. (Current
Protocols in Molecular Biology, Wiley Interscience, New York,
2001); Berger and Kimmel (Guide to Molecular Cloning Techniques,
1987, Academic Press, New York); and Sambrook et al., Molecular
Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press,
New York.
[0063] By "substantially identical" is meant a polypeptide or
nucleic acid molecule exhibiting at least 50% identity to a
reference amino acid sequence (for example, any one of the amino
acid sequences described herein) or nucleic acid sequence (for
example, any one of the nucleic acid sequences described herein).
Preferably, such a sequence is at least 60%, more preferably 80% or
85%, and more preferably 90%, 95% or even 99% identical at the
amino acid level or nucleic acid to the sequence used for
comparison.
[0064] Sequence identity is typically measured using sequence
analysis software (for example, Sequence Analysis Software Package
of the Genetics Computer Group, University of Wisconsin
Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705,
BLAST, BESTFIT, GAP, or PILEUP/PRETTYBOX programs). Such software
matches identical or similar sequences by assigning degrees of
homology to various substitutions, deletions, and/or other
modifications. Conservative substitutions typically include
substitutions within the following groups: glycine, alanine;
valine, isoleucine, leucine; aspartic acid, glutamic acid,
asparagine, glutamine; serine, threonine; lysine, arginine; and
phenylalanine, tyrosine. In an exemplary approach to determining
the degree of identity, a BLAST program may be used, with a
probability score between e.sup.-3 and e.sup.-100 indicating a
closely related sequence.
[0065] By "siRNA" is meant a double stranded RNA. Optimally, an
siRNA is 18, 19, 20, 21, 22, 23 or 24 nucleotides in length and has
a 2 base overhang at its 3' end. These dsRNAs can be introduced to
an individual cell or to a whole animal; for example, they may be
introduced systemically via the bloodstream. Such siRNAs are used
to downregulate mRNA levels or promoter activity.
[0066] By "specifically binds" is meant a compound or antibody that
recognizes and binds a polypeptide of the invention, but which does
not substantially recognize and bind other molecules in a sample,
for example, a biological sample, which naturally includes a
polypeptide of the invention.
[0067] By "subject" is meant a mammal, including, but not limited
to, a human or non-human mammal, such as a bovine, equine, canine,
ovine, or feline.
[0068] By "transgene" is meant any piece of DNA which is inserted
by artifice into a cell, and becomes part of the genome of the
organism which develops from that cell. Such a transgene may
include a gene which is partly or entirely heterologous (i.e.,
foreign) to the transgenic organism, or may represent a gene
homologous to an endogenous gene of the organism.
[0069] By "transgenic" is meant any cell which includes a DNA
sequence which is inserted by artifice into a cell and becomes part
of the genome of the organism which develops from that cell. As
used herein, the transgenic organisms are generally transgenic
mammalian (e.g., rodents such as rats or mice) and the DNA
(transgene) is inserted by artifice into the nuclear genome.
[0070] By "transformation" is meant any method for introducing
foreign molecules into a cell. Lipofection, calcium phosphate
precipitation, retroviral deliver, electroporation and biolistic
transformation are just a few of the teachings which may be used.
For example, Biolistic transformation is a method for introducing
foreign molecules into a cell using velocity driven
microprojectiles such as tungsten or gold particles. Such
velocity-driven methods originate from pressure bursts which
include, but are not limited to, helium-driven, air-driven, and
gunpowder-driven techniques. Biolistic transformation may be
applied to the transformation or transfection of a wide variety of
cell types and intact tissues including, without limitation,
intracellular organdies (e.g., and mitochondria and chloroplasts),
bacteria, yeast, fungi, algae, animal tissue, and cultured
cells.
[0071] By "positioned for expression" is meant that the DNA
molecule is positioned adjacent to a DNA sequence which directs
transcription and translation of the sequence (i.e., facilitates
the production of, e.g., an IAP polypeptide, a recombinant protein
or a RNA molecule).
[0072] By "promoter" is meant minimal sequence sufficient to direct
transcription. Also included in the invention are those promoter
elements which are sufficient to render promoter-dependent gene
expression controllable for cell-type specific, tissue-specific or
inducible by external signals or agents; such elements may be
located in the 5' or 3' regions of the native gene.
[0073] By "operably linked" is meant that a gene and a regulatory
sequence(s) are connected in such a way as to permit gene
expression when the appropriate molecules (e.g., transcriptional
activator proteins) are bound to the regulatory sequence(s).
[0074] Ranges provided herein are understood to be shorthand for
all of the values within the range. For example, a range of 1 to 50
is understood to include any number, combination of numbers, or
sub-range from the group consisting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,
28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44,
45, 46, 47, 48, 49, or 50.
[0075] As used herein, the terms "treat," treating," "treatment,"
and the like refer to reducing or ameliorating a disorder and/or
symptoms associated therewith. It will be appreciated that,
although not precluded, treating a disorder or condition does not
require that the disorder, condition or symptoms associated
therewith be completely eliminated.
[0076] Unless specifically stated or obvious from context, as used
herein, the term "or" is understood to be inclusive. Unless
specifically stated or obvious from context, as used herein, the
terms "a", "an", and "the" are understood to be singular or
plural.
[0077] Unless specifically stated or obvious from context, as used
herein, the term "about" is understood as within a range of normal
tolerance in the art, for example within 2 standard deviations of
the mean. About can be understood as within 10%, 9%, 8%, 7%, 6%,
5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated
value. Unless otherwise clear from context, all numerical values
provided herein are modified by the term about.
[0078] The recitation of a listing of chemical groups in any
definition of a variable herein includes definitions of that
variable as any single group or combination of listed groups. The
recitation of an embodiment for a variable or aspect herein
includes that embodiment as any single embodiment or in combination
with any other embodiments or portions thereof.
[0079] Any compositions or methods provided herein can be combined
with one or more of any of the other compositions and methods
provided herein.
BRIEF DESCRIPTION OF THE DRAWINGS
[0080] FIGS. 1A-1D provide a proteomic analysis of
lenalidomide-induced changes in ubiquitination, protein abundance
and CRBN interaction in MM1S cells. FIG. 1A is a schematic diagram
showing the experimental design for SILAC-based assessment of
global changes in ubiquitination and protein levels. Cells were
treated for 12 hours with DMSO, lenalidomide, or thalidomide. For
ubiquitination analysis 5 .mu.M MG132 were added for the last 3
hours. FIG. 1B is a ubiquitin analysis. Log.sub.2 ratios for
individual K-.epsilon.-GG sites of lenalidomide versus DMSO treated
cells for replicate 1 and 2. Each dot represents a unique
K-.epsilon.-GG site. FIG. 1C shows a proteome analysis. Log.sub.2
ratios of changes of protein abundance of lenalidomide versus DMSO
treated cells. Each dot represents a distinct protein group. FIG.
1D shows a CRBN interaction analysis in cells treated for 6 hours
with 1 .mu.M lenalidomide. Scatter plot shows log.sub.2 changes of
proteins pulled down by HA-CBRN in lenalidomide versus DMSO treated
control cells.
[0081] FIGS. 2A-2C are provided. FIG. 2A shows the synthesis of a
lenalidomide derivative immobilized to a bead that was used to pull
down proteins binding lenalidomide FIG. 28 provides two graphs
showing the viability (CellTiter-Glo.RTM. Luminescent Cell
Viability Assay, Promega) of lenalidomide sensitive MM1S cells and
lenalidomide insensitive K562 cells treated with lenalidomide or
lenalidomide derivative for 6 days. FIG. 2C shows a schematic
overview of pull down of candidate protein binders to lenalidomide
beads. K562 cells were cultured in light (R0K0) or heavy (R10K6)
SILAC media for 14 days to allow for quantitative assessment of
proteins binding the lenalidomide derivative bead by LC-MS/MS. In
the second condition cell lysates were additionally incubated with
soluble lenalidomide to compete off binding proteins. The ratio of
proteins pulled down in the lenalidomide beads only versus
lenalidomide beads with soluble lenalidomide represent proteins
that specifically bind lenalidomide. For a biological replicate
SILAC labeling for the two conditions was switched.
[0082] FIGS. 3A and 3B show the results of a proteomic assessment
of thalidomide induced in vivo changes of global ubiquitination and
proteome. FIG. 3A shows scatter plots for log.sub.2 ratios for
individual K-.epsilon.-GG sites of lenalidomide versus DMSO treated
cells for replicate 1 and 2. Each dot represents an individual KeGG
site. The table shows median log.sub.2 ratios from all 3
replicates. FIG. 3B shows Log.sub.2 ratios of changes of protein
abundance in lenalidomide versus DMSO treated cells. Each dot
represents an individual protein. The table shows median log.sub.2
ratios from 2 replicates.
[0083] FIG. 4 provides schematic diagrams illustrating the
experimental design for SILAC-based assessment of CRBN interaction
analysis in MM1S cells. HA-CRBN of DMSO and lenalidomide treated
cells was immunoprecipitated with anti-HA Sepharose conjugate
beads. Lysates of FLAG-CRBN expressing cells served as a negative
control to exclude non-specific binding to the antibody-sepharose
conjugate.
[0084] FIGS. 5A and 5B show results of CRBN co-immunoprecipitation,
continued from FIG. 1G. FIG. 5A shows Scatter plots with log.sub.2
ratios for (HA-CRBN expressing) DMSO treated versus (FLAG-CRBN
expressing) control cells (left) and lenalidomide versus DMSO
treated cells (both expressing HA-CRBN) (right). FIG. 5B shows a
list of top (co-)immunoprecipitated proteins from DMSO treated
versus control, lenalidomide treated versus control and
lenalidomide versus DMSO treated cells. For log.sub.2 ratios of
lenalidomide versus DMSO treated cells only proteins that bound to
CRBN in presence of lenalidomide and/or DMSO with a log.sub.2 ratio
>0.5 in both replicates were considered.
[0085] FIGS. 6A-6F show the effect of lenalidomide on IKZF1 and
IKZF3 protein levels. FIG. 6A is a graph. 293T cells transfected
with vectors expressing the indicated cDNA fused to firefly
luciferase and control renilla luciferase were treated with DMSO or
1 .mu.M lenalidomide for 24 hours. Bars represent the firefly to
renilla luciferase ratio, normalized to DMSO-treated cells. FIG. 6B
is a Western blot showing the effects of lenalidomide on endogenous
IKZF1 and IKZF3 in MM1S cells treated for 24 hours. FIG. 6C is a
Western blot showing a time course of lenalidomide treatment in
MM1S cells for IKZF1 and IKZF3 protein levels and FIG. 6D mRNA
levels. FIG. 6E provides immunoblots. Primary multiple myeloma
samples were treated for 6 hours and analyzed by immunoblot. FIG.
6F shows an in vivo ubiquitination analysis of HA-tagged IKZF1 and
IKZF3 expressed in MM1S cells treated for 1.5 hours with 100 nM
Epoxomicin and the indicated concentrations of lenalidomide. The
FK2 antibody detects covalently linked ubiquitin.
[0086] FIGS. 7A and 7B show that Lenalidomide induced decrease of
IKZF1 and IKZF3 in different cell lines. Cells were treated in the
presence of the respective lenalidomide concentrations for 24
hours. MM1S cells were treated with DMSO or 1 .mu.M lenalidomide in
the presence of 100 .mu.g/ml Cycloheximide.
[0087] FIGS. 8A-8C show the in vivo ubiquitination of IKZF1 and
IKZF3. Cells were treated with the indicated concentrations of
lenalidomide and/or 100 nM epoxomicin for 1.5 hours. FIG. 8A shows
results in 293T cells expressing stably transduced with a
retrovirus expressing FLAG-IKZF3. FIG. 8B shows results in MM1S
cells stably expressing HA-IKZF1. FIG. 8C shows endogenous IKZF3 of
MM1S cells was immunoprecipitated by an polyclonal IKZF3
antibody.
[0088] FIGS. 9A-9D show that CRBN is a substrate receptor for IKZF1
and IKZF3. FIG. 9A is a Western blot showing the
immunoprecipitation of endogenous CRBN in MM1S cells treated for 1
hour with the indicated drugs. FIG. 9B is shows the results of an
in vitro ubiquitination reaction of HA-IKZF3 co-immunoprecipitated
by FLAG-CRBN from 293T cells and incubated in the presence or
absence of E1 and E2 ubiquitin conjugating enzymes. FIG. 9C is a
schematic diagram showing the mapping of the degron that confers
lenalidomide sensitivity. Blue boxes in the IKZF3 protein represent
zinc finger domains. FIG. 9D shows a sequence alignment of the core
lenalidomide degron between the 5 Ikaros proteins. Western blots of
293T cells lysates 48 hours after co-transfection of FLAG-tagged
IKZF3 or IKZF4 with HA-tagged CRBN and 24 hours drug treatment.
[0089] FIG. 10 is a graph showing rescue of lenalidomide induced
growth inhibition by expression of CRBN.sup.YWAA that does not bind
lenalidomide. NCI-H929 cells were transduced with a retroviral
vector expressing CRBN wildtype and GFP or CRBN.sup.YWAA and
dTomato. Two days after transduction cells were mixed and treated
with the indicated concentrations of lenalidomide. The ratio of
dTomato versus GFP expressing cells was assessed by flow
cytometry.
[0090] FIG. 11A-11C shows deletion mapping of IKZF3. FIG. 11A
provides a representation of all IKZF3 mutants tested. Response to
lenalidomide was assessed with an ORF-luciferase reporter. The red
box indicates the critical peptide sequence (amino acids 140 to 180
of IKZF3) necessary for lenalidomide sensitivity. Substitution in
the H177P/L178F mutant is based on the sequence alignment of IKZF1
and IKZF3 versus IKZF2 and IKZF4 and does not affect lenalidomide
sensitivity in contrast to the Q147H substitution in IKZF3. FIG.
11B shows validation of lenalidomide response by western blot for
several IKZF3 mutants. FLAG-tagged versions were cloned into the
RSF91 vector, transfected into 293T cells together with a plasmid
expressing HA-CRBN. After 24 hours media was replaced with media
containing lenalidomide in the indicated concentrations and
incubated another 24 hours before lysis. FIG. 11C shows the
co-immunoprecipitation of FLAG-IKZF3 and its mutants by HA-CRBN.
293T cells were transfected with the indicated plasmids. After 48
hours 1 .mu.M lenalidomide was added for 1 hour before lysis and
HA-immunoprecipitation.
[0091] FIGS. 12A-12F shows the biological role of IKZF1 and IKZF3
in multiple myeloma cell lines and T cells. FIG. 12A includes two
graphs showing that Lenalidomide-sensitive and insensitive cell
lines were infected with lentivirus expressing IKZF1 or IKZF3
specific shRNAs and GFP. Relative depletion was assessed by flow
cytometry and normalized to day 2 post infection. FIG. 12B is a
graph showing that MM1S cells were transduced with retrovirus
expressing GFP and wild-type IKZF3 or a dominant negative IKZF3
Isoform with deletion of the complete DNA binding region. FIG. 12C
includes two graphs showing that MM1S cells were infected with
different retrovirus and competed against each other in media
containing DMSO or lenalidomide. Left panel: IKZF3.sup.wt/GFP
versus empty vector/dTomato. Right panel: IKZF3.sup.Q150H/GFP
versus IKZF3.sup.wt/dTomato. FIG. 12D shows results from human CD3+
T cells isolated from buffy coats of healthy blood donors were
stimulated with plate-bound anti-CD3 and anti-CD28 and treated with
different concentrations of lenalidomide for 24 hours. FIG. 12E is
a graph. T cells were infected with lentiviral vectors expressing
shRNAs targeting the indicated genes. After selection with
puromycin, T cells were stimulated with anti-CD3/CD28 Dynabeads and
treated with DMSO or 1 .mu.M lenalidomide for 12 hours before
lysis. IL-2 RNA expression levels were analyzed by quantitative
RT-PCR using GAPDH expression as an internal control. FIG. 12F is a
graph. IL2 expression was measured in lenalidomide treated T cells
expressing CRBN or control shRNAs.
[0092] FIG. 13 shows the Effect of a 2.sup.nd shRNA for IKZF1 and
IKZF3, respectively on cell growth of multiple myeloma and
lenalidomide-insensitive cell lines. Same experimental setup as in
FIG. 4A.
[0093] FIGS. 14A-14E show that lenalidomide and IKZF3 depletion
result in decreased expression of IRF4 in MM1S cells. In FIG. 14A
MM1S cells were treated for up to 48 hours with lenalidomide and
IRF4 protein levels determined by immunoblot. FIG. 14B shows IRF4,
IKZF1 and IKZF3 protein changes after 12 hours of lenalidomide
treatment assessed by quantitative MS. FIG. 14C is a graph showing
results of an RQ-PCR analysis of IRF4 RNA levels after 24 and 48
hour treatment with 1 .mu.M lenalidomide. FIG. 14D shows an IRF4
Immunoblot of MM1S cells that were transduced with lentivirus
expressing luciferase or IKZF3-specific shRNAs. FIG. 14E is a graph
showing IRF4 RNA expression levels after IKZF3 knockdown.
[0094] FIG. 15A-15C include graphs (left hand panel) and
immunoblots (right hand panel). Knockdown of shRNAs was assessed by
RQ-PCR and immunoblot in MM1S cells for (FIG. 15A) IKZF1, (FIG.
15B) IKZF3, and (FIG. 15C) CRBN.
[0095] FIG. 16 shows results of SILAC-based quantitative MS studies
used to characterize changes in the ubiquitinome and proteome in
the MM1S multiple myeloma cell line cultured in the presence of
lenalidomide.
[0096] FIG. 17 shows that lenalidomide treatment results in a
dose-dependent decrease in casein kinase 1A1 (CSNK1A) protein
levels in lenalidomide sensitive multiple myeloma cells. No
significant change is observed in RNA expression.
[0097] FIG. 18 shows that lenalidomide treatment did not alter
casein kinase 1A1 (CSNK1A) protein levels in mice.
[0098] FIG. 19 shows that expression of human CRBN in murine cells
was sufficient to confer lenalidomide sensitivity to CSNK1A1.
[0099] FIGS. 20A-20D show lenalidomide-induced changes in
ubiquitination and protein levels in KG-1 cells. FIG. 20A shows the
log.sub.2 ratios for individual K-.epsilon.-GG sites of
lenalidomide- (1 .mu.M) versus DMSO-treated cells for replicates 1
and 2. Each dot represents a unique K-.epsilon.-GG site. FIG. 20B
shows the log.sub.2 ratios of changes of protein abundance of
lenalidomide- (1 .mu.M) versus DMSO-treated cells for replicates 1
and 2. Each dot represents a unique protein group. FIG. 20C shows
the effects of lenalidomide on endogenous CSNK1A1 levels in KG-1
cells after 24-hour treatment. FIG. 20D shows a time course of
lenalidomide treatment in KG-1 cells for CSNK1A1 mRNA levels.
[0100] FIGS. 21A-21F show lenalidomide induces degradation of
CSNK1A1 by CRBN-CRL4. FIG. 21A shows CSNK1A1 protein levels in KG-1
cells treated with DMSO or 1 .mu.M or 10 .mu.M lenalidomide alone
or with MG-132 or MLN4924 for 6 hours. FIG. 21B shows CRBN knockout
293T cells were generated using CRISPR/Cas9-mediated deletion. The
effect of lenalidomide on CSNK1A1 protein was assessed in normal
and CRBN knockout 293T cells. FIG. 21C shows immunoprecipitation of
HA-CRBN in 293T cells treated for 4 hour with MG132 and DMSO or
lenalidomide. FIG. 21D shows in vivo ubiquitination analysis of
tagged CSNK1A1 transiently expressed in 293T cells with or without
CRBN. Cells were treated for 4 hours with the indicated
concentrations of lenalidomide. The K2 antibody was used to detect
ubiquitination of immunoprecipitated HA-CSNK1A1. FIG. 21E shows
CD34.sup.+ cells isolated from cord blood were transduced with
either luciferase control specific shRNA or CSNK1A1-specific shRNA
expressing GFP labeled lentivirus. After 48 hours cells were either
treated with DMSO or 1 .mu.M lenalidomide. FIG. 21F shows the
number of GFP positive cells as assessed by flow cytometry.
[0101] FIGS. 22A-22E show lenalidomide effects on murine cells.
FIG. 22A shows murine Baf3 cells or primary murine AML cells
transformed with an MLL-AF9 expressing retrovirus were treated with
lenalidomide for 24 hours in vitro. CSNK1A1 protein levels were
assessed by immunoblot. FIG. 22B shows murine Baf3 cells were
transduced with a retrovirus expressing murine CRBN (m), human CRBN
(h), human CRBN with single amino acid substitutions based on
corresponding residues in murine CRBN, or empty vector. After
selection with puromycin cells were treated for 24 hours with DMSO
(-) or 1 .mu.M lenalidomide (+) and CSNK1A1 protein levels were
assessed by immunoblot. FIG. 22C shows alignment of human and
murine CRBN. Non-conserved amino acids are indicated by red bars.
In the enlarged segment of the lenalidomide binding region the
critical non-conserved amino acid determining response to IMiDs
(human V387, murine I391) is indicated in red, the previously
described human CBRN mutant that does not bind IMiDs (Y383A/W385A)
is indicated in green. FIG. 22D shows murine Baf3 cells were
transduced with retrovirus expressing murine CRBN, human CRBN,
murine CRBN.sup.V3911, or empty vector. After selection with
puromycin cells were treated for 24 hours with DMSO or lenalidomide
and CSNK1A1 protein levels were assessed by immunoblot. FIG. 22E
shows human 293T cells were transfected with a IKZF3-luciferase
fusion protein together with a human or murine CRBN. Cells were
treated with DMSO or 1 .mu.M lenalidomide for 4 hours.
[0102] FIGS. 23A-23D show the evaluation of lenalidomide in murine
CSNK1A1.sup.+/- cells. FIG. 23A is an illustration showing the
experimental setup for in vitro competition experiments. Primary
hematopoietic progenitors (cKIT+) were isolated from the bone
marrow of CSNK1A1.sup.+/- MxCre.sup.+ or MxCre.sup.+ mice treated
with poly I:C 4 weeks before. When applicable, excision of exon 3
of CSNK1A1 on one allele was confirmed by excision PCR. One day
after harvest cells were transduced with a retrovirus expressing
murine CRBN.sup.V3911 and GFP. 72 hours after infection cells were
sorted, mixed with SJL cells and treated with DMSO or lenalidomide.
FIG. 23B is a graph showing the effects of 1 .mu.M and 10 .mu.M
lenalidomide on CSNK1A1.sup.+/-MxCre.sup.+ or MxCre.sup.+ cells as
analyzed by flow cytometry. FIG. 23C shows the quantitative RT-PCR
analysis of p21 expression in CSNK1A1.sup.+/-MxCre.sup.+ or
MxCre.sup.+ cells treated with DMSO or lenalidomide. FIG. 23D is a
graphs showing the effects of lenalidomide in p53.sup.+/- and
p53.sup.+/+ cells.
[0103] FIGS. 24A-24D show lenalidomide-induced changes in
ubiquitination and protein levels. FIG. 24A shows the log.sub.2
ratios for individual K-.epsilon.-GG sites of lenalidomide- (10
.mu.M) versus DMSO-treated cells for replicates 1 and 2. Each dot
represents a unique K-.epsilon.-GG site. FIG. 24B shows the
log.sub.2 ratios of changes of protein abundance of lenalidomide-
(10 .mu.M) versus DMSO-treated cells for replicates 1 and 2. Each
dot represents a unique protein group. FIG. 24C shows log.sub.2
ratios for different lysine residues in CK1.alpha., IKZF1, and CRBN
for 1 or 10 .mu.M lenalidomide treated cells versus DMSO treated
cells. FIG. 24D shows a list of significantly regulated
K-.epsilon.-GG sites with 1 .mu.M or 10 .mu.M lenalidomide vs.
DMSO. P-value is adjusted as described in the methods section.
[0104] FIGS. 25A-25C show the effect of lenalidomide in human
cells. FIG. 25A shows a time course of effect of lenalidomide
treatment on CK1.alpha. protein levels in KG-1 cells. FIG. 25B
shows the half-life of CK1.alpha. was assessed in 293T cells
treated with 100 .mu.g/ml cycloheximide in the absence or presence
of 1 .mu.M lenalidomide. FIG. 25C shows an immunoblot confirming
the loss of CRBN expression in 293T cells with the CRBN gene
disrupted by CRISPR/Cas genome editing.
[0105] FIGS. 26A-26D show sensitivity of human cells to growth
inhibition by lenalidomide. FIG. 26A shows 293T cells treated with
different concentrations of lenalidomide for 24 hours. CK1.alpha.
protein levels were detected by western blot. FIG. 26B shows
CSNK1A1 mRNA expression levels as measured by RQ-PCR. FIG. 26C
shows CK1.alpha. protein levels as detected hourly by western blot
in cells treated with 1 .mu.M lenalidomide. FIG. 26D shows MM1S and
MOLM13 cells treated with different concentrations of lenalidomide
for 24 hours and CSNK1A1 mRNA expression levels were measured by
RQ-PCR and CK1.alpha. protein levels were detected by western
blot.
[0106] FIGS. 27A-27C show the effects of lenalidomide on mouse
cells. FIG. 27A shows CK1.alpha. protein levels in Ba/F3 cells
transduced with empty vector, mouse CRBN or human CRBN and treated
with lenalidomide. FIG. 27B shows dual luciferase IKZF3 degradation
assay in 293T cells expressing different CRBN chimeras and mutants.
FIG. 27C shows the amino acid sequence alignment of mouse and human
CRBN.
DETAILED DESCRIPTION OF THE INVENTION
[0107] As described below, the present invention features
compositions and methods for identifying a subject having a B cell
neoplasia or myelodysplastic syndrome responsive to treatment with
lenalidomide and lenalidomide-related compounds.
[0108] The invention is based, at least in part, on the discovery
that lenalidomide causes selective ubiquitination and degradation
of two lymphoid transcription factors, IKZF1 and IKZF3, by the
CRBN-CRL4 ubiquitin ligase. IKZF1 and IKZF3 are essential
transcription factors for terminal B cell differentiation. A single
amino acid substitution of IKZF3 conferred resistance to
lenalidomide-induced degradation and rescued lenalidomide-induced
inhibition of cell growth. Similarly, it was found that
lenalidomide-induced IL2 production in T cells is due to depletion
of IKZF3. These findings reveal a novel mechanism of action for a
therapeutic agent, alteration of the activity of an E3 ubiquitin
ligase leading to selective degradation of specific targets.
[0109] In other aspects, the invention features the discovery that
casein kinase 1A1 (CSNK1A1) is a target of lenalidomide in del(5q)
myelodysplastic syndrome (MDS). Myelodysplastic syndrome (MDS) is a
heterogeneous clonal haematopoietic stem cell disorder
characterised by ineffective haematopoiesis and a high risk of
progression to acute myeloid leukemia (AML). Lenalidomide is often
used for the treatment of patients with MDS with 5q deletion
cytogenetic abnormalities. However, analysis of lenalidomide
activity has been hampered by the relative insensitivity of murine
cells to lenalidomide and related compounds. Expression of human
CRBN in murine cells was sufficient to confer lenalidomide
sensitivity to CSNK1A1. Accordingly, the present invention provides
murine cells and transgenic animals expressing human CRBN or mutant
CRBN.
Selection of Therapies for the Treatment of B Cell Neoplasia
[0110] As reported in detail below, lenalidomide causes the
selective ubiquitination and degradation of lymphoid transcription
factors, IKZF1 and IKZF3. IKZF1 and IKZF3 are expressed by B cell
neoplasias that are sensitive to treatment with lenalidomide or a
related compound, such as thalidomide or palidomide.
##STR00002##
Lenalidomide, pomalidomide, and thalidomide have been shown to have
immunomodulatory activity in multiple myeloma. Thus, these
compounds are termed IMiDs.
[0111] The invention provides methods for selecting IMiD therapy
for a subject having a B cell neoplasia by detecting an increased
level of biomarkers IKZF1 and/or IKZF3 in a biological sample of
the subject relative to the level present in a reference. Methods
for detecting IKZF1 and IKZF3 are known in the art and described
herein at Example 2.
[0112] The CRBN-CRL4 ubiquitin ligase selectively ubiquinates IKZF1
and IKZF3, thereby targeting IKZF1 and IKZF3 for
lenalidomide-induced degradation. In one embodiment, the invention
provides methods for selecting a therapy for a subject having a B
cell neoplasia by detecting the lenalidomide-induced ubiquitination
of IKZF1 and/or IKZF3 polypeptides in a biological sample from the
subject. In other embodiments, the method involves detecting a
decrease in ubiquitination of lysine residues of IKZF1 and IKZF3
prior to addition of a proteasome inhibitor (e.g., MG132). Methods
for detecting ubiquination are known in the art and described, for
example, herein at Example 1.
[0113] In other embodiments, the invention provides methods for
selecting lenalidomide as a therapy for a subject having a B cell
neoplasia. The method involves detecting a reduction in the level
of IKZF1 and/or IKZF3 polypeptides in response to lenalidomide in a
biological sample obtained from a subject.
[0114] Over time, many patients treated with lenalidomide acquire
resistance to the therapeutic effects of lenalidomide. The early
identification of lenalidomide resistance is important to patient
survival because it allows for the selection of alternate
therapies. As reported herein below, the anti-proliferative effect
of lenalidomide in B cell neoplasias is mediated by depletion of
IKZF1 and IKZF3. Accordingly, the invention provides methods for
identifying the presence of lenalidomide resistant B cells by
detecting IKZF1 and/or IKZF3 polypeptides that are resistant to
lenalidomide-induced degradation. In one embodiment, a lenalidomide
resistant B cell neoplasia is identified by detection of mutant
IKZF1 or IKZF3 proteins that are not degraded in response to
lenalidomide treatment or that are not ubiquitinated in response to
lenalidomide treatment.
