U.S. patent application number 13/701889 was filed with the patent office on 2013-06-06 for mek1 mutation conferring resistance to raf and mek inhibitors.
This patent application is currently assigned to Dana-Farber Cancer Institute, Inc.. The applicant listed for this patent is Caroline Emery, Levi A. Garraway, Nikhil Wagle. Invention is credited to Caroline Emery, Levi A. Garraway, Nikhil Wagle.
Application Number | 20130143911 13/701889 |
Document ID | / |
Family ID | 44357961 |
Filed Date | 2013-06-06 |
United States Patent
Application |
20130143911 |
Kind Code |
A1 |
Garraway; Levi A. ; et
al. |
June 6, 2013 |
MEK1 Mutation Conferring Resistance to RAF and MEK Inhibitors
Abstract
Nucleic acids and proteins having a mutant MEK sequence, and
methods concerning identification of patients having resistance to
treatment with anti-cancer agents, specifically inhibitors of RAF
or MEK are provided. Methods of treatment and for optimizing
treatment for patients having a mutation in a MEK1 sequence are
also provided.
Inventors: |
Garraway; Levi A.; (Newton,
MA) ; Emery; Caroline; (Brighton, MA) ; Wagle;
Nikhil; (Brookline, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Garraway; Levi A.
Emery; Caroline
Wagle; Nikhil |
Newton
Brighton
Brookline |
MA
MA
MA |
US
US
US |
|
|
Assignee: |
Dana-Farber Cancer Institute,
Inc.
Boston
MA
|
Family ID: |
44357961 |
Appl. No.: |
13/701889 |
Filed: |
June 9, 2011 |
PCT Filed: |
June 9, 2011 |
PCT NO: |
PCT/US2011/039789 |
371 Date: |
February 13, 2013 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
61352959 |
Jun 9, 2010 |
|
|
|
Current U.S.
Class: |
514/300 ;
435/194; 435/252.3; 435/252.33; 435/320.1; 435/366; 435/6.12;
514/394; 536/23.2 |
Current CPC
Class: |
A61P 35/00 20180101;
C12Q 2600/106 20130101; C12N 9/1205 20130101; G01N 33/5743
20130101; C12Q 1/6886 20130101; A61P 35/04 20180101; C12Y 207/12002
20130101; C12Q 2600/156 20130101; C12Q 2600/136 20130101; A61P
35/02 20180101; G01N 2500/10 20130101; G01N 2500/20 20130101; G01N
2333/91205 20130101 |
Class at
Publication: |
514/300 ;
536/23.2; 435/320.1; 435/252.3; 435/194; 435/6.12; 435/252.33;
435/366; 514/394 |
International
Class: |
A61K 31/4745 20060101
A61K031/4745; C07H 21/04 20060101 C07H021/04; C12N 9/12 20060101
C12N009/12; C12Q 1/68 20060101 C12Q001/68 |
Goverment Interests
FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
[0002] This invention was made with government support under
federal grant number K08 CA115927 awarded by National Institutes of
Health. The government has certain rights in the invention.
Claims
1. An isolated nucleic acid molecule encoding a mutant MEK1 protein
having MEK1 activity, wherein said mutant MEK1 protein comprises an
amino acid substitution at position 121 of wild-type MEK1 shown in
SEQ ID NO: 2, the amino acid substitution conferring resistance to
one or more RAF or MEK inhibitors on a cell expressing the mutant
MEK1 protein.
2. The nucleic acid molecule of claim 1, wherein the RAF inhibitor
is selected from the group consisting of PLX4720, PLX4032, BAY
43-9006 (Sorafenib), ZM 336372, RAF 265, AAL-881, LBT-613, and
CJS352.
3. The nucleic acid molecule of claim 1, wherein the MEK inhibitor
is selected from the group consisting of CI-1040, AZD6244,
PD318088, PD98059, PD334581, RDEA119, Compound A, and Compound
B.
4. The nucleic acid molecule of claim 1, wherein the amino acid
substitution is a 121C>S amino acid substitution.
5. The nucleic acid molecule of claim 1, wherein the mutant MEK1
protein comprises the amino acid sequence of SEQ ID NO: 4.
6. The nucleic acid molecule of claim 1, which comprises the
nucleotide sequence of SEQ ID NO: 3.
7. An expression vector comprising the nucleic acid of claim 1.
8. A host cell comprising the expression vector of claim 7.
9. A method of producing a mutant MEK1 protein comprising culturing
the host cell of claim 8 such that a mutant MEK1 protein is
produced by the cell.
10. An isolated mutant MEK1 protein having MEK1 activity, wherein
said mutant MEK1 protein comprises an amino acid substitution at
position 121 of wild-type MEK1 protein shown in SEQ ID NO: 2, the
amino acid substitution conferring resistance to one or more RAF or
MEK inhibitors on a cell expressing the mutant MEK1 protein.
11. The mutant MEK1 protein of claim 10, wherein the RAF inhibitor
is selected from the group consisting of PLX4720, PLX4032, BAY
43-9006 (Sorafenib), ZM 336372, RAF 265, AAL-881, LBT-613, and
CJS352.
12. The mutant MEK1 protein of claim 10, wherein the MEK inhibitor
is selected from the group consisting of CI-1040, AZD6244,
PD318088, PD98059, PD334581, RDEA119, Compound A, and Compound
B.
