U.S. patent application number 15/844751 was filed with the patent office on 2019-07-04 for phospholipase c gamma 2 and resistance associated mutations.
The applicant listed for this patent is Pharmacyclics LLC. Invention is credited to John C. Byrd, Jennifer A. Woyach.
Application Number | 20190203297 15/844751 |
Document ID | / |
Family ID | 54141528 |
Filed Date | 2019-07-04 |
United States Patent
Application |
20190203297 |
Kind Code |
A1 |
Byrd; John C. ; et
al. |
July 4, 2019 |
PHOSPHOLIPASE C GAMMA 2 AND RESISTANCE ASSOCIATED MUTATIONS
Abstract
Described herein is a mutation that confers resistance to the
treatment with a BTK inhibitor. Described herein is a modified
PLC.gamma.2 polypeptide that is modified at amino acid position
742, 845, or 1140 and the modified PLC.gamma.2 polypeptide exhibits
decreased inhibition (e.g., resistance) to a covalent and/or
irreversible BTK inhibitor. Described herein are diagnostic methods
for detecting the modified polypeptide and nucleic acid encoding
the modified polypeptide and applications of the methods thereof.
Described herein are compositions, combinations, and kits
containing the modified polypeptide and methods of using the
modified polypeptide. Also described herein are methods of using
the modified polypeptide as screening agents for the identification
and design of inhibitors of PLC.gamma.2.
Inventors: |
Byrd; John C.; (Columbus,
OH) ; Woyach; Jennifer A.; (Columbus, OH) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Pharmacyclics LLC |
Sunnyvale |
CA |
US |
|
|
Family ID: |
54141528 |
Appl. No.: |
15/844751 |
Filed: |
December 18, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14664663 |
Mar 20, 2015 |
9885086 |
|
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15844751 |
|
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62002743 |
May 23, 2014 |
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61968315 |
Mar 20, 2014 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01N 2800/52 20130101;
G01N 2333/916 20130101; C12Q 1/6886 20130101; C12Q 2600/156
20130101; G01N 33/57426 20130101; A61P 35/00 20180101; A61K 31/519
20130101; C12Q 2600/106 20130101; A61P 43/00 20180101 |
International
Class: |
C12Q 1/6886 20060101
C12Q001/6886; A61K 31/519 20060101 A61K031/519; G01N 33/574
20060101 G01N033/574 |
Claims
1. A method of assessing whether a subject having a hematologic
cancer is less responsive or likely to become less responsive to
therapy with a BTK inhibitor, comprising: a. testing a sample
containing a nucleic acid molecule encoding a PLC.gamma.2
polypeptide from the subject; b. determining whether the encoded
PLC.gamma.2 polypeptide is modified at an amino acid position
corresponding to amino acid position 742, 845, or 1140 of the amino
acid sequence set forth in SEQ ID NO: 2; and c. characterizing the
subject as resistant or likely to become resistant to therapy with
a BTK inhibitor if the subject has the modification at amino acid
position 742, 845, or 1140.
2. The method of claim 1, wherein the subject has been administered
a BTK inhibitor for treatment of a hematologic cancer.
3. A method of maintenance therapy in a subject having a
hematologic cancer, comprising: a. administering to the subject a
maintenance therapy regimen comprising administering a
therapeutically effective dose of a BTK inhibitor; and b.
monitoring the subject at predetermined intervals of time over the
course of the maintenance therapy regimen to determine whether the
subject has mutation in an endogenous gene encoding PLC.gamma.2
that results in a modification at an amino acid position
corresponding to amino acid position 742, 845, or 1140 of the amino
acid sequence set forth in SEQ ID NO: 2.
4. The method of claim 1, wherein the modification comprises a
substitution, an addition or a deletion of the amino acid at amino
acid position 742, 845, or 1140 in the PLC.gamma.2 polypeptide.
5. The method of claim 4, wherein the modification is: a. a
substitution of asparagine to an amino acid selected from among
leucine, cysteine, isoleucine, valine, alanine, glycine,
methionine, serine, threonine, phenylalanine, tryptophan, lysine,
arginine, histidine, proline, tyrosine, glutamine, aspartic acid
and glutamic acid at amino acid position 742 of the PLC.gamma.2
polypeptide; b. a substitution of leucine to an amino acid selected
from among cysteine, isoleucine, valine, alanine, glycine,
methionine, serine, threonine, phenylalanine, tryptophan, lysine,
arginine, histidine, proline, tyrosine, asparagine, glutamine,
aspartic acid and glutamic acid at amino acid position 845 of the
PLC.gamma.2 polypeptide; or c. a substitution of aspartic acid to
an amino acid selected from among leucine, cysteine, isoleucine,
valine, alanine, glycine, methionine, serine, threonine,
phenylalanine, tryptophan, lysine, arginine, histidine, proline,
tyrosine, asparagine, glutamine, aspartic acid and glutamic acid at
amino acid position 1140 of the PLC.gamma.2 polypeptide.
6. The method of claim 4, wherein the modification in the
PLC.gamma.2 polypeptide is selected from among R742P, L845F, and
D1140G.
7. The method of claim 1, wherein the nucleic acid encoding the
modified PLC.gamma.2 polypeptide has a mutation of adenine to
thymidine at B4823764.3 nucleic acid position corresponding to
nucleic acid position 2535 in the sequence of nucleotides set forth
in SEQ ID NO: 1.
8. The method of claim 1, wherein the PLC.gamma.2 polypeptide
further comprises modifications at additional amino acid
positions.
9. The method of claim 1, further comprising discontinuing
treatment with the BTK inhibitor if the subject has one or more
modifications with at least one modification at amino acid position
742, 845, or 1140 in the PLC.gamma.2 polypeptide.
10. The method of claim 1, further comprising administering an
inhibitor of PLC.gamma.2 if the subject has one or more
modifications with at least one modification at amino acid position
742, 845, or 1140 in the PLC.gamma.2 polypeptide.
11. The method of claim 1, wherein the subject possesses high-risk
cytogenetic features.
12. The method of claim 11, wherein the high-risk cytogenetic
features comprise del(11q22.3), del(17p13.1) or complex
karyotype.
13. The method of claim 1, further comprising testing a sample
containing a nucleic acid molecule encoding a PLC.gamma.2
polypeptide and an additional polypeptide and determining whether
the additional polypeptide contains mutations.
14. The method of claim 13, wherein the additional polypeptide is a
BTK polypeptide.
15. The method of claim 13, wherein the testing is by isothermal
amplification or polymerase chain reaction (PCR).
16. The method of claim 1, wherein the BTK inhibitor is
ibrutinib.
17. The method of claim 1, wherein the hematologic cancer is a
B-cell malignancy.
18. The method of claim 17, wherein the B-cell malignancy is
chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma
(SLL), diffuse large B-celllymphoma (DLBCL), activated B-cell
diffuse large B-celllymphoma (ABC-DLBCL), germinal center diffuse
large B-celllymphoma (GCB DLBCL), double-hit diffuse large
B-celllymphoma (DH-DLBCL), primary mediastinal B-celllymphoma
(PMBL), non-Hodgkin lymphoma, Burkitt's lymphoma, follicular
lymphoma, immunoblastic large cell lymphoma, precursor
B-lymphoblastic lymphoma, precursor B-cell acute lymphoblastic
leukemia, hairy cell leukemia, mantle cell lymphoma, B cell
prolymphocytic leukemia, lymphoplasmacytic lymphoma/Waldenstr6m
macroglobulinemia, splenic marginal zone lymphoma, plasma cell
myeloma, plasmacytoma, extranodal marginal zone B cell lymphoma,
nodal marginal zone B cell lymphoma, mediastinal (thymic) large B
cell lymphoma, intravascular large B cell lymphoma, primary
effusion lymphoma, or lymphomatoid granulomatosis.
19. A system of detecting a modified PLC.gamma.2 that confers
resistance to inhibition with an irreversible BTK inhibitor in a
subject, comprising: a. a sample containing a nucleic acid molecule
encoding a PLC.gamma.2 polypeptide from the subject; and b. a
microarray comprising nucleic acid encoding a modified PLC.gamma.2
polypeptide or a portion thereof that is modified at an amino acid
position corresponding to amino acid position 742, 845, or 1140 of
the amino acid sequence set forth in SEQ ID NO: 2.
20. The system of claim 19, wherein the microarray further
comprises comprising nucleic acid encoding a modified PLC.gamma.2
polypeptide or a portion thereof that is modified at additional
amino acid positions.
Description
CROSS-REFERENCE
[0001] This application is a continuation of U.S. patent
application Ser. No. 14/664,663, filed Mar. 20, 2015, which claims
the benefit of priority from U.S. Provisional Patent Application
Nos. 61/968,315, filed Mar. 20, 2014; and 62/002,743, filed May 23,
2014; which are herein incorporated by reference in their
entirety.
INCORPORATION BY REFERENCE OF SEQUENCE LISTING SUBMITTED AS A TEXT
FILE VIA EFS-WEB
[0002] The instant application contains a Sequence Listing, which
has been submitted as a computer readable text file in ASCII format
via EFS-Web and is hereby incorporated in its entirety by reference
herein. The text file, created date of May 20, 2015, is named
25922-307-201SEQ.txt and is 16,991 bytes in size.
BACKGROUND OF THE INVENTION
[0003] B-cell receptor (BCR) complex and its associated proteins
play a critical role in the development, proliferation and survival
of normal or malignant B cells. BCR function is required for normal
antibody production and abnormal BCR signal transduction is
implicated in B-cell malignancies. BCR signal transduction operates
through several signaling pathways, including the
PLC.gamma./calcium/NFAT pathway, the PI3K pathway, the
IKK/NF-.kappa.B pathway and the canonical ERK pathway.
[0004] Phospholipase C gamma 2 (PLC.gamma.2) is an enzyme of the
phospholipase C family that cleaves the phospholipid
phosphatidylinositol 4,5-bisphosphate (PIP2) into diacyl glycerol
(DAG) and inositol 1,4,5-trisphosphate (IP3). DAG remains bound to
the membrane, and IP3 is released as a soluble structure into the
cytosol. IP3 then diffuses through the cytosol to bind to IP3
receptors, particular calcium channels in the smooth endoplasmic
reticulum (ER). This causes the cytosolic concentration of calcium
to increase, causing a cascade of intracellular changes and
activity. In addition, calcium and DAG together work to activate
protein kinase C, which goes on to phosphorylate other molecules
within the pathway, leading to altered cellular activity. In some
cases, the mutant PLC.gamma.2 polypeptide are constitutively active
(i.e. does not require phosphorylation by BTK).
SUMMARY OF THE INVENTION
[0005] Disclosed herein is a method of assessing whether a subject
is less responsive or likely to become less responsive to therapy
with a BTK inhibitor, comprising: (a) testing a sample containing a
nucleic acid molecule encoding a PLC.gamma.2 polypeptide from the
subject; (b) determining whether the encoded PLC.gamma.2
polypeptide is modified at an amino acid position corresponding to
amino acid position 742, 845, or 1140 of the amino acid sequence
set forth in SEQ ID NO: 2; and (c) characterizing the subject as
resistant or likely to become resistant to therapy with a BTK
inhibitor if the subject has the modification at amino acid
position 742, 845, or 1140. In some embodiments, the subject has
been administered a covalent and/or irreversible BTK inhibitor for
treatment of a cancer. Disclosed herein is a method of monitoring
whether a subject receiving a BTK inhibitor for treatment of a
cancer has developed or is likely to develop resistance to the
therapy, comprising: (a) testing a sample containing a nucleic acid
molecule encoding a PLC.gamma.2 polypeptide from the subject; (b)
determining whether the encoded PLC.gamma.2 polypeptide is modified
at an amino acid position corresponding to amino acid position 742,
845, or 1140 of the amino acid sequence set forth in SEQ ID NO: 2;
and (c) characterizing the subject as resistant or is likely to
become resistant to therapy with a BTK inhibitor if the subject has
the modification at amino acid position 742, 845, or 1140.
Disclosed herein is a method of optimizing the therapy of a subject
receiving a BTK inhibitor for treatment of a cancer, comprising:
(a) testing a sample containing a nucleic acid molecule encoding a
PLC.gamma.2 polypeptide from the subject; and (b) determining
whether the encoded PLC.gamma.2 polypeptide is modified at an amino
acid position corresponding to amino acid position 742, 845, or
1140 of the amino acid sequence set forth in SEQ ID NO: 2. In some
embodiments, the modification comprises a substitution, an addition
or a deletion of the amino acid at amino acid position 742, 845, or
1140 in the PLC.gamma.2 polypeptide. In some embodiments, the
modification is a substitution of arginine to an amino acid
selected from among leucine, cysteine, isoleucine, valine, alanine,
glycine, methionine, serine, threonine, phenylalanine, tryptophan,
lysine, histidine, proline, tyrosine, asparagine, glutamine,
aspartic acid and glutamic acid at amino acid position 742 of the
PLC.gamma.2 polypeptide. In some embodiments, the modification is a
substitution of arginine to proline at amino acid position 742 of
the PLC.gamma.2 polypeptide. In some embodiments, the modification
is a substitution of leucine to an amino acid selected from among
cysteine, isoleucine, valine, alanine, glycine, methionine, serine,
threonine, phenylalanine, tryptophan, lysine, arginine, histidine,
proline, tyrosine, asparagine, glutamine, aspartic acid and
glutamic acid at amino acid position 845 of the PLC.gamma.2
polypeptide. In some embodiments, the modification is a
substitution of leucine to phenylalanine, tyrosine or tryptophan at
amino acid position 845 of the PLC.gamma.2 polypeptide. In some
embodiments, the modification is a substitution of leucine to
phenylalanine at amino acid position 845 of the PLC.gamma.2
polypeptide. In some embodiments, the modification is a
substitution of aspartic acid to an amino acid selected from among
arginine, leucine, cysteine, isoleucine, valine, alanine, glycine,
methionine, serine, threonine, phenylalanine, tryptophan, lysine,
histidine, proline, tyrosine, asparagine, glutamine, and glutamic
acid at amino acid position 742 of the PLC.gamma.2 polypeptide. In
some embodiments, the modification is a substitution of aspartic
acid to glycine at amino acid position 742 of the PLC.gamma.2
polypeptide. In some embodiments, the nucleic acid encoding the
modified PLC.gamma.2 polypeptide has a mutation of adenine to
thymidine at nucleic acid position corresponding to nucleic acid
position 2713 in the sequence of nucleotides set forth in SEQ ID
NO: 1. In some embodiments, the PLC.gamma.2 polypeptide further
comprises modifications at additional amino acid positions. In some
embodiments, the method of optimizing the therapy of a subject
receiving a BTK inhibitor for treatment of a cancer, further
comprising discontinuing treatment with the BTK inhibitor if the
subject has the modification at amino acid position 742, 845, or
1140 in the PLC.gamma.2 polypeptide. In some embodiments, the
method of optimizing the therapy of a subject receiving a BTK
inhibitor for treatment of a cancer, further comprising
discontinuing treatment with the BTK inhibitor if the subject has
one or more modifications with at least one modification at amino
acid position 742, 845, or 1140 in the PLC.gamma.2 polypeptide. In
some embodiments, the method of optimizing the therapy of a subject
receiving a BTK inhibitor for treatment of a cancer, further
comprising administering an inhibitor of PLC.gamma.2 if the subject
has one or more modifications with at least one modification at
amino acid position 742, 845, or 1140 in the PLC.gamma.2
polypeptide. In some embodiments, the method of optimizing the
therapy of a subject receiving a BTK inhibitor for treatment of a
cancer, further comprising administering an inhibitor of LYN, SYK,
JAK, PI3K, MAPK, MEK or NF.kappa.B if the subject has at least the
modification at amino acid position 742, 845, or 1140 in the
PLC.gamma.2 polypeptide. In some embodiments, the method of
optimizing the therapy of a subject receiving a BTK inhibitor for
treatment of a cancer, further comprising continuing treatment with
the BTK inhibitor if the subject does not have modifications in the
PLC.gamma.2 polypeptide. In some embodiments, the subject possesses
high-risk cytogenetic features. In some embodiments, the high-risk
cytogenetic features comprise del(11q22.3), del(17p13.1) or complex
karyotype. In some embodiments, the methods further comprising
testing a sample containing a nucleic acid molecule encoding a
PLC.gamma.2 polypeptide and an additional polypeptide and
determining whether the additional polypeptide contains mutations.
In some embodiments, the additional polypeptide is a BTK
polypeptide. In some embodiments, the nucleic acid molecule is RNA
or DNA. In some embodiments, the DNA is genomic DNA. In some
embodiments, the methods further comprises isolating mRNA from the
sample. In some embodiments, testing comprises amplifying the
nucleic acid encoding amino acid position 742, 845, or 1140 of the
PLC.gamma.2 polypeptide. In some embodiments, amplification is by
isothermal amplification or polymerase chain reaction (PCR). In
some embodiments, the amplification is by PCR. In some embodiments,
the PCR amplification comprises using oligonucleotide primer pairs
that flank the region encoding amino acid position 742, 845, or
1140 of the PLC.gamma.2 polypeptide. In some embodiments, testing
comprises sequencing the amplified nucleic acids. In some
embodiments, testing comprises contacting nucleic acids with
sequence specific nucleic acid probes, wherein the sequence
specific nucleic acid probes: (a) bind to either nucleic acid
encoding a modified PLC.gamma.2 that is modified at amino acid
position 742, 845, or 1140; and (b) do not bind to nucleic acid
encoding the wild-type PLC.gamma.2 having arginine at amino acid
position 742, do not bind to nucleic acid encoding the wild-type
PLC.gamma.2 having leucine at amino acid position 845, or do not
bind to nucleic acid encoding the wild-type PLC.gamma.2 having
aspartic acid at amino acid position 1140. In some embodiments,
testing comprises PCR amplification using the sequence specific
nucleic acid probes. In some embodiments, the methods further
comprise obtaining the sample from the subject. In some
embodiments, the sample contains one or more tumor cells from the
subject. In some embodiments, the sample contains circulating tumor
DNA (ctDNA). In some embodiments, the sample is a tumor biopsy
sample, a blood sample, a serum sample, a lymph sample or a bone
marrow aspirate. In some embodiments, the BTK inhibitor is a
covalent and/or irreversible BTK inhibitor. In some embodiments,
the covalent and/or irreversible BTK inhibitor is selected from
among ibrutinib, PCI-45292, PCI-45466, AVL-101/CC-101 (Avila
Therapeutics/Celgene Corporation), AVL-263/CC-263 (Avila
Therapeutics/Celgene Corporation), AVL-292/CC-292 (Avila
Therapeutics/Celgene Corporation), AVL-291/CC-291 (Avila
Therapeutics/Celgene Corporation), CNX 774 (Avila Therapeutics),
BMS-488516 (Bristol-Myers Squibb), BMS-509744 (Bristol-Myers
Squibb), CGI-1746 (CGI Pharma/Gilead Sciences), CGI-560 (CGI
Pharma/Gilead Sciences), CTA-056, GDC-0834 (Genentech), HY-11066
(also, CTK4I7891, HMS3265G21, HMS3265G22, HMS3265H21, HMS3265H22,
439574-61-5, AG-F-54930), ONO-4059 (Ono Pharmaceutical Co., Ltd.),
ONO-WG37 (Ono Pharmaceutical Co., Ltd.), PLS-123 (Peking
University), RN486 (Hoffmann-La Roche), HM71224 (Hanmi
Pharmaceutical Company Limited), LFM-A13, BGB-3111 (Beigene),
KBP-7536 (KBP BioSciences), ACP-196 (Acerta Pharma), JTE-051 (Japan
Tobacco Inc), PRN1008 (Principia), CTP-730 (Concert
Pharmaceuticals), or GDC-0853 (Genentech). In some embodiments, the
covalent and/or irreversible BTK inhibitor is ibrutinib. In some
embodiments, the subject has cancer. In some embodiments, the
cancer is a hematologic cancer. In some embodiments, the cancer is
a B-cell malignancy. In some embodiments, the cancer is selected
from among a leukemia, a lymphoma, or a myeloma. In some
embodiments, the B-cell malignancy is chronic lymphocytic leukemia
(CLL), small lymphocytic lymphoma (SLL), diffuse large B-cell
lymphoma (DLBCL), activated B-cell diffuse large B-cell lymphoma
(ABC-DLBCL), germinal center diffuse large B-cell lymphoma (GCB
DLBCL), double-hit diffuse large B-cell lymphoma (DH-DLBCL),
primary mediastinal B-cell lymphoma (PMBL), non-Hodgkin lymphoma,
Burkitt's lymphoma, follicular lymphoma, immunoblastic large cell
lymphoma, precursor B-lymphoblastic lymphoma, precursor B-cell
acute lymphoblastic leukemia, hairy cell leukemia, mantle cell
lymphoma, B cell prolymphocytic leukemia, lymphoplasmacytic
lymphoma/Waldenstrom macroglobulinemia, splenic marginal zone
lymphoma, plasma cell myeloma, plasmacytoma, extranodal marginal
zone B cell lymphoma, nodal marginal zone B cell lymphoma,
mediastinal (thymic) large B cell lymphoma, intravascular large B
cell lymphoma, primary effusion lymphoma, or lymphomatoid
granulomatosis. In some embodiments, the B-cell malignancy is CLL.
In some embodiments, the patient exhibits one or more symptoms of a
relapsed or refractory cancer. In some embodiments, the relapsed or
refractory cancer is a relapsed or refractory non-Hodgkin's
lymphoma. In some embodiments, the relapsed or refractory cancer is
a relapsed or refractory chronic lymphocytic leukemia (CLL), small
lymphocytic lymphoma (SLL), diffuse large B-cell lymphoma (DLBCL),
activated B-cell diffuse large B-cell lymphoma (ABC-DLBCL),
germinal center diffuse large B-cell lymphoma (GCB DLBCL),
double-hit diffuse large B-cell lymphoma (DH-DLBCL), primary
mediastinal B-cell lymphoma (PMBL), non-Hodgkin lymphoma, Burkitt's
lymphoma, follicular lymphoma, immunoblastic large cell lymphoma,
precursor B-lymphoblastic lymphoma, precursor B-cell acute
lymphoblastic leukemia, hairy cell leukemia, mantle cell lymphoma,
B cell prolymphocytic leukemia, lymphoplasmacytic
lymphoma/Waldenstrom macroglobulinemia, splenic marginal zone
lymphoma, plasma cell myeloma, plasmacytoma, extranodal marginal
zone B cell lymphoma, nodal marginal zone B cell lymphoma,
mediastinal (thymic) large B cell lymphoma, intravascular large B
cell lymphoma, primary effusion lymphoma, or lymphomatoid
granulomatosis. In some embodiments, the patient exhibits one or
more symptoms of Richter's transformation. In some embodiments, the
sample is a sample obtained at 1 week, 2 weeks, 3 weeks, 1 month, 2
months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months,
9 months, 10 months, 11 months, 12 months, 14 months, 16 months, 18
months, 20 months, 22 months, or 24 months following the first
administration of the covalent and/or irreversible BTK inhibitor.
In some embodiments, the sample is obtained 1, 2, 3, 4, 5, 6, 7, 8,
9, 10 times over the course of treatment with the irreversible BTK
inhibitor. In some embodiments, the subject is responsive to the
treatment with the irreversible BTK inhibitor when it is first
administered.
[0006] Disclosed herein is a method of maintenance therapy in a
patient having a hematologic cancer, comprising: (a) administering
to the patient a maintenance therapy regimen comprising
administering a therapeutically effective dose of a BTK inhibitor;
and (b) monitoring the patient at predetermined intervals of time
over the course of the maintenance therapy regimen to determine
whether the subject has mutation in an endogenous gene encoding
PLC.gamma.2 that results in a modification at an amino acid
position corresponding to amino acid position 742, 845, or 1140 of
the amino acid sequence set forth in SEQ ID NO: 2. In some
embodiments, the modification in the PLC.gamma.2 polypeptide is
R742P, L845F, or D1140G. In some embodiments, the modification in
the PLC.gamma.2 polypeptide further comprises additional
modifications. In some embodiments, the method further comprises
discontinuing maintenance therapy regimen if the subject has one or
more mutations with at least one mutation at amino acid position
742, 845, or 1140 in PLC.gamma.2 polypeptide. In some embodiments,
the method further comprises administering an inhibitor of
PLC.gamma.2 if the subject has one or more modifications with at
least one modification at amino acid position 742, 845, or 1140 in
the PLC.gamma.2 polypeptide. In some embodiments, the method
further comprises administering an inhibitor of LYN, SYK, JAK,
PI3K, MAPK, MEK or NF.kappa.B if the subject has at least the
modification at amino acid position 742, 845, or 1140 in the
PLC.gamma.2 polypeptide. In some embodiments, the method further
comprises continuing maintenance therapy regimen if the subject
does not have mutation at amino acid position 742, 845, or 1140 in
PLC.gamma.2 polypeptide. In some embodiments, the predetermined
interval of time is every week, every month, every 2 months, every
3 months, every 4 months, every 5 months, every 6 months, every 7
months, every 8 months, every 9 months, every 10 months, every 11
months, or every year. In some embodiments, the subject possesses
high-risk cytogenetic features. In some embodiments, the high-risk
cytogenetic features comprise del(11q22.3), del(17p13.1) or complex
karyotype. In some embodiments, the sample contains one or more
cancer cells. In some embodiments, the sample contains ctDNA. In
some embodiments, the method further comprises testing a sample
from the subject prior to treatment with the BTK inhibitor. In some
embodiments, the BTK inhibitor is a covalent and/or irreversible
BTK inhibitor. In some embodiments, the covalent and/or
irreversible BTK inhibitor is selected from among ibrutinib,
PCI-45292, PCI-45466, AVL-101/CC-101 (Avila Therapeutics/Celgene
Corporation), AVL-263/CC-263 (Avila Therapeutics/Celgene
Corporation), AVL-292/CC-292 (Avila Therapeutics/Celgene
Corporation), AVL-291/CC-291 (Avila Therapeutics/Celgene
Corporation), CNX 774 (Avila Therapeutics), BMS-488516
(Bristol-Myers Squibb), BMS-509744 (Bristol-Myers Squibb), CGI-1746
(CGI Pharma/Gilead Sciences), CGI-560 (CGI Pharma/Gilead Sciences),
CTA-056, GDC-0834 (Genentech), HY-11066 (also, CTK4I7891,
HMS3265G21, HMS3265G22, HMS3265H21, HMS3265H22, 439574-61-5,
AG-F-54930), ONO-4059 (Ono Pharmaceutical Co., Ltd.), ONO-WG37 (Ono
Pharmaceutical Co., Ltd.), PLS-123 (Peking University), RN486
(Hoffmann-La Roche), HM71224 (Hanmi Pharmaceutical Company
Limited), LFM-A13, BGB-3111 (Beigene), KBP-7536 (KBP BioSciences),
ACP-196 (Acerta Pharma), JTE-051 (Japan Tobacco Inc), PRN1008
(Principia), CTP-730 (Concert Pharmaceuticals), or GDC-0853
(Genentech). In some embodiments, the covalent and/or irreversible
BTK inhibitor is ibrutinib. In some embodiments, the maintenance
therapy regimen comprises administering the BTK inhibitor at a
daily dosage of about 10 mg per day to about 2000 mg per day, about
50 mg per day to about 1500 mg per day, about 100 mg per day to
about 1000 mg per day, about 250 mg per day to about 850 mg per
day, or about 300 mg per day to about 600 mg per day. In some
embodiments, the cancer is a hematologic cancer. In some
embodiments, the cancer is a B-cell malignancy. In some
embodiments, the cancer is selected from among a leukemia, a
lymphoma, or a myeloma. In some embodiments, the cancer is chronic
lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL),
diffuse large B-cell lymphoma (DLBCL), activated B-cell diffuse
large B-cell lymphoma (ABC-DLBCL), germinal center diffuse large
B-cell lymphoma (GCB DLBCL), double-hit diffuse large B-cell
lymphoma (DH-DLBCL), primary mediastinal B-cell lymphoma (PMBL),
non-Hodgkin lymphoma, Burkitt's lymphoma, follicular lymphoma,
immunoblastic large cell lymphoma, precursor B-lymphoblastic
lymphoma, precursor B-cell acute lymphoblastic leukemia, hairy cell
leukemia, mantle cell lymphoma, B cell prolymphocytic leukemia,
lymphoplasmacytic lymphoma/Waldenstrom macroglobulinemia, splenic
marginal zone lymphoma, plasma cell myeloma, plasmacytoma,
extranodal marginal zone B cell lymphoma, nodal marginal zone B
cell lymphoma, mediastinal (thymic) large B cell lymphoma,
intravascular large B cell lymphoma, primary effusion lymphoma, or
lymphomatoid granulomatosis. In some embodiments, the B-cell
malignancy is CLL. In some embodiments, the patient exhibits one or
more symptoms of a relapsed or refractory cancer. In some
embodiments, the relapsed or refractory cancer is a relapsed or
refractory non-Hodgkin's lymphoma. In some embodiments, the
relapsed or refractory cancer is a relapsed or refractory chronic
lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL),
diffuse large B-cell lymphoma (DLBCL), activated B-cell diffuse
large B-cell lymphoma (ABC-DLBCL), germinal center diffuse large
B-cell lymphoma (GCB DLBCL), double-hit diffuse large B-cell
lymphoma (DH-DLBCL), primary mediastinal B-cell lymphoma (PMBL),
non-Hodgkin lymphoma, Burkitt's lymphoma, follicular lymphoma,
immunoblastic large cell lymphoma, precursor B-lymphoblastic
lymphoma, precursor B-cell acute lymphoblastic leukemia, hairy cell
leukemia, mantle cell lymphoma, B cell prolymphocytic leukemia,
lymphoplasmacytic lymphoma/Waldenstrom macroglobulinemia, splenic
marginal zone lymphoma, plasma cell myeloma, plasmacytoma,
extranodal marginal zone B cell lymphoma, nodal marginal zone B
cell lymphoma, mediastinal (thymic) large B cell lymphoma,
intravascular large B cell lymphoma, primary effusion lymphoma, or
lymphomatoid granulomatosis. In some embodiments, the patient
exhibits one or more symptoms of Richter's transformation.
[0007] Disclosed herein is an isolated PLC.gamma.2 polypeptide or a
variant thereof having PLC.gamma.2 activity comprising a
modification at an amino acid position corresponding to amino acid
position 742, 845, or 1140 of the amino acid sequence set forth in
SEQ ID NO: 2, wherein the modification confers resistance of a
cancer cell to inhibition with a BTK inhibitor. In some
embodiments, the BTK inhibitor is ibrutinib. In some embodiments,
the isolated PLC.gamma.2 polypeptide comprises the sequence of
amino acids set forth in SEQ ID NO: 2 or a variant that has at
least or at least about 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%,
97%, 98%, 99% or more sequence identity with the polypeptide having
the sequence set forth in SEQ ID NO: 2, wherein the amino acid at
position 742 is not arginine, or wherein the amino acid at position
845 is not leucine, or wherein the amino acid at position 1140 is
not aspartic acid. In some embodiments, the amino acid at position
742 is proline. In some embodiments, the amino acid at position 845
is phenylalanine. In some embodiments, the amino acid at position
1140 is glycine. In some embodiments, disclosed herein is an
isolated nucleic acid molecule encoding the isolated PLC.gamma.2
polypeptide. In some embodiments, the nucleic acid is a DNA or an
RNA molecule. In some embodiments, the DNA is a cDNA molecule. In
some embodiments, the nucleic acid comprises the sequence of
nucleic acid set forth in SEQ ID NO: 1 or a variant that has at
least or at least about 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%,
97%, 98%, 99% or more sequence identity with the nucleic acid
having the sequence set forth in SEQ ID NO: 1, wherein the nucleic
acid codon encoding amino acid at position 742 does not encode
aspartic acid wherein the nucleic acid codon encoding amino acid at
position 845 does not encode leucine, or wherein the nucleic acid
codon encoding amino acid at position 1140 does not encode
glycine.
[0008] Disclosed herein is a system of detecting a modified
PLC.gamma.2 that confers resistance to inhibition with an
irreversible BTK inhibitor in a subject, comprising: (a) a sample
containing a nucleic acid molecule encoding a PLC.gamma.2
polypeptide from the subject; and (b) a microarray comprising
nucleic acid encoding a modified PLC.gamma.2 polypeptide or a
portion thereof that is modified at an amino acid position
corresponding to amino acid position 742, 845, or 1140 of the amino
acid sequence set forth in SEQ ID NO: 2. In some embodiments, the
microarray further comprises comprising nucleic acid encoding a
modified PLC.gamma.2 polypeptide or a portion thereof that is
modified at additional amino acid positions. In some embodiments,
the microarray is contained on a microchip.
[0009] Disclosed herein is a system of detecting a modified
PLC.gamma.2 that confers resistance to inhibition with an
irreversible BTK inhibitor in a subject, comprising: (a) a sample
containing a nucleic acid molecule encoding a PLC.gamma.2
polypeptide from the subject; and (b) a sequence specific nucleic
acid probe, wherein the sequence specific nucleic acid probe: (i)
binds to nucleic acid encoding a modified PLC.gamma.2 that is
modified at amino acid position 742, 845, or 1140; and (ii) does
not bind to nucleic acid encoding the wild-type PLC.gamma.2 having
arginine at amino acid position 742, or does not bind to nucleic
acid encoding the wild-type PLC.gamma.2 having leucine at amino
acid position 845, or does not bind to nucleic acid encoding the
wild-type PLC.gamma.2 having aspartic acid at amino acid position
1140. In some embodiments, the system further comprises additional
sequence specific nucleic acid probes, wherein the additional
sequence specific nucleic acid probes bind to nucleic acids
encoding a modified PLC.gamma.2 that is modified at amino acid
position 742, 845, or 1140 and at one or more additional
positions.
[0010] Disclosed herein is a system of detecting a modified
PLC.gamma.2 that confers resistance to inhibition with an
irreversible BTK inhibitor in a subject, comprising: (a) a sample
containing a nucleic acid molecule encoding a PLC.gamma.2
polypeptide from the subject; and (b) a pair of oligonucleotide
primers that flank the nucleic acid region encoding amino acid 742,
845, or 1140 of a PLC.gamma.2 polypeptide. In some embodiments, the
modification in the PLC.gamma.2 polypeptide is R742P, L845F, or
D1140G. In some embodiments, the system further comprises
additional oligonucleotide primers that flank nucleic acid regions
encoding additional amino acid modifications of the PLC.gamma.2
polypeptide.
[0011] Disclosed herein is a method of screening compounds that
inhibit a modified PLC.gamma.2, comprising: (a) providing a
modified PLC.gamma.2, wherein the modified PLC.gamma.2 is modified
at amino acid position corresponding to amino acid position 742,
845, or 1140 of the amino acid sequence set forth in SEQ ID NO: 2;
(b) contacting the modified PLC.gamma.2 with a test compound; and
(c) detecting the level of PLC.gamma.2 activity, wherein a decrease
in activity indicates that the compound inhibits the modified
PLC.gamma.2. In some embodiments, the modification is a
substitution, addition or deletion of the amino acid at position
742, 845, or 1140 of the PLC.gamma.2 polypeptide. In some
embodiments, the modification is a substitution of arginine to
proline at amino acid position 742 of the PLC.gamma.2 polypeptide.