[0115] Subjects identified as having a lenalidomide resistant B
cell neoplasia are identified as in need of alternative treatment.
Subjects identified as having a lenalidomide resistant myeloma, for
example, are treated with Velcade, corticosteroids, or other
anti-neoplastic therapy. For subjects identified as having
lenalidomide resistant myelodysplastic syndrome are treated, for
example, with azacitidine or decitabine.
[0116] Ubiquitination of IKZF1 and IKZF3 in response to
lenalidomide requires binding to CRBN. Mutations that reduce or
inhibit IKZF1 and IKZF3 binding to CRBN also render the B cell
neoplasia resistant to lenalidomide. Accordingly the invention
provides methods for detecting a reduction in IKZF1 and/or IKZF3
binding to CRBN. Methods for detecting CRBN binding to IKZF1 and/or
IKZF3 are known in the art and described, for example, at Examples
2 and 3. B cell neoplasias having a reduction in IKZF1 and/or IKZF3
binding to CRBN are identified as resistant to lenalidomide.
[0117] In still other embodiments, a lenalidomide resistant B cell
neoplasia is identified by detecting a mutation in an IKZF3 degron
sequence, such as a mutation in any one or more of amino acids
141-180 or 160-180. In particular embodiments, the invention
provides for the detection of a mutation at amino acid 147, 150,
161, or 162. In still other embodiments, the invention provides for
the detection of is Q147H, Q150H, L161R, or L162R. Methods for
detecting a mutation of the invention include immunoassay, direct
sequencing, and probe hybridization to a polynucleotide encoding
the mutant polypeptide.
Monitoring
[0118] Methods of monitoring the sensitivity of a B cell neoplasa
to lenalidomide in a subject are useful in managing subject
treatment. Provided are methods where alterations in a IKZF1 and/or
IKZF3 polypeptide (e.g, sequence, level, post-transcriptional
modification, biological activity) are analysed, such as before and
again after subject management or treatment. In these cases, the
methods are used to monitor the status of lenalidomide sensitivity
(e.g., response to lenalidomide treatment, resistance to
lenalidomide, amelioration of the disease or progression of the
disease).
[0119] For example, IKZF1 and/or IKZF3 polypeptide biomarkers can
be used to monitor a subject's response to certain treatments of B
cell neoplasia. The level, biological activity, sequence,
post-transcriptional modification, or sensitivity to lenalidomide
induced degradation of a IKZF1 and/or IKZF3 polypeptide may be
assayed before treatment, during treatment, or following the
conclusion of a treatment regimen. In some embodiments, multiple
assays (e.g., 2, 3, 4, 5) are made at one or more of those times to
assay resistance to lenalidomide.
Diagnostic Methods
[0120] Alterations in IKZF1 and/or IKZF3 polypeptides (e.g,
sequence, level, post-transcriptional modification, biological
activity) are detected in a biological sample obtained from a
patient that has or has a propensity to develop a B cell neoplasia.
Such biological samples include, but are not limited to, peripheral
blood, bone marrow, or lymphoid tissue obtained from the subject
relative to the level of such biomarkers in a reference.
[0121] Alterations in the levels of IKZF1 and/or IKZF3 polypeptide
biomarkers (or any other marker delineated herein) are detected
using standard methods. In one embodiment, the level of IKZF1 or
IKZF3 is detected using an antibody that specifically binds the
polypeptide. Exemplary antibodies that specifically bind such
polypeptides are known in the art and described herein. Such
antibodies are useful for the diagnosis of a B cell neoplasia that
is sensitive to treatment with lenalidomide. Methods for measuring
an antibody-biomarker complex include, for example, detection of
fluorescence, luminescence, chemiluminescence, absorbance,
reflectance, transmittance, birefringence or refractive index.
Optical methods include microscopy (both confocal and
non-confocal), imaging methods and non-imaging methods. Methods for
performing these assays are readily known in the art. Useful assays
include, for example, an enzyme immune assay (EIA), such as
enzyme-linked immunosorbent assay (ELISA), a radioimmune assay
(RIA), a Western blot assay, or a slot blot assay. Other assays
useful for detecting changes in IKZF1 or IKZF3 are
immunohistochemistry and quantitative fluorescent microscopy. These
methods are also described in, e.g., Methods in Cell Biology:
Antibodies in Cell Biology, volume 37 (Asai, ed. 1993); Basic and
Clinical Immunology (Stites & Ten, eds., 7th ed. 1991); and
Harlow & Lane, supra. Immunoassays can be used to determine the
quantity of marker in a sample, where an increase or decrease in
the level of the biomarker polypeptide is diagnostic of a patient
having a B cell neoplasia that is sensitive or resistant to
treatment with lenalidomide.
[0122] In general, the measurement of a IKZF1 and/or IKZF3
polypeptide in a subject sample is compared with an amount present
in a reference. A diagnostic amount distinguishes between a B cell
neoplasia that is sensitive to treatment with lenalidomide and a B
cell neoplasia that is resistant to treatment with lenalidomide.
The skilled artisan appreciates that the particular diagnostic
amount used can be adjusted to increase sensitivity or specificity
of the diagnostic assay depending on the preference of the
diagnostician. In general, any significant alteration (e.g., at
least about 10%, 15%, 30%, 50%, 60%, 75%, 80%, or 90%) in the level
of a biomarker polypeptide in the subject sample relative to a
reference may be used to diagnose a B cell neoplasia that is
sensitive or resistant to treatment with lenalidomide. In one
embodiment, the reference is the level of biomarker polypeptide
present in a corresponding control sample obtained from a patient
that does not have a B cell neoplasia. In another embodiment, the
reference is a baseline level of IKZF1 and/or IKZF3 markers present
in a biologic sample derived from a patient prior to, during, or
after treatment with lenalidomide. In yet another embodiment, the
reference is a standardized curve. In another example, levels of
IKZF1 or IKZF3 are measured relative to the level of other B cell
markers or actin.
Clinical Indicators
[0123] The present invention provides methods for detecting
alterations in an IKZF1 and/or IKZF3 polypeptide biomarker in a
biological sample (e.g., peripheral blood, bone marrow) derived
from a subject having a B cell neoplasia to determine whether the B
cell neoplasia is sensitive to treatment with lenalidomide or
whether it has acquired lenalidomide resistance. Alterations in
IKZF1 and/or IKZF3 are useful individually, or in combination with
other markers typically used in characterizing a B cell
neoplasia.
[0124] B-cell neoplasms typically recapitulate the normal stages of
B-cell differentiation, and can be classified according to their
putative cell of origin. Accordingly, alterations in IKZF1 and
IKZF3 may be assayed alone or in combination with the neoplasm's
cytogenetic profile, genotype, and immunophenotype. B cell markers
useful in the methods of the invention include, but are not limited
to, characterization of CD5, CD10, CD19, CD20, CD22, CD23, FMC7,
CD79a, CD40, CD38, and CD138.
Microarrays
[0125] The methods of the invention may also be used for
microarray-based assays that provide for the high-throughput
analysis of an IKZF1 and/or IKZF3 polypeptide or polynucleotide.
The IKZF1 and/or IKZF3 polypeptides, polynucleotides, or capture
molecules that specifically bind to IKZF1 and/or IKZF3 polypeptides
of the invention are useful as hybridizable array elements. If
desired, arrays of the invention include, for example, other
markers useful in the differential diagnosis of a B cell neoplasia
(e.g., CD5, CD10, CD19, CD20, CD22, CD23, FMC7, CD79a, CD40, and
CD38). The array elements are organized in an ordered fashion such
that each element is present at a specified location on the
substrate. Useful substrate materials include membranes, composed
of paper, nylon or other materials, filters, chips, glass slides,
and other solid supports. The ordered arrangement of the array
elements allows hybridization patterns and intensities to be
interpreted as expression levels of particular genes or proteins.
Methods for making nucleic acid microarrays are known to the
skilled artisan and are described, for example, in U.S. Pat. No.
5,837,832, Lockhart, et al. (Nat. Biotech. 14:1675-1680, 1996), and
Schena, et al. (Proc. Natl. Acad. Sci. 93:10614-10619, 1996),
herein incorporated by reference. Methods for making polypeptide
microarrays are described, for example, by Ge (Nucleic Acids Res.
28:e3.i-e3.vii, 2000), MacBeath et al., (Science 289:1760-1763,
2000), Zhu et al. (Nature Genet. 26:283-289), and in U.S. Pat. No.
6,436,665, hereby incorporated by reference.
[0126] IKZF1 and/or IKZF3 polypeptide may also be analyzed using
protein microarrays. Typically, protein microarrays feature a
protein, or fragment thereof, bound to a solid support. In
particular embodiments, the proteins are antibodies that
specifically bind a biomarker of the invention (e.g., IKZF1 and/or
IKZF3 polypeptide). Suitable solid supports include membranes
(e.g., membranes composed of nitrocellulose, paper, or other
material), polymer-based films (e.g., polystyrene), beads, or glass
slides. For some applications, biomarker polypeptides or antibodies
recognizing such biomarkers are spotted on a substrate using any
convenient method known to the skilled artisan (e.g., by hand or by
inkjet printer).
[0127] Biomarker levels present in a biological sample taken from a
patient, such as a bodily fluid (e.g., peripheral blood) may be
measured using an antibody or other molecule derived from a
peptide, nucleic acid, or chemical library. Hybridization
conditions (e.g., temperature, pH, protein concentration, and ionic
strength) are optimized to promote specific interactions. Such
conditions are known to the skilled artisan and are described, for
example, in Harlow, F. and Lane, D., Using Antibodies: A Laboratory
Manual. 1998, New York: Cold Spring Harbor Laboratories. After
removal of non-specific probes, specifically bound probes are
detected, for example, by fluorescence, enzyme activity (e.g., an
enzyme-linked calorimetric assay), direct immunoassay, radiometric
assay, or any other suitable detectable method known to the skilled
artisan.
Kits
[0128] In one aspect, the invention provides kits for monitoring
lenalidomide sensitivity, including the development of lenalidomide
resistance. For example, the kits can be used to detect an
alteration in an IKZF1 and/or IKZF3 polypeptide (e.g, sequence,
level, post-transcriptional modification, biological activity). If
desired a kit includes any one or more of the following: capture
molecules that bind IKZF1 and/or IKZF3. The kits have many
applications. For example, the kits can be used to determine if a
subject has a lenalidomide sensitive B cell neoplasia or if the
subject has developed resistance to lenalidomide.
[0129] The kits may include instructions for the assay, reagents,
testing equipment (test tubes, reaction vessels, needles, syringes,
etc.), standards for calibrating the assay, and/or equipment
provided or used to conduct the assay. The instructions provided in
a kit according to the invention may be directed to suitable
operational parameters in the form of a label or a separate
insert.
Inhibitory Nucleic Acids
[0130] As reported herein below, the anti-proliferative effect of
lenalidomide in B cell neoplasias is mediated by depletion of IKZF1
and/or IKZF3. Accordingly, the invention provides oligonucleotides
that inhibit the expression of IKZF1 and/or IKZF3. Such inhibitory
nucleic acid molecules include single and double stranded nucleic
acid molecules (e.g., DNA, RNA, and analogs thereof) that bind a
nucleic acid molecule that encodes an IKZF1 and/or IKZF3
polypeptide (e.g., antisense molecules, siRNA, shRNA).
[0131] siRNA
[0132] Short twenty-one to twenty-five nucleotide double-stranded
RNAs are effective at down-regulating gene expression (Zamore et
al., Cell 101: 25-33; Elbashir et al., Nature 411: 494-498, 2001,
hereby incorporated by reference). The therapeutic effectiveness of
an siRNA approach in mammals was demonstrated in vivo by McCaffrey
et al. (Nature 418: 38-39.2002).
[0133] Given the sequence of a target gene, siRNAs may be designed
to inactivate that gene. Such siRNAs, for example, could be
administered directly to an affected tissue, or administered
systemically. The nucleic acid sequence of a gene can be used to
design small interfering RNAs (siRNAs). The 21 to 25 nucleotide
siRNAs may be used, for example, as therapeutics to treat a B cell
neoplasia.
[0134] The inhibitory nucleic acid molecules of the present
invention may be employed as double-stranded RNAs for RNA
interference (RNAi)-mediated knock-down of IKZF1 and/or IKZF3
expression. RNAi is a method for decreasing the cellular expression
of specific proteins of interest (reviewed in Tuschl, Chembiochem
2:239-245, 2001; Sharp, Genes & Devel. 15:485-490, 2000;
Hutvagner and Zamore, Curr. Opin. Genet. Devel. 12:225-232, 2002;
and Hannon, Nature 418:244-251, 2002). The introduction of siRNAs
into cells either by transfection of dsRNAs or through expression
of siRNAs using a plasmid-based expression system is increasingly
being used to create loss-of-function phenotypes in mammalian
cells.
[0135] In one embodiment of the invention, a double-stranded RNA
(dsRNA) molecule is made that includes between eight and nineteen
consecutive nucleobases of a nucleobase oligomer of the invention.
The dsRNA can be two distinct strands of RNA that have duplexed, or
a single RNA strand that has self-duplexed (small hairpin (sh)RNA).
Typically, dsRNAs are about 21 or 22 base pairs, but may be shorter
or longer (up to about 29 nucleobases) if desired. dsRNA can be
made using standard techniques (e.g., chemical synthesis or in
vitro transcription). Kits are available, for example, from Ambion
(Austin, Tex.) and Epicentre (Madison, Wis.). Methods for
expressing dsRNA in mammalian cells are described in Brummelkamp et
al. Science 296:550-553, 2002; Paddison et al. Genes & Devel.
16:948-958, 2002. Paul et al. Nature Biotechnol. 20:505-508, 2002;
Sui et al. Proc. Natl. Acad. Sci. USA 99:5515-5520, 2002; Yu et al.
Proc. Natl. Acad. Sci. USA 99:6047-6052, 2002; Miyagishi et al.
Nature Biotechnol. 20:497-500, 2002; and Lee et al. Nature
Biotechnol. 20:500-505 2002, each of which is hereby incorporated
by reference.
[0136] Small hairpin RNAs (shRNAs) comprise an RNA sequence having
a stem-loop structure. A "stem-loop structure" refers to a nucleic
acid having a secondary structure that includes a region of
nucleotides which are known or predicted to form a double strand or
duplex (stem portion) that is linked on one side by a region of
predominantly single-stranded nucleotides (loop portion). The teen
"hairpin" is also used herein to refer to stem-loop structures.
Such structures are well known in the art and the term is used
consistently with its known meaning in the art. As is known in the
art, the secondary structure does not require exact base-pairing.
Thus, the stem can include one or more base mismatches or bulges.
Alternatively, the base-pairing can be exact, i.e. not include any
mismatches. The multiple stem-loop structures can be linked to one
another through a linker, such as, for example, a nucleic acid
linker, a miRNA flanking sequence, other molecule, or some
combination thereof.
[0137] As used herein, the term "small hairpin RNA" includes a
conventional stem-loop shRNA, which forms a precursor miRNA
(pre-miRNA). While there may be some variation in range, a
conventional stem-loop shRNA can comprise a stem ranging from 19 to
29 bp, and a loop ranging from 4 to 30 bp. "shRNA" also includes
micro-RNA embedded shRNAs (miRNA-based shRNAs), wherein the guide
strand and the passenger strand of the miRNA duplex are
incorporated into an existing (or natural) miRNA or into a modified
or synthetic (designed) miRNA. In some instances the precursor
miRNA molecule can include more than one stem-loop structure.
MicroRNAs are endogenously encoded RNA molecules that are about
22-nucleotides long and generally expressed in a highly tissue- or
developmental-stage-specific fashion and that
post-transcriptionally regulate target genes. More than 200
distinct miRNAs have been identified in plants and animals. These
small regulatory RNAs are believed to serve important biological
functions by two prevailing modes of action: (1) by repressing the
translation of target mRNAs, and (2) through RNA interference
(RNAi), that is, cleavage and degradation of mRNAs. In the latter
case, miRNAs function analogously to small interfering RNAs
(siRNAs). Thus, one can design and express artificial miRNAs based
on the features of existing miRNA genes.
[0138] shRNAs can be expressed from DNA vectors to provide
sustained silencing and high yield delivery into almost any cell
type. In some embodiments, the vector is a viral vector. Exemplary
viral vectors include retroviral, including lentiviral, adenoviral,
baculoviral and avian viral vectors, and including such vectors
allowing for stable, single-copy genomic integrations. Retroviruses
from which the retroviral plasmid vectors can be derived include,
but are not limited to, Moloney Murine Leukemia Virus, spleen
necrosis virus, Rous sarcoma Virus, Harvey Sarcoma Virus, avian
leukosis virus, gibbon ape leukemia virus, human immunodeficiency
virus, Myeloproliferative Sarcoma Virus, and mammary tumor virus. A
retroviral plasmid vector can be employed to transduce packaging
cell lines to form producer cell lines. Examples of packaging cells
which can be transfected include, but are not limited to, the
PE501, PA317, R-2, R-AM, PA12, T19-14x, VT-19-17-H2, RCRE, RCRIP,
GP+E-86, GP+envAm12, and DAN cell lines as described in Miller,
Human Gene Therapy 1:5-14 (1990), which is incorporated herein by
reference in its entirety. The vector can transduce the packaging
cells through any means known in the art. A producer cell line
generates infectious retroviral vector particles which include
polynucleotide encoding a DNA replication protein. Such retroviral
vector particles then can be employed, to transduce eukaryotic
cells, either in vitro or in vivo. The transduced eukaryotic cells
will express a DNA replication protein.
[0139] Catalytic RNA molecules or ribozymes that include an
antisense sequence of the present invention can be used to inhibit
expression of a IKZF1 and/or IKZF3 nucleic acid molecule in vivo.
The inclusion of ribozyme sequences within antisense RNAs confers
RNA-cleaving activity upon them, thereby increasing the activity of
the constructs. The design and use of target RNA-specific ribozymes
is described in Haseloff et al., Nature 334:585-591. 1988, and U.S.
Patent Application Publication No. 2003/0003469 A1, each of which
is incorporated by reference.
[0140] Accordingly, the invention also features a catalytic RNA
molecule that includes, in the binding arm, an antisense RNA having
between eight and nineteen consecutive nucleobases. In preferred
embodiments of this invention, the catalytic nucleic acid molecule
is formed in a hammerhead or hairpin motif. Examples of such
hammerhead motifs are described by Rossi et al., Aids Research and
Human Retroviruses, 8:183, 1992. Example of hairpin motifs are
described by Hampel et al., "RNA Catalyst for Cleaving Specific RNA
Sequences," filed Sep. 20, 1989, which is a continuation-in-part of
U.S. Ser. No. 07/247,100 filed Sep. 20, 1988, Hampel and Tritz,
Biochemistry, 28:4929, 1989, and Hampel et al., Nucleic Acids
Research, 18: 299, 1990. These specific motifs are not limiting in
the invention and those skilled in the art will recognize that all
that is important in an enzymatic nucleic acid molecule of this
invention is that it has a specific substrate binding site which is
complementary to one or more of the target gene RNA regions, and
that it have nucleotide sequences within or surrounding that
substrate binding site which impart an RNA cleaving activity to the
molecule.
[0141] Essentially any method for introducing a nucleic acid
construct into cells can be employed. Physical methods of
introducing nucleic acids include injection of a solution
containing the construct, bombardment by particles covered by the
construct, soaking a cell, tissue sample or organism in a solution
of the nucleic acid, or electroporation of cell membranes in the
presence of the construct. A viral construct packaged into a viral
particle can be used to accomplish both efficient introduction of
an expression construct into the cell and transcription of the
encoded shRNA. Other methods known in the art for introducing
nucleic acids to cells can be used, such as lipid-mediated carrier
transport, chemical mediated transport, such as calcium phosphate,
and the like. Thus the shRNA-encoding nucleic acid construct can be
introduced along with components that perform one or more of the
following activities: enhance RNA uptake by the cell, promote
annealing of the duplex strands, stabilize the annealed strands, or
otherwise increase inhibition of the target gene.
[0142] For expression within cells, DNA vectors, for example
plasmid vectors comprising either an RNA polymerase H or RNA
polymerase III promoter can be employed. Expression of endogenous
miRNAs is controlled by RNA polymerase II (Pol II) promoters and in
some cases, shRNAs are most efficiently driven by Pol II promoters,
as compared to RNA polymerase III promoters (Dickins et al., 2005,
Nat. Genet. 39: 914-921). In some embodiments, expression of the
shRNA can be controlled by an inducible promoter or a conditional
expression system, including, without limitation, RNA polymerase
type II promoters. Examples of useful promoters in the context of
the invention are tetracycline-inducible promoters (including
TRE-tight), IPTG-inducible promoters, tetracycline transactivator
systems, and reverse tetracycline transactivator (rtTA) systems.
Constitutive promoters can also be used, as can cell- or
tissue-specific promoters. Many promoters will be ubiquitous, such
that they are expressed in all cell and tissue types. A certain
embodiment uses tetracycline-responsive promoters, one of the most
effective conditional gene expression systems in in vitro and in
vivo studies. See International Patent Application
PCT/US2003/030901 (Publication No. WO 2004-029219 A2) and Fewell et
al., 2006, Drug Discovery Today 11: 975-982, for a description of
inducible shRNA.
Delivery of Polynucleotides
[0143] Naked polynucleotides, or analogs thereof, are capable of
entering mammalian cells and inhibiting expression of a gene of
interest. Nonetheless, it may be desirable to utilize a formulation
that aids in the delivery of oligonucleotides or other nucleobase
oligomers to cells (see, e.g., U.S. Pat. Nos. 5,656,611, 5,753,613,
5,785,992, 6,120,798, 6,221,959, 6,346,613, and 6,353,055, each of
which is hereby incorporated by reference).
Therapy
[0144] Therapy may be provided at home, the doctor's office, a
clinic, a hospital's outpatient department, or a hospital.
Treatment generally begins at a hospital so that the doctor can
observe the therapy's effects closely and make any adjustments that
are needed. The duration of the therapy depends on the kind of
cancer being treated, the age and condition of the patient, the
stage and type of the patient's disease, and how the patient's body
responds to the treatment. Drug administration may be performed at
different intervals (e.g., daily, weekly, or monthly).
Oligonucleotides and Other Nucleobase Oligomers
[0145] At least two types of oligonucleotides induce the cleavage
of RNA by RNase H: polydeoxynucleotides with phosphodiester (PO) or
phosphorothioate (PS) linkages. Although 2'-OMe-RNA sequences
exhibit a high affinity for RNA targets, these sequences are not
substrates for RNase H. A desirable oligonucleotide is one based on
2'-modified oligonucleotides containing oligodeoxynucleotide gaps
with some or all internucleotide linkages modified to
phosphorothioates for nuclease resistance. The presence of
methylphosphonate modifications increases the affinity of the
oligonucleotide for its target RNA and thus reduces the IC.sub.50.
This modification also increases the nuclease resistance of the
modified oligonucleotide. It is understood that the methods and
reagents of the present invention may be used in conjunction with
any technologies that may be developed, including covalently-closed
multiple antisense (CMAS) oligonucleotides (Moon et al., Biochem J.
346:295-303, 2000; PCT Publication No. WO 00/61595), ribbon-type
antisense (RiAS) oligonucleotides (Moon et al., J. Biol. Chem.
275:4647-4653, 2000; PCT Publication No. WO 00/61595), and large
circular antisense oligonucleotides (U.S. Patent Application
Publication No. US 2002/0168631 A1).
[0146] As is known in the art, a nucleoside is a nucleobase-sugar
combination. The base portion of the nucleoside is normally a
heterocyclic base. The two most common classes of such heterocyclic
bases are the purines and the pyrimidines. Nucleotides are
nucleosides that further include a phosphate group covalently
linked to the sugar portion of the nucleoside. For those
nucleosides that include a pentofuranosyl sugar, the phosphate
group can be linked to either the 2', 3' or 5' hydroxyl moiety of
the sugar. In forming oligonucleotides, the phosphate groups
covalently link adjacent nucleosides to one another to form a
linear polymeric compound. In turn, the respective ends of this
linear polymeric structure can be further joined to form a circular
structure; open linear structures are generally preferred. Within
the oligonucleotide structure, the phosphate groups are commonly
referred to as forming the backbone of the oligonucleotide. The
normal linkage or backbone of RNA and DNA is a 3' to 5'
phosphodiester linkage.
[0147] Specific examples of preferred nucleobase oligomers useful
in this invention include oligonucleotides containing modified
backbones or non-natural internucleoside linkages. As defined in
this specification, nucleobase oligomers having modified backbones
include those that retain a phosphorus atom in the backbone and
those that do not have a phosphorus atom in the backbone. For the
purposes of this specification, modified oligonucleotides that do
not have a phosphorus atom in their internucleoside backbone are
also considered to be nucleobase oligomers.
[0148] Nucleobase oligomers that have modified oligonucleotide
backbones include, for example, phosphorothioates, chiral
phosphorothioates, phosphorodithioates, phosphotriesters,
aminoalkyl-phosphotriesters, methyl and other alkyl phosphonates
including 3'-alkylene phosphonates and chiral phosphonates,
phosphinates, phosphoramidates including 3'-amino phosphoramidate
and aminoalkylphosphoramidates, thionophosphoramidates,
thionoalkylphosphonates, thionoalkylphosphotriest-ers, and
boranophosphates having normal 3'-5' linkages, 2'-5' linked analogs
of these, and those having inverted polarity, wherein the adjacent
pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to
5'-2'. Various salts, mixed salts and free acid forms are also
included. Representative United States patents that teach the
preparation of the above phosphorus-containing linkages include,
but are not limited to, U.S. Pat. Nos. 3,687,808; 4,469,863;
4,476,301; 5,023,243; 5,177,196; 5,188,897; 5,264,423; 5,276,019;
5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496;
5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,306;
5,550,111; 5,563,253; 5,571,799; 5,587,361; and 5,625,050, each of
which is herein incorporated by reference.
[0149] Nucleobase oligomers having modified oligonucleotide
backbones that do not include a phosphorus atom therein have
backbones that are formed by short chain alkyl or cycloalkyl
internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl
internucleoside linkages, or one or more short chain heteroatomic
or heterocyclic internucleoside linkages. These include those
having morpholino linkages (formed in part from the sugar portion
of a nucleoside); siloxane backbones; sulfide, sulfoxide and
sulfone backbones; formacetyl and thioformacetyl backbones;
methylene formacetyl and thioformacetyl backbones; alkene
containing backbones; sulfamate backbones; methyleneimino and
methylenehydrazino backbones; sulfonate and sulfonamide backbones;
amide backbones; and others having mixed N, O, S and CH.sub.2
component parts. Representative United States patents that teach
the preparation of the above oligonucleotides include, but are not
limited to, U.S. Pat. Nos. 5,034,506; 5,166,315; 5,185,444;
5,214,134; 5,216,141; 5,235,033; 5,264,562; 5,264,564; 5,405,938;
5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307; 5,561,225;
5,596,086; 5,602,240; 5,610,289; 5,602,240; 5,608,046; 5,610,289;
5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and
5,677,439, each of which is herein incorporated by reference.
[0150] In other nucleobase oligomers, both the sugar and the
internucleoside linkage, i.e., the backbone, are replaced with
novel groups. The nucleobase units are maintained for hybridization
with a gene listed in Table 2 or 3. One such nucleobase oligomer,
is referred to as a Peptide Nucleic Acid (PNA). In PNA compounds,
the sugar-backbone of an oligonucleotide is replaced with an amide
containing backbone, in particular an aminoethylglycine backbone.
The nucleobases are retained and are bound directly or indirectly
to aza nitrogen atoms of the amide portion of the backbone. Methods
for making and using these nucleobase oligomers are described, for
example, in "Peptide Nucleic Acids: Protocols and Applications" Ed.
P. E. Nielsen, Horizon Press, Norfolk, United Kingdom, 1999.
Representative United States patents that teach the preparation of
PNAs include, but are not limited to, U.S. Pat. Nos. 5,539,082;
5,714,331; and 5,719,262, each of which is herein incorporated by
reference. Further teaching of PNA compounds can be found in
Nielsen et al., Science, 1991, 254, 1497-1500.
[0151] In particular embodiments or the invention, the nucleobase
oligomers have phosphorothioate backbones and nucleosides with
heteroatom backbones, and in particular
--CH.sub.2--NH--O--CH.sub.2--,
--CH.sub.2--N(CH.sub.3)--O--CH.sub.2-- (known as a methylene
(methylimino) or MMI backbone),
--CH.sub.2--O--N(CH.sub.3)--CH.sub.2--,
--CH.sub.2--N(CH.sub.3)--N(CH.sub.3)--CH.sub.2--, and
--O--N(CH.sub.3)--CH.sub.2--CH.sub.2--. In other embodiments, the
oligonucleotides have morpholino backbone structures described in
U.S. Pat. No. 5,034,506.