13. The mutant MEK1 protein of claim 10, wherein the amino acid
substitution is a 121C>S amino acid substitution
14. The mutant MEK1 protein of claim 10, which comprises the amino
acid sequence of SEQ ID NO: 4.
15. A method of identifying a subject having cancer as having a
high risk of relapse during treatment or being unresponsive to
treatment--with a RAF inhibitor, comprising: (a) extracting nucleic
acid from cells of the cancer; and (b) sequencing a nucleic acid
molecule encoding a MEK1 protein; wherein the presence of
nucleotides that produce a 121C>S amino acid substitution in the
MEK1 protein, as compared to wild-type MEK1 protein, identifies the
subject as having a high risk of relapse during treatment with a
RAF inhibitor or identifies the subject as being unresponsive to
treatment with a RAF inhibitor.
16. (canceled)
17. The method according to claim 15, wherein the presence of
nucleotides that produce a 121C>S amino acid substitution in the
MEK1 protein, as compared to wild-type MEK1 protein, indicates a
need to treat the subject with a RAF inhibitor and a MEK inhibitor
wherein the MEK inhibitor targets a mutant MEK1 protein having a
121C>S amino acid substitution.
18. A method of treating a subject having cancer, comprising: (a)
extracting nucleic acid from cells of the cancer; (b) sequencing a
nucleic acid molecule encoding a MEK1 protein or subjecting the
sample to PCR and identifying the nucleotide sequence of a nucleic
acid molecule encoding a MEK1 protein; and (c) administering a RAF
inhibitor and administering a MEK inhibitor that targets a mutant
MEK1 protein having a 121C>S amino acid substitution. to the
subject when the nucleic acid molecule contains nucleotides that
produce a 121C>S amino acid substitution in the MEK1 protein, as
compared to wild-type MEK1 protein.
19. (canceled)
20. A method of identifying a subject having cancer who is likely
to benefit from treatment with a RAF inhibitor and a MEK inhibitor
that targets a mutant MEK1 protein having a 121C>S amino acid
substitution, comprising: (a) assaying a nucleic acid sample
obtained from the cancer for the presence one or more mutations in
a nucleic acid molecule encoding a MEK1 protein that produce a
121C>S amino acid substitution in the MEK1 protein, as compared
to wild-type MEK1 protein; and (b) correlating the presence of the
one or more mutations in a nucleic acid molecule encoding a MEK1
protein that produce a 121C>S amino acid substitution in the
MEK1 protein with a subject who is likely to benefit from treatment
with a RAF inhibitor and a MEK inhibitor that targets a mutant MEK1
protein having a 121C>S amino acid substitution; and treating
the subject with a MEK inhibitor that targets a mutant MEK1 protein
having a 121C>S amino acid substitution.
21. (canceled)
22. The method of claim 18, wherein the RAF inhibitor is selected
from the group consisting of PLX4720, PLX4032, BAY 43-9006
(Sorafenib), ZM 336372, RAF 265, AAL-881, LBT-613, and CJS352.
23. The method of claim 22, wherein the RAF inhibitor is PLX4720 or
PLX4032.
24. The method of claim 18, wherein the cancer is melanoma.
25. The method of claim 18, wherein the cancer is selected from the
group consisting of leukemias, lymphomas, myelomas, carcinomas,
metastatic carcinomas, sarcomas, adenomas, nervous system cancers
and geritourinary cancers.
Description
RELATED APPLICATIONS
[0001] This application claims the benefit of the filing date under
35 U.S.C. .sctn.119(e) of Provisional U.S. Patent Application Ser.
No. 61/352,959, filed Jun. 9, 2010, which is incorporated by
reference in its entirety.
BACKGROUND OF THE INVENTION
[0003] The treatment of cancer is one of the greatest challenges in
modern medicine. While chemotherapeutic agents are typically an
effective means of treating or reducing the symptoms associated
with cancer, in some cases, resistance to one or more
chemotherapeutic agents manifests during treatment. As a result, a
given chemotherapeutic agent can become ineffective in certain
individuals. The molecular mechanisms responsible for the
development of resistance in various types of cancer are poorly
understood. Elucidation of the mechanisms that underlie resistance
to specific agents is essential to discovering treatment approaches
that effectively circumvent drug resistance.
[0004] In particular, one type of cancer for which additional
treatment approaches are needed is malignant melanoma. Malignant
melanoma is the sixth most common cancer diagnosis in the US, with
68,729 estimated new cases in 2009. Metastatic melanoma is
associated with a very poor prognosis, with a median survival of 6
to 15 months. In melanoma, uncontrolled activity of the MAP kinase
pathway is nearly ubiquitous and occurs most commonly through
gain-of-function mutations involving codon 600 of the BRAF oncogene
(BRAF V600E). More than 50% of metastatic melanoma harbors BRAF
V600E mutations. Moreover, BRAF V600E mutations have been found in
10% of colorectal cancers and in 8% of all solid tumors.
[0005] Recently, efforts to specifically target mutated BRAF in
melanoma have yielded promising results. PLX4032 is an oral
targeted drug that specifically inhibits BRAF V600E. In the Phase 1
trial of melanoma patients with BRAF V600E mutations, 70% of
patients (19 of 27) had at least 30% tumor response by RECIST
criteria. Phase II and Phase III trials of PLX4032 are currently
underway. However, as with all other targeted therapies, resistance
to PLX4032 has begun to emerge, with patients relapsing after an
average of 9 months.
