U.S. patent application number 16/538232 was filed with the patent office on 2020-03-19 for ddr2 mutations in squamous cell lung cancer.
The applicant listed for this patent is The Broad Institute, Inc., Dana-Farber Cancer Institute, Inc.. Invention is credited to Peter Hammerman, Matthew Meyerson, Alexis Ramos.
Application Number | 20200087734 16/538232 |
Document ID | / |
Family ID | 49235826 |
Filed Date | 2020-03-19 |
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United States Patent
Application |
20200087734 |
Kind Code |
A1 |
Meyerson; Matthew ; et
al. |
March 19, 2020 |
DDR2 Mutations in Squamous Cell Lung Cancer
Abstract
Methods for treating patients with squamous cell lung cancer,
including detecting the presence of mutations in the discoidin
domain receptor 2 (DDR2) gene.
Inventors: |
Meyerson; Matthew; (Concord,
MA) ; Hammerman; Peter; (Boston, MA) ; Ramos;
Alexis; (Somerville, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The Broad Institute, Inc.
Dana-Farber Cancer Institute, Inc. |
Cambridge
Boston |
MA
MA |
US
US |
|
|
Family ID: |
49235826 |
Appl. No.: |
16/538232 |
Filed: |
August 12, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15294068 |
Oct 14, 2016 |
10385403 |
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16538232 |
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13875079 |
May 1, 2013 |
9499856 |
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15294068 |
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PCT/US2013/030292 |
Mar 11, 2013 |
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13875079 |
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61619273 |
Apr 2, 2012 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12Q 2600/156 20130101;
C12Q 1/68 20130101; C12Q 2600/106 20130101; C12Q 1/6886
20130101 |
International
Class: |
C12Q 1/6886 20060101
C12Q001/6886; C12Q 1/68 20060101 C12Q001/68 |
Goverment Interests
FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
[0002] This invention was made with Government support under Grant
No. LC090577 awarded by the Department of Defense and Grant No.
T32CA09172 awarded by the National Institutes of Health. The
Government has certain rights in the invention.
Claims
1. A method of treating a human subject diagnosed with squamous
cell carcinoma (SCC) of the lung, wherein the subject has a
sequence in a discoidin domain receptor 2 (DDR2) gene encoding at
least one amino acid variation relative to a reference amino acid
sequence, and wherein the at least one amino acid variation is
L63V, I120M, D125Y, G253C, G505S, C580Y, T765P, G774V, or G774E,
the method comprising administering a treatment comprising a
tyrosine kinase inhibitor (TKI).
2. (canceled)
3. The method of claim 1, wherein the subject is human and the
reference amino acid sequence comprises SEQ ID NO: 1.
4. The method of claim 1, wherein the reference sequence is
obtained from non-cancerous cells of the same subject.
5.-7. (canceled)
8. The method of claim 1, comprising determining a nucleic acid
sequence of a coding region of a discoidin domain receptor 2 (DDR2)
gene.
9. The method of claim 1, wherein the at least one amino acid
variation results in a decrease in expression levels, half-life, or
kinase activity of the DDR2 protein.
10. The method of claim 1, wherein the TKI is dasatinib, nilotinib,
imatinib, or ponatinib.
11. The method of claim 10, wherein the TKI is dasatinib.
12.-20. (canceled)
21. The method of claim 1, further comprising identifying the
subject as having at least one of said amino acid variations
relative to the reference amino acid sequence.
22. The method of claim 21, wherein identifying the subject
comprises performing an assay to determine a nucleic acid sequence
of all or part of a DDR2 gene in a sample comprising nucleated
cells from a SCC in the subject, wherein the assay comprises
contacting the DDR2 gene with a nucleic acid probe that
specifically hybridizes with a sequence encoding at least one amino
acid variation relative to a reference amino acid sequence, and
wherein the at least one amino acid variation is L63V, I120M,
D125Y, G253C, G505S, C580Y, T765P, G774V, or G774E; and detecting
in the sample the nucleic acid sequence encoding at least one amino
acid variation of L63V, I120M, D125Y, G253C, G505S, C580Y, T765P,
G774E, or G774V in the DDR2 gene.
Description
CLAIM OF PRIORITY
[0001] This application is a continuation of U.S. patent
application Ser. No. 15/294,068, filed Oct. 14, 2016, which is a
divisional of U.S. patent application Ser. No. 13/875,079, filed
May 1, 2013, now U.S. Pat. No. 9,499,856, which is a continuation
under 35 U.S.C. .sctn. 111 of International Patent Application No.
PCT/US2013/030292, filed on Mar. 11, 2013, and claims the benefit
of U.S. Provisional Patent Application Ser. No. 61/619,273, filed
on Apr. 2, 2012. The entire contents of the foregoing are hereby
incorporated by reference.
TECHNICAL FIELD
[0003] This invention relates to methods for treating patients with
squamous cell lung cancer, including detecting the presence of
mutations in the discoidin domain receptor 2 (DDR2) gene.
BACKGROUND
[0004] Lung cancer is the leading cause of cancer-related mortality
in the United States with over 157,000 deaths projected in 2010
(1). The more common type of lung cancer, non-small cell lung
cancer (NSCLC), accounts for 85% of cases and carries a grim
prognosis with approximately 70% of patients presenting with
advanced and often incurable disease at the time of diagnosis
(2).
[0005] Despite these statistics a great deal of progress has been
made in the targeted treatment of patients with NSCLC, largely due
to the development of small molecule inhibitors of the epidermal
growth factor receptor (EGFR) tyrosine kinase (3-5). Patients who
respond to EGFR kinase inhibitors are much more likely to have the
adenocarcinoma subtype of NSCLC (6). Patients with the other
principal subtype of NSCLC, lung squamous cell cancer (lung SCC),
very rarely respond to these agents and few advances have been made
in the treatment of this type of lung cancer which comprises 25% of
NSCLC. In addition to EGFR, several other promising therapeutic
targets have been identified in the laboratory such as EML4-ALK,
KRAS and MET; drugs directed against these proteins are being
tested in clinical trials (7-10). However, it appears that these
targets are likely limited to adenocarcinomas as well. A recent
report has suggested that targeting FGFR1 amplifications in SCC of
the lung may be a promising therapeutic strategy, though FGFR
inhibitors are not currently in clinical use for the treatment of
patients with lung cancer (11).
SUMMARY
[0006] At least in part, the present invention is based on the
discovery that mutations in the DDR2 kinase gene identify a novel
therapeutic target in squamous cell lung cancer, and can be used to
select therapy for patients whose tumors comprises cells harboring
DDR2 mutations, e.g., administration of tyrosine kinase inhibitors
such as dasatinib.
[0007] Thus in a first aspect, the invention provides methods for
selecting a treatment comprising administration of a tyrosine
kinase inhibitor (TKI) for a subject diagnosed with lung cancer,
e.g., non-small cell lung cancer (NSCLC), e.g., squamous cell
carcinoma (SCC). The methods include determining a nucleic acid
sequence of all or part of a discoidin domain receptor 2 (DDR2)
gene in a sample comprising nucleated cells from the SCC in the
subject; determining an expected amino acid translation of the
determined DDR2 sequence; and comparing the expected amino acid
translation with a reference amino acid sequence, wherein the
reference amino acid sequence is a wild type DDR2 sequence;
detecting the presence of at least one amino acid variation
relative to the reference amino acid sequence; and selecting a
treatment comprising administration of a TKI for the subject, based
on the presence of at least one amino acid variation relative to
the reference sequence.
[0008] In a further aspect, the invention provides methods for
predicting response to a treatment comprising administration of a
tyrosine kinase inhibitor (TKI) in a subject diagnosed with
squamous cell carcinoma (SCC). The methods include determining a
nucleic acid sequence of all or part of a discoidin domain receptor
2 (DDR2) gene in a sample comprising nucleated cells from the SCC
in the subject; determining an expected amino acid translation of
the determined DDR2 sequence; and comparing the expected amino acid
translation with a reference amino acid sequence, wherein the
reference amino acid sequence is a wild type DDR2 sequence;
detecting the presence of at least one amino acid variation
relative to the reference amino acid sequence; and selecting a
treatment comprising administration of a TKI for the subject, based
on the presence of the at least one amino acid variation relative
to the reference sequence.
[0009] In some embodiments of the methods described herein, the
subject is human and the reference amino acid sequence comprises
SEQ ID NO:1. In some embodiments of the methods described herein,
the reference sequence is obtained from non-cancerous cells of the
same subject.
[0010] In some embodiments of the methods described herein, the at
least one amino acid variation comprises a non-conservative amino
acid substitution.
[0011] In some embodiments of the methods described herein, the at
least one amino acid variation is within a kinase domain (amino
acids 563-849) or discoidin domain (amino acids 30-185) of
DDR2.
[0012] In some embodiments of the methods described herein, the at
least one amino acid variation is L63V, I120M, D125Y, L239R, G253C,
G5055, C580Y, I638F, T765P, G774E/V, or S768R. In some embodiments
of the methods described herein, the variation is due to a mutation
is shown in Tables 7 or 8.
[0013] In some embodiments of the methods described herein, the
methods include determining a nucleic acid sequence of a coding
region of a discoidin domain receptor 2 (DDR2) gene.
[0014] In some embodiments of the methods described herein, the at
least one amino acid variation results in a decrease in expression
levels, half-life, or kinase activity of the DDR2 protein.
[0015] In some embodiments of the methods described herein, the TKI
is dasatinib, nilotinib, imatinib, or ponatinib. In some
embodiments of the methods described herein, the TKI is
dasatinib.
[0016] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Methods
and materials are described herein for use in the present
invention; other, suitable methods and materials known in the art
can also be used. The materials, methods, and examples are
illustrative only and not intended to be limiting. All
publications, patent applications, patents, sequences, database
entries, and other references mentioned herein are incorporated by
reference in their entirety. In case of conflict, the present
specification, including definitions, will control.
[0017] Other features and advantages of the invention will be
apparent from the following detailed description and figures, and
from the claims.
DESCRIPTION OF DRAWINGS
[0018] FIGS. 1a-c: Sequencing of squamous lung cancer samples
identifies recurrent mutations in DDR2. (a) Schema depicted for the
primary, secondary and validation screens for DDR2 mutations in
squamous lung cancer samples. (b) Amino acid sequence of DDR2 with
the positions of the identified mutations shown in the context of
the known domain structure of DDR2. (c) Homology alignment of DDR2
amino acid sequence. Shown are the amino acid sequences of human
DDR2, listed as DDR2 HUMAN residues 54-264 (SEQ ID NO:4) and
468-786 (SEQ ID NO:5); mouse DDR2, listed as DDR2 MOUSE residues
54-264 (SEQ ID NO:6) and 468-786 (SEQ ID NO:7); and the closest
homologs in zebrafish, shown as ASWUM4 DANRE residues 27-236 (SEQ
ID NO:8) and 443-755 (SEQ ID NO:9) and C. elegans, shown as Q95ZV7
CAEEL residues 50-248 (SEQ ID NO:10) and 451-722 (SEQ ID NO:11).
Degree of homology is indicated by the bar graphs under each amino
acid and the position of the novel DDR2 mutations indicated.
[0019] FIGS. 2a-d: Lung cancer cell lines with DDR2 mutations are
sensitive to drugs and RNAi targeting DDR2. (a) Proliferation of
A549, NCI-H2286, HCC-366 and NCI-H1703 grown for six days in the
presence of various concentrations of dasatinib. Proliferation
shown relative to untreated cells at the same time point. Standard
errors are shown for triplicate samples. (b) Proliferation shown of
NCI-H2286 and HCC-366 cell lines ectopically expressing the T654M
gatekeeper mutation in DDR2, labeled as DDR2*. Six day
proliferation in the presence of dasatinib is shown as above. For
NCI-H2286 and HCC-366 the gatekeeper mutation is expressed in cis
with the DDR2 mutation found in the cell line. (c) Proliferation
measured as above for NCI-H2286, HCC-366 and NCI-H1703 cells stably
expressing sh-RNA vectors targeting either GFP or the 3' UTR of
DDR2 (DDR2 sh-RNA-2) or the coding sequence of DDR2 (DDR2
sh-RNA-5). Proliferation is measured after four days in culture as
compared to day 1. Standard errors are shown for triplicate
samples. Immunoblot showing relative levels of DDR2 in the cell
lines used in the experiment is shown in the inset. "G" indicates
cells expressing shGFP, and "2" and "5" the numbered DDR2 targeted
hairpins. (d) Four-day proliferation of the DDR2 mutant NCI-H2286
and HCC-366 cell lines stably expressing ectopic DDR2 following
knock-down of DDR2 by a sh-RNA targeting the 3' UTR of DDR2
(sh-RNA-2). Proliferation of triplicate samples is presented as
above relative to cells transduced with a sh-RNA targeting GFP.
Protein levels of DDR2 are shown in the immunoblot below, "G"
indicates sh-GFP, "2" indicates expression of sh-RNA 2 and "D"
indicates expression of sh-RNA2 and DDR2.
[0020] FIGS. 2e-i: Squamous lung cancer cell lines harboring DDR2
mutations are sensitive to tyrosine kinase inhibitor treatment. (e)
Proliferation of A549, NCIH2286, HCC-366 and NCI-H1703 grown for
six days in the presence of various concentrations of imatinib.
Proliferation is presented relative to untreated cells at the same
time point. Standard errors are presented for triplicate samples.
(f) Viability measured by trypan blue exclusion in A549, NCI-H2286,
HCC-366 and NCI-H1703 cells grown in the indicated concentrations
of dasatinib. Data are presented as viability relative to untreated
cells at the same time point and represent an average of fifty
independently acquired trypan blue images in each of three
replicates with standard errors shown. (g) Proliferation of A549,
NCIH2286, HCC-366 and NCI-H1703 cells grown for six days in the
presence of nilotinib. Data are presented as above. (h)
Proliferation of A549, NCI-H2286, HCC-366 and NCI-H1703 cells grown
in the presence of AP24534. Data are presented as above. (i)
Immunoblot showing DDR2 expression from cell lines used in the
experiment shown in FIG. 2c. DDR2* denotes the "gatekeeper"
transgene.
[0021] FIG. 3: Xenografts of squamous lung cancer cell lines
demonstrate anti-tumor effects of dasatinib in vivo. Athymic nu/nu
mice were injected subcutaneously with A549, NCI-H1703, HCC-366 and
NCI-H2286 cells (n=10) and treated with dasatinib or vehicle for
two weeks following tumor formation. Depicted are measurements of
tumor size in mice from each cohort. Tumors did not form in the
mice injected with HCC-366 and these mice could not be analyzed
further.
[0022] FIGS. 4a-d: Ectopic expression of DDR2 mutants leads to
cellular transformation which can be blocked by dasatinib or
combination tyrosine kinase inhibitor treatment. (a) Results from
soft agar assay in which 3T3 fibroblasts expressing the L63V DDR2
mutation, the L858R EGFR mutation or the KRAS G12V mutation were
plated in soft agar in the presence of various concentrations of
dasatinib. Colony number of six independent samples with standard
errors is shown. (b) Proliferation at four days of Ba/F3 cells
expressing vector only or one of six DDR2 mutations shown in cells
grown in the presence of dasatinib. For the vector control cells
are grown in the presence of IL-3 to maintain viability and in the
case of the DDR2 mutants all cells are IL-3 independent and
cultured in the absence of IL-3. Proliferation is shown relative to
untreated cells at the same time point for triplicate samples with
standard errors. (c) Proliferation of Ba/F3 cells expressing DDR2
L63V co-cultured with 50 nM of nilotinib, AP24534 or dasatinib with
or without 500 nM AZD0530. Proliferation is relative to untreated
cells grown in parallel. (d) Immunoblots of DDR2 L63V transformed
Ba/F3 cells treated for two days with the depicted concentrations
of AZD0530 (AZD) in addition to 50 nM nilotinib (N), AP24534 (AP)
or dasatinib (D). The first lane is an untreated sample. Shown are
immunoblots probed with antibodies against phospho-Src Y416,
phospho-STAT5 Y694, FLAG-DDR2 and actin.