In some embodiments, the modification is a substitution of leucine
to phenylalanine at amino acid position 845 of the PLC.gamma.2
polypeptide. In some embodiments, the modification is a
substitution of aspartic acid to glycine at amino acid position
1140 of the PLC.gamma.2 polypeptide. In some embodiments, detecting
the level of PLC.gamma.2 activity is assessed by an in vitro assay.
In some embodiments, the host cell stably expresses the modified
PLC.gamma.2 polypeptide. In some embodiments, the cell is deficient
for the expression of endogenous wild-type PLC.gamma.2. In some
embodiments, the cell is chicken DT40 PLC.gamma.2-/-B cell. In some
embodiments, the cell is a non B-cell. In some embodiments, the
cell is a mammalian non-B-cell. In some embodiments, the cell is a
293 cell. In some embodiments, the cell is a non-mammalian cell. In
some embodiments, the cell is an inset cell, a bacterial cell, a
yeast cell or a plant cell.
[0012] Disclosed herein is a method of assessing whether a subject
who possess high-risk cytogenetic features is less responsive or
likely to become less responsive to therapy with a BTK inhibitor,
comprising: (a) testing a sample containing a nucleic acid molecule
encoding a PLC.gamma.2 polypeptide from the subject; (b)
determining whether the encoded PLC.gamma.2 polypeptide is modified
at amino acid position corresponding to amino acid position 742,
845, or 1140 of the amino acid sequence set forth in SEQ ID NO: 2;
and (c) characterizing the subject as resistant or likely to become
resistant to therapy with a BTK inhibitor if the subject has the
modification. In some embodiments, the subject has been
administered a covalent and/or irreversible BTK inhibitor for
treatment of a cancer. Disclosed herein is a method of monitoring
whether a subject who possess high-risk cytogenetic features during
the course of a therapy with a BTK inhibitor has developed or is
likely to develop resistance to the therapy, comprising: (a)
testing a sample containing a nucleic acid molecule encoding a
PLC.gamma.2 polypeptide from the subject; (b) determining whether
the encoded PLC.gamma.2 polypeptide is modified at amino acid
position corresponding to amino acid position 742, 845, or 1140 of
the amino acid sequence set forth in SEQ ID NO: 2; and (c)
characterizing the subject as resistant or likely to become
resistant to therapy with a BTK inhibitor if the subject has the
modification. Disclosed herein is a method of optimizing the
therapy with a BTK inhibitor of a subject who possess high-risk
cytogenetic features, comprising: (a) testing a sample containing a
nucleic acid molecule encoding a PLC.gamma.2 polypeptide from the
subject; (b) determining whether the encoded PLC.gamma.2
polypeptide is modified at amino acid position corresponding to
amino acid position 742, 845, or 1140 of the amino acid sequence
set forth in SEQ ID NO: 2; and (c) discontinuing treatment with the
BTK inhibitor if the subject has the modification or continuing
treatment with the BTK inhibitor if the subject does not have the
modification. In some embodiments, the PLC.gamma.2 polypeptide is
modified at additional amino acid positions. In some embodiments,
the methods further comprise administering an inhibitor of
PLC.gamma.2 if the subject has one or more modifications with at
least one modification at amino acid position 742, 845, or 1140 in
the PLC.gamma.2 polypeptide. In some embodiments, the methods
further comprise administering an inhibitor of LYN, SYK, JAK, PI3K,
MAPK, MEK or NF.kappa.B if the subject has at least the
modification at amino acid position 742, 845, or 1140 in the
PLC.gamma.2 polypeptide. In some embodiments, the high-risk
cytogenetic features comprise del(11q22.3), del(17p13.1) or complex
karyotype. In some embodiments, the methods further comprise
testing a sample containing the nucleic acid molecule encoding the
PLC.gamma.2 polypeptide and a nucleic acid molecule encoding an
additional polypeptide. In some embodiments, the additional
polypeptide is a BTK polypeptide. In some embodiments, the sample
contains one or more cancer cells. In some embodiments, the sample
contains ctDNA. In some embodiments, the sample is a sample
obtained at 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months,
4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10
months, 11 months, 12 months, 14 months, 16 months, 18 months, 20
months, 22 months, or 24 months following the first administration
of the covalent and/or irreversible BTK inhibitor. In some
embodiments, the methods further comprise testing a sample from the
subject prior to treatment with the BTK inhibitor. In some
embodiments, the BTK inhibitor is a covalent and/or irreversible
BTK inhibitor. In some embodiments, the covalent and/or
irreversible BTK inhibitor is selected from among ibrutinib,
PCI-45292, PCI-45466, AVL-101/CC-101 (Avila Therapeutics/Celgene
Corporation), AVL-263/CC-263 (Avila Therapeutics/Celgene
Corporation), AVL-292/CC-292 (Avila Therapeutics/Celgene
Corporation), AVL-291/CC-291 (Avila Therapeutics/Celgene
Corporation), CNX 774 (Avila Therapeutics), BMS-488516
(Bristol-Myers Squibb), BMS-509744 (Bristol-Myers Squibb), CGI-1746
(CGI Pharma/Gilead Sciences), CGI-560 (CGI Pharma/Gilead Sciences),
CTA-056, GDC-0834 (Genentech), HY-11066 (also, CTK417891,
HMS3265G21, HMS3265G22, HMS3265H21, HMS3265H22, 439574-61-5,
AG-F-54930), ONO-4059 (Ono Pharmaceutical Co., Ltd.), ONO-WG37 (Ono
Pharmaceutical Co., Ltd.), PLS-123 (Peking University), RN486
(Hoffmann-La Roche), HM71224 (Hanmi Pharmaceutical Company
Limited), LFM-A13, BGB-3111 (Beigene), KBP-7536 (KBP BioSciences),
ACP-196 (Acerta Pharma), JTE-051 (Japan Tobacco Inc), PRN1008
(Principia), CTP-730 (Concert Pharmaceuticals), or GDC-0853
(Genentech). In some embodiments, the covalent and/or irreversible
BTK inhibitor is ibrutinib. In some embodiments, the maintenance
therapy regimen comprises administering the BTK inhibitor at a
daily dosage of about 10 mg per day to about 2000 mg per day, about
50 mg per day to about 1500 mg per day, about 100 mg per day to
about 1000 mg per day, about 250 mg per day to about 850 mg per
day, or about 300 mg per day to about 600 mg per day. In some
embodiments, the cancer is a hematologic cancer. In some
embodiments, the cancer is a B-cell malignancy. In some
embodiments, the cancer is selected from among a leukemia, a
lymphoma, or a myeloma. In some embodiments, the cancer is chronic
lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL),
diffuse large B-cell lymphoma (DLBCL), activated B-cell diffuse
large B-cell lymphoma (ABC-DLBCL), germinal center diffuse large
B-cell lymphoma (GCB DLBCL), double-hit diffuse large B-cell
lymphoma (DH-DLBCL), primary mediastinal B-cell lymphoma (PMBL),
non-Hodgkin lymphoma, Burkitt's lymphoma, follicular lymphoma,
immunoblastic large cell lymphoma, precursor B-lymphoblastic
lymphoma, precursor B-cell acute lymphoblastic leukemia, hairy cell
leukemia, mantle cell lymphoma, B cell prolymphocytic leukemia,
lymphoplasmacytic lymphoma/Waldenstrom macroglobulinemia, splenic
marginal zone lymphoma, plasma cell myeloma, plasmacytoma,
extranodal marginal zone B cell lymphoma, nodal marginal zone B
cell lymphoma, mediastinal (thymic) large B cell lymphoma,
intravascular large B cell lymphoma, primary effusion lymphoma, or
lymphomatoid granulomatosis. In some embodiments, the B-cell
malignancy is CLL. In some embodiments, the patient exhibits one or
more symptoms of a relapsed or refractory cancer. In some
embodiments, the relapsed or refractory cancer is a relapsed or
refractory non-Hodgkin's lymphoma. In some embodiments, the
relapsed or refractory cancer is a relapsed or refractory chronic
lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL),
diffuse large B-cell lymphoma (DLBCL), activated B-cell diffuse
large B-cell lymphoma (ABC-DLBCL), germinal center diffuse large
B-cell lymphoma (GCB DLBCL), double-hit diffuse large B-cell
lymphoma (DH-DLBCL), primary mediastinal B-cell lymphoma (PMBL),
non-Hodgkin lymphoma, Burkitt's lymphoma, follicular lymphoma,
immunoblastic large cell lymphoma, precursor B-lymphoblastic
lymphoma, precursor B-cell acute lymphoblastic leukemia, hairy cell
leukemia, mantle cell lymphoma, B cell prolymphocytic leukemia,
lymphoplasmacytic lymphoma/Waldenstrom macroglobulinemia, splenic
marginal zone lymphoma, plasma cell myeloma, plasmacytoma,
extranodal marginal zone B cell lymphoma, nodal marginal zone B
cell lymphoma, mediastinal (thymic) large B cell lymphoma,
intravascular large B cell lymphoma, primary effusion lymphoma, or
lymphomatoid granulomatosis. In some embodiments, the patient
exhibits one or more symptoms of Richter's transformation.
[0013] Disclosed herein is a kit comprising one or more reagents
for the detection of a mutant PLC.gamma.2 polypeptide, wherein the
mutant PLC.gamma.2 polypeptide comprises a modification at amino
acid position 742, 845, or 1140 or a nucleic acid encoding a mutant
PLC.gamma.2 polypeptide comprising modification at amino acid
position 742, 845, or 1140. In some embodiments, the kit comprises
oligonucleotide primer pairs that flank the nucleic acid region
encoding amino acid 742, 845, or 1140 of the PLC.gamma.2
polypeptide. In some embodiments, the kit comprises oligonucleotide
primers that (a) bind to nucleic acid encoding a modified
PLC.gamma.2 that is modified at amino acid position 742, 845, or
1140; and (b) do not bind to nucleic acid encoding the wild-type
PLC.gamma.2 having arginine at amino acid position 742, or do not
bind to nucleic acid encoding the wild-type PLC.gamma.2 having
leucine at amino acid position 845, or do not bind to nucleic acid
encoding the wild-type PLC.gamma.2 having aspartic acid at amino
acid position 1140. In some embodiments, the kit comprises a
microchip comprising (a) a modified PLC.gamma.2 polypeptide,
wherein the modified PLC.gamma.2 polypeptide has modifications at
amino acid position 742, 845, or 1140; or (b) a nucleic acid
molecule encoding a mutant PLC.gamma.2 polypeptide, wherein the
mutant PLC.gamma.2 polypeptide has a modification at amino acid
position 742, 845, or 1140 or a portion thereof comprising a
modification at amino acid position 742, 845, or 1140. In some
embodiments, the kit further comprises one or more reagents for the
detection of a mutant PLC.gamma.2 polypeptide, wherein the mutant
PLC.gamma.2 polypeptide comprises a modification at amino acid
position 742, 845, or 1140 and one or more additional
modifications.
BRIEF DESCRIPTION OF THE DRAWINGS
[0014] FIG. 1 illustrates characteristics of ibrutinib resistant
patients. Whole exome sequencing (WES) was performed on samples
from six patients. *Includes FISH for del(17p13.1), del(11q22.3),
centromere 12, and del(13q14.3) and complexity determined by
stimulated banded metaphase analysis. **A complete list of
functional mutations found only at relapse is shown.
[0015] FIG. 2 illustrates exome-seq analysis pipeline
flowchart.
[0016] FIG. 3 illustrates copy number profile for all samples. Data
were plotted using DNAcopy package of BioConductor.
[0017] FIG. 4 illustrates partial chromatographs generated by
chain-termination DNA sequencing of PLC.gamma.2 from peripheral
blood mononuclear cells (PBMC) of patients at relapse. Patient 5
had the A to T mutation in PLC.gamma.2 that results in a Leucine to
Phenylalanine substitution. This clone was very small on Sanger
sequencing.
[0018] FIG. 5A illustrates functional characterization of the L845F
mutation in PLC.gamma.2. pRetro X Tet-on Constructs containing
wild-type PLC.gamma.2 or the L845F mutant were transfected or
retro-virally delivered into 293 and PLC.gamma.2.sup.-/-DT40 cells.
After transfection, PLC.gamma.2 was present in these cells, and
Y1217 phosphorylation could be detected in 293 cells.
PLC.gamma.2.sup.-/-DT40 cells stably expressing either wild-type or
mutated pRetro-PLC.gamma.2 were treated with vehicle or 1 .mu.M
Ibrutinib for 30 minutes followed by stimulation for 15 minutes
with 5 .mu.g/ml anti-IgM and then lysed. Immunoblot analysis shows
that downstream BCR signaling as evidenced by phosphorylated AKT
and ERK are intact in these cells. In cells with the L845F
mutation, the repressions of these downstream signals are
diminished to a lesser degree by Ibrutinib after anti-IgM
stimulation as compared to the wild-type. All figures are
representative and are reflective of at least 3 independent
experiments.
[0019] FIG. 5B illustrates functional characterization of the L845F
mutation in PLC.gamma.2. pRetro X Tet-on Constructs containing
wild-type PLC.gamma.2 or the L845F mutant were transfected or
retro-virally delivered into 293 and PLC.gamma.2.sup.-/-DT40 cells.
After transfection, PLC.gamma.2 was present in these cells, and
Y1217 phosphorylation could be detected in 293 cells. All figures
are representative and are reflective of at least 3 independent
experiments.
[0020] FIG. 5C illustrates functional characterization of the L845F
mutation in PLC.gamma.2. pRetro X Tet-on Constructs containing
wild-type PLC.gamma.2 or the L845F mutant were transfected or
retro-virally delivered into 293 and PLC.gamma.2.sup.-/-DT40 cells.
After transfection, PLC.gamma.2 was present in these cells, and
Y1217 phosphorylation could be detected in 293 cells. All figures
are representative and are reflective of at least 3 independent
experiments.
[0021] FIG. 5D PLC.gamma.2.sup.-/-DT40 cells stably expressing
either wild-type or mutated pRetro-PLC.gamma.2 were treated with
vehicle or 1 .mu.M Ibrutinib for 30 minutes followed by stimulation
for 15 minutes with 5 .mu.g/ml anti-IgM and then lysed. Immunoblot
analysis shows that downstream BCR signaling as evidenced by
phosphorylated AKT and ERK are intact in these cells. In cells with
the L845F mutation, the repressions of these downstream signals are
diminished to a lesser degree by Ibrutinib after anti-IgM
stimulation as compared to the wild-type. All figures are
representative and are reflective of at least 3 independent
experiments. All figures are representative and are reflective of
at least 3 independent experiments.
[0022] FIG. 6A illustrates PLC.gamma.2 analysis by immunoblot at
relapse. At the time of relapse after drug had been discontinued,
fresh cells were treated with vehicle, plate-immobilized anti-IgM,
1 .mu.M ibrutinib, or ibrutinib+anti-IgM. Phosphorylation of
PLC.gamma.2 is not inhibited by ibrutinib. Samples were obtained
from patient 5.
[0023] FIG. 6B illustrates PLC.gamma.2 analysis by immunoblot at
relapse. At the time of relapse after drug had been discontinued,
fresh cells were treated with vehicle, plate-immobilized anti-IgM,
1 .mu.M ibrutinib, or ibrutinib+anti-IgM. Phosphorylation of ERK is
not inhibited by ibrutinib. Samples were obtained from patient
5.
[0024] FIG. 7 illustrates the cumulative incidence of CLL
progression, Richter's transformation, or other events among
patients with progressive disease during the course of ibrutinib
therapy.
[0025] FIG. 8 illustrates baseline characteristics associated with
study discontinuation among patients with progressive disease
(e.g., CLL, Richter's) or discontinuations for a non-progressive
disease reason (e.g., infection, toxicity or other).
[0026] FIG. 9 illustrates the identification of BTK and PLC.gamma.2
mutations in patients that experienced relapse on the Ibrutinib
therapy.
DETAILED DESCRIPTION OF THE INVENTION
[0027] Drug resistance is a problem affecting several areas of
medicine including infectious diseases and cancer. During the
course of cancer treatment, spontaneous random mutations occur as
the cancer cell population expands by repeated divisions, some of
which confer resistance and hence a survival advantage. The
acquisition of a resistance mutation has been described for all
major tyrosine kinase inhibitors in oncology, including imatinib
(Gleevec), and the EGFR inhibitors gefitinib, and erlotinib. The
emergence of resistance associated mutations forces patients to go
on to other therapies including dasatinib, nilotinib, etc., but
many of these eventually relapse with new resistance mutations. In
lung cancer, erlotinib and gefitinib have produced impressive and
durable clinical results, but nearly all become ineffective within
12-18 months due to resistance. .about.50% of these resistant
patients have a mutation in the target kinase (EGFR) called T790M,
which changes a single amino acid.
[0028] Described herein are mutations in PLC.gamma.2 gene that
arose during treatment with the irreversible BTK inhibitor
ibrutinib. In some embodiments, the mutation results in a modified
PLC.gamma.2 polypeptide that contain an amino acid substitution at
amino acid position 742, 845, or 1140 of the wild-type PLC.gamma.2
(e.g., R742P, L845F, D1140G). In some embodiments, the presence of
such mutation signals a development of resistance with BTK
inhibitor treatment such as ibrutinib. Also described herein, in
some embodiments, are modified PLC.gamma.2 polypeptides that
contain an amino acid substitution at amino acid position 742, 845,
or 1140 of the wild-type PLC.gamma.2 (e.g., R742P, L845F, D1140G)
and nucleic acids encoding the polypeptides.
[0029] As described herein, in some embodiments, subjects are
screened for the identification of a mutation at amino acid
position 742, 845, or 1140 in PLC.gamma.2. In some embodiments, the
subjects possess high-risk cytogenetic features (e.g.,
del(11q22.3), del(17p13.1) or complex karyotype). In some
embodiments, identification of mutation in PLC.gamma.2 allows for
the prescription of a cancer treatment or modification of a cancer
treatment. In some embodiments, identification of such a mutation
is used to stratify subjects for a particular therapy, such as for
example, therapy with an inhibitor that inhibits the activity of
the mutant PLC.gamma.2 (e.g., a PLC.gamma.2 inhibitor). In some
embodiments, identification of such a mutation is used to
characterize a subject as having a high risk of relapse of a
BTK-mediated disease or condition, such as, for example, a
hematologic cancer, such as a B-cell cancer. In some embodiments,
identification of such a mutation is used to characterize a subject
as lacking responsiveness to particular BTK inhibitor, such as for
example a covalent and/or irreversible BTK inhibitor, such as
ibrutinib.
[0030] As described herein, in some embodiments, subjects are
monitored throughout the course of a therapeutic regimen for the
development of the mutation in PLC.gamma.2 at amino acid position
742, 845, or 1140. In some embodiments, the therapeutic regimen is
a maintenance therapeutic regimen. In some embodiments, the
therapeutic regimen is optimized based on the identification of the
mutation in PLC.gamma.2.
[0031] Also described herein, in some embodiments, are designs and
screening of inhibitors effective for inhibition of a mutant
PLC.gamma.2 having one or more resistance mutations with at least
one mutation at amino acid position 742, 845, or 1140. Such
inhibitors are useful in clinical and therapeutic applications. In
some embodiments, the inhibitors are useful for the treatment of a
cancer, such as for example, a hematologic cancer, such as a B-cell
malignancy.
[0032] Further described herein, in some embodiments, are methods
of compositions, combinations and kits containing the modified
PLC.gamma.2 nucleic acids and polypeptides described herein and
reagents for detection of the modified PLC.gamma.2 nucleic acids
and polypeptides described herein. Also provided are methods of
using the modified PLC.gamma.2 polypeptides for identifying mutant
PLC.gamma.2 interacting molecules, including PLC.gamma.2
inhibitors. Also provided are methods of producing the modified
PLC.gamma.2 nucleic acids and polypeptides described herein.
Certain Terminology
[0033] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as is commonly understood by one
of skill in the art to which the claimed subject matter belongs. It
is to be understood that the foregoing general description and the
following detailed description are exemplary and explanatory only
and are not restrictive of any subject matter claimed. In this
application, the use of the singular includes the plural unless
specifically stated otherwise. It must be noted that, as used in
the specification and the appended claims, the singular forms "a,"
"an" and "the" include plural referents unless the context clearly
dictates otherwise. In this application, the use of "or" means
"and/or" unless stated otherwise. Furthermore, use of the term
"including" as well as other forms, such as "include", "includes,"
and "included," is not limiting.
[0034] As used herein, ranges and amounts can be expressed as
"about" a particular value or range. About also includes the exact
amount. Hence "about 5 .mu.g" means "about 5 .mu.g" and also "5
.mu.g." Generally, the term "about" includes an amount that would
be expected to be within experimental error.
[0035] The section headings used herein are for organizational
purposes only and are not to be construed as limiting the subject
matter described. All documents, or portions of documents, cited in
the application including, but not limited to, patents, patent
applications, articles, books, manuals, and treatises are hereby
expressly incorporated by reference in their entirety for any
purpose.
[0036] As used herein, the term "BTK inhibitor" or "BTK antagonist"
refers to an agent that inhibits or reduces at least one activity
of a BTK polypeptide. BTK activities include direct and indirect
activities. Exemplary direct activities include, but are not
limited to, association with a target molecule or phosphorylation
of a target substrate (i.e. kinase activity). Exemplary indirect
activities include, but are not limited to, activation or
inhibition of a downstream biological event, such as for example
activation of NF-.kappa.B-mediated gene transcription.
[0037] The term "irreversible inhibitor," as used herein, refers to
a compound that, upon contact with a target protein (e.g., a
kinase) causes the formation of a new covalent bond with or within
the protein, whereby one or more of the target protein's biological
activities (e.g., phosphotransferase activity) is diminished or
abolished notwithstanding the subsequent presence or absence of the
irreversible inhibitor.
[0038] The term "irreversible BTK inhibitor," as used herein,
refers to an inhibitor of BTK that can form a covalent bond with an
amino acid residue of BTK. In one embodiment, the irreversible
inhibitor of BTK can form a covalent bond with a Cysteine residue
of BTK; in particular embodiments, the irreversible inhibitor can
form a covalent bond with a Cysteine 481 residue (or a homolog
thereof) of BTK or a cysteine residue in the homologous
corresponding position of another tyrosine kinase.
[0039] As used herein, inhibition of BTK activity refers any
decrease in BTK activity in the presence of an inhibitor compared
to the same activity in the absence of the inhibitor.
[0040] As used herein, the term "PLC.gamma.2 inhibitor" refers to
an agent that inhibits at least one activity of a PLC.gamma.2
polypeptide containing an amino acid modification at position 742,
845, or 1140. In some embodiments, the agent inhibits at least one
activity of a PLC.gamma.2 polypeptide containing two or more amino
acid modifications at positions selected from 742, 845, or 1140 and
one or more additional positions. In some embodiments, the
PLC.gamma.2 inhibitor also inhibits the activity of a wild-type
PLC.gamma.2 polypeptide. In some embodiments, the PLC.gamma.2
inhibitor does not inhibit the activity of a wild-type PLC.gamma.2
polypeptide.
[0041] As used herein, "maintenance therapy" means the ongoing use
of chemotherapy or another treatment to assist in lowering the risk
of recurrence (return of cancer) following a beneficial response to
initial therapy, for example remission. Maintenance therapy also
may be used for patients with advanced cancer (e.g., cancer that
cannot be cured) to help keep it from growing and spreading
further.
[0042] The terms "cancer" and "cancerous" refer to or describe the
physiological condition in mammals that is typically characterized
by unregulated cell growth. Examples of cancer include, but are not
limited to, B-cell lymphoproliferative disorders (BCLDs), such as
lymphoma and leukemia, and solid tumors. By "B cell-related cancer"
or "cancer of B-cell lineage" is intended any type of cancer in
which the dysregulated or unregulated cell growth is associated
with B cells.
[0043] By "refractory" in the context of a cancer is intended the
particular cancer is resistant to, or non-responsive to, therapy
with a particular therapeutic agent. A cancer can be refractory to
therapy with a particular therapeutic agent either from the onset
of treatment with the particular therapeutic agent (i.e.,
non-responsive to initial exposure to the therapeutic agent), or as
a result of developing resistance to the therapeutic agent, either
over the course of a first treatment period with the therapeutic
agent or during a subsequent treatment period with the therapeutic
agent.
[0044] The term "nucleic acid" refers to deoxyribonucleotides,
deoxyribonucleosides, ribonucleosides, or ribonucleotides and
polymers thereof in either single- or double-stranded form. Unless
specifically limited, the term encompasses nucleic acids containing
known analogs of natural nucleotides which have similar binding
properties as the reference nucleic acid and are metabolized in a
manner similar to naturally occurring nucleotides. Unless
specifically limited otherwise, the term also refers to
oligonucleotide analogs including PNA (peptidonucleic acid),
analogs of DNA used in antisense technology (e.g.,
phosphorothioates, phosphoroamidates). Unless otherwise indicated,
a particular nucleic acid sequence also implicitly encompasses
conservatively modified variants thereof (including but not limited
to, degenerate codon substitutions) and complementary sequences as
well as the sequence explicitly indicated. Specifically, degenerate
codon substitutions are achieved by generating sequences in which
the third position of one or more selected (or all) codons is
substituted with mixed-base and/or deoxyinosine residues (Batzer et
al. (1991) Nucleic Acid Res. 19:5081; Ohtsuka et al. (1985) J.
Biol. Chem. 260:2605-2608; and Cassol et al. (1992) Mol. Cell.
Probes 6, 327-331; and Rossolini et al. (1994) Mol. Cell. Probes
8:91-98).
[0045] The term "amino acid" refers to naturally occurring and
non-naturally occurring amino acids, as well as amino acid analogs
and amino acid mimetics that function in a manner similar to the
naturally occurring amino acids. Naturally encoded amino acids are
the 20 common amino acids (alanine, arginine, asparagine, aspartic
acid, cysteine, glutamine, glutamic acid, glycine, histidine,
isoleucine, leucine, lysine, methionine, phenylalanine, proline,
serine, threonine, tryptophan, tyrosine, and valine) and pyrolysine
and selenocysteine. Amino acid analogs refers to agents that have
the same basic chemical structure as a naturally occurring amino
acid, i.e., an a carbon that is bound to a hydrogen, a carboxyl
group, an amino group, and an R group, such as, homoserine,
norleucine, methionine sulfoxide, methionine methyl sulfonium. Such
analogs have modified R groups (such as, norleucine) or modified
peptide backbones, but retain the same basic chemical structure as
a naturally occurring amino acid.
[0046] Amino acids are referred to herein by either their commonly
known three letter symbols or by the one-letter symbols recommended
by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides,
likewise, are referred to by their commonly accepted single-letter
codes.
[0047] The terms "polypeptide", peptide" and "protein" are used
interchangeably herein to refer to a polymer of amino acid
residues. The terms apply to naturally occurring amino acid
polymers as well as amino acid polymers in which one or more amino
acid residues is a non-naturally occurring amino acid, e.g., an
amino acid analog. The terms encompass amino acid chains of any
length, including full length proteins, wherein the amino acid
residues are linked by covalent peptide bonds.
[0048] As used herein, modification in reference to modification of
a sequence of amino acids of a polypeptide or a sequence of
nucleotides in a nucleic acid molecule and includes deletions,
insertions, and replacements of amino acids and nucleotides,
respectively.
[0049] To determine percent homology between two sequences, the
algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA
87:2264-2268, modified as in Karlin and Altschul (1993) Proc. Natl.
Acad. Sci. USA 90:5873-5877 is used. Such an algorithm is
incorporated into the NBLAST and XBLAST programs of Altschul, et
al. (1990) J. Mol. Biol. 215:403-410. BLAST nucleotide searches are
performed with the NBLAST program, score=100, wordlength=12 to
obtain nucleotide sequences homologous to a nucleic acid molecules
described or disclose herein. BLAST protein searches are performed
with the XBLAST program, score=50, wordlength=3. To obtain gapped
alignments for comparison purposes, Gapped BLAST is utilized as
described in Altschul et al. (1997) Nucleic Acids Res.
25:3389-3402. When utilizing BLAST and Gapped BLAST programs, the
default parameters of the respective programs (e.g., XBLAST and
NBLAST) are used. See the website of the National Center for
Biotechnology Information for further details (on the World Wide
Web at ncbi.nlm.nih.gov). Proteins suitable for use in the methods
described herein also includes proteins having between 1 to 15
amino acid changes, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,
13, 14, or 15 amino acid substitutions, deletions, or additions,
compared to the amino acid sequence of any protein described
herein. In other embodiments, the altered amino acid sequence is at
least 75% identical, e.g., 77%, 80%, 82%, 85%, 88%, 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino
acid sequence of any protein described herein. Such
sequence-variant proteins are suitable for the methods described
herein as long as the altered amino acid sequence retains
sufficient biological activity to be functional in the compositions
and methods described herein. Where amino acid substitutions are
made, the substitutions should be conservative amino acid
substitutions. Among the common amino acids, for example, a
"conservative amino acid substitution" is illustrated by a
substitution among amino acids within each of the following groups:
(1) glycine, alanine, valine, leucine, and isoleucine, (2)
phenylalanine, tyrosine, and tryptophan, (3) serine and threonine,
(4) aspartate and glutamate, (5) glutamine and asparagine, and (6)
lysine, arginine and histidine. Those of skill in this art
recognize that, in general, single amino acid substitutions in
non-essential regions of a polypeptide do not substantially alter
biological activity (see, e.g., Watson et al. Molecular Biology of
the Gene, 4th Edition, 1987, The Benjamin/Cummings Pub. co.,
p.224). The BLOSUM62 table is an amino acid substitution matrix
derived from about 2,000 local multiple alignments of protein
sequence segments, representing highly conserved regions of more
than 500 groups of related proteins (Henikoff et al (1992) Proc.
Natl. Acad. Sci. USA, 89:10915-10919). Accordingly, the BLOSUM62
substitution frequencies are used to define conservative amino acid
substitutions that, in some embodiments, are introduced into the
amino acid sequences described or disclosed herein. Although it is
possible to design amino acid substitutions based solely upon
chemical properties (as discussed above), the language
"conservative amino acid substitution" preferably refers to a
substitution represented by a BLOSUM62 value of greater than -1.
For example, an amino acid substitution is conservative if the
substitution is characterized by a BLOSUM62 value of 0, 1, 2, or 3.
According to this system, preferred conservative amino acid
substitutions are characterized by a BLOSUM62 value of at least 1
(e.g., 1, 2 or 3), while more preferred conservative amino acid
substitutions are characterized by a BLOSUM62 value of at least 2
(e.g., 2 or 3).
[0050] As used herein, corresponding residues refers to residues
that occur at aligned loci. Related or variant polypeptides are
aligned by any method known to those of skill in the art. Such
methods typically maximize matches, and include methods such as
using manual alignments and by using the numerous alignment
programs available (for example, BLASTP) and others known to those
of skill in the art. By aligning the sequences of polypeptides, one
skilled in the art can identify corresponding residues, using
conserved and identical amino acid residues as guides.
Corresponding positions also can be based on structural alignments,
for example by using computer simulated alignments of protein
structure. In other instances, corresponding regions can be
identified.
[0051] As used herein, the terms "subject", "individual" and
"patient" are used interchangeably. None of the terms are to be
interpreted as requiring the supervision of a medical professional
(e.g., a doctor, nurse, physician's assistant, orderly, hospice
worker). As used herein, the subject can be any animal, including
mammals (e.g., a human or non-human animal) and non-mammals. In one
embodiment of the methods and compositions provided herein, the
mammal is a human.
[0052] As used herein, the terms "treat," "treating" or
"treatment," and other grammatical equivalents, include
alleviating, abating or ameliorating one or more symptoms of a
disease or condition, ameliorating, preventing or reducing the
appearance, severity or frequency of one or more additional
symptoms of a disease or condition, ameliorating or preventing the
underlying metabolic causes of one or more symptoms of a disease or
condition, inhibiting the disease or condition, such as, for
example, arresting the development of the disease or condition,
relieving the disease or condition, causing regression of the
disease or condition, relieving a condition caused by the disease
or condition, or inhibiting the symptoms of the disease or
condition either prophylactically and/or therapeutically. In a
non-limiting example, for prophylactic benefit, a third-generation
BTK inhibitor compound disclosed herein is administered to an
individual at risk of developing a particular disorder, predisposed
to developing a particular disorder, or to an individual reporting
one or more of the physiological symptoms of a disorder. In some
embodiments, a third-generation BTK inhibitor compound disclosed
herein is administered to a subject following treatment with one or
more therapeutic agents. In some embodiments, a third-generation
BTK inhibitor compound disclosed herein is administered to a
subject in combination with treatment with one or more therapeutic
agents.
[0053] As used herein, "contacting" refers to refers to the act of
touching, making contact, or of bringing substances into immediate
proximity. "Contacting" can be achieved by mixing the components in
a fluid or semi-fluid mixture.
Mutant PLC.gamma.2 Polypeptides
[0054] Provided herein are mutant PLC.gamma.2 polypeptides. In some
embodiments, the mutant PLC.gamma.2 polypeptides are isolated
mutant PLC.gamma.2 polypeptides. In some embodiments, the isolated
mutant PLC.gamma.2 polypeptides are non-native mutant PLC.gamma.2
polypeptides. In some embodiments, the mutant PLC.gamma.2
polypeptides are recombinant proteins. In some embodiments, the
mutant PLC.gamma.2 polypeptides are purified from a host cell. In
some embodiments, the mutant PLC.gamma.2 polypeptides comprise one
or more mutations (e.g., substitution, deletion or addition). In
some embodiments, one or more mutations in the mutant PLC.gamma.2
polypeptides result in resistance of a patient to treatment with a
BTK inhibitor. In some embodiments, the one or more mutations are
gain of function mutations in PLC.gamma.2. In some embodiments, the
one or more mutations result in constitutive activation of
PLC.gamma.2. In some embodiments, constitutive activation of
PLC.gamma.2 results in mobilization of intracellular calcium,
activation of extracellular signal-regulated kinase (ERK) and c-Jun
NH2-terminal kinase (JNK) mitogen-activated protein kinase (MAPK)
pathways.
[0055] In some embodiments, the mutation results in a modification
at an amino acid position corresponding to amino acid position 742,
845, or 1140 of the amino acid sequence set forth in SEQ ID NO: 2.
In some embodiments, the mutation is a frame shift mutation that
results in a modification at an amino acid position corresponding
to amino acid position 742, 845, or 1140 of the amino acid sequence
set forth in SEQ ID NO: 2. In some embodiments, the mutation is a
frame shift mutation at that results in a truncation of the
PLC.gamma.2 polypeptide at or following amino acid position 742,
845, or 1140 of the amino acid sequence set forth in SEQ ID NO:
2.
[0056] Provided herein is an isolated PLC.gamma.2 polypeptide or a
variant thereof having PLC.gamma.2 activity comprising multiple
mutations. In some embodiments, the isolated PLC.gamma.2
polypeptide comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30
or more mutations. In some embodiments, the isolated PLC.gamma.2
polypeptide comprises one mutation. In some embodiments, the
mutations result in modifications at amino acid positions
corresponding to amino acid position 742, 845, or 1140 of the amino
acid sequence set forth in SEQ ID NO: 2.
[0057] In some embodiments, the modification comprises a
substitution, an addition or a deletion of the amino acid at amino
acid position 742, 845, or 1140 compared to a wild type PLC.gamma.2
set forth in SEQ ID NO: 2. In some embodiments, the modification
comprises substitution of the amino acid at position 742, 845, or
1140 compared to a wild type PLC.gamma.2 set forth in SEQ ID NO:
2.
