U.S. patent application number 16/520078 was filed with the patent office on 2019-12-12 for ddx43 as a biomarker of resistance to mek1/2 inhibitors.
This patent application is currently assigned to MEMORIAL SLOAN-KETTERING CANCER CENTER. The applicant listed for this patent is MEMORIAL SLOAN-KETTERING CANCER CENTER. Invention is credited to Grazia Ambrosini, Richard Carvajal, Raya Khanin, Gary K. Schwartz.
Application Number | 20190376149 16/520078 |
Document ID | / |
Family ID | 52629085 |
Filed Date | 2019-12-12 |
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United States Patent
Application |
20190376149 |
Kind Code |
A1 |
Ambrosini; Grazia ; et
al. |
December 12, 2019 |
DDX43 AS A BIOMARKER OF RESISTANCE TO MEK1/2 INHIBITORS
Abstract
The present invention relates to methods and compositions for
determining the likelihood that a subject suffering from a cancer
will benefit from treatment with a MEK inhibitor. It also relates
to methods of treatment based on such determination. The invention
is based, at least in part, on the discoveries that DDX43 mRNA and
protein are expressed at high levels in biopsies from
"non-responder" UM patients and that selumetinib-resistant cell
lines showed high DDX43 expression which correlated with increased
expression and activity of RAS. It was found that KRAS and HRAS but
not NRAS, mediated expression of pERK and pAKT, bypassing oncogenic
GNAQ. The invention is further based on the discovery that
selumetinib-resistant cells became sensitive to AKT inhibition,
suggesting alternative strategies for the treatment of cancer
patients with acquired resistance to MEK inhibitors.
Inventors: |
Ambrosini; Grazia; (Astoria,
NY) ; Khanin; Raya; (New York, NY) ; Carvajal;
Richard; (New York, NY) ; Schwartz; Gary K.;
(Briarcliff Manor, NY) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
MEMORIAL SLOAN-KETTERING CANCER CENTER |
New York |
NY |
US |
|
|
Assignee: |
MEMORIAL SLOAN-KETTERING CANCER
CENTER
New York
NY
|
Family ID: |
52629085 |
Appl. No.: |
16/520078 |
Filed: |
July 23, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15061578 |
Mar 4, 2016 |
10400285 |
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16520078 |
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PCT/US2014/054263 |
Sep 5, 2014 |
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15061578 |
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61874218 |
Sep 5, 2013 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01N 2333/914 20130101;
A61K 31/4375 20130101; C12Q 2600/106 20130101; C12Q 2600/136
20130101; C12Q 2600/158 20130101; C12Q 1/6886 20130101; A61K
31/4184 20130101; G01N 33/5743 20130101 |
International
Class: |
C12Q 1/6886 20060101
C12Q001/6886; G01N 33/574 20060101 G01N033/574; A61K 31/4375
20060101 A61K031/4375; A61K 31/4184 20060101 A61K031/4184 |
Goverment Interests
GRANT INFORMATION
[0002] This invention was made with government support under
CM062206 awarded by the National Cancer Institute of the National
Institutes of Health. The government has certain rights in the
invention.
Claims
1. A method of treating a subject having a cancer, comprising: (i)
determining whether an anti-cancer effect is unlikely to be
produced in a cancer by a MEK inhibitor, comprising determining
whether cells of the cancer comprise an increased level of mRNA
and/or protein corresponding to an exon of one or more of MFAPS,
DPYS, ACCN4, and DMKN relative to a normal value or normal values;
and (ii) treating the subject with a MEK inhibitor if the level of
mRNA and/or protein corresponding to an exon of one or more of
RHBG, MFAPS, DPYS, ACCN4, and DMKN is not increased, or (iii)
treating the subject with an anti-cancer agent other than a MEK
inhibitor if the level of mRNA and/or protein corresponding to an
exon of one or more RHBG, MFAPS, DPYS, ACCN4, and DMKN is
increased.
2. The method of claim 1, wherein the cancer is melanoma.
3. The method of claim 1, wherein the cancer is melanoma comprises
one or more mutations in GNAQ and/or GNA11.
4. The method of claim 1, wherein the cancer is uveal melanoma.
5. The method of claim 1, wherein the anti-cancer agent other than
a MEK inhibitor is an AKT inhibitor.
6. The method of claim 5, wherein the AKT inhibitor is selected
from the group consisting of VQD-002, perifosine, miltefosine,
AZD5363, and MK2206.
7. The method of claim 6, wherein the AKT inhibitor is MK2206.
8. The method of claim 1, wherein the MEK inhibitor is selected
from the group consisting of selumetinib, trametinib, MEK162,
PD-325901, XL518, and CI-1040.
9. The method of claim 8, wherein the MEK inhibitor is
selumetinib.
10. The method of claim 1, wherein (i) further comprises
determining whether the cells of the cancer comprise an increased
level of DDX43 mRNA and/or DDX43 protein relative to a normal value
or normal values.
11. The method of claim 10, wherein the level of DDX43 mRNA and/or
protein in the cells of the subject treated in (ii) is not
increased.
12. The method of claim 10, wherein the level of DDX43 mRNA and/or
protein in the cells of the subject treated in (iii) is
increased.
13. A method of treating a subject having a cancer comprising: (i)
determining whether an anti-cancer effect is unlikely to be
produced in the cancer by a MEK inhibitor, comprising determining
whether cells of the cancer comprise an increased level of mRNA or
protein corresponding to one or more genes or exons listed in Table
1 relative to a normal value or values, and (ii) treating the
subject with a MEK inhibitor if the level of mRNA or protein
corresponding to one or more gene or exon listed in Table 1 is not
increased; or (iii) treating the subject with an anti-cancer agent
other than a MEK inhibitor if the level of mRNA or protein
corresponding to one or more gene or exon listed in Table 1 is
increased.
14. The method of claim 13, wherein the cancer is melanoma.
15. The method of claim 13, wherein the cancer is melanoma
comprises one or more mutations in GNAQ and/or GNA11.
16. The method of claim 13, wherein the anti-cancer agent other
than a MEK inhibitor is an AKT inhibitor.
17. The method of claim 13, wherein the MEK inhibitor is selected
from the group consisting of selumetinib, trametinib, MEK162,
PD-325901, XL518, and CI-1040.
18. The method of claim 13, wherein (i) further comprises
determining whether the cells of the cancer comprise an increased
level of DDX43 mRNA and/or DDX43 protein relative to a normal value
or normal values.
19. The method of claim 18, wherein the level of DDX43 mRNA and/or
protein in the cells of the subject treated in (ii) is not
increased.
20. The method of claim 18, wherein the level of DDX43 mRNA and/or
protein in the cells of the subject treated in (iii) is increased.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of U.S. patent application
Ser. No. 15/061,578, filed Mar. 4, 2016, which is a Continuation of
International Patent Application No. PCT/US2014/054263, filed Sep.
5, 2014, which claims priority to U.S. Provisional Patent
Application Ser. No. 61/874,218, filed Sep. 5, 2013, the contents
of each of which are incorporated by reference in their entirety,
and to each of which priority is claimed.
1. INTRODUCTION
[0003] The present invention relates to methods and kits for
determining the likelihood that a subject suffering from a cancer
will benefit from treatment with a MEK inhibitor based on whether
or not DDX43 is over-expressed. It further relates to methods of
treatment based on such determination.
2. BACKGROUND OF THE INVENTION
[0004] The prognosis of patients with metastatic uveal melanoma
("UM") is poor with a median 1-year survival rate of less than 30%
[1] [2]. In 87% of patients, metastasis will develop primarily in
the liver, and there are limited therapeutic options for this
disease [3] [4]. Activating mutations in G-protein alpha subunits
GNAQ or GNA11 are early oncogenic events in UM development [5] and
result in the activation of the MAPK pathway [6]. We have reported
that the small molecule MEK inhibitor selumetinib can inhibit pERK
and cyclin D1, resulting in decreased viability of UM cell lines
[7]. Furthermore, in patients with UM, selumetinib can result in
tumor shrinkage, and the sustained inhibition of pERK may be
predictive of benefit [8].
[0005] MEK inhibitors have been reported to give a partial or
stable response in tumors with activated MAPK pathway, including
melanoma and solid malignancies [9], [10] [11]. However, the use of
small molecule MEK inhibitors has been undermined by acquired drug
resistance [12], which reduces the efficacy of these drugs in the
clinical setting (patients resistant to the drug being
"non-responders"). For example, resistance to selumetinib has been
described in colorectal cancer cells carrying BRAF and RAS
mutations, where resistance is mediated by the amplification of the
driving oncogene [13] [14]. In cutaneus melanoma, MEK1 mutations
have been found to confer resistance to MEK inhibitors [15]. In
uveal melanoma with GNAQ mutations, the mechanisms of acquired
resistance have been elusive and more effective therapies are
needed for the treatment of this disease.
[0006] The RNA helicase DDX43 was first identified as a
cancer/testis antigen, and it is highly expressed in many tumor
types compared to normal tissues [16, 52], including melanoma [19].
In particular, DDX43 was found to be overexpressed in 50% acute
myeloid leukemias (CML) [17], and its expression is associated with
advanced disease and poor prognosis [18]. It has been reported that
DDX43 promoted expression of RAS protein through RNA unwinding
[20].
[0007] DDX43, also called HAGE, is a member of the DEAD-box family
of ATP-dependent RNA helicases. These proteins browse RNA molecules
and promote the dissociation of the RNA from ribonucleoproteins to
which they have high affinity [21]. In this way RNA helicases
support processes like transcription, pre-mRNA splicing,
translation initiation/elongation, and RNA degradation [22] [23].
Their altered expression levels have been also implicated in tumor
initiation, progression and maintenance [24].
3. SUMMARY OF THE INVENTION
[0008] The present invention relates to methods and compositions
for determining the likelihood that a subject suffering from a
cancer will benefit from treatment with a MEK inhibitor. It also
relates to methods of treatment based on such determination. The
invention is based, at least in part, on the discoveries that DDX43
mRNA and protein are expressed at high levels in biopsies from
"non-responder" UM patients and that selumetinib-resistant cell
lines showed high DDX43 expression which correlated with increased
expression and activity of RAS. It was found that KRAS and HRAS but
not NRAS, mediated expression of pERK and pAKT, bypassing oncogenic
GNAQ. The invention is further based on the discovery that
selumetinib-resistant cells became sensitive to AKT inhibition,
suggesting alternative strategies for the treatment of cancer
patients with acquired resistance to MEK inhibitors.
4. BRIEF DESCRIPTION OF THE FIGURES
[0009] FIG. 1A and FIG. 1B. DDX43 is highly expressed at the mRNA
and protein levels in "non responder" biopsies. FIG. 1A, DDX43
expression was confirmed by real-time PCR in biopsies of 14
patients before selumetinib treatment. Triplicate values were
normalized with GADPH as housekeeping gene using the AACT method,
and reported as a Box plot showing significant association with
poor outcome in patients with uveal melanoma treated with
selumetinib *p=0.045. FIG. 1B, DDX43 expression was analyzed by
immunoblotting in liver biopsies of six representative patients (P)
before (-) and after (+) 14 days of selumetinib treatment.
[0010] FIG. 2A-FIG. 2D. UM cells become resistant to MEK inhibition
after long exposure to selumetinib. After continuous exposure, the
cell lines Res-Omm1.3 (FIG. 2A) and Res-Me1270 (FIG. 2B) became
resistant to selumetinib compared to their parental cell lines
Omm1.3 and Me1270. Cell viability on day 4 was calculated as
percent of untreated controls. Each point is a mean.+-.sd.
Immunoblot analysis of parental and resistant Omm1.3 (FIG. 2C) and
Me1270 (FIG. 2D) cells treated with increasing concentrations of
selumetinib for 24 hours. Both MEKi-resistant cell lines Res-Omm1.3
and Res-Me1270 showed sustained expression of pAKT, pRB, c-Jun,
DDX43 and RAS, independently of treatment.
[0011] FIG. 3A-FIG. 3C. DDX43 and RAS are highly expressed in the
MEKi-resistant cells. FIG. 3A, Parental and resistant cells were
treated with selumetinib for up to 48 hrs and analyzed for DDX43
expression. FIG. 3B, Resistant cells were transfected with an siRNA
control (-) and a DDX43-specific siRNA (+). Cell lysates were
analyzed by immunoblotting for expression of DDX43, total RAS,
pERK, pAKT and tubulin. FIG. 3C, Resistant cells with downregulated
DDX43 were assayed in cell viability assays, after 3 days from
transfection.