[0023] FIGS. 4e-h: Ectopic expression of DDR2 leads to cellular
transformation which is sensitive to AP24534 treatment. (e) Colony
formation in soft agar of NIH-3T3 fibroblasts stably expressing the
vector alone or wild-type or various mutant forms of DDR2. Colony
numbers with standard errors are shown for six independent samples.
Expression of FLAG-tagged DDR2 by immunoblotting of the cells used
in the transformation assay is shown in the inset and actin serves
as a loading control. 3T3 fibroblasts expressing the activating
L858R mutation of EGFR (EGFR*) are used as a positive control. (f)
Time to IL-3 independence is shown for Ba/F3 cells stably
expressing vector alone or wild-type or mutant forms of DDR2.
Expression level of the transgenes and KD DDR2 (K608E) is shown as
well as actin. The KD DDR2 was probed on a separate membrane which
is indicated by the separation bar.
(g) Proliferation at four days of Ba/F3 cells expressing vector
only or one of six DDR2 mutations is shown in cells grown in the
presence of imatinib. For the vector control the cells are grown in
the presence of IL-3 to maintain viability and in the case of the
DDR2 mutants all cells are IL-3 independent and cultured in the
absence of IL-3. Proliferation is shown relative to untreated cells
at the same time point for triplicate samples with standard errors.
(h) Experiment as above with AP24534.
[0024] FIGS. 4i-l: DDR2-transformed Ba/F3 cells maintain Src and
STAT5 phosphorylation and treatment of Ba/F3 cells expressing
mutant forms of DDR2 with nilotinib or AZD0530 results in minimal
toxicity. (i) Proliferation at four days of Ba/F3 cells expressing
vector only or one of six DDR2 mutations is shown in cells grown in
the presence of nilotinib. For the vector control the cells are
grown in the presence of IL-3 to maintain viability and in the case
of the DDR2 mutants all cells are IL-3 independent and cultured in
the absence of IL-3. Proliferation is shown relative to untreated
cells at the same time point for triplicate samples with standard
errors. (j) Immunoblots showing the levels of phospho-Src (Y416),
phospho-STAT5 (Y694) and actin in Ba/F3 cells expressing mutant
forms of DDR2 or the vector alone from the cell lines shown in
Supplementary FIG. 3b. All DDR2 mutant lines are grown in the
absence of IL-3 and the vector is shown in the presence and absence
of IL-3. (k) Experiment as above with Ba/F3 cells grown in the
presence of AZD0530. (1) Immunoblots of DDR2 L63V transformed Ba/F3
cells treated for two days with the depicted concentrations of
AZD0530, nilotinib (N), AP24534 (AP) or dasatinib (D). The first
lane is an untreated sample. Shown are immunoblots probed with
antibodies against phospho-Src, phospho-STAT5, FLAG-DDR2 and
actin.
[0025] FIGS. 4m-o: Combination treatment with nilotinib, AP24534 or
dasatinib and AZD0530 leads to increased killing of DDR2
transformed Ba/F3 cells. (m) Proliferation at four days of Ba/F3
cells expressing vector only or one of six DDR2 mutations is shown
in cells grown in the presence of a fixed concentration 4 of
nilotinib and the depicted amounts of AZD0530. For the vector
control the cells are grown in the presence of IL-3 to maintain
viability and in the case of the DDR2 mutants all cells are IL-3
independent and cultured in the absence of IL-3. Proliferation is
shown relative to untreated cells at the same time point for
triplicate samples with standard errors. (n) Experiment performed
as above with AP24534 and AZD0530. (o) Experiment performed as
above with dasatinib and AZD0530.
[0026] FIGS. 5a-b: Radiographic response of a patient with a S768R
DDR2 mutation treated with dasatinib plus erlotinib. (a) CT scan
images shown from a lung SCC patient who was treated with
chemotherapy and later with dasatinib plus erlotinib. Serial CT
scans are shown at the time of initiation of chemotherapy,
initiation of study treatment with dasatinib and erlotinib and
following two months of treatment with dasatinib plus erlotinib.
(b) Top panel: Tumor dimension measurements from the subject above
starting four months prior to chemotherapy treatment and extending
to the time at which combination therapy with dasatinib and
erlotinib was discontinued. Bottom panel: Bar graph depicting
measured tumor volume by RECIST criteria of the subject prior to
chemotherapy, following chemotherapy and following two months of
dasatinib plus erlotinib therapy.
DETAILED DESCRIPTION
[0027] Described herein is the identification of novel somatic
mutations in the discoidin domain receptor 2 (DDR2) tyrosine kinase
gene at a frequency of 3.8% (n=11) in a sample set of 290 squamous
cell lung cancer samples. DDR2 is a receptor tyrosine kinase which
binds collagen as its endogenous ligand and has been previously
shown to promote cell migration, proliferation and survival when
activated by ligand binding and phosphorylation (12-18). DDR1 and
DDR2 mutations have been reported in several cancer specimens,
including four DDR1 mutations (W385C, A496S, F866Y, F824W) and two
DDR2 mutations in lung cancer (R105S and N456S), but these reports
have not been confirmed in independent samples and functional
characterization of the mutations has not been reported (19-21). As
demonstrated herein, DDR2 mutation status is associated with
sensitivity to the tyrosine kinase inhibitor dasatinib or to sh-RNA
mediated depletion of DDR2. Additionally, DDR2 mutations are
oncogenic and their ability to transform cells can be blocked by
dasatinib treatment or by combination tyrosine kinase inhibitor
treatment. Furthermore, a DDR2 kinase domain mutation was observed
in a clinical trial subject with SCC of the lung who had a
radiographic response to combination therapy with erlotinib and
dasatinib and who did not have an EGFR mutation. Together, these
data indicate that DDR2 may be an important therapeutic target in
SCCs, and can be used to select therapy for patients with SCC.
Definitions
[0028] As used herein, an "allele" is one of a pair or series of
genetic variants at a specific genomic location. A "response
allele" is an allele that is associated with increased likelihood
of response; heterozygosity is sufficient as these are
gain-of-function. Where a SNP is biallelic, one allele (the
"response allele") will be associated with increased likelihood of
response, while the other allele is associated with average or
decreased likelihood of response, or some variation thereof.
[0029] As used herein, "genotype" refers to the diploid combination
of alleles for a given genetic polymorphism. A homozygous subject
carries two copies of the same allele and a heterozygous subject
carries two different alleles.
[0030] Microsatellites (sometimes referred to as a variable number
of tandem repeats or VNTRs) are short segments of DNA that have a
repeated sequence, usually about 2 to 5 nucleotides long (e.g.,
CACACA), that tend to occur in non-coding DNA. Changes in the
microsatellites sometimes occur during the genetic recombination of
sexual reproduction, increasing or decreasing the number of repeats
found at an allele, changing the length of the allele.
Microsatellite markers are stable, polymorphic, easily analyzed and
occur regularly throughout the genome, making them especially
suitable for genetic analysis.
[0031] The term "chromosome" as used herein refers to a gene
carrier of a cell that is derived from chromatin and comprises DNA
and protein components (e.g., histones). The conventional
internationally recognized individual human genome chromosome
numbering identification system is employed herein. The size of an
individual chromosome can vary from one type to another with a
given multi-chromosomal genome and from one genome to another. In
the case of the human genome, the entire DNA mass of a given
chromosome is usually greater than about 100,000,000 base
pairs.
[0032] The term "gene" refers to a DNA sequence in a chromosome
that codes for a product (either RNA or its translation product, a
polypeptide). A gene contains a coding region and includes regions
preceding and following the coding region (termed respectively
"leader" and "trailer"). The coding region is comprised of a
plurality of coding segments ("exons") and intervening sequences
("introns") between individual coding segments.
[0033] The term "reference sequence" refers to a sequence that is
present in a subject considered to be a reference or control
subject. The reference sequence as used in some embodiments is a
sequence that is present in the majority of a population, i.e., the
"wild-type" sequence. In some embodiments, the reference sequence
is nucleic acid (e.g., genomic DNA or mRNA/cDNA) or amino acid. In
some embodiments, the reference sequence is a sequence in the same
subject at an earlier time point, e.g., before treatment. In some
embodiments, the reference sequence is obtained from non-cancerous
cells of the same subject.
[0034] The term "probe" refers to an oligonucleotide. In some
embodiments, a probe is single stranded at the time of
hybridization to a target. As used herein, probes include primers,
i.e., oligonucleotides that can be used to prime a reaction, e.g.,
a PCR reaction.
[0035] The term "label" or "label containing moiety" refers in a
moiety capable of detection, such as a radioactive isotope or group
containing same, and non-isotopic labels, such as enzymes, biotin,
avidin, streptavidin, digoxygenin, luminescent agents, dyes,
haptens, and the like. Luminescent agents, depending upon the
source of exciting energy, can be classified as radioluminescent,
chemiluminescent, bioluminescent, and photoluminescent (including
fluorescent and phosphorescent). In some embodiments, a probe
described herein is bound, e.g., chemically bound to
label-containing moieties or can be suitable to be so bound. The
probe can be directly or indirectly labeled.
[0036] The term "direct label probe" (or "directly labeled probe")
refers to a nucleic acid probe whose label after hybrid formation
with a target is detectable without further reactive processing of
hybrid. The term "indirect label probe" (or "indirectly labeled
probe") refers to a nucleic acid probe whose label after hybrid
formation with a target is further reacted in subsequent processing
with one or more reagents to associate therewith one or more
moieties that finally result in a detectable entity.
[0037] The terms "target," "DNA target," or "DNA target region"
refers to a nucleotide sequence that occurs at a specific
chromosomal location. Each such sequence or portion is preferably
at least partially, single stranded (e.g., denatured) at the time
of hybridization. When the target nucleotide sequences are located
only in a single region or fraction of a given chromosome, the term
"target region" is sometimes used. In some embodiments, targets for
hybridization are derived from specimens which include, but are not
limited to, chromosomes or regions of chromosomes in normal,
diseased or malignant human cells, either interphase or at any
state of meiosis or mitosis, and either extracted or derived from
living or postmortem tissues, organs or fluids; germinal cells
including sperm and egg cells, or cells from zygotes, fetuses, or
embryos, or chorionic or amniotic cells, or cells from any other
germinating body; cells grown in vitro, from either long-term or
short-term culture, and either normal, immortalized or transformed;
inter- or intraspecific hybrids of different types of cells or
differentiation states of these cells; individual chromosomes or
portions of chromosomes, or translocated, deleted or other damaged
chromosomes, isolated by any of a number of means known to those
with skill in the art, including libraries of such chromosomes
cloned and propagated in prokaryotic or other cloning vectors, or
amplified in vitro by means well known to those with skill; or any
forensic material, including but not limited to blood, or other
samples.
[0038] The term "hybrid" refers to the product of a hybridization
procedure between a probe and a target.
[0039] The term "hybridizing conditions" has general reference to
the combinations of conditions that are employable in a given
hybridization procedure to produce hybrids, such conditions
typically involving controlled temperature, liquid phase, and
contact between a probe (or probe composition) and a target.
Conveniently and preferably, at least one denaturation step
precedes a step wherein a probe or probe composition is contacted
with a target. Guidance for performing hybridization reactions can
be found in Ausubel et al., Current Protocols in Molecular Biology,
John Wiley & Sons, N.Y. (2003), 6.3.1-6.3.6. Aqueous and
nonaqueous methods are described in that reference and either can
be used. Hybridization conditions referred to herein are a 50%
formamide, 2.times.SSC wash for 10 minutes at 45.degree. C.
followed by a 2.times.SSC wash for 10 minutes at 37.degree. C.
[0040] Calculations of "identity" between two sequences are
performed using methods known in the art, e.g., as follows. The
sequences are aligned for optimal comparison purposes (e.g., gaps
can be introduced in one or both of a first and a second nucleic
acid sequence for optimal alignment and non-identical sequences can
be disregarded for comparison purposes). The length of a sequence
aligned for comparison purposes is at least 30% (e.g., at least
30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%) of the length of the
reference sequence. The nucleotides at corresponding nucleotide
positions are then compared. When a position in the first sequence
is occupied by the same nucleotide as the corresponding position in
the second sequence, then the molecules are identical at that
position. The percent identity between the two sequences is a
function of the number of identical positions shared by the
sequences, taking into account the number of gaps, and the length
of each gap, which need to be introduced for optimal alignment of
the two sequences.
[0041] In some embodiments, the comparison of sequences and
determination of percent identity between two sequences is
accomplished using a mathematical algorithm. In some embodiments,
the percent identity between two nucleotide sequences is determined
using the GAP program in the GCG software package, using a Blossum
62 scoring matrix with a gap penalty of 12, a gap extend penalty of
4, and a frameshift gap penalty of 5.
[0042] As used herein, the term "substantially identical" is used
to refer to a first nucleotide sequence that contains a sufficient
number of identical nucleotides to a second nucleotide sequence
such that the first and second nucleotide sequences have similar
activities. Nucleotide sequences that are substantially identical
are at least 80% (e.g., 85%, 90%, 95%, 97% or more) identical.
[0043] The term "nonspecific binding DNA" refers to DNA which is
complementary to DNA segments of a probe, which DNA occurs in at
least one other position in a genome, outside of a selected
chromosomal target region within that genome. An example of
nonspecific binding DNA comprises a class of DNA repeated segments
whose members commonly occur in more than one chromosome or
chromosome region. Such common repetitive segments tend to
hybridize to a greater extent than other DNA segments that are
present in probe composition.
[0044] Methods of Selecting Treatment
[0045] Described herein are a number of methods of selecting a
treatment comprising administration of a tyrosine kinase inhibitor
for a subject who has squamous cell lung cancer.
[0046] As used herein, "detecting a variant DDR2" includes
obtaining information regarding the identity (i.e., of a specific
nucleotide), presence or absence of one or more specific sequences
or alleles in a subject. Detecting a variant DDR2 can, but need
not, include obtaining a sample comprising DNA from a subject,
e.g., from an SCC tumor cell, and/or assessing the identity,
presence or absence of a specific sequence or one or more genetic
markers in the sample. The individual or organization who detects
the variant DDR2 need not actually carry out the physical analysis
of a sample from a subject; the methods can include using
information obtained by analysis of the sample by a third party.
Thus the methods can include steps that occur at more than one
site. In some embodiments, a sample is obtained from a subject at a
first site, such as at a health care provider. In some embodiments,
the sample is analyzed at the same or a second site, e.g., at a
laboratory or other testing facility.
[0047] In some embodiments, to detect a variant DDR2, a biological
sample that includes nucleated cells (such as blood, a cheek swab
or mouthwash) is prepared and analyzed for sequence or the presence
or absence of pre-selected markers. Such diagnoses may be performed
by diagnostic laboratories, or, alternatively, in some embodiments,
diagnostic kits are manufactured and sold to health care providers
or to private individuals for self-diagnosis. In some embodiments,
diagnostic or prognostic tests are performed as described herein or
using well known techniques, such as described in U.S. Pat. No.
5,800,998.