SUMMARY OF THE INVENTION
[0006] The present invention pertains to mutation-mediated
resistance to chemotherapeutic treatment of cancer. In specific
embodiments, the present invention is directed to a mutation
identified in a MEK1 protein, and in nucleic acid molecules
encoding the MEK1 protein, wherein the mutation comprises a
substitution at amino acid position 121 of the wild-type MEK1
protein set forth in SEQ ID NO: 2. The mutation confers resistance
to RAF and MEK inhibitors currently in therapeutic use. The
identification of the mutation allows for the identification of
cancer patients that may be susceptible to resistance to
chemotherapeutic agents. Moreover, the identification of the
mutation allows for the development of second-generation MEK
inhibitors that exhibit activity against a MEK1 protein containing
the resistance mutation, such as the mutation described herein.
Such second-generation MEK inhibitors are useful in many clinical
and therapeutic applications, including the treatment of
cancer.
[0007] Accordingly, the invention features, in a first aspect, an
isolated nucleic acid molecule encoding a mutant MEK1 protein
having MEK1 activity, wherein said mutant MEK1 protein comprises an
amino acid substitution at position 121 of wild-type MEK1 shown in
SEQ ID NO: 2, the amino acid substitution conferring resistance to
one or more RAF or MEK inhibitors on a cell expressing the mutant
MEK1 protein. In various embodiments, the RAF inhibitor is selected
from the group consisting of PLX4720, PLX4032, BAY 43-9006
(Sorafenib), ZM 336372, RAF 265, AAL-881, LBT-613, and CJS352.
Preferred RAF inhibitors are the BRAF inhibitors PLX4720 and
PLX4032. In various embodiments, the MEK inhibitor is selected from
the group consisting of CI-1040, AZD6244, PD318088, PD98059,
PD334581, RDEA119, Compound A, and Compound B. In some embodiments,
the MEK inhibitor is the MEK1 inhibitor AZD6244. In some
embodiments, the amino acid substitution is a 121C>S amino acid
substitution. In some embodiments, the mutant MEK1 protein
comprises the amino acid sequence of SEQ ID NO: 4. In some
embodiments, the nucleic acid comprises nucleotide sequence of SEQ
ID NO: 3.
[0008] The isolated nucleic acid molecules encoding mutant MEK1
proteins can be inserted into an expression vector and expressed in
a host cell. Accordingly, in another aspect, the invention features
an expression vector comprising a nucleic acid molecule as set
forth herein. In another aspect, the invention features a host cell
comprising the foregoing expression vector. In another aspect, the
invention features a method of producing a mutant MEK1 protein,
comprising culturing a host cell containing an expression vector
encoding a mutant MEK1 protein, such that a mutant MEK protein is
produced by the cell.
[0009] In other aspects, the invention features an isolated mutant
MEK1 protein having MEK1 activity, wherein said mutant MEK1 protein
comprises an amino acid substitution at position 121 of wild-type
MEK1 protein shown in SEQ ID NO: 2, the amino acid substitution
conferring resistance to one or more RAF or MEK inhibitors on a
cell expressing the mutant MEK1 protein. In various embodiments,
the RAF inhibitor is selected from the group consisting of PLX4720,
PLX4032, BAY 43-9006 (Sorafenib), ZM 336372, RAF 265, AAL-881,
LBT-613, and CJS352. In some embodiments, the RAF inhibitors are
the BRAF inhibitors PLX4720 and PLX4032. In various embodiments,
the MEK inhibitor is selected from the group consisting of CI-1040,
AZD6244, PD318088, PD98059, PD334581, RDEA119, Compound A, and
Compound B. In some embodiments, the MEK inhibitor is the MEK1
inhibitor AZD6244. In some embodiments, the amino acid substitution
is a 121C>S amino acid substitution. In some embodiments, the
mutant MEK1 protein comprises the amino acid sequence of SEQ ID NO:
4.
[0010] In another aspect, the invention features a method of
identifying a compound that is useful in treating cancer,
comprising: providing an assay composition comprising a mutant MEK1
protein, wherein the mutant MEK1 protein comprises an amino acid
substitution at position 121 of wild-type MEK1 protein shown in SEQ
ID NO: 2, and a MEK1 substrate; contacting the assay composition
with a test compound under conditions that permit phosphorylation
of the MEK1 substrate in the absence of the test compound; and
determining the effect of the compound on phosphorylation of the
MEK1 substrate; wherein downmodulation of phosphorylation of the
MEK1 substrate as compared to a suitable control identifies the
compound as a compound that is useful in treating cancer. In some
embodiments, the mutant MEK1 protein comprises a 121C>S amino
acid substitution.
[0011] In a related aspect, the invention features a method of
identifying a compound as a second generation MEK1 inhibitor,
comprising: providing an assay composition comprising a mutant MEK1
protein, wherein the mutant MEK1 protein comprises an amino acid
substitution at position 121 of wild-type MEK1 protein shown in SEQ
ID NO: 2, and a MEK1 substrate; contacting the assay composition
with a test compound under conditions that permit phosphorylation
of the MEK1 substrate in the absence of the test compound; and
determining the effect of the compound on phosphorylation of the
MEK1 substrate; wherein downmodulation of phosphorylation of the
MEK1 substrate as compared to a suitable control identifies the
compound as a second generation MEK1 inhibitor. In some
embodiments, the mutant MEK1 protein comprises a 121C>S amino
acid substitution.
[0012] In exemplary embodiments of the foregoing aspects, the MEK1
substrate is ERK1/2. In other embodiments, the assay composition is
a cell extract.