[0012] FIG. 4A-FIG. 4D. DDX43 regulates RAS expression and mediates
MEK resistance. FIG. 4A, siRNA mediated knockdown of DDX43 (+) and
control siRNA (-) in the MEKi-resistant cell lines downregulates
KRAS, HRAS, NRAS and downstream signaling molecules like pERK, pAKT
and c-Jun. FIG. 4B, Cell viability of Res-Omm1.3 and Res-Me170 was
measured after 4 days from siRNA transfection. FIG. 4C, The
parental cell lines Omm1.3 and Me1270 were transfected with DDX43
or the empty vector pCMV. Cell lysates were subjected to Western
blot analysis for expression of RAS, pERK and pAKT. FIG. 4D, The
parental cell line Omm1.3 overexpressing DDX43 is more resistant to
selumetinib treatments. Columns, mean of three independent
experiments.
[0013] FIG. 5A-FIG. 5D. KRAS and HRAS, but not NRAS, mediate
ERK/AKT signaling in MEKi-resistant cells. Parental and
MEK-resistant cells where transfected with KRAS (FIG. 5A), HRAS
(FIG. 5B), NRAS (FIG. 5C) and GNAQ (FIG. 5D) siRNA and analyzed for
pMEK, pERK, pAKT, c-Jun, and cyclin D1 expression by
immunoblotting.
[0014] FIG. 6A-FIG. 6C. KRAS and HRAS are necessary for
MEKi-resistant cells survival. The cells depleted of each RAS
protein or GNAQ were tested in viability assays after 72 hr from
siRNA transfection in Res-Omm1.3 (FIG. 6A) and Res-Me1270 (FIG.
6B). * p<0.0001 and ** p<0.001 for comparison of siKRAS and
siHRAS versus control siRNA in both cell lines. FIG. 6B and FIG.
6C, Res-Omm1.3 and Res-Me270 cells are sensitive to AKT inhibition.
Sensitive and MEKi-resistant cells were exposed to increasing
concentrations of MK2206 with or without selumetinib and analyzed
in viability assays. Columns, mean of three independent
experiments. Mean.+-.sd
[0015] FIG. 7A-FIG. 7D. Resistant and parental cell lines were
treated with increasing doses of the MEK inhibitors PD0325901 (FIG.
7A, FIG. 7B) and GSK1120212 (FIG. 7C, FIG. 7D) for 4 days, and
analyzed in cell viability assays. Each experiment is
representative of three independent experiments. Mean.+-.sd.
[0016] FIG. 8A-FIG. 8B. The resistant cells Res-Omm1.3 (FIG. 8A)
and Res-Me1270 (FIG. 8B) escaped the G1 cell cycle arrest mediated
by selumetinib after 24 hours of treatment.
[0017] FIG. 9A-FIG. 9B. DDX43 mRNA expression is elevated in the
selumetinib-resistant cells compared to their parental cells.
Real-time PCR of sensitive and resistant cells (FIG. 9A) Omm1.3 and
(FIG. 9B) Me1270. Triplicate values were normalized to GADPH.
Mean.+-.sd.
[0018] FIG. 10. Parental and MEK inhibitor-resistant cell lines
were treated with 1 .mu.M selumetinib over the time. Immunoblots
show the expression levels of pERK, Spry2, Dusp6 and tubulin.
[0019] FIG. 11. A, MEKi-resistant cells were transfected with
control (-) and a DDX43-2 specific (+) siRNA as in Material and
Methods. After 48 hours, cell lysates were used in Western blots
for the expression of DDX43, pan-RAS, pERK pAKT and tubulin.
[0020] FIG. 12A-FIG. 12B. DDX43 does not regulate KRAS
transcription. The resistant cells were transfected with control
and DDX43 siRNA and real-time PCR for DDX43 (FIG. 12A) and KRAS
(FIG. 12B) expression were performed. Triplicate values were
normalized to GADPH. Mean.+-.sd.
5. DETAILED DESCRIPTION OF THE INVENTION
[0021] For clarity of disclosure and not by way of limitation the
detailed description of the invention is divided into the following
subsections:
[0022] (i) DDX43 nucleic acids and proteins;
[0023] (ii) cancers subject to the invention;
[0024] (iii) MEK inhibitors;
[0025] (iv) AKT inhibitors;
[0026] (v) methods of assessing sensitivity to MEK inhibitors;
[0027] (vi) methods of assessing sensitivity to AKT inhibitors;
[0028] (vii) methods of measuring mRNA or proteins
[0029] (viii) methods of treatment; and
[0030] (ix) kits.
[0031] "Responder" and "non-responder" are used herein to refer to
subjects having cancers that are antagonized by MEK inhibitors, and
also are used to refer to the responsive or non-responsive cancers
and cancer cells themselves.
5.1 DDX43 Nucleic Acids and Proteins
[0032] DDX43 nucleic acids include DNA and RNA comprising at least
a portion of a DDX43 gene, a DDX43 mRNA, or a DDX43 cDNA or a
sequence complementary or homologous thereto (including but not
limited to antisense or small interfering RNA). Said nucleic acid
may be comprised of natural nucleotides and may optionally comprise
nucleotide bases which are not naturally occurring. In certain
non-limiting embodiments, a DDX43 nucleic acid is present in or
obtained from a cell of a subject, which may be a cancer cell. In
certain other non-limiting embodiments, a DDX43 nucleic acid is a
primer or probe which may be used to measure the level of DDX43
expression.
[0033] In certain non-limiting embodiments, a DDX43 nucleic acid
may be between about 10 and 2707 bases long. In certain
non-limiting embodiments, a DDX43 nucleic acid may be at least 10,
or at least 15, or at least 20, or at least 30, and up to 30, or up
to 50, or up to 100, or up to 200; or between about 10 and 200 or
between about 15 and 100 or between about 15 and 50, bases in
length.
[0034] In certain non-limiting embodiments, a DDX43 nucleic acid
may be detectably labeled, for example with a fluorescent, or
radioactive, or colorimetric, or affinity label, using methods
known in the art.
[0035] In a specific non-limiting embodiment, a DDX43 nucleic acid
is a human DDX43 nucleic acid molecule which has the nucleic acid
sequence as set forth in GenBank/NCBI database accession no.
NM_018665 [40-42] or a portion thereof, which portion may be, for
example, at least 10, or at least 15, or at least 20, or at least
30, and up to 30, or up to 50, or up to 100, or up to 200; or
between about 10 and 200 or between about 15 and 100 or between
about 15 and 50, bases in length, or a nucleic acid which is at
least about 90 percent or at least about 95 percent or at least
about 98 percent homologous to the sequence set forth in NM_018665
or a portion thereof. Homology as referred to herein may be
determined using standard software, for example but not limited to
BLAST or FASTA.
[0036] In other specific non-limiting embodiments, a DDX43 nucleic
acid is a cat, chimpanzee, mouse or dog DDX43 nucleic acid molecule
which has, respectively, the nucleic acid sequence as set forth in
GenBank/NCBI accession nos. XM_003986327.1; XM_518584.3;
NM_001191044.1; or XM_848647 [43-46] or a portion thereof, which
portion may be, for example, at least 10, or at least 15, or at
least 20, or at least 30, or between about 10 and 200 or between
about 15 and 100 or between about 15 and 50, bases in length, or a
nucleic acid which is at least about 90 percent or at least about
95 percent or at least about 98 percent homologous to the sequence
set forth in XM_003986327.1; XM_518584.3; NM_001191044.1; or
XM_848647, or a portion thereof.
[0037] A DDX43 protein is present in, produced by or obtained from
a cell of a subject, which may be a cancer cell. In a specific
non-limiting embodiment, a DDX43 protein is a human DDX43 protein
molecule which has the amino acid sequence as set forth in
GenBank/NCBI database accession no. NM_018665 [40-42] or NP_061135
[47] or a variant thereof which is at least about 90 percent or at
least about 95 percent or at least about 98 percent or at least
about 99 percent homologous to the sequence set forth in NM_018665
or NP_061135. In other specific non-limiting embodiments, a DDX43
protein is a cat, chimpanzee, mouse or dog DDX43 protein which has,
respectively, the amino acid sequence as set forth in GenBank/NCBI
accession nos. XM_003986327.1 or XP_003986376 (cat); XM_518584.3 or
XP_518584.2 (chimpanzee); NM_001191044.1 or NP_001177973.1 (mouse);
or XM_848647 or XP_853740.1 (dog) [43-46, 48-51]
5.2 Cancers Subject to the Invention
[0038] In non-limiting embodiments, the invention may be applied to
cancers including uveal melanoma, cutaneous melanoma, metastatic
melanoma, sarcoma, bladder cancer (e.g. transitional cell
carcinoma), breast cancer (e.g., infiltrating ductal carcinoma),
astrocytoma, glioblastoma, colon cancer, lung cancer (e.g., lung
squamous cell carcinoma), esophageal cancer (e.g. small cell
carcinoma), renal cancer (e.g., clear cell carcinoma), liver
cancer, small intestine cancer (e.g. papillary adenocarcinoma), and
stomach cancer (e.g. adenocarcinoma) [52].
5.3 MEK Inhibitors
[0039] The present invention may be used to assess the likelihood
of therapeutic benefit to a MEK inhibitor. A MEK inhibitor is an
agent which inhibits activity of MEK (Mitogen-activated
protein/extracellular signal-regulated kinase kinase. Non-limiting
examples of MEK inhibitors include selumetinib, trametinib
(GSK1120212; GlaxoSmithKline), MEK162 (Array/Novartis), PD-325901
(Pfizer), XL518 (Exelixis), and CI-1040 (Selleck).
5.4 AKT Inhibitors
[0040] According to certain non-limiting embodiments of the
invention, if a cancer is resistant to treatment with a MEK
inhibitor associated with increased DDX43 expression, it may be
susceptible to treatment with an AKT (also known as Protein Kinase
B) inhibitor. Non-limiting examples of AKT inhibitors include
VQD-002 (VioQuest), perifosine (Selleck), miltefosine (Zentaris),
AZD5363 (Astrazeneca) and MK2206 (Merck).
5.5 Methods of Assessing Sensitivity to MEK Inhibitors
[0041] A subject may be a human or a non-human subject.
Non-limiting examples of non-human subjects include non-human
primates, dogs, cats, mice, rats, guinea pigs, rabbits, fowl, pigs,
horses, cows, goats, sheep, etc.
[0042] Cells for testing may be obtained by any method known in the
art, including but not limited to as a surgical resection, as a
biopsy for example but not limited to a needle biopsy, core biopsy,
or aspirate, or collection from a fluid sample, such as blood,
urine, cerebral spinal fluid, cystic fluid, etc.
[0043] Methods of measuring mRNA include but are not limited to
polymerase chain reaction, in situ hybridization, gel
electrophoresis, sequence analysis, and microarray analysis or a
combination thereof.
[0044] Methods of measuring protein include but are not limited to
mass spectrometry techniques, 1-D or 2-D gel-based analysis
systems, chromatography, enzyme linked immunosorbent assays
(ELISAs), radioimmunoassays (RIA), enzyme immunoassays (EIA),
Western Blotting, immunoprecipitation, and immunohistochemistry.
Antibody arrays or protein chips may also be employed.
[0045] That an anticancer effect is "likely" to be produced by an
agent in a subject means that the subject, in the parameter or
parameters being tested (e.g., level of DDX43 mRNA and/or protein,
expression of genes or exons listed in Table 1), is more similar to
other subjects in which the agent produces a significant anticancer
effect than to other subjects in which the agent does not produce a
significant anticancer effect.
[0046] That an anticancer effect is "unlikely" to be produced by an
agent in a subject means that the subject, in the parameter or
parameters being tested, is more similar to other subjects in which
the agent does not produce a significant anticancer effect than to
other subjects in which the agent does produce a significant
anticancer effect.
5.5.1 Using DDX43
[0047] In certain embodiments, the present invention provides for
measurement of expression of a DDX43 molecule, which may be a
measurement of DDX43 mRNA and/or measurement of DDX43 protein.
Measurement may be of intracellular levels of mRNA and/or protein.