[0048] In some embodiments, results of these tests, and optionally
interpretive information, are returned to the subject, the health
care provider or to a third party payor. The results can be used in
a number of ways. In some embodiments, the information is
communicated to the tested subject, e.g., with a prognosis and
optionally interpretive materials that help the subject understand
the test results and prognosis. In some embodiments, the
information is used, e.g., by a health care provider, to determine
whether to administer a treatment comprising a TKI for SCC, or
whether a subject should be assigned to a specific category, e.g.,
a category associated with a specific disease, or with drug
response or non-response. In some embodiments, the information is
used, e.g., by a third party payor such as a healthcare payer
(e.g., insurance company or HMO) or other agency, to determine
whether or not to reimburse a health care provider for services to
the subject, or whether to approve the provision of services to the
subject. For example, the healthcare payer may decide to reimburse
a health care provider for treatments comprising administration of
a TKI for SCC if the subject has a variant of DDR2 and this is
likely to response to a TKI. The presence or absence of a DDR2
variant, e.g., as described herein, in a subject may be ascertained
by using any of the methods described herein.
[0049] The methods can include obtaining a sample comprising cells
from the subject, e.g., cells from a SCC tumor biopsy, and
determining the sequence of all or part of the DDR2 gene. In some
embodiments, genomic DNA is sequenced; in some embodiments, mRNA is
sequenced (optionally after conversion to cDNA). In some
embodiments, rather than sequencing all or part of a gene, the
identity of a single nucleotide is determined.
[0050] Squamous Cell Lung Cancer
[0051] Non-small-cell lung cancer (NSCLC) is the leading cause of
cancer mortality in the United States and worldwide (Higgins and
Ettinger, Expert Rev Anticancer Ther 2009; 9:1365-1378).
Non-small-cell lung cancer can be divided into three major
histological subtypes: squamous cell carcinoma, adenocarcinoma (AC)
and large cell carcinoma (LCC). The vast majority of SCCs arise in
subsegmental or larger bronchi and grow centrally toward the main
bronchus, infiltrating the underlying bronchial cartilage, lymph
nodes, and adjacent lung parenchyma. The other types of NSCLC (AC
and LCC) are often peripherally located in the lungs. Although
exemplified in SCC, the methods described herein could be used in
the other types of lung cancer, e.g., SCLC and NSCLC.
[0052] A suspected case of lung cancer, e.g., SCC can be initially
identified based on symptoms, followed by the presence of findings
consistent with lung cancer, e.g., SCC on imaging studies, e.g.,
chest radiographs, Magnetic Resonance Imaging (MRI), positron
emission tomography (PET), or computed tomography (CT), and a
confirmed diagnosis obtained by histology, e.g., by sputum
cytologic studies, bronchoscopy, or CT-guided transthoracic needle
biopsy of the mass; selection of the method can depend on the
location of the tumor.
[0053] DDR2
[0054] DDR2 is a receptor tyrosine kinase that activates
intracellular signaling pathways including Ras/MAPK, ERK and NF-kB
upon stimulation by its endogenous ligand, extracellular collagen
(Vogel et al., J Biol Chem, 2000. 275(8):5779-84; Vogel et al., Mol
Cell, 1997. 1(1):13-23; Vogel et al., Mol Cell Biol, 2001.
21(8):2906-17). DDR2 is thought to be important in regulating cell
growth and survival, as a DDR2 null mouse exhibits dwarfism and
infertility and a family of humans with germline hypomophic DDR2
mutations have short limbs and a short stature (Bargal et al., Am J
Hum Genet, 2009. 84(1): 80-4). DDR2 mutations have been described
as rare events in a variety of cancers including a single report in
lung cancer, though the significance of these events is unknown.
(Rikova et al., Cell, 2007. 131(6):1190-203; Tomasson et al.,
Blood, 2008. 111(9):4797-808) Furthermore, DDR2 has been identified
as a target of the FDA-approved tyrosine kinase inhibitors
imatinib, nilotinib and dasatinib, with dasatinib the most potent
based on large scale phosphpopeptide screening assays. (Day et al.,
Eur J Pharmacol, 2008. 599(1-3): 44-53; Davies, H., et al., Cancer
Res, 2005. 65(17): 7591-5)
[0055] In some embodiments, the methods include determining the
sequence of all or part of the DDR2 gene in a sample comprising
cells, e.g., tumor cells, from the subject, and comparing the
sequence to a reference DDR2 gene or mRNA sequence. In some
embodiments, the methods include sequencing all or part of the
gene. The sequence of the DDR2 gene is known in the art and is
provided at GenBank Accession No. NG_016290.1. In some embodiments,
the methods include sequencing the coding region of the DDR2 gene;
two transcript variants exist, variant (1) (GenBank Acc. No.
NM_001014796.1) and variant (2) (GenBank Acc. No. NM_006182.2),
which differs in the 5' UTR compared to variant 1. Both encode the
same protein, i.e., GenBank Acc. No. NP_001014796.1 or NP_006173.2,
which both set forth the following sequence:
TABLE-US-00001 (SEQ ID NO: 1) 1 MILIPRMLLV LFLLLPILSS AKAQVNPAIC
RYPLGMSGGQ IPDEDITASS QWSESTAAKY 61 GRLDSEEGDG AWCPEIPVEP
DDLKEFLQID LHTLHFITLV GTQGRHAGGH GIEFAPMYKI 121 NYSRDGTRWI
SWRNRHGKQV LDGNSNPYDI FLKDLEPPIV ARFVRFIPVT DHSMNVCMRV 181
ELYGCVWLDG LVSYNAPAGQ QFVLPGGSII YLNDSVYDGA VGYSMTEGLG QLTDGVSGLD
241 DFTQTHEYHV WPGYDYVGWR NESATNGYIE IMFEFDRIRN FTTMKVHCNN
MFAKGVKIFK 301 EVQCYFRSEA SEWEPNAISF PLVLDDVNPS ARFVTVPLHH
RMASAIKCQY HFADTWMMFS 361 EITFQSDAAM YNNSEALPTS PMAPTTYDPM
LKVDDSNTRI LIGCLVAIIF ILLAIIVIIL 421 WRQFWQKMLE KASRRMLDDE
MTVSLSLPSD SSMFNNNRSS SPSEQGSNST YDRIFPLRPD 481 YQEPSRLIRK
LPEFAPGEEE SGCSGVVKPV QPSGPEGVPH YAEADIVNLQ GVTGGNTYSV 541
PAVTMDLLSG KDVAVEEFPR KLLTFKEKLG EGQFGEVHLC EVEGMEKFKD KDFALDVSAN
601 QPVLVAVKML RADANKNARN DFLKEIKIMS RLKDPNIIHL LAVCITDDPL
CMITEYMENG 661 DLNQFLSRHE PPNSSSSDVR TVSYTNLKFM ATQIASGMKY
LSSLNFVHRD LATRNCLVGK 721 NYTIKIADFG MSRNLYSGDY YRIQGRAVLP
IRWMSWESIL LGKFTTASDV WAFGVTLWET 781 FTFCQEQPYS QLSDEQVIEN
TGEFFRDQGR QTYLPQPAIC PDSVYKLMLS CWRRDTKNRP 841 SFQEIHLLLL
QQGDE
[0056] In some embodiments, all or part of the DDR2 nucleic acid
sequence is "translated" into a predicted amino acid sequence based
on known rules of codon-amino acid specification. This predicted
amino acid sequence can be evaluated for the presence of one or
more differences from the reference amino acid sequence. In some
embodiments, the presence of a missense mutation, e.g., a
non-conservative variant, as compared to a wild-type reference
(e.g., SEQ ID NO:1) in the amino acid sequence indicates an
increased likelihood of response to a TKI. In some embodiments, a
variant amino acid sequence is detected directly, e.g., using
methods known in the art, such as methods using antibodies that
bind specifically to a variant of DDR2 protein but not to
wild-type, or by direct sequencing of the protein, e.g., using mass
spectrometry analysis or other methods known in the art.
[0057] The methods can include the detection of specific mutations
described herein, e.g., as shown in FIG. 1B or 1C, that alter the
primary sequence of the DDR2 protein, e.g., L63V, I120M, D125Y,
L239R, G253C, G5055, C580Y, I638F, T765P, G774E/V, and/or
S768R.
[0058] Alternatively, the methods can include the detection of
other mutations that alter the primary sequence of the DDR2 protein
(e.g., missense or non-conservative mutations), or that do not
alter the primary sequence of the DDR2 protein but affect levels of
expression and/or half-life of the protein. In some embodiments,
the methods include detecting mutations that alter the primary
sequence of the DDR2 protein and affect kinase activity of the
protein. Mutations that affect expression levels, half-life, or
kinase activity can be identified readily by one of skill in the
art, e.g., using assays known in the art and/or described herein.
For example, in some embodiments a recombinant protein is produced
using known molecular biological techniques, e.g., obtaining a wild
type or reference sequence (e.g., genomic or cDNA), using
mutagenesis (e.g., site-directed mutagenesis) to alter the sequence
to reflect the mutation, and expressing the mutated protein in a
cell, e.g., a mammalian cell, and assaying for protein levels
and/or activity of the protein.
[0059] In some embodiments, the mutations are in the discoidin
domain, e.g., amino acids 30-185 or 32-184 of NP_001014796.1; or
the catalytic/kinase domain, e.g., amino acids 557-851 or 563-849
of NP_001014796.1.
[0060] As in preferred embodiments the methods described herein
will be performed on human subjects, the human DDR2 gene sequence
has been provided as an example. As one of skill in the art will
appreciate, if the methods are performed on subjects of other
species, a reference DDR2 sequence obtained from that species
should be used. In some embodiments, to identify a DDR2 reference
sequence, a biological sample that includes non-cancerous nucleated
cells (such as blood, a cheek swab, or mouthwash) is prepared and
analyzed for sequence, or for the presence or absence of
preselected markers. In some embodiments, direct analysis of DDR2
proteins is used to detect the presence of mutations, using samples
comprising DDR2 proteins from the subject, e.g., tissue samples,
e.g., from a tissue biopsy; in some embodiments, a SCC biopsy is
used. Such determinations can be performed using methods known in
the art by diagnostic laboratories, or, alternatively, diagnostic
kits can be manufactured and sold to health care providers or to
private individuals for self-diagnosis; such kits can include
primers, probes, or antibodies that bind specifically to a mutation
in DDR2. Diagnostic or prognostic tests can be performed as
described herein or using well known techniques, such as described
in U.S. Pat. No. 5,800,998. The presence or absence of a DDR2
variant in a subject may be ascertained by using any of the methods
described herein. In some cases, results of these tests, and
optionally interpretive information, can be returned to the subject
or the health care provider. Information gleaned from the methods
described herein can also be used to select or stratify subjects
for a clinical trial. For example, the presence of a selected
allelic variant described herein can be used to select or exclude a
subject for participation in a clinical trial, e.g., a trial of a
treatment for SCC, e.g., using a TKI.
[0061] The subject can be an adult, child, fetus, or embryo. In
some embodiments, the sample is obtained prenatally, either from a
fetus or embryo or from the mother (e.g., from fetal or embryonic
cells in the maternal circulation). Methods and reagents are known
in the art for obtaining, processing, and analyzing samples.
[0062] In some cases, the biological sample is processed for DNA
isolation. For example, DNA in a cell or tissue sample can be
separated from other components of the sample. Cells can be
harvested from a biological sample using standard techniques known
in the art. For example, cells can be harvested by centrifuging a
cell sample and resuspending the pelleted cells. The cells can be
resuspended in a buffered solution such as phosphate-buffered
saline (PBS). After centrifuging the cell suspension to obtain a
cell pellet, the cells can be lysed to extract DNA, e.g., gDNA.
See, e.g., Ausubel et al., Current Protocols in Molecular Biology,
eds., John Wiley & Sons (2003). The sample can be concentrated
and/or purified to isolate DNA. All samples obtained from a
subject, including those subjected to any sort of further
processing, are considered to be obtained from the subject. Routine
methods can be used to extract genomic DNA from a biological
sample, including, for example, phenol extraction. Alternatively,
genomic DNA can be extracted with kits such as the QIAAMP.RTM.
Tissue Kit (Qiagen, Chatsworth, Calif.) and the WIZARD.RTM. Genomic
DNA purification kit (Promega).
[0063] The absence or presence of a variant DDR2 associated with
TKI SENSITIVITY as described herein can be determined using methods
known in the art. For example, gel electrophoresis, capillary
electrophoresis, size exclusion chromatography, sequencing, and/or
arrays can be used to detect the presence or absence of a variant
DDR2. Amplification of nucleic acids, where desirable, can be
accomplished using methods known in the art, e.g., PCR. In one
example, a sample (e.g., a sample comprising genomic DNA), is
obtained from a subject. The DNA in the sample is then examined to
identify a variant DDR2 as described herein. The presence of the
variant DDR2 can be determined by any method described herein,
e.g., by sequencing or by hybridization of the gene in the genomic
DNA, RNA, or cDNA to a nucleic acid probe, e.g., a DNA probe (which
includes cDNA and oligonucleotide probes) or an RNA probe. The
nucleic acid probe can be designed to specifically or
preferentially hybridize with a particular variant.
[0064] Other methods of nucleic acid analysis can include direct
manual sequencing (Church and Gilbert, Proc. Natl. Acad. Sci. USA
81:1991-1995 (1988); Sanger et al., Proc. Natl. Acad. Sci. USA
74:5463-5467 (1977); Beavis et al., U.S. Pat. No. 5,288,644);
automated fluorescent sequencing; single-stranded conformation
polymorphism assays (SSCP) (Schafer et al., Nat. Biotechnol.
15:33-39 (1995)); clamped denaturing gel electrophoresis (CDGE);
two-dimensional gel electrophoresis (2DGE or TDGE); conformational
sensitive gel electrophoresis (CSGE); denaturing gradient gel
electrophoresis (DGGE) (Sheffield et al., Proc. Natl. Acad. Sci.
USA 86:232-236 (1989)); denaturing high performance liquid
chromatography (DHPLC, Underhill et al., Genome Res. 7:996-1005
(1997)); infrared matrix-assisted laser desorption/ionization
(IR-MALDI) mass spectrometry (WO 99/57318); mobility shift analysis
(Orita et al., Proc. Natl. Acad. Sci. USA 86:2766-2770 (1989));
restriction enzyme analysis (Flavell et al., Cell 15:25 (1978);
Geever et al., Proc. Natl. Acad. Sci. USA 78:5081 (1981));
quantitative real-time PCR (Raca et al., Genet Test 8(4):387-94
(2004)); heteroduplex analysis; chemical mismatch cleavage (CMC)
(Cotton et al., Proc. Natl. Acad. Sci. USA 85:4397-4401 (1985));
RNase protection assays (Myers et al., Science 230:1242 (1985));
use of polypeptides that recognize nucleotide mismatches, e.g., E.
coli mutS protein; allele-specific PCR, and combinations of such
methods. See, e.g., Gerber et al., U.S. Pat. Publication No.
2004/0014095 which is incorporated herein by reference in its
entirety.
[0065] Sequence analysis can also be used to detect specific
polymorphic variants. For example, polymorphic variants can be
detected by sequencing exons, introns, 5' untranslated sequences,
or 3' untranslated sequences. A sample comprising DNA or RNA is
obtained from the subject. PCR or other appropriate methods can be
used to amplify a portion encompassing the polymorphic site, if
desired. The sequence is then ascertained, using any standard
method, and the presence of a polymorphic variant is determined.
Real-time pyrophosphate DNA sequencing is yet another approach to
detection of polymorphisms and polymorphic variants (Alderborn et
al., Genome Research 10(8):1249-1258 (2000)). Additional methods
include, for example, PCR amplification in combination with
denaturing high performance liquid chromatography (dHPLC)
(Underhill et al., Genome Research 7(10):996-1005 (1997)).