[0152] Nucleobase oligomers may also contain one or more
substituted sugar moieties. Nucleobase oligomers comprise one of
the following at the 2' position: OH; F; O-, S-, or N-alkyl; O-,
S-, or N-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein
the alkyl, alkenyl, and alkynyl may be substituted or unsubstituted
C.sub.1 to C.sub.10 alkyl or C.sub.2 to C.sub.10 alkenyl and
alkynyl. Particularly preferred are
O[(CH.sub.2).sub.nO].sub.nCH.sub.3, O(CH.sub.2).sub.nOCH.sub.3,
O(CH.sub.2).sub.nNH.sub.2, O(CH.sub.2).sub.nCH.sub.3,
O(CH.sub.2).sub.nONH.sub.2, and
O(CH.sub.2)nON[(CH.sub.2).sub.nCH.sub.3)].sub.2, where n and m are
from 1 to about 10. Other preferred nucleobase oligomers include
one of the following at the 2' position: C.sub.1 to C.sub.10 lower
alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl, or
O-aralkyl, SH, SCH.sub.3, OCN, Cl, Br, CN, CF.sub.3, OCF.sub.3,
SOCH.sub.3, SO.sub.2CH.sub.3, ONO.sub.2, NO.sub.2, NH.sub.2,
heterocycloalkyl, heterocycloalkaryl, aminoalkylamino,
polyalkylamino, substituted silyl, an RNA cleaving group, a
reporter group, an intercalator, a group for improving the
pharmacokinetic properties of a nucleobase oligomer, or a group for
improving the pharmacodynamic properties of an nucleobase oligomer,
and other substituents having similar properties. Preferred
modifications are 2'-O-methyl and 2'-methoxyethoxy
(2'-O--CH.sub.2CH.sub.2OCH.sub.3, also known as
2'-O-(2-methoxyethyl) or 2'-MOE). Another desirable modification is
2'-dimethylaminooxyethoxy (i.e.,
O(CH.sub.2).sub.2ON(CH.sub.3).sub.2), also known as 2'-DMAOE. Other
modifications include, 2'-aminopropoxy
(2'-OCH.sub.2CH.sub.2CH.sub.2NH.sub.2) and 2'-fluoro (2'-F).
Similar modifications may also be made at other positions on an
oligonucleotide or other nucleobase oligomer, particularly the 3'
position of the sugar on the 3' terminal nucleotide or in 2'-5'
linked oligonucleotides and the 5' position of 5' terminal
nucleotide. Nucleobase oligomers may also have sugar mimetics such
as cyclobutyl moieties in place of the pentofuranosyl sugar.
Representative United States patents that teach the preparation of
such modified sugar structures include, but are not limited to,
U.S. Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044;
5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811;
5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873;
5,646,265; 5,658,873; 5,670,633; and 5,700,920, each of which is
herein incorporated by reference in its entirety.
[0153] Nucleobase oligomers may also include nucleobase
modifications or substitutions. As used herein, "unmodified" or
"natural" nucleobases include the purine bases adenine (A) and
guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and
uracil (U). Modified nucleobases include other synthetic and
natural nucleobases, such as 5-methylcytosine (5-me-C),
5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine,
6-methyl and other alkyl derivatives of adenine and guanine;
2-propyl and other alkyl derivatives of adenine and guanine;
2-thiouracil, 2-thiothymine and 2-thiocytosine; 5-halouracil and
cytosine; 5-propynyl uracil and cytosine; 6-azo uracil, cytosine
and thymine; 5-uracil (pseudouracil); 4-thiouracil; 8-halo,
8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted
adenines and guanines; 5-halo (e.g., 5-bromo), 5-trifluoromethyl
and other 5-substituted uracils and cytosines; 7-methylguanine and
7-methyladenine; 8-azaguanine and 8-azaadenine; 7-deazaguanine and
7-deazaadenine; and 3-deazaguanine and 3-deazaadenine. Further
nucleobases include those disclosed in U.S. Pat. No. 3,687,808,
those disclosed in The Concise Encyclopedia Of Polymer Science And
Engineering, pages 858-859, Kroschwitz, J. I., ed. John Wiley &
Sons, 1990, those disclosed by Englisch et al., Angewandte Chemie,
International Edition, 1991, 30, 613, and those disclosed by
Sanghvi, Y. S., Chapter 15, Antisense Research and Applications,
pages 289-302, Crooke, S. T. and Lebleu, B., ed., CRC Press, 1993.
Certain of these nucleobases are particularly useful for increasing
the binding affinity of an antisense oligonucleotide of the
invention. These include 5-substituted pyrimidines,
6-azapyrimidines, and N-2, N-6 and 0-6 substituted purines,
including 2-aminopropyladenine, 5-propynyluracil and
5-propynylcytosine. 5-methylcytosine substitutions have been shown
to increase nucleic acid duplex stability by 0.6-1.2.degree.C.
(Sanghvi, Y. S., Crooke, S. T. and Lebleu, B., eds., Antisense
Research and Applications, CRC Press, Boca Raton, 1993, pp.
276-278) and are desirable base substitutions, even more
particularly when combined with 2'-O-methoxyethyl or 2'-O-methyl
sugar modifications. Representative United States patents that
teach the preparation of certain of the above noted modified
nucleobases as well as other modified nucleobases include U.S. Pat.
Nos. 4,845,205; 5,130,302; 5,134,066; 5,175,273; 5,367,066;
5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711;
5,552,540; 5,587,469; 5,594,121, 5,596,091; 5,614,617; 5,681,941;
and 5,750,692, each of which is herein incorporated by
reference.
[0154] Another modification of a nucleobase oligomer of the
invention involves chemically linking to the nucleobase oligomer
one or more moieties or conjugates that enhance the activity,
cellular distribution, or cellular uptake of the oligonucleotide.
Such moieties include but are not limited to lipid moieties such as
a cholesterol moiety (Letsinger et al., Proc. Natl. Acad. Sci. USA,
86:6553-6556, 1989), cholic acid (Manoharan et al., Bioorg. Med.
Chem. Let, 4:1053-1060, 1994), a thioether, e.g.,
hexyl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci.,
660:306-309, 1992; Manoharan et al., Bioorg. Med. Chem. Let.,
3:2765-2770, 1993), a thiocholesterol (Oberhauser et al., Nucl.
Acids Res., 20:533-538: 1992), an aliphatic chain, e.g.,
dodecandiol or undecyl residues (Saison-Behmoaras et al., EMBO J.,
10:1111-1118, 1991; Kabanov et al., FEBS Lett., 259:327-330, 1990;
Svinarchuk et al., Biochimie, 75:49-54, 1993), a phospholipid,
e.g., di-hexadecyl-rac-glycerol or triethylammonium
1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al.,
Tetrahedron Lett., 36:3651-3654, 1995; Shea et al., Nucl. Acids
Res., 18:3777-3783, 1990), a polyamine or a polyethylene glycol
chain (Manoharan et al., Nucleosides & Nucleotides, 14:969-973,
1995), or adamantane acetic acid (Manoharan et al., Tetrahedron
Lett., 36:3651-3654, 1995), a palmityl moiety (Mishra et al.,
Biochim. Biophys. Acta, 1264:229-237, 1995), or an octadecylamine
or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J.
Pharmacol. Exp. Ther., 277:923-937, 1996. Representative United
States patents that teach the preparation of such nucleobase
oligomer conjugates include U.S. Pat. Nos. 4,587,044; 4,605,735;
4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,828,979; 4,835,263;
4,876,335; 4,904,582; 4,948,882; 4,958,013; 5,082,830; 5,109,124;
5,112,963; 5,118,802; 5,138,045; 5,214,136; 5,218,105; 5,245,022;
5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098;
5,371,241, 5,391,723; 5,414,077; 5,416,203, 5,451,463; 5,486,603;
5,510,475; 5,512,439; 5,512,667; 5,514,785; 5,525,465; 5,541,313;
5,545,730; 5,552,538; 5,565,552; 5,567,810; 5,574,142; 5,578,717;
5,578,718; 5,580,731; 5,585,481; 5,587,371; 5,591,584; 5,595,726;
5,597,696; 5,599,923; 5,599,928; 5,608,046: and 5,688,941, each of
which is herein incorporated by reference.
[0155] The present invention also includes nucleobase oligomers
that are chimeric compounds. "Chimeric" nucleobase oligomers are
nucleobase oligomers, particularly oligonucleotides, that contain
two or more chemically distinct regions, each made up of at least
one monomer unit, i.e., a nucleotide in the case of an
oligonucleotide. These nucleobase oligomers typically contain at
least one region where the nucleobase oligomer is modified to
confer, upon the nucleobase oligomer, increased resistance to
nuclease degradation, increased cellular uptake, and/or increased
binding affinity for the target nucleic acid. An additional region
of the nucleobase oligomer may serve as a substrate for enzymes
capable of cleaving RNA:DNA or RNA:RNA hybrids. By way of example,
RNase H is a cellular endonuclease which cleaves the RNA strand of
an RNA:DNA duplex. Activation of RNase H, therefore, results in
cleavage of the RNA target, thereby greatly enhancing the
efficiency of nucleobase oligomer inhibition of gene expression.
Consequently, comparable results can often be obtained with shorter
nucleobase oligomers when chimeric nucleobase oligomers are used,
compared to phosphorothioate deoxyoligonucleotides hybridizing to
the same target region.
[0156] Chimeric nucleobase oligomers of the invention may be formed
as composite structures of two or more nucleobase oligomers as
described above. Such nucleobase oligomers, when oligonucleotides,
have also been referred to in the art as hybrids or gapmers.
Representative United States patents that teach the preparation of
such hybrid structures include U.S. Pat. Nos. 5,013,830; 5,149,797;
5,220,007; 5,256,775; 5,366,878; 5,403,711; 5,491,133; 5,565,350;
5,623,065; 5,652,355; 5,652,356; and 5,700,922, each of which is
herein incorporated by reference in its entirety.
[0157] The nucleobase oligomers used in accordance with this
invention may be conveniently and routinely made through the
well-known technique of solid phase synthesis. Equipment for such
synthesis is sold by several vendors including, for example,
Applied Biosystems (Foster City, Calif.). Any other means for such
synthesis known in the art may additionally or alternatively be
employed. It is well known to use similar techniques to prepare
oligonucleotides such as the phosphorothioates and alkylated
derivatives.
[0158] The nucleobase oligomers of the invention may also be
admixed, encapsulated, conjugated or otherwise associated with
other molecules, molecule structures or mixtures of compounds, as
for example, liposomes, receptor targeted molecules, oral, rectal,
topical or other formulations, for assisting in uptake,
distribution and/or absorption. Representative United States
patents that teach the preparation of such uptake, distribution
and/or absorption assisting formulations include U.S. Pat. Nos.
5,108,921; 5,354,844; 5,416,016; 5,459,127; 5,521,291; 5,543,158;
5,547,932; 5,583,020; 5,591,721; 4,426,330; 4,534,899; 5,013,556;
5,108,921; 5,213,804; 5,227,170; 5,264,221; 5,356,633; 5,395,619;
5,416,016; 5,417,978; 5,462,854; 5,469,854; 5,512,295; 5,527,528;
5,534,259; 5,543,152; 5,556,948; 5,580,575; and 5,595,756, each of
which is herein incorporated by reference.
Casein Kinase 1A1
[0159] As reported in detail herein below, casein kinase 1A1
(CSNK1A1) was identified as a target of lenalidomide in del(5q)
myelodysplastic syndrome (MDS). Methods for characterizing the
biological activity of lenalidomide, thalidomide, and pomalidomide
have been hampered because mice have been largely unresponsive to
the activity of these compounds. Significantly, as reported herein
below, expression of human CRBN in murine cells was sufficient to
confer lenalidomide sensitivity to CSNK1A1. Moreover, mutation of
murine CRBN to include at least one of I391V, or any other
substitution, deletion or addition of the murine CRBN that confers
lenalidomide sensitivity to CSNK1A1, IKZF1 and IKZF3 is also
included. Accordingly, the present invention provides murine cells
and transgenic animals expressing human CRBN or mutant CRBN.
[0160] In other embodiments, the invention provides for the use of
casein kinase 1A1 inhibitors for the treatment of a B cell
neoplasia or related condition. Casein kinase 1A1 and casein kinase
1 inhibitors are useful in the methods of the invention. In
particular embodiments, casein kinase 1 inhibitors include, but are
not limited to, Casein Kinase I Inhibitor, D4476 (CAS 301836-43-1)
(Santa Cruz Biotechnology).
[0161] In yet other embodiments, the invention includes knock-down
or inhibition of casein kinase 1A1 expression for the treatment of
a B cell neoplasia or related condition. Knock-down or inhibition
of expression of casein kinase 1A1 is useful in the methods of the
invention to confer sensitivity to lenalidomide or a lenalidomide
analog. In particular embodiments, casein kinase 1 expression is
decreased by a method including, but are not limited to, antisense
nucleic acid molecule, siRNA molecule, shRNA, CRISPR, CRISPRi (Cell
152 (5): 1173-83, 2013) and other known method for decreasing gene
expression.
Generation of a Transgenic Mouse that is Responsive to Lenalidomide
and Other IMiDs
[0162] Generating transgenic mice involves five basic steps:
purification of a transgenic construct, harvesting donor zygotes,
microinjection of transgenic construct, implantation of
microinjected zygotes into the pseudo-pregnant recipient mice, and
genotyping and analysis of transgene expression in founder mice.
Methods for the generation of transgenic mice are known in the art
and described, for example, by Cho et al., Curr Protoc Cell Biol.
2009 March; CHAPTER: Unit-19.11, which is incorporated herein in
its entirety.
[0163] An expression vector, such as an expression vector encoding
human CRBN or an expression vector encoding a mutant CRBN (e.g.,
S369C, V380E, or I391V), is generated using standard methods known
in the art. Construction of transgenes can be accomplished using
any suitable genetic engineering technique, such as those described
in Ausubel et at (Current Protocols in Molecular Biology, John
Wiley & Sons, New York, 2000). Many techniques of transgene
construction and of expression constructs for transfection or
transformation in general are known and may be used to generate the
desired human CRBN-expressing construct.
[0164] One skilled in the art will appreciate that a promoter is
chosen that directs expression of the CRBN gene in all tissues or
in a preferred tissue. In particular embodiments, CRBN expression
is driven by a phosphoglycerate kinase 1 promoter (PGK1) (Qin et
al. (2010) PLoS ONE 5(5): e10611.
doi:10.1371/journal.pone.0010611), the spleen focus-forming virus
(SFFV) (Gonzalez-Murillo et al., Hum Gene Ther. 2010 May;
21(5):623-30, using knockin technology (Cohen-Tannoudji et al., Mol
Hum Reprod 4:929-938, 1998; Rossant et al., Nat Med 1:592-594,
1995; tet-off promoter (Clontech), human EF1s, CMV or endogenous
CRBN promotor. The modular nature of transcriptional regulatory
elements and the absence of position-dependence of the function of
some regulatory elements, such as enhancers, make modifications
such as, for example, rearrangements, deletions of some elements or
extraneous sequences, and insertion of heterologous elements
possible. Numerous techniques are available for dissecting the
regulatory elements of genes to determine their location and
function. Such information can be used to direct modification of
the elements, if desired. Preferably, an intact region that
includes all of the transcriptional regulatory elements of a gene
is used.
[0165] Following its construction, the transgene construct is
amplified by transforming bacterial cells using standard
techniques. Plasmid DNA is then purified and treated to remove
endogenous bacterial sequences. A fragment suitable for expression
of a transgenic CRBN under the control of a suitable promoter, such
as an endogenous murine CRBN promoter, and optionally additional
regulatory elements is purified (e.g., by a sucrose gradient or a
gel-purification method) in preparation for microinjection.
[0166] Foreign DNA is transferred into a mouse zygote by
microinjection into the pronucleus. A fragment of the transgene DNA
isolated above is microinjected into the male pronuclei of
fertilized mouse eggs derived from, for example, a C57BL/6 or C3136
F1 strain, using the techniques described in Gordon et al. (Proc.
Natl. Acad. Sci. USA 77:7380, 1980). The eggs are transplanted into
pseudopregnant female mice for full-term gestation, and resultant
litters are analysed to identify transgenic mice.
[0167] The practice of the present invention employs, unless
otherwise indicated, conventional techniques of molecular biology
(including recombinant techniques), microbiology, cell biology,
biochemistry and immunology, which are well within the purview of
the skilled artisan. Such techniques are explained fully in the
literature, such as, "Molecular Cloning: A Laboratory Manual",
second edition (Sambrook, 1989); "Oligonucleotide Synthesis" (Gait,
1984); "Animal Cell Culture" (Freshney, 1987); "Methods in
Enzymology" "Handbook of Experimental Immunology" (Weir, 1996);
"Gene Transfer Vectors for Mammalian Cells" (Miller and Calos,
1987); "Current Protocols in Molecular Biology" (Ausubel, 1987);
"PCR: The Polymerase Chain Reaction", (Mullis, 1994); "Current
Protocols in Immunology" (Coligan, 1991). These techniques are
applicable to the production of the polynucleotides and
polypeptides of the invention, and, as such, may be considered in
making and practicing the invention. Particularly useful techniques
for particular embodiments will be discussed in the sections that
follow.
[0168] The following examples are put forth so as to provide those
of ordinary skill in the art with a complete disclosure and
description of how to make and use the assay, screening, and
therapeutic methods of the invention, and are not intended to limit
the scope of what the inventors regard as their invention.
EXAMPLES
Example 1
DNA Damage Binding Protein 1 (DDB1) and Carbonyl Reductase 1 (CBR1)
Bind to Lenolidomide
[0169] Lenalidomide is a highly effective drug for the treatment of
multiple myeloma (Rajkumar et al., Blood 106, 4050 (Dec. 15,
2005).) and del(5q) MDS (List et al., N Engl J Med 352, 549 (Feb.
10, 2005)), and its use in a range of other conditions is being
actively explored, but the precise mechanism of action of
lenalidomide has not been established. In addition, lenalidomide
and its analogues thalidomide and pomalidomide have multiple
additional biological effects, including stimulation of IL-2
production by T cells, and inhibition of TNF production by
monocytes, but the molecular basis of these pleiotropic activities
is unknown.
[0170] In order to identify direct protein targets of lenalidomide,
a derivative of lenalidomide was synthesized that allowed
immobilization of the molecule to a bead (FIG. 2A). This derivative
retained the biological activity of lenalidomide, including
selective growth inhibition of multiple myeloma cells (FIG. 2B). To
identify proteins that bind to the lenalidomide derivative
immobilized on a solid support, SILAC (Stable Isotope Labeling of
Amino Acids in Cell Culture)-based quantitative mass spectrometry
(MS) was used to compare proteins pulled down by beads in the
presence or absence of 100-fold excess soluble lenalidomide,
enabling discrimination between proteins that bind lenalidomide
from those binding the bead or linker (FIG. 2C).
[0171] This approach identified two candidate proteins binding
specifically to lenalidomide, DNA damage binding protein 1 (DDB1)
and carbonyl reductase 1 (CBR1). DDB1 binds the lenalidomide
derivative-immobilized beads, and was competed off by lenalidomide,
thalidomide, and pomalidomide. Lenalidomide did not interact with
CBR1 in direct binding assays or inhibit CBR1 in biochemical assays
so it was not pursued further. Recently, Ito et al. reported a
similar proteomic strategy leading to the finding that thalidomide
binds to DDB1 via CRBN, and that this interaction is necessary for
thalidomide's teratogenic effects. DDB1 forms an E3 ubiquitin
ligase (CRL4) with Cullin 4A and 4B (Cul4A/4B) and regulator of
cullins 1 (RBX1). Consistent with these findings, it was found that
DDB1 and CRBN each bound the lenalidomide derivative beads and were
competed off by soluble lenalidomide. The finding that CRBN-DDB1
binds both lenalidomide and thalidomide in independent proteomic
studies provided powerful evidence that this ubiquitin ligase
complex is a major direct protein binding partner for this class of
molecules.
[0172] It was hypothesized that the pleiotropic effects of
lenalidomide might be caused by altered ubiquitination of target
proteins. Specificity of the CRL4 ubiquitin ligase is mediated by
an interchangeable substrate receptor, but no targets have been
identified for CRBN, a putative substrate receptor. To characterize
drug-induced modulation of CRL4-CRBN ubiquitin ligase activity,
SILAC-based quantitative MS studies were used to characterize
changes in the ubiquitinome and proteome in the MM1S multiple
myeloma cell line cultured in the presence of lenalidomide or
thalidomide for 12 hours (FIG. 1A, 1C). Ubiquitination profiling
was completed by enrichment of formerly ubiquitinated peptides with
an anti-K-.epsilon.-GG antibody (FIG. 1B). In parallel, the
landscape of lenalidomide-dependent CRBN protein interactions was
examined (FIG. 1D, 4).
Example 2
Lenalidomide Regulates Ikaros (IKZF1) and Aiolos (IKZF3)
[0173] Two proteins, Ikaros (IKZF1) and Aiolos (IKZF3), scored at
the top of the lists of proteins regulated by lenalidomide at both
the protein and ubiquitin-site level (FIG. 1C, 1D). Lenalidomide
decreased the abundance of IKZF3 (log.sub.2 ratio -2.09) and IKZF1
(log.sub.2 ratio -1.54). While increased ubiquitination would be
expected to be associated with decreased protein abundance, a
decrease in ubiquitination of multiple lysine residues of IKZF1 and
IKZF3 was observed after treating cells with lenalidomide for 12
hours prior to addition of the proteasome inhibitor MG132. A likely
interpretation of these results is that IKZF1 and IKZF3 are rapidly
ubiquitinated, targeting them for degradation and thereby resulting
in a decrease in abundance of both ubiquitinated and absolute
levels of these proteins. IKZF1 and IKZF3 also scored at the top of
the list of thalidomide-regulated proteins, consistent with the
similar biological activity of the molecules (FIGS. 3A and 3B).
[0174] Strikingly, the protein interaction study using HA-CRBN
revealed binding of IKZF1 and IKZF3 to the putative CRBN substrate
receptor in the presence of lenalidomide (FIGS. 5A and 5B). As
expected, all of the members of the CRBN-CRL4 ubiquitin ligase and
proteins known to interact with DDB1 including subunits 1 to 8 of
the COP9 signalosome complex CSN, DDA1, and DNA ligase 4 were
pulled down in both untreated or lenalidomide treated cells. No
other substrate receptors for DDB1 were co-immunoprecipitated,
Based on these results it is likely that CRBN is a substrate
receptor and precludes binding of alternative receptors to DDB1. In
aggregate, the proteomic data indicate that lenalidomide increases
the binding of IKZF1 and IKZF3 to the CRBN-DDB1 ubiquitin ligase
complex, leading to increased ubiquitination and consequent
degradation.
[0175] To validate this putative mechanism, the question of whether
lenalidomide causes post-transcriptional regulation of IKZF1 and
IKZF3 protein abundance was analyzed. The cDNAs of candidate genes,
fused to firefly luciferase (FFluc), were expressed in 293T cells.
IKZF1 and IKZF3 conferred a lenalidomide-regulated decrease in
protein abundance onto the fused FFLuc. In contrast, luciferase
levels were not altered after lenalidomide treatment when FFluc was
fused to RAB28, a protein that decreased in abundance after
lenalidomide treatment but did not bind to CRBN. Similarly,
lenalidomide did not alter the abundance of FFluc fused to three
other transcription factors of the Ikaros family, Helios (IKZF2),
Eos (IKZF4) and Pegasus (IKZF5); IRF4, a protein implicated in
lenalidomide activity; or the transcription factors HOXA9 and Myc
(FIG. 6A). It was confirmed that, in MM1S multiple myeloma cells
stably expressing HA-IKZF1 or HA-IKZF3, lenalidomide caused a
dose-dependent reduction of both proteins (FIG. 6B). Taken
together, these results demonstrate the selective regulation of
IKZF1 and IKZF3 levels in response to lenalidomide.
[0176] Endogenous protein expression was examined in response to
lenalidomide. Lenalidomide strongly decreased the abundance of
IKZF1 and IKZF3 in a dose-dependent manner in MM1S cells (FIG. 6C),
in primary cells (FIG. 6E) and other cell lines (FIGS. 7A and 7B).
Depletion of these proteins was evident in as little as 3 hours
after treatment. In contrast, IKZF1 and IKZF3 mRNA levels were not
altered by lenalidomide treatment (FIG. 6D). FIG. 6F shows an in
vivo ubiquitination analysis of HA-tagged IKZF1 and IKZF3 expressed
in MM1S cells treated for 1.5 hours with 100 nM Epoxomicin and the
indicated concentrations of lenalidomide. The FK2 antibody detects
covalently linked ubiquitin.
Example 3
Lenalidomide Induced Ubiquitination of IKZF1 and IKZF3
[0177] The direct effect of lenalidomide on ubiquitination of IKZF1
and IKZF3 was assessed. Lenalidomide induced dose-dependent
ubiquitination of tagged IKZF1 and IKZF3 in MM1S and 293' cells
(FIG. 8A-8C). Cain-RING ubiquitin ligase (CRL) activity depends on
NEDDylation and can be inhibited by the Nedd8 enzyme inhibitor
MLN4924. Treatment with 1 .mu.M MLN-4924 prevented the
lenalidomide-induced decrease of endogenous IKZF1 and IKZF3 in MM1S
cells and of FFluc-fused IKZF3 in 293T cells. These experiments
demonstrate that lenalidomide-induced degradation of IKZF1 and
IKZF3 involves ubiquitination by a cullin-based E3 ubiquitin
ligase.
[0178] Experiments were carried out to determine whether
lenalidomide-induced ubiquitination of IKZF1 and IKZF3 is caused by
altered binding of these proteins to CRBN, as observed in our
proteomic studies. These experiments confirmed that more IKZF1 and
IKZF3 co-irnmunoprecipitate with HA-CRBN after 3 hours of
lenalidomide treatment, despite a dramatic decrease of protein
levels in the whole cell lysate at the same time (FIG. 9A). If CRBN
is essential for lenalidomide-induced degradation of IKZF1 and
IKZF3, then loss or mutation of CRBN would inhibit the effect of
the drug. Consistent with this, it was found that shRNA knockdown
of CRBN prevented lenalidomide-induced degradation of luciferase
fusions of IKZF1 and IKZF3, and prevented degradation of HA-tagged
IKZF3 in 293T cells (FIG. 9B, 9C). Similarly, the CRBN.sup.YWAA
mutant that does not bind lenalidomide abrogated degradation of
IKZF1 and IKZF3 (FIG. 9D) and conferred lenalidomide resistance to
MM1S cells (FIG. 10), consistent with previous studies that have
shown CRBN to be essential for lenalidomide activity in multiple
myeloma. These studies demonstrate that lenalidomide causes
increased binding of IKZF1 and IKZF3 to CRBN, and that CRBN is
critical for the effects of lenalidomide on these proteins.
[0179] To assess whether IKZF3 is an enzymatic substrate of the
CRBN-DDB1 E3 ubiquitin ligase, an in vitro ubiquitination assay was
performed. HA-IKZF3 was co-immunoprecipitated by FLAG-CRBN from
293T cells treated with DMSO or lenalidomide. Lenalidomide was
added to the protein lysate in order to achieve efficient
co-immunoprecipitation of IKZF3. The eluted complex was then
incubated in the ubiquitin reaction mixture. Ubiquitinated IKZF3
could only be detected in reactions containing E1 and E2 ubiquitin
ligase enzymes and was increased in cells pre-treated with
lenalidomide, demonstrating that IKZF3 gets ubiquitinated when
bound to CRBN.
Example 4
Amino Acids 131 to 270 of IKZF3 Mediate Lenalidomide
Sensitivity
[0180] In order to identify a degron sequence in IKZF3 responsible
for lenalidomide sensitivity, a series of IKZF3 cDNA deletion
mutants was generated. Amino acids 131 to 270 of IKZF3 were
identified as necessary and sufficient for lenalidomide
sensitivity. Amino acids 141 to 180 were necessary for the
lenalidomide response. The critical amino acid sequence lies within
zinc finger domain 2, which is highly homologous between IKZF1 and
IKZF3. IKZF2, IKZF4, and IKZF5, proteins that are not sensitive to
lenalidomide-induced degradation, differ from IKZF1 and IKZF3 at
three amino acids within this region. Substitution of Q147 in IKZF3
with a histidine residue (IKZF3 Q147H), which is present at this
corresponding site in IKZF2 and IKZF4 resulted in resistance to
lenalidomide-induced degradation (FIG. 10). Conversely, when the
corresponding histidine (H188) in IKZF4 is changed to glutamine
(IKZF4 H188Q), IKZF4 was degraded after lenalidomide treatment
(FIG. 9G). In addition, Q150H and further point mutations in the
essential region of IKZF3 were identified that rendered IKZF3
resistant towards lenalidomide (FIG. 11A-11C). Binding to CRBN in
the presence of lenalidomide is decreased for Q147H and Q150H
mutants compared to wildtype IKZF3 (FIG. 11C). This domain is
therefore necessary and sufficient for lenalidomide-induced binding
to CRBN and subsequent protein degradation, and amino acid changes
in this region provide the basis for differential sensitivity to
lenalidomide between Ikaros family members.
Example 5
IKZF1 and IKZF3 Depletion Mediates Lenalidomide's
Anti-Proliferative Effect in Multiple Myeloma Cells
[0181] Having demonstrated that lenalidomide regulates IKZF1 and
IKZF3 ubiquitination and abundance, experiments were carried out to
determine whether these proteins mediate specific biological and
therapeutic effects of lenalidomide. IKZF1 and IKZF3 are essential
transcription factors for terminal differentiation of B and T cell
lineages. While IKZF1 is highly expressed in early lymphoid
progenitors, IKZF3 is expressed at high levels in more mature B
cell neoplasms, and murine studies have demonstrated that IKZF3 is
required for the generation of plasma cells, the physiologic
counterparts of multiple myeloma cells. Therefore, the dependence
of multiple myeloma cells on IKZF1 and IKZF3 expression by genetic
silencing of these proteins was assessed using RNA interference.
IKZF1 and IKZF3 shRNAs that effectively decreased expression of the
target proteins (FIG. 15A-15C) inhibited growth of
lenalidomide-sensitive multiple myeloma cell lines, while
lenalidomide insensitive cell lines were unaffected (FIG. 12A and
FIG. 13). Similarly, expression of a dominant negative IKZF3
isoform that lacks the complete DNA binding region resulted in
depletion of MM1S cells (FIG. 12B). Over-expression of IKZF3
conferred relative lenalidomide-resistance to MM1S cells when
competed with MM1S cells infected with a control retrovirus (FIG.