[0058] In some embodiments, the modification is a substitution of
arginine at position 742 to an amino acid selected from among
leucine, isoleucine, valine, alanine, glycine, methionine,
cysteine, serine, threonine, phenylalanine, tryptophan, lysine,
histidine, proline, tyrosine, asparagine, glutamine, aspartic acid
and glutamic acid at amino acid position 742 of the PLC.gamma.2
polypeptide. In some embodiments, the modification is a
substitution of arginine to proline at amino acid position 742 of
the PLC.gamma.2 polypeptide. In some embodiments, the substitution
is R742P.
[0059] In some embodiments, the modification is a substitution of
leucine at position 845 to an amino acid selected from among
isoleucine, valine, alanine, glycine, methionine, cysteine, serine,
threonine, phenylalanine, tryptophan, lysine, arginine, histidine,
proline, tyrosine, asparagine, glutamine, aspartic acid and
glutamic acid at amino acid position 845 of the PLC.gamma.2
polypeptide. In some embodiments, the modification is a
substitution of leucine to phenylalanine, tyrosine or tryptophan at
amino acid position 845 of the PLC.gamma.2 polypeptide. In some
embodiments, the modification is a substitution of leucine to
phenylalanine at amino acid position 845 of the PLC.gamma.2
polypeptide. In some embodiments, the substitution is L845F.
[0060] In some embodiments, the modification is a substitution of
aspartic acid at position 1140 to an amino acid selected from among
leucine, isoleucine, valine, alanine, glycine, methionine,
cysteine, serine, threonine, phenylalanine, tryptophan, lysine,
arginine, histidine, proline, tyrosine, asparagine, glutamine, and
glutamic acid at amino acid position 1140 of the PLC.gamma.2
polypeptide. In some embodiments, the modification is a
substitution of aspartic acid to glycine at amino acid position
1140 of the PLC.gamma.2 polypeptide. In some embodiments, the
substitution is D1140G.
[0061] In some embodiments, the mutant PLC.gamma.2 polypeptide
comprises a modification at amino acid position 742, 845, or 1140
and a modification at one or more additional amino acid positions.
In some embodiments, the modification at one or more additional
amino acid positions comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,
12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acid positions. In
some embodiments, the modification at amino acid position 742 is a
substitution that is R742P. In some embodiments, the modification
at amino acid position 845 is a substitution that is L845F. In some
embodiments, the modification at amino acid position 1140 is a
substitution that is D1140G.
[0062] In some embodiments, the mutant PLC.gamma.2 polypeptide
comprises a substitution of the amino acid at position 742, 845, or
1140 compared to a wild type PLC.gamma.2 set forth in SEQ ID NO: 2
and one or more additional amino acid substitutions. In some
embodiments, the mutant PLC.gamma.2 polypeptide comprises the
sequence of amino acids comprising a substitution of the amino acid
at position 742, 845, or 1140 or a variant that has at least or at
least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%,
99% or more sequence identity with the polypeptide having the
sequence set forth in SEQ ID NO: 2.
[0063] In some embodiments, the mutant PLC.gamma.2 polypeptide
comprises a substitution of the amino acid at position 742, 845, or
1140 compared to a wild type PLC.gamma.2 set forth in SEQ ID NO: 2
and one or more additional amino acid substitutions selected from
among a substitution of the amino acid at position 665 or 707
compared to a wild type PLC.gamma.2 set forth in SEQ ID NO: 2. In
some embodiments, the mutant PLC.gamma.2 polypeptide comprises a
substitution of the amino acid at position 742, 845, or 1140
compared to a wild type PLC.gamma.2 set forth in SEQ ID NO: 2 and
one or more additional amino acid substitutions selected from among
R665W, 5707F, 5707P, and 5707Y. In some embodiments, the mutant
PLC.gamma.2 polypeptide comprises an amino acid substitution
selected from among R742P, L845F, D1140G and one or more additional
amino acid substitutions selected from among R665W, 5707F, 5707P,
and 5707Y. In some embodiments, the mutant PLC.gamma.2 polypeptide
comprises one or more amino acid substitutions selected from among
R742P, L845F, D1140G, R665W, 5707F, 5707P, and 5707Y.
[0064] In some embodiments, the mutant PLC.gamma.2 polypeptide
comprises a portion of the mutant PLC.gamma.2 polypeptide set forth
in SEQ ID NO: 2. In some embodiments, the portion exhibits an
activity of a PLC.gamma.2 polypeptide. In some embodiments, the
portion comprises one or more domains of the PLC.gamma.2
polypeptide. The PLC.gamma.2 polypeptide comprises two SH2 domains
and one SH3 domain. In some embodiments, the two SH2 domains
comprise amino acid positions 498-636 and 636-744 set forth in SEQ
ID NO: 2. In some embodiments, the SH3 domain comprises amino acid
positions 762-877 set forth in SEQ ID NO: 2. In some embodiments,
the mutant PLC.gamma.2 polypeptide comprises one or both SH2
domains and SH3 domain of the PLC.gamma.2 polypeptide comprising
the modification at amino acid position 742, 845, or 1140 of the
mutant PLC.gamma.2 polypeptide set forth in SEQ ID NO: 2.
[0065] In some embodiments, a PLC.gamma.2 polypeptide is a fusion
protein comprising the domains of a PLC.gamma.2 polypeptide
comprising the modifications at amino acid position 742, 845, or
1140 of the mutant PLC.gamma.2 polypeptide set forth in SEQ ID NO:
2 linked to a heterologous polypeptide. Methods for the generation
of fusion proteins are known in the art and include standard
recombinant DNA techniques. For example, in some embodiments, DNA
fragments coding for the different polypeptide sequences are
ligated together in-frame in accordance with conventional
techniques, for example by employing blunt-ended or stagger-ended
termini for ligation, restriction enzyme digestion to provide for
appropriate termini, filling-in of cohesive ends as appropriate,
alkaline phosphatase treatment to avoid undesirable joining, and
enzymatic ligation. In some embodiments, the fusion gene can be
synthesized by conventional techniques including automated DNA
synthesizers. In some embodiments, PCR amplification of gene
fragments can be carried out using anchor primers which give rise
to complementary overhangs between two consecutive gene fragments
which can subsequently be annealed and reamplified to generate a
chimeric gene sequence (see, for example, Current Protocols in
Molecular Biology, eds. Ausubel et al. John Wiley & Sons:
1992). In some embodiments, expression vectors are commercially
available that encode a fusion moiety (e.g., a GST polypeptide). A
nucleic acid encoding a modified PLC.gamma.2 polypeptide can be
cloned into such an expression vector such that the fusion moiety
is linked in-frame to the modified PLC.gamma.2 polypeptide.
[0066] In some embodiments, a PLC.gamma.2 polypeptide comprising
modifications at amino acid position 742, 845, or 1140 of the
wild-type PLC.gamma.2 polypeptide set forth in SEQ ID NO: 2 linked
to a peptide tag. In some embodiments, the peptide tag is an
epitope tag recognized by a tag-specific antibody. In some
embodiments the tag is an epitope tag, such as, but not limited to
a c-myc, V-5, hemagglutinin (HA), FLAG, tag. In some embodiments
the tag is an affinity tag, such as, but not limited to, biotin,
strep-tag, chitin binding protein (CBP), maltose binding protein
(MBP), glutathione-S-transferase (GST), or a poly(His) tag. In some
embodiments, a PLC.gamma.2 polypeptide comprising modifications at
amino acid position 742, 845, or 1140 of the wild-type PLC.gamma.2
polypeptide set forth in SEQ ID NO: 2 linked to a detectable
protein or moiety, such a luminescent, chemiluminescent,
bioluminescent, or fluorescent protein or moiety. In some
embodiments, the fluorescent protein is a green (GFP), red (RFP),
cyan (CFP), yellow (YFP), or blue (BFP) fluorescent protein. In
some embodiments, a PLC.gamma.2 polypeptide comprising
modifications at amino acid position 742, 845, or 1140 of the
wild-type PLC.gamma.2 polypeptide set forth in SEQ ID NO: 2 linked
to an enzyme for example, a luciferase or a beta-galactosidase.
[0067] In some embodiments, provided herein is an array comprising
a mutant PLC.gamma.2 polypeptide provided herein. In some
embodiments, the mutant PLC.gamma.2 polypeptide is bound to a
microchip. In some embodiments, the mutant PLC.gamma.2 polypeptide
is bound directly to the microchip. In some embodiments, the mutant
PLC.gamma.2 polypeptide is bound indirectly to the microchip via a
linker. In some embodiments, provided herein is a microchip array
comprising a mutant PLC.gamma.2 polypeptide provided herein.
[0068] In some embodiments, the mutant PLC.gamma.2 polypeptide
contains one or more amino acid substitutions that confer
resistance to inhibition by a BTK inhibitor. In some embodiments,
the one or more amino acid substitutions comprise the substitution
at amino acid position 742, 845, or 1140. In some embodiments, the
mutant PLC.gamma.2 polypeptide contains one or more amino acid
substitutions that confer resistance to inhibition by a covalent
and/or irreversible BTK inhibitor that is ibrutinib, PCI-45292,
PCI-45466, AVL-101/CC-101 (Avila Therapeutics/Celgene Corporation),
AVL-263/CC-263 (Avila Therapeutics/Celgene Corporation),
AVL-292/CC-292 (Avila Therapeutics/Celgene Corporation),
AVL-291/CC-291 (Avila Therapeutics/Celgene Corporation), CNX 774
(Avila Therapeutics), BMS-488516 (Bristol-Myers Squibb), BMS-509744
(Bristol-Myers Squibb), CGI-1746 (CGI Pharma/Gilead Sciences),
CGI-560 (CGI Pharma/Gilead Sciences), CTA-056, GDC-0834
(Genentech), HY-11066 (also, CTK4I7891, HMS3265G21, HMS3265G22,
HMS3265H21, HMS3265H22, 439574-61-5, AG-F-54930), ONO-4059 (Ono
Pharmaceutical Co., Ltd.), ONO-WG37 (Ono Pharmaceutical Co., Ltd.),
PLS-123 (Peking University), RN486 (Hoffmann-La Roche), HM71224
(Hanmi Pharmaceutical Company Limited), LFM-A13, BGB-3111
(Beigene), KBP-7536 (KBP BioSciences), ACP-196 (Acerta Pharma),
JTE-051 (Japan Tobacco Inc), PRN1008 (Principia), CTP-730 (Concert
Pharmaceuticals), or GDC-0853 (Genentech). In some embodiments, the
mutant PLC.gamma.2 polypeptide contains one or more amino acid
substitutions that confer resistance to inhibition by a covalent
and/or irreversible BTK inhibitor that is ibrutinib, PCI-45292,
PCI-45466, AVL-101, AVL-291, AVL-292, ONO-WG-37 or
(R)-6-amino-9-(1-but-2-ynoylpyrrolidin-3-yl)-7-(4-phenoxyphenyl)-7H-purin-
-8(9H)-one. In some embodiments, the mutant PLC.gamma.2 polypeptide
contain one or more amino acid substitutions with at least one
substitution at amino acid position 742, 845, or 1140 that confer
resistance to inhibition by a covalent and/or irreversible BTK
inhibitor that is ibrutinib, PCI-45292, PCI-45466, AVL-101/CC-101
(Avila Therapeutics/Celgene Corporation), AVL-263/CC-263 (Avila
Therapeutics/Celgene Corporation), AVL-292/CC-292 (Avila
Therapeutics/Celgene Corporation), AVL-291/CC-291 (Avila
Therapeutics/Celgene Corporation), CNX 774 (Avila Therapeutics),
BMS-488516 (Bristol-Myers Squibb), BMS-509744 (Bristol-Myers
Squibb), CGI-1746 (CGI Pharma/Gilead Sciences), CGI-560 (CGI
Pharma/Gilead Sciences), CTA-056, GDC-0834 (Genentech), HY-11066
(also, CTK4I7891, HMS3265G21, HMS3265G22, HMS3265H21, HMS3265H22,
439574-61-5, AG-F-54930), ONO-4059 (Ono Pharmaceutical Co., Ltd.),
ONO-WG37 (Ono Pharmaceutical Co., Ltd.), PLS-123 (Peking
University), RN486 (Hoffmann-La Roche), HM71224 (Hanmi
Pharmaceutical Company Limited), LFM-A13, BGB-3111 (Beigene),
KBP-7536 (KBP BioSciences), ACP-196 (Acerta Pharma), JTE-051 (Japan
Tobacco Inc), PRN1008 (Principia), CTP-730 (Concert
Pharmaceuticals), or GDC-0853 (Genentech). In some embodiments, the
mutant PLC.gamma.2 polypeptide contain one or more amino acid
substitutions with at least one substitution at amino acid position
742, 845, or 1140 that confer resistance to inhibition by a
covalent and/or irreversible BTK inhibitor that is ibrutinib,
PCI-45292, PCI-45466, AVL-101, AVL-291, AVL-292, ONO-WG-37 or
(R)-6-amino-9-(1-but-2-ynoylpyrrolidin-3-yl)-7-(4-phenoxyphenyl)-7H-purin-
-8(9H)-one. In some embodiments, the mutant PLC.gamma.2 polypeptide
containing the substitution at amino acid position 742, 845, or
1140 that confer resistance to inhibition by a covalent and/or
irreversible BTK inhibitor that is ibrutinib, PCI-45292, PCI-45466,
AVL-101/CC-101 (Avila Therapeutics/Celgene Corporation),
AVL-263/CC-263 (Avila Therapeutics/Celgene Corporation),
AVL-292/CC-292 (Avila Therapeutics/Celgene Corporation),
AVL-291/CC-291 (Avila Therapeutics/Celgene Corporation), CNX 774
(Avila Therapeutics), BMS-488516 (Bristol-Myers Squibb), BMS-509744
(Bristol-Myers Squibb), CGI-1746 (CGI Pharma/Gilead Sciences),
CGI-560 (CGI Pharma/Gilead Sciences), CTA-056, GDC-0834
(Genentech), HY-11066 (also, CTK4I7891, HMS3265G21, HMS3265G22,
HMS3265H21, HMS3265H22, 439574-61-5, AG-F-54930), ONO-4059 (Ono
Pharmaceutical Co., Ltd.), ONO-WG37 (Ono Pharmaceutical Co., Ltd.),
PLS-123 (Peking University), RN486 (Hoffmann-La Roche), HM71224
(Hanmi Pharmaceutical Company Limited), LFM-A13, BGB-3111
(Beigene), KBP-7536 (KBP BioSciences), ACP-196 (Acerta Pharma),
JTE-051 (Japan Tobacco Inc), PRN1008 (Principia), CTP-730 (Concert
Pharmaceuticals), or GDC-0853 (Genentech). In some embodiments, the
mutant PLC.gamma.2 polypeptide containing the substitution at amino
acid position 742, 845, or 1140 that confer resistance to
inhibition by a covalent and/or irreversible BTK inhibitor that is
ibrutinib, PCI-45292, PCI-45466, AVL-101, AVL-291, AVL-292,
ONO-WG-37 or
(R)-6-amino-9-(1-but-2-ynoylpyrrolidin-3-yl)-7-(4-phenoxyphenyl)-7H-purin-
-8(9H)-one. In some embodiments, the covalent and/or irreversible
BTK inhibitor is ibrutinib. In some embodiments, the covalent
and/or irreversible BTK inhibitor is
(R)-6-amino-9-(1-but-2-ynoylpyrrolidin-3-yl)-7-(4-phenoxyphenyl)-7H-purin-
-8(9H)-one.
Nucleic Acids Encoding Mutant PLC.gamma.2 Polypeptides
[0069] Provided herein are nucleic acids encoding mutant
PLC.gamma.2 polypeptides. Provided herein are nucleic acids
encoding any of the mutant PLC.gamma.2 polypeptides described
herein. Methods for deducing nucleic acids that encode particular
polypeptides are known in the art and involve standard molecular
biology techniques. Exemplary nucleic acids encoding mutant
PLC.gamma.2 polypeptides provided herein are provided. It is
understood that due to the degeneracy of the genetic code multiple
variants nucleic acids exist that encode the same polypeptide.
Nucleic acids that encode the mutant PLC.gamma.2 polypeptides
provided herein encompass such variants. In some embodiments, the
mutant PLC.gamma.2 nucleic acids are synthetic nucleic acids. In
some embodiments, the mutant PLC.gamma.2 nucleic acids are cDNA
molecules. In some embodiments, the mutant PLC.gamma.2 nucleic
acids do not contain genomic DNA. In some embodiments, the mutant
PLC.gamma.2 nucleic acids are unmethylated. In some embodiments,
the mutant PLC.gamma.2 nucleic acids do not contain PLC.gamma.2
genomic intron sequences. In some embodiments, the mutant
PLC.gamma.2 nucleic acids comprise a sequence of nucleotides from
two or more exons of the PLC.gamma.2 genomic sequence, including
nucleic acid comprising the codon sequence encoding position 742,
845, or 1140 of the PLC.gamma.2 polypeptide. In some embodiments,
the mutant PLC.gamma.2 nucleic acids comprise a sequence of
nucleotides that encode a proline at a position corresponding to
position 742 of the wild-type PLC.gamma.2 polypeptide. In some
embodiments, the mutant PLC.gamma.2 nucleic acids comprise a
sequence of nucleotides that encode a phenylalanine at a position
corresponding to position 845 of the wild-type PLC.gamma.2
polypeptide. In some embodiments, the mutant PLC.gamma.2 nucleic
acids comprise a sequence of nucleotides that encode a glycine at a
position corresponding to position 1140 of the wild-type
PLC.gamma.2 polypeptide.$$
[0070] In some embodiments, the nucleic acid encoding a modified
PLC.gamma.2 polypeptide provided herein is a DNA or an RNA
molecule.
[0071] In some embodiments, the nucleic acid encoding a mutant
PLC.gamma.2 polypeptide comprises a modification where the encoded
polypeptide comprises a substitution of the amino acid proline at a
position corresponding to position 742 of the wild-type PLC.gamma.2
polypeptide set forth in SEQ ID NO: 2. In some embodiments, the
nucleic acid encoding a mutant PLC.gamma.2 polypeptide comprises
one or more modifications where the encoded polypeptide comprises
substitutions at position corresponding to amino acid position 742
and at one or more additional positions of the wild-type
PLC.gamma.2 polypeptide set forth in SEQ ID NO: 2. In some
embodiments, the nucleic acid comprises the sequence of nucleic
acids set forth in SEQ ID NO: 1, wherein the nucleic acid codon
encoding amino acid at position 742 is modified, whereby the codon
does not encode arginine, or a variant that has at least or at
least about 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%
or more sequence identity with the polypeptide having the sequence
set forth in SEQ ID NO: 2.
[0072] In some embodiments, the nucleic acid encoding a mutant
PLC.gamma.2 polypeptide comprises a modification where the encoded
polypeptide comprises a substitution of the amino acid
phenylalanine at a position corresponding to position 845 of the
wild-type PLC.gamma.2 polypeptide set forth in SEQ ID NO: 2. In
some embodiments, the nucleic acid encoding a mutant PLC.gamma.2
polypeptide comprises one or more modifications where the encoded
polypeptide comprises substitutions at position corresponding to
amino acid position 845 and at one or more additional positions of
the wild-type PLC.gamma.2 polypeptide set forth in SEQ ID NO: 2. In
some embodiments, the nucleic acid comprises the sequence of
nucleic acids set forth in SEQ ID NO: 1, wherein the nucleic acid
codon encoding amino acid at position 845 is modified, whereby the
codon does not encode leucine, or a variant that has at least or at
least about 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%
or more sequence identity with the polypeptide having the sequence
set forth in SEQ ID NO: 1.
[0073] In some embodiments, the nucleic acid encoding a mutant
PLC.gamma.2 polypeptide comprises a modification where the encoded
polypeptide comprises a substitution of the amino acid glycine at a
position corresponding to position 1140 of the wild-type
PLC.gamma.2 polypeptide set forth in SEQ ID NO: 2. In some
embodiments, the nucleic acid encoding a mutant PLC.gamma.2
polypeptide comprises one or more modifications where the encoded
polypeptide comprises substitutions at position corresponding to
amino acid position 1140 and at one or more additional positions of
the wild-type PLC.gamma.2 polypeptide set forth in SEQ ID NO: 2. In
some embodiments, the nucleic acid comprises the sequence of
nucleic acids set forth in SEQ ID NO: 1, wherein the nucleic acid
codon encoding amino acid at position 1140 is modified, whereby the
codon does not encode aspartic acid, or a variant that has at least
or at least about 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%,
99% or more sequence identity with the polypeptide having the
sequence set forth in SEQ ID NO: 1.
[0074] In some embodiments the nucleic acid modification is a
missense mutation or a deletion of one or more codons that encode
the PLC.gamma.2 polypeptide. In some embodiments, the modification
is a missense mutation that changes the nucleic acid codon that
encodes arginine at amino position 742 of the PLC.gamma.2
polypeptide. In some embodiments, the modification is a missense
mutation that changes the nucleic acid codon that encodes leucine
at amino position 845 of the PLC.gamma.2 polypeptide. In some
embodiments, the modification is a missense mutation that changes
the nucleic acid codon that encodes aspartic acid at amino position
1140 of the PLC.gamma.2 polypeptide.
[0075] In some embodiments the nucleic acid modification is a frame
shift mutation or a deletion of one or more codons that encode the
PLC.gamma.2 polypeptide. In some embodiments, the modification is a
frame shift mutation that changes the nucleic acid codon that
encodes arginine at amino position 742 of the PLC.gamma.2
polypeptide. In some embodiments, the modification is a missense
mutation that changes the nucleic acid codon that encodes leucine
at amino position 845 of the PLC.gamma.2 polypeptide. In some
embodiments, the modification is a frame shift mutation that
changes the nucleic acid codon that encodes aspartic acid at amino
position 1140 of the PLC.gamma.2 polypeptide.
[0076] In some embodiments, the nucleic acid codon that encodes
arginine at amino position 742 of the PLC.gamma.2 polypeptide is
CGT, CGC, CGA, CGG, AGA or AGG. In some embodiments, the
modification changes the nucleic acid codon that encodes arginine
at amino position 742 of the PLC.gamma.2 polypeptide to a nucleic
acid codon that encodes proline. In some embodiments, the nucleic
acid codon that encodes proline is CCT, CCC, CCA, or CCG.
[0077] In some embodiments, the nucleic acid codon that encodes
leucine at amino position 845 of the PLC.gamma.2 polypeptide is
TTA, TTG, CTT, CTC, CTA or CTG. In some embodiments, the
modification changes the nucleic acid codon that encodes leucine at
amino position 845 of the PLC.gamma.2 polypeptide to a nucleic acid
codon that encodes Phenylalanine. In some embodiments, the nucleic
acid codon that encodes Phenylalanine is TTT or TTC.
[0078] In some embodiments, the nucleic acid codon that encodes
aspartic acid at amino position 1140 of the PLC.gamma.2 polypeptide
is GAT or GAC. In some embodiments, the modification changes the
nucleic acid codon that encodes aspartic acid at amino position
1140 of the PLC.gamma.2 polypeptide to a nucleic acid codon that
encodes glycine. In some embodiments, the nucleic acid codon that
encodes glycine is GGT, GGC, GGA, or GGG.
[0079] In some embodiments, the nucleic acid provided herein
encoding a mutant PLC.gamma.2 polypeptide is an isolated nucleic
acid. In some embodiments, the nucleic acid provided herein
encoding a mutant PLC.gamma.2 polypeptide is a DNA molecule. In
some embodiments, the nucleic acid provided herein encoding a
mutant PLC.gamma.2 polypeptide is a cDNA molecule. In some
embodiments, the nucleic acid provided herein encoding a mutant
PLC.gamma.2 polypeptide is an RNA molecule. In some embodiments,
the nucleic acid provided herein encoding a mutant PLC.gamma.2
polypeptide is an inhibitory RNA molecule (i.e. RNAi). In some
embodiments, the nucleic acid provided herein is a nucleic acid
molecule that is complementary, or binds to, a nucleic acid
encoding a mutant PLC.gamma.2 polypeptide.
[0080] In some embodiments, the nucleic acid provided herein
encoding a mutant PLC.gamma.2 polypeptide encodes a portion of a
mutant PLC.gamma.2 polypeptide provided herein that comprises amino
acid position 742, 845, or 1140. In some embodiments, the nucleic
acid provided herein encoding a mutant PLC.gamma.2 polypeptide
encodes a portion of a mutant PLC.gamma.2 polypeptide provided
herein that comprises amino acid position 742, 845, or 1140. In
some embodiments, the nucleic acid provided herein encoding a
mutant PLC.gamma.2 polypeptide encodes one or more domains of a
mutant PLC.gamma.2 polypeptide provided herein. In some
embodiments, the nucleic acid provided herein encoding a mutant
PLC.gamma.2 polypeptide encodes one or both SH2 domains and SH3
domain of a mutant PLC.gamma.2 polypeptide provided herein.
[0081] In some embodiments, the nucleic acid provided herein
encoding a mutant PLC.gamma.2 polypeptide or a portion thereof
contains nucleic acid encoding an amino acid at position 742 that
is not arginine. In some embodiments, the nucleic acid provided
herein encoding a mutant PLC.gamma.2 polypeptide or a portion
thereof contains nucleic acid encoding proline at amino acid
position 742. In some embodiments, the nucleic acid provided herein
encoding a mutant PLC.gamma.2 polypeptide or a portion thereof
contains nucleic acid encoding amino acids at position 742.
[0082] In some embodiments, the nucleic acid provided herein
encoding a mutant PLC.gamma.2 polypeptide or a portion thereof
contains nucleic acid encoding an amino acid at position 845 that
is not leucine. In some embodiments, the nucleic acid provided
herein encoding a mutant PLC.gamma.2 polypeptide or a portion
thereof contains nucleic acid encoding phenylalanine at amino acid
position 845. In some embodiments, the nucleic acid provided herein
encoding a mutant PLC.gamma.2 polypeptide or a portion thereof
contains nucleic acid encoding amino acids at position 845.
[0083] In some embodiments, the nucleic acid provided herein
encoding a mutant PLC.gamma.2 polypeptide or a portion thereof
contains nucleic acid encoding an amino acid at position 1140 that
is not aspartic acid. In some embodiments, the nucleic acid
provided herein encoding a mutant PLC.gamma.2 polypeptide or a
portion thereof contains nucleic acid encoding glycine at amino
acid position 1140. In some embodiments, the nucleic acid provided
herein encoding a mutant PLC.gamma.2 polypeptide or a portion
thereof contains nucleic acid encoding amino acids at position
1140.
[0084] In some embodiments, the nucleic acid provide herein is an
oligonucleotide that encodes a portion of the mutant PLC.gamma.2
polypeptide. In some embodiments the nucleic acid provided herein
is an oligonucleotide that encodes a portion of the mutant
PLC.gamma.2 polypeptide that contains a nucleotide codon encoding
the amino acid corresponding to amino acid positions 742, 845, or
1140. In some embodiments, the codon encoding the amino acid
corresponding to amino acid position 742 encodes an amino acid that
is not arginine. In some embodiments, the codon encoding the amino
acid corresponding to amino acid position 742 encodes an amino acid
that is proline. In some embodiments, the codon encoding the amino
acid corresponding to amino acid position 845 encodes an amino acid
that is not leucine. In some embodiments, the codon encoding the
amino acid corresponding to amino acid position 845 encodes an
amino acid that is phenylalanine. In some embodiments, the codon
encoding the amino acid corresponding to amino acid position 1140
encodes an amino acid that is not aspartic acid. In some
embodiments, the codon encoding the amino acid corresponding to
amino acid position 1140 encodes an amino acid that is glycine.
[0085] In some embodiments, the nucleic acid provided herein is a
vector that comprises a nucleic acid molecule encoding a modified
PLC.gamma.2 polypeptide provided herein. In some embodiments, the
nucleic acid provided herein is a vector that comprises nucleic
acid encoding a mutant PLC.gamma.2 polypeptide provided herein is
an expression vector. In some embodiments, the nucleic acid
encoding a mutant PLC.gamma.2 polypeptide provided herein is
operably linked to a promoter. In some embodiments, the promoter is
a constitutive or an inducible promoter. In some embodiments,
provided herein is a host cell, comprising the vector or nucleic
acid molecule encoding a modified PLC.gamma.2 polypeptide provided
herein. In some embodiments, the cell is a prokaryotic cell or a
eukaryotic cell. Also provided herein is a mutant PLC.gamma.2
polypeptide expressed by the host cell.
[0086] In some embodiments, the vector is a viral or plasmid
vector. In some embodiments, the viral vector is a DNA or RNA viral
vector. Exemplary viral vectors include, but are not limited to, a
vaccinia, adenovirus, adeno-associated virus (AAV), retrovirus, or
herpesvirus vector.
[0087] In some embodiments, provided herein is an array comprising
a nucleic acid encoding any of the mutant PLC.gamma.2 polypeptides
provided herein. In some embodiments, the mutant PLC.gamma.2
nucleic acid is bound to a microchip. In some embodiments, the
mutant PLC.gamma.2 nucleic acid is bound directly to the microchip.
In some embodiments, the mutant PLC.gamma.2 nucleic acid is bound
indirectly to the microchip via a linker. In some embodiments,
provided herein is a microchip array comprising a nucleic acid
encoding any of the mutant PLC.gamma.2 polypeptides provided
herein.
Diagnostic Methods
[0088] Described herein, in certain embodiments, are diagnostic
methods that involve the detection of a mutant PLC.gamma.2
polypeptide in a subject or a nucleic acid encoding a mutant
PLC.gamma.2 polypeptide in a subject. In some embodiments, the
subject has a BTK-mediated disease or condition. In some
embodiments, the BTK-mediated disease or condition is a B-cell
cancer. In some embodiments, the diagnostic methods are employed
for the screening of subjects having a B-cell cancer that is
resistant to therapy with a covalent and/or irreversible BTK
inhibitor, identifying subjects for the treatment with a covalent
and/or irreversible BTK inhibitor, identifying subjects as likely
or unlikely to respond to treatment with a covalent and/or
irreversible BTK inhibitor, predicting whether a subject is likely
to develop resistance to treatment with a covalent and/or
irreversible BTK inhibitor, monitoring the therapy of subjects
receiving therapy with a covalent and/or irreversible BTK
inhibitor, optimizing the therapy of subjects receiving a covalent
and/or irreversible BTK inhibitor therapy, and any combinations
thereof. In some embodiments, the diagnostic methods involve the
detection of a mutant PLC.gamma.2 polypeptide. In some embodiments,
the methods comprise selecting a subject for therapy with an
inhibitor of PLC.gamma.2. In some embodiments, the methods further
comprise administering to the subject an inhibitor of PLC.gamma.2
that inhibits the mutant PLC.gamma.2. In some embodiments, the
PLC.gamma.2 modification confers resistance of a cancer cell to
treatment with a covalent and/or irreversible BTK inhibitor. In
some embodiments, the patient exhibits one or more symptoms of a
relapsed or refractory cancer. In some embodiments, the relapsed or
refractory cancer is a relapsed or refractory non-Hodgkin's
lymphoma. In some embodiments, the relapsed or refractory cancer is
a relapsed or refractory chronic lymphocytic leukemia (CLL), small
lymphocytic lymphoma (SLL), diffuse large B-cell lymphoma (DLBCL),
activated B-cell diffuse large B-cell lymphoma (ABC-DLBCL),
germinal center diffuse large B-cell lymphoma (GCB DLBCL),
double-hit diffuse large B-cell lymphoma (DH-DLBCL), primary
mediastinal B-cell lymphoma (PMBL), non-Hodgkin lymphoma, Burkitt's
lymphoma, follicular lymphoma, immunoblastic large cell lymphoma,
precursor B-lymphoblastic lymphoma, precursor B-cell acute
lymphoblastic leukemia, hairy cell leukemia, mantle cell lymphoma,
B cell prolymphocytic leukemia, lymphoplasmacytic
lymphoma/Waldenstrom macroglobulinemia, splenic marginal zone
lymphoma, plasma cell myeloma, plasmacytoma, extranodal marginal
zone B cell lymphoma, nodal marginal zone B cell lymphoma,
mediastinal (thymic) large B cell lymphoma, intravascular large B
cell lymphoma, primary effusion lymphoma, or lymphomatoid
granulomatosis. In some embodiments, the patient exhibits one or
more symptoms of Richter's transformation.
[0089] In some embodiments, provided is a method of assessing
whether a subject is less responsive or likely to become less
responsive to therapy with a BTK inhibitor, comprising: (a) testing
a sample containing a nucleic acid molecule encoding a PLC.gamma.2
polypeptide from the subject; (b) determining whether the encoded
PLC.gamma.2 polypeptide is modified at an amino acid position
corresponding to amino acid position 742, 845, or 1140 of the amino
acid sequence set forth in SEQ ID NO: 2; and (c) characterizing the
subject as resistant or likely to become resistant to therapy with
a BTK inhibitor if the subject has the modification at amino acid
position 742, 845, or 1140. In some embodiments, the modification
is R742P. In some embodiments, the modification is L845F. In some
embodiments, the modification is D1140G. In some embodiments, the
subject has been administered a covalent and/or irreversible BTK
inhibitor for the treatment of a cancer. In some embodiments, the
method further comprises determining whether the encoded
PLC.gamma.2 polypeptide is modified at one or more additional amino
acid positions. In some embodiments, the method further comprises
testing a sample and determining the presence of mutations in
PLC.gamma.2 and an additional polypeptide. In some embodiments, the
additional polypeptide is a polypeptide that encoded by a gene
associated in the BCR pathway. In some embodiments, the method
further comprises discontinuing treatment with the BTK inhibitor if
the subject has a modification at amino acid position 742, 845, or
1140 in the PLC.gamma.2 polypeptide. In some embodiments, the
method further comprises discontinuing treatment with the BTK
inhibitor if the subject has one or more modifications with at
least one modification at amino acid position 742, 845, or 1140 in
the PLC.gamma.2 polypeptide. In some embodiments, the method
further comprises discontinuing treatment with the BTK inhibitor if
the subject has one or more modifications with at least one
modification at amino acid positions 742, 845, or 1140 in the
PLC.gamma.2 polypeptide and modifications in an additional
polypeptide. In some embodiments, the method further comprises
discontinuing treatment with the BTK inhibitor if the subject has
no modification at amino acid position 742, 845, or 1140 in the
PLC.gamma.2 polypeptide but has additional modifications in the
PLC.gamma.2 polypeptide and/or has modifications in an additional
polypeptide. In some embodiments, the method further comprises
administering an inhibitor of PLC.gamma.2 if the subject has one or
more modifications with at least one modification at amino acid
position 742, 845, or 1140 in the PLC.gamma.2 polypeptide. In some
embodiments, the method further comprises administering an
inhibitor of LYN, SYK, JAK, PI3K, MAPK, MEK or NF.kappa.B if the
subject has one or more modifications with at least one
modification at amino acid position 742, 845, or 1140 in
PLC.gamma.2 polypeptide. In some embodiments, the method further
comprises continuing treatment with the covalent and/or
irreversible BTK inhibitor if the subject does not have
modifications in the PLC.gamma.2 polypeptide. In some embodiments,
the method further comprises continuing treatment with the covalent
and/or irreversible BTK inhibitor if the subject does not have
modifications in the PLC.gamma.2 polypeptide but has modifications
in an additional polypeptide. In some embodiments, the method
further comprises continuing treatment with the covalent and/or
irreversible BTK inhibitor if the subject does not have
modifications in the PLC.gamma.2 and an additional polypeptide. In
some embodiments, the additional polypeptide is a BTK polypeptide.