[0066] In some embodiments, the methods described herein include
determining the sequence of the entire region of the DDR2 locus
described herein as being of interest. For example, a method
provided herein can include determining a nucleic acid sequence of
a DDR2 gene in a sample from a human subject, determining an
expected amino acid translation of nucleic acid sequence; and
comparing the expected amino acid translation with a reference
amino acid sequence. In such a method, the presence of at least one
amino acid variant (e.g., a non-conservative amino acid
substitution) relative to the reference amino acid sequence can be
indicative of TKI sensitivity or an increased likelihood of
response to a TKI, or emergence of resistance to a TKI, in the
human subject. For example, an amino acid variant can comprise a
non-conservative substitution described herein. In some
embodiments, the sequence is determined on both strands of DNA.
[0067] In order to detect sequence variants, it may be desirable to
amplify a portion of genomic DNA (gDNA) or cDNA encompassing the
variant site. Such regions can be amplified and isolated by PCR
using oligonucleotide primers designed based on genomic and/or cDNA
sequences that flank the site. PCR refers to procedures in which
target nucleic acid (e.g., genomic DNA) is amplified in a manner
similar to that described in U.S. Pat. No. 4,683,195, and
subsequent modifications of the procedure described therein.
Generally, sequence information from the ends of the region of
interest or beyond are used to design oligonucleotide primers that
are identical or similar in sequence to opposite strands of a
potential template to be amplified. See e.g., PCR Primer: A
Laboratory Manual, Dieffenbach and Dveksler, (Eds.); McPherson et
al., PCR Basics: From Background to Bench (Springer Verlag, 2000);
Mattila et al., Nucleic Acids Res., 19:4967 (1991); Eckert et al.,
PCR Methods and Applications, 1:17 (1991); PCR (eds. McPherson et
al., IRL Press, Oxford); and U.S. Pat. No. 4,683,202. Other
amplification methods that may be employed include the ligase chain
reaction (LCR) (Wu and Wallace, Genomics 4:560 (1989), Landegren et
al., Science 241:1077 (1988), transcription amplification (Kwoh et
al., Proc. Natl. Acad. Sci. USA 86:1173 (1989)), self-sustained
sequence replication (Guatelli et al., Proc. Nat. Acad. Sci. USA
87:1874 (1990)), and nucleic acid based sequence amplification
(NASBA). Guidelines for selecting primers for PCR amplification are
well known in the art. See, e.g., McPherson et al., PCR Basics:
From Background to Bench, Springer-Verlag, 2000. A variety of
computer programs for designing primers are available, e.g.,
`Oligo` (National Biosciences, Inc, Plymouth, Minn.), MacVector
(Kodak/IBI), and the GCG suite of sequence analysis programs
(Genetics Computer Group, Madison, Wis. 53711).
[0068] In some cases, PCR conditions and primers can be developed
that amplify a product only when the variant allele is present or
only when the wild type allele is present (MSPCR or allele-specific
PCR). For example, subject DNA and a control can be amplified
separately using either a wild type primer or a primer specific for
the variant allele. Each set of reactions is then examined for the
presence of amplification products using standard methods to
visualize the DNA. For example, the reactions can be size-separated
by agarose gel electrophoresis and the DNA visualized by staining
with ethidium bromide or other DNA intercalating dye. In DNA
samples from heterozygous subjects, reaction products would be
detected in each reaction.
[0069] Real-time quantitative PCR can also be used to determine
copy number. Quantitative PCR permits both detection and
quantification of specific DNA sequence in a sample as an absolute
number of copies or as a relative amount when normalized to DNA
input or other normalizing genes. A key feature of quantitative PCR
is that the amplified DNA product is quantified in real-time as it
accumulates in the reaction after each amplification cycle. Methods
of quantification can include the use of fluorescent dyes that
intercalate with double-stranded DNA, and modified DNA
oligonucleotide probes that fluoresce when hybridized with a
complementary DNA.
[0070] In some embodiments, a peptide nucleic acid (PNA) probe can
be used instead of a nucleic acid probe in the hybridization
methods described above. PNA is a DNA mimetic with a peptide-like,
inorganic backbone, e.g., N-(2-aminoethyl)glycine units, with an
organic base (A, G, C, T or U) attached to the glycine nitrogen via
a methylene carbonyl linker (see, e.g., Nielsen et al.,
Bioconjugate Chemistry, The American Chemical Society, 5:1 (1994)).
The PNA probe can be designed to specifically hybridize to a
nucleic acid comprising a polymorphic variant conferring TKI
sensitivity or resistance.
[0071] In some cases, allele-specific oligonucleotides can also be
used to detect the presence of a variant. For example, variants can
be detected by performing allele-specific hybridization or
allele-specific restriction digests. Allele specific hybridization
is an example of a method that can be used to detect sequence
variants, including complete haplotypes of a subject (e.g., a
mammal such as a human). See Stoneking et al., Am. J. Hum. Genet.
48:370-382 (1991); and Prince et al., Genome Res. 11:152-162
(2001). An "allele-specific oligonucleotide" (also referred to
herein as an "allele-specific oligonucleotide probe") is an
oligonucleotide that is specific for a particular polymorphism can
be prepared using standard methods (see Ausubel et al., Current
Protocols in Molecular Biology, supra). Allele-specific
oligonucleotide probes typically can be approximately 10-50 base
pairs, preferably approximately 15-30 base pairs, that specifically
hybridize to a nucleic acid region that contains a polymorphism.
Hybridization conditions are selected such that a nucleic acid
probe can specifically bind to the sequence of interest, e.g., the
variant nucleic acid sequence. Such hybridizations typically are
performed under high stringency as some sequence variants include
only a single nucleotide difference. In some cases, dot-blot
hybridization of amplified oligonucleotides with allele-specific
oligonucleotide (ASO) probes can be performed. See, for example,
Saiki et al., Nature (London) 324:163-166 (1986).
[0072] In some embodiments, allele-specific restriction digest
analysis can be used to detect the existence of a variant, if the
variants result in the creation or elimination of a restriction
site. Allele-specific restriction digests can be performed in the
following manner. A sample containing genomic DNA is obtained from
the individual and genomic DNA is isolated for analysis. For
nucleotide sequence variants that introduce a restriction site,
restriction digest with the particular restriction enzyme can
differentiate the alleles. In some cases, polymerase chain reaction
(PCR) can be used to amplify a region comprising the polymorphic
site, and restriction fragment length polymorphism analysis is
conducted (see Ausubel et al., Current Protocols in Molecular
Biology, supra). The digestion pattern of the relevant DNA fragment
indicates the presence or absence of a variant and is therefore
indicative of the presence or absence of TKI sensitivity or
resistance. For sequence variants that do not alter a common
restriction site, mutagenic primers can be designed that introduce
a restriction site when the variant allele is present or when the
wild type allele is present. For example, a portion of a nucleic
acid can be amplified using the mutagenic primer and a wild type
primer, followed by digest with the appropriate restriction
endonuclease.
[0073] In some embodiments, fluorescence polarization
template-directed dye-terminator incorporation (FP-TDI) is used to
determine which of multiple polymorphic variants of a polymorphism
is present in a subject (Chen et al., Genome Research 9(5):492-498
(1999)). Rather than involving use of allele-specific probes or
primers, this method employs primers that terminate adjacent to a
variant site, so that extension of the primer by a single
nucleotide results in incorporation of a nucleotide complementary
to the polymorphic variant at the polymorphic site.
[0074] In some cases, DNA containing an amplified portion may be
dot-blotted, using standard methods (see Ausubel et al., Current
Protocols in Molecular Biology, supra), and the blot contacted with
the oligonucleotide probe. The presence of specific hybridization
of the probe to the DNA is then detected. Specific hybridization of
an allele-specific oligonucleotide probe to DNA from the subject is
indicative of TKI resistance or sensitivity.
[0075] The methods can include determining the genotype of a
subject with respect to both copies of the polymorphic site present
in the genome. For example, the complete genotype may be
characterized as -/-, as -/+, or as +/+, where a minus sign
indicates the presence of the reference or wild type sequence at
the polymorphic site, and the plus sign indicates the presence of a
variant other than the reference sequence. If multiple variants
exist at a site, this can be appropriately indicated by specifying
which ones are present in the subject. Any of the detection means
described herein can be used to determine the genotype of a subject
with respect to one or both copies of the variant present in the
subject's genome.
[0076] Methods of nucleic acid analysis to detect variants can
include, e.g., microarray analysis. Hybridization methods, such as
Southern analysis, Northern analysis, or in situ hybridizations,
can also be used (see Ausubel et al., Current Protocols in
Molecular Biology, eds., John Wiley & Sons (2003)). To detect
microdeletions, fluorescence in situ hybridization (FISH) using DNA
probes that are directed to a putatively deleted region in a
chromosome can be used. For example, probes that detect all or a
part of a microsatellite marker can be used to detect
microdeletions in the region that contains that marker.
[0077] In some embodiments, it is desirable to employ methods that
can detect the presence of multiple variants (e.g., variants at a
plurality of sites) in parallel or substantially simultaneously.
Oligonucleotide arrays represent one suitable means for doing so.
Other methods, including methods in which reactions (e.g.,
amplification, hybridization) are performed in individual vessels,
e.g., within individual wells of a multi-well plate or other vessel
can also be performed so as to detect the presence of multiple
variants (e.g., variants at a plurality of polymorphic sites) in
parallel or substantially simultaneously.
[0078] Nucleic acid probes can be used to detect and/or quantify
the presence of a particular target nucleic acid sequence within a
sample of nucleic acid sequences, e.g., as hybridization probes, or
to amplify a particular target sequence within a sample, e.g., as a
primer. Probes have a complimentary nucleic acid sequence that
selectively hybridizes to the target nucleic acid sequence. In
order for a probe to hybridize to a target sequence, the
hybridization probe must have sufficient identity with the target
sequence, i.e., at least 70% (e.g., 80%, 90%, 95%, 98% or more)
identity to the target sequence. The probe sequence must also be
sufficiently long so that the probe exhibits selectivity for the
target sequence over non-target sequences. For example, the probe
will be at least 20 (e.g., 25, 30, 35, 50, 100, 200, 300, 400, 500,
600, 700, 800, 900 or more) nucleotides in length. In some
embodiments, the probes are not more than 30, 50, 100, 200, 300,
500, 750, or 1000 nucleotides in length. Probes are typically about
20 to about 1.times.10.sup.6 nucleotides in length. Probes include
primers, which generally refers to a single-stranded
oligonucleotide probe that can act as a point of initiation of
template-directed DNA synthesis using methods such as polymerase
chain reaction (PCR), ligase chain reaction (LCR), etc., for
amplification of a target sequence.
[0079] Control probes can also be used. For example, a probe that
binds a less variable sequence, e.g., repetitive DNA associated
with a centromere of a chromosome, can be used as a control. Probes
that hybridize with various centromeric DNA and locus-specific DNA
are available commercially, for example, from Vysis, Inc. (Downers
Grove, Ill.), Molecular Probes, Inc. (Eugene, Oreg.), or from
Cytocell (Oxfordshire, UK). Probe sets are available commercially
such from Applied Biosystems, e.g., the Assays-on-Demand SNP kits.
Alternatively, probes can be synthesized, e.g., chemically or in
vitro, or made from chromosomal or genomic DNA through standard
techniques. For example, sources of DNA that can be used include
genomic DNA, cloned DNA sequences, somatic cell hybrids that
contain one, or a part of one, human chromosome along with the
normal chromosome complement of the host, and chromosomes purified
by flow cytometry or microdissection. The region of interest can be
isolated through cloning, or by site-specific amplification via
PCR. See, for example, Nath and Johnson, Biotechnic. Histochem.
73(1):6-22 (1998); Wheeless et al., Cytometry 17:319-326 (1994);
and U.S. Pat. No. 5,491,224.
[0080] In some embodiments, the probes are labeled, e.g., by direct
labeling, with a fluorophore, an organic molecule that fluoresces
after absorbing light of lower wavelength/higher energy. A directly
labeled fluorophore allows the probe to be visualized without a
secondary detection molecule. After covalently attaching a
fluorophore to a nucleotide, the nucleotide can be directly
incorporated into the probe with standard techniques such as nick
translation, random priming, and PCR labeling. Alternatively,
deoxycytidine nucleotides within the probe can be transaminated
with a linker. The fluorophore then is covalently attached to the
transaminated deoxycytidine nucleotides. See, e.g., U.S. Pat. No.
5,491,224.
[0081] Fluorophores of different colors can be chosen such that
each probe in a set can be distinctly visualized. For example, a
combination of the following fluorophores can be used:
7-amino-4-methylcoumarin-3-acetic acid (AMCA), TEXAS RED.TM.
(Molecular Probes, Inc., Eugene, Oreg.),
5-(and-6)-carboxy-X-rhodamine, lissamine rhodamine B,
5-(and-6)-carboxyfluorescein, fluorescein-5-isothiocyanate (FITC),
7-diethylaminocoumarin-3-carboxylic acid,
tetramethylrhodamine-5-(and-6)-isothiocyanate,
5-(and-6)-carboxytetramethylrhodamine,
7-hydroxycoumarin-3-carboxylic acid, 6-[fluorescein
5-(and-6)-carboxamido]hexanoic acid,
N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a diaza-3-indacenepropionic
acid, eosin-5-isothiocyanate, erythrosin-5-isothiocyanate, and
CASCADE.TM. blue acetylazide (Molecular Probes, Inc., Eugene,
Oreg.). Fluorescently labeled probes can be viewed with a
fluorescence microscope and an appropriate filter for each
fluorophore, or by using dual or triple band-pass filter sets to
observe multiple fluorophores. See, for example, U.S. Pat. No.
5,776,688. Alternatively, techniques such as flow cytometry can be
used to examine the hybridization pattern of the probes.
Fluorescence-based arrays are also known in the art.
[0082] In other embodiments, the probes can be indirectly labeled
with, e.g., biotin or digoxygenin, or labeled with radioactive
isotopes such as .sup.32P and .sup.3H. For example, a probe
indirectly labeled with biotin can be detected by avidin conjugated
to a detectable marker. For example, avidin can be conjugated to an
enzymatic marker such as alkaline phosphatase or horseradish
peroxidase. Enzymatic markers can be detected in standard
colorimetric reactions using a substrate and/or a catalyst for the
enzyme. Catalysts for alkaline phosphatase include
5-bromo-4-chloro-3-indolylphosphate and nitro blue tetrazolium.
Diaminobenzoate can be used as a catalyst for horseradish
peroxidase.
[0083] Oligonucleotide probes that exhibit differential or
selective binding to polymorphic sites may readily be designed by
one of ordinary skill in the art. For example, an oligonucleotide
that is perfectly complementary to a sequence that encompasses a
polymorphic site (i.e., a sequence that includes the polymorphic
site, within it or at one end) will generally hybridize
preferentially to a nucleic acid comprising that sequence, as
opposed to a nucleic acid comprising an alternate polymorphic
variant.
[0084] In another aspect, the invention features arrays that
include a substrate having a plurality of addressable areas, and
methods of using them. At least one area of the plurality includes
a nucleic acid probe that binds specifically to a sequence
comprising a variant described herein, and can be used to detect
the absence or presence of said variant, e.g., one or more SNPs,
microsatellites, minisatellites, or indels, as described herein,
e.g., to determine a haplotype comprising a plurality of variants.