[0013] In another aspect, the invention features a method of
identifying a compound that is useful in treating cancer,
comprising: providing a cell comprising a mutant MEK1 protein,
wherein the mutant MEK1 protein comprises an amino acid
substitution at position 121 of wild-type MEK1 protein shown in SEQ
ID NO: 2; contacting the cell with a test compound; and determining
the effect of the compound on ERK1/2 phosphorylation; wherein
downmodulation of ERK1/2 phosphorylation as compared to an
appropriate control identifies the compound as a compound that is
useful in treating cancer. In a related aspect, the invention
provides a method of identifying a compound that is a second
generation MEK1 inhibitor, comprising: providing a cell comprising
a mutant MEK1 protein, wherein the mutant MEK1 protein comprises an
amino acid substitution at position 121 of wild-type MEK1 protein
shown in SEQ ID NO: 2; contacting the cell with a test compound;
and determining the effect of the compound on ERK1/2
phosphorylation; wherein downmodulation of ERK1/2 phosphorylation
as compared to an appropriate control identifies the compound as a
second generation MEK1 inhibitor. For some embodiments of both of
these methods, the mutant MEK1 protein comprises a 121C>S amino
acid substitution.
[0014] In another aspect, the invention features a method of
identifying a compound that is useful in treating cancer,
comprising: providing a cell comprising a mutant MEK1 protein,
wherein the mutant MEK1 protein comprises an amino acid
substitution at position 121 of wild-type MEK1 protein shown in SEQ
ID NO: 2; contacting the cell with a test compound; and determining
the effect of the compound on cell proliferation; wherein reduction
in cell proliferation as compared to an appropriate control
identifies the compound as a compound that is useful in treating
cancer. In a related aspect, the invention provides a method of
identifying a compound that is a second generation MEK1 inhibitor,
comprising: providing a cell comprising a mutant MEK1 protein,
wherein the mutant MEK1 protein comprises an amino acid
substitution at position 121 of wild-type MEK1 protein shown in SEQ
ID NO: 2; contacting the cell with a test compound; and determining
the effect of the compound on cell proliferation; wherein reduction
in cell proliferation as compared to an appropriate control
identifies the compound as a second generation MEK1 inhibitor. For
some embodiments of both of these methods, the mutant MEK1 protein
comprises a 121C>S amino acid substitution.
[0015] In another aspect, the invention features a cell-based
screening method for identifying a test compound as a second
generation MEK1 inhibitor, the method comprising contacting a host
cell with a test compound, wherein the host cell comprises a mutant
MEK1 protein, wherein the mutant MEK1 protein comprises an amino
acid substitution at position 121 of wild-type MEK1 protein shown
in SEQ ID NO: 2, wherein sensitivity of the host cell to the test
compound identifies the compound as a second-generation MEK1
inhibitor. In one embodiment of this aspect, sensitivity of the
host cell to the test compound is measured using an assay selected
from the group consisting of a cell proliferation assay, a cell
viability assay, and a ERK1/2 phosphorylation assay, wherein a
reduction in cell proliferation, cell viability, or ERK1/2
phosphorylation in the presence of the test compound identifies the
compound as a second-generation MEK1 inhibitor. In some
embodiments, the mutant MEK1 protein comprises a 121C>S amino
acid substitution.
[0016] In another aspect, the invention features a method of
identifying a second-generation MEK1 inhibitor, comprising:
selecting a potential drug using computer-assisted modeling with a
three-dimensional crystal or solution structure of a mutant MEK1
protein, wherein said mutant MEK1 protein comprises an amino acid
substitution at position 121 of wild-type MEK1 protein shown in SEQ
ID NO: 2; contacting said potential drug with the mutant MEK1
protein; and detecting the interaction of said potential drug with
the mutant MEK1 protein; wherein a compound that is capable of
interacting with the mutant MEK1 protein is identified as a
second-generation MEK1 inhibitor. In some embodiments, the mutant
MEK1 protein comprises a 121C>S amino acid substitution.
[0017] In one embodiment of the foregoing aspects, the test
compound is a member of a library of compounds.
[0018] In another aspect, the invention features a compound
identified by one of the foregoing methods. Such compounds are
useful, for example, in inhibiting the activity of a mutant MEK1
protein comprising an amino acid substitution at position 121 of
wild-type MEK1 protein shown in SEQ ID NO: 2, in some embodiments,
a 121C>S amino acid substitution. Accordingly, in another
aspect, the invention features a method of inhibiting the activity
of a mutant MEK1 protein, wherein the mutant MEK1 protein comprises
an amino acid substitution at position 121 of wild-type MEK1
protein shown in SEQ ID NO: 2 (preferably a 121C>S amino acid
substitution), the method comprising contacting the mutant MEK1
protein with a compound identified according to one of the
foregoing methods. In an exemplary embodiment, the compound
inhibits the activity of a mutant MEK1 protein and a wild-type MEK1
protein. In one embodiment, said contacting occurs in vitro. In
another embodiment, said contacting occurs in vivo. In another
embodiment, said contacting occurs in a subject. In an exemplary
embodiment, said contacting occurs in a subject having a cancer. In
one embodiment, the subject having a cancer has relapsed from
treatment with a RAF inhibitor, such as a RAF inhibitor selected
from the group consisting of PLX4720, PLX4032, BAY 43-9006
(Sorafenib), ZM 336372, RAF 265, AAL-881, LBT-613, and CJS352
(preferably the BRAF inhibitors PLX4720 and PLX4032). Additionally
or alternatively, the subject having cancer has relapsed from
treatment with a first generation MEK inhibitor, such as a MEK
inhibitor selected from the group consisting of CI-1040, AZD6244,
PD318088, PD98059, PD334581, RDEA119, Compound A, and Compound B In
some embodiments, the MEK1 inhibitor is AZD6244. In an exemplary
embodiment, the cancer is a melanoma.