In some embodiments, the subject possesses high-risk cytogenetic
features. In some embodiments, the high-risk cytogenetic features
comprise del(11q22.3), del(17p13.1) or complex karyotype. In some
embodiments, the subject has a hematologic cancer or a B-cell
malignancy. In some embodiments, the cancer is selected from among
a leukemia, a lymphoma or a myeloma. In some embodiments, the
B-cell malignancy is CLL. In some embodiments, the subject has
lymphocytosis. In some embodiments, the subject has prolonged
lymphocytosis. In some embodiments, the subject with prolonged
lymphocytosis does not have the 742, 845, or 1140 mutation in the
PLC.gamma.2 polypeptide. In some embodiments, the patient exhibits
one or more symptoms of a relapsed or refractory cancer. In some
embodiments, the relapsed or refractory cancer is a relapsed or
refractory non-Hodgkin's lymphoma. In some embodiments, the
relapsed or refractory cancer is a relapsed or refractory chronic
lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL),
diffuse large B-cell lymphoma (DLBCL), activated B-cell diffuse
large B-cell lymphoma (ABC-DLBCL), germinal center diffuse large
B-cell lymphoma (GCB DLBCL), double-hit diffuse large B-cell
lymphoma (DH-DLBCL), primary mediastinal B-cell lymphoma (PMBL),
non-Hodgkin lymphoma, Burkitt's lymphoma, follicular lymphoma,
immunoblastic large cell lymphoma, precursor B-lymphoblastic
lymphoma, precursor B-cell acute lymphoblastic leukemia, hairy cell
leukemia, mantle cell lymphoma, B cell prolymphocytic leukemia,
lymphoplasmacytic lymphoma/Waldenstrom macroglobulinemia, splenic
marginal zone lymphoma, plasma cell myeloma, plasmacytoma,
extranodal marginal zone B cell lymphoma, nodal marginal zone B
cell lymphoma, mediastinal (thymic) large B cell lymphoma,
intravascular large B cell lymphoma, primary effusion lymphoma, or
lymphomatoid granulomatosis. In some embodiments, the patient
exhibits one or more symptoms of Richter's transformation.
[0090] In some embodiments, provided is a method of monitoring
whether a subject receiving a BTK inhibitor for treatment of a
cancer has developed or is likely to develop resistance to the
therapy, comprising: (a) testing a sample containing a nucleic acid
molecule encoding a PLC.gamma.2 polypeptide from the subject; (b)
determining whether the encoded PLC.gamma.2 polypeptide is modified
at an amino acid position corresponding to amino acid position 742,
845, or 1140 of the amino acid sequence set forth in SEQ ID NO: 2;
and (c) characterizing the subject as resistant or is likely to
become resistant to therapy with a BTK inhibitor if the subject has
the modification at amino acid position 742, 845, or 1140. In some
embodiments, the modification is R742P. In some embodiments, the
modification is L845F. In some embodiments, the modification is
D1140G. In some embodiments, the method further comprises
determining whether the encoded PLC.gamma.2 polypeptide is modified
at one or more additional amino acid positions. In some
embodiments, the method further comprises testing a sample and
determining the presence of mutations in PLC.gamma.2 and an
additional polypeptide. In some embodiments, the additional
polypeptide is a polypeptide that encoded by a gene associated in
the BCR pathway. In some embodiments, the method further comprises
discontinuing treatment with the BTK inhibitor if the subject has a
modification at amino acid position 742, 845, or 1140 in the
PLC.gamma.2 polypeptide. In some embodiments, the method further
comprises discontinuing treatment with the BTK inhibitor if the
subject has one or more modifications at amino acid positions with
at least one modification at amino acid position 742, 845, or 1140
in the PLC.gamma.2 polypeptide. In some embodiments, the method
further comprises discontinuing treatment with the BTK inhibitor if
the subject has one or more modifications at amino acid positions
in the PLC.gamma.2 polypeptide with at least one modification at
amino acid position 742, 845, or 1140 and modifications in an
additional polypeptide. In some embodiments, the method further
comprises discontinuing treatment with the BTK inhibitor if the
subject has no modifications at amino acid position 742, 845, or
1140 in the PLC.gamma.2 polypeptide but has additional
modifications in the PLC.gamma.2 polypeptide and/or has
modifications in an additional polypeptide. In some embodiments,
the method further comprises administering an inhibitor of
PLC.gamma.2 if the subject has one or more modifications with at
least one modification at amino acid position 742, 845, or 1140 in
the PLC.gamma.2 polypeptide. In some embodiments, the method
further comprises administering an inhibitor of LYN, SYK, JAK,
PI3K, MAPK, MEK or NF.kappa.B if the subject has one or more
modifications with at least one modification at amino acid position
742, 845, or 1140 in the PLC.gamma.2 polypeptide. In some
embodiments, the method further comprises continuing treatment with
the covalent and/or irreversible BTK inhibitor if the subject does
not have modifications in the PLC.gamma.2 polypeptide. In some
embodiments, the method further comprises continuing treatment with
the covalent and/or irreversible BTK inhibitor if the subject does
not have modifications in the PLC.gamma.2 polypeptide but has
modifications in an additional polypeptide. In some embodiments,
the method further comprises continuing treatment with the covalent
and/or irreversible BTK inhibitor if the subject does not have
modifications in the PLC.gamma.2 and an additional polypeptide. In
some embodiments, the additional polypeptide is a BTK polypeptide.
In some embodiments, the subject possesses high-risk cytogenetic
features. In some embodiments, the high-risk cytogenetic features
comprise del(11q22.3), del(17p13.1) or complex karyotype. In some
embodiments, the subject has a hematologic cancer or a B-cell
malignancy. In some embodiments, the cancer is selected from among
a leukemia, a lymphoma or a myeloma. In some embodiments, the
B-cell malignancy is CLL. In some embodiments, the subject has
lymphocytosis. In some embodiments, the subject has prolonged
lymphocytosis. In some embodiments, the subject with prolonged
lymphocytosis does not have mutations in the PLC.gamma.2
polypeptide. In some embodiments, the patient exhibits one or more
symptoms of a relapsed or refractory cancer. In some embodiments,
the relapsed or refractory cancer is a relapsed or refractory
non-Hodgkin's lymphoma. In some embodiments, the relapsed or
refractory cancer is a relapsed or refractory chronic lymphocytic
leukemia (CLL), small lymphocytic lymphoma (SLL), diffuse large
B-cell lymphoma (DLBCL), activated B-cell diffuse large B-cell
lymphoma (ABC-DLBCL), germinal center diffuse large B-cell lymphoma
(GCB DLBCL), double-hit diffuse large B-cell lymphoma (DH-DLBCL),
primary mediastinal B-cell lymphoma (PMBL), non-Hodgkin lymphoma,
Burkitt's lymphoma, follicular lymphoma, immunoblastic large cell
lymphoma, precursor B-lymphoblastic lymphoma, precursor B-cell
acute lymphoblastic leukemia, hairy cell leukemia, mantle cell
lymphoma, B cell prolymphocytic leukemia, lymphoplasmacytic
lymphoma/Waldenstrom macroglobulinemia, splenic marginal zone
lymphoma, plasma cell myeloma, plasmacytoma, extranodal marginal
zone B cell lymphoma, nodal marginal zone B cell lymphoma,
mediastinal (thymic) large B cell lymphoma, intravascular large B
cell lymphoma, primary effusion lymphoma, or lymphomatoid
granulomatosis. In some embodiments, the patient exhibits one or
more symptoms of Richter's transformation.
[0091] In some embodiments, provided herein is a method of
optimizing the therapy of a subject receiving a BTK inhibitor for
treatment of a cancer, comprising: (a) testing a sample containing
a nucleic acid molecule encoding a PLC.gamma.2 polypeptide from the
subject; and (b) determining whether the encoded PLC.gamma.2
polypeptide is modified at an amino acid position corresponding to
amino acid position 742, 845, or 1140 of the amino acid sequence
set forth in SEQ ID NO: 2. In some embodiments, the modification is
R742P. In some embodiments, the modification is L845F. In some
embodiments, the modification is D1140G. In some embodiments, the
method further comprises determining whether the encoded
PLC.gamma.2 polypeptide is modified at additional amino acid
positions. In some embodiments, the method further comprises
testing a sample and determining the presence of mutations in
PLC.gamma.2 and an additional polypeptide. In some embodiments, the
additional polypeptide is a polypeptide that encoded by a gene
associated in the BCR pathway. In some embodiments, the method
further comprises discontinuing treatment with the BTK inhibitor if
the subject has a modification at amino acid position 742, 845, or
1140 in the PLC.gamma.2 polypeptide. In some embodiments, the
method further comprises discontinuing treatment with the BTK
inhibitor if the subject has one or more modifications with at
least one modification at amino acid position 742, 845, or 1140 in
the PLC.gamma.2 polypeptide and modifications in an additional
polypeptide. In some embodiments, the method further comprises
discontinuing treatment with the BTK inhibitor if the subject has
no modification at amino acid position 742, 845, or 1140 in the
PLC.gamma.2 polypeptide but has additional modifications in the
PLC.gamma.2 polypeptide and/or has modifications in an additional
polypeptide. In some embodiments, the method further comprises
administering an inhibitor of PLC.gamma.2 if the subject has one or
more modifications with at least one modification at amino acid
position 742, 845, or 1140 in the PLC.gamma.2 polypeptide. In some
embodiments, the method further comprises administering an
inhibitor of LYN, SYK, JAK, PI3K, MAPK, MEK or NF.kappa.B if the
subject has one or more modifications with at least one
modification at amino acid position 742, 845, or 1140 in the
PLC.gamma.2 polypeptide. In some embodiments, the method further
comprises continuing treatment with the covalent and/or
irreversible BTK inhibitor if the subject does not have
modifications in the PLC.gamma.2 polypeptide. In some embodiments,
the method further comprises continuing treatment with the covalent
and/or irreversible BTK inhibitor if the subject does not have
modifications in the PLC.gamma.2 polypeptide but has modifications
in an additional polypeptide. In some embodiments, the method
further comprises continuing treatment with the covalent and/or
irreversible BTK inhibitor if the subject does not have
modifications in the PLC.gamma.2 and an additional polypeptide. In
some embodiments, the additional polypeptide is a BTK polypeptide.
In some embodiments, the subject possesses high-risk cytogenetic
features. In some embodiments, the high-risk cytogenetic features
comprise del(11q22.3), del(17p13.1) or complex karyotype. In some
embodiments, the subject has a hematologic cancer or a B-cell
malignancy. In some embodiments, the cancer is selected from among
a leukemia, a lymphoma or a myeloma. In some embodiments, the
B-cell malignancy is CLL. In some embodiments, the subject has
lymphocytosis. In some embodiments, the subject has prolonged
lymphocytosis. In some embodiments, the subject with prolonged
lymphocytosis does not have mutations in the PLC.gamma.2
polypeptide. In some embodiments, the patient exhibits one or more
symptoms of a relapsed or refractory cancer. In some embodiments,
the relapsed or refractory cancer is a relapsed or refractory
non-Hodgkin's lymphoma. In some embodiments, the relapsed or
refractory cancer is a relapsed or refractory chronic lymphocytic
leukemia (CLL), small lymphocytic lymphoma (SLL), diffuse large
B-cell lymphoma (DLBCL), activated B-cell diffuse large B-cell
lymphoma (ABC-DLBCL), germinal center diffuse large B-cell lymphoma
(GCB DLBCL), double-hit diffuse large B-cell lymphoma (DH-DLBCL),
primary mediastinal B-cell lymphoma (PMBL), non-Hodgkin lymphoma,
Burkitt's lymphoma, follicular lymphoma, immunoblastic large cell
lymphoma, precursor B-lymphoblastic lymphoma, precursor B-cell
acute lymphoblastic leukemia, hairy cell leukemia, mantle cell
lymphoma, B cell prolymphocytic leukemia, lymphoplasmacytic
lymphoma/Waldenstrom macroglobulinemia, splenic marginal zone
lymphoma, plasma cell myeloma, plasmacytoma, extranodal marginal
zone B cell lymphoma, nodal marginal zone B cell lymphoma,
mediastinal (thymic) large B cell lymphoma, intravascular large B
cell lymphoma, primary effusion lymphoma, or lymphomatoid
granulomatosis. In some embodiments, the patient exhibits one or
more symptoms of Richter's transformation.
[0092] In some embodiments, provided is a method of assessing
whether a subject who possess high-risk cytogenetic features is
less responsive or likely to become less responsive to therapy with
a BTK inhibitor, comprising: (a) testing a sample containing a
nucleic acid molecule encoding a PLC.gamma.2 polypeptide from the
subject; (b) determining whether the encoded PLC.gamma.2
polypeptide is modified at amino acid position corresponding to
amino acid position 742, 845, or 1140 of the amino acid sequence
set forth in SEQ ID NO: 2; and (c) characterizing the subject as
resistant or likely to become resistant to therapy with a BTK
inhibitor if the subject has the modification. In some embodiments,
the method further comprises testing a sample and determining the
presence of additional mutations in the PLC.gamma.2 polypeptide. In
some embodiments, the method further comprises testing a sample and
determining the presence of mutations in PLC.gamma.2 and an
additional polypeptide. In some embodiments, the additional
polypeptide is a polypeptide that encoded by a gene associated in
the BCR pathway. In some embodiments, the method further comprises
discontinuing treatment with the BTK inhibitor if the subject has a
modification at amino acid position 742, 845, or 1140 in the
PLC.gamma.2 polypeptide. In some embodiments, the method further
comprises discontinuing treatment with the BTK inhibitor if the
subject has one or more modifications with at least one
modification at amino acid position 742, 845, or 1140 in the
PLC.gamma.2 polypeptide. In some embodiments, the method further
comprises discontinuing treatment with the BTK inhibitor if the
subject has one or more modifications in the PLC.gamma.2
polypeptide and modifications in an additional polypeptide. In some
embodiments, the method further comprises discontinuing treatment
with the BTK inhibitor if the subject has no modification at amino
acid position 742, 845, or 1140 in the PLC.gamma.2 polypeptide but
has additional modifications in the PLC.gamma.2 polypeptide and/or
has modifications in an additional polypeptide. In some
embodiments, the method further comprises administering an
inhibitor of PLC.gamma.2 if the subject has one or more
modifications with at least one modification at amino acid position
742, 845, or 1140 in the PLC.gamma.2 polypeptide. In some
embodiments, the method further comprises administering an
inhibitor of LYN, SYK, JAK, PI3K, MAPK, MEK or NF.kappa.B if the
subject has one or more modifications with at least one
modification at amino acid position 742, 845, or 1140 in
PLC.gamma.2 and/or BTK polypeptide. In some embodiments, the method
further comprises continuing treatment with the covalent and/or
irreversible BTK inhibitor if the subject does not have
modifications in the PLC.gamma.2 polypeptide. In some embodiments,
the method further comprises continuing treatment with the covalent
and/or irreversible BTK inhibitor if the subject does not have
modifications in the PLC.gamma.2 but has modifications in an
additional polypeptide. In some embodiments, the method further
comprises continuing treatment with the covalent and/or
irreversible BTK inhibitor if the subject does not have
modifications in the PLC.gamma.2 and an additional polypeptide. In
some embodiments, the additional polypeptide is a BTK polypeptide.
In some embodiments, the subject has a hematologic cancer or a
B-cell malignancy. In some embodiments, the cancer is selected from
among a leukemia, a lymphoma or a myeloma. In some embodiments, the
B-cell malignancy is CLL. In some embodiments, the subject has
lymphocytosis. In some embodiments, the subject has prolonged
lymphocytosis. In some embodiments, the subject with prolonged
lymphocytosis does not have mutations in the PLC.gamma.2
polypeptide. In some embodiments, the patient exhibits one or more
symptoms of a relapsed or refractory cancer. In some embodiments,
the relapsed or refractory cancer is a relapsed or refractory
non-Hodgkin's lymphoma. In some embodiments, the relapsed or
refractory cancer is a relapsed or refractory chronic lymphocytic
leukemia (CLL), small lymphocytic lymphoma (SLL), diffuse large
B-cell lymphoma (DLBCL), activated B-cell diffuse large B-cell
lymphoma (ABC-DLBCL), germinal center diffuse large B-cell lymphoma
(GCB DLBCL), double-hit diffuse large B-cell lymphoma (DH-DLBCL),
primary mediastinal B-cell lymphoma (PMBL), non-Hodgkin lymphoma,
Burkitt's lymphoma, follicular lymphoma, immunoblastic large cell
lymphoma, precursor B-lymphoblastic lymphoma, precursor B-cell
acute lymphoblastic leukemia, hairy cell leukemia, mantle cell
lymphoma, B cell prolymphocytic leukemia, lymphoplasmacytic
lymphoma/Waldenstrom macroglobulinemia, splenic marginal zone
lymphoma, plasma cell myeloma, plasmacytoma, extranodal marginal
zone B cell lymphoma, nodal marginal zone B cell lymphoma,
mediastinal (thymic) large B cell lymphoma, intravascular large B
cell lymphoma, primary effusion lymphoma, or lymphomatoid
granulomatosis. In some embodiments, the patient exhibits one or
more symptoms of Richter's transformation.
[0093] In some embodiments, provided is a method of monitoring
whether a subject who possess high-risk cytogenetic features during
the course of a therapy with a BTK inhibitor has developed or is
likely to develop resistance to the therapy, comprising: (a)
testing a sample containing a nucleic acid molecule encoding a BTK
polypeptide and a nucleic acid molecule encoding a PLC.gamma.2
polypeptide from the subject; (b) determining whether the encoded
PLC.gamma.2 polypeptide is modified at the amino acid position
corresponding to amino acid position 742, 845, or 1140 of the amino
acid sequence set forth in SEQ ID NO: 2; and (c) characterizing the
subject as resistant or likely to become resistant to therapy with
a BTK inhibitor if the subject has the modification. In some
embodiments, the method further comprises testing a sample and
determining the presence of additional mutations in the PLC.gamma.2
polypeptide. In some embodiments, the method further comprises
testing a sample and determining the presence of mutations in
PLC.gamma.2 and an additional polypeptide. In some embodiments, the
additional polypeptide is a polypeptide that encoded by a gene
associated in the BCR pathway. In some embodiments, the method
further comprises discontinuing treatment with the BTK inhibitor if
the subject has the modification at amino acid position 742, 845,
or 1140 in the PLC.gamma.2 polypeptide. In some embodiments, the
method further comprises discontinuing treatment with the BTK
inhibitor if the subject has one or more modifications with at
least one modification at amino acid position 742, 845, or 1140 in
the PLC.gamma.2 polypeptide and modifications in an additional
polypeptide. In some embodiments, the method further comprises
discontinuing treatment with the BTK inhibitor if the subject has
no modification at amino acid position 742, 845, or 1140 in the
PLC.gamma.2 polypeptide but has additional modifications in the
PLC.gamma.2 polypeptide and/or has modifications in an additional
polypeptide. In some embodiments, the method further comprises
administering an inhibitor of PLC.gamma.2 if the subject has one or
more modifications with at least one modification at amino acid
position 742, 845, or 1140 in the PLC.gamma.2 polypeptide. In some
embodiments, the method further comprises administering an
inhibitor of LYN, SYK, JAK, PI3K, MAPK, MEK or NF.kappa.B if the
subject has one or more modifications with at least one
modification at amino acid position 742, 845, or 1140 in
PLC.gamma.2 and/or BTK polypeptide. In some embodiments, the method
further comprises continuing treatment with the covalent and/or
irreversible BTK inhibitor if the subject does not have
modifications in the PLC.gamma.2 polypeptide. In some embodiments,
the method further comprises continuing treatment with the covalent
and/or irreversible BTK inhibitor if the subject does not have
modifications in the PLC.gamma.2 polypeptide but has modifications
in an additional polypeptide. In some embodiments, the method
further comprises continuing treatment with the covalent and/or
irreversible BTK inhibitor if the subject does not have
modifications in the PLC.gamma.2 polypeptide and an additional
polypeptide. In some embodiments, the additional polypeptide is a
BTK polypeptide. In some embodiments, the subject has a hematologic
cancer or a B-cell malignancy. In some embodiments, the cancer is
selected from among a leukemia, a lymphoma or a myeloma. In some
embodiments, the B-cell malignancy is CLL. In some embodiments, the
subject has lymphocytosis. In some embodiments, the subject has
prolonged lymphocytosis. In some embodiments, the subject with
prolonged lymphocytosis does not have mutations in the PLC.gamma.2
polypeptide. In some embodiments, the patient exhibits one or more
symptoms of a relapsed or refractory cancer. In some embodiments,
the relapsed or refractory cancer is a relapsed or refractory
non-Hodgkin's lymphoma. In some embodiments, the relapsed or
refractory cancer is a relapsed or refractory chronic lymphocytic
leukemia (CLL), small lymphocytic lymphoma (SLL), diffuse large
B-cell lymphoma (DLBCL), activated B-cell diffuse large B-cell
lymphoma (ABC-DLBCL), germinal center diffuse large B-cell lymphoma
(GCB DLBCL), double-hit diffuse large B-cell lymphoma (DH-DLBCL),
primary mediastinal B-cell lymphoma (PMBL), non-Hodgkin lymphoma,
Burkitt's lymphoma, follicular lymphoma, immunoblastic large cell
lymphoma, precursor B-lymphoblastic lymphoma, precursor B-cell
acute lymphoblastic leukemia, hairy cell leukemia, mantle cell
lymphoma, B cell prolymphocytic leukemia, lymphoplasmacytic
lymphoma/Waldenstrom macroglobulinemia, splenic marginal zone
lymphoma, plasma cell myeloma, plasmacytoma, extranodal marginal
zone B cell lymphoma, nodal marginal zone B cell lymphoma,
mediastinal (thymic) large B cell lymphoma, intravascular large B
cell lymphoma, primary effusion lymphoma, or lymphomatoid
granulomatosis. In some embodiments, the patient exhibits one or
more symptoms of Richter's transformation.
[0094] In some embodiments, provided is a method of optimizing the
therapy with a BTK inhibitor of a subject who possess high-risk
cytogenetic features, comprising: (a) testing a sample containing a
nucleic acid molecule encoding a PLC.gamma.2 polypeptide from the
subject; (b) determining whether the encoded PLC.gamma.2
polypeptide is modified at amino acid position corresponding to
amino acid position 742, 845, or 1140 of the amino acid sequence
set forth in SEQ ID NO: 2; and (c) discontinuing treatment with the
BTK inhibitor if the subject has the modification or continuing
treatment with the BTK inhibitor if the subject does not have the
modification in the PLC.gamma.2 polypeptide. In some embodiments,
the method further comprises testing a sample and determining the
presence of additional mutations in the PLC.gamma.2 polypeptide. In
some embodiments, the method further comprises testing a sample and
determining the presence of mutations in the PLC.gamma.2
polypeptide and an additional polypeptide. In some embodiments, the
additional polypeptide is a polypeptide that encoded by a gene
associated in the BCR pathway. In some embodiments, the method
further comprises discontinuing treatment with the BTK inhibitor if
the subject has the modification at amino acid position 742, 845,
or 1140 in the PLC.gamma.2 polypeptide. In some embodiments, the
method further comprises discontinuing treatment with the BTK
inhibitor if the subject has one or more modifications with at
least one modification at amino acid position 742, 845, or 1140 in
the PLC.gamma.2 polypeptide and modifications in an additional
polypeptide. In some embodiments, the method further comprises
discontinuing treatment with the BTK inhibitor if the subject has
no modifications at amino acid position 742, 845, or 1140 in the
PLC.gamma.2 polypeptide but has additional modifications in the
PLC.gamma.2 polypeptide and/or has modifications in an additional
polypeptide. In some embodiments, the method further comprises
administering an inhibitor of PLC.gamma.2 if the subject has one or
more modifications with at least one modification at amino acid
position 742, 845, or 1140 in the PLC.gamma.2 polypeptide. In some
embodiments, the method further comprises administering an
inhibitor of LYN, SYK, JAK, PI3K, MAPK, MEK or NF.kappa.B if the
subject has one or more modifications with at least one
modification at amino acid position 742, 845, or 1140 in
PLC.gamma.2 and/or BTK polypeptide. In some embodiments, the method
further comprises continuing treatment with the covalent and/or
irreversible BTK inhibitor if the subject does not have
modifications in the PLC.gamma.2 polypeptide. In some embodiments,
the method further comprises continuing treatment with the covalent
and/or irreversible BTK inhibitor if the subject does not have
modifications in the PLC.gamma.2 polypeptide but has modifications
in an additional polypeptide. In some embodiments, the method
further comprises continuing treatment with the covalent and/or
irreversible BTK inhibitor if the subject does not have
modifications in the PLC.gamma.2 polypeptide and an additional
polypeptide. In some embodiments, the additional polypeptide is a
BTK polypeptide. In some embodiments, the subject has a hematologic
cancer or a B-cell malignancy. In some embodiments, the cancer is
selected from among a leukemia, a lymphoma or a myeloma. In some
embodiments, the B-cell malignancy is CLL. In some embodiments, the
subject has lymphocytosis. In some embodiments, the subject has
prolonged lymphocytosis. In some embodiments, the subject with
prolonged lymphocytosis does not have mutations in the PLC.gamma.2
polypeptide. In some embodiments, the patient exhibits one or more
symptoms of a relapsed or refractory cancer. In some embodiments,
the relapsed or refractory cancer is a relapsed or refractory
non-Hodgkin's lymphoma. In some embodiments, the relapsed or
refractory cancer is a relapsed or refractory chronic lymphocytic
leukemia (CLL), small lymphocytic lymphoma (SLL), diffuse large
B-cell lymphoma (DLBCL), activated B-cell diffuse large B-cell
lymphoma (ABC-DLBCL), germinal center diffuse large B-cell lymphoma
(GCB DLBCL), double-hit diffuse large B-cell lymphoma (DH-DLBCL),
primary mediastinal B-cell lymphoma (PMBL), non-Hodgkin lymphoma,
Burkitt's lymphoma, follicular lymphoma, immunoblastic large cell
lymphoma, precursor B-lymphoblastic lymphoma, precursor B-cell
acute lymphoblastic leukemia, hairy cell leukemia, mantle cell
lymphoma, B cell prolymphocytic leukemia, lymphoplasmacytic
lymphoma/Waldenstrom macroglobulinemia, splenic marginal zone
lymphoma, plasma cell myeloma, plasmacytoma, extranodal marginal
zone B cell lymphoma, nodal marginal zone B cell lymphoma,
mediastinal (thymic) large B cell lymphoma, intravascular large B
cell lymphoma, primary effusion lymphoma, or lymphomatoid
granulomatosis. In some embodiments, the patient exhibits one or
more symptoms of Richter's transformation.
[0095] In some embodiments, the subject possesses cytogenetic
features. In some embodiments, the cytogenetic features is further
categorized as low-risk or favorable, intermediate or high-risk or
unfavorable cytogenetic features. In some embodiments, the subject
possesses high-risk cytogenetic features. In some embodiments,
cytogenetic features are associated with cytogenetic abnormalities.
In some embodiments, high-risk cytogenetic features are associated
with cytogenetic abnormalities. In some embodiments, the subject
possessing high-risk cytogenetic features have cytogenetic
abnormalities.
[0096] In some embodiments, cytogenetic abnormalities are
associated with aberrant chromosomes or aberrant chromosome number.
In some embodiments, aberrant chromosomes refer to chromosomes
comprising deletion, duplication, inversion, insertion,
translocation or any combinations thereof. In some embodiments,
aberrant chromosome number refers to addition or deletion of a
chromosome. In some embodiments, multiple cytogenetic abnormalities
are associated with aberrant chromosomes or chromosome numbers. In
some embodiments, the multiple cytogenetic abnormalities are
referred to as a complex karyotype. In some embodiments, the
complex karyotype comprises about 2, 3, 4, 5, 6, 7, 8, 9 10 or more
cytogenetic abnormalities. In some embodiments, the cytogenetic
abnormalities occurs on chromosome 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, X, Y or any
combinations thereof.
[0097] In some embodiments, cytogenetic abnormalities result in
gene alterations. In some embodiments, gene alterations comprise
insertion, deletion or substitution of one or more amino acids. In
some embodiments, gene alterations results in mutations. In some
embodiments, mutations comprise nonsense mutation, missense
mutation, silent mutation, frameshift mutation, dynamic mutation or
any combinations thereof. In some embodiments, the mutations occur
on chromosome 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,
16, 17, 18, 19, 20, 21, 22, X, Y or any combinations thereof.
[0098] In some embodiments, different cancers are associated with
different cytogenetic abnormalities. In some embodiments, a subject
who has a particular cancer is associated with a particular set of
cytogenetic abnormalities. In some embodiments, a subject who has
high-risk cytogenetic features has a particular set of cytogenetic
abnormalities. In some embodiments, the cancer is a hematologic
cancer or a B-cell malignancy. In some embodiments, the cancer is
selected from among a leukemia, a lymphoma or a myeloma. In some
embodiments, the B-cell malignancy is CLL. In some embodiments,
cytogenetic abnormalities associated with CLL comprise trisomy 12,
del(11q22.3), del(13q14.3), del(17p13.1), t(11;14)(q13;q32),
t(14;19)(q32;q13) or t(2;14)(p13;q32). In some embodiments, the
complex karyotype comprises about two or more cytogenetic
abnormalities selected from trisomy 12, del(11q22.3), del(13q14.3),
del(17p13.1), t(11;14)(q13;q32), t(14;19)(q32;q13) or
t(2;14)(p13;q32). In some embodiments, high-risk cytogenetic
features comprise of cytogenetic abnormalities selected from
trisomy 12, del(11q22.3), del(13q14.3), del(17p13.1),
t(11;14)(q13;q32), t(14;19)(q32;q13) or t(2;14)(p13;q32). In some
embodiments, the subject has CLL. In some embodiments, the subject
having CLL possesses high-risk cytogenetic features. In some
embodiments, the subject possessing high-risk cytogenetic features
has cytogenetic abnormalities selected from trisomy 12,
del(11q22.3), del(13q14.3), del(17p13.1), t(11;14)(q13;q32),
t(14;19)(q32;q13) or t(2;14)(p13;q32). In some embodiments, the
subject possessing high-risk cytogenetic features has del(11q22.3),
del(17p13.1) or a complex karyotype. In some embodiments, the
subject possessing high-risk cytogenetic features has del(11q22.3).
In some embodiments, the subject possessing high-risk cytogenetic
features has del(17p13.1). In some embodiments, the subject
possessing high-risk cytogenetic features has a complex
karyotype.
[0099] In some embodiments, a subject possessing high-risk
cytogenetic features is associated with CLL relapse. In some
embodiments, CLL relapse is associated with ibrutinib resistance.
In some embodiments, the subject possessing high-risk cytogenetic
features is associated with ibrutinib resistance. In some
embodiments, ibrutinib resistance is associated with mutations in
the PLC.gamma.2 gene. In some embodiments, ibrutinib resistance is
associated with mutations in only PLC.gamma.2. The PLC.gamma.2 gene
is located on chromosome 16. In some embodiments, ibrutinib
resistance is associated with mutations in PLC.gamma.2 and an
additional gene. In some embodiments, ibrutinib resistance is not
associated with mutations in PLC.gamma.2. In some embodiments,
ibrutinib resistance is associated with mutation at amino acid
position 742, 845, or 1140 in the PLC.gamma.2 gene (those amino
acid sequence is set forth in SEQ ID NO: 2).
[0100] In some embodiments, the subject possessing high-risk
cytogenetic features having ibrutinib resistance has mutations in
the PLC.gamma.2. In some embodiments, the subject possessing
high-risk cytogenetic features having ibrutinib resistance has
mutations in only PLC.gamma.2. In some embodiments, the subject
possessing high-risk cytogenetic features having ibrutinib
resistance has mutations in PLC.gamma.2 and an additional gene. In
some embodiments, the subject possessing high-risk cytogenetic
features having ibrutinib resistance has mutation at amino acid
position 742, 845, or 1140 in the PLC.gamma.2 gene. In some
embodiments, the subject possessing high-risk cytogenetic features
having ibrutinib resistance does not have mutations in PLC.gamma.2.
In some embodiments, the subject possessing high-risk cytogenetic
features having ibrutinib resistance does not have mutation at
amino acid position 742, 845, or 1140 in PLC.gamma.2.
[0101] In some embodiments, ibrutinib resistance is associated with
mutations in PLC.gamma.2 and an additional gene. In some
embodiments, the additional gene is selected from CSF1, DAB1, ARTN,
COL8A2 or LDLRAP1 located on chromosome 1; PRR21, NDUFA10, ASIC4,
POTEE or XPO1 located on chromosome 2; RAB6B, TMPRSS7 or CACNA1D
located on chromosome 3; GUCY1B3, MAML3, FRAS1 or EVC2 located on
chromosome 4; NPM1, G3BP1, H2AFY, HEATR7B2 or ADAMTS12 located on
chromosome 5; KIAA1244, ENPP1, NKAIN2, REV3L, COL12A1 or IRF4
located on chromosome 6; ZNF775, SSPO, ZNF777 or ABCA13 located on
chromosome 7; TRPS1 located on chromosome 8; UAP1L1, AGPAT2,
SNAPC4, RALGPS1 or GNAQ located on chromosome 9; PIK3AP1, EGR2 or
NRP1 located on chromosome 10; KRTAP5-9, CAPN1 or MUC2 located on
chromosome 11; DPY19L2, KRT73, SLC11A2, MLL2, SYT10 or OVOS2
located on chromosome 12; TRPC4 located on chromosome 13; SLC8A3
located on chromosome 14; BLM, DISP2 or C15orf55 located on
chromosome 15; MMP25 or MAPK8IP3 located on chromosome 16; LLGL2,
KRTAP9-3, TRAF4, CENPV or TP53 located on chromosome 17; CEACAM18,
SPIB, TPRX1, DMKN, LSM4, CACNA1A, CCDC151, LONP1 or STAP2 located
on chromosome 19; TSPEAR, KCNJ15, DYRK1A or IFNAR1 located on
chromosome 21; SLC5A4 or HIRA located on chromosome 22; or BTK,
IL13RA2, MAGEE1, SHROOM4 or NYX located on chromosome X. In some
embodiments, the subject possessing high-risk cytogenetic features
has mutations in PLC.gamma.2 and BTK. In some embodiments, the
subject possessing high-risk cytogenetic features has mutations in
PLC.gamma.2, BTK and an additional gene.
[0102] In some embodiments of the methods, the nucleic acid
molecule for use in the assay is RNA or DNA. In some embodiments of
the methods, the nucleic acid molecule for use in the assay is
genomic DNA. In some embodiments of the methods, the nucleic acid
molecule for use in the assay is total RNA. In some embodiments of
the methods, the nucleic acid molecule for use in the assay is
mRNA. In some embodiments of the methods, the method further
comprises isolating mRNA from the RNA sample. In some embodiments
of the methods, the nucleic acid molecule for use in the assay is
cDNA. In some embodiments of the methods, the method further
comprises reverse transcribing an RNA sample into cDNA. In some
embodiments of the methods, the method comprises analyzing the
cDNA. In some embodiments, the sample is a plasma or serum sample
containing circulating tumor DNA (ctDNA), RNA (ctRNA) or microRNA
(see e.g., Chan et al. (2007) Br J Cancer. 96(5):681-5).