For example, the array can include one or more nucleic acid probes
that can be used to detect a variant described herein. In some
embodiments, the array further includes at least one area that
includes a nucleic acid probe that can be used to specifically
detect another marker associated with TKI resistance or
sensitivity, as described herein. In some embodiments, the probes
are nucleic acid capture probes.
[0085] Generally, microarray hybridization is performed by
hybridizing a nucleic acid of interest (e.g., a nucleic acid
encompassing a polymorphic site) with the array and detecting
hybridization using nucleic acid probes. In some cases, the nucleic
acid of interest is amplified prior to hybridization. Hybridization
and detecting are generally carried out according to standard
methods. See, e.g., Published PCT Application Nos. WO 92/10092 and
WO 95/11995, and U.S. Pat. No. 5,424,186. For example, the array
can be scanned to determine the position on the array to which the
nucleic acid hybridizes. The hybridization data obtained from the
scan is typically in the form of fluorescence intensities as a
function of location on the array.
[0086] Arrays can be formed on substrates fabricated with materials
such as paper, glass, plastic (e.g., polypropylene, nylon, or
polystyrene), polyacrylamide, nitrocellulose, silicon, optical
fiber, or any other suitable solid or semisolid support, and can be
configured in a planar (e.g., glass plates, silicon chips) or three
dimensional (e.g., pins, fibers, beads, particles, microtiter
wells, capillaries) configuration. Methods for generating arrays
are known in the art and include, e.g., photolithographic methods
(see, e.g., U.S. Pat. Nos. 5,143,854; 5,510,270; and 5,527,681),
mechanical methods (e.g., directed-flow methods as described in
U.S. Pat. No. 5,384,261), pin-based methods (e.g., as described in
U.S. Pat. No. 5,288,514), and bead-based techniques (e.g., as
described in PCT US/93/04145). The array typically includes
oligonucleotide probes capable of specifically hybridizing to
different polymorphic variants. Oligonucleotide probes forming an
array may be attached to a substrate by any number of techniques,
including, without limitation, (i) in situ synthesis (e.g.,
high-density oligonucleotide arrays) using photolithographic
techniques; (ii) spotting/printing at medium to low density on
glass, nylon or nitrocellulose; (iii) by masking, and (iv) by
dot-blotting on a nylon or nitrocellulose hybridization membrane.
Oligonucleotides also can be non-covalently immobilized on a
substrate by hybridization to anchors, by means of magnetic beads,
or in a fluid phase such as in microtiter wells or capillaries.
[0087] Arrays can include multiple detection blocks (i.e., multiple
groups of probes designed for detection of particular variants).
Such arrays can be used to analyze multiple different variants.
Detection blocks may be grouped within a single array or in
multiple, separate arrays so that varying conditions (e.g.,
conditions optimized for particular variants) may be used during
the hybridization. For example, it may be desirable to provide for
the detection of those variants that fall within G-C rich stretches
of a genomic sequence, separately from those falling in A-T rich
segments. General descriptions of using oligonucleotide arrays for
detection of variants can be found, for example, in U.S. Pat. Nos.
5,858,659 and 5,837,832. In addition to oligonucleotide arrays,
cDNA arrays may be used similarly in certain embodiments of the
invention.
[0088] The methods described herein can include providing an array
as described herein; contacting the array with a sample (e.g., a
portion of genomic DNA that includes at least a portion of human
chromosome 14q32 (e.g., a region between SNPs rs3783397 and
r56576201) and/or optionally, a different portion of genomic DNA
(e.g., a portion that includes a different portion of a human
chromosome (e.g., including another region associated with TKI
resistance or sensitivity)), and detecting binding of a nucleic
acid from the sample to the array. Optionally, the method includes
amplifying nucleic acid from the sample, e.g., genomic DNA that
includes at least a portion of an IFN2 gene, and, optionally, a
region that includes another region associated with TKI resistance
or sensitivity, prior to or during contact with the array.
[0089] In some aspects, the methods described herein can include
using an array that can ascertain differential expression patterns
or copy numbers of one or more genes in samples from normal and
affected individuals (see, e.g., Redon et al., Nature
444(7118):444-54 (2006)). For example, arrays of probes to a marker
described herein can be used to measure variants between DNA from a
subject having TKI sensitive SCC and control DNA, e.g., DNA
obtained from an individual that does not have TKI sensitive SCC.
Since the clones on the array contain sequence tags, their
positions on the array are accurately known relative to the genomic
sequence. Different hybridization patterns between DNA from an
individual with TKI sensitive SCC and DNA from a normal individual
at areas in the array corresponding to markers in the human
chromosome 14q32 locus as described herein, and, optionally, one or
more other regions associated with TKI sensitive SCC, are
indicative of sensitivity to the TKI. Methods for array production,
hybridization, and analysis are described, e.g., in Snijders et
al., Nat. Genet. 29:263-264 (2001); Klein et al., Proc. Natl Acad.
Sci. USA 96:4494-99 (1999); Albertson et al., Breast Cancer Res.
and Treatment 78:289-298 (2003); and Snijders et al. "BAC
microarray based comparative genomic hybridization," in Zhao et al.
(eds), Bacterial Artificial Chromosomes: Methods and Protocols,
Methods in Molecular Biology, Humana Press, 2002.
[0090] Tyrosine Kinase Inhibitors (TKIs)
[0091] Upon identification of a subject as having a tumor with one
or more DDR2 mutations as described herein, the methods can include
administering a therapeutically effective amount of one or more
tyrosine kinase inhibitors. TKIs useful in the methods described
herein can include Axitinib (INLYTA; Pfizer), Critozinib (XALKORI;
Pfizer), Dasatinib (SPRYCEL; BMS), Erlotinib (TARCEVA; Roche,
Astellas), Gefitinib (IRESSA; Astra Zeneca), Imatinib (GLEEVEC;
Novartis), Lapatinib (TYKERB; GSK), Nilotinib (TASIGNA; Novartis),
Pazopanib (VOTRIENT; GSK), Ruxolitinib (JAKAFI; Incyte, Novartis),
Sorafenib (NEXAVAR; Bayer/Onyx), Sunitinib (SUTENT; Pfizer),
Vandetanib (CAPRELSA/ZACTIMA; Astra Zeneca), ponatinib (AP24534;
ARIAD); Vemurafanib (ZELBORAF; Roche/Daiichi Sankyo), lapatinib
(GW-572016), canertinib (CI-1033), semaxinib (SU5416), vatalanib
(PTK787/ZK222584), sorafenib (BAY 43-9006), sutent (SU11248), and
leflunomide (SU101). In preferred embodiments, the TKI is
dasatinib, nilotinib, imatinib, and/or ponatinib.
[0092] SPRYCEL (dasatinib, Bristol-Myers Squibb [BMS]-354825) is a
potent, broad spectrum inhibitor of 5 critical oncogenic tyrosine
kinases/kinase family members (BCR-ABL, SRC, c-KIT, PDGF receptor
.beta. [PDGFR.beta.], and ephrin [EPH] receptor kinases), each of
which are activated in multiple forms of human malignancies, and
was discovered and developed by BMS. The chemical name for
dasatinib is N-(2
chloro-6-methylphenyl)-2-[[6-[4-(2-hydroxyethyl)-1-piperazinyl]-2-methyl--
4 pyrimidinyl]amino]-5-thiazolecarboxamide, monohydrate. The
molecular formula is C.sub.22H.sub.26ClN.sub.7O.sub.2S.H.sub.2O,
which corresponds to a formula weight of 506.02 (monohydrate). The
anhydrous free base has a molecular weight of 488.01. SPRYCEL is
approved in the United States (US)i, Europe (EU), and several other
countries for the treatment of adults in all phases of chronic
myeloid leukemia (CML) with resistance or intolerance to prior
therapy including imatinib, and in patients with Philadelphia
chromosome positive acute lymphoblastic leukemia (Ph+ ALL) who are
resistant or intolerant to prior therapy.
[0093] Pharmaceutical compositions including TKIs, as well as
dosage and routes of administration, are known in the art or can be
determined using routine experimentation.
[0094] For example, the recommended starting dosage of dasatinib
for adults with chronic phase CML is 100 mg administered orally
once daily (QD). The recommended starting dosage for accelerated
phase CIVIL, myeloid or lymphoid blast phase CIVIL, or Ph+ ALL is
140 mg/day administered orally once daily. In clinical studies of
adult CML and Ph+ ALL patients, dose escalation to 140 mg once
daily (chronic phase CIVIL) or 180 mg once daily (advanced phase
CML and Ph+ ALL) was allowed in patients who did not achieve a
hematologic or cytogenetic response at the recommended starting
dosage.
Examples
[0095] The invention is further described in the following
examples, which do not limit the scope of the invention described
in the claims.
[0096] Materials and Methods
[0097] The following materials and methods were used in the
Examples described herein.
[0098] Collection of Squamous Cell Lung Cancer Samples:
[0099] For the primary and secondary screens tumor samples were
obtained under a general tissue collection protocol for patients
with lung cancer who are consented to tissue collection for
research, including DNA sequencing, prior to surgery. All patients
with resectable biopsy-proven squamous cell lung cancers (as
diagnosed by a board-certified Anatomic Pathologist) were eligible
and eligible subjects underwent a detailed informed consent
procedure prior to enrollment on the protocol which included a
discussion of the use of tissue samples for DNA sequencing studies
and written documentation of consent. In addition, DNA samples from
de-identified squamous cell lung cancer patients were obtained from
the Ontario Cancer Institute for the primary and secondary screens
as part of a Dana Farber/Harvard Cancer Center IRB-approved
collection of de-identified tumor samples for DNA sequencing
studies. IRB approval for collection of de-identified samples was
subject to a review of the local IRB-approved protocols for all
external sites to ensure an adequate informed consent process had
taken place.
[0100] The validation screen was performed as follows. Samples were
collected in accordance with a tissue collection protocol approved
by the University of Cologne Ethics Committee which involved a
detailed informed consent process including a discussion of genetic
testing prior to the subject's surgery with written documentation
of consent. Again, all patients with biopsy-proven squamous cell
lung cancer were eligible regardless of disease stage as long as
their tumors were considered resectable. De-identified samples were
also collected from additional European sites including Haukeland
University Hospital, University Hospital Zurich, Universite Joseph
Fourier, Oslo University Hospital, Jena University Hospital and
University Medical Centre Groningen. At all sites samples were
obtained in accordance with an IRB approved tissue collection
protocol and the collection of de-identified samples from these
sites was approved by the University of Cologne Ethics Committee
after a review of local collection protocols.
[0101] For the single patient sample obtained from the recent
clinical trial of combination therapy with dasatinib and erlotinib
for advanced lung cancer DNA was obtained under an IRB-approved
protocol and the sequencing of the de-identified sample by both 454
and Sanger sequencing was performed with approval after a review of
the collection protocol.
[0102] In all instances specimens were continuously selected at the
site of surgery in order to avoid sampling bias and all samples
were de-identified prior to processing for DDR2 sequencing. When
available, de-identified correlative clinical data was provided
with the samples, though this data was not available to the
investigators prior to sample genotyping. Patients with a prior
history of tumors involving a visceral organ site were excluded to
avoid the inclusion of metastases.
[0103] DDR2 Sequencing.
[0104] DDR2 was sequenced from genomic DNA obtained from squamous
lung cancer cell lines and patient samples by conventional Sanger
sequencing. In the discovery set 20 patient samples and matched
normal DNA were used for sequencing 201 genes including 90 kinases.
All mutations were verified as somatic. Mutations were identified
using an automated mutation caller and then verified manually with
comparison made to the matched normal sequence in the case of all
primary tumor samples. In the secondary screen 35 additional
patient samples and 13 SCC cell lines were used for sequencing the
six mutated tyrosine kinases identified in the primary screen
(DDR2, FGFR2, NTRK2, JAK2, CDK8 and FLT3). In the validation screen
222 total samples underwent sequencing of the DDR2 gene. In all
cases except D125Y matched normal DNA was available to verify the
mutation as somatic.
[0105] Cell Culture.
[0106] A549, NCI-H2286, HCC-366 and NCI-H1703 cells were obtained
from the core collection at the Dana Farber Cancer Institute,
having previously been purchased from the ATCC and used to
establish a collection of early passage lung cancer cell lines
which were analyzed by fingerprinting and SNP arrays (32). All
cells used for the experiments described in this manuscript were
obtained from freezes made at that time. Lung cancer cell lines
were grown in RPMI (Invitrogen) with 10% fetal calf serum, NIH-3T3
cells were grown in DMEM (Mediatech) with 10% serum and Ba/F3 cells
in RPMI supplemented with 10% serum and IL-3 (BD Biosciences) at 10
ng/ml. For IL-3 withdrawal experiments Ba/F3 cells were collected
via centrifugation, washed once in sterile PBS and then resuspended
in media without IL-3. Colony formation assays in NIH-3T3 cells
were performed in six-well plates in which 25,000 NIH-3T3 cells
were plated in triplicate in 1 ml of 0.33% top agar on top of 2 ml
of 0.5% bottom agar. After three weeks colonies were counted using
the NIH ImageJ software.
[0107] Vectors.
[0108] The full-length DDR2 cDNA was obtained from Origene and
cloned into the EcoRI site of the retroviral vector p-Wzl-Blast and
p-Babe-puro following the addition of a c-terminal FLAG tag by PCR.
Mutants were generated by site-directed mutagenesis using the
Quickchange site directed mutagenesis kit (Strategene). All
mutations were verified by sequencing. sh-RNA lentiviral vectors
for DDR2 were obtained from The RNAi Consortium at the Broad
Institute (46, 47). DDR2 sh-RNA-2 corresponds to TRC clone
TRCN0000121117 with hairpin sequence
5'-CCGG-CCCATGCCTATGCCACTCCAT-CTCGAG-ATGGAGTGGCATAGGCATGGG-TTTTTG-3'
(SEQ ID NO:2) targeting the 3' UTR of DDR2. DDR2 sh-RNA-5
corresponds to TRC clone TRCN0000121121 with hairpin sequence
5'-CCGG-CCCTGGAGGTTCCATCATTTA-CTCGAG-TAAATGATGGAACCTCCAGGG-TTTTTG-3'
(SEQ ID NO:3) targeting the coding sequence of DDR2. Both hairpins
were provided in the pLKO vector. A hairpin targeting GFP (shGFP)
was obtained from TRC as well and used as a control.
[0109] Viral Infections.
[0110] The DDR2 transgene was expressed in the lung cancer cell
lines, NIH-3T3 cells and Ba/F3 cells using retroviral transduction
with the pWzl vector, as has been previously described. Briefly,
293T cells were used to generate the virus with the appropriate
pWzl or pBabe vector and packaging vector transfected using Fugene
(Roche). Cells were subjected to two rounds of overnight infection
in the presence of polybrene and stable cells generated using
blasticidin selection at 10 mg/ml for 3T3, Ba/F3 and A549, 2 mg/ml
NCI-H2286 and for NCI-H1703 and 1 mg/ml for HCC-366. Lentiviral
infections were performed per the on-line TRC protocol (48) with
293T cells transfected with the suggested three vector combination
of pLKO, VSVG and delta 8.9. Virus was collected and used to infect
the lung cancer cell lines for six hours in the presence of
polybrene. Stable cell lines were generated using puromycin
selection at a concentration of 2 mg/ml for NCI-H2286 and 4 mg/ml
for NCI-H1703, A549 and HCC-366.
[0111] Cell Proliferation and Viability Assays.
[0112] Cell proliferation was measured with the Cell-Titer-Glo
reagent (Promega) per the manufacturer's instructions. For
experiments with the SCC cell lines cells were plated in
clear-bottomed 96 well plates at a density of 1500 cells per well.