12C). Moreover, MM1S cells expressing the lenalidomide-resistant
IKZF3 Q150H mutation were relatively resistant towards lenalidomide
when competed to MM1S cells expressing wild-type IKZF3. These
studies indicate that the anti-proliferative effect of lenalidomide
in multiple myeloma cells is mediated by depletion of IKZF1 and
IKZF3.
[0182] The transcription factor IRF4 was previously reported to be
an important gene in multiple myeloma, and was implicated in the
activity of lenalidomide in this disease (Y. Yang et al., Cancer
Cell 21, 723 (Jun. 12, 2012)., A. L. Shaffer et al., Nature 454,
226 (Jul. 10, 2008).). While IRF4 levels were only slightly
decreased in a proteomic analysis, performed on cells treated with
lenalidomide for 12 hours, a significant decrease of IRF4 mRNA and
protein was observed when cells were treated for 24 hours and
longer. Knockdown of IKZF3 also suppressed IRF4 mRNA levels,
suggesting that lenalidomide regulates IRF4 through Ikaros-mediated
transcriptional repression (FIG. 14A-14E).
Example 6
Knockdown of IKZF3 Induced IL2 Expression
[0183] IKZF3 binds the IL2 gene promoter and repressed IL2
transcription in T cells. Experiments were carried out to determine
whether lenalidomide regulates IL2 levels by modulating IKZF3
expression. Both IKZF1 and IKZF3 protein levels decreased markedly
in primary human T cells treated with lenalidomide (FIG. 12D).
Lentiviral shRNA-mediated knockdown of IKZF3 induced IL2
expression. Lenalidomide induced IL2 mRNA expression by 3.3-fold in
T cells expressing a control shRNA, and this induction was blocked
by IKZF3 knockdown (FIG. 12E). Similarly, the effect of
lenalidomide on IL2 expression was abrogated by shRNA knockdown of
CRBN (FIG. 12F). These studies demonstrated that one of the primary
immunomodulatory activities of lenalidomide, induction of IL2, is
mediated by de-repression of the IL2 promoter by depletion of
IKZF3.
[0184] In aggregate, the studies reported herein above demonstrate
that lenalidomide acts via a novel mechanism of drug activity,
enforced binding of the substrate receptor CRBN to IKZF1 and IKZF3,
resulting in selective ubiquitination and degradation of the target
proteins. IKZF1 and IKZF3 play central roles in the biology of B
and T cells, and ablation of protein expression for these
transcription factors explains the activity of lenalidomide in
lymphoid cells. In particular, IKZF3 is critical for plasma cell
development, and these data indicate that IKZF3 is important in
multiple myeloma, a plasma cell malignancy, providing a mechanistic
basis for therapeutic efficacy in this disorder. Moreover, the
activity of lenalidomide in other B cell neoplasms, including
mantle cell lymphoma and chronic lymphocytic leukemia, may be
explained by high IKZF3 expression in these disorders. In contrast
to the high expression and essentiality of IKZF1 and IKZF3 in
mature B cells, somatic genetic inactivation of the IKZF1 and IKZF3
occurs in acute lymphoblastic leukemia, resulting in an
accumulation of immature lymphoid progenitor cells (C. G. Mullighan
et al., Nature 446, 758 (Apr. 12, 2007); S. Winandy et al., Cell
83, 289 (Oct. 20, 1995)). In T cells, ablation of IKZF3-mediated
repression of IL2 gene expression provides a mechanism for
increased IL2 production in response to lenalidomide. The
teratogenicity of thalidomide and the efficacy of lenalidomide in
MDS may be mediated by alternative substrates in different cellular
lineages.
[0185] RING-based E3 ubiquitin ligases are characterized by a high
specificity for their substrates and therefore represent promising
drug targets in cancer and other diseases. Following the
identification of an E3 ubiquitin ligase as a target of thalidomide
and lenalidomide, inhibition of enzymatic activity would have
seemed a more likely mechanism of action. The results reported
herein reveal that lenalidomide modulates the activity of the
CRL4-CRBN complex to increase ubiquitination of two transcription
factors, IKZF1 and IKZF3 that would otherwise be considered
"undruggable." A plant hormone, auxin, appears to act similarly,
increasing the interaction between a ubiquitin ligase and a
specific substrate, suggesting that this mechanism might be
operative in additional biological contexts. Selective
ubiquitination and degradation of specific targets provides a novel
mechanism of therapeutic activity for proteins that are not
otherwise amenable to small-molecule inhibition.
Example 7
Lenalidomide Treatment Reduced CSNK1A1 Levels in Murine Cells
Over-Expressing Human CRBN
[0186] To determine whether mouse and human cells responded
similarly, cell lines were treated with lenalidomide (FIG. 16).
Lenalidomide decreased CSNK1A levels in all human cell lines
expressing CRBN (see FIG. 17). FIG. 18 shows that levels of the
short and long forms of casein kinase were not reduced in bone
marrow from mice treated with lenalidomide. Similarly, murine
casein kinase 1 levels were not reduced in spleen in response to
lenalidomide (FIG. 18). No change in CSKN1A levels was seen in
Murine baf-3 cells or in primary MLL-AF9 transformed mouse cells.
In contrast, lenalidomide treatment reduced CSNK1A1 levels in
murine cells over-expressing human CRBN (FIG. 19). CSNK1A1 was used
as a readout because CSNK1A1 decreased after being treated with
Lenolidomide. hCRBN was clearly more sensitive to Lenalidomide than
mCRBN (FIG. 19).
Example 8
Lenalidomide Induces Ubiquitination and Degradation of Casein
Kinase 1A1 Via CRL4.sup.CRBN
[0187] Lenalidomide is a highly effective treatment for
myelodysplastic syndrome (MDS) with deletion of chromosome 5q
(del(5q)), inducing cytogenetic remission in more than 50% of
patients. No biallelic deletions or loss of function mutations on
the remaining allele have been detected in any of the genes located
in the commonly deleted regions of in del(5q) MDS, implying that
del(5q) MDS is a haploinsufficiency disease. MDS patients without
del(5q) are much less sensitive to lenalidomide, suggesting that
haploinsufficiency for a gene on chromosome 5q causes selective
sensitivity of the MDS cells to the drug. Recently, it has been
demonstrated that lenalidomide acts to modulate CRBN-CRL4 E3
ubiquitin ligase. Ubiquitination and degradation of the
transcription factors, IKZF1 and IKZF3, by lenalidomide is
responsible for two major properties of IMiDs: growth inhibition of
multiple myeloma cells and interleukin-2 release from T-cells.
However, it is unlikely that degradation of these lymphoid
transcription factors also accounts for therapeutic activity in del
(5q) MDS. Instead, it is possible that ubiquitination of a
different CRBN substrate in myeloid cells accounts for the efficacy
of lenalidomide in del(5q) MDS.
[0188] In order to identify such substrates. SILAC (stable isotope
labeling of amino acids in cell culture)-based quantitative mass
spectrometry was applied to assess global changes in ubiquitination
and protein levels in the myeloid cell line KG-1. Similar to the
analysis in multiple myeloma, lenalidomide altered the
ubiquitination and protein levels of a strikingly low number of
proteins, demonstrating the highly specific effects of the drug on
ubiquitin ligase function. Consistent with previous studies,
lenalidomide treatment decreased ubiquitination of CRBN and
increased ubiquitination of IKZF1, followed by the corresponding
changes in protein levels. Aside from IKZF1, casein kinase 1A1
(CSNK1A1, also known as CK1.alpha.) had the greatest increase in
ubiquitination and decrease in protein abundance following
lenalidomide treatment. CSNK1A1 is encoded by a gene in the del(5q)
commonly deleted region and has been shown to be a therapeutic
target in AML, and is thus an attractive candidate for mediating
the effects of lenalidomide in del(5q) MDS (FIGS. 20A, 20B and
FIGS. 24A-24C)
[0189] Based on the proteomics results, validation that CSNK1A1 is
a lenalidomide-dependent target of the CRBN-CRL4 ubiquitin ligase
was sought. It was confirmed that lenalidomide treatment decreased
CSNK1A1 protein levels in multiple human cell lines in a
dose-dependent fashion (FIGS. 20C, 24D, 25A-25B), decreased the
half-life of the CSNK1A1 protein, and did not alter CSNK1A1 mRNA
levels (FIGS. 20D and 25C). The lenalidomide-induced decrease in
CSNK1A1 protein levels was abrogated by treatment with the
proteasome inhibitor MG132 and the NEDD8-activating enzyme
inhibitor, MLN-4924, which interferes with the activity of
cullin-RING E3 ubiquitin ligases (FIG. 21A). Cells with homozygous
genetic inactivation of the CRBN gene by CRISPR-Cas genome
engineering were not responsive to lenalidomide (FIG. 21B).
Finally, it was demonstrated that CSNK1A1 co-immunoprecipitates
with hemagglutinin (HA)-tagged CRBN, and that lenalidomide
increases this association (FIG. 21D). Co-transfection of CRBN with
HA-CSNK1A1 promoted lenalidomide-induced ubiquitination of the
tagged CSNK1A1 in 293T cells (FIGS. 21B, 21D). In aggregate, these
experiments indicate that CSNK1A1 is a CRBN-CRL4 E3 ligase
substrate that is ubiquitinated and degraded in the presence of
lenalidomide.
[0190] Next, it was examined whether CK1.alpha. binds CRBN and is
ubiquitinated by the CRL4.sup.CRBN E3 ubiquitin ligase. CK1.alpha.
co-immunoprecipitated with FLAG-tagged CRBN only in the presence of
lenalidomide (FIG. 21C). Lenalidomide treatment increased the
ubiquitination of FLAG-CK1.alpha. in 293T cells (FIG. 21D).
[0191] The effects of CSNK1A1 depletion on cell proliferation were
assessed. CSNK1A1 is a serine/threonine kinase with multiple
cellular activities, including the suppression of TP53 and
.beta.-catenin activity. Complete loss of CSNK1A1 induces apoptosis
in normal and leukemic stem cells via p53 activation, while
heterozygous loss of CSNK1A1 causes stem cell expansion with
.beta.-catenin activation. Since p53 activation occurs when CSNK1A1
levels are less than 50% of normal, haploinsufficiency of CSNK1A1
in del (5q) MDS was thought to sensitize cells to a further
decrease in CSNK1A1 expression. To address this hypothesis, primary
human CD34.sup.+ hematopoietic stem and progenitor cells were
transduced with lentiviral vectors expressing GFP, as well as
CSNK1A1 or control shRNAs. Cells expressing CSNK1A1 shRNAs were
depleted in the absence of treatment, confirming that CSNK1A1
depletion inhibited growth of hematopoietic cells. (FIGS. 21E, 21F)
The addition of lenalidomide enhanced the depletion of CSNK1A1
shRNA expressing cells, but not cells expressing control shRNAs,
demonstrating that reduced CSNK1A1 levels sensitized hematopoietic
cells to lenalidomide (FIGS. 21E, 21F, 26B, 26C).
[0192] It was determined whether haploinsufficiency for Csnk1a1
sensitizes cells to lenalidomide in a genetically engineered mouse
model. In initial experiments, it was found that lenalidomide did
not decrease Csnk1a1 protein levels in murine Baf3 cells or primary
murine leukemia cells treated with lenalidomide (FIG. 22A, 27A,
27B). Mice did not develop the specific limb deformations observed
in human embryos exposed to thalidomide and primary murine multiple
myeloma did not respond to lenalidomide, suggesting that murine
cells were intrinsically resistant to IMiDs. Since CRBN is a direct
protein target of lenalidomide, it was examined whether expression
of the human CRBN could confer drug sensitivity onto murine cells.
Overexpression of human, but not murine CRBN, in murine cells
resulted in a decrease of CSNK1A1 protein levels, implying amino
acid differences between murine CRBN (mCRBN) and human CBRN (hCRBN)
were responsible for species-specific response to lenalidomide
(FIGS. 22B, 22C, 27A, 27B).
[0193] In order to determine the amino acids responsible for the
differential sensitivity to lenalidomide between species, a series
of human/mouse CRBN chimeric cDNAs and point mutations were tested.
A single amino acid in the C-terminus of CRBN (residue I391
(murine) and V387 (human)) was identified that determined the
response to lenalidomide. (FIG. 22C, 27C) Expression of a murine
CRBN mutant (mCRBN.sup.I391V) conferred sensitivity to
lenalidomide-induced degradation of CSNK1A1 in Baf3 cells. (FIG.
22D) Conversely, expression of the reciprocal human CRBN.sup.V387I
abrogated sensitivity to lenalidomide in human cells (FIG. 22E).
Amino acid 387 of CRBN is located in the IMiD-binding region
described by Ito et al., and in close proximity to the
CRBN.sup.Y383A,W385A mutant that does not bind to IMiDs.
[0194] Having determined the mechanism of lenalidomide resistance
in murine cells, the mCRBN.sup.I391V cDNA was expressed in
hematopoietic cells from Csnk1a1 conditional knockout mice to
determine the effects of Csnk1a1 haploinsufficiency on drug
sensitivity. c-Kit.sup.+ hematopoietic stem and progenitor cells
were isolated from Csnk1a1.sup.+/- and control littermates,
transduced with a retroviral vector expressing mCRBN.sup.I391V, and
cultured in competition with a neutral comparator line, SJL, in the
presence or absence of lenalidomide (FIG. 23A). Lenalidomide had no
effect on the control cells, but Csnk1a1.sup.+/- cells were
significantly depleted in the presence of lenalidomide (FIG. 23B).
The enhanced sensitivity of Csnk1a1.sup.+/- cells to lenalidomide
was associated with induction of the p53 target gene p21 (FIG. 23C)
and rescued by heterozygous deletion of p53 (FIG. 23D),
demonstrating a critical down-stream role for p53. These results
were consistent with the clinical observation that p53 mutations
conferred lenalidomide resistance in MDS with del(5q).
[0195] This study demonstrated that the efficacy of lenalidomide in
del(5q) MDS was mediated by targeted degradation of a
haploinsufficient protein, CSNK1A1. Loss of CSNK1A1 induces p53
activity, and other deleted genes on chromosome 5q, such as RPS14,
which may further sensitize cells to p53 activation. Degradation of
CSNK1A1 may also contribute to other clinical effects of
lenalidomide such as myelosuppression. CSNK1A1 degradation may be
involved in the clinical activity of lenalidomide in lymphoid
malignancies, including the activated B-cell (ABC) subtype of
diffuse large B-cell lymphoma, which requires CSNK1A1 for
constitutive NF-.kappa.B activity, and multiple myeloma cells.
[0196] The concept that genes within heterozygous deletions could
cause vulnerabilities in cancer cells has been confirmed in these
cell lines. Heterozygous deletion of CSNK1A1 was demonstrated to
create such vulnerability in del(5q) MDS cells, and that
lenalidomide-induced degradation of this protein resulted in major
significant clinical efficacy. Induction of ubiquitination and
degradation of other haploinsufficient proteins may provide a basis
for the development of new targeted therapies in cancer.
[0197] The results described in Examples 1-6 were carried out using
the following methods and materials.
Synthesis of Lenalidomide Derivative
[0198] NMR spectra were recorded on Bruker DRX-600, DRX-500, and
AMX-400 instruments and calibrated using residual undeuterated
solvent as an internal reference (CHCl.sub.3 @ 7.26 ppm 1H NMR,
77.16 ppm 13C NMR). The following abbreviations (or combinations
thereof) were used to explain the multiplicities: s=singlet,
d=doublet, t=triplet, ap=apparent, m=multiplet, b=broad, ABq=AB
quartet.
[0199] Compounds were purified by mass-directed purification on a
Waters Autopurification system (Milford, Mass.). Collection was
triggered on the (M+H)+ and (M+Na)+ ions on a ZQ mass spectrometer
using positive electrospray ionization. Mobile phase A consisted of
0.2% ammonium hydroxide in water, while mobile phase B consisted of
0.2% ammonium hydroxide in acetonitrile. An initial hold at 0%
mobile phase B for 1.0 minutes was followed by a gradient from 0%
to 100% mobile phase B over 11.0 minutes at 24 mL/min. A 2.0 ml/min
at-column dilution was present using 100% acetonitrile as well as a
2.0 mL/min make-up flow using 90/10/0.1 methanol/water/formic acid.
An XBridge OBD Prep C18, 5 um, 19.times.100 mm column was used at
room temperature.
Preparation of
N-butyl-4-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)amino)butana-
mide (lenaderivative)
[0200] Lenalidomide (30 mg, 0.116 mmol) and succinic semialdehyde
(0.075 ml, 0.116 mmol) (15% in water) were dissolved in DMF (0.4
ml) and AcOH (8.16 .mu.l). The reaction was stirred at room
temperature for 1 hour. Sodium triacetoxyborohydride (36.8 mg,
0.174 mmol) was then added and the reaction is maintained at room
temperature. After 4 hours, additional succinic semialdehyde (0.075
ml, 0.116 mmol) and sodium triacetoxyborohydride (36.8 mg, 0.174
mmol) were added and the reaction was stirred for a further 16
hours at room temperature. The reaction mixture was diluted with
MeOH, concentrated and purified by HPLC purification to afford the
desired carboxylic acid
(4-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)amino)butanoic
acid) (16.2 mg, 41%). MS (ESI) calcd for
C.sub.17H.sub.19N.sub.3O.sub.5 [M+H]+: 345. Found: 346. The
Lenalidomide carboxylic acid derivative (7 mg, 0.020 mmol) was
dissolved in DMF (0.5 mL). N-Hydroxysuccinimide (2.333 mg, 0.020
mmol) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (5.83 mg,
0.030 mmol) were then added. After 15 minutes, n-butylamine (10.02
.mu.L, 0.101 mmol) was added. The reaction mixture was concentrated
and purified by HPLC purification to afford the desired amide
(lenaderivative) (2.6 mg, 32%). MS (ESI) calcd for
C.sub.21H.sub.28N.sub.4O.sub.4 [M+H]+: 400. Found: 401. .sup.1H NMR
(300 MHz, M CD3OD) .delta. 8.51 (bs, 1H), 7.31 (ap t, J=7.8 Hz,
1H), 7.06 (d, J=7.5 Hz, 1H), 6.81 (d, J=8.1 Hz, 1H), 5.14 (dd,
J=13.3, 5.2 Hz, 1H), 4.30, 4.23 (ABq, J.sub.AB 16.9 Hz, 2H),
(3.29-3.02 (m, 4H), 2.97-2.69 (m, 2H), 2.57-2.35 (m, 1H), 2.29 (ap
t, J=7.3 Hz, 2H), 2.21-2.07 (m, 1H), 1.99-1.84 (m, 2H), 1.61-1.17
(m, 4H), 0.90 (t, J=7.2 Hz, 3H).
Immobilization of the Lenalidomide Derivative onto Affigel
Beads
[0201] The solid-phase beads used in small molecule immobilization
were Affigel 102 (Bio-Rad) with a loading level of 12 .mu.mol/mL
suspension. The bead suspension (1.0 mL) was transferred to a 2.0
mL eppendorf tube and washed with DMSO (6.times.1.5 mL). The beads
were then suspended in anhydrous DMSO (0.5 mL).
[0202] The lenalidomide-derived carboxylic acid
(4-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)amino)butanoic
acid) (0.277 mL, 10 .mu.mol) was dissolved in DMSO (0.5 mL) and to
this were added N-hydroxysuccinimide (1.151 mg, 10.00 .mu.mol) and
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (2.88 mg, 15.00
.mu.mol). After 45 minutes further N-hydroxysuccinimide (4 mg, 35
.mu.mol) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (6 mg,
31 .mu.mol) were added and the reaction mixture was stirred for a
further 60 minutes. At this point LC-MS indicated 60% of the
carboxylic acid had been activation with N-hydroxysuccinimide. To
achieve a 12.5% loading level of the Affigel beads, 1.5 .mu.mol of
activated compound was added to the suspended beads. Thus the
activated acid solution was added to the bead suspension followed
by triethylamine (8.36 .mu.L, 60.0 .mu.mol). The suspension was
then vortexed at room temperature for 1 hour and the depletion of
free activated bait molecule was monitored by LC-MS. After the
immobilization, the vials were centrifuged, the supernatant was
removed and the beads were washed with DMSO (3.times.2 mL) and
1-120 (3.times.2 mL). The heads were subsequently suspended in PBS
(0.8 mL) and stored at 4.degree. C. before use.
SILAC Media Preparation and Cell Culture Conditions
[0203] All standard SILAC media preparation and labeling steps were
as previously described (E.Ong, Nature protocols 1, 2650 (2006))
with the addition of light proline to prevent the conversion of
arginine to proline (S. C. Bendall et al., Mol Cell Proteomics 7,
1587 (September, 2008)). Briefly, L-methionine and 200 mg/L of
L-Proline were added to base media according to standard
formulations for RPM (Caisson Labs) or DMEM (Caisson Labs). This
base media was divided into three and to each added 1-arginine
(Arg0) and 1-lysine (Lys0) (light), 13C614N4-1-arginine (Arg6) and
4,4,5,5-D4-1-lysine (Lys4) (medium) or 13C615N4-1-arginine (Arg10)
and 13C615N2-1-Lysine (Lys8) (heavy) to generate the three SILAC
labeling mediums. Each medium with the full complement of amino
acids at the standard concentration for each media, was sterile
filtered through a 0.22.mu. filter (Milipore, Bedford Mass.). Each
cell type was grown in the corresponding labeling media, prepared
as described above, supplemented with 2 mM L-glutamine (Gibco), and
10% dialyzed fetal bovine serum (Sigma) plus antibiotics (Gibco),
in a humidified atmosphere with 5% CO2 in air. Cells were grown for
at least six cell divisions in labeling media.
Biochemical Purification with Lenalidomide-Derivative Beads
[0204] Separate cultures of K562 cells SILAC labeled either with
L-arginine and L-lysine (light) or L-arginine-13C6 and
L-lysine-13C6-15N2 (heavy) were lysed in ice-chilled ModRIPA buffer
containing 1% NP-40, 0.1% Na deoxycholate, 150 mM NaCl, 1 mM EDTA,
50 mM Tris, pH 7.5, and protease inhibitors (Complete.TM. tablets,
RocheApplied Science, Indianapolis, Ind.). Lysates were vortexed
intermittently while chilled on ice for 10 min and clarified by
spinning at 14,000.times.g. Protein concentrations of light and
heavy lysates were estimated with the Protein Assay Dye Reagent
Concentrate (Biorad, Hercules Calif.) and equalized. The protein
concentrations of lysates varied between 1.7 to 2.2 mg/mL, affinity
enrichments were performed in lysate volumes of 1.4 mL in a 1.5 mL
microcentrifuge tube.
[0205] Lenalidomide (in DMSO) at 100-fold excess over the amount of
lenalidomide-derivative on beads was added to 2 mg of light lysate.
An equal volume of DMSO was then added to 2 mg of heavy lysate as a
control and pre-incubated for 30 minutes. Thirty microliters of a
50% slurry in phosphate buffered saline (PBS) of
lenalidomide-derivative bead was added to both light and heavy
lysates.
[0206] Affinity enrichments were incubated overnight (approx. 16
hrs) on an end-over-end rotator at 4.degree. C. Following
incubation, the tubes were spun at 1000.times.g on a benchtop
centrifuge to pellet the beads. The supernatant was aspirated,
taking care to avoid disturbing the beads. Each tube in a set was
washed with ModRIPA buffer twice to remove excess soluble small
molecule competitor. Beads from the two tubes were then be combined
for an extra washing step in ModRIPA. After the third and final
wash, beads were collected by spinning at 1000.times.g and the wash
aspirated leaving approximately 20 .mu.L of buffer in the tube.
[0207] The experiment was done in process replicate in which the
labels were swapped, with lenalidomide being pre-incubated in the
heavy and DMSO in the light.
1D-SDS-PAGE and MS Analysis for Lenalidomide-Protein Interaction
Studies.
[0208] Proteins enriched in SILAC affinity pull-downs were reduced
and alkylated, on bead, in 2 mM DTT and 10 mM iodoacetamide
respectively. One part LDS buffer (Invitrogen) was added to three
parts sample (including beads) and tubes heated to 70.degree. C.
for 10 minutes. Proteins were resolved on a 4-12% gradient 1.5 mm
thick Bis-Tris gel with MES running buffer (Nupage, Invitrogen) and
Coomassie stained (Simply Blue, Invitrogen). Gel lanes were excised
into six pieces and then further cut into 1.5 mm cubes. The gel
pieces were further destained in a solution containing 50% EtOH and
50% 50 mM ammonium bicarbonate, then dehydrated in 100% EtOH before
addition of sufficient trypsin (12.5 ng/.mu.L) to swell the gel
pieces completely. An additional 100 .mu.L of 50 mM ammonium
bicarbonate was added before incubating at 37.degree. C. overnight
on a thermomixer (Eppendorf). Enzymatic digestion was stopped by
the addition of 100 .mu.L of 1% TFA to tubes. A second extraction
with 300 .mu.L of 0.1% TFA was combined with the first extract and
the peptides from each gel slice cleaned up on C18 StageTips.
(Rappsilber et al., Nature protocols 2, 1896 (2007)) Peptides were
eluted in 50 .mu.L of 80% acetonitrile/0.1% TFA and dried down in
an evaporative centrifuge to remove organic solvents. The peptides
were then resuspended by vortexing in 7 .mu.L of 0.1% TFA and
analyzed by nanoflow-LCMS with an Agilent 1100 with autosampler and
a LTQ Orbitrap. Peptides were resolved on a 10 cm column, made
in-house by packing a self-pulled 75 .mu.m I.D. capillary, 15 .mu.m
tip (P-2000 laser based puller, Sutter Instruments) column with 3
.mu.m Reprosil-C18-AQ beads (Dr. Maisch GmbH, Ammerbuch-Entringen,
Germany) with an analytical flowrate of 200 nL/min and a 58 min
linear gradient (.about.0.57% B/min) from 0.1% formic acid in water
to 0.1% formic acid/90% acetonitrile. The run time was 108 min for
a single sample, including sample loading and column
reconditioning. An MS method was used a with a master Orbitrap full
scan (60,000 resolution) and data dependent LTQ MS/MS scans for the
top five precursors (excluding z=1) from the Orbitrap scan. Each
cycle was approximately 2 secs long.
Identification and Quantification of Proteins for
Lenalidomide-Protein Interaction Studies
[0209] All mass spectra were analyzed with MaxQuant software
version 1.1.1.36.sup.4. using a human IPI database v3.68. MS/MS
searches for the proteome data sets were performed with the
following parameters: Oxidation of methionine and protein
N-terminal acetylation as variable modifications;
carbamidomethylation as fixed modification. Trypsin/P was selected
as the digestion enzyme, and a maximum of 3 labeled amino acids and
2 missed cleavages per peptide were allowed. The mass tolerance for
precursor ions was set to 20 p.p.m. for the first search (used for
nonlinear mass re-calibration) and 6 p.p.m. for the main search.
Fragment ion mass tolerance was set to 20 p.p.m. For identification
a maximum FDR of 1% was applied separately on protein, peptide and
PTM-site level. 2 or more unique/razor peptides were required for
protein identification and a ratio count of 2 or more for protein
quantification per replicate measurement.
CRBN-Protein Interaction Studies
[0210] MMS1 cells stably expressing FLAG- or HA-tagged CRBN were
grown for 2 weeks (.about.6 cell doublings) in RPMI depleted of
L-arginine and L-lysine (Caisson Labs Inc.) and supplemented with
10% dialyzed FBS (Sigma) and amino acids as described above to
generate light-, medium- and heavy-labeled cells. FLAG-CRBN
expressing cells were cultured in light media, HA-CRBN expressing
cells were grown in medium and heavy media. On day 14, HA-CRBN
expressing cells grown in medium media were treated with DMSO and
HA-CRBN cells grown in heavy media with 1 .mu.M lenalidomide for 6
hours. For a second replicate labels were swapped such that
HA-tagged CRBN expressing cells grown in medium media were treated
with lenalidomide and cells grown in heavy media treated with DMSO.
Cells were lysed in IP lysis buffer (Pierce) containing protease
and phosphatase inhibitor cocktail (Pierce). For
immunoprecipitation of HA-tagged proteins, 1000 .mu.g protein was
incubated together with HA-Tag Rabbit mAb Sepharose (C29F4) Bead
Conjugate (Cell Signaling) over night at 4.degree. C. in the
presence of 1 .mu.M lenalidomide or DMSO. Lysates of FLAG-CRBN
expressing cells served as negative control to exclude non-specific
binding to the anti-HA sepharose conjugates used for
immunoprecipitation. For a schematic presentation of the experiment
see FIG. 4.
1D-SDS-PAGE and MS Analysis for CRBN-Protein Interaction
Studies.