Measurement may be in vitro in a sample, for example a cell sample
such as from a biopsy of a cancer (primary or metastatic), from a
subject, or may be in vivo using a labeled probe.
[0048] In certain non-limiting embodiments, the present invention
provides for a method of determining whether an anti-cancer effect
is unlikely to be produced in a cancer by a MEK inhibitor,
comprising determining whether cells of the cancer contain an
increased level of DDX43 mRNA and/or DDX43 protein, where if the
level of DDX43 mRNA and/or protein is increased, it is unlikely
that a MEK inhibitor would have an anti-cancer effect on the
cancer.
[0049] In certain non-limiting embodiments, the present invention
provides for a method of determining whether an anti-cancer effect
is unlikely to be produced in a cancer by a MEK inhibitor,
comprising obtaining a sample of the cancer, and determining, in
the sample, whether cells of the cancer contain an increased level
of DDX43 mRNA and/or DDX43 protein, where if the level of DDX43
mRNA and/or protein is increased, it is unlikely that a MEK
inhibitor would have an anti-cancer effect on the cancer.
[0050] In certain non-limiting embodiments, the present invention
provides for a method of determining whether an anti-cancer effect
is unlikely to be produced in a subject having a cancer by a MEK
inhibitor, comprising obtaining a sample of the cancer, and
determining, in the sample, whether cells of the cancer contain an
increased level of DDX43 mRNA and/or DDX43 protein, where if the
level of DDX43 mRNA and/or protein is increased, it is unlikely
that a MEK inhibitor would have an anti-cancer effect on the
cancer.
[0051] An increased level of DDX43 mRNA or protein is a significant
increase relative to the level of DDX43 mRNA or protein in a normal
tissue (a "normal value"). In specific, non-limiting examples, the
level of DDX43 mRNA and/or protein may be increased by at least a
factor of 10, or at least a factor of 15, or at least a factor of
20, or at least a factor of 30, or at least a factor of 40, or at
least a factor of 50, relative to the level in a normal healthy
subject or normal tissue in the same subject. In particular
non-limiting embodiments the level of DDX43 mRNA and/or protein may
be expressed as a ratio relative to the expression of a reference
gene, such as a housekeeping gene, which is expected to be
expressed at about the same level in normal versus cancer tissue.
In non-limiting examples suitable reference genes may be GAPDH,
beta-actin or beta-tubulin. In particular non-limiting examples,
the ratio of the expression level of DDX43 mRNA or protein,
relative to GADPH mRNA or protein (DDX43/GAPDH ratio) or to mRNA or
protein expression of another housekeeping gene such as beta actin
or beta tubulin, in a responder may be less than 0.5 or less than
0.1 and in a non-responder may be at least 1. In one specific
non-limiting example the DDX43/GAPDH ratio in responders may be up
to 0.078 and in non-responders may be at least 1.457.
[0052] In non-limiting embodiments, a control value may be
predetermined or may be determined in parallel or subsequent to an
assay determining mRNA and/or protein level in a subject.
[0053] An anti-cancer effect means one or more of a reduction in
aggregate cancer cell mass, a reduction in cancer cell growth rate,
a reduction in cancer cell proliferation, a reduction in tumor
mass, a reduction in tumor volume, a reduction in tumor cell
proliferation, a reduction in tumor growth rate, or a reduction in
tumor metastasis.
[0054] In certain non-limiting embodiments, the present invention
provides for a method of determining whether an anti-cancer effect
is unlikely to be produced in a cancer by selumetinib, comprising
determining whether cells of the cancer contain an increased level
of DDX43 mRNA and/or DDX43 protein, where if the level of DDX43
mRNA and/or protein is increased, it is unlikely that selumetinib
would have an anti-cancer effect on the cancer.
[0055] In certain non-limiting embodiments, the present invention
provides for a method of determining whether an anti-cancer effect
is unlikely to be produced in a cancer by selumetinib, comprising
obtaining a sample of the cancer, and determining, in the sample,
whether cells of the cancer contain an increased level of DDX43
mRNA and/or DDX43 protein, where if the level of DDX43 mRNA and/or
protein is increased, it is unlikely that selumetinib would have an
anti-cancer effect on the cancer.
[0056] In certain non-limiting embodiments, the present invention
provides for a method of determining whether an anti-cancer effect
is unlikely to be produced in a subject having a cancer by
selumetinib, comprising obtaining a sample of the cancer, and
determining, in the sample, whether cells of the cancer contain an
increased level of DDX43 mRNA and/or DDX43 protein, where if the
level of DDX43 mRNA and/or protein is increased, it is unlikely
that selumetinib would have an anti-cancer effect on the
cancer.
[0057] In certain non-limiting embodiments, the present invention
provides for a method of determining whether an anti-cancer effect
is unlikely to be produced in melanoma by a MEK inhibitor,
comprising determining whether cells of the melanoma contain an
increased level of DDX43 mRNA and/or DDX43 protein, where if the
level of DDX43 mRNA and/or protein is increased, it is unlikely
that a MEK inhibitor would have an anti-cancer effect on the
melanoma.
[0058] In certain non-limiting embodiments, the present invention
provides for a method of determining whether an anti-cancer effect
is unlikely to be produced in a melanoma by a MEK inhibitor,
comprising obtaining a sample of the melanoma, and determining, in
the sample, whether cells of the melanoma contain an increased
level of DDX43 mRNA and/or DDX43 protein, where if the level of
DDX43 mRNA and/or protein is increased, it is unlikely that a MEK
inhibitor would have an anti-cancer effect on the melanoma.
[0059] In certain non-limiting embodiments, the present invention
provides for a method of determining whether an anti-cancer effect
is unlikely to be produced in a subject having a melanoma by a MEK
inhibitor, comprising obtaining a sample of the melanoma, and
determining, in the sample, whether cells of the melanoma contain
an increased level of DDX43 mRNA and/or DDX43 protein, where if the
level of DDX43 mRNA and/or protein is increased, it is unlikely
that a MEK inhibitor would have an anti-cancer effect on the
melanoma.
[0060] In certain non-limiting embodiments, the present invention
provides for a method of determining whether an anti-cancer effect
is unlikely to be produced in melanoma by selumetinib, comprising
determining whether cells of the melanoma contain an increased
level of DDX43 mRNA and/or DDX43 protein, where if the level of
DDX43 mRNA and/or protein is increased, it is unlikely that
selumetinib would have an anti-cancer effect on the melanoma.
[0061] In certain non-limiting embodiments, the present invention
provides for a method of determining whether an anti-cancer effect
is unlikely to be produced in a melanoma by selumetinib, comprising
obtaining a sample of the melanoma, and determining, in the sample,
whether cells of the melanoma contain an increased level of DDX43
mRNA and/or DDX43 protein, where if the level of DDX43 mRNA and/or
protein is increased, it is unlikely that selumetinib would have an
anti-cancer effect on the melanoma.
[0062] In certain non-limiting embodiments, the present invention
provides for a method of determining whether an anti-cancer effect
is unlikely to be produced in a subject having a melanoma by
selumetinib, comprising obtaining a sample of the melanoma, and
determining, in the sample, whether cells of the melanoma contain
an increased level of DDX43 mRNA and/or DDX43 protein, where if the
level of DDX43 mRNA and/or protein is increased, it is unlikely
that selumetinib would have an anti-cancer effect on the
melanoma.
5.5.2 Using Markers Other Than DDX43
[0063] As demonstrated in the working example below, differential
expression levels of other genes (or gene exons) were associated
with decreased response to MEK inhibition. In non-limiting
embodiments of the invention, differential expression of these
genes and/or exons may be used instead of DDX43 or in addition to
DDX43 to assess the likelihood that a subject will have a favorable
response to treatment.
[0064] In certain non-limiting embodiments, the present invention
provides for a method of determining whether an anti-cancer effect
is unlikely to be produced in a subject cancer by a MEK inhibitor,
comprising determining whether cells of the subject cancer contain
an increased level, relative to a responder cancer cell, of mRNA
and/or protein corresponding to one or more gene or exon listed in
Table 1 below, where if the level of mRNA and/or protein is
increased, it is unlikely that a MEK inhibitor would have an
anti-cancer effect on the subject cancer.
[0065] In certain non-limiting embodiments, the present invention
provides for a method of determining whether an anti-cancer effect
is unlikely to be produced in a subject cancer by a MEK inhibitor,
comprising determining whether cells of the subject cancer contain
a decreased level, relative to a responder cancer cell, of mRNA
and/or protein corresponding to an exon of one or more of the
following: RHBG, MFAPS, DPYS, ACCN4, or DMKN, where if the level of
mRNA and/or protein is decreased, it is unlikely that a MEK
inhibitor would have an anti-cancer effect on the subject
cancer.
[0066] In certain non-limiting embodiments, the present invention
provides for a method of determining whether an anti-cancer effect
is unlikely to be produced in a subject cancer by a MEK inhibitor,
comprising obtaining a sample of the subject cancer, and
determining, in the sample, whether cells of the subject cancer
contain an increased level, relative to a responder cancer cell, of
mRNA and/or protein corresponding to one or more gene or exon
listed in Table 1 below, where if the level of mRNA and/or protein
is increased, it is unlikely that a MEK inhibitor would have an
anti-cancer effect on the subject cancer.
[0067] In certain non-limiting embodiments, the present invention
provides for a method of determining whether an anti-cancer effect
is unlikely to be produced in a subject cancer by a MEK inhibitor,
comprising obtaining a sample of the subject cancer, and
determining, in the sample, whether cells of the subject cancer
contain a decreased level, relative to a responder cancer cell, of
mRNA and/or protein corresponding to an exon of one or more of the
following: RHBG, MFAPS, DPYS, ACCN4, or DMKN, where if the level of
mRNA and/or protein is decreased, it is unlikely that a MEK
inhibitor would have an anti-cancer effect on the subject
cancer.
[0068] In certain non-limiting embodiments, the present invention
provides for a method of determining whether an anti-cancer effect
is unlikely to be produced in a subject having a cancer by a MEK
inhibitor, comprising obtaining a sample of the subject cancer, and
determining, in the sample, whether cells of the subject cancer
contain an increased level, relative to a responder cancer cell, of
mRNA and/or protein corresponding to one or more gene or exon
listed in Table 1 below, where if the level of mRNA and/or protein
is increased, it is unlikely that a MEK inhibitor would have an
anti-cancer effect on the subject cancer.
[0069] In certain non-limiting embodiments, the present invention
provides for a method of determining whether an anti-cancer effect
is unlikely to be produced in a subject having a cancer by a MEK
inhibitor, comprising obtaining a sample of the subject cancer, and
determining, in the sample, whether cells of the subject cancer
contain a decreased level, relative to a responder cancer cell, of
mRNA and/or protein corresponding to an exon of one or more of the
following: RHBG, MFAPS, DPYS, ACCN4, or DMKN, where if the level of
mRNA and/or protein is increased, it is unlikely that a MEK
inhibitor would have an anti-cancer effect on the subject
cancer.
TABLE-US-00001 TABLE 1 List of genes associated with lack of
clinical benefit to selumetinib by differential expression (left)
and differential exon levels (right), by comparing "responders"
versus "non responders", independently of treatment. p values and
log.sub.2 fold changes for each gene are also shown. Genes
Differentially Expressed Exons Differentially Expressed Gene Gene
Symbol p Value Log2 FC Symbol p Value Log2 FC RIMS2 4.30E-10
10.2024 CAPN3 3.33E-16 2.8463 DDX43 1.67E-08 5.8753 ITLN2 2.75E-08
6.8585 ADAMTS14 2.62E-08 4.8677 PCDHGA11 1.83E-06 4.6135 GTF2I
5.33E-12 4.7563 FIBCD1 3.35E-05 3.7236 MRC2 0.00011 3.4279 SERPINE2
0.00013 3.4407 XIST 7.56E-10 28.1751 DDIT4L 0.00015 3.2857 FMN2
9.16E-09 -4.9091 ICAM5 0.00023 3.8144 RPS24 3.62E-07 -2.2390
[0070] In particular, non-limiting embodiments, the one or more
gene for which expression is evaluated according to this section is
RIMS2, ITLN2, PCHGA11 and/or DDIT4L.
[0071] In particular, non-limiting embodiments, the one or more
exon for which expression is evaluated according to this section is
CAPN3, RHBG, MFAP5, DPYS and/or GTF2I.