The following day the drug was added and cell proliferation was
measured six days later for the SCC cell lines. For Ba/F3 cells
were plated at 5000 cells per well and the drug added the same day.
Proliferation was measured four days later. Proliferation
measurements were made using a standard 96 well plate
luminometer/plate reader. Data are shown as relative values in
which the luminescence at a given drug concentration is compared to
untreated cells of the same cell type. Kinase inhibitors were
purchased from LC Labs or were synthesized by Nathanael Gray's
laboratory at Harvard Medical School. In vitro IC50s for DDR2 were
determined for all compounds by LanthaScreen TR-FRET kinase
activity assays performed by Invitrogen. Cell viability was
measured using a vi-Cell reader to stain cells with trypan blue and
to generate 50 independent images for each measured sample. Annexin
V (BD Biosciences) analysis was performed on dasatinib treated
cells 48 hours after addition of drug per the manufacturer's
protocol. For sh-RNA experiments cells were plated at a density of
1500 cells per well in 96 well plates following puromycin
selection. Proliferation was measured four days later as compared
to cells expressing a hairpin targeting GFP.
[0113] Immunoblots:
[0114] Immunoblots were performed using the Nupage system
(Invitrogen) per the manufacturer's protocol. Cells were lysed in
1% NP-40 with protease (Roche) and phosphatase inhibitors
(Calbiochem) and protein concentration assayed with the Bradford
reagent (Bio-Rad). Primary antibodies used were Flag-M2 (Sigma),
phospho-Y417-Src (Cell Signaling Technologies), phospho-Y694-STAT5
(Cell Signaling) and Actin (Santa Cruz). A DDR2 antibody was
generated for this project by Bethyl Labs. Secondary HRP-conjugated
antibodies were all obtained from Pierce and proteins detected by
pico-ECL (Thermo Scientific). Images were imported into Adobe
Illustrator using an Epson 4490 scanner. In some cases, brightness
and/or contrast of the scanned images was adjusted for clarity and
blots were cropped to display the area of interest in the displayed
figures. In all cases adjustment of brightness or contrast the
adjustment was applied uniformly to the image as a whole.
[0115] Xenografts:
[0116] All animal experiments were performed according to
institutional guidelines regarding animal safety. Nude mice were
injected with the lung cancer cell lines at a density of 2.5
(A549), 3.0 (NCI-H1703) or 5.0 (NCI-H2286 and HCC-366) million
cells per injection to try to control for the variable rates of
tumor growth in the animals. Cohorts of ten mice were injected at
three sites for each cell type and the mice were observed until the
tumor volume approached 150 cubic milliliters for A549 and
NCI-H1703 or 100 cubic milliliters for NCI-H1703. At that time mice
were treated with dasatinib at 50 mg/kg or vehicle control daily
for two weeks and tumor size measured during the treatment
period.
[0117] Statistics:
[0118] For proliferation and colony formation assays mean values
from a minimum of triplicate samples are reported as well as
standard errors as calculated by Microsoft Excel. IC.sub.50 values
were obtained using Graph Pad Prism software. Power and sample size
calculations were performed using the Interactive Statistics Web
Resource (43).
Example 1. DDR2 is Mutated in Squamous Cell Lung Cancer
[0119] Sanger sequencing of 201 genes, including the entire
tyrosine kinome, was performed in an initial set of 20 primary lung
SCC samples and matched normal controls. Somatic missense mutations
were identified in 25 genes in this discovery sample set including
six in tyrosine kinase genes (FIG. 1a). Recurrent somatic mutations
were identified in TP53 (n=8), and in the tyrosine kinase genes:
Discoidin Domain Receptor 2 (DDR2; n=2) and Kinase insert Domain
Receptor (KDR; n=2) (FIG. 1a). Subsequent sequencing of six of the
mutated tyrosine kinase genes (DDR2, FGFR2, NTRK2, JAK2, FLT3 and
CDK8), selected on the basis of being possible therapeutic targets,
in a secondary screen of 48 squamous cell lung cancer samples
including 13 cell lines revealed four additional DDR2 mutations
(FIG. 1a) as well as three FLT3 mutations, two NTRK2 and JAK2
mutations and one mutation in each of FGFR2 and CDK8.
[0120] Given that DDR2 was the most frequently mutated gene in the
primary and secondary screen, DDR2 was sequenced in a validation
cohort of 222 primary lung SCC samples which yielded an additional
five samples with mutation, resulting in an overall frequency of
3.8% (n=11) in 290 total samples and an overall frequency of 3.2%
in primary lung SCC samples when cell lines were excluded (n=9/277)
(FIG. 1a). Mutations were found both in the kinase domain and in
other regions of the protein sequence and two mutations were
identified at G774 (FIG. 1b). The L239R and I638F mutations were
identified in the HCC-366 and NCI-H2286 SCC cell lines,
respectively, and the remainder of the mutations was found in
primary SCC samples. The majority of the mutations resided in
regions of high degrees of amino acid conservation as compared to
the murine, zebrafish and C. elegans homologs of DDR2 (FIG. 1c).
Additional genomic analysis of previously reported copy number and
gene expression datasets did not reveal any evidence of DDR2
overexpression in SCCs as compared to normal lung or lung
adenocarcinoma nor were any copy number alterations in DDR2 found
(19, 22-24). A query of the limited clinical information
accompanying the sequenced samples did not Identify any significant
correlation of DDR2 mutation status with the age, sex or smoking
status of the patients.
Example 2. DDR2 Mutant Cell Lines are Selectively Sensitive to
Tyrosine Kinase Inhibitors and to Sh-RNA-Mediated Depletion of
DDR2
[0121] To assess whether targeting DDR2 might be a promising
therapeutic strategy in lung SCC, several tyrosine kinase
inhibitors reported to inhibit DDR2 including imatinib and
dasatinib, drugs which are FDA-approved for clinical use for
targeting BCR-Abl in chronic myelogenous leukemia and acute
lymphoblastic leukemia, c-KIT in gastrointestinal stromal tumors
and PDGFR in chronic myelomonocytic leukemia (21, 25-28) were
analyzed. Fluorescence resonance energy transfer (FRET)
measurements provided in vitro K.sub.d values of dasatinib (5.4 nM)
and imatinib (71.6 nM) for recombinant DDR2 (Table 1).
TABLE-US-00002 TABLE 1 Chemical structures and Kd for DDR2 for the
compounds described in the manuscript. Compound Structure Kd(nM)
Dasatinib ##STR00001## 5.4 Imatinib ##STR00002## 71.6 Nilotinib
##STR00003## 35.4 AZD0530 ##STR00004## 291 AP24534 ##STR00005##
8.99
[0122] Dasatinib showed particular efficacy against SCC cell lines
bearing DDR2 mutations, as dasatinib inhibited proliferation of the
DDR2-mutant NCI-H2286 and HCC-366 cells with calculated IC.sub.50s
of 139 and 140 nM respectively (FIG. 2a). Of note, a recent
pharmacokinetic analysis of dasatinib in lung cancer patients
demonstrated that peak concentrations of dasatinib were in the
range of 300 ng/ml (615 nM) at the maximum tolerated dose of 140 mg
daily, a dose approved for use in leukemias (29). Imatinib was less
potent when tested in the same cell lines with respective
IC.sub.50s of 1.2 and 1.0 mM for the DDR2-mutant NCI-H2286 and
HCC-366 cell lines (FIG. 2e). Dasatinib and imatinib were less
effective against the A549 cell line which is known to harbor a
KRAS mutation and does not have any DDR2 mutations (calculated
IC.sub.50 of 7.4 mM for dasatinib and 2.3 mM for imatinib).
Consistent with previous reports, the NCI-H1703 SCC cell line,
which contains a PDGFRA amplification, was sensitive to both drugs,
serving as a positive control for our assay (30, 31). Notably, no
other somatic mutations have been reported in the COSMIC database
for NCI-H2286 or HCC-366 lines to suggest alternative dasatinib
targets and a previous report examining the drug sensitivities of
83 NSCLC cell lines identified HCC-366 as the most sensitive
squamous cell lung cancer line to dasatinib, though NCI-H2286 and
NCI-H1703 were not assayed (32). Treatment of the DDR2 mutant cell
lines with dasatinib appeared to lead to cell death as opposed to
cell cycle arrest as measured by trypan blue exclusion (FIG. 2f).
Dasatinib treatment was associated with an increase in cellular
annexin V staining, suggesting that the treated cells died by
apoptosis.
[0123] To validate DDR2 as a relevant target of dasatinib in SCCs a
DDR2 transgene with a threonine to methionine mutation at amino
acid 654, a mutation site shown previously to render DDR2
dasatinib-insensitive in a manner similar to the ability of the
T790M mutation in EGFR to confer acquired resistance to the
tyrosine kinase inhibitors erlotinib and gefitinib (33), was
ectopically expressed. The dasatinib-insensitive DDR2 "gatekeeper"
mutant was introduced in cis with the observed L239R and I638F
mutations in the HCC-366 and NCI-H2286 cell lines respectively as
well as alone in NCI-H1703. Expression of the gatekeeper mutation
led to a decrease in dasatinib sensitivity in both DDR2 mutant cell
lines and had a modest effect on NCI-H1703 (FIG. 2b; transgene
expression is shown in FIG. 2i). While the calculated IC.sub.50 for
NCI-H1703 did not change with ectopic expression of the gatekeeper,
the IC.sub.50 increased by 35-fold for NCI-H2286 and 209-fold for
HCC-366, respectively. Interestingly, a parallel sequencing project
in our lab identified a T654I mutation in DDR2 in a primary
endometrial carcinoma sample.
[0124] Dasatinib was originally designed as an inhibitor of Src and
is a multi-targeted tyrosine kinase inhibitor (34). Dasatinib
treatment is associated with toxicity in patients including
myelosuppression and the development of pleural and pericardial
effusions (35, 36). In an attempt to identify additional agents
which could potently inhibit DDR2 with less associated toxicity we
screened a panel of 20 tyrosine kinase inhibitors which were
predicted to have the potential to inhibit DDR2 based on their
respective structures. We found that nilotinib, a second-generation
BCR-Abl inhibitor, as well as with AP24534, a third generation
BCR-Abl inhibitor which displays activity against BCR-Abl and
imatinib-resistant BCR-Abl (36), inhibited the proliferation of SCC
lines harboring DDR2 mutations (FIGS. 2g and 2h). We observed that
AP24534 treatment resulted in a greater degree of inhibition than
nilotinib which was agreement with calculated in vitro K.sub.d
values of 35.4 nM for nilotinib and 9.0 nM for AP24534 as compared
to 5.4 nM for dasatinib (Table 1).
Example 3. Sh-RNAs Targeting DDR2 Kill DDR2-Mutant SCC Cell
Lines
[0125] As an independent measure of DDR2-dependency short-hairpin
RNAs targeting DDR2 were expressed using lentiviral vectors in the
NCI-H2286, HCC-366 and NCI-H1703 cell lines. A set of
sh-RNA-expressing plasmids was screened for the ability to
knock-down DDR2 mRNA expression by real-time PCR in NCI-H2286 cells
and selected two hairpins for further analysis given their ability
to reduce DDR2 mRNA levels by approximately 50%. Knock-down of DDR2
by these two sh-RNAs led to a reduction in proliferation of the two
DDR2 mutant cell lines but not of PDGFRA-amplified NCI-H1703 cells
which had been sensitive to imatinib and dasatinib in our
proliferation assays (FIG. 2c). The reduction in proliferation
appeared to correlate with the degree of knock-down as the observed
phenotype was greater with sh-RNA-2 than sh-RNA-5 (FIG. 2c) and
appeared to be caused by cell death and not cell cycle arrest.
[0126] To assess the specificity of the observed knock-down
phenotype a similar experiment was performed in NCI-H2286 and
HCC-366 cells ectopically expressing their described mutated forms
of DDR2 (I638F and L239R respectively); endogenous DDR2 was
knocked-down with sh-RNA-2 which targets the 3' UTR of DDR2 and so
would not be expected to interfere with ectopic expression of DDR2.
For both NCI-H2286 and HCC-366, ectopic expression of DDR2
attenuated the anti-proliferative effect of endogenous DDR2
knock-down and the effect was of greater magnitude in NCI-H2286,
perhaps due to a greater degree of off-target effects in HCC-366
(FIG. 2d).
Example 4. DDR2 Mutations are Associated with Dasatinib Sensitivity
In Vivo
[0127] To analyze the effects of dasatinib treatment in a somewhat
more physiological setting, xenograft studies were performed in
athymic nude mice in which cohorts of mice were injected with
NCI-H2286, HCC-366, NCI-H1703 and A549 cells. HCC-366 cells did not
form tumors in the mice and could not be analyzed further.
Following tumor formation of the three tested lines mice were
treated with dasatinib at 50 mg/kg by oral gavage for two weeks or
vehicle control. Dasatinib treatment led to a decrease in tumor
size in the NCI-H1703 and NCI-H2286 lines but not in A549,
consistent with the in vitro results (FIG. 3).
Example 5. DDR2 Mutations are Oncogenic and DDR2-Driven
Transformation is Dasatinib-Sensitive
[0128] Next, the ability of DDR2 mutations to confer an oncogenic
gain-of-function phenotype was examined. Ectopic expression of a
subset of the DDR2 mutants identified in our primary and secondary
screens (n=2/6) promoted the formation of colonies in soft agar of
NIH-3T3 cells (FIG. 4e). Colony formation was greatest in the L63V
and I638F mutants at a level comparable to that driven by
expression of the gain-of-function L858R mutation in EGFR and
modest in the remainder of the genotypes. Colony formation could be
inhibited with a single application of dasatinib at the time of
plating in the case of the L63V mutant, the mutant which
reproducibly formed the most colonies in our assay (FIG. 4a).
Dasatinib treatment also inhibited the colony formation of NIH-3T3
cells expressing the L858R mutation in EGFR, consistent with
previous reports, and did so to a lesser extent in NIH-3T3 cells
stably expressing the activating G12V KRAS mutation (FIG. 4a) (32,
37).
[0129] As the observed gain-of-function phenotype was modest for
many of the DDR2 mutants in NIH-3T3 cells, the transforming
potential of DDR2 was evaluated in the interleukin-3
(IL-3)-dependent hematopoietic cell line Ba/F3. Ectopic expression
of all six DDR2 mutants identified in the primary and secondary
screens led to IL-3-independent growth of Ba/F3 cells as did high
levels of expression of wild-type DDR2 and no differences were
observed in the time to transformation or the rate of IL-3
independent proliferation (FIG. 4f). A kinase-dead DDR2 transgene
(K608E) did not support the IL-3-independent growth of Ba/F3 cells
(FIG. 4f). While culture with the less potent DDR2 inhibitor
imatinib did not lead to significant killing of Ba/F3 cells
expressing DDR2 mutations as compared to cells grown in the
presence of IL-3, culture with dasatinib led to cell death in all
cell lines expressing DDR2 mutants with a mean calculated IC.sub.50
of 680 nM for the mutants and 30 mM for the control (FIGS. 4b and
4g). The third-generation BCR-Abl inhibitor AP24534 was also
effective in killing the IL-3-independent Ba/F3 cells expressing
mutant forms of DDR2, suggesting that this class of drugs may be
effective against DDR2-driven neoplasms while the second generation
BCR-Abl inhibitor nilotinib demonstrated modest activity against
the DDR2-tranformed Ba/F3 cells (FIGS. 4h and 4i). Survival of
Ba/F3 cells in the absence of IL-3 was associated with maintenance
of STAT5 phosphorylation as has been previously shown (FIG.