[0103] In some embodiments, the genomic nucleic acid sample is
amplified by a nucleic acid amplification method. In some
embodiments, the nucleic acid amplification method is polymerase
chain reaction (PCR). In some embodiments, the genomic nucleic acid
sample is amplified using a set of nucleotide primers specific for
the PLC.gamma.2 gene. In some embodiments, the set of nucleotide
primers flank the nucleic acid sequence encoding amino acid
position 742, 845, or 1140 of the PLC.gamma.2 polypeptide. In some
embodiments, the amplification product is a nucleic acid encoding
amino acid position 742, 845, or 1140 of the PLC.gamma.2
polypeptide. In some embodiments, a sequence specific primer is
conjugated to a detectable molecule, such as a fluorescent label, a
bioluminescent label, a chemiluminescent label, a radiolabel, an
enzyme label, a detectable substrate, or a peptide or molecule that
binds to a second detectable molecule.
[0104] A variety of methods are available in the art for the
detection of single point mutations in nucleic acids encoding
mutant PLC.gamma.2 polypeptides and amino acid changes in the
PLC.gamma.2 polypeptide in a sample. The following methods for
detection of mutations in nucleic acids and mutant polypeptides are
meant to be exemplary and are not exclusive.
[0105] In some embodiments of the methods, testing comprises
performing polymerase chain reaction (PCR) amplification of nucleic
acid encoding amino acid position 742, 845, or 1140 of the
PLC.gamma.2 polypeptide. In some embodiments, PCR amplification
comprises using a pair of oligonucleotide primers that flank the
region encoding amino acid position 742, 845, or 1140 of the
PLC.gamma.2 polypeptide. In some embodiments, the method comprises
sequencing the amplified nucleic acid using a sequence specific
primer. In some embodiments, the method comprises ligating the
amplified PCR fragment into a vector and then sequencing the
nucleic acid encoding the PLC.gamma.2 polypeptide or portion
thereof containing amino acid position 742, 845, or 1140. In some
embodiments, the method comprises sequencing the amplified nucleic
acid in a vector using a vector sequence specific primer. In some
embodiments, the sequencing method is a high-throughput method. In
some embodiments, the sequencing method is a next-generation
sequencing method.
[0106] As described elsewhere herein, exemplary sequencing methods
for use in the methods provide herein include, but are not limited
to, dideoxy or chain termination methods, Maxam-Gilbert sequencing,
massively parallel signature sequencing (or MPSS), polony
sequencing, pyrosequencing, Illumina dye sequencing, SOLiD (or
sequencing by ligation) sequencing, ion semiconductor sequencing,
DNA nanoball sequencing, heliscope sequencing, single molecule real
time (SMRT) sequencing, whole-exome sequencing, Ion Torrent
sequencing, Helicos True Single Molecule Sequencing (tSMS) (Harris
T. D. et al. (2008) Science 320:106-109); 454 sequencing (Roche)
(Margulies, M. et al. 2005, Nature, 437, 376-380); SOLiD technology
(Applied Biosystems); SOLEXA sequencing (Illumina); single
molecule, real-time (SMRT.TM.) technology of Pacific Biosciences;
nanopore sequencing (Soni G V and Meller A. (2007) Clin Chem 53:
1996-2001); semiconductor sequencing (Ion Torrent; Personal Genome
Machine); DNA nanoball sequencing; sequencing using technology from
Dover Systems (Polonator), and technologies that do not require
amplification or otherwise transform native DNA prior to sequencing
(e.g., Pacific Biosciences and Helicos), such as nanopore-based
strategies (e.g. Oxford Nanopore, Genia Technologies, and
Nabsys).
[0107] In some embodiments of the methods, testing comprises
contacting the nucleic acid molecule encoding a PLC.gamma.2
polypeptide with a sequence specific nucleic acid probe, wherein
the sequence specific nucleic acid probe: (a) binds to nucleic acid
encoding a modified PLC.gamma.2 that is modified at amino acid
position 742, 845, or 1140; and (b) does not bind to nucleic acid
encoding the wild-type PLC.gamma.2 having leucine at amino acid
position 742, 845, or 1140. In some embodiments of the methods,
testing comprises PCR amplification using the sequence specific
nucleic acid probe. In some embodiments, testing further comprises
additional sequence specific nucleic acid probes. In some
embodiments, the sequence specific probe is conjugated to a
detectable molecule, such as a fluorescent label, a bioluminescent
label, a chemiluminescent label, a radiolabel, an enzyme label, a
detectable substrate, or a peptide or molecule that binds to a
second detectable molecule.
[0108] In some embodiments of the methods, testing the sample
comprises contacting the nucleic acid with a pair of
oligonucleotide primers that flank the nucleic acid region encoding
amino acid 742, 845, or 1140 of a PLC.gamma.2 polypeptide. In some
embodiments, testing the sample further comprises oligonucleotide
primers that flank the nucleic acid regions encoding additional
amino acid positions of the PLC.gamma.2 polypeptide. In some
embodiments, testing the sample further comprises oligonucleotide
primers that flank the nucleic acid regions encoding additional
polypeptides.
[0109] In some embodiments of the methods, testing comprises using
allele specific PCR. In some embodiments, single nucleotide changes
are detectable PCR using PCR-based cleaved amplified polymorphic
sequences (CAPS) markers which create restriction sites in the
mutant sequences (Michaels et al (1998) Plant J. 4(3):381-5) or
sequence specific hairpin probes attached to detectable moieties,
such as, but not limited to, a fluorophore (Mhlanga and Malmberg
(2001) Methods 25:463-471). In some embodiments, the sequence
specific probe is conjugated to a detectable molecule, such as a
fluorescent label, a bioluminescent label, a chemiluminescent
label, a radiolabel, an enzyme label, a detectable substrate, or a
peptide or molecule that binds to a second detectable molecule. In
some embodiments, the oligonucleotide probe is specific for nucleic
acid encoding serine at a position corresponding to amino acid 742,
845, or 1140 of a PLC.gamma.2 polypeptide.
[0110] In some embodiments, the DNA encoding the mutant PLC.gamma.2
is assessed by BEAMing (beads, amplification, emulsion, magnetic)
PCR sequencing method (see, e.g., Li et al. (2006) Nat Methods.
3(2):95-7; Li et al. (2006) Nat Methods. 3(7):551-9; and Diehl et
al. (2008) Nat Med. 14(9): 985-990). BEAMing is a technique in
which individual DNA molecules are attached to magnetic beads in
water-in-oil emulsions and then subjected to compartmentalized PCR
amplification. The mutational status of DNA bound to beads is then
determined by hybridization to fluorescent allele-specific probes
for, for example, mutant or wild-type PLC.gamma.2. Flow cytometry
is then used to quantify the level of mutant DNA present in the
plasma or serum (see e.g., Higgins et al. (2012) Clin Cancer Res
18: 3462-3469).
[0111] In some embodiments, testing the sample comprises denaturing
high performance liquid chromatography (D-HPLC). D-HPLC relies upon
the differential retention kinetics of heteroduplex/homoduplex DNA
species within a cartridge matrix designed to separate DNA
fragments according to charge density against an electrolyte
gradient. (see e.g., Frueh et al (2003) Clin Chem Lab Med.
41(4):452-61).
[0112] In some embodiments, testing the sample comprises
nanofluidics, including using NanoPro to determine the pI
differences in a wild-type or mutant polypeptide bound to an
inhibitor. For example, NanoPro can be used to determine the pI
differences in a wild-type PLC.gamma.2 polypeptide covalently bound
to a PLC.gamma.2 inhibitor at amino acid position 742, 845, or 1140
and mutant PLC.gamma.2 polypeptide (e.g., having a modification
that is R742P, L845F, D1140G) that does not covalently bind to the
PLC.gamma.2 inhibitor. NanoPro is an instrument that can separate
proteins based on small differences in isoelectric points. The
covalent modification of amino acid position 742, 845, or 1140 with
the PLC.gamma.2 inhibitor compared to the unconjugated mutant
PLC.gamma.2 will change its isoelectric point, which is used to
detect drug binding to PLC.gamma.2.
[0113] In some embodiments, testing the sample comprises using a
microarray. In some embodiments, the presence of DNA encoding the
mutant PLC.gamma.2 is assessed using an oligonucleotide array (see
e.g., Hastia et al. (1999) J Med Genet. 36(10):730-6). In some
embodiments, the microarray comprising nucleic acid encoding a
modified PLC.gamma.2 polypeptide or a portion thereof that is
modified at an amino acid position corresponding to amino acid
position 742, 845, or 1140 of the amino acid sequence set forth in
SEQ ID NO: 2. In some embodiments, the microarray further comprises
comprising nucleic acid encoding a modified PLC.gamma.2 polypeptide
or a portion thereof that is modified at additional amino acid
positions. In some embodiments, the oligonucleotide array is
contained on a microchip. In some embodiments, single nucleotide
changes are detectable using microchips.
[0114] In some embodiments of the method, the sample for detection
of a mutant PLC.gamma.2 is a protein sample that contains a
PLC.gamma.2 polypeptide. In such examples, testing comprises
detection of the mutation with an antibody specific for the mutant
polypeptides. In some embodiments, the method of detecting a mutant
PLC.gamma.2 polypeptide comprises providing a sample from a
subject, wherein the sample comprises a PLC.gamma.2 polypeptide and
testing the sample for the presence of a mutant PLC.gamma.2
polypeptide by contacting the sample with an antibody that is
specific for binding to the mutant PLC.gamma.2 polypeptide, and
does not bind or binds with decreased affinity for the wild-type
PLC.gamma.2 polypeptide, wherein the presence of the mutant
PLC.gamma.2 polypeptide creates an antibody-mutant PLC.gamma.2
polypeptide complex. In some embodiments, the method further
comprises detecting the antibody-mutant PLC.gamma.2 polypeptide
complex. In some embodiments, the method further comprises
detecting the antibody-mutant PLC.gamma.2 polypeptide complex with
a detection reagent. In some embodiments, the mutant PLC.gamma.2
specific antibody is conjugated to a detectable molecule, such as a
fluorescent label, a bioluminescent label, a chemiluminescent
label, a radiolabel, an enzyme label, a detectable substrate, or a
peptide or molecule that binds to a second detectable protein
(e.g., a secondary antibody). In some embodiments, binding of the
mutant PLC.gamma.2 specific antibody is detected by assaying for
the detectable molecule. In some embodiments, binding of the mutant
PLC.gamma.2 specific antibody is detected by using a secondary
(e.g., anti-IgG) antibody.
[0115] In some embodiments of the methods, the subject has a
BTK-mediated disease or disorder. In some embodiments of the
methods, the subject has a B-cell proliferative disorder. In some
embodiments of the methods, the subject has cancer. In some
embodiments, the cancer is a hematologic cancer. In some
embodiments, cancer is a B-cell malignancy. In some embodiments,
cancer is selected from among a leukemia, a lymphoma, or a myeloma.
In some embodiments, the B-cell malignancy is chronic lymphocytic
leukemia (CLL), small lymphocytic lymphoma (SLL), diffuse large
B-cell lymphoma (DLBCL), activated B-cell diffuse large B-cell
lymphoma (ABC-DLBCL), germinal center diffuse large B-cell lymphoma
(GCB DLBCL), double-hit diffuse large B-cell lymphoma (DH-DLBCL),
primary mediastinal B-cell lymphoma (PMBL), non-Hodgkin lymphoma,
Burkitt's lymphoma, follicular lymphoma, immunoblastic large cell
lymphoma, precursor B-lymphoblastic lymphoma, precursor B-cell
acute lymphoblastic leukemia, hairy cell leukemia, mantle cell
lymphoma, B cell prolymphocytic leukemia, lymphoplasmacytic
lymphoma/Waldenstrom macroglobulinemia, splenic marginal zone
lymphoma, plasma cell myeloma, plasmacytoma, extranodal marginal
zone B cell lymphoma, nodal marginal zone B cell lymphoma,
mediastinal (thymic) large B cell lymphoma, intravascular large B
cell lymphoma, primary effusion lymphoma, or lymphomatoid
granulomatosis. In some embodiments, the subject has a solid
tumor.
[0116] In some embodiments, the subject has a solid tumor. In some
embodiments, the subject has a sarcoma, carcinoma, a
neurofibromatoma or a lymphoma.
[0117] In some embodiments, the subject has a cancer of the lung,
breast, colon, brain, prostate, liver, pancreas, esophagus, kidney,
stomach, thyroid, bladder, uterus, cervix or ovary. In some
embodiments, the subject has a metastatic cancer. In some
embodiments, the subject has a cancer that is acute lymphoblastic
leukemia, acute lymphoblastic leukemia, acute myeloid leukemia,
acute promyelocytic leukemia, adenocarcinoma, adenoma, adrenal
cancer, adrenocortical carcinoma, AIDS-related cancer, AIDS-related
lymphoma, anal cancer, appendix cancer, astrocytoma, basal cell
carcinoma, bile duct cancer, bladder cancer, bone cancer,
osteosarcoma/malignant fibrous histiocytoma, brainstem glioma,
brain cancer, carcinoma, cerebellar astrocytoma, cerebral
astrocytoma/malignant glioma, ependymoma, medulloblastoma,
supratentorial primitive neuroectodermal tumor, visual pathway or
hypothalamic glioma, breast cancer, bronchial adenoma/carcinoid,
Burkitt lymphoma, carcinoid tumor, carcinoma, central nervous
system lymphoma, cervical cancer, chronic lymphocytic leukemia,
chronic myelogenous leukemia, chronic myeloproliferative disorder,
colon cancer, cutaneous T-cell lymphoma, desmoplastic small round
cell tumor, endometrial cancer, ependymoma. epidermoid carcinoma,
esophageal cancer, Ewing's sarcoma, extracranial germ cell tumor,
extragonadal germ cell tumor, extrahepatic bile duct cancer, eye
cancer/intraocular melanoma, eye cancer/retinoblastoma, gallbladder
cancer, gallstone tumor, gastric/stomach cancer, gastrointestinal
carcinoid tumor, gastrointestinal stromal tumor, giant cell tumor,
glioblastoma multiforme, glioma, hairy-cell tumor, head and neck
cancer, heart cancer, hepatocellular/liver cancer, Hodgkin
lymphoma, hyperplasia, hyperplastic corneal nerve tumor, in situ
carcinoma, hypopharyngeal cancer, intestinal ganglioneuroma, islet
cell tumor, Kaposi's sarcoma, kidney/renal cell cancer, laryngeal
cancer, leiomyoma tumor, lip and oral cavity cancer, liposarcoma,
liver cancer, non-small cell lung cancer, small cell lung cancer,
lymphomas, macroglobulinemia, malignant carcinoid, malignant
fibrous histiocytoma of bone, malignant hypercalcemia, malignant
melanomas, marfanoid habitus tumor, medullary carcinoma, melanoma,
merkel cell carcinoma, mesothelioma, metastatic skin carcinoma,
metastatic squamous neck cancer, mouth cancer, mucosal neuromas,
multiple myeloma, mycosis fungoides, myelodysplastic syndrome,
myeloma, myeloproliferative disorder, nasal cavity and paranasal
sinus cancer, nasopharyngeal carcinoma, neck cancer, neural tissue
cancer, neuroblastoma, oral cancer, oropharyngeal cancer,
osteosarcoma, ovarian cancer, ovarian epithelial tumor, ovarian
germ cell tumor, pancreatic cancer, parathyroid cancer, penile
cancer, pharyngeal cancer, pheochromocytoma, pineal astrocytoma,
pineal germinoma, pineoblastoma, pituitary adenoma, pleuropulmonary
blastoma, polycythemia vera, primary brain tumor, prostate cancer,
rectal cancer, renal cell tumor, reticulum cell sarcoma,
retinoblastoma, rhabdomyosarcoma, salivary gland cancer, seminoma,
Sezary syndrome, skin cancer, small intestine cancer, soft tissue
sarcoma, squamous cell carcinoma, squamous neck carcinoma, stomach
cancer, supratentorial primitive neuroectodermal tumor, testicular
cancer, throat cancer, thymoma, thyroid cancer, topical skin
lesion, trophoblastic tumor, urethral cancer, uterine/endometrial
cancer, uterine sarcoma, vaginal cancer, vulvar cancer,
Waldenstrom's macroglobulinemia or Wilm's tumor.
[0118] In some embodiments, the subject has a relapsed cancer. In
some embodiments, the subject has a refractory cancer. In some
embodiments, the subject has a refractory cancer where the cancer
is refractory to treatment with a covalent and/or irreversible BTK
inhibitor. In some embodiments, the subject has a refractory cancer
where the subject exhibits a decrease in sensitivity to treatment
with a covalent and/or irreversible BTK inhibitor. In some
embodiments, the subject has a refractory cancer where the subject
exhibits a decrease in sensitivity to a particular dosage of a
covalent and/or irreversible BTK inhibitor. In some embodiments,
the subject has a refractory cancer where the subject exhibits an
increase in severity or the appearance of one or more symptoms of a
cancer (i.e. disease progression). In some embodiments, the subject
exhibits a decrease in the regression of a cancer. In some
embodiments, the regression of a cancer ceases. In some
embodiments, the subject has a relapsed or refractory hematologic
cancer. In some embodiments, the subject has a relapsed or
refractory B-cell malignancy.
[0119] In some embodiments the subject is suspected of having a
hematologic cancer or is at high risk of having a hematologic
cancer. In some embodiments the subject is suspected of having a
B-cell malignancy or is at high risk of having a B-cell malignancy.
In some embodiments the subject is suspected of having or is at
high risk of having a leukemia, a lymphoma, or a myeloma.
[0120] In some embodiments, the subject exhibits one or more
symptoms of a hematologic cancer. In some embodiments, the subject
exhibits one or more symptoms of a B-cell malignancy. In some
embodiments, the subject exhibits one or more symptoms of a
leukemia, a lymphoma, or a myeloma. In some embodiments, the
subject exhibits one or more symptoms such as, but not limited to,
abnormal B-cell function, abnormal B-cell size or shape, abnormal
B-cell count, fatigue, fever, night sweats, frequent infection,
enlarged lymph nodes, paleness, anemia, easy bleeding or bruising,
loss of appetite, weight loss, bone or joint pain, headaches, and
petechie.
[0121] In some embodiments, the subject is suffering from an
autoimmune disease, e.g., inflammatory bowel disease, arthritis,
lupus, rheumatoid arthritis, psoriatic arthritis, osteoarthritis,
Still's disease, juvenile arthritis, diabetes, myasthenia gravis,
Hashimoto's thyroiditis, Ord's thyroiditis, Graves' disease
Sjogren's syndrome, multiple sclerosis, Guillain-Barre syndrome,
acute disseminated encephalomyelitis, Addison's disease,
opsoclonus-myoclonus syndrome, ankylosing spondylitisis,
antiphospholipid antibody syndrome, aplastic anemia, autoimmune
hepatitis, coeliac disease, Goodpasture's syndrome, idiopathic
thrombocytopenic purpura, optic neuritis, scleroderma, primary
biliary cirrhosis, Reiter's syndrome, Takayasu's arteritis,
temporal arteritis, warm autoimmune hemolytic anemia, Wegener's
granulomatosis, psoriasis, alopecia universalis, Behcet's disease,
chronic fatigue, dysautonomia, endometriosis, interstitial
cystitis, neuromyotonia, scleroderma, or vulvodynia.
[0122] In other embodiments, the subject is suffering from a
heteroimmune condition or disease, e.g., graft versus host disease,
transplantation, transfusion, anaphylaxis, allergy, type I
hypersensitivity, allergic conjunctivitis, allergic rhinitis, or
atopic dermatitis.
[0123] In some embodiments, the subject has an inflammatory
disease, e.g., asthma, appendicitis, blepharitis, bronchiolitis,
bronchitis, bursitis, cervicitis, cholangitis, cholecystitis,
colitis, conjunctivitis, cystitis, dacryoadenitis, dermatitis,
dermatomyositis, encephalitis, endocarditis, endometritis,
enteritis, enterocolitis, epicondylitis, epididymitis, fasciitis,
fibrositis, gastritis, gastroenteritis, hepatitis, hidradenitis
suppurativa, laryngitis, mastitis, meningitis, myelitis
myocarditis, myositis, nephritis, oophoritis, orchitis, osteitis,
otitis, pancreatitis, parotitis, pericarditis, peritonitis,
pharyngitis, pleuritis, phlebitis, pneumonitis, pneumonia,
proctitis, prostatitis, pyelonephritis, rhinitis, salpingitis,
sinusitis, stomatitis, synovitis, tendonitis, tonsillitis, uveitis,
vaginitis, vasculitis, or vulvitis.
[0124] In further embodiments, the subject is suffering from a
thromboembolic disorder, e.g., myocardial infarct, angina pectoris,
reocclusion after angioplasty, restenosis after angioplasty,
reocclusion after aortocoronary bypass, restenosis after
aortocoronary bypass, stroke, transitory ischemia, a peripheral
arterial occlusive disorder, pulmonary embolism, or deep venous
thrombosis.
[0125] In some embodiments, the subject is administered or has been
administered one or more therapeutic agents for treatment of a
disease or condition. In some embodiments, the subject is
administered or has been administered a BTK inhibitor for treatment
of a disease or condition. In some embodiments, the subject is
administered or has been administered one or more therapeutic
agents in addition to a BTK inhibitor for treatment of a disease or
condition.
[0126] In some embodiments, the subject is administered or has been
administered one or more chemotherapeutic agents for treatment of
cancer. In some embodiments, the subject is administered or has
been administered a BTK inhibitor for treatment of a cancer. In
some embodiments, the subject is administered or has been
administered one or more chemotherapeutic agents in addition to a
BTK inhibitor for treatment of cancer.
[0127] In some embodiments, the sample for use in the methods is
from any tissue or fluid from an organism. Samples include, but are
not limited, to whole blood, dissociated bone marrow, bone marrow
aspirate, pleural fluid, peritoneal fluid, central spinal fluid,
abdominal fluid, pancreatic fluid, cerebrospinal fluid, brain
fluid, ascites, pericardial fluid, urine, saliva, bronchial lavage,
sweat, tears, ear flow, sputum, hydrocele fluid, semen, vaginal
flow, milk, amniotic fluid, and secretions of respiratory,
intestinal or genitourinary tract. In particular embodiments, the
sample is a tumor biopsy sample. In particular embodiments, the
sample is from a fluid or tissue that is part of, or associated
with, the lymphatic system or circulatory system. In some
embodiments, the sample is a blood sample that is a venous,
arterial, peripheral, tissue, cord blood sample. In particular
embodiments, the sample is a blood cell sample containing one or
more peripheral blood mononuclear cells (PBMCs). In some
embodiments, the sample contains one or more circulating tumor
cells (CTCs). In some embodiments, the sample contains one or more
disseminated tumor cells (DTC, e.g., in a bone marrow aspirate
sample).
[0128] Methods for the isolation of nucleic acids and proteins from
cells contained in tissue and fluid samples are well-known in the
art. In particular embodiments, the sample obtained from the
subject is isolated from cells contained in a tumor biopsy from the
subject. In particular embodiments, the sample obtained from the
subject is isolated from cells in a bone marrow aspirate. In
particular embodiments, the sample obtained from the subject is
isolated from cells contained a serum sample. In particular
embodiments, the sample obtained from the subject is isolated from
cells contained in a lymph sample. In particular embodiments, the
sample contains circulating tumor nucleic acid not contained in a
cell.
[0129] In some embodiments, the samples are obtained from the
subject by any suitable means of obtaining the sample using
well-known and routine clinical methods. Procedures for obtaining
fluid samples from a subject are well known. For example,
procedures for drawing and processing whole blood and lymph are
well-known and can be employed to obtain a sample for use in the
methods provided. Typically, for collection of a blood sample, an
anti-coagulation agent (e.g., EDTA, or citrate and heparin or CPD
(citrate, phosphate, dextrose) or comparable substances) is added
to the sample to prevent coagulation of the blood. In some
examples, the blood sample is collected in a collection tube that
contains an amount of EDTA to prevent coagulation of the blood
sample.
[0130] In some embodiments, the sample is a tissue biopsy and is
obtained, for example, by needle biopsy, CT-guided needle biopsy,
aspiration biopsy, endoscopic biopsy, bronchoscopic biopsy,
bronchial lavage, incisional biopsy, excisional biopsy, punch
biopsy, shave biopsy, skin biopsy, bone marrow biopsy, and the Loop
Electrosurgical Excision Procedure (LEEP). Typically, a
non-necrotic, sterile biopsy or specimen is obtained that is
greater than 100 mg, but which can be smaller, such as less than
100 mg, 50 mg or less, 10 mg or less or 5 mg or less; or larger,
such as more than 100 mg, 200 mg or more, or 500 mg or more, 1 gm
or more, 2 gm or more, 3 gm or more, 4 gm or more or 5 gm or more.
The sample size to be extracted for the assay depends on a number
of factors including, but not limited to, the number of assays to
be performed, the health of the tissue sample, the type of cancer,
and the condition of the patient. In some embodiments, the tissue
is placed in a sterile vessel, such as a sterile tube or culture
plate, and is optionally immersed in an appropriate media.
Typically, the cells are dissociated into cell suspensions by
mechanical means and/or enzymatic treatment as is well known in the
art. Typically, the cells are collected and then subjected to
standard procedures for the isolation of nucleic acid for the
assay.
[0131] In some embodiments, the collection of a sample from the
subject is performed at regular intervals, such as, for example,
one day, two days, three days, four days, five days, six days, one
week, two weeks, weeks, four weeks, one month, two months, three
months, four months, five months, six months, one year, daily,
weekly, bimonthly, quarterly, biyearly or yearly.
[0132] In some embodiments, the collection of a sample is performed
at a predetermined time or at regular intervals relative to
treatment with one or more anti-cancer agents. In some embodiments,
anticancer agent is administered for the treatment of a leukemia,
lymphoma or a myeloma. Exemplary anti-cancer agents for the
treatment of a leukemia, lymphoma or a myeloma include but are not
limited to adriamycin (doxorubicin), bexxar, bendamustine,
bleomycin, blenoxane, bortezomib, dacarbazine, deltasone,
cisplatin, cyclophosphamide, cytoxan, DTIC dacarbazine, dasatinib,
doxorubicin, etoposide, fludarabine, granisetron, kytril,
lenalidomide, matulane, mechlorethamine, mustargen, mustine,
natulan, Rituxan (rituximab, anti-CD20 antibody), VCR, neosar,
nitrogen mustard, oncovin, ondansetron, orasone, prednisone,
procarbazine, thalidomide, VP-16, velban, velbe, velsar, VePesid,
vinblastine, vincristine, Zevalin.RTM., zofran, stem cell
transplantation, radiation therapy or combination therapies, such
as, for example, ABVD (adriamycin, bleomycin, vinblastine and
dacarbazine), ChlvPP (chlorambucil, vinblastine, procarbazine and
prednisolone), Stanford V (mustine, doxorubicin, vinblastine,
vincristine, bleomycin, etoposide and steroids), BEACOPP
(bleomycin, etoposide, doxorubicin, cyclophosphamide, vincristine,
procarbazine and prednisolone), BEAM (carmustine (BiCNU) etoposide,
cytarabine (Ara-C, cytosine arabinoside), and melphalan), CHOP
(cyclophosphamide, doxorubicin, vincristine, and prednisone),
R-CHOP (rituximab, doxorubicin, cyclophosphamide, vincristine, and
prednisone), EPOCH (etoposide, vincristine, doxorubicin,
cyclophosphamide, and prednisone), CVP (cyclophosphamide,
vincristine, and prednisone), ICE
(ifosfamide-carboplatin-etoposide), R-ACVBP (rituximab,
doxorubicin, cyclophosphamide, vindesine, bleomycin, and
prednisone), DHAP (dexamethasone, high-dose cytarabine, (Ara C),
cisplatin), R-DHAP(rituximab, dexamethasone, high-dose cytarabine,
(Ara C), cisplatin), ESHAP (etoposide (VP-16), methyl-prednisolone,
and high-dose cytarabine (Ara-C), cisplatin), CDE
(cyclophosphamide, doxorubicin and etoposide), Velcade.RTM.
(bortezomib) plus Doxil.RTM. (liposomal doxorubicin), Revlimid.RTM.
(lenalidomide) plus dexamethasone, and bortezomib plus
dexamethasone. In some embodiments, anticancer agent is
fludarabine. In some embodiments, anticancer agent is bendamustine.
In some embodiments, the anticancer agent is Rituxan. In some
embodiments, the anticancer agent is dasatinib. In some
embodiments, a sample is collected at a predetermined time or at
regular intervals prior to, during, or following treatment or
between successive treatments with the anti-cancer agent. In
particular examples, a sample is obtained from the subject prior to
administration of an anti-cancer therapy and then again at regular
intervals after treatment has been effected.
[0133] In some embodiments, the collection of a sample is performed
at a predetermined time or at regular intervals relative to
treatment with a covalent and/or irreversible BTK inhibitor. For
example, a sample is collected at a predetermined time or at
regular intervals prior to, during, or following treatment or
between successive treatments. In particular examples, a sample is
obtained from the subject prior to administration of a covalent
and/or irreversible BTK inhibitor and then again at regular
intervals after treatment with the irreversible BTK inhibitor has
been effected. In some embodiments, the subject is administered a
covalent and/or irreversible BTK inhibitor and one or more
additional anti-cancer agents. In some embodiments, the subject is
administered a covalent and/or irreversible BTK inhibitor and one
or more additional anti-cancer agents that are not irreversible BTK
inhibitors. In some embodiments, the subject is administered one or
more irreversible BTK inhibitors. In some embodiments, the
irreversible BTK inhibitor is ibrutinib, PCI-45292, PCI-45466,
AVL-101/CC-101 (Avila Therapeutics/Celgene Corporation),
AVL-263/CC-263 (Avila Therapeutics/Celgene Corporation),
AVL-292/CC-292 (Avila Therapeutics/Celgene Corporation),
AVL-291/CC-291 (Avila Therapeutics/Celgene Corporation), CNX 774
(Avila Therapeutics), BMS-488516 (Bristol-Myers Squibb), BMS-509744
(Bristol-Myers Squibb), CGI-1746 (CGI Pharma/Gilead Sciences),
CGI-560 (CGI Pharma/Gilead Sciences), CTA-056, GDC-0834
(Genentech), HY-11066 (also, CTK417891, HMS3265G21, HMS3265G22,
HMS3265H21, HMS3265H22, 439574-61-5, AG-F-54930), ONO-4059 (Ono
Pharmaceutical Co., Ltd.), ONO-WG37 (Ono Pharmaceutical Co., Ltd.),
PLS-123 (Peking University), RN486 (Hoffmann-La Roche), HM71224
(Hanmi Pharmaceutical Company Limited), LFM-A13, BGB-3111
(Beigene), KBP-7536 (KBP BioSciences), ACP-196 (Acerta Pharma),
JTE-051 (Japan Tobacco Inc), PRN1008 (Principia), CTP-730 (Concert
Pharmaceuticals), or GDC-0853 (Genentech). In some embodiments, the
irreversible BTK inhibitor is ibrutinib, PCI-45292, PCI-45466,
AVL-101, AVL-291, AVL-292, ONO-WG-37 or
(R)-6-amino-9-(1-but-2-ynoylpyrrolidin-3-yl)-7-(4-phenoxyphenyl)-7H-purin-
-8(9H)-one. In some embodiments, the irreversible BTK inhibitor is
ibrutinib. In some embodiments, the irreversible BTK inhibitor is
(R)-6-amino-9-(1-but-2-ynoylpyrrolidin-3-yl)-7-(4-phenoxyphenyl)-7H-purin-
-8(9H)-one.
[0134] Additional BTK inhibitors for use in any of the methods
provided herein can be found, for example, in U.S. Pat. Nos.
7,547,689, 7,960,396 and U.S. Patent Publication Nos. US
2009-0197853 A1 and US 2012-0065201 A1, all of which are
incorporated by reference in their entirety. Additional BTK
inhibitors for use in any of the methods provided herein also can
be found, for example, in US20100029610, WO09051822, WO10123870,
WO09158571, WO11034907, WO12021444, WO11029046, WO08110624,
WO10080481, WO10144647, WO10056875, WO05047290, WO06053121,
WO06099075, WO08033834, WO08033857, WO08033858, WO09137596,
WO10056875, WO10068788, WO10068806, WO10068810, WO11140488,
WO12030990, WO12031004, WO2010056875, WO05066156, WO10056875,
US20120316148, WO09048307, WO09147190, WO11162515, WO11162515,
WO06036941, WO10126960, WO07136790, WO12025186, WO2013010380,
WO2013010868, WO2013010869, WO2013008095, WO11152351, WO2013060098,
WO2013060098, WO07002325, WO07002433, WO07013896, WO09143024,
WO10065898, WO2012158764, WO2012158785, WO2012158795, WO2012158810,
WO09053269, WO09156284, WO2012020008, WO2012156334, WO2013024078,
WO08057252, WO03081210, WO03087051, US20130059847A1, WO06065946,
WO07027594, and WO08092199 all of which are incorporated by
reference in their entirety.
[0135] Further BTK inhibitors for use in any of the methods
provided herein can be found, for example, in U.S. Pat. Nos.
7,514,444; 7,960,396; 8,236,812; 8,497,277; 8,563,563; 8,399,470;
8,088,781; 8,501,751; 8,008,309; 8,552,010; 7,732,454; 7,825,118;
8,377,946; 8,501,724; US Patent Pub. No. 2011-0039868; U.S. Pat.
Nos. 8,232,280; 8,158,786; US Patent Pub. No. 2011-0281322; US
Patent Pub. No. 2012-0088912; US Patent Pub. No. 2012-0108612; US
Patent Pub. No. 2012-0115889; US Patent Pub. No. 2013-0005745; US
Patent Pub. No. 2012-0122894; US Patent Pub. No. 2012-0135944; US
Patent Pub. No. 2012-0214826; US Patent Pub. No. 2012-0252821; US
Patent Pub. No. 2012-0252822; US Patent Pub. No. 2012-0277254; US
Patent Pub. No. 2010-0022561; US Patent Pub. No. 2010-0324050; US
Patent Pub. No. 2012-0283276; US Patent Pub. No. 2012-0065201; US
Patent Pub. No. 2012-0178753; US Patent Pub. No. 2012-0101113; US
Patent Pub. No. 2012-0101114; US Patent Pub. No. 2012-0165328; US
Patent Pub. No. 2012-0184013; US Patent Pub. No. 2012-0184567; US
Patent Pub. No. 2012-0202264; US Patent Pub. No. 2012-0277225; US
Patent Pub. No. 2012-0277255; US Patent Pub. No. 2012-0296089; US
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App. No. PCT/US2012/72043; U.S. Patent App. No. 61/593,146; U.S.
Patent App. No. 61/637,765; PCT App. No. PCT/US2013/23918; U.S.
Patent App. No. 61/781,975; U.S. Patent App. No. 61/727,031; PCT
App. No. PCT/US2013/7016; U.S. Patent App. No. 61/647,956; PCT App.
No. PCT/US2013/41242; U.S. Patent App. No. 61/769,103; U.S. Patent
App. No. 61/842,321; and U.S. Patent App. No. 61/884,888, all of
which are incorporated herein in their entirety by reference.