5.6 Methods of Assessing Sensitivity to AKT Inhibitors
[0072] Cell viability and proliferation rate in response to an AKT
inhibitor may optionally be evaluated using standard techniques to
determine whether a cell, such as a cancer cell (for example a
cancer cell collected from a subject) is sensitive to AKT
inhibition.
5.7 Methods of Measuring mRNA or Proteins
[0073] In certain non-limiting embodiments, the invention comprises
measuring the level of DDX43 mRNA and/or protein, the level of mRNA
and/or protein corresponding to one or more gene or exon listed in
Table 1, or the level of mRNA and/or protein corresponding to an
exon of one or more of the following: RHBG, MFAPS, DPYS, ACCN4, or
DMKN. Any methods for measuring the level of mRNA and/or the level
of proteins known in the art for can be used for the measurements
of the invention. In non-limiting examples, one or more of the
following: quantitative real-time PCR, reverse transcriptase PCR,
Northern blot, Western blot, immunohistochemistry, and
antibody-binding may be used to measure the level of DDX43 mRNA
and/or protein, the level of mRNA and/or protein corresponding to
one or more gene or exon listed in Table 1, and the level of mRNA
and/or protein corresponding to an exon of one or more of the
following: RHBG, MFAPS, DPYS, ACCN4, or DMKN.
5.8 Methods of Treatment
[0074] In certain non-limiting embodiments, the present invention
provides for a method of treating a subject having a cancer
comprising (i) determining whether an anti-cancer effect is
unlikely to be produced in the cancer by a MEK inhibitor,
comprising determining whether cells of the cancer contain an
increased level of DDX43 mRNA and/or DDX43 protein; and (ii)
treating the subject with a therapeutic amount of a MEK inhibitor
if the level of DDX43 mRNA and/or protein is not increased or (iii)
treating the subject with a therapeutic amount of an anticancer
agent other than a MEK inhibitor where the level of DDX43 mRNA
and/or protein is increased. In one specific non-limiting
embodiment the anticancer agent other than a MEK inhibitor is an
AKT inhibitor.
[0075] In certain non-limiting embodiments, the present invention
provides for a method of treating a subject having a cancer
comprising (i) determining whether an anti-cancer effect is
unlikely to be produced in a cancer by a MEK inhibitor, comprising
obtaining a sample of the cancer, and determining, in the sample,
whether cells of the cancer contain an increased level of DDX43
mRNA and/or DDX43 protein, and (ii) treating the subject with a
therapeutic amount of a MEK inhibitor if the level of DDX43 mRNA
and/or protein is not increased or (iii) treating the subject with
a therapeutic amount of an anticancer agent other than a MEK
inhibitor where the level of DDX43 mRNA and/or protein is
increased. In one specific non-limiting embodiment the anticancer
agent other than a MEK inhibitor is an AKT inhibitor.
[0076] In certain non-limiting embodiments, the present invention
provides for a method of treating a subject having a cancer
comprising (i) determining whether an anti-cancer effect is
unlikely to be produced in the subject by a MEK inhibitor,
comprising obtaining a sample of the cancer, and determining, in
the sample, whether cells of the cancer contain an increased level
of DDX43 mRNA and/or DDX43 protein, and (ii) treating the subject
with a therapeutic amount of a MEK inhibitor if the level of DDX43
mRNA and/or protein is not increased or (iii) treating the subject
with a therapeutic amount of an anticancer agent other than a MEK
inhibitor where the level of DDX43 mRNA and/or protein is
increased. In one specific non-limiting embodiment the anticancer
agent other than a MEK inhibitor is an AKT inhibitor.
[0077] In certain non-limiting embodiments, the present invention
provides for a method of treating a subject having a cancer
comprising (i) determining whether an anti-cancer effect is
unlikely to be produced in the cancer by a MEK inhibitor,
comprising determining whether cells of the cancer contain an
increased level of DDX43 mRNA and/or DDX43 protein and/or an
increased level of mRNA or protein corresponding to one or more
gene or exon listed in Table 1, and (ii) treating the subject with
a therapeutic amount of a MEK inhibitor if the level of DDX43 mRNA
and/or protein, and/or the level of mRNA or protein corresponding
to one or more gene or exon listed in Table 1, is not increased or
(iii) treating the subject with a therapeutic amount of an
anticancer agent other than a MEK inhibitor where the level of
DDX43 mRNA and/or protein, and/or the level of mRNA or protein
corresponding to one or more gene or exon listed in Table 1, is
increased. In one specific non-limiting embodiment the anticancer
agent other than a MEK inhibitor is an AKT inhibitor.
[0078] In certain non-limiting embodiments, the present invention
provides for a method of treating a subject having a cancer
comprising (i) determining whether an anti-cancer effect is
unlikely to be produced in the cancer by a MEK inhibitor,
comprising determining whether cells of the cancer contain an
increased level of DDX43 mRNA and/or DDX43 protein and/or a
decreased level of mRNA or protein corresponding to an exon of one
or more of the following: RHBG, MFAPS, DPYS, ACCN4, or DMKN, and
(ii) treating the subject with a therapeutic amount of a MEK
inhibitor if the level of DDX43 mRNA and/or protein is not
increased, and/or the level of mRNA or protein corresponding to an
exon of one or more of the following: RHBG, MFAPS, DPYS, ACCN4, or
DMKN, is not decreased or (iii) treating the subject with a
therapeutic amount of an anticancer agent other than a MEK
inhibitor where the level of DDX43 mRNA and/or protein is
increased, and/or the level of mRNA or protein corresponding to an
exon of one or more of the following: RHBG, MFAPS, DPYS, ACCN4, or
DMKN, is decreased. In one specific non-limiting embodiment the
anticancer agent other than a MEK inhibitor is an AKT
inhibitor.
[0079] In certain non-limiting embodiments, the present invention
provides for a method of treating a subject having a cancer
comprising (i) determining whether an anti-cancer effect is
unlikely to be produced in the cancer by a MEK inhibitor,
comprising obtaining a sample of the cancer, and determining, in
the sample, whether cells of the cancer contain an increased level
of DDX43 mRNA and/or DDX43 protein and/or an increased level of
mRNA or protein corresponding to one or more gene or exon listed in
Table 1, and (ii) treating the subject with a therapeutic amount of
a MEK inhibitor if the level of DDX43 mRNA and/or protein, and/or
the level of mRNA or protein corresponding to one or more of the
gene or exon listed in Table 1, is not increased or (iii) treating
the subject with a therapeutic amount of an anticancer agent other
than a MEK inhibitor where the level of DDX43 mRNA and/or protein,
and/or the level of mRNA or protein corresponding to one or more
gene or exon listed in Table 1, is increased. In one specific
non-limiting embodiment the anticancer agent other than a MEK
inhibitor is an AKT inhibitor.
[0080] In certain non-limiting embodiments, the present invention
provides for a method of treating a subject having a cancer
comprising (i) determining whether an anti-cancer effect is
unlikely to be produced in the cancer by a MEK inhibitor,
comprising obtaining a sample of the cancer, and determining, in
the sample, whether cells of the cancer contain an increased level
of DDX43 mRNA and/or DDX43 protein and/or a decreased level of mRNA
or protein corresponding to an exon of one or more of the
following: RHBG, MFAPS, DPYS, ACCN4, or DMKN, and (ii) treating the
subject with a therapeutic amount of a MEK inhibitor if the level
of DDX43 mRNA and/or protein is not increased, and/or the level of
mRNA or protein corresponding to an exon of one or more of the
following: RHBG, MFAPS, DPYS, ACCN4, or DMKN, is not decreased or
(iii) treating the subject with a therapeutic amount of an
anticancer agent other than a MEK inhibitor where the level of
DDX43 mRNA and/or protein is increased, and/or the level of mRNA or
protein corresponding to an exon of one or more of the following:
RHBG, MFAPS, DPYS, ACCN4, or DMKN, is decreased. In one specific
non-limiting embodiment the anticancer agent other than a MEK
inhibitor is an AKT inhibitor.
[0081] In certain non-limiting embodiments, the present invention
provides for a method of treating a subject having a cancer
comprising (i) determining whether an anti-cancer effect is
unlikely to be produced in the subject by a MEK inhibitor,
comprising obtaining a sample of the cancer, and determining, in
the sample, whether cells of the cancer contain an increased level
of DDX43 mRNA and/or DDX43 protein and/or an increased level of
mRNA or protein corresponding to one or more gene or exon listed in
Table 1, and (ii) treating the subject with a therapeutic amount of
a MEK inhibitor if the level of DDX43 mRNA and/or protein, and/or
the level of mRNA or protein corresponding to one or more gene or
exon listed in Table 1, is not increased or (iii) treating the
subject with a therapeutic amount of an anticancer agent other than
a MEK inhibitor where the level of DDX43 mRNA and/or protein,
and/or the level of mRNA or protein corresponding to one or more
gene or exon listed in Table 1, is increased. In one specific
non-limiting embodiment the anticancer agent other than a MEK
inhibitor is an AKT inhibitor.
[0082] In certain non-limiting embodiments, the present invention
provides for a method of treating a subject having a cancer
comprising (i) determining whether an anti-cancer effect is
unlikely to be produced in the subject by a MEK inhibitor,
comprising obtaining a sample of the cancer, and determining, in
the sample, whether cells of the cancer contain an increased level
of DDX43 mRNA and/or DDX43 protein and/or a decreased level of mRNA
or protein corresponding to an exon of one or more of the
following: RHBG, MFAPS, DPYS, ACCN4, or DMKN, and (ii) treating the
subject with a therapeutic amount of a MEK inhibitor if the level
of DDX43 mRNA and/or protein is not increased and/or the level of
mRNA or protein corresponding to an exon of one or more of the
following: RHBG, MFAPS, DPYS, ACCN4, or DMKN, is not decreased or
(iii) treating the subject with a therapeutic amount of an
anticancer agent other than a MEK inhibitor where the level of
DDX43 mRNA and/or protein is increased, and/or the level of mRNA or
protein corresponding to an exon of one or more of the following:
RHBG, MFAPS, DPYS, ACCN4, or DMKN, is decreased. In one specific
non-limiting embodiment the anticancer agent other than a MEK
inhibitor is an AKT inhibitor.
[0083] In certain non-limiting embodiments, the present invention
provides for a method of treating a subject having a cancer
comprising (i) determining whether an anti-cancer effect is
unlikely to be produced in the cancer by selumetinib, comprising
determining whether cells of the cancer contain an increased level
of DDX43 mRNA and/or DDX43 protein; and (ii) treating the subject
with a therapeutic amount of selumetinib if the level of DDX43 mRNA
and/or protein is not increased or (iii) treating the subject with
a therapeutic amount of an anticancer agent other than a MEK
inhibitor where the level of DDX43 mRNA and/or protein is
increased. In one specific non-limiting embodiment the anticancer
agent other than a MEK inhibitor is an AKT inhibitor.
[0084] In certain non-limiting embodiments, the present invention
provides for a method of treating a subject having a cancer
comprising (i) determining whether an anti-cancer effect is
unlikely to be produced in the cancer by selumetinib, comprising
obtaining a sample of the cancer, and determining, in the sample,
whether cells of the cancer contain an increased level of DDX43
mRNA and/or DDX43 protein, and (ii) treating the subject with a
therapeutic amount of selumetinib if the level of DDX43 mRNA and/or
protein is not increased or (iii) treating the subject with a
therapeutic amount of an anticancer agent other than a MEK
inhibitor where the level of DDX43 mRNA and/or protein is
increased. In one specific non-limiting embodiment the anticancer
agent other than a MEK inhibitor is an AKT inhibitor.
[0085] In certain non-limiting embodiments, the present invention
provides for a method of treating a subject having a cancer
comprising (i) determining whether an anti-cancer effect is
unlikely to be produced in the subject by selumetinib, comprising
obtaining a sample of the cancer, and determining, in the sample,
whether cells of the cancer contain an increased level of DDX43
mRNA and/or DDX43 protein, and (ii) treating the subject with a
therapeutic amount of selumetinib if the level of DDX43 mRNA and/or
protein is not increased or (iii) treating the subject with a
therapeutic amount of an anticancer agent other than a MEK
inhibitor where the level of DDX43 mRNA and/or protein is
increased. In one specific non-limiting embodiment the anticancer
agent other than a MEK inhibitor is an AKT inhibitor.