[0211] The beads from immunopurification samples were washed once
with IP lysis buffer (Pierce), then the three different lysates of
each replicate combined, washed again and reduced and alkylated, on
bead, in 2 mM DTT and 10 mM iodoacetamide respectively. One part
LDS buffer (Invitrogen) was added to three parts sample (including
beads) and tubes heated to 70.degree. C. for 10 minutes. Proteins
were resolved on a 4-12% gradient 1.5 mm thick Bis-Tris gel with
MES running buffer: 50 mM (2-[N-morpholino]ethanesulfonic acid); 50
mM Tris base; 1 mM EDTA; 1% (w/v) SDS (Nupage, Invitrogen) and
Coomassie stained (Simply Blue, Invitrogen). Gel lanes were excised
into nine pieces and then further cut into 1.5 mm cubes. The gel
pieces were further destained in a solution containing 50% EtOH and
50% 50 mM ammonium bicarbonate, then dehydrated in 100% EtOH before
addition of sufficient trypsin (12.5 ng/.mu.L) to swell the gel
pieces completely. An additional 100 .mu.L of 50 mM ammonium
bicarbonate was added before incubating at 37.degree. C. overnight
on a thermomixer (Eppendorf). Enzymatic digestion was stopped by
the addition of 100 .mu.L, of 1% trifluoracetic acid (TFA) to
tubes. A second extraction with 300 .mu.L of 0.1% TFA was combined
with the first extract and the peptides from each gel slice cleaned
up on C18 StageTips (Rappsilber et al., Nature protocols 2, 1896
(2007)). Peptides were eluted in 50 .mu.L of 80% acetonitrile/0.1%
TFA and dried down in an evaporative centrifuge to remove organic
solvents. The peptides were then reconstituted with 3% ACN in 0.1%
formic acid. Reconstituted peptides were separated on an online
nanoflow EASY-nLC 1000 UHPLC system (Thermo Fisher Scientific) and
analyzed on a benchtop Orbitrap Q Exactive mass spectrometer
(Thermo Fisher Scientific). The peptide samples were injected onto
a capillary column (Picofrit with 10 .mu.m tip opening/75 .mu.m
diameter, New Objective, PF360-75-10-N-5) packed in-house with 20
cm C18 silica material (1.9 .mu.m ReproSil-Pur C18-AQ medium, Dr.
Maisch GmbH, r119.aq). The UHPLC setup was connected with a
custom-fit microadapting tee (360 .mu.m, IDEX Health & Science,
UH-753), and capillary columns were heated to 50.degree. C. in
column heater sleeves (Phoenix-ST) to reduce backpressure during
UHPLC separation. Injected peptides were separated at a flow rate
of 200 nL/min with a linear 80 min gradient from 100% solvent A (3%
acetonitrile, 0.1% formic acid) to 30% solvent B (90% acetonitrile,
0.1% formic acid), followed by a linear 6 min gradient from 30%
solvent B to 90% solvent B. Each sample was run for 150 min,
including sample loading and column equilibration times.
Data-dependent acquisition was obtained using Xcalibur 2.2 software
in positive ion mode at a spray voltage of 2.00 kV. MS1 Spectra
were measured with a resolution of 70,000, an AGC target of 3e6 and
a mass range from 300 to 1800 m/z. Up to 12 MS2 spectra per duty
cycle were triggered at a resolution of 17,500, an AGC target of
5e4, an isolation window of 2.5 m/z and a normalized collision
energy of 25. Peptides that triggered MS2 scans were dynamically
excluded from further MS2 scans for 20 s.
Identification and Quantification of Proteins for CRBN-Protein
Interaction Studies.
[0212] All mass spectra were analyzed with MaxQuant software
version 1.3.0.5. (J. Cox et al., Journal of proteome research 10,
1794 (Apr. 1, 2011)) using a human Uniprot database. MS/MS searches
for the proteome data sets were performed with the following
parameters: Oxidation of methionine and protein N-terminal
acetylation as variable modifications; carbamidomethylation as
fixed modification. Trypsin/P was selected as the digestion enzyme,
and a maximum of 3 labeled amino acids and 2 missed cleavages per
peptide were allowed. The mass tolerance for precursor ions was set
to 20 p.p.m. for the first search (used for nonlinear mass
re-calibration) and 6 p.p.m. for the main search. Fragment ion mass
tolerance was set to 20 p.p.m. For identification a maximum FDR of
1% was applied separately on protein, peptide and PTM-site level. 2
or more unique/razor peptides were required for protein
identification and a ratio count of 2 or more for protein
quantification per replicate measurement. To assign interacting
proteins the Limma package was used in the R environment to
calculate moderated t-test p, as described previously (9).
Cell Culture and Treatment for K-.epsilon.-GG and Proteome
Profiling
[0213] MM1S cells were cultured for 2 weeks (.about.6 cell
doublings) in RPMI depleted of L-arginine and L-lysine (Caisson
Labs Inc.) and supplemented with 10% dialyzed FBS (Sigma) and amino
acids as described above to generate light-, medium- and
heavy-labeled cells. Media was exchanged every 3rd day. On day 14
cells were treated for 12 hours with 1 .mu.M lenalidomide, 20 .mu.M
thalidomide or DMSO. For each of the three replicates SILAC labels
were flipped:
TABLE-US-00010 SILAC labelling Light Medium Heavy Replicate 1 DMSO
Thal 20 uM Len 1 uM Replicate 2 Len 1 uM DMSO Thal 20 uM Replicate
3 Thal 20 uM Len 1 uM DMSO
For the last 3 hours cells determined for K-.epsilon.-GG profiling
were treated with 5 .mu.M MG132 together with lenalidomide,
thalidomide or DMSO. K-.epsilon.-GG profiling was later performed
for all 3 replicates and proteome profiling for replicate 1 and
2.
Cell Lysis and Trypsin Digestion for K-.epsilon.-GG and Proteome
Profiling
[0214] SILAC-labeled cell pellets were lysed in 8 M urea, 50 mM
Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 2 ug/ml aprotinin
(Sigma-Aldrich), 10 ug/ml leupeptin (Roche Applied Science), 1 mM
phenylmethylsulfonyl fluoride (PMSF), 50 uM PR-619, and 1 mM
chloroacetamide at 4 C. Following lysis, samples were centrifuged
at 20,000.times.g for 15 minutes at 4 C to remove insoluble
material. Protein concentrations were determined using a
bicincohoninic acid (BCA) protein assay (Pierce) and samples were
mixed equitably at 10 mg per SILAC state. Proteins were reduced
with 5 mM dithiothreitol for 45 minutes at room temperature (RT)
and subsequently carbamidomethylated with 10 mM iodoacetamide for
30 min at RT in the dark. Samples were diluted to 2 M urea with 50
mM Tris-HCl, pH 7.5, and digested with sequencing grade trypsin
(Promega) at 25 C o/n using an enzyme to substrate ratio of 1:50.
Digested samples were acidified to 1% formic acid (FA)
(Sigma-Aldrich).
[0215] Tryptic peptides were desalted on 500-mg tC18 Sep-Pak SPE
cartridges (Waters). Cartridges were conditioned with 5 ml of 100%
acetonitrile (MeCN), 5 ml of 50% MeCN/0.1% FA, and four times with
5 ml of 0.1% trifluoroacetic acid (TFA). Up to 15 mg of sample was
loaded onto a single cartridge, and subsequently washed 3.times.
with 5 ml of 0.1% TFA. Samples were eluted from cartridges by
washing 2.times. with 3 ml of 50% MeCN/0.1% FA. Desalted samples
were dried overnight in a Savant SC210A SpeedVac concentrator
(Thermo Scientific).
Basic pH Reverse Phase (bRP) Fractionation
[0216] Offline bRP fractionation was completed using a
custom-manufactured Zorbax 300 Extend-C18 column (9.4.times.250 mm,
300 .ANG., 5 um, Agilent) on an Agilent 1100 series HPLC system.
Approximately 15 mg of peptide sample was resuspended in 1.8 ml of
basic RP solvent A (2% MeCN, 5 mM ammonium formate, pH 10),
separated into 2 HPLC vials and injected with Solvent A at flow
rate of 3 ml/min. A 64-min method was used for fractionation. The
gradient was composed of an initial increase to 8% Solvent B (1.1%
B/min) (90% MeCN, 5 mM ammonium formate), followed by a 38-minute
linear phase (0.5% B/min) where the amount of solvent B was
increased form 8% to 27% and ramp phases where the Solvent B amount
was increased from 31% (1% B/min) to 39% (0.5% B/min), and finally
to 60% (3% B/min). A total of 96 2 ml fractions were collected
every 0.66 min at a flow rate of 3 ml/min. For the proteome
profiling, 5% of each fraction was pooled into 22 fractions. For
ubiquitination profiling, 95% of each fraction was pooled into 8
fractions using a concatenated pooling strategy. Pooled samples
were dried using a SpeedVac concentrator.
K-.epsilon.-GG Enrichment
[0217] The anti-K-.epsilon.-GG antibody was obtained from the
PTMScan.RTM. ubiquitin remnant motif (K-.epsilon.-GG) kit (Cell
Signaling Technology). Prior to enrichment, the antibody was
covalently coupled to Protein A agarose beads by chemical
cross-linking with DMP. For cross-linking, the antibody bound beads
were first washed 3.times. with 1 ml of 100 mM sodium borate, pH 9
and then incubated in 1 ml of 20 mM dimethyl pimelimidate (DMP) for
30 minutes with rotation at RT. The reaction was stopped by washing
beads 2.times. with 1 ml of 200 mM ethanolamine, pH 8 followed by
incubation for 2 hours at 4 C with rotation. Antibody-bound beads
were washed three times in 1.5 ml of ice cold immunoprecipitation
(IAP) buffer (50 mM MOPS, pH 7.2, 10 mM sodium phosphate, 50 mM
NaCl), resuspended in IAP buffer, and stored at 4 C.
[0218] For K-.epsilon.-GG enrichment, bRP fractions were
reconstituted in 1.5 ml of IAP buffer and each fraction was
incubated with 32 ug of cross-linked anti-K-.epsilon.-GG antibody
for 1 hour, at 4 C, while rotating. Following incubation, samples
were spun down at 2000.times.g and the supernatant was removed.
Antibody-bound beads were washed 4.times. with 1.5 ml of ice cold
PBS and peptides were then eluted from the beads with 2.times.50 ul
of 0.15% TFA. Eluted peptides were desalted using C18 StageTips.
Each StageTip was packed with two plugs of C18 material (Empore.TM.
C18 Extraction Disk; 3M) and then conditioned with 100 ul of MeOH,
100 ul of 50% MeCN/0.1% FA, and 2.times. with 100 ul of 0.1% FA.
K-.epsilon.-GG peptides were loaded onto the condition StageTips,
washed 2.times. with 100 ul of 0.1% FA, eluted with 50 ul of 50%
MeCN/0.1% FA, and dried to completeness.
LC-MS/MS Analysis
[0219] K-.epsilon.-GG and global proteome fractions were
reconstituted in 8 ul and 20 ul of 3% MeCN/1% FA, respectively, and
analyzed by nanoflow-UPLC-HCD-MS/MS using Q Exactive mass
spectrometer (Thermo Fishes Scientific) coupled on-line to a
Proxeon Easy-nLC 1000 system. 4 ul and 1 ul of K-.epsilon.-GG and
global proteome samples was injected, respectively, for each
analysis. Samples were injected onto a microcapillary column (360
um OD.times.75 um ID) packed with 24 cm of ReproSil-Pul C18-AQ 1.9
um beads (Dr. Maisch GmbH) that was equipped with an integrated
electrospray emitter tip (10 um). For online analyses, the column
was heated to 50 C using a 20 cm column heater (Phoenix S&T).
For LC separation, solvent A was 0.1% FA/3% MeCN and solvent B was
90% MeCN/0.1% FA. Peptides were eluted on the mass spectrometer at
a flow rate of 200 nl/min using a gradient consisting of a linear
phase at 0.3% B/min, followed by a ramp to 60% B (10% B/min). The
total analysis time for each sample was 150 minutes. The Q Exactive
instrument was operated in the data-dependent mode acquiring HCD
MS/MS scans (R=17,500) after each MS1 scan (R=70,000) on the 12 top
most abundant ions using an MS1 ion target of 3.times.10.sup.6 ions
and an MS2 target of 5.times.10.sup.4 ions. The maximum ion time
utilized for the MS/MS scans was 120 ms; the HCD-normalized
collision energy was set to 25; the dynamic exclusion time was set
to 20 s, and the peptide match and isotope exclusion functions were
enabled.
K-.epsilon.-GG and Proteome MS Data Analysis
[0220] MS data was analyzed with the MaxQuant software version
1.3.0.5 and searched against the human Uniprot database that
contained 248 common laboratory contaminants was provided by the
MaxQuant software package. The search parameters were as follows:
enzyme specificity was set to trypsin, maximum number of mixed
cleavages to 2, precursor mass tolerance was at 20 ppm for the
first search (used for nonlinear mass re-calibration), and set to 6
ppm for the main search. Oxidized methionines and N-terminal
protein acetylation were searched as variable modifications, with
carbamidomethylation of cysteines set to fixed modification. For
searching K-.epsilon.-GG data files, Gly-Gly addition to lysines
was also searched as a variable modification. The minimum peptide
length was set to 6, and false discovery rate for peptide, protein,
and side identification was set to 1%. The filter labeled amino
acids and peptide quantification functions were enabled. For
proteome data, proteins were considered in the dataset if they were
identified by 2 or more razor/unique peptides and quantified by 3
or more ratio counts in bot biological replicates. For the
K-.epsilon.-GG data, K-.epsilon.-GG sites were considered if they
were confidently localized (>0.75) and quantified in all three
biological replicates.
Cell Lines and Primary Cells
[0221] MM15, NCI-H929, U266, Namalwa, Jurakat, K562, KIEL and 293T
cells were obtained from American Type Culture Collection. Cells
were cultured in RPMI 1640 (Mediatech) or DMEM (Mediatech)
supplemented with 10-20% heat-inactivated fetal bovine serum (Omega
Scientific) and 1% penicillin, streptomycin, and L-glutamine
(Mediatech). Cells were grown at 37.degree. C. in a humidified
incubator under 5% CO.sub.2.
[0222] Primary T cells were obtained from healthy donors under an
Institutional Review Board approved protocol at the Dana-Farber
Cancer Institute. PBMCs were isolated using Ficoll (Ficoll-Paque
PLUS, GE Healthcare) according to the protocol. After positive
selection with CD3+ MACS beads (Miltenyi), T cells were cultured in
RPMI with 10% human Serum (Sigma) and 100 U/ml recombinant IL-2
(Miltenyi). For stimulation, tissue culture plates were pre-coated
with 2.5 .mu.g/ml CD3 (OKT3, Biolegend) and CD28 (CD28.2,
Pharmingen).
Antibodies
[0223] The following antibodies were used: HA-HRP (Miltenyi,
GG8-1F3.3), Flag-HRP (M2, Sigma Aldrich), Actin-HRP (Abcam), rabbit
IKZF3 (Imginex), IKZF1 (H-100, Santa Cruz), FK2-HRP (Enzo
Lifescience), DDB1 (Abcam), and p27 (Cell Signaling).
Virus Constructs
[0224] For cDNA over-expression, the RSF91 retrovirus backbone
(kind gift of Prof. Dr. Christopher Baum of Hanover Medical School)
was used. For certain constructs GFP was replaced by GFP-T2A-Puro
or dTomato. The Gateway Vector Conversion System (Invitrogen) was
used to converted RSF91 to a Gateway Destination vector. Entry
clones were obtained from the Broad Institute Orfeome collection
and cloned into RSF91-Gateway with LR clonase enzyme mix II
(Invitrogen). IKZF4 cDNA was obtained from GeneCopeia. The CRBN
YWAA mutant, IKZF3 and IKZF4 mutants, IKZF2 Isoform 1 were cloned
by PCR using overlapping primers containing the respective
mutations.
TABLE-US-00011 ORF Origin Clone IKZF1 Broad Institute
ORF016074.1_s300c1 IKZF2 Broad Institute ORF018485.1_s300c1 IZKF3
Broad Institute ORF000952.1_s304c1 IKZF4 GeneCopoeia # GC-Z2828
IZKF5 Broad Institute ORF004130.1_s300c1 RAB28 Broad Institute
ORF011035.1_s304c1 IRF4 Broad Institute ORF002494.1_s304c1 HOXA9
Broad Institute ORF016570.1_s300c1 CRBN Broad Institute
ORF007943.1_s300c1
Lentivrial vectors expressing shRNAs were obtained from the RNAi
consortium (TRC) of the Broad Institute:
TABLE-US-00012 shRNA Clone Name Target sequence Luciferase#1
TRCN0000072254 ATGTTTACTACACTCGGATAT Luciferase#2 TRCN0000072243
CTTCGAAATGTCCGTTCGGTT CRBN#1 TRCN0000141562 CGCTGGCTGTATTCCTTATAT
CRBN#2 TRCN0000144360 CAGGATAGTAAAGAAGCCAAA CRBN#3 TRCN0000139091
CTTAACGCGATCTGCTCTGTT IKZF1#1 TRCN0000236419 CCGCTTCCACATGAGCTAAAG
IKZF1#2 TRCN0000236420 GCATTTGGAAACGGGAATAAA IKZF1#3 TRCN0000244221
GTGATATCTGTGGGATCATTT IKZF3#1 TRCN0000236419 CCGCTTCCACATGAGCTAAAG
IKZF3#2 TRCN0000236420 GCATTTGGAAACGGGAATAAA IKZF3#3 TRCN0000244221
GTGATATCTGTGGGATCATTT
[0225] The luciferase reporter plasmid
CMV-IRES-RenillaLUC-IRES-Gateway-FireflyLUC (11) was a kind gift
from William G. Kaelin (Dana-Farber Cancer Institute). Cloning of
cDNAs was performed using Gateway LR reaction (invitrogen). 24
hours after transfection of 40 ng reporter plasmid in 10.times.e4
293T cells, the media was changed to media containing lenalidomide
or the vehicle control. After an additional 24 hours, dual
luciferase assays were performed using the Dual-Glo Luciferase
Assay System (Promega) according to the manufacturer's
protocol.
Transfections, Virus Production and Infections
[0226] Retro- or lentiviral vectors were transfected using
TRANS-LTI (Mirrus) into 293T cells together with packaging plasmid
(retrogagpol or pSPAX2) and envelope plasmids expressing VSV-G. The
media was changed after 12 hours, and the viral supernatant was
collected 36 hours and 48 hours after transfection.
[0227] For viral infection, cells were seeded in high density and
supplemented with Polybrene (Sigma) at concentrations of 1 to 2
.mu.g/ml. Primary T cells were stimulated on plates pre-coated with
anti-C3/CD28 for 48 h before lentiviral transduction. Puromycin
selection was started 1 day after transduction at concentrations
between 0.5 and 2 .mu.g/ml.
Quantitative RT-PCR
[0228] Gene expression was measured by reverse transcription
quantitative PCR. (RQ-PCR). cDNA was synthesized using the cDNA
synthesis Kit for Multimacs (Miltenyi) according to the
manufacturer's protocol, and used 1 ul of the product per RQ-PCR
reaction in a 384-well plate. The following primer-probe sets from
fife Technologies were used: GAPDH ( ), IKZF1 ( ), IKZF3 ( ), CRBN
(Hs00372271_m1), IL-2 ( ). Analysis was performed on a 7900HT Fast
Real-Time PCR System (Applied Biosystems). Expression levels were
calculated using the .DELTA..DELTA.CT method.
Western Blot
[0229] Protein lysates were run on Tris-HCl, 1 mm Criterion.TM.
Precast gels (Bio-Rad) at a constant voltage. Proteins were
transferred onto Imobilon-P transfer membranes (Millipore) at a
constant amperage. Before staining, blots were blocked in 5% BSA in
TBST for 30 minutes.
Immunoprecipitation
[0230] For immunoprecipitation of HA-tagged proteins, the
HA-protein Isolation Kit from Miltenyi was used according to the
manufacturer's protocol using an MultiMACS M96 Separator
(Miltenyi). Proteins tagged with the FLAG peptide were
immunoprecipitated using anti-FLAG M2 Affinity Gel (Sigma-Aldrich)
according to the manufacturer's protocol. 500 to 1000 .mu.g protein
was incubated together with the specific bead-bound antibody
overnight at 4.degree. C. The samples were washed 4 times with RIPA
buffer or IP lysis buffer (Pierce) and protein was eluted from the
affinity gel or Multimacs columns with 98.degree. C. laemmli buffer
(Bio-Rad).
In Vivo Ubiquitination
[0231] MM1S or 293T cells expressing tagged IKZF1 or IKZF3 were
treated with the respective concentrations of lenalidomide and/or
epoxomicin for 1.5 hours. Cells were then washed twice with
ice-cold PBS and lysed under denaturing conditions using 2%
SDS-containing lysis buffer and boiled for 10 minutes. SDS was
diluted with addition of 10.times. Ip lysis buffer (Pierce),
incubated at 4.degree. C. for 30 minutes prior addition of IP
antibodies. Immunoprecipitation was performed over night and then
washed 4.times. with 1 ml RIPA buffer. Proteins were eluted from
the beads by addition of Laemmli buffer and incubation at
95.degree. C. for 5 minutes. The supernatant was then loaded on a
gel and analyzed by Western Blot.
In Vitro Ubiquitination
[0232] 293T cells were co-transfected with HA-IKZF3 and FLAG-CRBN.
After 48 hours cells were treated with DMSO or 1 .mu.M lenalidomide
for 20 minutes, lysed in IP lysis buffer (Pierce) and
immunoprecipitation was performed overnight with anti-FLAG M2
sepharose beads (Sigma) to obtain CRBN together with CRBN-bound
IKZF3. The beads were washed 3.times. with IP lysis buffer,
1.times. ubiquitination buffer (Boston Biochem) and eluted with 250
.mu.g/ml FLAG peptide (Sigma) for 30 min at 4.degree. C. The
CRBN-IKZF3 complex was incubated for 90 min at 30.degree. C. in
ubiquitination reaction mixture containing 200 nM E1, 500 nM E2
(UbcH5a and UbcH5b), 20 .mu.g ubiquitin, 1 .mu.M ubiquitin
aldehyde, 1.times. ubiquitin reaction buffer, 1.times. Energy
Restoration System (all Boston Biochem), and 100 nM MG101 in a
total volume of 75 .mu.l. Negative controls did not include E1 and
E2 enzymes. 20 .mu.l of the reaction was denatured by adding
5.times.SDS containing loading buffer (Boston Biochem) and boiling
at 95.degree. C. for 5 minutes, separated by SDS-PAGE and
transferred to a PVDF membrane in order to detect HA-IKZF3 and its
ubiquitinated forms with an HA specific antibody. The remaining 55
.mu.l reaction mix were denatured by adding SDS to a final
concentration of 1% and boiling for 10 minutes. 500 .mu.l IP lysis
buffer was added for 30 minutes before adding anti-HA magnetic
beads (Miltenyi) for 1 hour. After purification on Multimacs
columns (Miltenyi) eluates were separated by SDS-PAGE, transferred
to a PVDF membrane and stained with anti-ubiquitin antibodies
(FK2).
Flow Cytometry
[0233] Flow cytometry was performed on a FACS Canto II (BD
Bioscience) using PE channel for detection of dTomato-, and FITC
for GFP-expressing cells. DAPI staining was performed to exclude
dead cells.
[0234] For investigating shRNA effects on proliferation 50,000
cells were infected in a 96-well plate with 50 .mu.l lentivirus
containing medium in the presence of polybrene. Media was changed
after 24 hours. The number of infected cells was determined on day
2 when GFP was fully expressed in all infected cells. The number of
viable GFP positive cells on day 2 was set to 100% to normalize for
transduction efficiency and every consecutive assessment was
calculated in relation to day 2.
[0235] To investigate the effect of IKZF3 over-expression on
lenalidomide sensitivity, MM1S were separately infected with an
empty backbone expressing dTomato or an IKZF3 and GFP expressing
vector. After two days cells were washed, combined in a 96-well
plate and analyzed by flow cytometry for the relative number of GFP
and Tomato expressing cells before start of lenalidomide treatment.
Media was changed every 3.sup.rd day containing the drug. Every
experiment was performed in triplicate.
Viability
[0236] For assessing the effects of lenalidomide on cell growth
cells were plated in a 96-well plate and treated with lenalidomide.
On the respective days, total cellular ATP content was assessed
using CellTiter-Glo.RTM. Luminescent Cell Viability Assay (Promega)
according to the protocol. Luminescence was assessed by a multimode
detector DTX880 (Beckman Coulter).
[0237] The results described in Example 8 were carried out using
the following methods and materials.
Reagents
[0238] Lenalidomide (Toronto Research Chemicals and Selleck
Chemicals), Thalidomide (Milipore), Pomalidomide (Selleck
Chemicals), MG-132 (Selleck Chemicals), CC-122 (Celgene), PR619
(Lifesensors) and MLN4924 (Active Biochem) were dissolved in DMSO
at 10 to 100 mM and stored at -20.degree. C. for up to 6 months.
For cell culture experiments drugs were diluted at least by 1:1000
so that the final DMSO concentration was 0.1% or lower.
Cell Lines
[0239] KG-1, Ba/F3, K562, MM1S, Jurkat, and 293T cells were
obtained from American Type Culture Collection (ATCC). Cells were
cultured in RPMI 1640 (Mediatech) or DMEM (Mediatech) supplemented
with 10-20% heat-inactivated fetal bovine serum (FBS)(Omega
Scientific) and 1% penicillin, streptomycin, and L-glutamine
(Mediatech). Cells were grown at 37.degree. C. in a humidified
incubator under 5% CO.sub.2. Ba/F3 cells were cultured in the
presence of 10 ng/ml murine IL-3 (Miltenyi) and MDS-L cells were
cultured with 10 ng/ml human GM-CSF. 293T cells were transfected
using TransIT-LT1 (Mirius Bio) according to the manufacturer's
protocol.
Cell Culture and Treatment for K-.epsilon.-GG and Proteome
Profiling
[0240] KG-1 cells were cultured for 2 weeks (.about.6 cell
doublings) in RPMI depleted of L-arginine and L-lysine (Caisson
Labs Inc.) and supplemented with 10% dialyzed FBS (Sigma) and
L-arginine (Arg0) and L-lysine (Lys0) (light),
.sup.13C.sub.6.sup.14N.sub.4-L-arginine (Arg6) and
4,4,5,5-D.sub.4-L-lysine (Lys4) (medium) or
.sup.13C.sub.6.sup.15N.sub.4-L-arginine (Arg10) and
.sup.13C.sub.6.sup.15N.sub.2-L-Lysine (Lys8) (heavy) to generate
light-, medium- and heavy-labeled cells. Media was exchanged every
3.sup.rd day. On day 14 cells were treated with 1 .mu.M
lenalidomide, 10 .mu.M lenalidomide or DMSO for 4 hours for
ubiquitination profiling and 24 hours for protein level assessment.
Experiments were performed in two biological replicates with
flipped SILAC labeling: Relicate 1: DMSO/light, lenalidomide 1
.mu.M/medium; lenalidomide 10 .mu.M/heavy; replicate 2:
lenalidomide 10 .mu.M/light, DMSO/medium; lenalidomide 1
.mu.M/heavy.
SILAC Based K-.epsilon.-GG and Proteome Profiling of KG-1 Cells
[0241] Cell lysis and trypsin digestion, basic pH reversed phase
fractionation, K-.epsilon.-GG enrichment, and LC-MS/MS analysis for
KG-1 cells were performed as recently described (Science 343,
301-305, (2014)). For this work, 10 mg of protein was input per
SILAC state for the ubiquitin workflow. For proteome profiling, 1.5
mg of protein was input per SILAC state and samples were
fractionated by bRP using a 4.6 mm.times.250 mm column (Agilent,
3.5 urn bead size) using the method previously described (Nature
methods 10, 634-637, (2013)).
[0242] For data analysis, normalized SILAC ratios for the 2
biological replicates were filtered to retain only those deemed
reproducible. Reproducibility was based on replicates being
confined within the 95% limits of agreement of a Bland-Altman plot.
In the Bland-Altman plot, differences of the replicates are plotted
against the average values and the limits of agreement correspond
to the prediction confidence interval for a regression line with
unit slope. Reproducible replicates were then subjected to a
moderated T-test to assess statistical significance. This statistic
is similar to the ordinary t-statistic, with the exception that the
standard errors are calculated using an empirical Bayes method
utilizing information across all proteins, thereby making inference
about each individual protein more robust. The nominal p-values
arising from the moderated t-statistic are corrected for multiple
testing by controlling the false discovery rate (FDR). Proteins
with an FDR adjusted p-value of less than 0.05 were deemed to be
reproducibly regulated. Figures containing scatter plots of SILAC
data show all points regardless of the reproducibility measure.
Statistical significance was assessed using only reproducible data
points.
Plasmids and Virus Constructs
[0243] The following cDNAs were cloned in the RSF91 retrovirus
backbone (kind gift of Christopher Baum, Hanover Medical School) or
EF1a-IRES-GFP lentiviral backbone: CSNK1A1 Isoform 2
(ccsbBroadEN_06055), CSNK1E (ccsbBroadEN_00379), murine CRBN
Isoform 2 (Thermo Scientific), and human CRBN Isoform 2
(ccsbBroadEn_08244). For certain experiments GFP was replaced by
dTomato for competition experiments or GFP-T2A-Puro to allow for
drug selection of positively transduced cells. Chimeric cDNAs and
point mutations were cloned with overlapping PCR primers.
Lentivirus was concentrated by ultracentrifugation for transduction
of primary cells.
[0244] Lentiviral vectors (TRC005 backbone) expressing shRNAs
targeting luciferase (TRCN0000072254: ATGTTTACTACACTCGGATAT) and
CSNK1A1 (#1: TRCN0000342505, CATCTATTTGGCGATCAACAT; #2:
TRCN0000342507, GCAGAATTTGCGATGTACTTA) were obtained from The RNAi
Consortium (TRC) of the Broad Institute. For certain experiments,
the puromycin resistance gene was replaced by GFP.
[0245] The luciferase reporter plasmid
CMV-IRES-RenillaLUC-IRES-Gateway-FireflyLU was a kind gift from
William G. Kaelin (Dana-Farber Cancer Institute). Cloning of cDNAs
was performed using Gateway LR reaction (Invitrogen).
[0246] CRISPR mediated genetic deletion was performed with the
sgRNA-CAS9-T2A-Puro plasmid..sup.29 A CRBN exon 1-specific guide
RNA was cloned in the BsmBI site.