[0019] In another aspect, the invention features a method of
treating a subject having a cancer, comprising administering to the
subject a compound identified according to one of the foregoing
methods. In an exemplary embodiment, the compound inhibits the
activity of a mutant MEK1 protein, comprising an amino acid
substitution at position 121 of wild-type MEK1 protein shown in SEQ
ID NO: 2 (preferably a 121C>S amino acid substitution), and a
wild-type MEK1 protein. In one embodiment, the subject having a
cancer has relapsed from treatment with a RAF inhibitor, such as a
RAF inhibitor selected from the group consisting of PLX4720,
PLX4032, BAY 43-9006 (Sorafenib), ZM 336372, RAF 265, AAL-881,
LBT-613, and CJS352. In some embodiments the BRAF inhibitors are
selected from PLX4720 and PLX4032. Additionally or alternatively,
the subject having cancer has relapsed from treatment with a first
generation MEK inhibitor, such as a MEK inhibitor selected from the
group consisting of CI-1040, AZD6244, PD318088, PD98059, PD334581,
RDEA119, Compound A, and Compound B. In some embodiments, the MEK1
inhibitor is AZD6244. In a particular embodiment, the cancer
comprises a MEK1 protein having a 121C>S amino acid substitution
with respect to a wild type MEK1 protein, and/or a MEK1 nucleic
acid molecule encoding a MEK1 protein having a 121C>S amino acid
substitution with respect to a wild type MEK1 protein, wherein the
121C>S amino acid substitution confers resistance to one or more
RAF or MEK inhibitors on cells expressing the mutant MEK1 protein.
In an exemplary embodiment, the cancer is a melanoma.
[0020] In another aspect, the invention features a method of
screening a subject having cancer, the method comprising obtaining
a cancer cell-containing sample from the subject; and determining
the presence or absence of an amino acid substitution at position
121 of wild type MEK1 protein, shown in SEQ ID NO: 2, in the cancer
cell-containing sample. In some embodiments, the presence or
absence of a 121C>S amino acid substitution is determined.
[0021] In one embodiment of the foregoing aspect, detection of the
mutation at position 121 in the MEK1 protein identifies the subject
as having a relatively high risk of relapse during treatment with a
RAF inhibitor. In another embodiment, detection of the mutation at
position 121 in the MEK protein identifies the subject as being
unresponsive to treatment with a RAF inhibitor. In exemplary
embodiments, the RAF inhibitor is selected from the group
consisting of PLX4720, PLX4032, BAY 43-9006 (Sorafenib), ZM 336372,
RAF 265, AAL-881, LBT-613, and CJS352. In some embodiments the BRAF
inhibitor is selected from PLX4720 and PLX4032). In another
embodiment of the foregoing aspect, the presence of the mutation at
position 121 (preferably C121S) in the MEK1 protein stratifies the
subject to treatment with a second generation MEK1 inhibitor that
targets the mutant MEK1 protein. In another embodiment, the
presence of the mutation at position 121 in the MEK1 protein
stratifies the subject to treatment with a combination therapy that
includes administration of both a RAF inhibitor and a second
generation MEK1 inhibitor that targets the mutant MEK1 protein. In
some embodiments, the mutation at position 121 is C121S.
[0022] In another aspect, the invention provides a method of
identifying a subject having cancer as having a high risk of
relapse during treatment with a RAF inhibitor, comprising:
[0023] (a) extracting nucleic acid from cells of the cancer;
and
[0024] (b) sequencing a nucleic acid molecule encoding a MEK1
protein;
wherein the presence of nucleotides that produce a 121C>S amino
acid substitution in the MEK1 protein, as compared to wild-type
MEK1 protein, identifies the subject as having a high risk of
relapse during treatment with a RAF inhibitor.
[0025] In another aspect, the invention provides a method of
identifying a subject having cancer as being unresponsive to
treatment with a RAF inhibitor, comprising:
[0026] (a) extracting nucleic acid from cells of the cancer;
and
[0027] (b) sequencing a nucleic acid molecule encoding a MEK1
protein;
wherein the presence of nucleotides that produce a 121C>S amino
acid substitution in the MEK1 protein, as compared to wild-type
MEK1 protein, identifies the subject as being unresponsive to
treatment with a RAF inhibitor.
[0028] In another aspect, the invention provides a method of
optimizing treatment of a subject having cancer, comprising:
[0029] (a) extracting nucleic acid from cells of the cancer;
and
[0030] (b) sequencing a nucleic acid molecule encoding a MEK1
protein;
[0031] wherein the presence of nucleotides that produce a 121C>S
amino acid substitution in the MEK1 protein, as compared to
wild-type MEK1 protein, indicates a need to treat the subject with
a RAF inhibitor and a MEK inhibitor that targets a mutant MEK1
protein having a 121C>S amino acid substitution.