[0086] In certain non-limiting embodiments, the present invention
provides for a method of treating a subject having a cancer
comprising (i) determining whether an anti-cancer effect is
unlikely to be produced in the cancer by selumetinib, comprising
determining whether cells of the cancer contain an increased level
of DDX43 mRNA and/or DDX43 protein and/or an increased level of
mRNA or protein corresponding to one or more gene or exon listed in
Table 1, and (ii) treating the subject with a therapeutic amount of
selumetinib if the level of DDX43 mRNA and/or protein, and/or the
level of mRNA or protein corresponding to one or more gene or exon
listed in Table 1, is not increased or (iii) treating the subject
with a therapeutic amount of an anticancer agent other than a MEK
inhibitor where the level of DDX43 mRNA and/or protein, and/or the
level of mRNA or protein corresponding to one or more gene or exon
listed in Table 1, is increased. In one specific non-limiting
embodiment the anticancer agent other than a MEK inhibitor is an
AKT inhibitor.
[0087] In certain non-limiting embodiments, the present invention
provides for a method of treating a subject having a cancer
comprising (i) determining whether an anti-cancer effect is
unlikely to be produced in the cancer by selumetinib, comprising
determining whether cells of the cancer contain an increased level
of DDX43 mRNA and/or DDX43 protein and/or a decreased level of mRNA
or protein corresponding to an exon of one or more of the
following: RHBG, MFAPS, DPYS, ACCN4, or DMKN, and (ii) treating the
subject with a therapeutic amount of selumetinib if the level of
DDX43 mRNA and/or protein is not increased, and/or the level of
mRNA or protein corresponding to an exon of one or more of the
following: RHBG, MFAPS, DPYS, ACCN4, or DMKN, is not decreased or
(iii) treating the subject with a therapeutic amount of an
anticancer agent other than a MEK inhibitor where the level of
DDX43 mRNA and/or protein is increased, and/or the level of mRNA or
protein corresponding to an exon of one or more of the following:
RHBG, MFAPS, DPYS, ACCN4, or DMKN, is decreased. In one specific
non-limiting embodiment the anticancer agent other than a MEK
inhibitor is an AKT inhibitor.
[0088] In certain non-limiting embodiments, the present invention
provides for a method of treating a subject having a cancer
comprising (i) determining whether an anti-cancer effect is
unlikely to be produced in the cancer by selumetinib, comprising
obtaining a sample of the cancer, and determining, in the sample,
whether cells of the cancer contain an increased level of DDX43
mRNA and/or DDX43 protein and/or an increased level of mRNA or
protein corresponding to one or more gene or exon listed in Table
1, and (ii) treating the subject with a therapeutic amount of
selumetinib if the level of DDX43 mRNA and/or protein, and/or the
level of mRNA or protein corresponding to one or more gene or exon
listed in Table 1, is not increased or (iii) treating the subject
with a therapeutic amount of an anticancer agent other than a MEK
inhibitor where the level of DDX43 mRNA and/or protein, and/or the
level of mRNA or protein corresponding to one or more gene or exon
listed in Table 1, is increased. In one specific non-limiting
embodiment the anticancer agent other than a MEK inhibitor is an
AKT inhibitor.
[0089] In certain non-limiting embodiments, the present invention
provides for a method of treating a subject having a cancer
comprising (i) determining whether an anti-cancer effect is
unlikely to be produced in the cancer by selumetinib, comprising
obtaining a sample of the cancer, and determining, in the sample,
whether cells of the cancer contain an increased level of DDX43
mRNA and/or DDX43 protein and/or a decreased level of mRNA or
protein corresponding to an exon of one or more of the following:
RHBG, MFAPS, DPYS, ACCN4, or DMKN, and (ii) treating the subject
with a therapeutic amount of selumetinib if the level of DDX43 mRNA
and/or protein is not increased, and/or the level of mRNA or
protein corresponding to an exon of one or more of the following:
RHBG, MFAPS, DPYS, ACCN4, or DMKN, is not decreased or (iii)
treating the subject with a therapeutic amount of an anticancer
agent other than a MEK inhibitor where the level of DDX43 mRNA
and/or protein is increased, and/or the level of mRNA or protein
corresponding to an exon of one or more of the following: RHBG,
MFAPS, DPYS, ACCN4, or DMKN, is decreased. In one specific
non-limiting embodiment the anticancer agent other than a MEK
inhibitor is an AKT inhibitor.
[0090] In certain non-limiting embodiments, the present invention
provides for a method of treating a subject having a cancer
comprising (i) determining whether an anti-cancer effect is
unlikely to be produced in the subject by selumetinib, comprising
obtaining a sample of the cancer, and determining, in the sample,
whether cells of the cancer contain an increased level of DDX43
mRNA and/or DDX43 protein and/or an increased level of mRNA or
protein corresponding to one or more of the gene or exon listed in
Table 1, and (ii) treating the subject with a therapeutic amount of
selumetinib if the level of DDX43 mRNA and/or protein, and/or the
level of mRNA or protein corresponding to one or more gene or exon
listed in Table 1, is not increased or (iii) treating the subject
with a therapeutic amount of an anticancer agent other than a MEK
inhibitor where the level of DDX43 mRNA and/or protein, and/or the
level of mRNA or protein corresponding to one or more gene or exon
listed in Table 1, is increased. In one specific non-limiting
embodiment the anticancer agent other than a MEK inhibitor is an
AKT inhibitor.
[0091] In certain non-limiting embodiments, the present invention
provides for a method of treating a subject having a cancer
comprising (i) determining whether an anti-cancer effect is
unlikely to be produced in the subject by selumetinib, comprising
obtaining a sample of the cancer, and determining, in the sample,
whether cells of the cancer contain an increased level of DDX43
mRNA and/or DDX43 protein and/or a decreased level of mRNA or
protein corresponding to an exon of one or more of the following:
RHBG, MFAPS, DPYS, ACCN4, or DMKN, and (ii) treating the subject
with a therapeutic amount of selumetinib if the level of DDX43 mRNA
and/or protein is not increased, and/or the level of mRNA or
protein corresponding to an exon of one or more of the following:
RHBG, MFAPS, DPYS, ACCN4, or DMKN, is not decreased or (iii)
treating the subject with a therapeutic amount of an anticancer
agent other than a MEK inhibitor where the level of DDX43 mRNA
and/or protein is increased, and/or the level of mRNA or protein
corresponding to an exon of one or more of the following: RHBG,
MFAPS, DPYS, ACCN4, or DMKN, is decreased. In one specific
non-limiting embodiment the anticancer agent other than a MEK
inhibitor is an AKT inhibitor.
[0092] In certain non-limiting embodiments, the present invention
provides for a method of treating a subject having a melanoma
comprising (i) determining whether an anti-cancer effect is
unlikely to be produced in the melanoma by a MEK inhibitor,
comprising determining whether cells of the melanoma contain an
increased level of DDX43 mRNA and/or DDX43 protein; and (ii)
treating the subject with a therapeutic amount of a MEK inhibitor
if the level of DDX43 mRNA and/or protein is not increased or (iii)
treating the subject with a therapeutic amount of an anticancer
agent other than a MEK inhibitor where the level of DDX43 mRNA
and/or protein is increased. In one specific non-limiting
embodiment the anticancer agent other than a MEK inhibitor is an
AKT inhibitor.
[0093] In certain non-limiting embodiments, the present invention
provides for a method of treating a subject having a melanoma
comprising (i) determining whether an anti-cancer effect is
unlikely to be produced in the melanoma by a MEK inhibitor,
comprising obtaining a sample of the melanoma, and determining, in
the sample, whether cells of the melanoma contain an increased
level of DDX43 mRNA and/or DDX43 protein, and (ii) treating the
subject with a therapeutic amount of a MEK inhibitor if the level
of DDX43 mRNA and/or protein is not increased or (iii) treating the
subject with a therapeutic amount of an anticancer agent other than
a MEK inhibitor where the level of DDX43 mRNA and/or protein is
increased. In one specific non-limiting embodiment the anticancer
agent other than a MEK inhibitor is an AKT inhibitor.
[0094] In certain non-limiting embodiments, the present invention
provides for a method of treating a subject having a melanoma
comprising (i) determining whether an anti-cancer effect is
unlikely to be produced in the subject by a MEK inhibitor,
comprising obtaining a sample of the melanoma, and determining, in
the sample, whether cells of the melanoma contain an increased
level of DDX43 mRNA and/or DDX43 protein, and (ii) treating the
subject with a therapeutic amount of a MEK inhibitor if the level
of DDX43 mRNA and/or protein is not increased or (iii) treating the
subject with a therapeutic amount of an anticancer agent other than
a MEK inhibitor where the level of DDX43 mRNA and/or protein is
increased. In one specific non-limiting embodiment the anticancer
agent other than a MEK inhibitor is an AKT inhibitor.
[0095] In certain non-limiting embodiments, the present invention
provides for a method of treating a subject having a melanoma
comprising (i) determining whether an anti-cancer effect is
unlikely to be produced in the melanoma by a MEK inhibitor,
comprising determining whether cells of the melanoma contain an
increased level of DDX43 mRNA and/or DDX43 protein and/or an
increased level of mRNA or protein corresponding to one or more
gene or exon listed in Table 1, and (ii) treating the subject with
a therapeutic amount of a MEK inhibitor if the level of DDX43 mRNA
and/or protein, and/or the level of mRNA or protein corresponding
to one or more gene or exon listed in Table 1, is not increased or
(iii) treating the subject with a therapeutic amount of an
anticancer agent other than a MEK inhibitor where the level of
DDX43 mRNA and/or protein, and/or the level of mRNA or protein
corresponding to one or more gene or exon listed in Table 1, is
increased. In one specific non-limiting embodiment the anticancer
agent other than a MEK inhibitor is an AKT inhibitor.
[0096] In certain non-limiting embodiments, the present invention
provides for a method of treating a subject having a melanoma
comprising (i) determining whether an anti-cancer effect is
unlikely to be produced in the melanoma by a MEK inhibitor,
comprising determining whether cells of the melanoma contain an
increased level of DDX43 mRNA and/or DDX43 protein and/or a
decreased level of mRNA or protein corresponding to an exon of one
or more of the following: RHBG, MFAPS, DPYS, ACCN4, or DMKN, and
(ii) treating the subject with a therapeutic amount of a MEK
inhibitor if the level of DDX43 mRNA and/or protein is not
increased, and/or the level of mRNA or protein corresponding to an
exon of one or more of the following: RHBG, MFAPS, DPYS, ACCN4, or
DMKN, is not decreased or (iii) treating the subject with a
therapeutic amount of an anticancer agent other than a MEK
inhibitor where the level of DDX43 mRNA and/or protein is
increased, and/or the level of mRNA or protein corresponding to an
exon of one or more of the following: RHBG, MFAPS, DPYS, ACCN4, or
DMKN, is decreased. In one specific non-limiting embodiment the
anticancer agent other than a MEK inhibitor is an AKT
inhibitor.
[0097] In certain non-limiting embodiments, the present invention
provides for a method of treating a subject having a melanoma
comprising (i) determining whether an anti-cancer effect is
unlikely to be produced in the melanoma by a MEK inhibitor,
comprising obtaining a sample of the melanoma, and determining, in
the sample, whether cells of the melanoma contain an increased
level of DDX43 mRNA and/or DDX43 protein and/or an increased level
of mRNA or protein corresponding to one or more gene or exon listed
in Table 1, and (ii) treating the subject with a therapeutic amount
of a MEK inhibitor if the level of DDX43 mRNA and/or protein,
and/or the level of mRNA or protein corresponding to one or more
gene or exon listed in Table 1, is not increased or (iii) treating
the subject with a therapeutic amount of an anticancer agent other
than a MEK inhibitor where the level of DDX43 mRNA and/or protein,
and/or the level of mRNA or protein corresponding to one or more
gene or exon listed in Table 1, is increased. In one specific
non-limiting embodiment the anticancer agent other than a MEK
inhibitor is an AKT inhibitor.