[0247] 1.times.10.sup.5 293 T cells were transfected in a 12-well
with 1 .mu.g plasmid using TransLTI (Mirrus). After 24 hours
transfected cells were selected with 2 .mu.g/ml puromycin for 4
days. Then 293T cells were diluted to single cell and plated in
96-well. Colonies were tested by western blot and sanger sequencing
of the endogenous CRBN exon1 locus for inactivating biallelic
out-of-frame mutations.
Western Blot and Antibodies
[0248] Protein lysates were run on Tris-HCl, 1 mm Criterion.TM.
Precast gels (Bio-Rad) or NuPAGE Bis-Tris gels (Novex) gels at a
constant voltage. Proteins were transferred onto Immobilon-P
transfer membranes (Millipore) at a constant amperage. Before
staining with primary antibodies, blots were blocked in 5% non-fat
dry milk (Santa Cruz) or BSA in TBST for 30 minutes.
[0249] For protein detection primary antibodies detecting
CK1.alpha. (C-19, Santa Cruz or Abcam ab108296), HA (HRP-conjugate,
Miltenyi, GG8-1F3.3), FLAG (M2, HRP-conjugate Sigma Aldrich),
ubiquitin (FK2, HRP-conjugate Enzo Life Sciences), Actin
(HRP-conjugate, Abcam), and GAPDH (Santa Cruz sc-47724) were used.
Secondary antibodies were HRP conjugated Bovine anti-Goat (Jackson
ImmunoResearch) and HRP conjugated donkey anti-rabbit (GE
Healthcare). Supersignal chemi-luminescent substrate was used for
detection. For re-probing, blots were stripped in Restore Western
Blot Stripping Buffer (Thermo Scientific), activated in methanol,
and re-blocked.
Flow Cytometry
[0250] Flow cytometry was performed on a FACS Canto II (BD
Bioscience) using the PE and FITC channels for the detection of
dTomato and GFP, respectively. DAPI staining was performed to
exclude dead cells. A High-Throughput Sampler (BD) was used for
some experiments.
Quantitative RT-PCR
[0251] Gene expression was measured by reverse transcription
quantitative PCR (RQ-PCR). For RNA isolation and reverse
transcription a cDNA Synthesis Kit for MultiMacs (Miltenyi) was
used according to the manufacturer's protocol. The following
primer-probe sets from Life Technologies were used with TaqMan Gene
Expression Master Mix (Life Technologies): human GAPDH (402869),
human CSNK1A1 (Hs00793391_m1), murine GAPDH (Mm99999915_g1), murine
p21 (Mm04205640_g1). Analysis was performed on a 7900HT Fast
Real-Time PCR System (Applied Biosystems) in a 384-well plate.
Relative expression levels were calculated using the
.DELTA..DELTA.CT method.
Immunoprecipitation of FLAG-CRBN
[0252] 3.times.10.sup.6 293 T cells were plated in a 10 cm dish and
transfected with 10 .mu.g FLAG-hCRBN or empty vector. Cells were
treated with DMSO or 1 .mu.M lenalidomide and 10 .mu.M MG132 for 3
hours. Cells were lysed in Pierce IP Lysis Buffer and lysates were
cleared by centrifugation. FLAG-CRBN was immunoprecipitated
overnight using anti-FLAG M2 Affinity Gel (Sigma-Aldrich) in the
presence of 10 .mu.M MG132 and DMSO or 1 .mu.M lenalidomide. The
beads were washed 3 times with IP lysis buffer (Pierce) and protein
was eluted from the affinity gel with 250 .mu.g/ml FLAG peptide
(Sigma) after incubation for 30 min at 4.degree. C. Protein lysates
were then analyzed as described above.
In Vivo Ubiquitination
[0253] For in vivo ubiquitination analysis 300,000 293T cells were
plated in a 6-well. The next day, cells were transfected with 100
ng FLAG-CRBN and 300 ng HA-CK1.alpha. using TransLTI (Mirus). After
48 hours, cells were treated with lenalidomide or DMSO and 10 .mu.M
MG132 for 4 hours. Cells were then washed twice with ice-cold PBS
and lysed under denaturing conditions using 1% SDS-containing lysis
buffer and boiled for 10 minutes at 95.degree. C. The SDS was
diluted with the addition of 9 volumes of IP lysis buffer (Pierce)
followed by incubation at 4.degree. C. for 30 minutes. Lysates were
cleared from debris by centrifugation and incubated with anti-HA
microbeads (Miltenyi) in the presence of lenalidomide or DMSO, 10
.mu.M MG132, and 50 .mu.M PR-619 for 1 hour. Samples were applied
to columns on a MultiMacs 96 Separation Unit (Miltenyi), washed
four times with RIPA buffer, and eluted by addition of 95.degree.
C. Lamelli Buffer (Bio Rad) with .beta.-mercaptoethanol (Sigma).
Samples were separated by SDS-PAGE, transferred to a PVDF membrane
and probed with anti-CK1A antibody, anti-FK2 for polyubiquitinated
proteins and anti-actin as a loading control
In Vitro Ubiquitination
[0254] 293T cells were transfected with either HA-CK1A or FLAG-CRBN
vectors. After 48 hours, cells were lysed in Pierce IP lysis buffer
(Thermo Scientific) and immunoprecipitated overnight with
FLAG-Sepharose beads (Anti-FLAG M2 Affinity Gel, Sigma) or
HA-Sepharose beads (EZView Red anti-HA affinity gel, Sigma). The
beads were washed 3.times. in IP lysis buffer and 2.times. in E3
Ligase Reaction buffer (Boston Biochem) and eluted with 250
.mu.g/ml FLAG peptide (Sigma) or 100 .mu.g/ml HA peptide for 30 min
at 4.degree. C. The eluates were mixed in a 1:1 ratio and added to
a ubiquitination reaction mixture containing 200 nM E1 (UBE1), 2
.mu.M UbcH5a, 1 .mu.M UbcH5c, 1 .mu.g/.mu.L K.sub.0 ubiquitin, 1
.mu.M ubiquitin aldehyde, 1.times.Mg-ATP, 1.times.E3 Ligase
Reaction Buffer (all Boston Biochem), 10 .mu.M MG132, 100 nM MG101
and 1 .mu.M lenalidomide, 10 .mu.M lenalidomide, or DMSO (1:1000)
as appropriate in a total volume of 25 .mu.l.
[0255] Negative controls did not include E1 and P2 enzymes. After a
90 minute incubation at 30.degree. C., the reaction was denatured
by adding 5.times.SDS containing loading buffer (Boston Biochem),
boiled at 95.degree. C. for 5 minutes, separated by SDS-PAGE and
transferred to a PVDF membrane in order to detect HA-CK1A and its
ubiquitinated forms with CK1A antibody. The membrane was then
stripped and re-probed with anti-FLAG antibody.
Purification, Culture, and Lentiviral Infection of Human CD34+
Cells for shRNA Experiments
[0256] Research cord blood units were obtained from The New York
Blood Center according to an Institutional Review Board-approved
protocol. Cord blood CD34.sup.+ hematopoietic cells were isolated
from Ficoll purified PBMCs with an Indirect CD34 MicroBead kit
(Miltenyi) and an Auto MACS Pro (Miltenyi) according to the
manufacturer's protocol. Cells were cultured in serum free media
(SFEM, stem span) containing 50 ng/ml recombinant human SCF
(Miltenyi), 40 ng/.mu.l human FLT3 ligand (Miltenyi), 25 ng/.mu.l
recombinant human thrombopoietin (Miltenyi), and 10 ng/.mu.l IL-3
(Miltenyi). For shRNA experiments, CD34.sup.+ cells were transduced
with a VSV-G pseudotyped TRC pLKO.005 lentiviral vector expressing
GFP instead of puromycin resistance gene. Infection was performed
after 24 hours in culture in a 96-well using spinfection in the
presence of 2 .mu.g/ml polybrene (hexadimethrine bromide, Sigma).
48 hours after transduction the number of transduced cells was
analyzed by flow cytometry and was used as baseline. Then cells
were cultured in 1 .mu.M lenalidomide or DMSO and the relative
number of infected cells was assessed by flow cytometry for 3
weeks.
Purification, Culture, and Lentiviral Infection of Patient
Samples
[0257] Viably frozen bone marrow mononuclear cells were obtained
from healthy donors or patient with del(5q) MDS according to IRB
approved protocols at the University of Pennsylvania and Roswell
Park Cancer Institute. Samples were thawed and CD34.sup.+
hematopoietic cells were isolated 20-24 hours later using an
Indirect CD34 MicroBead kit (Miltenyi) and an Auto MACS Pro
(Miltenyi). Cells were grown in serum free media (SFEM, StemSpan)
supplemented with 25 ng/ml SCF, 40 ng/ml FLT3 ligand, 50 ng/ml
thrombopoietin, 40 .mu.g/mL lipids, 100 U/ml Pen/Strep and 2 mM
glutamine. 6-8 hours after CD34.sup.+ isolation, cells were
transduced with concentrated VSV-G pseuotyped
EF1a-GFP-IRES-hCSNK1A1 cDNA virus or empty vector control via
spinfection in the presence of 4 .mu.g/ml polybrene (Sigma, diluted
to 2 .mu.g/ml after spinfection). After 3 days, the initial
percentage of transduced cells was determined by flow cytometry and
remaining cells were split to treatment with either DMSO or 1 .mu.M
lenalidomide. The relative abundance of transduced cells in each
condition was assessed by after 5 days by flow cytometry. Control
cord-blood CD34.sup.+ cells were isolated as above. Adult bone
marrow CD34.sup.+ cells were purchased as single-donor lots from
AllCells (Alameda, Calif.).
[0258] For qPCR validation of CSNK1A1 expression, cord blood
CD34.sup.+ cells were transduced with lentivirus expressing GFP and
hCSNK1A1 or empty vector. After 3 days, transduced GFP.sup.+ cells
were FACS sorted and RNA extraction and qPCR was performed as
above.
Expressing Different CRBN Proteins in Ba/F3 Cells
[0259] Variants of human and mouse CRBN were cloned into a modified
pRSF91 backbone to generate SFFV-CRBN-IRES-GFP-T2A-Puro retroviral
constructs. 200,000 Ba/F3 cells were infected with ecotropic
retrovirus in the presence of 2 .mu.g/ml polybrene. After 24 hours,
1 .mu.g/ml puromycin (Gibco) was added and cells were selected for
3-4 days. Cells were confirmed to be >90% GFP+ by flow cytometry
and 1,000,000 cells were plated per 6-well and treated with DMSO or
lenalidomide for 24 hours. Protein lysates were harvested and
immunoblotted for CK1.alpha. as described above.
IKZF3 Luciferase Reporter Assay
[0260] 10,000 293T cells were transfected with 40 ng of
CMV-IRES-RenillaLUC-IRES-IKZF3-FireflyLUC reporter plasmid. After
24 hours, cells were treated with DMSO and lenalidomide. 4 hours
following treatment, luciferase activity was measured using the
Dual-Glo Luciferase Assay System (Promega) according to the
manufacturer's protocol.
Mouse Experiments
[0261] Mouse experiments were performed according to an IUCAC
approved protocol at Children's Hospital Boston. Generation and
characterization of the conditional Csnk1a1 knockout mouse has been
described previously (Cancer Cell, 13; 26(4):509-20, 2014).
Csnk1a1.sup.flox/flox mice were crossed with Mx1Cre mice to obtain
Csnk1a1.sup.flox/flox Mx1Cre.sup.+ mice. Csnk1a1.sup.flox/flox
Mx1Cre.sup.+ or control Csnk1a1.sup.+/+ Mx1Cre.sup.+ mice were
treated with 3 doses of 200 .mu.g poly (I:C) (Invivogen HMW) at
8-10 weeks of age and gene excision was confirmed where applicable.
At least 2 weeks following poly(I:C) treatment, the long bones and
spines were harvested and crushed and RBC were lysed. CKit.sup.+
cells were isolated with a CD117 MicroBead Kit (Miltenyi) and an
AutoMacs Pro and grown in SFEM (StemSpan) supplemented with
antibiotics and 50 ng/ml mTPO (Peprotech) and 50 ng/ml mSCF
(Peprotech) for 24 hours. Ecotropic pseudotyped retrovirus was spun
onto Retronectin (Clontech) coated 6 well plates and cells were
added in 1 ml of media with 2 .mu.g/ml polybrene. An addition 1 mL
media was added after 24 hours. After 48 hours, GFP+ or dTomato+
cells were isolated by FACS sorting (BD FACS Aria II) and CD45.1
and CD45.2 cells were mixed. Cells were treated with various doses
of lenalidomide and the percent CD45.1 and CD45.2 cells expressing
the fluorescent marker was followed by flow cytometry over time
following cell surface staining. Antibodies for flow cytometry were
as follows: CD45.1 APC/Cy7 (A20, BioLegend), CD45.2 PE (104,
eBioscience), and CD45.2 FITC (104, eBioscience)
Other Embodiments
[0262] From the foregoing description, it will be apparent that
variations and modifications may be made to the invention described
herein to adopt it to various usages and conditions. Such
embodiments are also within the scope of the following claims.
[0263] The recitation of a listing of elements in any definition of
a variable herein includes definitions of that variable as any
single element or combination (or subcombination) of listed
elements. The recitation of an embodiment herein includes that
embodiment as any single embodiment or in combination with any
other embodiments or portions thereof.
[0264] All patents and publications, including U.S. Ser. No.
61/902,066, mentioned in this specification are herein incorporated
by reference to the same extent as if each independent patent and
publication was specifically and individually indicated to be
incorporated by reference.
Sequence CWU 1
1
331477PRTHomo sapiens 1Met Asp Ala Asp Glu Gly Gln Asp Met Ser Gln
Val Ser Gly Lys Glu 1 5 10 15 Ser Pro Pro Val Ser Asp Thr Pro Asp
Glu Gly Asp Glu Pro Met Pro 20 25 30 Ile Pro Glu Asp Leu Ser Thr
Thr Ser Gly Gly Gln Gln Ser Ser Lys 35 40 45 Ser Asp Arg Val Val
Ala Ser Asn Val Lys Val Glu Thr Gln Ser Asp 50 55 60 Glu Glu Asn
Gly Arg Ala Cys Glu Met Asn Gly Glu Glu Cys Ala Glu 65 70 75 80 Asp
Leu Arg Met Leu Asp Ala Ser Gly Glu Lys Met Asn Gly Ser His 85 90
95 Arg Asp Gln Gly Ser Ser Ala Leu Ser Gly Val Gly Gly Ile Arg Leu
100 105 110 Pro Asn Gly Lys Leu Lys Cys Asp Ile Cys Gly Ile Ile Cys
Ile Gly 115 120 125 Pro Asn Val Leu Met Val His Lys Arg Ser His Thr
Gly Glu Arg Pro 130 135 140 Phe Gln Cys Asn Gln Cys Gly Ala Ser Phe
Thr Gln Lys Gly Asn Leu 145 150 155 160 Leu Arg His Ile Lys Leu His
Ser Gly Glu Lys Pro Phe Lys Cys His 165 170 175 Leu Cys Asn Tyr Ala
Cys Arg Arg Arg Asp Ala Leu Thr Gly His Leu 180 185 190 Arg Thr His
Ser Val Ile Lys Glu Glu Thr Asn His Ser Glu Met Ala 195 200 205 Glu
Asp Leu Cys Lys Ile Gly Ser Glu Arg Ser Leu Val Leu Asp Arg 210 215
220 Leu Ala Ser Asn Val Ala Lys Arg Lys Ser Ser Met Pro Gln Lys Phe
225 230 235 240 Leu Gly Asp Lys Gly Leu Ser Asp Thr Pro Tyr Asp Ser
Ser Ala Ser 245 250 255 Tyr Glu Lys Glu Asn Glu Met Met Lys Ser His
Val Met Asp Gln Ala 260 265 270 Ile Asn Asn Ala Ile Asn Tyr Leu Gly
Ala Glu Ser Leu Arg Pro Leu 275 280 285 Val Gln Thr Pro Pro Gly Gly
Ser Glu Val Val Pro Val Ile Ser Pro 290 295 300 Met Tyr Gln Leu His
Lys Pro Leu Ala Glu Gly Thr Pro Arg Ser Asn 305 310 315 320 His Ser
Ala Gln Asp Ser Ala Val Glu Asn Leu Leu Leu Leu Ser Lys 325 330 335
Ala Lys Leu Val Pro Ser Glu Arg Glu Ala Ser Pro Ser Asn Ser Cys 340
345 350 Gln Asp Ser Thr Asp Thr Glu Ser Asn Asn Glu Glu Gln Arg Ser
Gly 355 360 365 Leu Ile Tyr Leu Thr Asn His Ile Ala Pro His Ala Arg
Asn Gly Leu 370 375 380 Ser Leu Lys Glu Glu His Arg Ala Tyr Asp Leu
Leu Arg Ala Ala Ser 385 390 395 400 Glu Asn Ser Gln Asp Ala Leu Arg
Val Val Ser Thr Ser Gly Glu Gln 405 410 415 Met Lys Val Tyr Lys Cys
Glu His Cys Arg Val Leu Phe Leu Asp His 420 425 430 Val Met Tyr Thr
Ile His Met Gly Cys His Gly Phe Arg Asp Pro Phe 435 440 445 Glu Cys
Asn Met Cys Gly Tyr His Ser Gln Asp Arg Tyr Glu Phe Ser 450 455 460
Ser His Ile Thr Arg Gly Glu His Arg Phe His Met Ser 465 470 475
2519PRTHomo sapiens 2Met Asp Ala Asp Glu Gly Gln Asp Met Ser Gln
Val Ser Gly Lys Glu 1 5 10 15 Ser Pro Pro Val Ser Asp Thr Pro Asp
Glu Gly Asp Glu Pro Met Pro 20 25 30 Ile Pro Glu Asp Leu Ser Thr
Thr Ser Gly Gly Gln Gln Ser Ser Lys 35 40 45 Ser Asp Arg Val Val
Ala Ser Asn Val Lys Val Glu Thr Gln Ser Asp 50 55 60 Glu Glu Asn
Gly Arg Ala Cys Glu Met Asn Gly Glu Glu Cys Ala Glu 65 70 75 80 Asp
Leu Arg Met Leu Asp Ala Ser Gly Glu Lys Met Asn Gly Ser His 85 90
95 Arg Asp Gln Gly Ser Ser Ala Leu Ser Gly Val Gly Gly Ile Arg Leu
100 105 110 Pro Asn Gly Lys Leu Lys Cys Asp Ile Cys Gly Ile Ile Cys
Ile Gly 115 120 125 Pro Asn Val Leu Met Val His Lys Arg Ser His Thr
Gly Glu Arg Pro 130 135 140 Phe Gln Cys Asn Gln Cys Gly Ala Ser Phe
Thr Gln Lys Gly Asn Leu 145 150 155 160 Leu Arg His Ile Lys Leu His
Ser Gly Glu Lys Pro Phe Lys Cys His 165 170 175 Leu Cys Asn Tyr Ala
Cys Arg Arg Arg Asp Ala Leu Thr Gly His Leu 180 185 190 Arg Thr His
Ser Val Gly Lys Pro His Lys Cys Gly Tyr Cys Gly Arg 195 200 205 Ser
Tyr Lys Gln Arg Ser Ser Leu Glu Glu His Lys Glu Arg Cys His 210 215
220 Asn Tyr Leu Glu Ser Met Gly Leu Pro Gly Thr Leu Tyr Pro Val Ile
225 230 235 240 Lys Glu Glu Thr Asn His Ser Glu Met Ala Glu Asp Leu
Cys Lys Ile 245 250 255 Gly Ser Glu Arg Ser Leu Val Leu Asp Arg Leu
Ala Ser Asn Val Ala 260 265 270 Lys Arg Lys Ser Ser Met Pro Gln Lys
Phe Leu Gly Asp Lys Gly Leu 275 280 285 Ser Asp Thr Pro Tyr Asp Ser
Ser Ala Ser Tyr Glu Lys Glu Asn Glu 290 295 300 Met Met Lys Ser His
Val Met Asp Gln Ala Ile Asn Asn Ala Ile Asn 305 310 315 320 Tyr Leu
Gly Ala Glu Ser Leu Arg Pro Leu Val Gln Thr Pro Pro Gly 325 330 335
Gly Ser Glu Val Val Pro Val Ile Ser Pro Met Tyr Gln Leu His Lys 340
345 350 Pro Leu Ala Glu Gly Thr Pro Arg Ser Asn His Ser Ala Gln Asp
Ser 355 360 365 Ala Val Glu Asn Leu Leu Leu Leu Ser Lys Ala Lys Leu
Val Pro Ser 370 375 380 Glu Arg Glu Ala Ser Pro Ser Asn Ser Cys Gln
Asp Ser Thr Asp Thr 385 390 395 400 Glu Ser Asn Asn Glu Glu Gln Arg
Ser Gly Leu Ile Tyr Leu Thr Asn 405 410 415 His Ile Ala Pro His Ala
Arg Asn Gly Leu Ser Leu Lys Glu Glu His 420 425 430 Arg Ala Tyr Asp
Leu Leu Arg Ala Ala Ser Glu Asn Ser Gln Asp Ala 435 440 445 Leu Arg
Val Val Ser Thr Ser Gly Glu Gln Met Lys Val Tyr Lys Cys 450 455 460
Glu His Cys Arg Val Leu Phe Leu Asp His Val Met Tyr Thr Ile His 465
470 475 480 Met Gly Cys His Gly Phe Arg Asp Pro Phe Glu Cys Asn Met
Cys Gly 485 490 495 Tyr His Ser Gln Asp Arg Tyr Glu Phe Ser Ser His
Ile Thr Arg Gly 500 505 510 Glu His Arg Phe His Met Ser 515
33572DNAHomo sapiens 3ggcagcagag gaaccttttg gaggaggaag aggacacaga
ggccctgtag ccaggcacca 60agatccctcc caggtggctg ggtctgaggg gaactccgag
cagccctagg tcctcaaagt 120ctggatttgt gtggaaaagg cagctctcac
ttggccttgg cgaggcctcg gttggttgat 180aacctgagga ccatggatgc
tgatgagggt caagacatgt cccaagtttc agggaaggaa 240agcccccctg
taagcgatac tccagatgag ggcgatgagc ccatgccgat ccccgaggac
300ctctccacca cctcgggagg acagcaaagc tccaagagtg acagagtcgt
ggccagtaat 360gttaaagtag agactcagag tgatgaagag aatgggcgtg
cctgtgaaat gaatggggaa 420gaatgtgcgg aggatttacg aatgcttgat
gcctcgggag agaaaatgaa tggctcccac 480agggaccaag gcagctcggc
tttgtcggga gttggaggca ttcgacttcc taacggaaaa 540ctaaagtgtg
atatctgtgg gatcatttgc atcgggccca atgtgctcat ggttcacaaa
600agaagccaca ctggagaacg gcccttccag tgcaatcagt gcggggcctc
attcacccag 660aagggcaacc tgctccggca catcaagctg cattccgggg
agaagccctt caaatgccac 720ctctgcaact acgcctgccg ccggagggac
gccctcactg gccacctgag gacgcactcc 780gtcattaaag aagaaactaa
tcacagtgaa atggcagaag acctgtgcaa gataggatca 840gagagatctc
tcgtgctgga cagactagca agtaacgtcg ccaaacgtaa gagctctatg
900cctcagaaat ttcttgggga caagggcctg tccgacacgc cctacgacag
cagcgccagc 960tacgagaagg agaacgaaat gatgaagtcc cacgtgatgg
accaagccat caacaacgcc 1020atcaactacc tgggggccga gtccctgcgc
ccgctggtgc agacgccccc gggcggttcc 1080gaggtggtcc cggtcatcag
cccgatgtac cagctgcaca agccgctcgc ggagggcacc 1140ccgcgctcca
accactcggc ccaggacagc gccgtggaga acctgctgct gctctccaag
1200gccaagttgg tgccctcgga gcgcgaggcg tccccgagca acagctgcca
agactccacg 1260gacaccgaga gcaacaacga ggagcagcgc agcggtctca
tctacctgac caaccacatc 1320gccccgcacg cgcgcaacgg gctgtcgctc
aaggaggagc accgcgccta cgacctgctg 1380cgcgccgcct ccgagaactc
gcaggacgcg ctccgcgtgg tcagcaccag cggggagcag 1440atgaaggtgt
acaagtgcga acactgccgg gtgctcttcc tggatcacgt catgtacacc
1500atccacatgg gctgccacgg cttccgtgat ccttttgagt gcaacatgtg
cggctaccac 1560agccaggacc ggtacgagtt ctcgtcgcac ataacgcgag
gggagcaccg cttccacatg 1620agctaaagcc ctcccgcgcc cccaccccag
accccgagcc accccaggaa aagcacaagg 1680actgccgcct tctcgctccc
gccagcagca tagactggac tggaccagac aatgttgtgt 1740ttggatttgt
aactgttttt tgttttttgt ttgagttggt tgattggggt ttgatttgct
1800tttgaaaaga tttttatttt tagaggcagg gctgcattgg gagcatccag
aactgctacc 1860ttcctagatg tttccccaga ccgctggctg agattccctc
acctgtcgct tcctagaatc 1920cccttctcca aacgattagt ctaaattttc
agagagaaat agataaaaca cgccacagcc 1980tgggaaggag cgtgctctac
cctgtgctaa gcacggggtt cgcgcaccag gtgtcttttt 2040ccagtcccca
gaagcagaga gcacagcccc tgctgtgtgg gtctgcaggt gagcagacag
2100gacaggtgtg ccgccaccca agtgccaaga cacagcaggg ccaacaacct
gtgcccaggc 2160cagcttcgag ctacatgcat ctagggcgga gaggctgcac
ttgtgagaga aaatactatt 2220tcaagtcata ttctgcgtag gaaaatgaat
tggttgggga aagtcgtgtc tgtcagactg 2280ccctgggtgg agggagacgc
cgggctagag cctttgggat cgtcctggat tcactggctt 2340tgcggaggct
gctcagatgg cctgagcctc ccgaggcttg ctgccccgta ggaggagact
2400gtcttcccgt gggcatatct ggggagccct gttccccgct ttttcactcc
cataccttta 2460atggccccca aaatctgtca ctacaattta aacaccagtc