4j)(38).
Example 6. DDR2 Transformed Cell Lines Maintain Src Phosphorylation
and are Especially Sensitive to Dual Inhibition of DDR2 and Src
[0130] Given that the type I kinase inhibitor dasatinib was more
potent in DDR2-transformed Ba/F3 cells than the more
target-specific type II inhibitors nilotinib and imatinib, whether
the potency of dasatinib in this system might be due to effects of
dasatinib on other kinases in addition to DDR2 was evaluated. DDR2
has previously been shown to require Src for maximal kinase
activity (16) and levels of phosphorylated Src were maintained in
Ba/F3 cells expressing DDR2 mutants in the absence of IL-3 (FIG.
4j). To test whether the ability of DDR2 mutations to confer
IL-3-independent proliferation in Ba/F3 cells might depend on both
DDR2 and Src activity, Ba/F3 cells expressing DDR2 were treated
with AZD0530, a highly selective Src-family kinase inhibitor which
displays minimal activity against DDR2 as compared to the other
inhibitors described in this manuscript (in vitro K.sub.d 291 nM,
Table 1) (39). Similar to nilotinib treatment, AZD0530 had a modest
effect on the proliferation of the IL-3-independent DDR2-expressing
Ba/F3 cells (FIGS. 4i and 4k). However, when Ba/F3 cells expressing
the L63V DDR2 mutation were grown in 50 nM nilotinib, a
concentration associated with little effect on proliferation of
wild-type Ba/F3 cells or Ba/F3 cells expressing DDR2 mutations
(FIG. 4i), the addition of AZD0530 led to a marked reduction in
proliferation of Ba/F3 cells expressing DDR2 L63V, suggesting that
the coordinated activity of DDR2 and Src-family kinases may be
required for the ability of DDR2 mutated Ba/F3 cells to grow in the
absence of IL-3 and thereby providing a possible explanation for
the potency of dasatinib in this system (FIGS. 4c and 4m). A
similar additive effect of AZD0530 was observed when the Ba/F3
cells were co-treated with AZD0530 and 50 nM of either AP24534 or
dasatinib (FIG. 4c for L63V; for a more detailed version of this
experiment including additional DDR2 mutants see FIGS. 4m-o).
AZD0530 reduced Src and STAT5 phosphorylation in a dose-dependent
fashion in the DDR2 L63V-expressing Ba/F3 cells when used as a
single agent or in combination with nilotinib, AP24534 or dasatinib
(FIGS. 4d and 4l).
Example 7. Observation of a DDR2 Kinase Domain Mutation in a
Clinical Trial Subject with a Radiographic Response to Combination
Therapy with Dasatinib and Erlotinib
[0131] Two recent early-phase clinical trials of dasatinib have
been reported in which subjects with advanced stage lung cancer
were treated with either dasatinib or a combination of dasatinib
and erlotinib (29, 40). One of seven subjects with a squamous cell
lung cancer exhibited a significant shrinkage in tumor size while
undergoing therapy with a combination of dasatinib and erlotinib,
and unlike the other subject on study with lung adenocarcinoma who
exhibited a response to treatment, there was no evidence of EGFR
mutation in the subject with squamous cell lung cancer. The patient
was a 59 year old Caucasian woman with a 1/3 pack per day smoking
history for 38 years who quit one year before her diagnosis of lung
cancer. She was found to have a left lower lobe stage I (T2N0M0)
squamous cell lung cancer and received primary treatment with
weekly carboplatin and paclitaxel with concomitant 70 Gy of
radiation resulting in a complete response. However, approximately
one year later she developed progression of disease within the
radiation field and treatment was initiated with s standard dose
carboplatin and paclitaxel without response. She then began
combination dasatinib and erlotinib therapy on protocol. A
restaging CT scan after nearly 2 months indicated tumor shrinkage
and the patient experienced improved symptoms (resolved dyspnea and
cough) (FIGS. 5A and 5B). She remained on treatment for 14 months
until therapy had to be discontinued secondary to treatment-induced
airspace disease and pleural effusions.
[0132] Directed sequencing of DDR2 was performed in a pre-treatment
tumor specimen derived from this individual and a novel DDR2 kinase
domain mutation, S768R, was identified that was present in 844 of
3020 (28%) of reads obtained by 454 sequencing and independently
verified by Sanger sequencing. The mutation could not be verified
as somatic as no normal DNA was available for this individual who
is deceased. There were no other SCC subjects who responded to
therapy on this study or a subsequent study of dasatinib alone
(n=13 total) to further explore this correlation.
[0133] Three-dimensional modeling of the S768R mutation in the
context of the DDR2 kinase domain was performed. A structural model
of the kinase domain of DDR2 (residues 545-854) was generated based
on the crystal structure of the Abl kinase domain in the active
(DFG-in) conformation (PDB code: 3DK6) in complex with a small
molecule inhibitor. Dasatinib was modeled into the ATP-binding site
of DDR2 based on the crystal structure of dasatinib in complex with
cSrc (PDB code: 3G5D). The proposed binding mode showed the
inhibitor core within hydrogen bonding distance to the backbone of
the hinge region of the kinase domain. The terminal
ethanol-piperazine was solvent exposed. The activating mutation
Ser768Arg was found at the N-terminal end of a helix below the 5
activation loop. The structural model indicated that the side chain
of Ser768 in wild type DDR2 likely resides in a packed environment
flanked by Glu213 and Phe220. The extra charge and steric bulk of
an Arg at position 768 in mutant DDR2 is likely to introduce
structural changes in this region which could potentially alter the
kinase activity and/or regulatory mechanisms of DDR2. These results
suggest that the S768R substitution is likely to alter the kinase
activity of DDR2.
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Available on the internet at
broadinstitute.org/rnai/public/resources/protocols
Other Embodiments
[0182] It is to be understood that while the invention has been
described in conjunction with the detailed description thereof, the
foregoing description is intended to illustrate and not limit the
scope of the invention, which is defined by the scope of the
appended claims. Other aspects, advantages, and modifications are
within the scope of the following claims.
Sequence CWU 1
1
111855PRTHomo sapiens 1Met Ile Leu Ile Pro Arg Met Leu Leu Val Leu
Phe Leu Leu Leu Pro1 5 10 15Ile Leu Ser Ser Ala Lys Ala Gln Val Asn
Pro Ala Ile Cys Arg Tyr 20 25 30Pro Leu Gly Met Ser Gly Gly Gln Ile
Pro Asp Glu Asp Ile Thr Ala 35 40 45Ser Ser Gln Trp Ser Glu Ser Thr
Ala Ala Lys Tyr Gly Arg Leu Asp 50 55 60Ser Glu Glu Gly Asp Gly Ala
Trp Cys Pro Glu Ile Pro Val Glu Pro65 70 75 80Asp Asp Leu Lys Glu
Phe Leu Gln Ile Asp Leu His Thr Leu His Phe 85 90 95Ile Thr Leu Val
Gly Thr Gln Gly Arg His Ala Gly Gly His Gly Ile 100 105 110Glu Phe
Ala Pro Met Tyr Lys Ile Asn Tyr Ser Arg Asp Gly Thr Arg 115 120
125Trp Ile Ser Trp Arg Asn Arg His Gly Lys Gln Val Leu Asp Gly Asn
130 135 140Ser Asn Pro Tyr Asp Ile Phe Leu Lys Asp Leu Glu Pro Pro
Ile Val145 150 155 160Ala Arg Phe Val Arg Phe Ile Pro Val Thr Asp
His Ser Met Asn Val 165 170 175Cys Met Arg Val Glu Leu Tyr Gly Cys
Val Trp Leu Asp Gly Leu Val 180 185 190Ser Tyr Asn Ala Pro Ala Gly
Gln Gln Phe Val Leu Pro Gly Gly Ser 195 200 205Ile Ile Tyr Leu Asn
Asp Ser Val Tyr Asp Gly Ala Val Gly Tyr Ser 210 215 220Met Thr Glu
Gly Leu Gly Gln Leu Thr Asp Gly Val Ser Gly Leu Asp225 230 235
240Asp Phe Thr Gln Thr His Glu Tyr His Val Trp Pro Gly Tyr Asp Tyr
245 250 255Val Gly Trp Arg Asn Glu Ser Ala Thr Asn Gly Tyr Ile Glu
Ile Met 260 265 270Phe Glu Phe Asp Arg Ile Arg Asn Phe Thr Thr Met
Lys Val His Cys 275 280 285Asn Asn Met Phe Ala Lys Gly Val Lys Ile
Phe Lys Glu Val Gln Cys 290 295 300Tyr Phe Arg Ser Glu Ala Ser Glu
Trp Glu Pro Asn Ala Ile Ser Phe305 310 315 320Pro Leu Val Leu Asp
Asp Val Asn Pro Ser Ala Arg Phe Val Thr Val 325 330 335Pro Leu His
His Arg Met Ala Ser Ala Ile Lys Cys Gln Tyr His Phe 340 345 350Ala
Asp Thr Trp Met Met Phe Ser Glu Ile Thr Phe Gln Ser Asp Ala 355 360
365Ala Met Tyr Asn Asn Ser Glu Ala Leu Pro Thr Ser Pro Met Ala Pro
370 375 380Thr Thr Tyr Asp Pro Met Leu Lys Val Asp Asp Ser Asn Thr
Arg Ile385 390 395 400Leu Ile Gly Cys Leu Val Ala Ile Ile Phe Ile
Leu Leu Ala Ile Ile 405 410 415Val Ile Ile Leu Trp Arg Gln Phe Trp
Gln Lys Met Leu Glu Lys Ala 420 425 430Ser Arg Arg Met Leu Asp Asp
Glu Met Thr Val Ser Leu Ser Leu Pro 435 440 445Ser Asp Ser Ser Met
Phe Asn Asn Asn Arg Ser Ser Ser Pro Ser Glu 450 455 460Gln Gly Ser
Asn Ser Thr Tyr Asp Arg Ile Phe Pro Leu Arg Pro Asp465 470 475
480Tyr Gln Glu Pro Ser Arg Leu Ile Arg Lys Leu Pro Glu Phe Ala Pro
485 490 495Gly Glu Glu Glu Ser Gly Cys Ser Gly Val Val Lys Pro Val
Gln Pro 500 505 510Ser Gly Pro Glu Gly Val Pro His Tyr Ala Glu Ala
Asp Ile Val Asn 515 520 525Leu Gln Gly Val Thr Gly Gly Asn Thr Tyr
Ser Val Pro Ala Val Thr 530 535 540Met Asp Leu Leu Ser Gly Lys Asp
Val Ala Val Glu Glu Phe Pro Arg545 550 555 560Lys Leu Leu Thr Phe
Lys Glu Lys Leu Gly Glu Gly Gln Phe Gly Glu 565 570 575Val His Leu
Cys Glu Val Glu Gly Met Glu Lys Phe Lys Asp Lys Asp 580 585 590Phe
Ala Leu Asp Val Ser Ala Asn Gln Pro Val Leu Val Ala Val Lys 595 600
605Met Leu Arg Ala Asp Ala Asn Lys Asn Ala Arg Asn Asp Phe Leu Lys
610 615 620Glu Ile Lys Ile Met Ser Arg Leu Lys Asp Pro Asn Ile Ile
His Leu625 630 635 640Leu Ala Val Cys Ile Thr Asp Asp Pro Leu Cys
Met Ile Thr Glu Tyr 645 650 655Met Glu Asn Gly Asp Leu Asn Gln Phe
Leu Ser Arg His Glu Pro Pro 660 665 670Asn Ser Ser Ser Ser Asp Val
Arg Thr Val Ser Tyr Thr Asn Leu Lys 675 680 685Phe Met Ala Thr Gln
Ile Ala Ser Gly Met Lys Tyr Leu Ser Ser Leu 690 695 700Asn Phe Val
His Arg Asp Leu Ala Thr Arg Asn Cys Leu Val Gly Lys705 710 715
720Asn Tyr Thr Ile Lys Ile Ala Asp Phe Gly Met Ser Arg Asn Leu Tyr
725 730 735Ser Gly Asp Tyr Tyr Arg Ile Gln Gly Arg Ala Val Leu Pro
Ile Arg 740 745 750Trp Met Ser Trp Glu Ser Ile Leu Leu Gly Lys Phe
Thr Thr Ala Ser 755 760 765Asp Val Trp Ala Phe Gly Val Thr Leu Trp
Glu Thr Phe Thr Phe Cys 770 775 780Gln Glu Gln Pro Tyr Ser Gln Leu
Ser Asp Glu Gln Val Ile Glu Asn785 790 795 800Thr Gly Glu Phe Phe
Arg Asp Gln Gly Arg Gln Thr Tyr Leu Pro Gln 805 810 815Pro Ala Ile
Cys Pro Asp Ser Val Tyr Lys Leu Met Leu Ser Cys Trp 820 825 830Arg
Arg Asp Thr Lys Asn Arg Pro Ser Phe Gln Glu Ile His Leu Leu 835 840
845Leu Leu Gln Gln Gly Asp Glu 850 855258DNAArtificial
Sequencelaboratory synthesized primer 2ccggcccatg cctatgccac