[0098] In certain non-limiting embodiments, the present invention
provides for a method of treating a subject having a melanoma
comprising (i) determining whether an anti-cancer effect is
unlikely to be produced in the melanoma by a MEK inhibitor,
comprising obtaining a sample of the melanoma, and determining, in
the sample, whether cells of the melanoma contain an increased
level of DDX43 mRNA and/or DDX43 protein and/or a decreased level
of mRNA or protein corresponding to an exon of one or more of the
following: RHBG, MFAPS, DPYS, ACCN4, or DMKN, and (ii) treating the
subject with a therapeutic amount of a MEK inhibitor if the level
of DDX43 mRNA and/or protein is not increased, and/or the level of
mRNA or protein corresponding to an exon of one or more of the
following: RHBG, MFAPS, DPYS, ACCN4, or DMKN, is not decreased or
(iii) treating the subject with a therapeutic amount of an
anticancer agent other than a MEK inhibitor where the level of
DDX43 mRNA and/or protein is increased, and/or the level of mRNA or
protein corresponding to an exon of one or more of the following:
RHBG, MFAPS, DPYS, ACCN4, or DMKN, is decreased. In one specific
non-limiting embodiment the anticancer agent other than a MEK
inhibitor is an AKT inhibitor.
[0099] In certain non-limiting embodiments, the present invention
provides for a method of treating a subject having a melanoma
comprising (i) determining whether an anti-cancer effect is
unlikely to be produced in the subject by a MEK inhibitor,
comprising obtaining a sample of the melanoma, and determining, in
the sample, whether cells of the melanoma contain an increased
level of DDX43 mRNA and/or DDX43 protein and/or an increased level
of mRNA or protein corresponding to one or more gene or exon listed
in Table 1, and (ii) treating the subject with a therapeutic amount
of a MEK inhibitor if the level of DDX43 mRNA and/or protein,
and/or the level of mRNA or protein corresponding to one or more
gene or exon listed in Table 1, is not increased or (iii) treating
the subject with a therapeutic amount of an anticancer agent other
than a MEK inhibitor where the level of DDX43 mRNA and/or protein,
and/or the level of mRNA or protein corresponding to one or more
gene or exon listed in Table 1, is increased. In one specific
non-limiting embodiment the anticancer agent other than a MEK
inhibitor is an AKT inhibitor.
[0100] In certain non-limiting embodiments, the present invention
provides for a method of treating a subject having a melanoma
comprising (i) determining whether an anti-cancer effect is
unlikely to be produced in the subject by a MEK inhibitor,
comprising obtaining a sample of the melanoma, and determining, in
the sample, whether cells of the melanoma contain an increased
level of DDX43 mRNA and/or DDX43 protein and/or a decreased level
of mRNA or protein corresponding to an exon of one or more of the
following: RHBG, MFAPS, DPYS, ACCN4, or DMKN, and (ii) treating the
subject with a therapeutic amount of a MEK inhibitor if the level
of DDX43 mRNA and/or protein is not increased, and/or the level of
mRNA or protein corresponding to an exon of one or more of the
following: RHBG, MFAPS, DPYS, ACCN4, or DMKN, is not decreased or
(iii) treating the subject with a therapeutic amount of an
anticancer agent other than a MEK inhibitor where the level of
DDX43 mRNA and/or protein is increased, and/or the level of mRNA or
protein corresponding to an exon of one or more of the following:
RHBG, MFAPS, DPYS, ACCN4, or DMKN, is decreased. In one specific
non-limiting embodiment the anticancer agent other than a MEK
inhibitor is an AKT inhibitor.
[0101] In certain non-limiting embodiments, the present invention
provides for a method of treating a subject having a melanoma
comprising (i) determining whether an anti-cancer effect is
unlikely to be produced in the cancer by selumetinib, comprising
determining whether cells of the melanoma contain an increased
level of DDX43 mRNA and/or DDX43 protein; and (ii) treating the
subject with a therapeutic amount of selumetinib if the level of
DDX43 mRNA and/or protein is not increased or (iii) treating the
subject with a therapeutic amount of an anticancer agent other than
a MEK inhibitor where the level of DDX43 mRNA and/or protein is
increased. In one specific non-limiting embodiment the anticancer
agent other than a MEK inhibitor is an AKT inhibitor.
[0102] In certain non-limiting embodiments, the present invention
provides for a method of treating a subject having a melanoma
comprising (i) determining whether an anti-cancer effect is
unlikely to be produced in the melanoma by selumetinib, comprising
obtaining a sample of the melanoma, and determining, in the sample,
whether cells of the melanoma contain an increased level of DDX43
mRNA and/or DDX43 protein, and (ii) treating the subject with a
therapeutic amount of selumetinib if the level of DDX43 mRNA and/or
protein is not increased or (iii) treating the subject with a
therapeutic amount of an anticancer agent other than a MEK
inhibitor where the level of DDX43 mRNA and/or protein is
increased. In one specific non-limiting embodiment the anticancer
agent other than a MEK inhibitor is an AKT inhibitor.
[0103] In certain non-limiting embodiments, the present invention
provides for a method of treating a subject having a melanoma
comprising (i) determining whether an anti-cancer effect is
unlikely to be produced in the subject by selumetinib, comprising
obtaining a sample of the melanoma, and determining, in the sample,
whether cells of the melanoma contain an increased level of DDX43
mRNA and/or DDX43 protein, and (ii) treating the subject with a
therapeutic amount of selumetinib if the level of DDX43 mRNA and/or
protein is not increased or (iii) treating the subject with a
therapeutic amount of an anticancer agent other than a MEK
inhibitor where the level of DDX43 mRNA and/or protein is
increased. In one specific non-limiting embodiment the anticancer
agent other than a MEK inhibitor is an AKT inhibitor.
[0104] In certain non-limiting embodiments, the present invention
provides for a method of treating a subject having a melanoma
comprising (i) determining whether an anti-cancer effect is
unlikely to be produced in the melanoma by selumetinib, comprising
determining whether cells of the melanoma contain an increased
level of DDX43 mRNA and/or DDX43 protein and/or an increased level
of mRNA or protein corresponding to one or more gene or exon listed
in Table 1, and (ii) treating the subject with a therapeutic amount
of selumetinib if the level of DDX43 mRNA and/or protein, and/or
the level of mRNA or protein corresponding to one or more gene or
exon listed in Table 1, is not increased or (iii) treating the
subject with a therapeutic amount of an anticancer agent other than
a MEK inhibitor where the level of DDX43 mRNA and/or protein,
and/or the level of mRNA or protein corresponding to one or more
gene or exon listed in Table 1, is increased. In one specific
non-limiting embodiment the anticancer agent other than a MEK
inhibitor is an AKT inhibitor.
[0105] In certain non-limiting embodiments, the present invention
provides for a method of treating a subject having a melanoma
comprising (i) determining whether an anti-cancer effect is
unlikely to be produced in the melanoma by selumetinib, comprising
determining whether cells of the melanoma contain an increased
level of DDX43 mRNA and/or DDX43 protein and/or a decreased level
of mRNA or protein corresponding to an exon of one or more of the
following: RHBG, MFAPS, DPYS, ACCN4, or DMKN, and (ii) treating the
subject with a therapeutic amount of selumetinib if the level of
DDX43 mRNA and/or protein is not increased, and/or the level of
mRNA or protein corresponding to an exon of one or more of the
following: RHBG, MFAPS, DPYS, ACCN4, or DMKN, is not decreased or
(iii) treating the subject with a therapeutic amount of an
anticancer agent other than a MEK inhibitor where the level of
DDX43 mRNA and/or protein is increased, and/or the level of mRNA or
protein corresponding to an exon of one or more of the following:
RHBG, MFAPS, DPYS, ACCN4, or DMKN, is decreased. In one specific
non-limiting embodiment the anticancer agent other than a MEK
inhibitor is an AKT inhibitor.
[0106] In certain non-limiting embodiments, the present invention
provides for a method of treating a subject having a melanoma
comprising (i) determining whether an anti-cancer effect is
unlikely to be produced in the melanoma by selumetinib, comprising
obtaining a sample of the melanoma, and determining, in the sample,
whether cells of the melanoma contain an increased level of DDX43
mRNA and/or DDX43 protein and/or an increased level of mRNA or
protein corresponding to one or more gene or exon listed in Table
1, and (ii) treating the subject with a therapeutic amount of
selumetinib if the level of DDX43 mRNA and/or protein, and/or the
level of mRNA or protein corresponding to one or more gene or exon
listed in Table 1, is not increased or (iii) treating the subject
with a therapeutic amount of an anticancer agent other than a MEK
inhibitor where the level of DDX43 mRNA and/or protein, and/or the
level of mRNA or protein corresponding to one or more gene or exon
listed in Table 1, is increased. In one specific non-limiting
embodiment the anticancer agent other than a MEK inhibitor is an
AKT inhibitor.
[0107] In certain non-limiting embodiments, the present invention
provides for a method of treating a subject having a melanoma
comprising (i) determining whether an anti-cancer effect is
unlikely to be produced in the melanoma by selumetinib, comprising
obtaining a sample of the melanoma, and determining, in the sample,
whether cells of the melanoma contain an increased level of DDX43
mRNA and/or DDX43 protein and/or a decreased level of mRNA or
protein corresponding to an exon of one or more of the following:
RHBG, MFAPS, DPYS, ACCN4, or DMKN, and (ii) treating the subject
with a therapeutic amount of selumetinib if the level of DDX43 mRNA
and/or protein is not increased, and/or the level of mRNA or
protein corresponding to an exon of one or more of the following:
RHBG, MFAPS, DPYS, ACCN4, or DMKN, is not decreased or (iii)
treating the subject with a therapeutic amount of an anticancer
agent other than a MEK inhibitor where the level of DDX43 mRNA
and/or protein is increased, and/or the level of mRNA or protein
corresponding to an exon of one or more of the following: RHBG,
MFAPS, DPYS, ACCN4, or DMKN, is decreased. In one specific
non-limiting embodiment the anticancer agent other than a MEK
inhibitor is an AKT inhibitor.
[0108] In certain non-limiting embodiments, the present invention
provides for a method of treating a subject having a melanoma
comprising (i) determining whether an anti-cancer effect is likely
to be produced in the subject by selumetinib, comprising obtaining
a sample of the melanoma, and determining, in the sample, whether
cells of the melanoma contain an increased level of DDX43 mRNA
and/or DDX43 protein and/or an increased level of mRNA or protein
corresponding to one or more gene or exon listed in Table 1, and
(ii) treating the subject with a therapeutic amount of selumetinib
if the level of DDX43 mRNA and/or protein, and/or the level of mRNA
or protein corresponding to one or more gene or exon listed in
Table 1, is not increased or (iii) treating the subject with a
therapeutic amount of an anticancer agent other than a MEK
inhibitor where the level of DDX43 mRNA and/or protein, and/or the
level of mRNA or protein corresponding to one or more gene or exon
listed in Table 1, is increased. In one specific non-limiting
embodiment the anticancer agent other than a MEK inhibitor is an
AKT inhibitor.
[0109] In certain non-limiting embodiments, the present invention
provides for a method of treating a subject having a melanoma
comprising (i) determining whether an anti-cancer effect is likely
to be produced in the subject by selumetinib, comprising obtaining
a sample of the melanoma, and determining, in the sample, whether
cells of the melanoma contain an increased level of DDX43 mRNA
and/or DDX43 protein and/or a decreased level of mRNA or protein
corresponding to an exon of one or more of the following: RHBG,
MFAPS, DPYS, ACCN4, or DMKN, and (ii) treating the subject with a
therapeutic amount of selumetinib if the level of DDX43 mRNA and/or
protein is not increased, and/or the level of mRNA or protein
corresponding to an exon of one or more of the following: RHBG,
MFAPS, DPYS, ACCN4, or DMKN, is not decreased or (iii) treating the
subject with a therapeutic amount of an anticancer agent other than
a MEK inhibitor where the level of DDX43 mRNA and/or protein is
increased, and/or the level of mRNA or protein corresponding to an
exon of one or more of the following: RHBG, MFAPS, DPYS, ACCN4, or
DMKN, is decreased. In one specific non-limiting embodiment the
anticancer agent other than a MEK inhibitor is an AKT
inhibitor.
[0110] A therapeutically effective amount is an amount that is able
to achieve one or more of an anticancer effect, prolongation of
survival, and/or prolongation of period until relapse.