[0136] In some embodiments, the subject is administered a covalent
and/or irreversible BTK inhibitor that covalently binds to cysteine
481 of the wild-type BTK in combination with one or more reversible
BTK inhibitors. For example, in some embodiments, the subject is
administered a covalent and/or irreversible BTK inhibitor that
covalently binds to cysteine 481 of the wild-type BTK in
combination with one or more reversible BTK inhibitors that are not
dependent on cysteine 481 for binding. Reversible BTK inhibitors
are known in the art and include, but are not limited to,
dasatinib, PC-005, RN486, PCI-29732 or terreic acid. In some
embodiments, the irreversible BTK inhibitor ibrutinib is
administered in combination with the reversible BTK inhibitor
dasatinib. In some embodiments, the irreversible BTK inhibitor
(R)-6-amino-9-(1-but-2-ynoylpyrrolidin-3-yl)-7-(4-phenoxyphenyl)-7H-purin-
-8(9H)-one is administered in combination with the reversible BTK
inhibitor dasatinib.
[0137] In some embodiments, the sample is obtained at 1 week, 2
weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6
months, 7 months, 8 months, 9 months, 10 months, 11 months, 12
months, 14 months, 16 months, 18 months, 20 months, 22 months, 24
months, 26 months, 28 months, 30 months, 32 months, 34 months, 36
months or longer following the first administration of the
irreversible BTK inhibitor. In some embodiments, the sample is
obtained at 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months,
4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10
months, 11 months, 12 months, 14 months, 16 months, 18 months, 20
months, 22 months, 24 months, 26 months, 28 months, 30 months, 32
months, 34 months, 36 months or longer following the first
administration of the irreversible BTK inhibitor to a subject naive
for exposure to the irreversible BTK inhibitor. In some
embodiments, the sample is obtained at 1 week, 2 weeks, 3 weeks, 1
month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months,
8 months, 9 months, 10 months, 11 months, 12 months, 14 months, 16
months, 18 months, 20 months, 22 months, 24 months, 26 months, 28
months, 30 months, 32 months, 34 months, 36 months or longer
following the first administration of the irreversible BTK
inhibitor to a subject having a relapsed or refractory cancer. In
some embodiments, the sample is obtained 1, 2, 3, 4, 5, 6, 7, 8, 9,
10 times or more over the course of treatment with the irreversible
BTK inhibitor. In some embodiments, the subject is responsive the
treatment with the irreversible BTK inhibitor when it is first
administered. Sequencing methods
[0138] In some embodiments, a method described herein utilizes an
amplification method. In some instances, the amplification is a PCR
method. In some instances, the method described herein is a
high-throughput method. In some instances, the method is a
next-generation sequencing method. In some instances, the
next-generation sequencing method includes, but is not limited to,
semiconductor sequencing (Ion Torrent; Personal Genome Machine);
Helicos True Single Molecule Sequencing (tSMS) (Harris T. D. et al.
(2008) Science 320:106-109); 454 sequencing (Roche) (Margulies, M.
et al. 2005, Nature, 437, 376-380); SOLiD technology (Applied
Biosystems); SOLEXA sequencing (Illumina); single molecule,
real-time (SMRT.TM.) technology of Pacific Biosciences; nanopore
sequencing (Soni G V and Meller A. (2007) Clin Chem 53: 1996-2001);
DNA nanoball sequencing; sequencing using technology from Dover
Systems (Polonator), and technologies that do not require
amplification or otherwise transform native DNA prior to sequencing
(e.g., Pacific Biosciences and Helicos), such as nanopore-based
strategies (e.g. Oxford Nanopore, Genia Technologies, and
Nabsys).
[0139] In some aspects, the next generation sequencing comprises
ion semiconductor sequencing (e.g., using technology from Life
Technologies (Ion Torrent)). In some cases, ion semiconductor
sequencing takes advantage of the fact that when a nucleotide is
incorporated into a strand of DNA, an ion can be released. To
perform ion semiconductor sequencing, a high density array of
micromachined wells is formed. Each well holds a single DNA
template. Beneath the well is an ion sensitive layer, and beneath
the ion sensitive layer is an ion sensor. When a nucleotide is
added to a DNA, H+ is released, which is measured as a change in
pH. The H+ ion is converted to voltage and is recorded by the
semiconductor sensor. An array chip is sequentially flooded with
one nucleotide after another. No scanning, light, or cameras are
required. In some embodiments, an IONPROTON.TM. Sequencer is used
to sequence nucleic acid. In some embodiments, an IONPGM.TM.
Sequencer is used.
[0140] In some instances, the next generation sequencing technique
is 454 sequencing (Roche) (see e.g., Margulies, M et al. (2005)
Nature 437: 376-380). In some cases, 454 sequencing involves two
steps. In the first step, DNA is sheared into fragments of
approximately 300-800 base pairs, and the fragments is blunt ended.
Oligonucleotide adaptors is then ligated to the ends of the
fragments. The adaptors serve as sites for hybridizing primers for
amplification and sequencing of the fragments. The fragments are
attached to DNA capture beads, e.g., streptavidin-coated beads
using, e.g., Adaptor B, which contains 5'-biotin tag. The fragments
are attached to DNA capture beads through hybridization. A single
fragment is captured per bead. The fragments attached to the beads
are PCR amplified within droplets of an oil-water emulsion. The
result is multiple copies of clonally amplified DNA fragments on
each bead. The emulsion is broken while the amplified fragments
remain bound to their specific beads. In a second step, the beads
are captured in wells (pico-liter sized; PicoTiterPlate (PTP)
device). The surface is designed so that only one bead fits per
well. The PTP device is loaded into an instrument for sequencing.
Pyrosequencing is performed on each DNA fragment in parallel.
Addition of one or more nucleotides generates a light signal that
is recorded by a CCD camera in a sequencing instrument. The signal
strength is proportional to the number of nucleotides
incorporated.
[0141] Pyrosequencing uses pyrophosphate (PPi) which is released
upon nucleotide addition. PPi is converted to ATP by ATP
sulfurylase in the presence of adenosine 5' phosphosulfate.
Luciferase then uses ATP to convert luciferin to oxyluciferin, and
this reaction generates light that is detected and analyzed. In
some instances, the 454 Sequencing system used includes GS FLX+
system or the GS Junior System.
[0142] In some instances, the next generation sequencing technique
is SOLiD technology (Applied Biosystems; Life Technologies). In
SOLiD sequencing, genomic DNA is sheared into fragments, and
adaptors are attached to the 5' and 3' ends of the fragments to
generate a fragment library. Alternatively, internal adaptors are
introduced by ligating adaptors to the 5' and 3' ends of the
fragments, circularizing the fragments, digesting the circularized
fragment to generate an internal adaptor, and attaching adaptors to
the 5' and 3' ends of the resulting fragments to generate a
mate-paired library. Next, clonal bead populations are prepared in
microreactors containing beads, primers, template, and PCR
components. Following PCR, the templates are denatured and beads
are enriched to separate the beads with extended templates.
Templates on the selected beads are subjected to a 3' modification
that permits bonding to a glass slide. A sequencing primer binds to
adaptor sequence. A set of four fluorescently labeled di-base
probes competes for ligation to the sequencing primer. Specificity
of the di-base probe is achieved by interrogating every first and
second base in each ligation reaction. The sequence of a template
is determined by sequential hybridization and ligation of partially
random oligonucleotides with a determined base (or pair of bases)
that is identified by a specific fluorophore. After a color is
recorded, the ligated oligonucleotide is cleaved and removed and
the process is then repeated. Following a series of ligation
cycles, the extension product is removed and the template is reset
with a primer complementary to the n-1 position for a second round
of ligation cycles. Five rounds of primer reset are completed for
each sequence tag. Through the primer reset process, most of the
bases are interrogated in two independent ligation reactions by two
different primers. In some instances, up to 99.99% accuracy are
achieved by sequencing with an additional primer using a multi-base
encoding scheme.
[0143] In some embodiments, the next generation sequencing
technique is SOLEXA sequencing (ILLUMINA sequencing). ILLUMINA
sequencing is based on the amplification of DNA on a solid surface
using fold-back PCR and anchored primers. ILLUMINA sequencing
involves a library preparation step, a cluster generation step, and
a sequencing step. During the library preparation step, genomic DNA
is fragmented, and sheared ends is repaired and adenylated.
Adaptors are added to the 5' and 3' ends of the fragments. The
fragments are then size selected and purified. During the cluster
generation step, DNA fragments are attached to the surface of flow
cell channels by hybridizing to a lawn of oligonucleotides attached
to the surface of the flow cell channel. The fragments are extended
and clonally amplified through bridge amplification to generate
unique clusters. The fragments become double stranded, and the
double stranded molecules are denatured. Multiple cycles of the
solid-phase amplification followed by denaturation create several
million clusters of approximately 1,000 copies of single-stranded
DNA molecules of the same template in each channel of the flow
cell. Reverse strands are cleaved and washed away. Ends are
blocked, and primers hybridized to DNA templates. During the
sequencing step, hundreds of millions of clusters are sequenced
simultaneously. Primers, DNA polymerase and four
fluorophore-labeled, reversibly terminating nucleotides are used to
perform sequential sequencing. All four bases compete with each
other for the template. After nucleotide incorporation, a laser is
used to excite the fluorophores, and an image is captured and the
identity of the first base is recorded. The 3' terminators and
fluorophores from each incorporated base are removed and the
incorporation, detection and identification steps are repeated. A
single base is read each cycle. In some instances, a HiSeq system
(e.g., HiSeq 2500, HiSeq 1500, HiSeq 2000, or HiSeq 1000) is used
for sequencing. In some instances, a MiSeq personal sequencer is
used. In some instances, a NextSeq system is used. In some
instances, a Genome Analyzer IIx is used.
[0144] In some embodiments, the next generation sequencing
technique comprises real-time (SMRT.TM.) technology by Pacific
Biosciences. In SMRT, each of four DNA bases is attached to one of
four different fluorescent dyes. These dyes are phospholinked. A
single DNA polymerase is immobilized with a single molecule of
template single stranded DNA at the bottom of a zero-mode waveguide
(ZMW). A ZMW is a confinement structure which enables observation
of incorporation of a single nucleotide by DNA polymerase against
the background of fluorescent nucleotides that is rapidly diffuse
in an out of the ZMW (in microseconds). It takes several
milliseconds to incorporate a nucleotide into a growing strand.
During this time, the fluorescent label is excited and produces a
fluorescent signal, and the fluorescent tag is cleaved off. The ZMW
is illuminated from below. Attenuated light from an excitation beam
penetrates the lower 20-30 nm of each ZMW. A microscope with a
detection limit of 20 zeptoliters (10.sup.-21 liters) is created.
The tiny detection volume provides 1000-fold improvement in the
reduction of background noise. Detection of the corresponding
fluorescence of the dye indicates which base is incorporated.
[0145] In some instances, the next generation sequencing is
nanopore sequencing (See e.g., Soni G V and Meller A. (2007) Clin
Chem 53: 1996-2001). A nanopore is a small hole, of the order of
about one nanometer in diameter. Immersion of a nanopore in a
conducting fluid and application of a potential across results in a
slight electrical current due to conduction of ions through the
nanopore. The amount of current which flows is sensitive to the
size of the nanopore. As a DNA molecule passes through a nanopore,
each nucleotide on the DNA molecule obstructs the nanopore to a
different degree. Thus, the change in the current passing through
the nanopore as the DNA molecule passes through the nanopore
represents a reading of the DNA sequence. In some instances, the
nanopore sequencing technology is from Oxford Nanopore
Technologies; e.g., a GridION system. A single nanopore is inserted
in a polymer membrane across the top of a microwell. Each microwell
has an electrode for individual sensing. The microwells are
fabricated into an array chip, with 100,000 or more microwells
(e.g., more than about 200,000, 300,000, 400,000, 500,000, 600,000,
700,000, 800,000, 900,000, or 1,000,000) per chip. An instrument
(or node) is used to analyze the chip. Data is analyzed in
real-time. One or more instruments are operated at a time. In some
cases, the nanopore is a protein nanopore, e.g., the protein
alpha-hemolysin, a heptameric protein pore. In some instances, the
nanopore is a solid-state nanopore made, e.g., a nanometer sized
hole formed in a synthetic membrane (e.g., SiNx, or SIO.sub.2). In
some instances, the nanopore is a hybrid pore (e.g., an integration
of a protein pore into a solid-state membrane). In some cases, the
nanopore is a nanopore with an integrated sensors (e.g., tunneling
electrode detectors, capacitive detectors, or graphene based
nano-gap or edge state detectors (see e.g., Garaj et al. (2010)
Nature vol. 67, doi:10.1038/nature09379)). In some aspects, the
nanopore is functionalized for analyzing a specific type of
molecule (e.g., DNA, RNA, or protein). In some cases, nanopore
sequencing comprises "strand sequencing" in which intact DNA
polymers pass through a protein nanopore with sequencing in real
time as the DNA translocates the pore. An enzyme separates strands
of a double stranded DNA and feed a strand through a nanopore. In
some cases, the DNA has a hairpin at one end, and the system reads
both strands. In some embodiments, nanopore sequencing is
"exonuclease sequencing" in which individual nucleotides is cleaved
from a DNA strand by a processive exonuclease, and the nucleotides
are passed through a protein nanopore. The nucleotides transiently
bind to a molecule in the pore (e.g., cyclodextran). A
characteristic disruption in current is used to identify bases.
[0146] In some instances, nanopore sequencing technology from GENIA
is used. An engineered protein pore is embedded in a lipid bilayer
membrane. "Active Control" technology is used to enable efficient
nanop ore-membrane assembly and control of DNA movement through the
channel. In some embodiments, the nanopore sequencing technology is
from NABsys. Genomic DNA is fragmented into strands of average
length of about 100 kb. The 100 kb fragments are made single
stranded and subsequently hybridized with a 6-mer probe. The
genomic fragments with probes are driven through a nanopore, which
creates a current-versus-time tracing. The current tracing provides
the positions of the probes on each genomic fragment. The genomic
fragments are lined up to create a probe map for the genome. The
process is done in parallel for a library of probes. A
genome-length probe map for each probe is generated. Errors are
fixed with a process termed "moving window Sequencing By
Hybridization (mwSBH)." In some embodiments, the nanopore
sequencing technology is from IBM/Roche. An electron beam is used
to make a nanopore sized opening in a microchip. An electrical
field is used to pull or thread DNA through the nanopore. A DNA
transistor device in the nanopore comprises alternating nanometer
sized layers of metal and dielectric. Discrete charges in the DNA
backbone get trapped by electrical fields inside the DNA nanopore.
Turning off and on gate voltages allow the DNA sequence to be
read.
[0147] In some instances, the next generation sequencing is DNA
nanoball sequencing (as performed, e.g., by Complete Genomics; see
e.g., Drmanac et al. (2010) Science 327: 78-81). DNA is isolated,
fragmented, and size selected. For example, DNA is fragmented
(e.g., by sonication) to a mean length of about 500 bp. Adaptors
(Ad1) are attached to the ends of the fragments. The adaptors are
used to hybridize to anchors for sequencing reactions. DNA with
adaptors bound to each end is PCR amplified. The adaptor sequences
are modified so that complementary single strand ends bind to each
other forming circular DNA. The DNA is methylated to protect it
from cleavage by a type IIS restriction enzyme used in a subsequent
step. An adaptor (e.g., the right adaptor) has a restriction
recognition site, and the restriction recognition site remains
non-methylated. The non-methylated restriction recognition site in
the adaptor is recognized by a restriction enzyme (e.g., Acul), and
the DNA is cleaved by Acul 13 bp to the right of the right adaptor
to form linear double stranded DNA. A second round of right and
left adaptors (Ad2) are ligated onto either end of the linear DNA,
and all DNA with both adapters bound can be PCR amplified (e.g., by
PCR). Ad2 sequences are modified to allow them to bind each other
and form circular DNA. The DNA is methylated, but a restriction
enzyme recognition site remains non-methylated on the left Ad1
adapter. A restriction enzyme (e.g., Acul) is applied, and the DNA
is cleaved 13 bp to the left of the Ad1 to form a linear DNA
fragment. A third round of right and left adaptor (Ad3) is ligated
to the right and left flank of the linear DNA, and the resulting
fragment is PCR amplified. The adaptors are modified so that they
bind to each other and form circular DNA. A type III restriction
enzyme (e.g., EcoP15) is added; EcoP15 cleaves the DNA 26 bp to the
left of Ad3 and 26 bp to the right of Ad2. This cleavage removes a
large segment of DNA and linearize the DNA once again. A fourth
round of right and left adaptors (Ad4) is ligated to the DNA, the
DNA is amplified (e.g., by PCR), and modified so that they bind
each other and form the completed circular DNA template. Rolling
circle replication (e.g., using Phi 29 DNA polymerase) is used to
amplify small fragments of DNA. The four adaptor sequences contain
palindromic sequences that hybridize and a single strand fold onto
itself to form a DNA nanoball (DNB.TM.) which in some cases, is
approximately 200-300 nanometers in diameter on average. A DNA
nanoball is attached (e.g., by adsorption) to a microarray
(sequencing flowcell). The flow cell is a silicon wafer coated with
silicon dioxide, titanium and hexamehtyldisilazane (HMDS) and a
photoresist material. Sequencing is performed by unchained
sequencing by ligating fluorescent probes to the DNA. The color of
the fluorescence of an interrogated position is visualized by a
high resolution camera. The identity of nucleotide sequences
between adaptor sequences is determined.
[0148] In some embodiments, the next generation sequencing
technique is Helicos True Single Molecule Sequencing (tSMS) (see
e.g., Harris T. D. et al. (2008) Science 320:106-109). In the tSMS
technique, a DNA sample is cleaved into strands of approximately
100 to 200 nucleotides, and a polyA sequence is added to the 3' end
of each DNA strand. Each strand is labeled by the addition of a
fluorescently labeled adenosine nucleotide. The DNA strands are
then hybridized to a flow cell, which contain millions of oligo-T
capture sites immobilized to the flow cell surface. The templates
are at a density of about 100 million templates/cm.sup.2. The flow
cell is then loaded into an instrument, e.g., HELISCOPE.TM.
sequencer, and a laser illuminate the surface of the flow cell,
revealing the position of each template. A CCD camera maps the
position of the templates on the flow cell surface. The template
fluorescent label is cleaved and washed away. The sequencing
reaction begins by introducing a DNA polymerase and a fluorescently
labeled nucleotide. The oligo-T nucleic acid serves as a primer.
The DNA polymerase incorporates the labeled nucleotides to the
primer in a template directed manner. The DNA polymerase and
unincorporated nucleotides are removed. The templates that have
directed incorporation of the fluorescently labeled nucleotide are
detected by imaging the flow cell surface. After imaging, a
cleavage step removes the fluorescent label, and the process is
repeated with other fluorescently labeled nucleotides until a
desired read length is achieved. Sequence information is collected
with each nucleotide addition step. The sequencing is asynchronous.
The sequencing comprises at least 1 billion bases per day or per
hour.
[0149] In some embodiments, a sequencing technique comprises
paired-end sequencing in which both the forward and reverse
template strand is sequenced. In some embodiments, the sequencing
technique comprises mate pair library sequencing. In mate pair
library sequencing, DNA comprises fragments, and 2-5 kb fragments
are end-repaired (e.g., with biotin labeled dNTPs). The DNA
fragments are circularized, and non-circularized DNA are removed by
digestion. Circular DNA are fragmented and purified (e.g., using
the biotin labels). Purified fragments are end-repaired and ligated
to sequencing adaptors.
[0150] In some embodiments, a sequencing method comprises Sanger
sequencing, Maxam-Gilbert sequencing, Shotgun sequencing, bridge
PCR, mass spectrometry based sequencing, microfluidic based Sanger
sequencing, microscopy-based sequencing, RNAP sequencing, or
hybridization based sequencing. Sanger sequencing utilizes a
chain-termination method which relies on selective incorporation of
chain-terminating dideoxynucleotides by DNA polymerases during
replication. Maxam-Gilbert sequencing utilizes chemical
modification of DNA and subsequent cleavage at specific bases. In a
shotgun sequencing method, DNA is randomly fragmented and then
sequenced using chain termination methods to obtain reads. Multiple
overlapping reads are pooled and assembled into a continuous
sequence. In a bridge PCR method, DNA is fragmented and then
amplified by solid surface tethered primers to form "DNA colonies"
or "DNA clusters". Multiple overlapping "DNA colonies" or "DNA
clusters" are pooled and assembled into a continuous sequence. In a
mass spectrometry-based sequencing, DNA fragments are generated by
chain-termination sequencing methods and the fragments are read by
mass spectrometries such as matrix-assisted laser desorption
ionization time-of-flight mass spectrometry (MALDI-TOF MS). In a
microfluidic Sanger sequencing method, amplification of the DNA
fragments and their separation are achieved on a single glass
wafer. In a microscopy-based method, electron microscopy such as
transmission electron microscopy DNA sequencing are used to
visualize DNA bases labeled with heavy atoms. A RNAP sequencing
method utilizes the distinct motions that RNA polymerase generates
during transcription of each nucleotide base and generates a
sequence based on this motion. A hybridization-based sequencing
utilizes a DNA microarray in which a single pool of DNA of interest
is fluorescently labeled and hybridized to an array containing
known sequences. Strong hybridization signals from a particular
spot on the array allow identification of the sequence of the DNA
of interest.
[0151] In some instances, amplification methodologies are used to
amplify the nucleic acid sequences. Exemplary amplification
methodologies include, but are not limited to, polymerase chain
reaction (PCR), nucleic acid sequence based amplification (NASBA),
self-sustained sequence replication (3 SR), loop mediated
isothermal amplification (LAMP), strand displacement amplification
(SDA), whole genome amplification, multiple displacement
amplification, strand displacement amplification, helicase
dependent amplification, nicking enzyme amplification reaction,
recombinant polymerase amplification, reverse transcription PCR,
ligation mediated PCR, or methylation specific PCR.
[0152] In some instances, additional methods that are used to
obtain a nucleic acid sequence include, e.g., array-based
comparative genomic hybridization, detecting single nucleotide
polymorphisms (SNPs) with arrays, subtelomeric fluorescence in situ
hybridization (ST-FISH) (e.g., to detect submicroscopic copy-number
variants (CNVs)), DNA microarray, high-density oligonucleotide
microarray, whole-genome RNA expression array, peptide microarray,
enzyme-linked immunosorbent assay (ELISA), genome sequencing, de
novo sequencing, Pacific Biosciences SMRT sequencing, Genia
Technologies nanopore single-molecule DNA sequencing, Oxford
Nanopore single-molecule DNA sequencing, polony sequencing, copy
number variation (CNV) analysis sequencing, small nucleotide
polymorphism (SNP) analysis, immunohistochemistry (IHC),
immunoctyochemistry (ICC), mass spectrometry, tandem mass
spectrometry, matrix-assisted laser desorption ionization time of
flight mass spectrometry (MALDI-TOF MS), in-situ hybridization,
fluorescent in-situ hybridization (FISH), chromogenic in-situ
hybridization (CISH), silver in situ hybridization (SISH), digital
PCR (dPCR), reverse transcription PCR, quantitative PCR (Q-PCR),
single marker qPCR, real-time PCR, nCounter Analysis (Nanostring
technology), Western blotting, Southern blotting, SDS-PAGE, gel
electrophoresis, and Northern blotting.
Maintenance Therapy
[0153] Provided herein are methods for maintenance therapy of
subject having a B-cell proliferative disorder. In some
embodiments, B-cell proliferative disorder is cancer. In some
embodiments, the cancer is hematologic cancer. In some embodiments,
the methods for maintenance therapy comprise treating a hematologic
cancer with a covalent and/or irreversible BTK inhibitor for an
initial treatment period, followed by a maintenance therapy
regimen. In some embodiments, the methods for maintenance therapy
comprise treating a hematologic cancer with a covalent and/or
irreversible BTK inhibitor for a period of six months or longer,
such as, for example, 6 months, 7 months, 8 months, 9 months, 10
months, 11 months, 12 months, 13 months, 14 months, 15 months, 16
months, 17 months, 18 months, 19 months, 20 months, 21 months, 22
months, 23 months, 24 months, 25 months, 26 months, 27 months, 28
months, 29 months, 30 months, 31 months, 32 months, 33 months, 34
months, 35 months, 3 years, 4 years, 5 years, 6 years, 7 years, 8
years, 9 years, 10 years or longer. In some embodiments, the
irreversible BTK inhibitor covalently binds to cysteine 481 of the
wild-type BTK. In some embodiments, the irreversible BTK inhibitor
is selected from among ibrutinib, PCI-45292, PCI-45466,
AVL-101/CC-101 (Avila Therapeutics/Celgene Corporation),
AVL-263/CC-263 (Avila Therapeutics/Celgene Corporation),
AVL-292/CC-292 (Avila Therapeutics/Celgene Corporation),
AVL-291/CC-291 (Avila Therapeutics/Celgene Corporation), CNX 774
(Avila Therapeutics), BMS-488516 (Bristol-Myers Squibb), BMS-509744
(Bristol-Myers Squibb), CGI-1746 (CGI Pharma/Gilead Sciences),
CGI-560 (CGI Pharma/Gilead Sciences), CTA-056, GDC-0834
(Genentech), HY-11066 (also, CTK4I7891, HMS3265G21, HMS3265G22,
HMS3265H21, HMS3265H22, 439574-61-5, AG-F-54930), ONO-4059 (Ono
Pharmaceutical Co., Ltd.), ONO-WG37 (Ono Pharmaceutical Co., Ltd.),
PLS-123 (Peking University), RN486 (Hoffmann-La Roche), HM71224
(Hanmi Pharmaceutical Company Limited), LFM-A13, BGB-3111
(Beigene), KBP-7536 (KBP BioSciences), ACP-196 (Acerta Pharma),
JTE-051 (Japan Tobacco Inc), PRN1008 (Principia), CTP-730 (Concert
Pharmaceuticals), or GDC-0853 (Genentech). In some embodiments, the
irreverisble BTK inhibitor is selected from among ibrutinib,
PCI-45292, PCI-45466, AVL-101, AVL-291, AVL-292, ONO-WG-37 or
(R)-6-amino-9-(1-but-2-ynoylpyrrolidin-3-yl)-7-(4-phenoxyphenyl)-7H-purin-
-8(9H)-one. In some embodiments, the irreversible BTK inhibitor is
ibrutinib. In some embodiments, the irreversible BTK inhibitor is
(R)-6-amino-9-(1-but-2-ynoylpyrrolidin-3-yl)-7-(4-phenoxyphenyl)-7H-purin-
-8(9H)-one.
[0154] In some embodiments, provided is a method of maintenance
therapy in a patient having a hematologic cancer, comprising: (a)
administering to the patient a maintenance therapy regimen
comprising administering a therapeutically effective dose of a BTK
inhibitor; and (b) monitoring the patient at predetermined
intervals of time over the course of the maintenance therapy
regimen to determine whether the subject has mutation in an
endogenous gene encoding PLC.gamma.2 that results in a modification
at an amino acid position corresponding to amino acid position 742,
845, or 1140 of the amino acid sequence set forth in SEQ ID NO: 2.
In some embodiments, the modification in the PLC.gamma.2
polypeptide is R742P. In some embodiments, the modification in the
PLC.gamma.2 polypeptide is L845F. In some embodiments, the
modification in the PLC.gamma.2 polypeptide is D1140G. In some
embodiments, the modification in the PLC.gamma.2 polypeptide
further comprises additional modifications. In some embodiments,
the method further comprises determining whether the subject has
mutations in PLC.gamma.2 and an additional polypeptide. In some
embodiments, the additional polypeptide is a polypeptide that
encoded by a gene associated in the BCR pathway. In some
embodiments, the additional polypeptide is BTK. In some
embodiments, the additional mutation results in a modified BTK
polypeptide having an amino acid substitution at C481. In some
embodiments, the additional mutation results in a modified BTK
polypeptide having an amino acid substitution selected from among
C481F, C481S, C481Y, and C481R. In some embodiments, the additional
mutation results in a modified BTK polypeptide having an amino acid
substitution at L527. In some embodiments, the additional mutation
results in a modified BTK polypeptide having an amino acid
substitution that is L527W. In some embodiments, the addition
mutation is in a gene is selected from TP53, c-MYC, BCL6, IGHV,
CD38, CSF1, DAB1, ARTN, COL8A2 or LDLRAP1 located on chromosome 1;
PRR21, NDUFA10, ASIC4, POTEE or XPO1 located on chromosome 2;
RAB6B, TMPRSS7 or CACNA1D located on chromosome 3; GUCY1B3, MAML3,
FRAS1 or EVC2 located on chromosome 4; NPM1, G3BP1, H2AFY, HEATR7B2
or ADAMTS12 located on chromosome 5; KIAA1244, ENPP1, NKAIN2,
REV3L, COL12A1 or IRF4 located on chromosome 6; ZNF775, SSPO,
ZNF777 or ABCA13 located on chromosome 7; TRPS1 located on
chromosome 8; UAP1L1, AGPAT2, SNAPC4, RALGPS1 or GNAQ located on
chromosome 9; PIK3AP1, EGR2 or NRP1 located on chromosome 10;
KRTAP5-9, CAPN1 or MUC2 located on chromosome 11; DPY19L2, KRT73,
SLC11A2, MLL2, SYT10 or OVOS2 located on chromosome 12; TRPC4
located on chromosome 13; SLC8A3 located on chromosome 14; BLM,
DISP2 or C15orf55 located on chromosome 15; MMP25 or MAPK8IP3
located on chromosome 16; LLGL2, KRTAP9-3, TRAF4, CENPV or TP53
located on chromosome 17; CEACAM18, SPIB, TPRX1, DMKN, LSM4,
CACNA1A, CCDC151, LONP1 or STAP2 located on chromosome 19; TSPEAR,
KCNJ15, DYRK1A or IFNAR1 located on chromosome 21; SLC5A4 or HIRA
located on chromosome 22; or BTK, IL13RA2, MAGEE1, SHROOM4 or NYX
located on chromosome X. In some embodiments, the method further
comprises discontinuing maintenance therapy regimen if the subject
has one or more mutations with at least one modification at amino
acid position 742, 845, or 1140 in PLC.gamma.2 polypeptide. In some
embodiments, the method further comprises discontinuing maintenance
therapy regimen if the subject has no mutation at amino acid
position 742, 845, or 1140 in PLC.gamma.2 polypeptide but has
additional mutations in PLC.gamma.2 polypeptide and/or has
mutations in an additional polypeptide. In some embodiments, the
method further comprises administering an inhibitor of PLC.gamma.2
if the subject has one or more modifications with at least one
modification at amino acid position 742, 845, or 1140 in the
PLC.gamma.2 polypeptide. In some embodiments, the method further
comprises administering an inhibitor of LYN, SYK, JAK, PI3K, MAPK,
MEK or NF.kappa.B if the subject has one or more modifications with
at least one modification at amino acid position 742, 845, or 1140
in PLC.gamma.2 polypeptide. In some embodiments, the method further
comprises continuing maintenance therapy regimen if the subject has
no mutations in the PLC.gamma.2 polypeptide. In some embodiments,
the method further comprises continuing maintenance therapy regimen
if the subject has no mutations in the PLC.gamma.2 polypeptide but
has mutations in an additional polypeptide. In some embodiments,
the method further comprises continuing maintenance therapy regimen
if the subject has no mutations in the PLC.gamma.2 polypeptide or
in the additional polypeptide. In some embodiments, the additional
polypeptide is a BTK polypeptide. In some embodiments, the
predetermined interval of time is every month, every 2 months,
every 3 months, every 4 months, every 5 months, every 6 months,
every 7 months or every 8 months.
[0155] In some embodiments, the BTK inhibitor is administered at a
daily dosage of about 10 mg per day to about 2000 mg per day, about
50 mg per day to about 1500 mg per day, about 100 mg per day to
about 1000 mg per day, about 250 mg per day to about 850 mg per
day, or about 300 mg per day to about 600 mg per day. In some
embodiments, ibrutinib is administered at a daily dosage of about
140 mg per day, 420 mg per day, 560 mg per day or 840 mg per day.
In some embodiments, the BTK inhibitor is a covalent and/or
irreversible BTK inhibitor. In some embodiments, the BTK inhibitor
is selected from among ibrutinib, PCI-45292, PCI-45466,
AVL-101/CC-101 (Avila Therapeutics/Celgene Corporation),
AVL-263/CC-263 (Avila Therapeutics/Celgene Corporation),
AVL-292/CC-292 (Avila Therapeutics/Celgene Corporation),
AVL-291/CC-291 (Avila Therapeutics/Celgene Corporation), CNX 774
(Avila Therapeutics), BMS-488516 (Bristol-Myers Squibb), BMS-509744
(Bristol-Myers Squibb), CGI-1746 (CGI Pharma/Gilead Sciences),
CGI-560 (CGI Pharma/Gilead Sciences), CTA-056, GDC-0834
(Genentech), HY-11066 (also, CTK417891, HMS3265G21, HMS3265G22,
HMS3265H21, HMS3265H22, 439574-61-5, AG-F-54930), ONO-4059 (Ono
Pharmaceutical Co., Ltd.), ONO-WG37 (Ono Pharmaceutical Co., Ltd.),
PLS-123 (Peking University), RN486 (Hoffmann-La Roche), HM71224
(Hanmi Pharmaceutical Company Limited), LFM-A13, BGB-3111
(Beigene), KBP-7536 (KBP BioSciences), ACP-196 (Acerta Pharma),
JTE-051 (Japan Tobacco Inc), PRN1008 (Principia), CTP-730 (Concert
Pharmaceuticals), or GDC-0853 (Genentech). In some embodiments, the
BTK inhibitor is selected from among ibrutinib (PCI-32765),
PCI-45292, PCI-45466, AVL-101, AVL-291, AVL-292, ONO-WG-37 or
(R)-6-amino-9-(1-but-2-ynoylpyrrolidin-3-yl)-7-(4-phenoxyphenyl)-7H-purin-
-8(9H)-one. In some embodiments, the BTK inhibitor is ibrutinib. In
some embodiments, the BTK inhibitor is
(R)-6-amino-9-(1-but-2-ynoylpyrrolidin-3-yl)-7-(4-phenoxyphenyl)-7H-purin-
-8(9H)-one.
[0156] In some embodiments, the subject is monitored every month,
every 2 months, every 3 months, every 4 months, every 5 months,
every 6 months, every 7 months, every 8 months, every 9 months,
every 10 months, every 11 months, or every year to determine
whether the subject acquires mutation in an endogenous gene
encoding PLC.gamma.2 that results in modifications at R742, L845,
D1140 of the PLC.gamma.2 polypeptide.
[0157] In some embodiments, the subject possesses high-risk
cytogenetic features. In some embodiments, the subject possessing
high-risk cytogenetic features has cytogenetic abnormalities
selected from trisomy 12, del(11q22.3), del(13q14.3), del(17p13.1),
t(11;14)(q13;q32), t(14;19)(q32;q13) or t(2;14)(p13;q32). In some
embodiments, the subject possessing high-risk cytogenetic features
has del(11q22.3), del(17p13.1) or a complex karyotype. In some
embodiments, the subject possessing high-risk cytogenetic features
has del(11q22.3). In some embodiments, the subject possessing
high-risk cytogenetic features has del(17p13.1). In some
embodiments, the subject possessing high-risk cytogenetic features
has a complex karyotype.