[0032] In another aspect, the invention provides a method of
treating a subject having cancer, comprising: [0033] (a) extracting
nucleic acid from cells of the cancer; [0034] (b) sequencing a
nucleic acid molecule encoding a MEK1 protein; and [0035] (c)
administering a RAF inhibitor and a MEK inhibitor that targets a
mutant MEK1 protein having a 121C>S amino acid substitution to
the subject when the nucleic acid molecule contains nucleotides
that produce a 121C>S amino acid substitution in the MEK1
protein, as compared to wild-type MEK1 protein.
[0036] In another aspect, the invention provides a method of
treating a subject having cancer, comprising: [0037] (a) extracting
nucleic acid from cells of the cancer; [0038] (b) subjecting the
sample to PCR and identifying the nucleotide sequence of a nucleic
acid molecule encoding a MEK1 protein; [0039] (c) administering a
RAF inhibitor and a MEK inhibitor that targets a mutant MEK1
protein having a 121C>S amino acid substitution to the subject
when the nucleic acid molecule contains nucleotides that produce a
121C>S amino acid substitution in the MEK1 protein, as compared
to wild-type MEK1 protein.
[0040] In another aspect, the invention provides a method of
identifying a subject having cancer who is likely to benefit from
treatment with a RAF inhibitor and a MEK inhibitor that targets a
mutant MEK1 protein having a 121C>S amino acid substitution,
comprising: [0041] (a) assaying a nucleic acid sample obtained from
the cancer for the presence one or more mutations in a nucleic acid
molecule encoding a MEK1 protein that produce a 121C>S amino
acid substitution in the MEK1 protein, as compared to wild-type
MEK1 protein; and [0042] (b) correlating the presence of the one or
more mutations in a nucleic acid molecule encoding a MEK1 protein
that produce a 121C>S amino acid substitution in the MEK1
protein with a subject who is likely to benefit from treatment with
a RAF inhibitor and a MEK inhibitor that targets a mutant MEK1
protein having a 121C>S amino acid substitution.
[0043] In another aspect, the invention provides a method of
identifying a subject having cancer who is likely to benefit from
treatment with a MEK inhibitor that targets a mutant MEK1 protein
having a 121C>S amino acid substitution, comprising: [0044] (a)
extracting nucleic acid from cells of the cancer; and [0045] (b)
sequencing a nucleic acid molecule encoding a MEK1 protein; wherein
the presence of nucleotides that produce a 121C>S amino acid
substitution in the MEK1 protein, as compared to wild-type MEK1
protein, identifies the subject as being likely to benefit from
treatment with a MEK inhibitor that targets a mutant MEK1 protein
having a 121C>S amino acid substitution.
[0046] In various embodiments of the foregoing aspects, the RAF
inhibitor is selected from the group consisting of PLX4720,
PLX4032, BAY 43-9006 (Sorafenib), ZM 336372, RAF 265, AAL-881,
LBT-613, and CJS35. In some embodiments, the RAF inhibitor is
PLX4720 or PLX4032. In some embodiments of the foregoing aspects,
the cancer is a leukemia, a lymphoma, a myeloma, a carcinoma, a
metastatic carcinoma, a sarcoma, an adenoma, a nervous system
cancer or a geritourinary cancer. In an exemplary embodiment, the
cancer is a melanoma.
[0047] In another aspect, the invention provides kits for
identifying a subject having cancer who has a high risk of relapse
during treatment with a RAF inhibitor, or for identifying a subject
having cancer who is unresponsive to treatment with a RAF
inhibitor, or for optimizing treatment of a subject having cancer,
or for identifying a subject having cancer who is likely to benefit
from treatment with a RAF inhibitor and a MEK inhibitor that
targets a mutant MEK1 protein having a 121C>S amino acid
substitution, or for identifying a subject having cancer who is
likely to benefit from treatment with a MEK inhibitor that targets
a mutant MEK1 protein having a 121C>S amino acid, the kits
comprising: (a) a detection reagent useful for identifying the
presence or absence of a mutation at amino acid position 121 in a
MEK1 protein of SEQ ID NO:1 or SEQ ID NO:2; and (b) instructions
describing a method set forth herein. In an exemplary embodiment,
the detection reagent comprises nucleic acid primers useful for
amplification of a MEK1 protein of SEQ ID NOs: 1 or 2.
[0048] In various embodiments of the foregoing aspects, the
presence of a mutation at amino acid position 121 (e.g., a C121S
mutation) in a MEK1 protein e.g., a MEK1 protein in a cancer
cell-containing sample, is determined by a method comprising
determining the sequence of a nucleic acid molecule encoding the
MEK1 protein. In other embodiments, the presence of a mutation at
amino acid position 121 in a MEK1 protein is determined using an
antibody that recognizes a MEK1 protein comprising the mutation. In
another embodiment, the foregoing methods further comprise
administering a compound of identified according to a method
described herein to a subject in whom the presence of one or more
mutations in a MEK1 protein was detected.
[0049] In another aspect, the invention features a method of
inhibiting a mutant MEK1 protein in a subject, the mutant MEK1
protein comprising an amino acid substitution at position 21 of
wild-type MEK1 protein shown in SEQ ID NO: 2, the method comprising
administering a compound identified by one of the foregoing methods
to a subject in whom the presence of the mutant MEK1 protein was
detected. In some embodiments the amino acid substitution is a
121C>S amino acid substitution.