[0111] In certain non-limiting embodiments, a therapeutically
effective amount of selumetinib is between 50 mg and 200 mg/day,
for example, 50 mg taken orally twice a day or 75 mg taken orally
twice a day.
[0112] In non-limiting embodiments, the present invention provides
for a kit for determining whether an anti-cancer effect is unlikely
to be produced in a cancer by a MEK inhibitor, comprising a means
for determining the level of DDX43 mRNA and/or protein in a cell or
cells of the cancer.
[0113] In other non-limiting embodiments, the present invention
provides for a kit for determining whether an anti-cancer effect is
unlikely to be produced in a cancer by a MEK inhibitor, comprising
a means for determining the level of mRNA and/or protein
corresponding to one or more gene or exon listed in Table 1 or an
exon of one or more of the following: RHBG, MFAPS, DPYS, ACCN4, or
DMKN, including but not limited to RIMS2, ITLN2, PCHGA11, DDIT4L,
CAPN3, RHBG, MFAPS, DPYS and/or GTF2I (these, and DDX43, referred
to as "biomarkers").
[0114] Types of kits include, but are not limited to, packaged
probe and primer sets (e.g. TaqMan probe/primer sets),
arrays/microarrays, biomarker-specific antibodies and beads, which
further contain one or more probes, primers, or other detection
reagents for detecting one or more biomarkers of the present
invention.
[0115] In a specific, non-limiting embodiment, a kit may comprise a
pair of oligonucleotide primers, suitable for polymerase chain
reaction (PCR) or nucleic acid sequencing, for measuring levels of
mRNA. A pair of primers may comprise nucleotide sequences
complementary to a biomarker set forth above, and be of sufficient
length to selectively hybridize with said biomarker. Alternatively,
the complementary nucleotides may selectively hybridize to a
specific region in close enough proximity 5' and/or 3' to the
biomarker position to perform PCR and/or sequencing. Multiple
biomarker-specific primers may be included in the kit. The kit may
also comprise one or more polymerases, reverse transcriptase, and
nucleotide bases, wherein the nucleotide bases can be further
detectably labeled.
[0116] In non-limiting embodiments, a primer may be at least about
10 nucleotides or at least about 15 nucleotides or at least about
20 nucleotides in length and/or up to about 200 nucleotides or up
to about 150 nucleotides or up to about 100 nucleotides or up to
about 75 nucleotides or up to about 50 nucleotides in length.
[0117] In a further non-limiting embodiment, the oligonucleotide
primers may be immobilized on a solid surface or support, for
example, on a nucleic acid microarray, wherein the position of each
oligonucleotide primer bound to the solid surface or support is
known and identifiable.
[0118] In a specific, non-limiting embodiment, a kit may comprise
at least one nucleic acid probe, suitable for in situ hybridization
or fluorescent in situ hybridization, for measuring mRNA. Such kits
will generally comprise one or more oligonucleotide probes that
have specificity for various biomarkers.
[0119] In other non-limiting embodiments, a kit may comprise at
least one antibody for immunodetection of the biomarker(s).
Antibodies, both polyclonal and monoclonal, specific for a
biomarker, may be prepared using conventional immunization
techniques, as will be generally known to those of skill in the
art. The immunodetection reagents of the kit may include detectable
labels that are associated with, or linked to, the given antibody
or antigen itself. Such detectable labels include, for example,
chemiluminescent or fluorescent molecules (rhodamine, fluorescein,
green fluorescent protein, luciferase, Cy3, Cy5, or ROX),
radiolabels (3H, 35S, 32P, 14C, 131I) or enzymes (alkaline
phosphatase, horseradish peroxidase).
[0120] In a further non-limiting embodiment, the biomarker-specific
antibody may be provided bound to a solid support, such as a column
matrix, an array, or well of a microtiter plate. Alternatively, the
support may be provided as a separate element of the kit.
[0121] In certain non-limiting embodiments, where the measurement
means in the kit employs an array, the set of biomarkers set forth
above may constitute at least 10 percent or at least 20 percent or
at least 30 percent or at least 40 percent or at least 50 percent
or at least 60 percent or at least 70 percent or at least 80
percent of the species of markers represented on the
microarray.
[0122] In certain non-limiting embodiments, a kit may comprise one
or more detection reagents and other components (e.g. a buffer,
enzymes such as DNA polymerases or ligases, chain extension
nucleotides such as deoxynucleotide triphosphates, and in the case
of Sanger-type DNA sequencing reactions, chain terminating
nucleotides, positive control sequences, negative control
sequences, and the like) necessary to carry out an assay or
reaction to detect a biomarker.
[0123] In certain non-limiting embodiments, a kit may comprise a
normal control sample or may disclose, in a written material, a
normal control value.
[0124] A kit may further contain means for comparing the biomarker
with a standard, and can include instructions for using the kit to
detect the biomarker of interest. Specifically, the instructions
indicate that an increased level, in a cancer cell or cells, of
mRNA or protein corresponding to DDX43, or another biomarker listed
in Table 1, or a decreased level of mRNA or protein corresponding
to an exon of one or more of the following: RHBG, MFAPS, DPYS,
ACCN4, or DMKN, indicates that it is unlikely that a MEK inhibitor
such as selumetinib would have an anti-cancer effect on the cancer.
In certain non-limiting embodiments the invention provides for a
kit for determining whether an anti-cancer effect is likely to be
produced in a cancer by a MEK inhibitor, comprising a means for
determining the level of DDX43 mRNA and/or protein in a cell or
cells of the cancer together with a disclosure that an increased
level of DDX43 expression in a cancer is associated with a lower
likelihood that a MEK inhibitor will have an anticancer effect
against the cancer. In certain non-limiting embodiments the
invention provides for a kit for determining whether an anti-cancer
effect is likely to be produced in a cancer by a MEK inhibitor,
comprising a means for determining the level of mRNA or protein
corresponding to one or more gene or exon listed in Table 1
together with a disclosure that an increased level of said gene(s)
and/or exon(s) in a cancer is associated with a lower likelihood
that a MEK inhibitor will have an anticancer effect against the
cancer. In certain non-limiting embodiments the invention provides
for a kit for determining whether an anti-cancer effect is likely
to be produced in a cancer by a MEK inhibitor, comprising a means
for determining the level of mRNA or protein corresponding to an
exon of one or more of the following: RHBG, MFAPS, DPYS, ACCN4, or
DMKN, together with a disclosure that an decreased level of exon(s)
in a cancer is associated with a lower likelihood that a MEK
inhibitor will have an anticancer effect against the cancer.
6. EXAMPLE: DDX43 MEDIATES RESISTANCE TO MEK1/2 BY REGULATING RAS
EXPRESSION IN GNAQ MUTANT UVEAL MELANOMA
6.1 Materials and Methods
[0125] Whole-Transcriptome Sequencing.
[0126] Matched tumor biopsies were collected from patients with
metastatic uveal melanoma carrying GNA11 or GNAQ mutations
(clinicaltrials.gov # NCT01143402), before and after 14 days of
selumetinib treatment. Total RNA was extracted from flash frozen
specimens using Trizol (Invitrogen) followed by DNase digestion and
Qiagen RNeasy (Qiagen, Valencia, Calif.) column purification
following the manufacture's protocol. The RNA integrity was
verified using an Agilent Bioanalyzer 2100 (Agilent). RNA was
processed using an Illumina RNA-seq sample prep kit following the
manufacturer's instructions (Illumina, San Diego, Calif.).
[0127] RNA-Seq on Applied Biosystem SOLiD.
[0128] Taqman gene expression assay primers and probes for DDX43
and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were purchased
from Applied Biosystems, and real-time PCR assays were performed
using Applied Biosystem 7500.
[0129] Data Analysis and Bio-Informatics.
[0130] RNA-seq reads were aligned using the Tophat2 tool [39]. The
reads were further assigned to the exonic regions of each gene by
running htseq.py [25]. Counts data was normalized using default
DESeq normalization and clustered using hierarchical clustering
with Euclidian distance measure on log 2 counts. Differential
analysis of genes was performed using DESeq R package [25]. Genes
that have at least 2 fold change and False Discovery Rate (FDR) of
0.05 were flagged as significant.
[0131] Quantitative Real-Time PCR.
[0132] RNA was extracted with Trizol Reagent (Invitrogen). The
quality of RNA was checked with the RNA 6000 NanoAssay and a
Bioanalyzer 2100 (Agilent). 1 .mu.g of total RNA was
reverse-transcribed using the Thermoscript RT-PCR system (Life
Technologies). The resultant cDNA was used in a RT-PCR reactions
using an iCycler (Bio-Rad) and pre-designed TaqMan Gene expression
Assays for DDX43 and glyceraldehyde-3-phosphate dehydrogenase
(GAPDH) genes (Life Technologies). Triplicates CT values were
averaged, amounts of target were interpolated from the standard
curves and normalized to GAPDH.
[0133] Cell Culture.
[0134] Omm1.3 and Me1270 (Dr Bruce Ksander). UM cell lines have
been sequenced for the presence of activating mutations in codons
209 (exon 5) and 183 (exon 4) of GNAQ and GNA11. Cells were
cultured in RPMI medium supplemented with 10% fetal bovine serum,
100 units/ml penicillin and 100 .mu.g/ml streptomycin, and
maintained at 37.degree. C. in 5% CO2. Cells were treated with
selumetinib (AZD6244, ARRY-142866, AstraZeneca) and MK2206
(Merck).
[0135] Cell Viability Assays.
[0136] Cells were plated in 96-well plates, and treated with the
indicated concentrations of drugs. Viability was assessed after
five days of treatment using the Cell Counting Kit 8 (CCK8) from
Dojindo Molecular Technologies (Gaithersburg, Md.) according to the
manufacturer's instructions. Cell viability is expressed as a
percentage of untreated cells.
[0137] Immunoblotting.
[0138] Biopsy tissues or cells were lysed in RIPA buffer
supplemented with protease inhibitor cocktail tablets (Roche
Diagnostics) and 1 mM NaVO.sub.3. Total protein concentration of
the lysates was measured by Bio-Rad protein assay (Bio-Rad), and
equal amounts of protein were loaded on 4-12% PAGE gels
(Invitrogen). PVDF membranes were blocked with 5% nonfat dried milk
in PBS buffer containing 0.1% Tween-20 (PBST) for 1 hour and probed
with antibody for pERK, ERK, pAKT, pan-AKT, c-Jun, cyclin D1,
tubulin (Cell Signaling), DDX43 (Abcam), KRAS, HRAS (Abnova) and
NRAS (Santa Cruz Biotechnology).
[0139] RNAi-Mediated Gene Knockdown.
[0140] Small interfering RNA against GNAQ (sc-35429), DDX43, KRAS
(sc-35731), NRAS (sc-36004), HRAS (sc-29340) and control siRNA
(sc-37007) were purchased from Santa Cruz Biotechnology. DDX43-1
and -2 siRNA were from OriGene. They were transfected using
Lipofectamine RNAiMAX reagent (Invitrogen) following the
manufacturer's instructions. The human DDX43 cDNA clone in
pCMV6-XL5 (or PCMV6-Neo) was from OriGene.
6.2 Results
[0141] Differential Expression of Genes in Liver Biopsies of
Patients with Uveal melanoma.
[0142] It has been reported that selumetinib inhibits pERK and
induces cell cycle arrest in UM cells with GNAQ mutations [7].
Selumetinib is currently in Phase II clinical trial for patients
with metastatic uveal melanoma [8]. In order to identify potential
markers of drug resistance, whole-transcriptome sequencing
technology (RNA seq) was performed on liver metastases of patients
with uveal melanoma carrying GNAQ or GNA11 mutations.
[0143] Transcription expression profiles of biopsies from patients
who had partial response or stable disease were compared with the
profiles of patients with progressive disease. The analysis of
differentially expressed genes was performed using DESeq R package
[25]. Genes that had at least log 2 fold change>2 and False
Discovery Rate (FDR) of 0.05 were flagged as significant. Several
genes, including RIMS2, DDX43, ITLN2, PCHGA11, and DDIT4L, were
significantly up-regulated in both pre- and post-treatment
specimens obtained from "non-responders" when compared to samples
obtained from those from "responders" (Table 1). Similarly, genes
with differentially expressed exons (novel transcriptional hybrids)
were identified as being up-regulated in biopsies of the
"non-responders" as opposed to the "responders", like CAPN3, RHBG,
MFAPS, DPYS, GTF2I. The top 20 genes differentially expressed and
ranked according to level of significance (highest to lowest) are
shown in Table 1.