[0158] In some embodiments, the subject has cancer. In some
embodiments, the cancer is a hematologic cancer. In some
embodiments, the cancer is a B-cell malignancy. In some
embodiments, cancer is selected from among a leukemia, a lymphoma,
or a myeloma. In some embodiments, the hematologic cancer is a
B-cell malignancy. In some embodiments, the B-cell malignancy is
chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma
(SLL), diffuse large B-cell lymphoma (DLBCL), activated B-cell
diffuse large B-cell lymphoma (ABC-DLBCL), germinal center diffuse
large B-cell lymphoma (GCB DLBCL), double-hit diffuse large B-cell
lymphoma (DH-DLBCL), primary mediastinal B-cell lymphoma (PMBL),
non-Hodgkin lymphoma, Burkitt's lymphoma, follicular lymphoma,
immunoblastic large cell lymphoma, precursor B-lymphoblastic
lymphoma, precursor B-cell acute lymphoblastic leukemia, hairy cell
leukemia, mantle cell lymphoma, B cell prolymphocytic leukemia,
lymphoplasmacytic lymphoma/Waldenstrom macroglobulinemia, splenic
marginal zone lymphoma, plasma cell myeloma, plasmacytoma,
extranodal marginal zone B cell lymphoma, nodal marginal zone B
cell lymphoma, mediastinal (thymic) large B cell lymphoma,
intravascular large B cell lymphoma, primary effusion lymphoma, or
lymphomatoid granulomatosis. In some embodiments, the subject has a
solid tumor. In some embodiments, the B-cell malignancy is chronic
lymphocytic leukemia (CLL).
[0159] In some embodiments, maintenance therapy comprises multiple
cycles of administration. In some embodiments, a cycle of
administration is one month, 2 months, 3 months, 4 months, 6
months, 6 months, 7 months, 8 months, 9 months, 10 months, 11
months, 12 months or longer. In some embodiments, a cycle of
administration comprises administration of a single therapeutic
dosage of the irreversible BTK inhibitor over the cycle. In some
embodiments, a cycle of administration comprises two or more
different dosages of the irreversible BTK inhibitor over the cycle.
In some embodiments, the dosage of the irreversible BTK inhibitor
differs over consecutive cycles. In some embodiments, the dosage of
the irreversible BTK inhibitor increases over consecutive cycles.
In some embodiments, the dosage of the irreversible BTK inhibitor
is the same over consecutive cycles.
[0160] In some embodiments, maintenance therapy comprises
administration of a daily dosage of the irreversible BTK inhibitor.
In some embodiments, the daily dosage of the irreversible BTK
inhibitor administered is at or about 10 mg per day to about 2000
mg per day, such as for example, about 50 mg per day to about 1500
mg per day, such as for example about 100 mg per day to about 1000
mg per day, such as for example about 250 mg per day to about 850
mg per day, such as for example about 300 mg per day to about 600
mg per day. In a particular embodiment, the maintenance dosage of
the irreversible BTK inhibitor is about 840 mg per day. In a
particular embodiment, where the irreversible inhibitor is
ibrutinib, the maintenance dosage is about 840 mg ibrutinib per
day. In a particular embodiment, the maintenance dosage of the
irreversible BTK inhibitor is about 560 mg per day. In a particular
embodiment, where the irreversible inhibitor is ibrutinib, the
maintenance dosage is about 560 mg ibrutinib per day. In a
particular embodiment, the maintenance dosage is about 420 mg per
day. In a particular embodiment, where the irreversible inhibitor
is ibrutinib, the maintenance dosage is about 420 mg ibrutinib per
day. In a particular embodiment, the maintenance dosage of the
irreversible BTK inhibitor is about 140 mg per day. In a particular
embodiment, where the irreversible inhibitor is ibrutinib, the
maintenance dosage is about 140 mg ibrutinib per day.
[0161] In some embodiments, the irreversible BTK inhibitor is
administered once per day, two times per day, three times per day
or more frequent. In a particular embodiment, the irreversible BTK
inhibitor is administered once per day. In some embodiments, the
irreversible BTK inhibitor that is ibrutinib is administered once
per day, two times per day, three times per day or more frequent.
In a particular embodiment, the irreversible BTK inhibitor that is
ibrutinib is administered once per day.
[0162] In some embodiments, the dosage of the irreversible BTK
inhibitor is escalated over time. In some embodiments, the dosage
of the irreversible BTK inhibitor that is ibrutinib is escalated
over time. In some embodiments, the dosage of the irreversible BTK
inhibitor is escalated from at or about 1.25 mg/kg/day to at or
about 12.5 mg/kg/day over a predetermined period of time. In some
embodiments, the dosage of the irreversible BTK inhibitor that is
ibrutinib is escalated from at or about 1.25 mg/kg/day to at or
about 12.5 mg/kg/day over a predetermined period of time. In some
embodiments the predetermined period of time is over 1 month, over
2 months, over 3 months, over 4 months, over 5 months, over 6
months, over 7 months, over 8 months, over 9 months, over 10
months, over 11 months, over 12 months, over 18 months, over 24
months or longer.
[0163] In some embodiments, a cycle of administration comprises
administration of the irreversible BTK inhibitor in combination
with an additional therapeutic agent. In some embodiments the
additional therapeutic is administered simultaneously,
sequentially, or intermittently with the irreversible BTK
inhibitor. In some embodiments the additional therapeutic agent is
an anti-cancer agent. In some embodiments the additional
therapeutic agent is an anti-cancer agent for the treatment of a
leukemia, lymphoma or a myeloma. Exemplary anti-cancer agents for
administration in a combination with a covalent and/or irreversible
BTK inhibitor are provided elsewhere herein. In a particular
embodiment, the anti-cancer agent is an anti-CD 20 antibody (e.g.,
Rituxan). In a particular embodiment, the anti-cancer agent
bendamustine. In some embodiments, the additional anti-cancer agent
is a reversible BTK inhibitor. In some embodiments, the additional
anti-cancer agent is a reversible BTK inhibitor that does not
depend on cysteine 481 for binding to BTK. In some embodiments, the
additional anti-cancer agent is dasatinib.
Identification of Molecules that Interact with Mutant
PLC.gamma.2
[0164] Provided herein are methods of screening compounds that
inhibit a modified PLC.gamma.2, comprising: (a) providing a
modified PLC.gamma.2, wherein the modified PLC.gamma.2 is modified
at amino acid position corresponding to amino acid position 742,
845, or 1140 of the amino acid sequence set forth in SEQ ID NO: 2;
(b) contacting the modified PLC.gamma.2 with a test compound; and
(c) detecting the level of PLC.gamma.2 activity, wherein a decrease
in activity indicates that the compound inhibits the modified
PLC.gamma.2. In some embodiments, the modification is a
substitution, addition or deletion of the amino acid at position
742, 845, or 1140 of the PLC.gamma.2 polypeptide. In some
embodiments, detecting the level of PLC.gamma.2 activity is
assessed by an in vitro assay (e.g., calcium flux assay,
co-localization assay or kinase assay). In some embodiments,
detecting the level of PLC.gamma.2 activity is assessed by
measuring the level of calcium within a cell. In some embodiments,
the cell is a B lymphocyte, a monocyte, or a macrophage. In some
embodiments, the cell is a cancer cell line, such as a lymphoma,
leukemia, or myeloma cell line. In some embodiments, the cell line
is a MCL, DBCL or a follicular lymphoma cell line. In some
embodiments, the cell line is a BTK knockout B lymphoma cell line,
such as the DT40 BTK knockout cell line. In some embodiments,
antibodies are used to detect the level and location of particular
PLC.gamma.2 targets. In some embodiments, the cells are first
stimulated to activate BCR signaling pathway prior to, during or
following exposure to the test compound. In some embodiments, the
cells are first stimulated with anti-IgM or anti-IgG to activate
BCR signaling pathway prior to, during or following exposure to the
test compound.
[0165] In some embodiments, a host cell line that can be
transfected with nucleic acid encoding the modified PLC.gamma.2
polypeptide and in which PLC.gamma.2 activity can be monitored is
used in the method. In some embodiments, the host cell does not
express wild-type PLC.gamma.2. In some embodiments, the host cell
is deficient for the expression of endogenous wild-type
PLC.gamma.2. In some embodiments, the host cell expressing the
modified PLC.gamma.2 polypeptide stably expresses the modified
PLC.gamma.2 polypeptide. In some embodiments, the nucleic acid
encoding the modified PLC.gamma.2 polypeptide is integrated into
the genome of the cell.
[0166] In some embodiments, the host cell is a chicken DT40 BTK-/-B
cell. In some embodiments, the cell is a non B-cell. In some
embodiments, the cell is a mammalian non-B-cell. In some
embodiments, the cell is a 293 cell. In some embodiments, the cell
is a non-mammalian cell. In some embodiments, the cell is an insect
cell, a bacterial cell, a yeast cell, or a plant cell.
[0167] Cellular functional assays for BTK inhibition include
measuring one or more cellular endpoints in response to stimulating
a PLC.gamma.2-mediated pathway in a cell line in the absence or
presence of a range of concentrations of a candidate PLC.gamma.2
inhibitor compound. Useful endpoints for determining a response to
BCR activation include, e.g., inhibition of IP2 into IP3 or
cytoplasmic calcium flux.
[0168] In some embodiments, a downstream transcription target assay
is employed to determine BTK activity in the presence or absence of
the test compounds. In some embodiments, the downstream
transcription target assay is an NF-.kappa.B based assay. In some
example, a gene encoding a reporter protein is operably linked to
an NF-.kappa.B responsive promoter that is sensitive to BCR pathway
signaling and is inhibited when BTK is inhibited. In some
embodiments, the reporter gene encodes a protein selected from
among a luciferase, a fluorescent protein, a bioluminescent
protein, or an enzyme. In some embodiments, the assay comprises a
host cell that contains the reporter and the mutant BTK. Detection
of the level of gene expression in the presence or absence of the
test compound indicates whether the test compound inhibits the BCR
pathway in the presence of the mutant BTK. In some embodiments, the
test compound inhibits the mutant PLC.gamma.2 directly.
[0169] High throughput assays for many cellular biochemical assays
(e.g., kinase assays) and cellular functional assays (e.g., calcium
flux) are well known to those of ordinary skill in the art. In
addition, high throughput screening systems are commercially
available (see, e.g., Zymark Corp., Hopkinton, Mass.; Air Technical
Industries, Mentor, Ohio; Beckman Instruments, Inc. Fullerton,
Calif.; Precision Systems, Inc., Natick, Mass., etc.). These
systems typically automate entire procedures including sample and
reagent pipetting, liquid dispensing, timed incubations, and final
readings of the microplate in detector(s) appropriate for the
assay. Automated systems thereby allow the identification and
characterization of a large number of BTK inhibitor compounds
without undue effort.
[0170] In some embodiments, detecting the level of PLC.gamma.2
activity is assessed by an in vivo assay. In some embodiments,
detecting the level of PLC.gamma.2 activity is assessed in animal
model. In some embodiments the animal model is one that is a mouse
model of leukemia. Such animal model is well-known in the art and
includes, for example, mouse models, of AML and CLL (see, e.g.,
Zuber, (2009) Genes and Development 23(7):877-89 and Pekarsky et
al. (2007) J Cell Biochem. 100(5):1109-18. In some embodiments the
animal model is a transgenic animal that expresses a modified
PLC.gamma.2 that is modified at R742, L845, or D1140. In some
embodiments, a test compound is administered to a transgenic animal
that expresses a modified PLC.gamma.2 that is modified at R742,
L845, or D1140 and the activity of PLC.gamma.2 is assessed by one
or more assays described herein. In some embodiments, the assay is
performed with the mutant PLC.gamma.2 polypeptide isolated from the
transgenic animal administered the test compound and compared to a
control. In some embodiments, the level of phosphorylation,
translocation or calcium flux of one or more BTK targets is
assessed in a B-cell sample from the transgenic animal administered
the test compound and compared to a control. In some embodiments,
the control is a sample from an animal not administered the test
compound. In some embodiments, the control is a sample from an
animal administered a covalent and/or irreversible BTK
inhibitor.
Kits and Articles of Manufacture
[0171] For use in the diagnostic and therapeutic applications
described herein, kits and articles of manufacture are also
described herein. Such kits can comprise a carrier, package, or
container that is compartmentalized to receive one or more
containers such as vials, tubes, and the like, each of the
container(s) comprising one of the separate elements to be used in
a method described herein. Suitable containers include, for
example, bottles, vials, syringes, and test tubes. The containers
are formed from any acceptable material including, e.g., glass or
plastic.
[0172] Described herein, in certain embodiments, is a kit
comprising one or more reagents for the detection of a modified
PLC.gamma.2 polypeptide comprising a modification at amino acid
position 742, 845, or 1140. In some embodiments, the kit comprises
a microchip comprising a mutant PLC.gamma.2 polypeptide having a
modification that is R742P, L845F, or D1140G.
[0173] Described herein, in certain embodiments, is a kit
comprising one or more reagents for the detection of nucleic acid
encoding a mutant PLC.gamma.2 polypeptide comprising a modification
at amino acid position 742, 845, or 1140. In some embodiments, the
kit comprises a microchip comprising nucleic acid encoding a mutant
PLC.gamma.2 polypeptide having a modification that is R742P, L845F,
or D1140G.
[0174] In some embodiments, the kits provided herein are for use in
detecting nucleic acid encoding modified PLC.gamma.2 polypeptide in
a subject or for detecting modified PLC.gamma.2 polypeptide in a
subject. In some embodiments, the kits provided herein are for use
as a companion diagnostic with one or more covalent and/or
irreversible BTK inhibitors. In some embodiments the kits are
employed for selecting patients for treatment with a PLC.gamma.2
inhibitor, for identifying subjects as resistant or likely to
become resistant to a covalent and/or irreversible BTK inhibitor,
for monitoring the development of resistance to a covalent and/or
irreversible BTK inhibitor, or combinations thereof. The kits
provided herein contain one or more reagents for the detection of
the nucleic acid encoding modified PLC.gamma.2 polypeptide, for the
detection of modified PLC.gamma.2 polypeptide, for detection of
PLC.gamma.2 activities in cells from the subject, for detection of
PLC.gamma.2 activities in vitro or in vivo or combinations thereof.
Exemplary reagents include but are not limited to, oligonucleotide,
PCR reagents, buffers, antibodies, BTK substrates for determining
kinase activity, substrates for enzymatic staining, chromagens or
other materials, such as slides, containers, microtiter plates, and
optionally, instructions for performing the methods. Those of skill
in the art will recognize many other possible containers and plates
and reagents that can be used for contacting the various materials.
Kits also can contain control samples, such as for example, nucleic
acids or proteins, such as for example a mutant PLC.gamma.2
polypeptide provided herein or nucleic acids encoding a modified
PLC.gamma.2 polypeptide provided herein. In some embodiments, kits
contain one or more set of oligonucleotide primers for detection of
mutant PLC.gamma.2 expression.
[0175] In some embodiments, the container(s) can comprise one or
more inhibitors of PLC.gamma.2 identified by the methods described
herein, optionally in a composition or in combination with another
agent as disclosed herein. The container(s) optionally have
materials, such as syringes, needles, dosing cups or vials, for
administration. Such kits optionally comprise a compound with an
identifying description or label or instructions relating to its
use in the methods described herein.
[0176] In some embodiment, a kit comprises a modified PLC.gamma.2
polypeptide or a variant thereof having PLC.gamma.2 activity
comprising a modification at amino acid position corresponding to
amino acid position 742, 845, or 1140 of the amino acid sequence
set forth in SEQ ID NO: 2. In some embodiments, a kit comprises an
isolated nucleic acid of any encoding a modified BTK polypeptide
provided herein or a vector comprising such nucleic acid.
[0177] In some embodiment, a kit comprises a microchip comprising
the modified PLC.gamma.2 polypeptide provided herein or the nucleic
acid encoding modified PLC.gamma.2 polypeptide provided herein. In
some embodiments, the modification is a substitution at amino acid
position 742, 845, or 1140 of the PLC.gamma.2 polypeptide.
Production of Nucleic Acids and Polypeptides
[0178] In some embodiments, an isolated nucleic acid molecule
encoding a mutant PLC.gamma.2 polypeptide provided herein is
generated by standard recombinant methods. In some embodiments, an
isolated nucleic acid molecule encoding a mutant PLC.gamma.2
polypeptide provided herein is generated by amplification of a
mutant PLC.gamma.2 sequence from genomic DNA. In some embodiments,
an isolated nucleic acid molecule encoding a mutant PLC.gamma.2
polypeptide provided herein is generated by polymerase chain
reaction using PLC.gamma.2 sequence specific primers.
[0179] In some embodiments, an isolated nucleic acid molecule
encoding a mutant PLC.gamma.2 polypeptide provided herein is
inserted into an expression vector and expressed in a host cell or
a non-cell extract. In some embodiments, an isolated nucleic acid
molecule encoding a mutant PLC.gamma.2 polypeptide provided herein
is operatively linked to a promoter for expression of the encoding
polypeptide in a cell or non-cell extract. In some embodiments, the
promoter is a constitutive promoter. In some embodiments, the
promoter is an inducible promoter.
[0180] In some embodiments, the nucleic acid molecule encoding a
mutant PLC.gamma.2 polypeptide provided herein is "exogenous" to a
cell, which means that it is foreign to the cell into which the
vector is being introduced or that the sequence is homologous to a
sequence in the cell but in a position within the host cell nucleic
acid in which the sequence is ordinarily not found. Vectors include
plasmids, cosmids, viruses (bacteriophage, animal viruses, and
plant viruses), and artificial chromosomes (e.g., YACs). One of
skill in the art would be well equipped to construct a vector
through standard recombinant techniques, which are described in
Sambrook et al., 1989 and Ausubel et al., 1996, both incorporated
herein by reference.
[0181] Methods for the expression of a protein in a cell are well
known in the art and include, for example, expression in cells,
such as animal and plant cells. Exemplary animal cells for the
expression of mutant PLC.gamma.2 polypeptide provided herein
include but are not limited to bacteria, yeast, insect cells,
amphibian, and mammalian cells, such as for example, human,
primate, rodent, bovine, and ovine cells. In some embodiments, the
nucleic acid encoding the mutant PLC.gamma.2 is integrated into the
genome of the host cell.
[0182] In some embodiments, a method for the expression of a mutant
PLC.gamma.2 polypeptide provided herein comprises culturing a host
cell containing an expression vector encoding a mutant PLC.gamma.2
polypeptide such that the mutant PLC.gamma.2 polypeptide is
produced by the cell. In some methods, the nucleic acid encoding as
mutant polypeptide is connected to nucleic acid encoding a signal
sequence such that the signal sequence is expressed as a fusion
peptide with the mutant PLC.gamma.2 polypeptide. In some
embodiments the signal sequence allows for the secretion of the
mutant PLC.gamma.2 polypeptide by the host cell.
[0183] In some embodiments the mutant PLC.gamma.2 polypeptide is
isolated from a host cell expressing the mutant polypeptide. In
some embodiments an extract is prepared from the host cell and the
mutant PLC.gamma.2 polypeptide is isolated by purification methods
such as but not limited to chromatography or immunoaffinity with an
antibody that is specific for PLC.gamma.2 polypeptide or specific
to the mutant PLC.gamma.2 polypeptide in particular.
Antibodies
[0184] Provided herein are isolated antibodies that bind to a
modified PLC.gamma.2 polypeptide. In some embodiments, the
antibodies do not bind to or bind with lower affinity to a
wild-type PLC.gamma.2 polypeptide. In some embodiments, the
modified PLC.gamma.2 polypeptide has modifications at amino acid
position 742, 845, or 1140.
[0185] In some embodiments, mutant PLC.gamma.2 polypeptide provided
herein are detected using antibodies that specifically recognize
the mutant PLC.gamma.2 polypeptide, but do not recognize wild-type
PLC.gamma.2 polypeptide. In some embodiments, mutant PLC.gamma.2
polypeptides provided herein are detected using antibodies that
specifically recognize a mutant PLC.gamma.2 polypeptide having a
phenylalanine at amino acid position 742, 845, or 1140 but do not
recognize the wild-type PLC.gamma.2 polypeptides. In some
embodiments, antibodies are raised against one or more allelic
forms of the mutant PLC.gamma.2 polypeptide provided herein.
Techniques for using a specific protein or an oligopeptide as an
antigen to elicit antibodies that specifically recognize epitopes
on the peptide or protein are well known. In one embodiment, the
DNA sequence of the desired allelic form of the target gene is
cloned by insertion into an appropriate expression vector and
translated into protein in a prokaryotic or eukaryotic host cell.
The protein is recovered and used as an antigen to elicit the
production of specific antibodies. In another embodiment, the DNA
of the desired allelic form of the target gene is amplified by PCR
technology and is subsequently translated in vitro into protein to
be used as the antigen to elicit the production of specific
antibodies. In another embodiment, the DNA sequence of the
alternative alleles is used as a basis for the generation of
synthetic peptides representing the amino acid sequence of the
alleles for use as the antigen to elicit the production of specific
antibodies.
[0186] In some embodiments, antibodies are generated either by
standard monoclonal antibody techniques or generated through
recombinant based expression systems. See generally, Abbas,
Lichtman, and Pober, Cellular and Molecular Immunology, W. B.
Saunders Co. (1991). The term "antibodies" is meant to include
intact antibody molecules as well as antibody fragments or
derivatives, such as Fab and F(ab')2, which are capable of
specifically binding to antigen. The antibodies so produced
preferentially bind only the mutant protein produced in the allelic
form which was used as an antigen to create the antibody. Methods
of generating allele-specific antibodies are also described in U.S.
Pat. Nos. 6,200,754 and 6,054,273, the entire contents of which are
incorporated herein by reference.
[0187] In some embodiments, the antibody provided herein is a
humanized antibody. A "humanized antibody" refers to a type of
engineered antibody having its CDRs derived from a non-human donor
immunoglobulin, the remaining immunoglobulin-derived parts of the
molecule being derived from one or more human immunoglobulin(s). In
some embodiments, framework support residues are altered to
preserve binding affinity (see, e.g., Queen et al. Proc. Natl. Acad
Sci USA, 86:10029-10032 (1989), Hodgson et al. Bio/Technology,
9:421 (1991)). In some embodiments, a suitable human acceptor
antibody is one selected from a conventional database, e.g., the
KABAT.RTM. database, Los Alamos database, and Swiss Protein
database, by homology to the nucleotide and amino acid sequences of
the donor antibody. In some embodiments, a human antibody
characterized by a homology to the framework regions of the donor
antibody (on an amino acid basis) is suitable to provide a heavy
chain constant region and/or a heavy chain variable framework
region for insertion of the donor CDRs. In some embodiments, a
suitable acceptor antibody capable of donating light chain constant
or variable framework regions is selected in a similar manner. In
some embodiments, the acceptor antibody heavy and light chains
originate from the same acceptor antibody. In some embodiments, the
acceptor antibody heavy and light chains originate from the
different acceptor antibodies. The prior art describes several ways
of producing such humanized antibodies--see, for example,
EP-A-0239400 and EP-A-054951.
[0188] In some embodiments, antibodies specific for mutant
PLC.gamma.2 polypeptide provided herein are used to detect the
presence of a mutant PLC.gamma.2 polypeptide provided herein in a
sample, e.g., an assay sample, a cell sample, a cell extract, a
biological sample, or a patient sample, using techniques known in
the art. These techniques include, for example, Western blot,
immunohistochemistry, indirect immunofluorescence, and antibody
microarray. In some embodiments, an antibody which specifically
recognizes a mutant PLC.gamma.2 polypeptide is an inhibitor of
PLC.gamma.2.
EXAMPLES
[0189] These examples are provided for illustrative purposes only
and not to limit the scope of the claims provided herein.
Example 1: DNA Constructs and Cell Culture
PLC.gamma.2 Mutant Cell Lines
[0190] The pRetro-X Tet-On (Clontech, Mountainview, Calif.) or
pBABE vectors were used to generate DNA constructs of PLC.gamma.2
that were introduced through retroviral infection. R665W or L845F
mutated PLC.gamma.2 was derived by site-directed mutagenesis
(QuikChange II, Stratagene-Agilent Technologies, Santa Clara,
Calif.). Both constructs have puromycin selection markers. The
expression of wild-type or mutant PLC.gamma.2 was controlled by
promotors of either CMV in pRetro-X tet-On or SV40 promotor in
pBABE vectors. Cells were maintained in RPMI 1640 (Life
Technologies, Grand Island, N.Y.) with 2 mM L-glutamine and 10%
fetal bovine serum in addition to Penicillin/Streptomycin
antibiotics. Stably infected cells were selected and maintained by
adding puromycin (1.0 .mu.g/mL) into the cell culture medium.
Primary CLL Cells
[0191] For primary CLL cell experiments, peripheral blood
mononuclear cells were obtained using Ficoll density gradient
centrifugation. B cells were not specifically selected, but at the
time of blood acquisition, clinical flow cytometry revealed 85-98%
B cells as a percentage of total blood lymphocytes.
Example 2: Methods
DNA Sequencing
[0192] Blood was obtained from patients enrolled on Institutional
Review Board approved trials of ibrutinib. Tumor DNA was isolated
from blood mononuclear cells using AllPrep DNA/RNA Mini kit
(Qiagen). Sample preparation and whole-exome sequencing using
Agilent SureSelect Human All Exon V4 and Illumina HiSeq2000
technology was performed by Expression Analysis (Durham, N.C.).
Data Analysis Workflow
[0193] Copy number analysis of exome-seq data was performed using
VarScan 2.3.6 and the BioConductor package DNAcopy. Sequence
alignment files of primary and relapse samples were provided to
VarScan as pairs. The ratio of uniquely mapped reads were provided
to correct potential biases between primary and relapse samples.
VarScan generated relapse specific candidate regions with potential
copy number alteration. DNAcopy library was used to apply circular
binary segmentation (CBS) algorithm to identify unified regions
with copy number alterations. This generated a list of chromosomal
regions and relapse vs. primary log 2 ratios of coverage. A cutoff
of 0.59 was applied on absolute value of log 2 ratios, suggesting
at least a copy gain or loss. To identify genes affected by copy
number alterations Bedtools intersect function on RefGene
annotations was used.
[0194] FIG. 2 illustrates the exome-seq analysis pipeline
flowchart. Sequencing reads were aligned to the human reference
genome (1000 Genomes Project human assembly/GRCh37) with BWA
(v0.7.5). After potential PCR/optical duplicates were marked with
Picard (v1.94, picard.sourceforge.net), local realignment around
indels were performed with the Genome Analysis Toolkit (GATK
v2.8.1), relapse-specific single point mutations and indels were
detected with MuTect (v1.1.4) and GATK Somatic Indel Detector,
respectively. After filtering out variants previously reported in
dbSNP (build 137), variants were annotated and their potential
mutational effects predicted with SnpEff (v3.4,). Finally, newly
acquired relapse-specific high quality nonsynonymous mutations were
verified by Sanger or Ion Torrent sequencing.
Ion Torrent Analysis
[0195] DNA was extracted from cryopreserved cells using QIAmp DNA
Mini kit (Qiagen; Hilden, Germany). PLC.gamma.2 gene was analyzed
using the Ion Torrent platform from Life Technologies (Carlsbad,
Calif.). Library was prepared with Ion AmpliSeq Library kit2.0
(4475345) with custom designed panel of AmpliSeq primers that
covers the entire coding sequence and intronic splice acceptor and
donor sites for both genes and IonExpress barcode adapters
(kit#4471250). DNA was amplified on GeneAmp PCR system 9700 Dual
96-well thermal cycler from Applied Biosystems. PCR product was
purified with Agencourt AMPure XP kit (A63881 Beckman Coulter,
Indianapolis, Ind.). Library was quantified using real time PCR
with Ion Library TAQMAN Quantitation kit 44688022 on (Applied
Biosystems ViiA7 Real Time PCR System) instrument to allow for
optimal final dilution of library for template preparation on One
Touch DL version instrument with Ion One Touch 200Template Kit
v2DL(4480285). The ISPs enrichment and purification was performed
on One Touch ES using One Touch 200Template Kit v2DL(4480285).
Purified ISPs were analyzed on Ion Torrent personal Genome Machine
using IonPGM 200 Sequencing kit (4474004) and 316 chips (4466616).
Data was collected and analyzed using Torrent Server (4462616) with
Torrent Suite 3.6.2. Final analysis of sequence data was performed
using combination of software: Variant Caller v.3.6.63335,
IonTorrent IGV3.6.033 and IonReporterUploader v.3.6.2-r62834. The
following reference sequence was used for analysis; PLCG2
NM002661.3 (SEQ ID NO: 2). The entire length of sequences was
reviewed manually using these programs to assets for deviation from
reference sequence and to evaluate the quality of sequence and the
depth of coverage.
Phosphoflow and Immunoblot Assays
[0196] HEK293T cells were transiently transfected with the
indicated expression constructs, treated with ibrutinib for 1 hour,
and fixed with paraformaldehyde or washed into fresh media and then
fixed. Cells were permeabilized, stained, and analyzed on a BD FACS
Canto II.
[0197] For immunoblots, whole cell lysates were prepared and
equivalent amounts of protein were separated on polyacrylamide gels
and transferred onto nitrocellulose membranes. After antibody
incubations, proteins were detected with chemiluminescent substrate
(SuperSignal; Pierce). Antibodies against phospho-BTK(Tyr223),
phospho-AKT (Ser473), AKT, ERK1/2, phospho-PLC.gamma.2(Tyr759;
Tyr1217) were obtained from Cell Signaling Technologies (Danvers,
Mass.). Phospho-Erk(Thr202/Tyr204) and total PLC.gamma.2 were
obtained from Cell Signaling Technologies or Santa Cruz
Biotechnology (Santa Cruz, Calif.). Tubulin was obtained from
Abcam, and Actin was obtained from Santa Cruz Biotechnologies.
Calcium Flux Assays
[0198] DT40 cells stably expressing either wild-type or mutated
PLC.gamma.2 were treated with DMSO or 1 .mu.M Ibrutinib at
37.degree. C. for 30 min. The intracellular calcium level was
detected by Calcium Assay Kit (BD Biosciences, San Diego, Calif.)
and measured by Beckman Coulter DTX880 microplate reader. After 195
seconds of acquisition to determine the baseline, 3 .mu.g/ml
anti-chicken IgM (SouthernBiotech, Birmingham, Ala.) was added to
stimulate the cells.
Statistical Methods
[0199] Linear mixed models with fixed and random effects were used
to model all data from different experiments. In experiments
designed to determine if autophosphorylation was inhibited in
mutated versus wild-type cells and if this inhibition was different
under treatment with ibrutinib or dasatinib, interaction contrasts
at each concentration of interest were used to directly test the
inhibitory hypotheses, including random effects associated with
these contrasts. In the experiments testing if the increase in
calcium flux over time and following stimulation was different in
mutated cell lines treated with ibrutinib or vehicle control,
models were fit with treatment and time as fixed effects, allowing
for random intercepts and slopes for each condition and replicate.
Only data from time points where the effects of stimulation had
been observed were included (i.e. time >39 seconds). Statistical
significance was declared at .alpha.=0.05. All analyses were
performed using SAS 9.3 (SAS Institute, Cary N.C.).
Example 3: Whole Exome Sequencing Reveals Mutations in BTK and
PLC.gamma.2
[0200] Peripheral blood samples were available from patients with
progressive CLL at baseline and at the time of relapse. Whole exome
sequencing (WES) was performed on each sample. FIG. 1 illustrates
the clinical characteristics and new mutations identified at
relapse in the patients with matched samples. Table 1 illustrates
alignment statistics. On average 99 million reads were generated
for each sample. While 98% mapped to the reference genome, on
average 78% of them mapped to unique chromosomal positions and used
for further analysis. These reads provide approximately 60.times.
coverage of exonic regions. Copy number analysis was performed to
ensure identified variants were not result of potential copy number
alterations (Table 2, FIG. 3). All patients possess high-risk
cytogenetic features including del(11q22.3), del(17p13.1), or
complex karyotype. In the tested patient population, the relapse
sample revealed distinct PLC.gamma.2 mutations including a leucine
to phenylalanine mutation at position 845 (L845F; FIG. 4). In this
patient, the PLC.gamma.2 L845F mutation was found by WES. To verify
this clone, Ion Torrent sequencing was performed again at a sample
1 month following relapse and the mutation was still present (Table
3). The mutation identified by WES was confirmed by Sanger
sequencing and/or Ion Torrent deep sequencing. At baseline, no
patient had evidence of mutation in PLC.gamma.2 by WES. In patient
5, Ion Torrent sequencing was performed, and no mutation was
>0.5% of reads (Table 4). No other high-confidence recurrent
mutation was noted in any of the patients examined from diagnosis
to relapse.
Example 4: Identified Mutations of PLC.gamma.2 are Potentially Gain
of Function in the Presence of BCR Stimulation and Represent
Resistance Mechanisms in Patients
[0201] WT PLC.gamma.2 and L845F PLC.gamma.2 were stably transfected
into 293 cells and DT40 cells which lack endogenous PLC.gamma.2
expression (FIG. 5). Calcium flux was examined in DT40 cells after
anti-IgM stimulation in the presence of WT or mutant PLC.gamma.2.
The PLC.gamma.2 mutant showed enhanced IgM-mediated calcium flux
that was not inhibited by ibrutinib (FIG. 5A). This showed that the
mutation allowed for BCR-mediated signaling which was independent
of BTK. Similarly, after stimulation with anti-IgM, cells with
L845F mutation showed less inhibition in the presence of ibrutinib
than WT cells as measured by phosphorylation of ERK and AKT (FIG.
5B-5D). These data demonstrated that L845F PLC.gamma.2 is
potentially a gain of function mutation in the presence of BCR
stimulation and could be relevant resistance mutations to ibrutinib
in patients. An additional mutation in PLC.gamma.2 was also tested
and was shown to be another potential gain of function mutation in
the presence of BCR stimulation.
[0202] CLL cells were examined at baseline and at the time of
relapse from patients #5. In patient 5 who possessed a L845F
mutation in PLC.gamma.2, in vitro ibrutinib did not inhibit
PLC.gamma.2 phosphorylation (FIG. 6). These data suggest that the
gain of function phenotype seen in vitro is also relevant in
patients.
Example 5: Patients with Prolonged Lymphocytosis on Ibrutinib do
not have PLC.gamma.2 Mutation
[0203] Patients treated with ibrutinib develop a characteristic
lymphocytosis as CLL cells are mobilized from lymph nodes and
spleen. While most patients resolve their lymphocytosis within 8
months, a subset of patients have lymphocytosis that lasts >12
months in the presence of continued response to ibrutinib. To
determine whether these patients developed new mutations in
PLC.gamma.2 and may therefore be at risk for relapse, the
PLC.gamma.2 gene was sequenced using Ion Torrent technology on 9
patients with at least 12 months of lymphocytosis at 12 months
after the initiation of ibrutinib. Sequencing depth for PLC.gamma.2
at L845 was >100.times.. No patient had evidence of any mutation
of PLC.gamma.2. This suggests that known resistance mutations are
not present in patients with persistent lymphocytosis.