tccatctcga gatggagtgg cataggcatg ggtttttg 58358DNAArtificial
Sequencelaboratory synthesized primer 3ccggccctgg aggttccatc
atttactcga gtaaatgatg gaacctccag ggtttttg 584211PRTHomo
sapiensVARIANT(1)...(211)NCBI Reference Sequence NP_001014796.1
residues 54-264 4Glu Ser Thr Ala Ala Lys Tyr Gly Arg Leu Asp Ser
Glu Glu Gly Asp1 5 10 15Gly Ala Trp Cys Pro Glu Ile Pro Val Glu Pro
Asp Asp Leu Lys Glu 20 25 30Phe Leu Gln Ile Asp Leu His Thr Leu His
Phe Ile Thr Leu Val Gly 35 40 45Thr Gln Gly Arg His Ala Gly Gly His
Gly Ile Glu Phe Ala Pro Met 50 55 60Tyr Lys Ile Asn Tyr Ser Arg Asp
Gly Thr Arg Trp Ile Ser Trp Arg65 70 75 80Asn Arg His Gly Lys Gln
Val Leu Asp Gly Asn Ser Asn Pro Tyr Asp 85 90 95Ile Phe Leu Lys Asp
Leu Glu Pro Pro Ile Val Ala Arg Phe Val Arg 100 105 110Phe Ile Pro
Val Thr Asp His Ser Met Asn Val Cys Met Arg Val Glu 115 120 125Leu
Tyr Gly Cys Val Trp Leu Asp Gly Leu Val Ser Tyr Asn Ala Pro 130 135
140Ala Gly Gln Gln Phe Val Leu Pro Gly Gly Ser Ile Ile Tyr Leu
Asn145 150 155 160Asp Ser Val Tyr Asp Gly Ala Val Gly Tyr Ser Met
Thr Glu Gly Leu 165 170 175Gly Gln Leu Thr Asp Gly Val Ser Gly Leu
Asp Asp Phe Thr Gln Thr 180 185 190His Glu Tyr His Val Trp Pro Gly
Tyr Asp Tyr Val Gly Trp Arg Asn 195 200 205Glu Ser Ala
2105320PRTHomo sapiensVARIANT(1)...(320)NCBI Reference Sequence
NP_001014796.1 residues 468-787 5Asn Ser Thr Tyr Asp Arg Ile Phe
Pro Leu Arg Pro Asp Tyr Gln Glu1 5 10 15Pro Ser Arg Leu Ile Arg Lys
Leu Pro Glu Phe Ala Pro Gly Glu Glu 20 25 30Glu Ser Gly Cys Ser Gly
Val Val Lys Pro Val Gln Pro Ser Gly Pro 35 40 45Glu Gly Val Pro His
Tyr Ala Glu Ala Asp Ile Val Asn Leu Gln Gly 50 55 60Val Thr Gly Gly
Asn Thr Tyr Ser Val Pro Ala Val Thr Met Asp Leu65 70 75 80Leu Ser
Gly Lys Asp Val Ala Val Glu Glu Phe Pro Arg Lys Leu Leu 85 90 95Thr
Phe Lys Glu Lys Leu Gly Glu Gly Gln Phe Gly Glu Val His Leu 100 105
110Cys Glu Val Glu Gly Met Glu Lys Phe Lys Asp Lys Asp Phe Ala Leu
115 120 125Asp Val Ser Ala Asn Gln Pro Val Leu Val Ala Val Lys Met
Leu Arg 130 135 140Ala Asp Ala Asn Lys Asn Ala Arg Asn Asp Phe Leu
Lys Glu Ile Lys145 150 155 160Ile Met Ser Arg Leu Lys Asp Pro Asn
Ile Ile His Leu Leu Ala Val 165 170 175Cys Ile Thr Asp Asp Pro Leu
Cys Met Ile Thr Glu Tyr Met Glu Asn 180 185 190Gly Asp Leu Asn Gln
Phe Leu Ser Arg His Glu Pro Pro Asn Ser Ser 195 200 205Ser Ser Asp
Val Arg Thr Val Ser Tyr Thr Asn Leu Lys Phe Met Ala 210 215 220Thr
Gln Ile Ala Ser Gly Met Lys Tyr Leu Ser Ser Leu Asn Phe Val225 230
235 240His Arg Asp Leu Ala Thr Arg Asn Cys Leu Val Gly Lys Asn Tyr
Thr 245 250 255Ile Lys Ile Ala Asp Phe Gly Met Ser Arg Asn Leu Tyr
Ser Gly Asp 260 265 270Tyr Tyr Arg Ile Gln Gly Arg Ala Val Leu Pro
Ile Arg Trp Met Ser 275 280 285Trp Glu Ser Ile Leu Leu Gly Lys Phe
Thr Thr Ala Ser Asp Val Trp 290 295 300Ala Phe Gly Val Thr Leu Trp
Glu Thr Phe Thr Phe Cys Gln Glu Gln305 310 315 3206211PRTMus
musculusVARIANT(1)...(211)NP_072075.2 residues 54-264 6Glu Ser Thr
Ala Ala Lys Tyr Gly Arg Leu Asp Ser Glu Glu Gly Asp1 5 10 15Gly Ala
Trp Cys Pro Glu Ile Pro Val Gln Pro Asp Asp Leu Lys Glu 20 25 30Phe
Leu Gln Ile Asp Leu Arg Thr Leu His Phe Ile Thr Leu Val Gly 35 40
45Thr Gln Gly Arg His Ala Gly Gly His Gly Ile Glu Phe Ala Pro Met
50 55 60Tyr Lys Ile Asn Tyr Ser Arg Asp Gly Thr Arg Trp Ile Ser Trp
Arg65 70 75 80Asn Arg His Gly Lys Gln Val Leu Asp Gly Asn Ser Asn
Pro Tyr Asp 85 90 95Val Phe Leu Lys Asp Leu Glu Pro Pro Ile Val Ala
Arg Phe Val Arg 100 105 110Leu Ile Pro Val Thr Asp His Ser Met Asn
Val Cys Met Arg Val Glu 115 120 125Leu Tyr Gly Cys Val Trp Leu Asp
Gly Leu Val Ser Tyr Asn Ala Pro 130 135 140Ala Gly Gln Gln Phe Val
Leu Pro Gly Gly Ser Ile Ile Tyr Leu Asn145 150 155 160Asp Ser Val
Tyr Asp Gly Ala Val Gly Tyr Ser Met Thr Glu Gly Leu 165 170 175Gly
Gln Leu Thr Asp Gly Val Ser Gly Leu Asp Asp Phe Thr Gln Thr 180 185
190His Glu Tyr His Val Trp Pro Gly Tyr Asp Tyr Val Gly Trp Arg Asn
195 200 205Glu Ser Ala 2107319PRTMus
musculusVARIANT(1)...(319)NP_072075.2 residues 468-786 7Asn Ser Thr
Tyr Asp Arg Ile Phe Pro Leu Arg Pro Asp Tyr Gln Glu1 5 10 15Pro Ser
Arg Leu Ile Arg Lys Leu Pro Glu Phe Ala Pro Gly Glu Glu 20 25 30Glu
Ser Gly Cys Ser Gly Val Val Lys Pro Ala Gln Pro Asn Gly Pro 35 40
45Glu Gly Val Pro His Tyr Ala Glu Ala Asp Ile Val Asn Leu Gln Gly
50 55 60Val Thr Gly Gly Asn Thr Tyr Cys Val Pro Ala Val Thr Met Asp
Leu65 70 75 80Leu Ser Gly Lys Asp Val Ala Val Glu Glu Phe Pro Arg
Lys Leu Leu 85 90 95Ala Phe Lys Glu Lys Leu Gly Glu Gly Gln Phe Gly
Glu Val His Leu 100 105 110Cys Glu Val Glu Gly Met Glu Lys Phe Lys
Asp Lys Asp Phe Ala Leu 115 120 125Asp Val Ser Ala Asn Gln Pro Val
Leu Val Ala Val Lys Met Leu Arg 130 135 140Ala Asp Ala Asn Lys Asn
Ala Arg Asn Asp Phe Leu Lys Glu Ile Lys145 150 155 160Ile Met Ser
Arg Leu Lys Asp Pro Asn Ile Ile Arg Leu Leu Ala Val 165 170 175Cys
Ile Thr Glu Asp Pro Leu Cys Met Ile Thr Glu Tyr Met Glu Asn 180 185
190Gly Asp Leu Asn Gln Phe Leu Ser Arg His Glu Pro Leu Ser Ser Cys
195 200 205Ser Ser Asp Ala Thr Val Ser Tyr Ala Asn Leu Lys Phe Met
Ala Thr 210 215 220Gln Ile Ala Ser Gly Met Lys Tyr Leu Ser Ser Leu
Asn Phe Val His225 230 235 240Arg Asp Leu Ala Thr Arg Asn Cys Leu
Val Gly Lys Asn Tyr Thr Ile 245 250 255Lys Ile Ala Asp Phe Gly Met
Ser Arg Asn Leu Tyr Ser Gly Asp Tyr 260 265 270Tyr Arg Ile Gln Gly
Arg Ala Val Leu Pro Ile Arg Trp Met Ser Trp 275 280 285Glu Ser Ile
Leu Leu Gly Lys Phe Thr Thr Ala Ser Asp Val Trp Ala 290 295 300Phe
Gly Val Thr Leu Trp Glu Thr Phe Thr Phe Cys Gln Glu Gln305 310
3158210PRTDanio rerioVARIANT(1)...(210)NCBI Reference Sequence
XP_687859.4 residues 27-236 8Glu Ser Thr Ala Ala Arg Tyr Gly Arg
Leu Asp Phe Asp Asp Gly Asp1 5 10 15Gly Ala Trp Cys Pro Asp Val Met
Ala Glu Ala His Ser Leu Lys Glu 20 25 30Phe Leu Gln Ile Asp Leu Arg
Thr Leu His Phe Val Thr Leu Val Gly 35 40 45Thr Gln Gly Arg His Ala
Asp Gly Val Gly Asn Glu Phe Ala Gln Arg 50 55 60Tyr Arg Ile Lys Tyr
Ser Arg Asp Gly Thr Arg Trp Val Ser Trp Arg65 70 75 80Asp Arg Gln
Gly Arg Gln Ile Ile Glu Gly Asn Ser Asn Ala Tyr Asp 85 90 95Ile Val
Leu Lys Asp Leu Glu Pro Pro Ile Ile Ala Arg Phe Val Arg 100 105
110Phe Met Pro Val Thr Asp Pro Ser Met Ile Val Cys Met Arg Val Glu
115 120 125Leu Tyr Gly Cys Glu Trp Leu Asp Gly Leu Val Ser Tyr Ser
Ala Pro 130 135 140Ala Gly Gln Gln Met Thr Phe Arg Gly Gln Gln Ile
Tyr Leu Asn Asp145 150 155 160Thr Val Tyr Asp Gly Ala Val Ser Ser
Ser Met Ala Glu Gly Leu Gly 165 170 175Gln Leu Thr Asp Gly Ser Trp
Gly Leu Asp Asp Phe Thr Lys Ser Gln 180 185 190Val Tyr Gly Val Trp
Pro Gly Tyr Asp Tyr Val Gly Trp Thr Asn Ala 195 200 205Ser Leu
2109313PRTDanio rerioVARIANT(1)...(313)NCBI Reference Sequence
XP_687859.4 residues 443-755 9Thr Ser Thr Tyr Glu Arg Ile Phe Pro
Leu Gly Ser Asp Tyr Gln Glu1 5 10 15Pro Ser Gln Leu Leu Arg Lys Leu
Pro Glu Phe Gln Ala Met Glu Asn 20 25 30Leu Ala Met Ile Asp Gly Ala
Ser Glu Pro Ser Pro Ala Gln Gly Thr 35 40 45Asp Glu Ala Pro His Tyr
Ala Glu Ala Asp Ile Pro Gly Ala His Ser 50 55 60Tyr Arg Val Thr Val
Val Asn Met Pro Leu Ser Pro Gly Arg Asp Gly65 70 75 80Ala Leu Glu
Glu Phe Pro Arg Asp Arg Leu Thr Phe Lys Glu Lys Leu 85 90 95Gly Glu
Gly Gln Phe Gly Glu Val His Leu Cys Glu Ala Glu Gly Met 100 105
110Gln Glu Phe Met Lys Asp His Cys Asp Asp Ile Cys Val Asp Pro Met
115 120 125Leu Val Ala Val Lys Thr Leu Arg Glu Asp Ala Asp Lys Asn
Ala Arg 130 135 140Asn Asp Phe Met Lys Glu Ile Arg Ile Ile Ser Arg
Leu Arg Asp Pro145 150 155 160Asn Ile Ile Arg Leu Leu Ala Val Cys
Val Glu Ser Asp Pro Leu Cys 165 170 175Met Ile Thr Glu Tyr Met Glu
Asn Gly Asp Leu Asn Gln Phe Leu Ser 180 185 190Arg His Gln Leu Gln
Glu Asp Gly Val Gln Ala Asp Ser Thr Ser Ile 195 200 205Ser Tyr Gly
Thr Leu Ile Asn Met Ala Ser Gln Ile Ser Ser Gly Met 210 215 220Lys
Tyr Leu Ser Ser Leu Asn Phe Val His Arg Asp Leu Ala Thr Arg225
230
235 240Asn Cys Leu Val Gly Met Asn Asn Ile Ile Lys Ile Ala Asp Phe
Gly 245 250 255Met Ser Arg Asn Leu Tyr Arg Gly Asp Tyr Tyr Arg Ile
Gln Gly Arg 260 265 270Ala Val Leu Pro Ile Arg Trp Met Ser Trp Glu
Ser Ile Leu Leu Gly 275 280 285Lys Phe Thr Met Ala Ser Asp Val Trp
Ala Phe Gly Val Thr Leu Trp 290 295 300Glu Ile Leu Thr Leu Cys Lys
Glu Gln305 31010199PRTCaenorhabditis
elegansVARIANT(1)...(199)UniProtKB/Swiss-Prot Q95ZV7 residues
50-248 10Gln Ser Thr Gly Pro Gln His Ala Arg Ala His Gln Glu Ser
Gly Ser1 5 10 15Gly Ala Trp Cys Pro Lys Asn Gln Ile Asn Ser Leu Ser
Lys Glu Trp 20 25 30Leu Gln Ile Ser Phe Ser Val Asp Thr Val Ile Thr
Ser Val Glu Thr 35 40 45Gln Gly Arg Phe Asp Asp Gly Arg Gly Met Glu
Tyr Ala Thr Ala Phe 50 55 60Lys Ile Gln Tyr Trp Arg Pro Ser Leu Asn
Ala Trp Ala Ser Tyr Lys65 70 75 80Asp Asp Phe Glu Leu Glu Thr Ile
Pro Ala Asn Asn Asp Thr Glu His 85 90 95Ala Ile Arg Arg His Leu Asp
Arg Ala Ile Ile Ala Arg Arg Ile Arg 100 105 110Ile Val Pro Val Ser
Asn Ser Thr Arg Thr Val Cys Met Arg Val Glu 115 120 125Val Phe Gly
Cys Pro Phe Asp Asp Ser Leu Val Phe Tyr Asn Val Asp 130 135 140Gln
Gly Asp Leu Gln Ser Gly Ile Ser Tyr His Asp Phe Ser Tyr Asp145 150
155 160Gly Asn Leu Ala Asn Ser Pro His Leu Thr Gly Gly Ile Gly Lys
Leu 165 170 175Tyr Asp Gly Glu Val Gly Lys Asn Asn Val Phe Val Asn
His His Lys 180 185 190Trp Val Gly Trp Arg Arg Lys
19511272PRTCaenorhabditis
elegansVARIANT(1)...(272)UniProtKB/Swiss-Prot Q95ZV7 residues
451-722 11Leu Gln Asn Ala Leu Ile Glu Lys Met Pro Met Ser Pro Ile
Ile Ser1 5 10 15Asp Tyr Ala Glu Pro Asp Ile Ser Val Cys Ser Asp Val
Thr Ala Asn 20 25 30Thr Pro Leu Leu Tyr Gly Ile Asp Gly Pro Tyr Asp
Thr Gln Lys Arg 35 40 45Ser Asn Pro Leu Ser Ser Met Val Lys Tyr Ser
Asp Tyr Gly Glu Val 50 55 60Tyr Cys Thr Thr Leu Pro Glu Ile Ala Arg
Asp Lys Leu Ile Cys Val65 70 75 80Ser Arg Ile Gly Gln Gly Glu Phe
Gly Glu Val Asp Leu Cys Gln Leu 85 90 95Glu Asn Arg Lys Val Ala Val
Lys Lys Leu His Gly Ile Ser Gln Ala 100 105 110Asp Glu Phe Ser Phe
His Arg Glu Ile Arg Val Leu Gly Ser Leu Lys 115 120 125His Pro Asn
Val Val Glu Val Val Gly Val Cys Thr Ile Gln Lys Pro 130 135 140Ile
Leu Cys Ile Met Glu Tyr Met Glu Asn Gly Asp Leu Lys Ser Tyr145 150
155 160Ile Leu Lys Asn Pro Thr Ile Gln Thr Ser Gln Cys Ile Ser Ile
Cys 165 170 175Thr Gln Leu Ala Ala Gly Leu Ala Tyr Leu Glu Ser Cys
Asn Phe Val 180 185 190His Arg Asp Ile Ala Ala Arg Asn Cys Leu Val
Asp Gly Glu Gly Asn 195 200 205Val Lys Ile Ala Asp Phe Gly Met Ala
Arg Ser Leu Tyr Ser Gln Glu 210 215 220Tyr Tyr Lys Val Glu Gly Lys
Phe Val Leu Pro Ile Arg Trp Met Ala225 230 235 240Trp Glu Ala Leu
Leu Leu Gly Lys Phe Ser Thr Ala Ser Asp Val Trp 245 250 255Gly Phe
Gly Val Thr Met Trp Glu Ile Phe Ser Leu Cys Ser Glu Lys 260 265
270
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