[0144] In order to investigate possible genes associated with MEK
inhibitor resistance, particular focus was placed on the DEAD-box
helicase antigen DDX43. The expression of DDX43 was 60-fold higher
in the "non-responders" compared to "responders". DDX43 has been
reported to be highly expressed in many tumor types including
melanoma when compared to normal tissues [16, 17, 19]. In order to
validate the RNAseq results for DDX43, we performed real-time PCR
and expanded our analysis to include 14 biopsy samples (6
"responders" and 8 "non-responders") for DDX43 expression at
pre-treatment condition (FIG. 1A). Patients who responded to
therapy ("responders") had low expression of DDX43. In contrast,
DDX43 was highly expressed in the "non responders", and there was a
statistically significant association with poor outcome in these
patients (p=0.045). Similarly, protein levels of DDX43 were low in
"responders" at baseline, and slightly induced post-selumetinib
treatment (FIG. 1B), while it was highly expressed in "non
responders" regardless of treatment.
[0145] DDX43 is Highly Expressed in UM Cell Lines with Acquired
Resistance to Selumetinib.
[0146] To explore mechanisms of drug resistance, MEKi-resistant
GNAQ-mutant cell lines were generated by exposing the cells to
increasing concentrations of selumetinib for at least four weeks
and routinely grown in 1 .mu.M selumetinib, without clonal
selection. After continuous exposure, the cell lines Res-Omm1.3 and
Res-Me1270 became resistant to selumetinib compared to their
parental cells Omm1.3 and Me1270 (FIG. 2A, B). The resistant cells
showed IC50 that were 10 fold higher than their sensitive
counterparts. Furthermore, these cells showed cross resistance to
the MEK inhibitor PD0325901 and GSK1120212 (FIG. 7).
[0147] The resistant cells showed no cell cycle arrest with
increasing doses of selumetinib, compared to the dose-dependent
arrest in G1 population in the parental cells (FIG. 8). The
expression level and phosphorylation status of different components
of the MAPK pathway were then examined by immunoblotting. In
parental Omm1.3 and Me1270 cells, treatment with selumetinib caused
a decrease in pERK, cyclin D1 and phospho-retinoblastoma protein
(pRB) (FIGS. 2C and 2D). Res-Omm1.3 and Res-Me1270 cells were
maintained in drug-free media for 24 h, and when treated again
their pERK decreased, while cyclin D1 and pRB remained unchanged.
Of note, selumetinib induced pMEK in both parental and resistant
cells, suggesting that MEK feedback mechanisms are still intact in
the resistant cells, while downstream signaling is de-regulated,
with low but sustained expression of cyclin D1 and constitutive
activation of pRB. Higher levels of pAKT were observed at baseline
and with the treatments, when compared to the parental cells (FIGS.
2B and 2D). It has been reported that mutant GNAQ signals to both
ERK and AKT [26], and the increase in pAKT suggests that the
GNAQ-mutant resistant cells adapt to MEK inhibition by increasing
the flux through the PI3K/AKT pathways to maintain cell
proliferation. We have also reported that c-Jun is uniquely
upregulated by selumetinib in GNAQ mutant cells, as opposed to
BRAF-mutant or wild-type cells, and it mediated intrinsic
resistance to selumetinib [7]. In addition, Little et al. reported
an increase in c-Jun in colorectal cancer cells with acquired
resistance to selumetinib [14]. Indeed, c-Jun was induced by
selumetinib in the parental cells and highly expressed in
Res-Omm1.3 and Res-Me1270 resistant cells.
[0148] Finally, the cell lines were analyzed for DDX43 expression.
These cells showed high levels of DDX43 protein compared to their
parental cells (FIG. 3A), and the expression did not change with
treatments, or over time (FIG. 3B). The increased expression of
DDX43 was also confirmed at the mRNA levels in the resistant cell
lines (FIG. 9). Since DDX43 was reported to regulate NRAS
translation [20], RAS levels were analyzed in all the cell lines.
Pan-RAS expression was slightly induced by selumetinib in the
parental cells at higher doses (FIG. 3A) and over time (FIG. 3B),
while it was constitutively expressed in both resistant cell lines
(FIG. 3A, B). This has important implications as RAS regulates
multiple pathways [27, 28] and could explain the increased flux
into both the ERK and AKT in the resistant cells.
[0149] Next, experiments were performed to determine whether RAS
protein levels correlated with RAS activity, by pull down assays.
In the parental Omm1.3 and Me1270 cells, basal RAS expression and
activity were relatively low and were slightly induced by the
selumetinib treatment (FIG. 3C). However, RAS activation was
temporary, and it returned to normal levels after 24 hours of drug
removal. This activation of RAS is possibly mediated by the
downregulation of the Sprouty proteins by MEK inhibition as
reported [29]. It has also been shown that GNAQ mutant cells have
an elevated Spry2 expression that is downregulated by selumetinib
treatment ([30] and FIG. 10). In contrast, RAS expression and
activity was elevated in the resistant cells, and it remained
active even when selumetinib was removed for up to 48 and 96 hours
in Res-Me1270 and Res-Omm1.3 cells, respectively (FIG. 3C). In
these cells Spry2 was also downregulated by the treatment or not
expressed (FIG. 10), which could contribute to the elevated
activity of RAS, but not to its expression. Other possible
mechanisms of acquired resistance to MEK inhibition were explored
by performing genomic sequencing and/or by FISH analysis of MEK,
KRAS, NRAS, HRAS, but neither mutations nor gene amplification of
the oncogene GNAQ or RAS proteins were observed. Assays failed to
detect activation of receptor tyrosine kinases (RTK) resulted also
negative.
[0150] Acquired Resistance to Selumetinib is Mediated by
DDX43-Mediated RAS Expression in GNAQ Mutant Cells.
[0151] In order to determine whether DDX43 regulates RAS
expression, DDX43 was silenced in the resistant cells using two
siRNA sequences (siDDX43-1, FIG. 4; and siDDX43-2, FIG. 11).
Downregulation of DDX43 in both resistant cell lines corresponded
to decreased expression of KRAS, NRAS, HRAS and the downstream
effectors pERK, pAKT and c-Jun (FIG. 4A). DDX43 downregulation also
induced PARP cleavage (FIG. 4A), which corresponded to decreased
cell viability of both the resistant cell lines (FIG. 4B),
suggesting that DDX43 is required for sustained RAS expression and
cell survival. DDX43 depletion did not affect RAS mRNA expression,
as detected by real-time PCR analysis (FIG. 12) as previously
reported [20], nor RAS protein stability.
[0152] To further characterize these mechanisms of drug resistance,
DDX43 was over-expressed in the parental cells. In these cells,
DDX43 induced expression of RAS and pAKT, but not pERK (FIG. 4C).
In addition, the parental cells overexpressing DDX43 became more
resistant to selumetinib treatments, compared to mock cells (FIG.
4D), confirming that DDX43 renders cells resistant to the MEK
inhibitor through RAS and AKT activation.
[0153] In order to confirm that the effects of DDX43 silencing are
mediated by RAS downregulation, each RAS protein was directly
downregulated in both sensitive and resistant cells by
gene-specific siRNA. Interestingly, KRAS and HRAS knockdown caused
downregulation of pERK, pAKT in all the cell lines (FIG. 5A, B),
while NRAS did not (FIG. 5C). c-Jun was downregulated in cells
depleted of KRAS and NRAS, while HRAS regulated expression of c-Jun
in one of the resistant cell lines (Res-Me1270).
[0154] Since RAS proteins regulate signaling in the MEK-resistant
cells, the contribution of mutant GNAQ on downstream signaling
pathways was evaluated in these cells. GNAQ silencing inhibited
pERK and pAKT in the parental cells as previously reported [26],
while none or minimal inhibition was seen in the resistant cells
(FIG. 5D). Expression of c-Jun was not affected by GNAQ siRNA in
any of the cell lines.
[0155] Moreover, KRAS and HRAS downregulation decreased cell
viability of the resistant cell lines, while NRAS had partial
effects, and GNAQ had minimal effects (FIG. 6A, B).
[0156] These results indicate that KRAS and HRAS signal to MEK and
AKT and mediate c-Jun expression in the resistant cells. This is
also supported by the lack of inhibition of pMEK by GNAQ siRNA in
the resistant cells but not in the sensitive cells (FIG. 5D), which
suggests that mutant GNAQ loses its capacity to activate MEK in the
resistant cells, while RAS proteins become the dominant mediators
of cell signaling. This is further confirmed by the general
resistance of these cells to inhibition of cell viability by GNAQ
depletion (FIG. 6A), as opposed to the parental cells, which are
sensitive to GNAQ silencing (26, 6).
[0157] AKT inhibition suppresses cell viability of
selumetinib-resistant GNAQ mutant cells. The increased RAS
expression and activity mediates activation of downstream survival
pathways, especially pAKT (FIG. 2C, D and FIG. 4C.). Accordingly
the effects of the AKT inhibitor MK2206 (AKTi) were evaluated in
these MEKi-resistant cell lines. Decreased viability was observed
with AKTi in both Res-Omm1.3 and Res-Me1270 when compared to the
parental lines (FIGS. 6B and C). This sensitivity to AKTi was
sustained when combined with MEKi (FIGS. 6B and C), suggesting that
AKT inhibition may be a means of overcoming MEKi-resistance.
6.3 Discussion
[0158] The preliminary results of the single agent phase II study
with selumetinib in patients with metastatic uveal melanoma appear
promising with inhibition of pERK, suppression of cyclin D1, and
partial radiologic responses [8].
[0159] However, acquired resistance to MEK inhibitors is common and
undermines the efficacy of these treatments [12, 31]. Using a
whole-transcriptome sequencing technology (RNA-seq) of paired tumor
samples, and validation in cell lines with acquired MEK
inhibitor-resistance, we identified DDX43 as a mediator of MEK
resistance.
[0160] MEK resistant cells became sensitive to AKT inhibition, by
overcoming MEK resistance and providing an alternative treatment
for patient who fail a MEK inhibitor regiment. MEK resistance has
been described in the setting of BRAF and RAS mutations, by
amplification of the driving oncogene [14] or by dimerization of
aberrantly spliced mutant BRAF [32]. Recently, Lito et al, have
described that BRAF inhibitors cause relief of negative feedback
with the activation of RAS and rebound of ERK activity [29].
[0161] A similar activation of RAS was induced by MEK inhibition in
GNAQ mutant cells. This effect was possibly due to feedback
activation, and it was reversible in cells exposed to the drug for
a short period of time. On the contrary, cells continuously exposed
to the MEK inhibitor exhibited sustained expression of DDX43 and
RAS. DDX43 silencing decreased RAS expression and its downstream
effectors pERK and pAKT, thus making DDX43 a novel mediator of MEK
resistance that could represent a class effect to all MEK
inhibitors.
[0162] DDX43 is a member of the D-E-A-D (Asp-Glu-Ala-Asp) box
family of RNA helicases, which comprises more than 60 members [33].
Another member of this family, DDX5, regulates alternative splicing
of HRAS [34], while DDX11 in involved in sister chromatin cohesion,
and is essential for the survival of advanced melanoma [35].
Altered expression of DDX43 has been reported in other human tumors
[36] [24]. For example, DDX43 has been identified as a tumor
specific gene expressed in human sarcoma [16], and its expression
has been reported as a biomarker of poor clinical outcome of breast
cancer [37]. In chronic myeloid leukemia, DDX43 over-expression was
associated with gene demethylation, and it correlated with advanced
disease and poor outcome [18]. Finally, DDX43 was required for
tumor cell proliferation of malignant melanoma-initiating cells
through RAS protein expression [20]. In the experiments described
herein, DDX43 has been associated with MEK resistance in uveal
melanoma through the activation of RAS and downstream pathways. In
particular, overexpression of DDX43 led to the activation of pAKT.
It has been reported, in BRAF-mutant cutaneous melanoma, that basal
and treatment-induced activation of AKT mediates resistance to
selumetinib [38]. In the experiments described herein, the
treatment with an AKT inhibitor alone or in combination with a MEK
inhibitor seemed to overcome resistance in our cells.
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are hereby incorporated by reference in their entireties.
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