ccgaaatttg gatcttcttt 2520ctttttgaat ctctcaaacg gcaacattcc
tcagaaacca aagctttatt tcaaatctct 2580tccttccctg gctggttcca
tctagtacca gaggcctctt ttcctgaaga aatccaatcc 2640tagccctcat
tttaattatg tacatctgtt tgtagccaca agcctgaatt tctcagtgtt
2700ggtaagtttc tttacctacc ctcactatat attattctcg ttttaaaacc
cataaaggag 2760tgatttagaa cagtcattaa ttttcaactc aatgaaatat
gtgaagccca gcatctctgt 2820tgctaacaca cagagctcac ctgtttgaaa
ccaagctttc aaacatgttg aagctcttta 2880ctgtaaaggc aagccagcat
gtgtgtccac acatacatag gatggctggc tctgcacctg 2940taggatattg
gaatgcacag ggcaattgag ggactgagcc agaccttcgg agagtaatgc
3000caccagatcc cctaggaaag aggaggcaaa tggcactgca ggtgagaacc
ccgcccatcc 3060gtgctatgac atggaggcac tgaagcccga ggaaggtgtg
tggagattct aatcccaaca 3120agcaagggtc tccttcaaga ttaatgctat
caatcattaa ggtcattact ctcaaccacc 3180taggcaatga agaatatacc
atttcaaata tttacagtac ttgtcttcac caacactgtc 3240ccaaggtgaa
atgaagcaac agagaggaaa ttgtacataa gtacctcagc atttaatcca
3300aacaggggtt cttagtctca gcactatgac attttgggct gactacttat
ttgttaggca 3360ggagctctcc tgtgcattgt aggataatta gcagtatccc
tggtggctac ccaatagacg 3420ccagtagcac cccgaattga caacccaaac
tctccagaca tcaccaactg tcccctgcga 3480ggagaaatca ctcctggggg
agaaccactg acccaaatga attctaaacc aatcaaatgt 3540ctgggaagcc
ctccaagaaa aaaaaaaaaa aa 35724509PRTHomo sapiens 4Met Glu Asp Ile
Gln Thr Asn Ala Glu Leu Lys Ser Thr Gln Glu Gln 1 5 10 15 Ser Val
Pro Ala Glu Ser Ala Ala Val Leu Asn Asp Tyr Ser Leu Thr 20 25 30
Lys Ser His Glu Met Glu Asn Val Asp Ser Gly Glu Gly Pro Ala Asn 35
40 45 Glu Asp Glu Asp Ile Gly Asp Asp Ser Met Lys Val Lys Asp Glu
Tyr 50 55 60 Ser Glu Arg Asp Glu Asn Val Leu Lys Ser Glu Pro Met
Gly Asn Ala 65 70 75 80 Glu Glu Pro Glu Ile Pro Tyr Ser Tyr Ser Arg
Glu Tyr Asn Glu Tyr 85 90 95 Glu Asn Ile Lys Leu Glu Arg His Val
Val Ser Phe Asp Ser Ser Arg 100 105 110 Pro Thr Ser Gly Lys Met Asn
Cys Asp Val Cys Gly Leu Ser Cys Ile 115 120 125 Ser Phe Asn Val Leu
Met Val His Lys Arg Ser His Thr Gly Glu Arg 130 135 140 Pro Phe Gln
Cys Asn Gln Cys Gly Ala Ser Phe Thr Gln Lys Gly Asn 145 150 155 160
Leu Leu Arg His Ile Lys Leu His Thr Gly Glu Lys Pro Phe Lys Cys 165
170 175 His Leu Cys Asn Tyr Ala Cys Gln Arg Arg Asp Ala Leu Thr Gly
His 180 185 190 Leu Arg Thr His Ser Val Glu Lys Pro Tyr Lys Cys Glu
Phe Cys Gly 195 200 205 Arg Ser Tyr Lys Gln Arg Ser Ser Leu Glu Glu
His Lys Glu Arg Cys 210 215 220 Arg Thr Phe Leu Gln Ser Thr Asp Pro
Gly Asp Thr Ala Ser Ala Glu 225 230 235 240 Ala Arg His Ile Lys Ala
Glu Met Gly Ser Glu Arg Ala Leu Val Leu 245 250 255 Asp Arg Leu Ala
Ser Asn Val Ala Lys Arg Lys Ser Ser Met Pro Gln 260 265 270 Lys Phe
Ile Gly Glu Lys Arg His Cys Phe Asp Val Asn Tyr Asn Ser 275 280 285
Ser Tyr Met Tyr Glu Lys Glu Ser Glu Leu Ile Gln Thr Arg Met Met 290
295 300 Asp Gln Ala Ile Asn Asn Ala Ile Ser Tyr Leu Gly Ala Glu Ala
Leu 305 310 315 320 Arg Pro Leu Val Gln Thr Pro Pro Ala Pro Thr Ser
Glu Met Val Pro 325 330 335 Val Ile Ser Ser Met Tyr Pro Ile Ala Leu
Thr Arg Ala Glu Met Ser 340 345 350 Asn Gly Ala Pro Gln Glu Leu Glu
Lys Lys Ser Ile His Leu Pro Glu 355 360 365 Lys Ser Val Pro Ser Glu
Arg Gly Leu Ser Pro Asn Asn Ser Gly His 370 375 380 Asp Ser Thr Asp
Thr Asp Ser Asn His Glu Glu Arg Gln Asn His Ile 385 390 395 400 Tyr
Gln Gln Asn His Met Val Leu Ser Arg Ala Arg Asn Gly Met Pro 405 410
415 Leu Leu Lys Glu Val Pro Arg Ser Tyr Glu Leu Leu Lys Pro Pro Pro
420 425 430 Ile Cys Pro Arg Asp Ser Val Lys Val Ile Asn Lys Glu Gly
Glu Val 435 440 445 Met Asp Val Tyr Arg Cys Asp His Cys Arg Val Leu
Phe Leu Asp Tyr 450 455 460 Val Met Phe Thr Ile His Met Gly Cys His
Gly Phe Arg Asp Pro Phe 465 470 475 480 Glu Cys Asn Met Cys Gly Tyr
Arg Ser His Asp Arg Tyr Glu Phe Ser 485 490 495 Ser His Ile Ala Arg
Gly Glu His Arg Ala Leu Leu Lys 500 505 59686DNAHomo sapiens
5gcaggagcac gtggagaggc cgagtagcca cagcggcagc tccagcccgg cccggcagcg
60acatggaaga tatacaaaca aatgcggaac tgaaaagcac tcaggagcag tctgtgcccg
120cagaaagtgc agcggttttg aatgactaca gtttaaccaa atctcatgaa
atggaaaatg 180tggacagtgg agaaggccca gccaatgaag atgaagacat
aggagatgat tcaatgaaag 240tgaaagatga atacagtgaa agagatgaga
atgttttaaa gtcagaaccc atgggaaatg 300cagaagagcc tgaaatccct
tacagctatt caagagaata taatgaatat gaaaacatta 360agttggagag
acatgttgtc tcattcgata gtagcaggcc aaccagtgga aagatgaact
420gcgatgtgtg tggattatcc tgcatcagct tcaatgtctt aatggttcat
aagcgaagcc 480atactggtga acgcccattc cagtgtaatc agtgtggggc
atcttttact cagaaaggta 540acctcctccg ccacattaaa ctgcacacag
gggaaaaacc ttttaagtgt cacctctgca 600actatgcatg ccaaagaaga
gatgcgctca cggggcatct taggacacat tctgtggaga 660aaccctacaa
atgtgagttt tgtggaagga gttacaagca gagaagttcc cttgaggagc
720acaaggagcg ctgccgtaca tttcttcaga gcactgaccc aggggacact
gcaagtgcgg 780aggcaagaca catcaaagca gagatgggaa gtgaaagagc
tctcgtactg gacagattag 840caagcaatgt ggcaaaacga aaaagctcaa
tgcctcagaa attcattggt gagaagcgcc 900actgctttga tgtcaactat
aattcaagtt acatgtatga gaaagagagt gagctcatac 960agacccgcat
gatggaccaa gccatcaata acgccatcag ctatcttggc gccgaagccc
1020tgcgcccctt ggtccagaca ccgcctgctc ccacctcgga gatggttcca
gttatcagca 1080gcatgtatcc catagccctc acccgggctg agatgtcaaa
cggtgcccct caagagctgg 1140aaaagaaaag catccacctt ccagagaaga
gcgtgccttc tgagagaggc ctctctccca 1200acaatagtgg ccacgactcc
acggacactg acagcaacca tgaagaacgc cagaatcaca 1260tctatcagca
aaatcacatg gtcctgtctc gggcccgcaa tgggatgcca cttctgaagg
1320aggttccccg ctcttacgaa ctcctcaagc ccccgcccat ctgcccaaga
gactccgtca 1380aagtgatcaa caaggaaggg gaggtgatgg atgtgtatcg
gtgtgaccac tgccgcgtcc 1440tcttcctgga ctatgtgatg ttcacgattc
acatgggctg ccacggcttc cgtgaccctt 1500tcgagtgtaa catgtgtgga
tatcgaagcc atgatcggta
tgagttctcg tctcacatag 1560ccagaggaga acacagagcc ctgctgaagt
gaatatctgg tctcagggat tgctcctatg 1620tattcagcat cgtttctaaa
aaccaatgac ctcgcctaac agattgctct caaaacatac 1680tcagttccaa
acttcttttc ataccatttt tagctgtgtt cacaggggta gccagggaaa
1740cactgtcttc cttcagaaat tattcgcagg tctagcatat tattactttt
gtgaaacctt 1800tgttttccca tcagggactt gaattttatg gaatttaaaa
gccaaaaagg tatttggtca 1860ttatcttcta cagcagtgga atgagtggtc
ccggagatgt gctatatgaa acattctttc 1920tgagatatat caaccacacg
tggaaaagcc tttcagtcat acatgcaaat ccacaaagag 1980gaagagctga
ccagctgacc ttgctgggaa gcctcaccct tctgcccttc acaggctgaa
2040gggttaagat ctaatctccc taatctaaat gacagtctaa gagtaagtaa
aagaacagcc 2100ataaaataag tatctgttac gagtaactga agaccccatt
ctccaagcat cagatccatt 2160tcctatcaca acatttttaa aaaatgtcat
ctgatggcac ttctgcttct gtcctttacc 2220ttcccatctc cagtgaaaag
ctgagctgct ttgggctaaa ccagttgtct atagaagaaa 2280atctatgcca
gaagaactca tggttttaaa tatagaccat catcgaaact ccagaaattt
2340atccactgtg gatgatgaca tcgctttcct ttggtcaagg ttggcagagc
aagggtataa 2400agggggaaat tgtttggcag caccaacaga aaacaaacaa
acaaaaaaca gctacctaaa 2460acttcttgaa agagttcatg gagaattggt
gatacagacc caaagcaaat ttgccaatga 2520tattttccac aaaaaaagtc
caaaaagtat ggctcagcct ccccctcccc acaggagagg 2580aattggagat
agatggcatg tgtgtttaga tcggagttga gctccggaat ggggtgagga
2640gggacacctc tattgagagg ttctccttga tcaggcaggc ttcggccctt
tttttcccat 2700ttaaatggaa ctgctgtatt ccatgaaaat tcctgaaagt
ctgatcacgg ttctgcagat 2760gtataagtca tccttgtcac tcataatatg
tacatactat caggaggagt gctgttatca 2820tggtaaaatt agcactggaa
taggaggtca caaaatgctg gctaattagc tatgtgactt 2880tgagaaatcg
tttaactttt tttttttttt tttttttgag acaggatctc actctgttgc
2940ccaggctgga gtgcagtggt gcaatcatgg ctcagtgcag cctcgacctc
cccaggctca 3000ggtgatcctc ccacctcagc ctcttgagta ctgggacaac
aagtgcacac caccatgtct 3060ggctacattt tgttcttttt gtagagatag
gggtctcact atgttgccca tgctggtctt 3120gaactcctgg gctcaagcaa
tcagcccgcc tcagcctcct aaagtgctgg gattacaggt 3180gtgagccacc
acacccagcc ttatttaact cttaaaactc agtttccggc caggctcggt
3240ggctcacacc tgtaatccca acactttggg aagccgaggc aggcgcatca
tttgaggtca 3300ggagttcgag accagcctga cccacatggt gaaaccctgt
ctctactaaa aatacaaaaa 3360ttagctgggc agtagtggca catgcctgta
atcccagcta ctccggaggc tgaggcagaa 3420aaatcgctta agcctgggag
gttgaggttg cggtgagtgg agatcacact actgcactcc 3480agtctgggcg
acagagtgag accctgtctc aaacaaaaca aaacaaaaac aaacaaacaa
3540aaacaaaaaa aactcagttt cctcatccat aaaataggaa ttagatttca
atgttctctt 3600aggtcccttc tagctttaat tcatatgtga ttatgcagta
accacaaggt attttttaaa 3660cctcctaatg tatggatatt aagcagaaga
gtatttatat gaatacatgt ttcacattcc 3720tttggtatga aaatggtgtg
ttaagttttt cctttaacca ctgagttgtg aatgtgaaga 3780aggtggtgga
gaggaacaaa aaacagaaag gtattttgat cttgccacaa agcatacaca
3840caaattggca catgcagctg tttgccaaag ccttcttttt ttttttactt
tttaagaaat 3900tatgttaggg aaaataaatt ctgcttccag ggacaacttc
atggagccta tttacaaatt 3960aagagtcagc ttaatttgta acatttctac
cagagccaag aatcccaaat tcctggtaga 4020ttagtgtttt atttctaagg
ggcttatgca ttcggctcca actcaactcg tctatgtgct 4080gccagtaatt
aaaatgttcc acctcagact gcacaaatgg cttatccttc tttgtggcat
4140ggcgtctgtc tcaggaaaaa aggttttatg aaattccatg gcaacagtcc
caacatgttt 4200gagacttcag ctaaaggaat ggatgtattt tggtgtgtag
tcttcagtat atcactgtat 4260ttccgtaata ctagactcca agctatgcca
gattgcttat tccctttgtg aaagaggagt 4320tgctcattac gttcttgaaa
tatcgcacat cctgttggtt cttcaaggga caagagaaag 4380agaatttgga
agcagggatt agtagaagag aaaacgaggg aaaggaagcc tttccaccag
4440attagtgttc aagtctttgc agaggagacc aacttttttt gttttctttt
gttttgagac 4500agtctctcgc tctgttgccc aggctggagt gcagtggcgc
gatctcggct cacggcaacc 4560tccgcctccc gggttcaagc aattctcctg
cctcagcctc ccaagtagct gggattacag 4620gtgctcacca ccaagcccgg
ctaatttttg tatttttagt agagacaagg tttcaccatg 4680ttggccaggc
cagtctcaaa ctcctgacct caggtgatct gcccgccttg gcctcccaca
4740gtgctgggat tacaggcatg agctaccgca cccagcctga gaccaccttt
tgcatctcaa 4800gattgtgaaa ccaaggccca ttccaccagc ctggggactc
tttttataga tatgatcctc 4860ctttttcctg tgactaatga atttgctgca
tgatttctat tcttctgagg ttagttttct 4920gagtaaggtg accactcaca
aaggcacttt ctttgtggca ttctgagcct agattggggc 4980ccatcaattc
cagaaaaaat ttatgtgtgg aaactctgca tccttaagtc ttgaagttga
5040accagatatg cagtggttac catcacacag ataaacgctg ccttctgtac
atacccctta 5100tgctgtacta attaacaaac cccttgccag ggctggggag
gtgagggtga aggagaatct 5160tagcagaagg gcagagtcag gacttgcatc
tgccactgct gggcactgaa gccctggagc 5220agcttcagat agtacctgta
ctttctcatg cagactccct ctgaacaaga gccttgtagg 5280cccctctcct
tcatttccca ccagcctctt atcaggcggg ctttccacca tacacccagg
5340aggccacggt ctgaggaaca accaaaccca tgcaaagggc cgggcgcgat
agctcacgcc 5400tgtaatgcca gcactttggg aggctggggc aggcagatca
cctgaggttg ggagttcgag 5460acctgcctga ccaacatgga gaaaccccca
tctctactaa aaatacaaaa ttagccgggc 5520gtgatggcac atgcctgtaa
tcccagctac tcaggaggct gaggcaggag aatcgcttga 5580acccgggagg
cggaggttgc ggtgagccga gatggcacca ctgcactcca gcctcggcaa
5640caagagcgaa actctgtcta aaacaaaaac aaacaaacaa acaaaaaaac
ccaggcaaag 5700tttccttgca gccaaggtga cagaactggg ctgagggtgg
aaaagaaaca gaaccagtgc 5760tccaggtgtt ttttaatttt ttaatttatt
tttatttttt ttgtatatgt atatatatgt 5820atgtatattt tagaggacca
gggtctcact atgttgccta ggccagactc aaactcctgt 5880gctcaagcaa
tcctgcctca gcctcccaag tagctgggat tacaggcatg cacaaacaat
5940gcccagctct ccaaatgttt tctgtcacta cctgaagtgt tgcatcggta
cttcctacgg 6000aaagaaaact aaatagaagt gtctctcccg tgagccccca
ccactaccac cagaaaaaaa 6060aaagagagaa aatgaactca tcagtcttta
gtttcctcaa gttattctcc caaaaagaca 6120ttcgccttgg cacagataag
ccagctaatc ttatgcttta tgacccactg tgagctgttc 6180ctgacacagc
ttctgacttt gtcagtgaca aaatttctca ccttttaaat gcagtgctta
6240acattttgtt aggcccatac tcaaaatcgg ccagatataa aatgacctca
gattttgatc 6300tcctaggctc aaacaatcct cctacctcag cctcccaagt
agctgggact ataggcacac 6360caccatgcac agctaatttt ttttgtattt
ttctgcagag atggcgtttc gccatactgc 6420ccaggctagt ctcaaaatcc
tgggctcaag caatctgccc acctcagcct cccaaagtgc 6480tggaactaca
ggcaagagcc actgcgccca gccacaacct cagatttctt tggcaaacag
6540aaatgtttaa aaacacaaaa ttttgctcag gtgaaacact gtgttactat
caaatctcac 6600atccacataa agtttttctt ttcggctttg tttcgtgagg
aacagacaga acaaagtttt 6660tccaggtagc atctgtatca ctattattct
cctatttcct gtaccacccc cacctcccca 6720agccctactg aatgtgaggt
ttagaatgtt ttaaggaggg tcaggtgcgg tggctcacgc 6780ctgtaatccc
agcactttgg gaggccaagg cgggcggatc acctgagttt gggagttcga
6840gaccagcctg accaacatgg agaaaccctg tctctactaa aaatacaaaa
ttagccaggc 6900gtggtggcac atgcctgtaa tcccagctac ttaggaggct
gaggcaggag aatcgcttga 6960acccaggagg aggaggttgt ggtgagccga
gatcgtgcca ttgcactcca gcctgggtga 7020cagagtgaga ctccatctcg
aaaaaaaaaa tacaaaaatt agctgggtgt ggtggtgcac 7080acctgtaatc
ccagctactc gggaggctga cgcaggagaa ttgcttgaac ctgggaggtg
7140gaggttgcag tgagccgaga tcgcgccatt gcaatccagc ctggacaaca
gagtgagact 7200ccatctcaaa aaaaaaaaaa aaaagaatgt tttaaggaaa
aaaatagtac tgttacatat 7260aatcccaggt gataagacca caatggaaat
gtttaagtcc tcactttaaa gagtacccca 7320ctgagaagag gtatgttgga
ctctagcaga gatttggaaa ctctgggaca ctcaagatgt 7380gaaagagcct
ggctatctga ggactcaaag agtcagcatc gggacttgtg agctcaagaa
7440gagaaaaggg agtggtgaaa ctttgtccta aaagttagca ccaggaacag
aagaaaaaaa 7500cccgatatat agtgatacct catcttttag agaatgggaa
gctatttttg tgttcacaca 7560gaaagtatag ttcaaaaaac ctctatatcc
agagttcaga caaggagaat gatttgagat 7620ataagtgccg atgaaggagg
tcaattttga tctgaaacca gcagctggac ctgggccacc 7680tcaggaaaag
gactctgttc tccaaggcag cacgactgaa tggttctgag aataagccag
7740ggttcaggac tcctgaccct ttaggaccat ggactcagaa gagcctgaag
gacaattgtg 7800ggctttaaac ttctgagagc ttgtaaagta acacaagact
gtgcctctcc cttgccccag 7860ctgtagatag tctttgcccc accattgtta
tgaagataca cagggttttg cagtttgaat 7920aaattggata caagtttcct
cttttttttt ttctttttga gacaaagtct cgctctgttt 7980ccccaggctg
agtgcagtgg cacaatcaag gcttacttgc cgcctcaacc tcctgggctc
8040aagcaacgag ccatcctccc gtcttagcct cccaactagc tgagactaca
ggcgtgggtc 8100accacaccca gctaattttt gtactttttg tagagacagg
gtctcaccat gttgcccagg 8160ctggtcctga actcctgggc tcaagtaatc
tgcccacctc agcctcccaa agtgttgggg 8220ttacaggcgt gaggcaccgc
ggctggcctg agtttcttct taatactgta tcacaattgt 8280gggctgtctt
atgtgttgat atcgattgag ctatttgaaa taggaatgtt aatgggtgta
8340ttaaattttt gtaaggatat aacaatatct accttccaag gatgttgtga
ggttttccat 8400gattttgtat atgagctaat gttacctttg aggggtggtg
tgcattatgt tggatgattg 8460taaattttca gtggaaaatg taccgtgtcc
taaatttaaa gacatgaaaa atatcccaag 8520atcatactag atcataatag
caattccttt acaaatgaat tatggaggta actgatctct 8580aacagtttcc
ttcatgttgt tttaatgcac aagggcagag gatctgctga cccttggaac
8640cagcgtgagc taaccacgtg ctatagacac ttcatggtgt cgcacccagg
gaagtcaaag 8700cgctttgctc cctcactgtc tgtgagtcct cagccattag
taccccaccc cccgctgctc 8760caaaacttga gttatttcaa atgtttctca
ctgttcatct ctccactgac cccactccag 8820aaagcctgga gagagtccca
agatgccacc caccttcccc aatccctcgc cacagatctg 8880tgtctatctc
acactctgta agtgccgctt tgcttcttcc tctcttgaaa agactgagaa
8940cacacatttt aacatgttag gaaaatgggg cagcctaaaa aatgactgat
cccaccgcca 9000gtgactcatg tatactccag gctagcagac aaggcccttt
ttggtgggcc tgcttctgtg 9060ggttcacaga aaccaaatta ctgtgggttg
caaagaatta gcaggtcatt tacaaagcag 9120acatcccttc acccagactg
tggttttgca tgctcaggtt ctcagtctat gagctttggt 9180gcaggatcat
tttggctact ggaaaaacca tagcttattt taaatttctg gttgccaaag
9240ccaccacacg tgtggtctgt ggatgaccat tgtctgcaga atgacgagga
aggaacagaa 9300tgtggtttgg ggctcagggt ggccttccca ctgggaggga
aggcgggagg gagcccttgc 9360cctgggtttt gacacagcct gtgctcacag
cctctcctct catctgcatt tctcagaaat 9420gccctccctg cccagtggtg
actttccctc gtcactccta tggagttcta cctggagccc 9480agccatgtgt
ggaactgtga agtttactcc tctgtaaaga tggtttaaag aaagtcagct
9540tctgaaatgt aacaatgcta acccttgctg gaaccctgta agaaatagcc
ctgctgatag 9600ttttctaggt ttatcatgtt tgatttttac actgaaaaat
aaaaaaatcc tggtatgttt 9660gaaattaaaa aaaaaaaaaa aaaaaa
96866441PRTHomo sapiens 6Met Ala Gly Glu Gly Asp Gln Gln Asp Ala
Ala His Asn Met Gly Asn 1 5 10 15 His Leu Pro Leu Leu Pro Glu Ser
Glu Glu Glu Asp Glu Met Glu Val 20 25 30 Glu Asp Gln Asp Ser Lys
Glu Ala Lys Lys Pro Asn Ile Ile Asn Phe 35 40 45 Asp Thr Ser Leu
Pro Thr Ser His Thr Tyr Leu Gly Ala Asp Met Glu 50 55 60 Glu Phe
His Gly Arg Thr Leu His Asp Asp Asp Ser Cys Gln Val Ile 65 70 75 80
Pro Val Leu Pro Gln Val Met Met Ile Leu Ile Pro Gly Gln Thr Leu 85
90 95 Pro Leu Gln Leu Phe His Pro Gln Glu Val Ser Met Val Arg Asn
Leu 100 105 110 Ile Gln Lys Asp Arg Thr Phe Ala Val Leu Ala Tyr Ser
Asn Val Gln 115 120 125 Glu Arg Glu Ala Gln Phe Gly Thr Thr Ala Glu
Ile Tyr Ala Tyr Arg 130 135 140 Glu Glu Gln Asp Phe Gly Ile Glu Ile
Val Lys Val Lys Ala Ile Gly 145 150 155 160 Arg Gln Arg Phe Lys Val
Leu Glu Leu Arg Thr Gln Ser Asp Gly Ile 165 170 175 Gln Gln Ala Lys
Val Gln Ile Leu Pro Glu Cys Val Leu Pro Ser Thr 180 185 190 Met Ser
Ala Val Gln Leu Glu Ser Leu Asn Lys Cys Gln Ile Phe Pro 195 200 205
Ser Lys Pro Val Ser Arg Glu Asp Gln Cys Ser Tyr Lys Trp Trp Gln 210
215 220 Lys Tyr Gln Arg Arg Lys Phe His Cys Ala Asn Leu Thr Ser Trp
Pro 225 230 235 240 Arg Trp Leu Tyr Ser Leu Tyr Asp Ala Glu Thr Leu
Met Asp Arg Ile 245 250 255 Lys Lys Gln Leu Arg Glu Trp Asp Glu Asn
Leu Lys Asp Asp Ser Leu 260 265 270 Pro Ser Asn Pro Ile Asp Phe Ser
Tyr Arg Val Ala Ala Cys Leu Pro 275 280 285 Ile Asp Asp Val Leu Arg
Ile Gln Leu Leu Lys Ile Gly Ser Ala Ile 290 295 300 Gln Arg Leu Arg
Cys Glu Leu Asp Ile Met Asn Lys Cys Thr Ser Leu 305 310 315 320 Cys
Cys Lys Gln Cys Gln Glu Thr Glu Ile Thr Thr Lys Asn Glu Ile 325 330
335 Phe Ser Leu Ser Leu Cys Gly Pro Met Ala Ala Tyr Val Asn Pro His
340 345 350 Gly Tyr Val His Glu Thr Leu Thr Val Tyr Lys Ala Cys Asn
Leu Asn 355 360 365 Leu Ile Gly Arg Pro Ser Thr Glu His Ser Trp Phe
Pro Gly Tyr Ala 370 375 380 Trp Thr Val Ala Gln Cys Lys Ile Cys Ala
Ser His Ile Gly Trp Lys 385 390 395 400 Phe Thr Ala Thr Lys Lys Asp
Met Ser Pro Gln Lys Phe Trp Gly Leu 405 410 415 Thr Arg Ser Ala Leu
Leu Pro Thr Ile Pro Asp Thr Glu Asp Glu Ile 420 425 430 Ser Pro Asp
Lys Val Ile Leu Cys Leu 435 440 7441PRTHomo sapiens 7Met Ala Gly
Glu Gly Asp Gln Gln Asp Ala Ala His Asn Met Gly Asn 1 5 10 15 His
Leu Pro Leu Leu Pro Glu Ser Glu Glu Glu Asp Glu Met Glu Val 20 25
30 Glu Asp Gln Asp Ser Lys Glu Ala Lys Lys Pro Asn Ile Ile Asn Phe
35 40 45 Asp Thr Ser Leu Pro Thr Ser His Thr Tyr Leu Gly Ala Asp
Met Glu 50 55 60 Glu Phe His Gly Arg Thr Leu His Asp Asp Asp Ser
Cys Gln Val Ile 65 70 75 80 Pro Val Leu Pro Gln Val Met Met Ile Leu
Ile Pro Gly Gln Thr Leu 85 90 95 Pro Leu Gln Leu Phe His Pro Gln
Glu Val Ser Met Val Arg Asn Leu 100 105 110 Ile Gln Lys Asp Arg Thr
Phe Ala Val Leu Ala Tyr Ser Asn Val Gln 115 120 125 Glu Arg Glu Ala
Gln Phe Gly Thr Thr Ala Glu Ile Tyr Ala Tyr Arg 130 135 140 Glu Glu
Gln Asp Phe Gly Ile Glu Ile Val Lys Val Lys Ala Ile Gly 145 150 155
160 Arg Gln Arg Phe Lys Val Leu Glu Leu Arg Thr Gln Ser Asp Gly Ile
165 170 175 Gln Gln Ala Lys Val Gln Ile Leu Pro Glu Cys Val Leu Pro
Ser Thr 180 185 190 Met Ser Ala Val Gln Leu Glu Ser Leu Asn Lys Cys
Gln Ile Phe Pro 195 200 205 Ser Lys Pro Val Ser Arg Glu Asp Gln Cys
Ser Tyr Lys Trp Trp Gln 210 215 220 Lys Tyr Gln Lys Arg Lys Phe His
Cys Ala Asn Leu Thr Ser Trp Pro 225 230 235 240 Arg Trp Leu Tyr Ser
Leu Tyr Asp Ala Glu Thr Leu Met Asp Arg Ile 245 250 255 Lys Lys Gln
Leu Arg Glu Trp Asp Glu Asn Leu Lys Asp Asp Ser Leu 260 265 270 Pro
Ser Asn Pro Ile Asp Phe Ser Tyr Arg Val Ala Ala Cys Leu Pro 275 280
285 Ile Asp Asp Val Leu Arg Ile Gln Leu Leu Lys Ile Gly Ser Ala Ile
290 295 300 Gln Arg Leu Arg Cys Glu Leu Asp Ile Met Asn Lys Cys Thr
Ser Leu 305 310 315 320 Cys Cys Lys Gln Cys Gln Glu Thr Glu Ile Thr
Thr Lys Asn Glu Ile 325 330 335 Phe Ser Leu Ser Leu Cys Gly Pro Met
Ala Ala Tyr Val Asn Pro His 340 345 350 Gly Tyr Val His Glu Thr Leu
Thr Val Tyr Lys Ala Cys Asn Leu Asn 355 360 365 Leu Ile Gly Arg Pro
Ser Thr Glu His Ser Trp Phe Pro Gly Tyr Ala 370 375 380 Trp Thr Val
Ala Gln Cys Lys Ile Cys Ala Ser His Ile Gly Trp Lys 385 390 395 400
Phe Thr Ala Thr Lys Lys Asp Met Ser Pro Gln Lys Phe Trp Gly Leu 405
410 415 Thr Arg Ser Ala Leu Leu Pro Thr Ile Pro Asp Thr Glu Asp Glu
Ile 420 425 430 Ser Pro Asp Lys Val Ile Leu Cys Leu 435 440
82213DNAHomo sapiens 8gcgtgtaaac agacatggcc ggcgaaggag atcagcagga
cgctgcgcac aacatgggca 60accacctgcc gctcctgcct gagagtgagg aagaagatga
aatggaagtt gaagaccagg 120atagtaaaga agccaaaaaa ccaaacatca
taaattttga caccagtctg ccgacatcac 180atacatacct aggtgctgat
atggaagaat ttcatggcag gactttgcac gatgacgaca 240gctgtcaggt
gattccagtt cttccacaag tgatgatgat cctgattccc ggacagacat
300tacctcttca gctttttcac cctcaagaag tcagtatggt gcggaattta
attcagaaag 360atagaacctt tgctgttctt gcatacagca atgtacagga
aagggaagca cagtttggaa 420caacagcaga gatatatgcc tatcgagaag
aacaggattt tggaattgag atagtgaaag 480tgaaagcaat tggaagacaa
aggttcaaag tccttgagct aagaacacag tcagatggaa 540tccagcaagc