TABLE-US-00001 TABLE 1 Alignment Statistics # Uniquely # Mapped #
Duplicate Mapped Uniquely Exome Patient State # Reads Reads Mapped
% Reads Reads Mapped % Coverage X 1 primary 79, 983, 792 79, 473,
899 0.99 14, 216, 155 65, 257, 744 0.82 59.4 1 relapse 92, 261, 016
91, 723, 753 0.99 15, 369, 175 76, 354, 578 0.83 69.7 2 primary 83,
615, 748 83, 013, 800 0.99 13, 503, 169 69, 510, 631 0.83 63.0 2
relapse 82, 729, 482 82, 256, 076 0.99 15, 685, 173 66, 570, 903
0.80 59.5 3 primary 103, 691, 442 102, 676, 127 0.99 48, 551, 111
54, 125, 016 0.52 52.0 3 relapse 85, 019, 380 84, 190, 701 0.99 15,
568, 341 68, 622, 360 0.81 62.6 4 primary 100, 604, 310 99, 982,
483 0.99 29, 263, 406 70, 719, 077 0.70 67.0 4 relapse 103, 968,
204 103, 334, 312 0.99 27, 245, 541 76, 088, 771 0.73 71.5 5
primary 149, 140, 122 138, 197, 068 0.93 15, 773, 113 122, 423, 955
0.82 43.0 5 relapse 133, 058, 006 121, 507, 870 0.91 14, 330, 393
107, 177, 477 0.81 36.5 6 primary 90, 076, 314 89, 600, 955 0.99
21, 305, 687 68, 295, 268 0.76 61.0 6 relapse 85, 427, 322 85, 034,
356 1.00 10, 225, 110 74, 809, 246 0.88 68.3 Average 99, 131, 262
96, 749, 283 0.98 20, 086, 365 76, 662, 919 0.78 59.5
TABLE-US-00002 TABLE 2 Copy number analysis and genes affected by
copy number alterations. Log 2 ratio (relapse vs Patient Chr Start
End Width primary) Genes 2 3 20136589 24398068 4261479 -0.5996
KAT2B, LOC152024, NKIRAS1, NR1D2, RPL15, SGOL1, THRB, UBE2E1,
UBE2E2, VENTXP7, ZNF385D 2 8 133918901 135651823 1732922 -0.6082
NDRG1, SLA, ST3GAL1, TG, WISP1, ZFAT, ZFATAS 2 8 127569519
133905776 6336257 -0.6033 ADCY8, ASAP1, ASAP1IT, EFR3A, FAM49B,
FAM84B, GSDMC, HHLA1, HPYR1, KCNQ3, LOC727677, LOC728724, LRRC6,
MIR1204, MIR1205, MIR1206, MIR1207, MIR1208, MYC, OC90, PHF20L1,
POU5F1B, PVT1, TG, TMEM71 3 8 141930840 142138799 207959 -0.8497
DENND3, PTK2 3 Y 8493559 9097882 604323 -0.9623 TTTY11, TTTY18,
TTTY19 4 7 5364714 5364847 133 -1.2545 TNRC18 4 14 74766221
74769554 3333 -1.0545 ABCD4 6 1 161640950 170916399 9275449 -0.821
ADCY10, ALDH9A1, ANKRD36BP1, ATF6, ATP1B1, BLZF1, BRP44, C1orf110,
C1orf111, C1orf112, C1orf114, C1orf129, C1orf156, C1orf226, CD247,
CREG1, DCAF6, DDR2, DPT, DUSP12, DUSP27, F5, FAM78B, FCGR2B, FCRLA,
FCRLB, FMO9P, GORAB, GPA33, GPR161, HSD17B7, ILDR2, KIFAP3, LMX1A,
LOC284688, LOC400794, LOC440700, LRRC52, MAEL, METTL11B, MGC4473,
MGST3, MIR3119- 1, MIR3119-2, MIR556, MIR557, MIR921, MPZL1, NME7,
NOS1AP, NUF2, OLFML2B, PBX1, POGK, POU2F1, PRRX1, RCSD1, RGS4,
RGS5, RPL31P11, RXRG, SCYL3, SELE, SELL, SELP, SFT2D2, SH2D1B,
SLC19A2, TADA1, TBX19, TIPRL, TMCO1, UAP1, UCK2, UHMK1, XCL1, XCL2
6 1 161559078 161600706 41628 -1.267 FCGR2C, FCGR3B, HSPA7 6 1
156721019 161519393 4798374 -0.8325 ADAMTS4, AIM2, APCS, APOA2,
ARHGAP30, ARHGEF11, ATP1A2, ATP1A4, B4GALT3, C1orf192, C1orf204,
C1orf92, CADM3, CASQ1, CCDC19, CD1A, CD1B, CD1C, CD1D, CD1E, CD244,
CD48, CD5L, CD84, COPA, CRP, CYCSP52, DARC, DCAF8, DEDD, DUSP23,
ETV3, ETV3L, F11R, FCER1A, FCER1G, FCGR2A, FCGR3A, FCRL1, FCRL2,
FCRL3, FCRL4, FCRL5, FCRL6, HDGF, HSPA6, IFI16, IGSF8, IGSF9,
INSRR, ITLN1, ITLN2, KCNJ10, KCNJ9, KIRREL, KLHDC9, LOC646268, LY9,
MIR765, MNDA, MPZ, NCSTN, NDUFS2, NHLH1, NIT1, NR1I3, NTRK1,
OR10J1, OR10J3, OR10J5, OR10K1, OR10K2, OR10R2, OR10T2, OR10X1,
OR10Z1, OR6K2, OR6K3, OR6K6, OR6N1, OR6N2, OR6P1, OR6Y1, PCP4L1,
PEA15, PEAR1, PEX19, PFDN2, PIGM, PPOX, PRCC, PVRL4, PYHIN1, SDHC,
SH2D2A, SLAMF1, SLAMF6, SLAMF7, SLAMF8, SLAMF9, SPTA1, SUMO1P3,
TAGLN2, TOMM40L, TSTD1, UFC1, USF1, USP21, VANGL2, VSIG8 6 1
156646068 156714751 68683 0.6946 C1orf66, CRABP2, HDGF, ISG20L2,
MRPL24, NES 6 1 156506937 156644828 137891 0.9182 APOA1BP, BCAN,
GPATCH4, HAPLN2, IQGAP3, NES, TTC24 6 1 145301664 146737497 1435833
0.5901 ANKRD34A, ANKRD35, CD160, CHD1L, FMO5, GNRHR2, GPR89A,
GPR89C_dup1, HFE2, ITGA10, LIX1L, LOC728989, NBPF10, NBPF11_dup1,
NBPF24_dup1, NUDT17, PDIA3P, PDZK1, PDZK1P1_dup1, PEX11B, PIAS3,
POLR3C, POLR3GL, PRKAB2, RBM8A, RNF115, TXNIP 6 7 141794234
159025990 17231756 -0.8421 ABCB8, ABCF2, ABP1, ACCN3, ACTR3B,
ACTR3C, AGAP3, ARHGEF35, ARHGEF5, ASB10, ATG9B, ATP6V0E2, C7orf13,
C7orf29, C7orf33, C7orf34, CASP2, CDK5, CHPF2, CLCN1, CNPY1,
CNTNAP2, CRYGN, CTAGE15P, CTAGE4_dup1, CTAGE4_dup2, CTAGE6P, CUL1,
DNAJB6, DPP6, EN2, EPHA1, EPHB6, ESYT2, EZH2, FABP5P3, FAM115A,
FAM115C, FAM131B, FASTK, GALNT11, GALNTL5, GBX1, GIMAP1, GIMAP2,
GIMAP4, GIMAP5, GIMAP6, GIMAP7, GIMAP8, GSTK1, HTR5A, INSIG1,
KCNH2, KEL, KRBA1, LMBR1, LOC100124692, LOC100128542, LOC100128822,
LOC100131176, LOC100132707, LOC154761, LOC154822, LOC155060,
LOC202781, LOC285965, LOC285972, LOC401431, LOC728377, LOC728743,
LOC730441, LOC93432, LRRC61, MGAM, MIR153-2, MIR548F3, MIR548F4,
MIR548I4, MIR548T, MIR595, MIR671, MLL3, MNX1, MOXD2P, MTRNR2L6,
NCAPG2, NCRNA00244, NOBOX, NOM1, NOS3, NUB1, OR2A12, OR2A14,
OR2A1_dup1, OR2A1_dup2, OR2A2, OR2A20P_dup1, OR2A20P_dup2, OR2A25,
OR2A42_dup1, OR2A42_dup2, OR2A5, OR2A7, OR2A9P_dup1, OR2A9P_dup2,
OR2F1, OR2F2, OR6B1, OR6V1, OR6W1P, OR9A2, PAXIP1, PDIA4, PIP,
PRKAG2, PRSS1, PRSS2, PTPRN2, RARRES2, RBM33, REPIN1, RHEB, RNF32,
SHH, SLC4A2, SMARCD3, SSPO, TAS2R39, TAS2R40, TAS2R41, TAS2R60,
TMEM139, TMEM176A, TMEM176B, TMUB1, TPK1, TRPV5, TRPV6, TRY6,
TRYX3, UBE3C, VIPR2, WDR60, WDR86, XRCC2, ZNF212, ZNF282, ZNF398,
ZNF425, ZNF467, ZNF746, ZNF767, ZNF775, ZNF777, ZNF783, ZNF786,
ZNF862, ZYX 6 7 130562110 141764144 11202034 -0.8339 ADCK2, AGBL3,
AGK, AKR1B1, AKR1B10, AKR1B15, AKR1D1, ATP6V0A4, BPGM, BRAF,
C7orf49, C7orf55, CALD1, CHCHD3, CHRM2, CLEC2L, CLEC5A, CNOT4,
CREB3L2, DENND2A, DGKI, EXOC4, FAM180A, FLJ40852, FLJ43663, HIPK2,
JHDM1D, KIAA1147, KIAA1549, KLRG2, LOC100129148, LOC100131199,
LOC100134229, LOC100134713, LOC646329, LRGUK, LUC7L2, LUZP6, MGAM,
MIR29B1, MIR490, MKLN1, MKRN1, MRPS33, MTPN, NDUFB2, NUP205, OR9A4,
PARP12, PL-5283, PLXNA4, PODXL, PRSS37, PTN, RAB19, SLC13A4,
SLC35B4, SLC37A3, SSBP1, STRA8, SVOPL, TAS2R3, TAS2R38, TAS2R4,
TAS2R5, TBXAS1, TMEM140, TMEM213, TRIM24, TTC26, UBN2, WDR91, WEE2,
ZC3HAV1, ZC3HAV1L 6 12 10370520 23893778 13523258 -0.8412 ABCC9,
AEBP2, APOLD1, ARHGDIB, ART4, ATF7IP, BCL2L14, C12orf36, C12orf39,
C12orf60, C12orf69, CAPZA3, CDKN1B, CMAS, CREBL2, CSDA, DDX47,
DERA, DUSP16, EMP1, EPS8, ERP27, ETNK1, ETV6, GABARAPL1, GOLT1B,
GPR19, GPRC5A, GPRC5D, GRIN2B, GSG1, GUCY2C, GYS2, H2AFJ, HEBP1,
HIST4H4, HTR7P1, IAPP, KCNJ8, KIAA0528, KIAA1467, KLRA1, KLRC1,
KLRC2, KLRC3, KLRC4, KLRD1, KLRK1, LDHB, LMO3, LOC100129361,
LOC338817, LOC728622, LOH12CR1, LOH12CR2, LRP6, LST-3TM12, MAGOHB,
MANSC1, MGP, MGST1, MIR1244-1_dup2, MIR1244-2_dup2, MIR1244-3_dup2,
MIR613, MIR614, PDE3A, PDE6H, PIK3C2G, PLBD1, PLCZ1, PLEKHA5, PRB1,
PRB2, PRB3, PRB4, PRH1, PRH2, PRR4, PTPRO, PYROXD1, RECQL, RERG,
RERGL, RPL13AP20, SLC15A5, SLCO1A2, SLCO1B1, SLCO1B3, SLCO1C1,
SOX5, ST8SIA1, STRAP, STYK1, TAS2R10, TAS2R13, TAS2R14, TAS2R19,
TAS2R20, TAS2R30, TAS2R31, TAS2R42, TAS2R43, TAS2R46, TAS2R50,
TAS2R7, TAS2R8, TAS2R9, WBP11 6 14 106922029 107034811 112782
-1.1707 NCRNA00221
TABLE-US-00003 TABLE 3 Patient 5 Ion Torrent sequencing at relapse
and 1 month post-relapse Chro- Relapse 1 month post-relapse mo- AA
Variant Variant some Gene change Coverage Frequency Coverage
Frequency 16 PLC.gamma.2 R665W 278 5.4% 614 3.7% 16 PLC.gamma.2
S707Y 1570 8% 1287 6.8% 16 PLC.gamma.2 L845F 579 17.4% 806 23.8% X
BTK C481S 992 2.6% 1011 3.5%
TABLE-US-00004 TABLE 4 Baseline data for patients deep sequenced
with Ion Torrent AA Variant ID Chrom Position Gene change Reference
Variant Coverage Frequency 3 X 100611164 BTK C481S C G 693 0 5 16
81946260 PLC.gamma.2 R665W C T 928 0.1% 5 16 81953154 PLC.gamma.2
S707Y C A 2839 0 5 16 81962183 PLC.gamma.2 L845F A T 207 0 5 X
100611164 BTK C481S C G 875 0 6 16 81946260 PLC.gamma.2 R665W C T
1758 0.2%
Example 6: Acquisition of Resistance Mutations Associated with
Disease Progression on Ibrutinib Therapy: Single Center Study
[0204] 267 patients from The Ohio State University Comprehensive
Cancer Center participating in 3 Institutional Review Board
approved trials of ibrutinib were included; 196 patients received
single agent ibrutinib and 71 received ibrutinib plus ofatumumab. A
subset of patients with PD had Ion Torrent deep sequencing
performed on peripheral blood at baseline and relapse.
[0205] Fine and Gray models of cumulative incidence were fit to
identify variables associated with a particular type of
discontinuation and in the presence of competing risks. Patients
who had not discontinued study were censored at date of last
contact; patients who went off study for transplant or to continue
treatment elsewhere (n=7) were also censored at that time. Final
models included variables significant at p<0.05 using forward
selection or variables of borderline significance that were deemed
clinically meaningful, while controlling for type of therapy.
[0206] The treatment regimens of the patient groups were as
follows:
[0207] 1. OSU 10032 (PCYC 1102) N=50
[0208] Ibrutinib 420 mg or 840 mg daily until disease
progression
[0209] 2. OSU 10053 (PCYC 1109) N=71
[0210] Cohort 1: Ibrutinib 420 mg daily starting C1D1 until disease
progression; Ofatumumab start C2D1 weekly x 8 weeks, then monthly x
4 months
[0211] Cohort 2: Ibrutinib 420 mg daily starting C1D2 until disease
progression; Ofatumumab start C1D1 weekly x 8 weeks, then monthly x
4 months
[0212] Cohort 3: Ofatumumab start C1D1 weekly x 8 weeks, then
monthly x 4 months. Ibrutinib start C3D1 daily until disease
progression.
[0213] 3. OSU 11133 N=146
[0214] Ibrutinib 420 mg daily until disease progression
[0215] Results
[0216] At median follow-up of 20.2 months (range 2.6-46 months),
factors related to discontinuation on the study included
progressive disease (n=29), infection (n=25), toxicity (n=8), or
other complications (n=7) or receipt of therapy elsewhere (n=7).
FIG. 7 summarizes the cumulative incidence of CLL progression,
Richter's transformation, or other event among patients with
progressive disease. FIG. 8 summarizes baseline characteristics
associated with study discontinuation among patients with
progressive disease (e.g., CLL, Richter's) or discontinuations for
a non-progressive disease reason (e.g., infection, toxicity or
other). Both models were adjusted for type of therapy: combination
versus monotherapy with Ibrutinib. FIG. 9 illustrates the
identification of BTK and PLC.gamma.2 mutations in patients that
experienced relapse on the Ibrutinib therapy.
[0217] For the patients that were characterized as having Richter's
Transformation, 5 patients received no additional therapy and died
within 1 month of transformation and 10 patients with DLBCL
received additional therapy: R-EPOCH (N=5) 4 PD, 1 PR, R-CHOP (N=1)
PD, R-ICE (N=1) PD, OFAR (N=1) PD. Over the course of the study to
date 14 of 17 patients with Richter's Transformation have died. The
Median Survival from date off ibrutinib study was 120 days.
[0218] For the patients that were characterized as having CLL
progression, 2 patients received no additional therapy and 10
patients received further therapy <2 months post-PD, most within
2 weeks. Over the course of the study to date 4 of 12 patients
having CLL progression have died. The Median Survival from date off
ibrutinib study was 535 days.
[0219] From this study it was concluded that Ibrutinib was a well
tolerated and effective therapy, and discontinuation was uncommon
with the study length of follow up. Disease progression on
ibrutinib was associated with karyotypic complexity and BCL6 on
FISH. Richter's transformation was more common than progressive CLL
and tended to occur earlier in therapy. Progressive CLL was
commonly associated with mutations in BTK and PLC.gamma.. Both
Richter's and progressive CLL tended to progress rapidly,
especially after discontinuation of ibrutinib.
[0220] The examples and embodiments described herein are for
illustrative purposes only and various modifications or changes
suggested to persons skilled in the art are to be included within
the spirit and purview of this application and scope of the
appended claims.
Sequence CWU 1
1
214289DNAHomo sapiensmisc_feature(1)..(4289)PLCgamma2 1agtagcgagc
gccggcggcg gagggcgtga gcggcgctga gtgacccgag tcgggacgcg 60ggctgcgcgc
gcgggacccc ggagcccaaa cccggggcag gcgggcagct gtgcccgggc
120ggcacggcca gcttcctgat ttctcccgat tccttccttc tccctggagc
ggccgacaat 180gtccaccacg gtcaatgtag attcccttgc ggaatatgag
aagagccaga tcaagagagc 240cctggagctg gggacggtga tgactgtgtt
cagcttccgc aagtccaccc ccgagcggag 300aaccgtccag gtgatcatgg
agacgcggca ggtggcctgg agcaagaccg ccgacaagat 360cgagggcttc
ttggatatca tggaaataaa agaaatccgc ccagggaaga actccaaaga
420tttcgagcga gcaaaagcag ttcgccagaa agaagactgc tgcttcacca
tcctatatgg 480cactcagttc gtcctcagca cgctcagctt ggcagctgac
tctaaagagg atgcagttaa 540ctggctctct ggcttgaaaa tcttacacca
ggaagcgatg aatgcgtcca cgcccaccat 600tatcgagagt tggctgagaa
agcagatata ttctgtggat caaaccagaa gaaacagcat 660cagtctccga
gagttgaaga ccatcttgcc cctgatcaac tttaaagtga gcagtgccaa
720gttccttaaa gataagtttg tggaaatagg agcacacaaa gatgagctca
gctttgaaca 780gttccatctc ttctataaaa aacttatgtt tgaacagcaa
aaatcgattc tcgatgaatt 840caaaaaggat tcgtccgtgt tcatcctggg
gaacactgac aggccggatg cctctgctgt 900ttacctgcat gacttccaga
ggtttctcat acatgaacag caggagcatt gggctcagga 960tctgaacaaa
gtccgtgagc ggatgacaaa gttcattgat gacaccatgc gtgaaactgc
1020tgagcctttc ttgtttgtgg atgagttcct cacgtacctg ttttcacgag
aaaacagcat 1080ctgggatgag aagtatgacg cggtggacat gcaggacatg
aacaaccccc tgtctcatta 1140ctggatctcc tcgtcacata acacgtacct
tacaggtgac cagctgcgga gcgagtcgtc 1200cccagaagct tacatccgct
gcctgcgcat gggctgtcgc tgcattgaac tggactgctg 1260ggacgggccc
gatgggaagc cggtcatcta ccatggctgg acgcggacta ccaagatcaa
1320gtttgacgac gtcgtgcagg ccatcaaaga ccacgccttt gttacctcga
gcttcccagt 1380gatcctgtcc atcgaggagc actgcagcgt ggagcaacag
cgtcacatgg ccaaggcctt 1440caaggaagta tttggcgacc tgctgttgac
gaagcccacg gaggccagtg ctgaccagct 1500gccctcgccc agccagctgc
gggagaagat catcatcaag cataagaagc tgggcccccg 1560aggcgatgtg
gatgtcaaca tggaggacaa gaaggacgaa cacaagcaac agggggagct
1620gtacatgtgg gattccattg accagaaatg gactcggcac tactgcgcca
ttgccgatgc 1680caagctgtcc ttcagtgatg acattgaaca gactatggag
gaggaagtgc cccaggatat 1740accccctaca gaactacatt ttggggagaa
atggttccac aagaaggtgg agaagaggac 1800gagtgccgag aagttgctgc
aggaatactg catggagacg gggggcaagg atggcacctt 1860cctggttcgg
gagagcgaga ccttccccaa tgactacacc ctgtccttct ggcggtcagg
1920ccgggtccag cactgccgga tccgctccac catggagggc gggaccctga
aatactactt 1980gactgacaac ctcaccttca gcagcatcta tgccctcatc
cagcactacc gcgagacgca 2040cctgcgctgc gccgagttcg agctgcggct
cacggaccct gtgcccaacc ccaaccccca 2100cgagtccaag ccgtggtact
atgacagcct gagccgcgga gaggcagagg acatgctgat 2160gaggattccc
cgggacgggg ccttcctgat ccggaagcga gaggggagcg actcctatgc
2220catcaccttc agggctaggg gcaaggtaaa gcattgtcgc atcaaccggg
acggccggca 2280ctttgtgctg gggacctccg cctattttga gagtctggtg
gagctcgtca gttactacga 2340gaagcattca ctctaccgaa agatgagact
gcgctacccc gtgacccccg agctcctgga 2400gcgctacaat atggaaagag
atataaactc cctctacgac gtcagcagaa tgtatgtgga 2460tcccagtgaa
atcaatccgt ccatgcctca gagaaccgtg aaagctctgt atgactacaa
2520agccaagcga agcgatgagc tgagcttctg ccgtggtgcc ctcatccaca
atgtctccaa 2580ggagcccggg ggctggtgga aaggagacta tggaaccagg
atccagcagt acttcccatc 2640caactacgtc gaggacatct caactgcaga
cttcgaggag ctagaaaagc agattattga 2700agacaatccc ttagggtctc
tttgcagagg aatattggac ctcaatacct ataacgtcgt 2760gaaagcccct
cagggaaaaa accagaagtc ctttgtcttc atcctggagc ccaagcagca
2820gggcgatcct ccggtggagt ttgccacaga cagggtggag gagctctttg
agtggtttca 2880gagcatccga gagatcacct ggaagattga caccaaggag
aacaacatga agtactggga 2940gaagaaccag tccatcgcca tcgagctctc
tgacctggtt gtctactgca aaccaaccag 3000caaaaccaag gacaacttag
aaaatcctga cttccgagaa atccgctcct ttgtggagac 3060gaaggctgac
agcatcatca gacagaagcc cgtcgacctc ctgaagtaca atcaaaaggg
3120cctgacccgc gtctacccaa agggacaaag agttgactct tcaaactacg
accccttccg 3180cctctggctg tgcggttctc agatggtggc actcaatttc
cagacggcag ataagtacat 3240gcagatgaat cacgcattgt tttctctcaa
tgggcgcacg ggctacgttc tgcagcctga 3300gagcatgagg acagagaaat
atgacccgat gccacccgag tcccagagga agatcctgat 3360gacgctgaca
gtcaaggttc tcggtgctcg ccatctcccc aaacttggac gaagtattgc
3420ctgtcccttt gtagaagtgg agatctgtgg agccgagtat gacaacaaca
agttcaagac 3480gacggttgtg aatgataatg gcctcagccc tatctgggct
ccaacacagg agaaggtgac 3540atttgaaatt tatgacccaa acctggcatt
tctgcgcttt gtggtttatg aagaagatat 3600gttcagcgat cccaactttc
ttgctcatgc cacttacccc attaaagcag tcaaatcagg 3660attcaggtcc
gttcctctga agaatgggta cagcgaggac atagagctgg cttccctcct
3720ggttttctgt gagatgcggc cagtcctgga gagcgaagag gaactttact
cctcctgtcg 3780ccagctgagg aggcggcaag aagaactgaa caaccagctc
tttctgtatg acacacacca 3840gaacttgcgc aatgccaacc gggatgccct
ggttaaagag ttcagtgtta atgagaacca 3900gctccagctg taccaggaga
aatgcaacaa gaggttaaga gagaagagag tcagcaacag 3960caagttttac
tcatagaagc tggggtatgt gtgtaagggt attgtgtgtg tgcgcatgtg
4020tgtttgcatg taggagaacg tgccctattc acactctggg aagacgctaa
tctgtgacat 4080cttttcttca agcctgccat caaggacatt tcttaagacc
caactggcat gagttggggt 4140aatttcctat tattttcatc ttggacaact
ttcttaactt atattcttta tagaggattc 4200cccaaaatgt gctcctcatt
tttggcctct catgttccaa acctcattga ataaaagcaa 4260tgaaaacctt
gaaaaaaaaa aaaaaaaaa 428921265PRTHomo
sapiensMISC_FEATURE(1)..(1265)PLCgamma2 2Met Ser Thr Thr Val Asn
Val Asp Ser Leu Ala Glu Tyr Glu Lys Ser1 5 10 15Gln Ile Lys Arg Ala
Leu Glu Leu Gly Thr Val Met Thr Val Phe Ser 20 25 30Phe Arg Lys Ser
Thr Pro Glu Arg Arg Thr Val Gln Val Ile Met Glu 35 40 45Thr Arg Gln
Val Ala Trp Ser Lys Thr Ala Asp Lys Ile Glu Gly Phe 50 55 60Leu Asp
Ile Met Glu Ile Lys Glu Ile Arg Pro Gly Lys Asn Ser Lys65 70 75
80Asp Phe Glu Arg Ala Lys Ala Val Arg Gln Lys Glu Asp Cys Cys Phe
85 90 95Thr Ile Leu Tyr Gly Thr Gln Phe Val Leu Ser Thr Leu Ser Leu
Ala 100 105 110Ala Asp Ser Lys Glu Asp Ala Val Asn Trp Leu Ser Gly
Leu Lys Ile 115 120 125Leu His Gln Glu Ala Met Asn Ala Ser Thr Pro
Thr Ile Ile Glu Ser 130 135 140Trp Leu Arg Lys Gln Ile Tyr Ser Val
Asp Gln Thr Arg Arg Asn Ser145 150 155 160Ile Ser Leu Arg Glu Leu
Lys Thr Ile Leu Pro Leu Ile Asn Phe Lys 165 170 175Val Ser Ser Ala
Lys Phe Leu Lys Asp Lys Phe Val Glu Ile Gly Ala 180 185 190His Lys
Asp Glu Leu Ser Phe Glu Gln Phe His Leu Phe Tyr Lys Lys 195 200
205Leu Met Phe Glu Gln Gln Lys Ser Ile Leu Asp Glu Phe Lys Lys Asp
210 215 220Ser Ser Val Phe Ile Leu Gly Asn Thr Asp Arg Pro Asp Ala
Ser Ala225 230 235 240Val Tyr Leu His Asp Phe Gln Arg Phe Leu Ile
His Glu Gln Gln Glu 245 250 255His Trp Ala Gln Asp Leu Asn Lys Val
Arg Glu Arg Met Thr Lys Phe 260 265 270Ile Asp Asp Thr Met Arg Glu
Thr Ala Glu Pro Phe Leu Phe Val Asp 275 280 285Glu Phe Leu Thr Tyr
Leu Phe Ser Arg Glu Asn Ser Ile Trp Asp Glu 290 295 300Lys Tyr Asp
Ala Val Asp Met Gln Asp Met Asn Asn Pro Leu Ser His305 310 315
320Tyr Trp Ile Ser Ser Ser His Asn Thr Tyr Leu Thr Gly Asp Gln Leu
325 330 335Arg Ser Glu Ser Ser Pro Glu Ala Tyr Ile Arg Cys Leu Arg
Met Gly 340 345 350Cys Arg Cys Ile Glu Leu Asp Cys Trp Asp Gly Pro
Asp Gly Lys Pro 355 360 365Val Ile Tyr His Gly Trp Thr Arg Thr Thr
Lys Ile Lys Phe Asp Asp 370 375 380Val Val Gln Ala Ile Lys Asp His
Ala Phe Val Thr Ser Ser Phe Pro385 390 395 400Val Ile Leu Ser Ile
Glu Glu His Cys Ser Val Glu Gln Gln Arg His 405 410 415Met Ala Lys
Ala Phe Lys Glu Val Phe Gly Asp Leu Leu Leu Thr Lys 420 425 430Pro
Thr Glu Ala Ser Ala Asp Gln Leu Pro Ser Pro Ser Gln Leu Arg 435 440
445Glu Lys Ile Ile Ile Lys His Lys Lys Leu Gly Pro Arg Gly Asp Val
450 455 460Asp Val Asn Met Glu Asp Lys Lys Asp Glu His Lys Gln Gln
Gly Glu465 470 475 480Leu Tyr Met Trp Asp Ser Ile Asp Gln Lys Trp
Thr Arg His Tyr Cys 485 490 495Ala Ile Ala Asp Ala Lys Leu Ser Phe
Ser Asp Asp Ile Glu Gln Thr 500 505 510Met Glu Glu Glu Val Pro Gln
Asp Ile Pro Pro Thr Glu Leu His Phe 515 520 525Gly Glu Lys Trp Phe
His Lys Lys Val Glu Lys Arg Thr Ser Ala Glu 530 535 540Lys Leu Leu
Gln Glu Tyr Cys Met Glu Thr Gly Gly Lys Asp Gly Thr545 550 555
560Phe Leu Val Arg Glu Ser Glu Thr Phe Pro Asn Asp Tyr Thr Leu Ser
565 570 575Phe Trp Arg Ser Gly Arg Val Gln His Cys Arg Ile Arg Ser
Thr Met 580 585 590Glu Gly Gly Thr Leu Lys Tyr Tyr Leu Thr Asp Asn
Leu Thr Phe Ser 595 600 605Ser Ile Tyr Ala Leu Ile Gln His Tyr Arg
Glu Thr His Leu Arg Cys 610 615 620Ala Glu Phe Glu Leu Arg Leu Thr
Asp Pro Val Pro Asn Pro Asn Pro625 630 635 640His Glu Ser Lys Pro
Trp Tyr Tyr Asp Ser Leu Ser Arg Gly Glu Ala 645 650 655Glu Asp Met
Leu Met Arg Ile Pro Arg Asp Gly Ala Phe Leu Ile Arg 660 665 670Lys
Arg Glu Gly Ser Asp Ser Tyr Ala Ile Thr Phe Arg Ala Arg Gly 675 680
685Lys Val Lys His Cys Arg Ile Asn Arg Asp Gly Arg His Phe Val Leu
690 695 700Gly Thr Ser Ala Tyr Phe Glu Ser Leu Val Glu Leu Val Ser
Tyr Tyr705 710 715 720Glu Lys His Ser Leu Tyr Arg Lys Met Arg Leu
Arg Tyr Pro Val Thr 725 730 735Pro Glu Leu Leu Glu Arg Tyr Asn Met
Glu Arg Asp Ile Asn Ser Leu 740 745 750Tyr Asp Val Ser Arg Met Tyr
Val Asp Pro Ser Glu Ile Asn Pro Ser 755 760 765Met Pro Gln Arg Thr
Val Lys Ala Leu Tyr Asp Tyr Lys Ala Lys Arg 770 775 780Ser Asp Glu
Leu Ser Phe Cys Arg Gly Ala Leu Ile His Asn Val Ser785 790 795
800Lys Glu Pro Gly Gly Trp Trp Lys Gly Asp Tyr Gly Thr Arg Ile Gln
805 810 815Gln Tyr Phe Pro Ser Asn Tyr Val Glu Asp Ile Ser Thr Ala
Asp Phe 820 825 830Glu Glu Leu Glu Lys Gln Ile Ile Glu Asp Asn Pro
Leu Gly Ser Leu 835 840 845Cys Arg Gly Ile Leu Asp Leu Asn Thr Tyr
Asn Val Val Lys Ala Pro 850 855 860Gln Gly Lys Asn Gln Lys Ser Phe
Val Phe Ile Leu Glu Pro Lys Gln865 870 875 880Gln Gly Asp Pro Pro
Val Glu Phe Ala Thr Asp Arg Val Glu Glu Leu 885 890 895Phe Glu Trp
Phe Gln Ser Ile Arg Glu Ile Thr Trp Lys Ile Asp Thr 900 905 910Lys
Glu Asn Asn Met Lys Tyr Trp Glu Lys Asn Gln Ser Ile Ala Ile 915 920
925Glu Leu Ser Asp Leu Val Val Tyr Cys Lys Pro Thr Ser Lys Thr Lys
930 935 940Asp Asn Leu Glu Asn Pro Asp Phe Arg Glu Ile Arg Ser Phe
Val Glu945 950 955 960Thr Lys Ala Asp Ser Ile Ile Arg Gln Lys Pro
Val Asp Leu Leu Lys 965 970 975Tyr Asn Gln Lys Gly Leu Thr Arg Val
Tyr Pro Lys Gly Gln Arg Val 980 985 990Asp Ser Ser Asn Tyr Asp Pro
Phe Arg Leu Trp Leu Cys Gly Ser Gln 995 1000 1005Met Val Ala Leu
Asn Phe Gln Thr Ala Asp Lys Tyr Met Gln Met 1010 1015 1020Asn His
Ala Leu Phe Ser Leu Asn Gly Arg Thr Gly Tyr Val Leu 1025 1030
1035Gln Pro Glu Ser Met Arg Thr Glu Lys Tyr Asp Pro Met Pro Pro
1040 1045 1050Glu Ser Gln Arg Lys Ile Leu Met Thr Leu Thr Val Lys
Val Leu 1055 1060 1065Gly Ala Arg His Leu Pro Lys Leu Gly Arg Ser
Ile Ala Cys Pro 1070 1075 1080Phe Val Glu Val Glu Ile Cys Gly Ala
Glu Tyr Asp Asn Asn Lys 1085 1090 1095Phe Lys Thr Thr Val Val Asn
Asp Asn Gly Leu Ser Pro Ile Trp 1100 1105 1110Ala Pro Thr Gln Glu
Lys Val Thr Phe Glu Ile Tyr Asp Pro Asn 1115 1120 1125Leu Ala Phe
Leu Arg Phe Val Val Tyr Glu Glu Asp Met Phe Ser 1130 1135 1140Asp
Pro Asn Phe Leu Ala His Ala Thr Tyr Pro Ile Lys Ala Val 1145 1150
1155Lys Ser Gly Phe Arg Ser Val Pro Leu Lys Asn Gly Tyr Ser Glu
1160 1165 1170Asp Ile Glu Leu Ala Ser Leu Leu Val Phe Cys Glu Met
Arg Pro 1175 1180 1185Val Leu Glu Ser Glu Glu Glu Leu Tyr Ser Ser
Cys Arg Gln Leu 1190 1195 1200Arg Arg Arg Gln Glu Glu Leu Asn Asn
Gln Leu Phe Leu Tyr Asp 1205 1210 1215Thr His Gln Asn Leu Arg Asn
Ala Asn Arg Asp Ala Leu Val Lys 1220 1225 1230Glu Phe Ser Val Asn
Glu Asn Gln Leu Gln Leu Tyr Gln Glu Lys 1235 1240 1245Cys Asn Lys
Arg Leu Arg Glu Lys Arg Val Ser Asn Ser Lys Phe 1250 1255 1260Tyr
Ser 1265
* * * * *