BRIEF DESCRIPTION OF THE DRAWINGS
[0050] FIGS. 1A and 1B are graphs showing the percent growth
inhibition of A375 melanoma cells treated with increasing
concentrations (in .mu.M) of either the BRAF inhibitor PLX4720
(FIG. 1A) or the MEK1 inhibitor AZD6244 (FIG. 1B), wherein the A375
cells were either untransfected (A375), transfected with wild-type
MEK1 (MEK-WT), transfected with the MEK1 C121S mutant (MEK-C121S)
or transfected with a constitutively active MEK variant
(MEK-DD).
[0051] FIGS. 2A and 2B show the kinase activity of the MEK1 C121S
mutant as compared wild-type MEK1 (MEK-WT) and a constitutively
active MEK variant (MEK-DD).
[0052] FIG. 2A is a photograph of an immunoblot. FIG. 2B is a bar
graph quantitating the relative percent of phosphorylated ERK
(pERK).
[0053] FIG. 3 depicts the wild-type human MEK1 nucleic acid
sequence (SEQ ID NO:1) (Accession No. NM.sub.--002755;
gi:169790828).
[0054] FIG. 4 depicts the wild-type human MEK1 protein sequence
(SEQ ID NO:2) (Accession No. NP.sub.--002746; gi:5579478).
DETAILED DESCRIPTION OF THE INVENTION
[0055] The present invention relates to the development of
resistance to chemotherapeutic therapy for cancer, particularly
resistance in malignant melanoma treated with a RAF inhibitor.
Described herein is a patient with metastatic melanoma who
developed resistance to the BRAF inhibitor PLX4032 after an initial
dramatic response. As described in Example 1, massively parallel
sequencing was used to conduct a comparative genomic analysis of 3
different DNA samples from the patient: (i) tumor that was
sensitive to PLX4032, (ii) tumor that was resistant to PLX4032, and
(iii) normal skin. A MEK1 mutation at amino acid position 121, in
particular a C121S mutation, was identified as conferring
resistance to the RAF inhibitor, as well as resistance to a MEK
inhibitor, on cells expressing the mutant MEK1 protein. Thus, the
development of a MEK1 mutation in response to BRAF-inhibition
represents the first reported example in a patient of an acquired
resistance mechanism in which the tumor develops an activating
mutation downstream of the target kinase.
[0056] In an exemplary embodiment, the mutation at amino acid
position 121 in the MEK1 protein confers resistance to the RAF
inhibitors PLX4032 and PLX4720, as well as to the MEK inhibitor
AZD6244. Accordingly, a "resistance mutation," as used herein, is a
mutation in a MEK1 protein which confers resistance to one or more
RAF or MEK inhibitors. As used herein, the term "resistance to one
or more RAF or MEK inhibitors" is intended to mean that the
resistance can be to one or more RAF inhibitors, one or more MEK
inhibitors, or a combination of RAF and MEK inhibitors (e.g., a RAF
inhibitor and a MEK inhibitor).
[0057] The identification of the mutation at amino acid position
121 in the MEK1 protein that confers resistance to RAF inhibitors,
as well as MEK inhibitors, allows the development of
"second-generation MEK inhibitors" the specifically target this
mutant MEK1 protein. As used herein, the term "second-generation
MEK inhibitor" refers to an agent that inhibits a biological
activity of a MEK1 protein containing a mutation at amino acid
position 121, such as the mutation described herein (C121S). In a
preferred embodiment, a second-generation MEK inhibitor also
inhibits a biological activity of a wild-type MEK1 protein.
Accordingly, a second generation MEK inhibitor of the invention can
inhibit a biological activity of a MEK1 protein containing a
resistance mutation and can inhibit a biological activity of a
wild-type MEK protein. Such second-generation MEK inhibitors are
useful in many clinical and therapeutic applications, for example,
in the treatment of cancer. In contrast, the term "first-generation
MEK inhibitor," as used herein, refers to an agent that inhibits a
biological activity of a wild-type MEK protein, but does not
inhibit a biological activity of a MEK1 protein containing a
resistance mutation at amino acid position 121, such as the
mutation described herein.
[0058] Non-limiting examples of RAF inhibitors include PLX4720,
PLX4032, BAY 43-9006 (Sorafenib), ZM 336372, RAF 265, AAL-881
(Novartis), LBT-613 (Novartis), and CJS352. PLX4720, PLX4032, BAY
43-9006 (Sorafenib), ZM 336372, RAF 265 are shown in Table 1.
Additional RAF inhibitors known in the art may also be used.
TABLE-US-00001 TABLE 1 Exemplary RAF Inhibitors Name CAS No.
Structure 1 PLX4720 918505-84-7 ##STR00001## 2 PLX4032 1029872-54-5
##STR00002## 3 Bay 43-9006 Sorafenib Tosylate Nexavar 475207-59-1
##STR00003## 4 ZM 336372 208260-29-1 ##STR00004## 5 RAF265
927880-90- ##STR00005##
[0059] Non-limiting examples of MEK inhibitors include CI-1040,
AZD6244, PD318088, PD98059, PD334581, RDEA119, Compound A, and
Compound B (Table 2). Compounds A and B
(6-Methoxy-7-(3-morpholin-4-yl-propoxy)-4-(4-phenoxy-phenylamino)-quinoli-
ne-3-carbonitrile and
4-[3-Chloro-4-(1-methyl-1H-imidazol-2-ylsulfanyl)-phenylamino]-6-methoxy--
7-(3-morpholin-4-yl-propoxy)-quinoline-3-carbonitrile,
respectively) are further described in Zhang et al., Bioorganic
& Medicinal Chemistry Letters, 11(11):1407-1410 (2001) and
Mallon et al., Mol Cancer Ther. June; 3(6):755-62 (2004), the
entire contents of which are incorporated herein by reference.