taaagtgcaa attcttcccg aatgtgtgtt gccttcaacc atgtctgcag
600ttcaattaga atccctcaat aagtgccaga tatttccttc aaaacctgtc
tcaagagaag 660accaatgttc atataaatgg tggcagaaat accagaggag
aaagtttcat tgtgcaaatc 720taacttcatg gcctcgctgg ctgtattcct
tatatgatgc tgagacctta atggacagaa 780tcaagaaaca gctacgtgaa
tgggatgaaa atctaaaaga tgattctctt ccttcaaatc 840caatagattt
ttcttacaga gtagctgctt gtcttcctat tgatgatgta ttgagaattc
900agctccttaa aattggcagt gctatccagc gacttcgctg tgaattagac
attatgaata 960aatgtacttc cctttgctgt aaacaatgtc aagaaacaga
aataacaacc aaaaatgaaa 1020tattcagttt atccttatgt gggccgatgg
cagcttatgt
gaatcctcat ggatatgtgc 1080atgagacact tactgtgtat aaggcttgca
acttgaatct gataggccgg ccttctacag 1140aacacagctg gtttcctggg
tatgcctgga ctgttgccca gtgtaagatc tgtgcaagcc 1200atattggatg
gaagtttacg gccaccaaaa aagacatgtc acctcaaaaa ttttggggct
1260taacgcgatc tgctctgttg cccacgatcc cagacactga agatgaaata
agtccagaca 1320aagtaatact ttgcttgtaa acagatgtga tagagataaa
gttagttatc taacaaattg 1380gttatattct aagatctgct ttggaaatta
ttgcctctga tacataccta agtaaacata 1440acattaatac ctaagtaaac
ataacattac ttggagggtt gcagtttcta agtgaaactg 1500tatttgaaac
ttttaagtat actttaggaa acaagcatga acggcagtct agaataccag
1560aaacatctac ttgggtagct tggtgccatt atcctgtgga atctgatatg
tctggtagcg 1620tgtcattgat gggacatgaa gacatctttg gaaatgatga
gattatttcc tgtgttaaaa 1680aaaaaaaaaa aatcttaaat tcctacaatg
tgaaactgaa actaataatt tgatcctgat 1740gtatgggaca gcgtatctgt
accagtgctc taaataacaa aagctagggt gacaagtaca 1800tgttcctttt
ggaaagaagc aaggcaatgt atattaatta ttctaaaagg gctttgttcc
1860tttccatttt ctttaacttc tctgagatac tgatttgtaa attttgaaaa
ttagttaaaa 1920tatgcagttt tttgagccca cgaatagttg tcatttcctt
tatgtgcctg ttagtaaaaa 1980gtagtattgt gtatttgctc agtatctgaa
ctataagccc atttatactg ttccatacaa 2040aagctatttt tcaaaaatta
atttgaacca aaactactac tatagggaaa agatgccaaa 2100acatgtcccc
tcacccaggc taaacttgat actgtattat tttgttcaat gtaaattgaa
2160gaaaatctgt aagtaagtaa accttaagtg tgaaactaaa aaaaaaaaaa aaa
22139431PRTMus musculus 9Met Gly Asn His Leu Pro Leu Leu Pro Asp
Ser Glu Asp Glu Asp Asp 1 5 10 15 Glu Ile Glu Met Glu Val Glu Asp
Gln Asp Ser Lys Glu Ala Arg Lys 20 25 30 Pro Asn Ile Ile Asn Phe
Asp Thr Ser Leu Pro Thr Ser His Thr Tyr 35 40 45 Leu Gly Ala Asp
Met Glu Glu Phe His Gly Arg Thr Leu His Asp Asp 50 55 60 Asp Ser
Cys Gln Val Ile Pro Val Leu Pro Glu Val Leu Met Ile Leu 65 70 75 80
Ile Pro Gly Gln Thr Leu Pro Leu Gln Leu Ser His Pro Gln Glu Val 85
90 95 Ser Met Val Arg Asn Leu Ile Gln Lys Asp Arg Thr Phe Ala Val
Leu 100 105 110 Ala Tyr Ser Asn Val Gln Glu Arg Glu Ala Gln Phe Gly
Thr Thr Ala 115 120 125 Glu Ile Tyr Ala Tyr Arg Glu Glu Gln Glu Phe
Gly Ile Glu Val Val 130 135 140 Lys Val Lys Ala Ile Gly Arg Gln Arg
Phe Lys Val Leu Glu Leu Arg 145 150 155 160 Thr Gln Ser Asp Gly Ile
Gln Gln Ala Lys Val Gln Ile Leu Pro Glu 165 170 175 Cys Val Leu Pro
Ser Thr Met Ser Ala Val Gln Val Glu Ser Leu Asn 180 185 190 Lys Cys
Gln Val Phe Pro Ser Lys Pro Ile Ser Trp Glu Asp Gln Tyr 195 200 205
Ser Cys Lys Trp Trp Gln Lys Tyr Gln Lys Arg Lys Phe His Cys Ala 210
215 220 Asn Leu Thr Ser Trp Pro Arg Trp Leu Tyr Ser Leu Tyr Asp Ala
Glu 225 230 235 240 Thr Leu Met Asp Arg Ile Lys Lys Gln Leu Arg Glu
Trp Asp Glu Asn 245 250 255 Leu Lys Asp Asp Ser Leu Pro Glu Asn Pro
Ile Asp Phe Ser Tyr Arg 260 265 270 Val Ala Ala Cys Leu Pro Ile Asp
Asp Val Leu Arg Ile Gln Leu Leu 275 280 285 Lys Ile Gly Ser Ala Ile
Gln Arg Leu Arg Cys Glu Leu Asp Ile Met 290 295 300 Asn Lys Cys Thr
Ser Leu Cys Cys Lys Gln Cys Gln Glu Thr Glu Ile 305 310 315 320 Thr
Thr Lys Asn Glu Ile Phe Ser Leu Ser Leu Cys Gly Pro Met Ala 325 330
335 Ala Tyr Val Asn Pro His Gly Tyr Val His Glu Thr Leu Thr Val Tyr
340 345 350 Lys Ala Ser Asn Leu Asn Leu Ile Gly Arg Pro Ser Thr Val
His Ser 355 360 365 Trp Phe Pro Gly Tyr Ala Trp Thr Ile Ala Gln Cys
Lys Ile Cys Ala 370 375 380 Ser His Ile Gly Trp Lys Phe Thr Ala Thr
Lys Lys Asp Met Ser Pro 385 390 395 400 Gln Lys Phe Trp Gly Leu Thr
Arg Ser Ala Leu Leu Pro Thr Ile Pro 405 410 415 Glu Thr Glu Asp Glu
Ile Ser Pro Asp Lys Val Ile Leu Cys Leu 420 425 430 10445PRTMus
musculus 10Met Ala Gly Glu Gly Asp Gln Gln Asp Ala Ala His Asn Met
Gly Asn 1 5 10 15 His Leu Pro Leu Leu Pro Ala Asp Ser Glu Asp Glu
Asp Asp Glu Ile 20 25 30 Glu Met Glu Val Glu Asp Gln Asp Ser Lys
Glu Ala Arg Lys Pro Asn 35 40 45 Ile Ile Asn Phe Asp Thr Ser Leu
Pro Thr Ser His Thr Tyr Leu Gly 50 55 60 Ala Asp Met Glu Glu Phe
His Gly Arg Thr Leu His Asp Asp Asp Ser 65 70 75 80 Cys Gln Val Ile
Pro Val Leu Pro Glu Val Leu Met Ile Leu Ile Pro 85 90 95 Gly Gln
Thr Leu Pro Leu Gln Leu Ser His Pro Gln Glu Val Ser Met 100 105 110
Val Arg Asn Leu Ile Gln Lys Asp Arg Thr Phe Ala Val Leu Ala Tyr 115
120 125 Ser Asn Val Gln Glu Arg Glu Ala Gln Phe Gly Thr Thr Ala Glu
Ile 130 135 140 Tyr Ala Tyr Arg Glu Glu Gln Glu Phe Gly Ile Glu Val
Val Lys Val 145 150 155 160 Lys Ala Ile Gly Arg Gln Arg Phe Lys Val
Leu Glu Leu Arg Thr Gln 165 170 175 Ser Asp Gly Ile Gln Gln Ala Lys
Val Gln Ile Leu Pro Glu Cys Val 180 185 190 Leu Pro Ser Thr Met Ser
Ala Val Gln Leu Glu Ser Leu Asn Lys Cys 195 200 205 Gln Val Phe Pro
Ser Lys Pro Ile Ser Trp Glu Asp Gln Tyr Ser Cys 210 215 220 Lys Trp
Trp Gln Lys Tyr Gln Lys Arg Lys Phe His Cys Ala Asn Leu 225 230 235
240 Thr Ser Trp Pro Arg Trp Leu Tyr Ser Leu Tyr Asp Ala Glu Thr Leu
245 250 255 Met Asp Arg Ile Lys Lys Gln Leu Arg Glu Trp Asp Glu Asn
Leu Lys 260 265 270 Asp Asp Ser Leu Pro Glu Asn Pro Ile Asp Phe Ser
Tyr Arg Val Ala 275 280 285 Ala Cys Leu Pro Ile Asp Asp Val Leu Arg
Ile Gln Leu Leu Lys Ile 290 295 300 Gly Ser Ala Ile Gln Arg Leu Arg
Cys Glu Leu Asp Ile Met Asn Lys 305 310 315 320 Cys Thr Ser Leu Cys
Cys Lys Gln Cys Gln Glu Thr Glu Ile Thr Thr 325 330 335 Lys Asn Glu
Ile Phe Ser Leu Ser Leu Cys Gly Pro Met Ala Ala Tyr 340 345 350 Val
Asn Pro His Gly Tyr Val His Glu Thr Leu Thr Val Tyr Lys Ala 355 360
365 Ser Asn Leu Asn Leu Ile Gly Arg Pro Ser Thr Val His Ser Trp Phe
370 375 380 Pro Gly Tyr Ala Trp Thr Ile Ala Gln Cys Lys Ile Cys Ala
Ser His 385 390 395 400 Ile Gly Trp Lys Phe Thr Ala Thr Lys Lys Asp
Met Ser Pro Gln Lys 405 410 415 Phe Trp Gly Leu Thr Arg Ser Ala Leu
Leu Pro Thr Ile Pro Glu Thr 420 425 430 Glu Asp Glu Ile Ser Pro Asp
Lys Val Ile Leu Cys Leu 435 440 445 114064DNAMus musculus
11tttcccaggc tcctttgcgg gtaaacagac atggccggcg agggagatca gcaggacgct
60gcgcacaaca tgggaaacca cctgccgctt ctgcctgaca gtgaagatga agatgatgaa
120attgaaatgg aagttgaaga ccaagatagt aaagaagcca gaaaaccgaa
tatcataaac 180tttgacacca gtctgccaac ctcacataca tacctgggag
ctgatatgga ggagttccac 240gggagaactt tgcatgacga cgacagctgc
caggtgatcc cagtccttcc tgaggtgctg 300atgatcctga ttcctgggca
gacactccca ctgcagctct ctcacccaca ggaagtcagc 360atggtgcgga
acttaatcca gaaagacagg acctttgcag tccttgcata cagtaatgtg
420caagaaaggg aagcacagtt tgggacaaca gcagagatct atgcctatcg
agaagagcag 480gagtttggaa ttgaagtagt gaaagtgaaa gcaattggaa
ggcagcggtt caaggtcctc 540gaacttcgaa cacagtcaga tggaatccag
caagctaaag tgcagatttt gccagagtgt 600gtgttgccgt caaccatgtc
tgcagtgcag ttagaatcac tcaataagtg ccaggtattt 660ccttcaaaac
ccatctcctg ggaagaccag tattcatgta aatggtggca gaaataccag
720aagagaaagt ttcactgtgc aaatctaaca tcatggcctc gctggctgta
ttcattatat 780gatgctgaaa cattaatgga tagaattaag aaacagctac
gtgaatggga tgaaaatctc 840aaagatgatt ctcttcctga aaatccaata
gacttttctt acagagtagc tgcttgtctt 900cctattgatg atgtattgag
aattcagctc cttaaaatcg gcagtgctat tcaacggctt 960cgctgtgaat
tggacatcat gaacaaatgt acttcccttt gctgtaaaca atgtcaagaa
1020acagaaataa cgacaaagaa tgaaatattt agtttatcct tatgtggtcc
aatggcagca 1080tatgtgaatc ctcatggata tgtacatgag acactgactg
tgtataaagc gtccaacctg 1140aatctgatag gccggccttc tacagtgcac
agctggtttc ccgggtatgc atggaccatt 1200gcccagtgca agatctgtgc
aagccatatt ggatggaaat ttacagccac aaaaaaagac 1260atgtcacctc
aaaaattttg gggcttaact cgctctgctc tgttacccac aattccagag
1320actgaagatg aaataagtcc agacaaagta atactttgtt tataagtgca
cctgtaggag 1380tgacttcctg acagatattt cctcaagtca gatctgccca
gtcatcactg cctctgatat 1440atgtgtatag tgggttacag catttgccta
ccaagttcaa gagcatattt agggaatgag 1500aaagcagtat aaaacataag
gctgggttcc aaaatacttg ctttttagta gcttggtgcc 1560atggattatc
ctgttgagtc tatgtcatga caggatagga aaacacagtt gaaataatgg
1620gaatggccat ggaacaggat aggggcacca ctgctctaaa tgatgaagct
ctaaatgatg 1680aatgctccag aaactgggtt ggtaagcaca agatagaggc
aaggcagtgt aattttaaaa 1740ggactttgct cctttcaatt ttccttagct
tgtctgagat actgacctgt acattttgaa 1800catattaaag agtaactaag
tattctgagc agaaatagca gcatttggtg tagttgcact 1860tttgatttga
tgagcctgtg atgtgctaga tccctttaac taatgtatat gtccattttg
1920cattttattt gcaaatataa gtgaacagta tatatttcta ggattatacc
atttaggaaa 1980caggtttaca taaacataaa tatccaaatc tattctattt
ggctgaatta tgtcaaagta 2040atcaagtaga atactgaaaa gtgtaagtac
gtaataaaat gcaactcaag aataggctgc 2100tccttaatgt cattttttca
aaagttctac ttgtgtttca ttcaagctgc tgtgatggag 2160tggggaatta
tgcctttact gctgcagtat aatctgatga tccatggact gtttaccatt
2220actttcagat aggactgttt aaaggaatct tacacaatat agcagctttg
atgtcactcc 2280atctgtgcag atgacaacag cagaaactcc atagtttaaa
atccaggtat ttactgacct 2340gggtgaagta gattttgaca cgccctttta
tagcacatca ccttatttga cttcaagaaa 2400attcaaaatc caaaagctgc
tgtttacttg tacagtacac agatatctat gagcagctat 2460gcagtaagta
actatgtaag ctatcagaaa gctaagccat atccatctaa cttgtaaaat
2520aaacaatgtg ttcactatct gtggcacctg atataaaggc aagagtctca
gcacaagccc 2580tcctgttatt cctgcaactt tctgaaatca gaacaatcct
gttataaata gatgctacta 2640tggactcatt caggaaacca ctaagaaaac
atagtttctc ttcaacagtt actacatttt 2700aagatcaaca gcactgctcc
acaagcattg ggaaattcag gaggtagact tgagcttagt 2760ttttctacct
acactcatgc tggttttggg gtctcagtaa cacaggaggg gagaacacca
2820gccttaccaa gacttcccct gtttcataca gggctcatct ttaggtcttc
tttatgtaac 2880ttagtagttc atctttttcc atccggtaac cacttttctt
ccactgttca cgcaactgct 2940gtagcagggc cccaatttcc ttccctgaag
aaatacccac tttcctgatg tcatgtccac 3000tcacagggaa cggcgggaca
gaccactgct gcatctcttt gagaagacca tgctctcctt 3060ggtacttcag
cagctcacaa acacgggcag ttgcatctgg ttccctagac taaatgacac
3120agttttatca catactaaac actcacaatt tcattctact atttaaatac
ttacatcaaa 3180tctacagtgt gaaaaagtta cttttctcta gttagtgaga
actattttct gctcagacct 3240aatacatact tacgtctatc acaaagtctt
ggtatggttt caatggttct gaactatctg 3300ttgctttaat caagtctttc
ctgtttttaa ctataaataa acccaggttt ttctcctctt 3360ttgaaatttt
caatctcaag tccaattttg tgacatcatc ttgtactttg aataaagaag
3420ccaaaagagt cattggtttt ggtgaaaagc cttcaacatt tttactgact
ttgttaaatt 3480cttctaaatt tgcattagca ggtaaacctg taaacaaaag
agaaaagtca tttttttctt 3540aactacaaaa ccctcactca cctcttaaac
tatgcagatt tttaagaatg tgtagtgttc 3600tttctccact gcttattatc
agcccattcg tcactccctt aactcctaga agaaatctat 3660catgttcctg
tttcctgtag cagcatgtct tgtgaagctc aggagctgtg atcatatcag
3720gtaccagcat atgccttctc agtcatgatc ctgtctgcac acattcccta
ctcagcaatt 3780gtatgttctt gtaaaacagt caaagttact gtctaaaata
tactggctat agttattaat 3840ttcctttcta tatattaagt gttttgtgaa
agagcttatt atacattaac ttattgcttc 3900atcctcctct ctatgaagta
gcttttattt tgaacccttt gtgattataa accaacccaa 3960cctgcaaaac
cagtaagctt catcaaattc aggtgttctc tctgaactat tctttaccaa
4020taaataaact atttccatct ttaatcccaa aaaaaaaaaa aaaa
4064124067DNAMus musculus 12tttcccaggc tcctttgcgg gtaaacagac
atggccggcg agggagatca gcaggacgct 60gcgcacaaca tgggaaacca cctgccgctt
ctgcctgcag acagtgaaga tgaagatgat 120gaaattgaaa tggaagttga
agaccaagat agtaaagaag ccagaaaacc gaatatcata 180aactttgaca
ccagtctgcc aacctcacat acatacctgg gagctgatat ggaggagttc
240cacgggagaa ctttgcatga cgacgacagc tgccaggtga tcccagtcct
tcctgaggtg 300ctgatgatcc tgattcctgg gcagacactc ccactgcagc
tctctcaccc acaggaagtc 360agcatggtgc ggaacttaat ccagaaagac
aggacctttg cagtccttgc atacagtaat 420gtgcaagaaa gggaagcaca
gtttgggaca acagcagaga tctatgccta tcgagaagag 480caggagtttg
gaattgaagt agtgaaagtg aaagcaattg gaaggcagcg gttcaaggtc
540ctcgaacttc gaacacagtc agatggaatc cagcaagcta aagtgcagat
tttgccagag 600tgtgtgttgc cgtcaaccat gtctgcagtg cagttagaat
cactcaataa gtgccaggta 660tttccttcaa aacccatctc ctgggaagac
cagtattcat gtaaatggtg gcagaaatac 720cagaagagaa agtttcactg
tgcaaatcta acatcatggc ctcgctggct gtattcatta 780tatgatgctg
aaacattaat ggatagaatt aagaaacagc tacgtgaatg ggatgaaaat
840ctcaaagatg attctcttcc tgaaaatcca atagactttt cttacagagt
agctgcttgt 900cttcctattg atgatgtatt gagaattcag ctccttaaaa
tcggcagtgc tattcaacgg 960cttcgctgtg aattggacat catgaacaaa
tgtacttccc tttgctgtaa acaatgtcaa 1020gaaacagaaa taacgacaaa
gaatgaaata tttagtttat ccttatgtgg tccaatggca 1080gcatatgtga
atcctcatgg atatgtacat gagacactga ctgtgtataa agcgtccaac
1140ctgaatctga taggccggcc ttctacagtg cacagctggt ttcccgggta
tgcatggacc 1200attgcccagt gcaagatctg tgcaagccat attggatgga
aatttacagc cacaaaaaaa 1260gacatgtcac ctcaaaaatt ttggggctta
actcgctctg ctctgttacc cacaattcca 1320gagactgaag atgaaataag
tccagacaaa gtaatacttt gtttataagt gcacctgtag 1380gagtgacttc
ctgacagata tttcctcaag tcagatctgc ccagtcatca ctgcctctga
1440tatatgtgta tagtgggtta cagcatttgc ctaccaagtt caagagcata
tttagggaat 1500gagaaagcag tataaaacat aaggctgggt tccaaaatac
ttgcttttta gtagcttggt 1560gccatggatt atcctgttga gtctatgtca
tgacaggata ggaaaacaca gttgaaataa 1620tgggaatggc catggaacag
gataggggca ccactgctct aaatgatgaa gctctaaatg 1680atgaatgctc
cagaaactgg gttggtaagc acaagataga ggcaaggcag tgtaatttta
1740aaaggacttt gctcctttca attttcctta gcttgtctga gatactgacc
tgtacatttt 1800gaacatatta aagagtaact aagtattctg agcagaaata
gcagcatttg gtgtagttgc 1860acttttgatt tgatgagcct gtgatgtgct
agatcccttt aactaatgta tatgtccatt 1920ttgcatttta tttgcaaata
taagtgaaca gtatatattt ctaggattat accatttagg 1980aaacaggttt
acataaacat aaatatccaa atctattcta tttggctgaa ttatgtcaaa
2040gtaatcaagt agaatactga aaagtgtaag tacgtaataa aatgcaactc
aagaataggc 2100tgctccttaa tgtcattttt tcaaaagttc tacttgtgtt
tcattcaagc tgctgtgatg 2160gagtggggaa ttatgccttt actgctgcag
tataatctga tgatccatgg actgtttacc 2220attactttca gataggactg
tttaaaggaa tcttacacaa tatagcagct ttgatgtcac 2280tccatctgtg
cagatgacaa cagcagaaac tccatagttt aaaatccagg tatttactga
2340cctgggtgaa gtagattttg acacgccctt ttatagcaca tcaccttatt
tgacttcaag 2400aaaattcaaa atccaaaagc tgctgtttac ttgtacagta
cacagatatc tatgagcagc 2460tatgcagtaa gtaactatgt aagctatcag
aaagctaagc catatccatc taacttgtaa 2520aataaacaat gtgttcacta
tctgtggcac ctgatataaa ggcaagagtc tcagcacaag 2580ccctcctgtt
attcctgcaa ctttctgaaa tcagaacaat cctgttataa atagatgcta
2640ctatggactc attcaggaaa ccactaagaa aacatagttt ctcttcaaca
gttactacat 2700tttaagatca acagcactgc tccacaagca ttgggaaatt
caggaggtag acttgagctt 2760agtttttcta cctacactca tgctggtttt
ggggtctcag taacacagga ggggagaaca 2820ccagccttac caagacttcc
cctgtttcat acagggctca tctttaggtc ttctttatgt 2880aacttagtag
ttcatctttt tccatccggt aaccactttt cttccactgt tcacgcaact
2940gctgtagcag ggccccaatt tccttccctg aagaaatacc cactttcctg
atgtcatgtc 3000cactcacagg gaacggcggg acagaccact gctgcatctc
tttgagaaga ccatgctctc 3060cttggtactt cagcagctca caaacacggg
cagttgcatc tggttcccta gactaaatga 3120cacagtttta tcacatacta
aacactcaca atttcattct actatttaaa tacttacatc 3180aaatctacag
tgtgaaaaag ttacttttct ctagttagtg agaactattt tctgctcaga
3240cctaatacat acttacgtct atcacaaagt cttggtatgg tttcaatggt
tctgaactat 3300ctgttgcttt aatcaagtct ttcctgtttt taactataaa
taaacccagg tttttctcct 3360cttttgaaat tttcaatctc aagtccaatt
ttgtgacatc atcttgtact ttgaataaag 3420aagccaaaag agtcattggt
tttggtgaaa agccttcaac atttttactg actttgttaa 3480attcttctaa
atttgcatta gcaggtaaac ctgtaaacaa aagagaaaag tcattttttt
3540cttaactaca aaaccctcac tcacctctta aactatgcag atttttaaga
atgtgtagtg 3600ttctttctcc actgcttatt atcagcccat tcgtcactcc
cttaactcct agaagaaatc 3660tatcatgttc ctgtttcctg tagcagcatg
tcttgtgaag ctcaggagct gtgatcatat 3720caggtaccag catatgcctt
ctcagtcatg atcctgtctg cacacattcc ctactcagca 3780attgtatgtt
cttgtaaaac agtcaaagtt actgtctaaa atatactggc tatagttatt
3840aatttccttt ctatatatta agtgttttgt gaaagagctt attatacatt
aacttattgc 3900ttcatcctcc tctctatgaa gtagctttta ttttgaaccc
tttgtgatta taaaccaacc 3960caacctgcaa aaccagtaag cttcatcaaa
ttcaggtgtt ctctctgaac tattctttac
4020caataaataa actatttcca tctttaatcc caaaaaaaaa aaaaaaa
406713337PRTHomo sapiens 13Met Ala Ser Ser Ser Gly Ser Lys Ala Glu
Phe Ile Val Gly Gly Lys 1 5 10 15 Tyr Lys Leu Val Arg Lys Ile Gly
Ser Gly Ser Phe Gly Asp Ile Tyr 20 25 30 Leu Ala Ile Asn Ile Thr
Asn Gly Glu Glu Val Ala Val Lys Leu Glu 35 40 45 Ser Gln Lys Ala
Arg His Pro Gln Leu Leu Tyr Glu Ser Lys Leu Tyr 50 55 60 Lys Ile
Leu Gln Gly Gly Val Gly Ile Pro His Ile Arg Trp Tyr Gly 65 70 75 80
Gln Glu Lys Asp Tyr Asn Val Leu Val Met Asp Leu Leu Gly Pro Ser 85
90 95 Leu Glu Asp Leu Phe Asn Phe Cys Ser Arg Arg Phe Thr Met Lys
Thr 100 105 110 Val Leu Met Leu Ala Asp Gln Met Ile Ser Arg Ile Glu
Tyr Val His 115 120 125 Thr Lys Asn Phe Ile His Arg Asp Ile Lys Pro
Asp Asn Phe Leu Met 130 135 140 Gly Ile Gly Arg His Cys Asn Lys Leu
Phe Leu Ile Asp Phe Gly Leu 145 150 155 160 Ala Lys Lys Tyr Arg Asp
Asn Arg Thr Arg Gln His Ile Pro Tyr Arg 165 170 175 Glu Asp Lys Asn
Leu Thr Gly Thr Ala Arg Tyr Ala Ser Ile Asn Ala 180 185 190 His Leu
Gly Ile Glu Gln Ser Arg Arg Asp Asp Met Glu Ser Leu Gly 195 200 205
Tyr Val Leu Met Tyr Phe Asn Arg Thr Ser Leu Pro Trp Gln Gly Leu 210
215 220 Lys Ala Ala Thr Lys Lys Gln Lys Tyr Glu Lys Ile Ser Glu Lys
Lys 225 230 235 240 Met Ser Thr Pro Val Glu Val Leu Cys Lys Gly Phe
Pro Ala Glu Phe 245 250 255 Ala Met Tyr Leu Asn Tyr Cys Arg Gly Leu
Arg Phe Glu Glu Ala Pro 260 265 270 Asp Tyr Met Tyr Leu Arg Gln Leu
Phe Arg Ile Leu Phe Arg Thr Leu 275 280 285 Asn His Gln Tyr Asp Tyr
Thr Phe Asp Trp Thr Met Leu Lys Gln Lys 290 295 300 Ala Ala Gln Gln
Ala Ala Ser Ser Ser Gly Gln Gly Gln Gln Ala Gln 305 310 315 320 Thr
Pro Thr Gly Lys Gln Thr Asp Lys Thr Lys Ser Asn Met Lys Gly 325 330
335 Phe 1421DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 14atgtttacta cactcggata t
211521DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 15cttcgaaatg tccgttcggt t
211621DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 16cgctggctgt attccttata t
211721DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 17caggatagta aagaagccaa a
211821DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 18cttaacgcga tctgctctgt t
211921DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 19ccgcttccac atgagctaaa g
212021DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 20gcatttggaa acgggaataa a
212121DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 21gtgatatctg tgggatcatt t
212221DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 22ccgcttccac atgagctaaa g
212321DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 23gcatttggaa acgggaataa a
212421DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 24gtgatatctg tgggatcatt t
212521DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 25catctatttg gcgatcaaca t
212621DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 26gcagaatttg cgatgtactt a 212730PRTHomo
sapiens 27His Thr Gly Glu Arg Pro Phe Gln Cys Asn Gln Cys Gly Ala
Ser Phe 1 5 10 15 Thr Gln Lys Gly Asn Leu Leu Arg His Ile Lys Leu
His Thr 20 25 30 2830PRTHomo sapiens 28His Thr Gly Glu Arg Pro Phe
Gln Cys Asn Gln Cys Gly Ala Ser Phe 1 5 10 15 Thr Gln Lys Gly Asn
Leu Leu Arg His Ile Lys Leu His Ser 20 25 30 2930PRTHomo sapiens
29His Thr Gly Glu Arg Pro Phe His Cys Asn Gln Cys Gly Ala Ser Phe 1
5 10 15 Thr Gln Lys Gly Asn Leu Leu Arg His Ile Lys Leu His Ser 20
25 30 3030PRTHomo sapiens 30His Thr Gly Glu Arg Pro Phe His Cys Asn
Gln Cys Gly Ala Ser Phe 1 5 10 15 Thr Gln Lys Gly Asn Leu Leu Arg
His Ile Lys Leu His Ser 20 25 30 3130PRTHomo sapiens 31His Thr Gly
Glu Lys Pro His Arg Cys His Leu Cys Pro Phe Ala Ser 1 5 10 15 Ala
Tyr Glu Arg His Leu Glu Ala His Met Arg Ser His Thr 20 25 30
3260PRTHomo sapiens 32Glu Thr Leu Thr Val Tyr Lys Ala Cys Asn Leu
Asn Leu Ile Gly Arg 1 5 10 15 Pro Ser Thr Glu His Ser Trp Phe Pro
Gly Tyr Ala Trp Thr Val Ala 20 25 30 Gln Cys Lys Ile Cys Ala Ser
His Ile Gly Trp Lys Phe Thr Ala Thr 35 40 45 Lys Lys Asp Met Ser
Pro Gln Lys Phe Trp Gly Leu 50 55 60 3360PRTMus musculus 33Glu Thr
Leu Thr Val Tyr Lys Ala Ser Asn Leu Asn Leu Ile Gly Arg 1 5 10 15
Pro Ser Thr Val His Ser Trp Phe Pro Gly Tyr Ala Trp Thr Ile Ala 20
25 30 Gln Cys Lys Ile Cys Ala Ser His Ile Gly Trp Lys Phe Thr Ala
Thr 35 40 45 Lys Lys Asp Met Ser Pro Gln Lys Phe Trp Gly Leu 50 55
60
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