TABLE-US-00002 TABLE 2 Exemplary MEK Inhibitors Name CAS No.
Structure 1 CI-1040/PD184352 212631-79-3 ##STR00006## 2 AZD6244
606143-52-6 ##STR00007## 3 PD318088 391210-00-7 ##STR00008## 4
PD98059 167869-21-8 ##STR00009## 5 PD334581 ##STR00010## 6 RDEA119
N-[3,4-difluoro-2-[(2-fluoro- 4-iodophenyl)amino]-6-
methoxyphenyl]-1-[(2R)- 2,3-dihydroxypropyl]-
Cyclopropanesulfonamide 923032-38-6 ##STR00011## 7
6-Methoxy-7-(3-morpholin- 4-yl-propoxy)-4-(4- phenoxy-phenylamino)-
quinoline-3-carbonitrile ##STR00012## 8 4-[3-Chloro-4-(1-methyl-
1H-imidazol-2-ylsulfanyl)- phenylamino]-6-methoxy-7-
(3-morpholin-4-yl-propoxy)- quinoline-3-carbonitrile
##STR00013##
[0060] The MEK inhibitors shown in Table 2 as well as additional
MEK inhibitors known in the art may be tested using the methods
described herein to determine if they are "first-generation MEK
inhibitors" or "second-generation MEK inhibitors."
[0061] Identification of resistance mutations in the MEK1 protein
also allows for the screening of patients having a cancer in order
to determine the presence or absence of a MEK1 resistance mutation
at amino acid position 121 in the cancer. Determining the presence
or absence of one or more MEK resistance mutations in a cancer
allows for alteration of the treatment strategy of a cancer
patient. Such alterations can include, for example, starting or
stopping treatment with a first generation MEK1 inhibitor or a RAF
inhibitor, or starting or stopping treatment with a second
generation MEK1 inhibitor.
[0062] Various aspects of the invention are described in further
detail in the following subsections. Unless otherwise defined, all
technical and scientific terms used herein have the same meaning as
is commonly understood by one of ordinary skill in the art to which
this invention belongs. In case of conflict, the present
specification, including definitions, will control. Although
methods and materials similar or equivalent to those described
herein can be used in the practice of the invention, examples of
suitable methods and materials are described below. The materials,
methods, and examples described herein are illustrative only and
are not intended to be limiting. All publications, patent
applications, patents, and other references mentioned herein are
incorporated by reference in their entirety.
I. MEK Biological Activity
[0063] As used herein, the terms "MEK1 protein" refers to a
protein, also known as MKK1 (MAP kinase kinase 1), which is a
dual-specific tyrosine and serine/threonine kinase that plays a key
role in mitogen-activated protein kinase (MAPK) intracellular
signaling. MEK1 is approximately 45 kDa in size, and is expressed
ubiquitously in mammalian cells. MEK1 contains an activation loop
that includes two serine residues at positions 217 and 221.
Phosphorylation of these residues by the protein kinase Raf results
in MEK1 activation during MAPK signaling. MEK1 also contains two
regulatory phosphorylation sites outside the activation loop.
Phosphorylation at Serine 298 may help prime MEK1 for activation.
Conversely, phosphorylation at Serine 212 may decrease MEK1
activity.
[0064] The mitogen-activated protein kinase (MAPK) cascade is a
critical intracellular signaling pathway that regulates signal
transduction in response to diverse extracellular stimuli,
including growth factors, cytokines, and proto-oncogenes.
Activation of this pathway results in transcription factor
activation and alterations in gene expression, which ultimately
lead to changes in cellular functions including cell proliferation,
cell cycle regulation, cell survival, angiogenesis and cell
migration. Classical MAPK signaling is initiated by receptor
tyrosine kinases at the cell surface, however many other cell
surface molecules are capable of activating the MAPK cascade,
including integrins, heterotrimeric G-proteins, and cytokine
receptors.
[0065] Ligand binding to a cell surface receptor, e.g., a receptor
tyrosine kinase, typically results in phosphorylation of the
receptor. The adaptor protein Grb2 associates with the
phosphorylated intracellular domain of the activated receptor, and
this association recruits guanine nucleotide exchange factors
including SOS-1 and CDC25 to the cell membrane. These guanine
nucleotide exchange factors interact with and activate the GTPase
Ras. Common Ras isoforms include K-Ras, N-Ras, H-Ras and others.
Following Ras activation, the serine/threonine kinase Raf (e.g.,
A-Raf, B-Raf or Raf-1) is recruited to the cell membrane through
interaction with Ras. Raf is then phosphorylated. Raf directly
activates MEK1 and MEK2 by phosphorylation of two serine residues
at positions 217 and 221. Following activation, MEK1 and MEK2
phosphorylate tyrosine (Tyr-185) and threonine (Thr-183) residues
in serine/threonine kinases Erk1 and Erk2, resulting in Erk
activation. Activated Erk regulates many targets in the cytosol and
also translocates to the nucleus, where it phosphorylates a number
of transcription factors regulating gene expression. Erk kinase has
numerous targets, including Elk-1, c-Ets1, c-Ets2, p90RSK1, MNK1,
MNK2, MSK1, MSK2 and TOB. While the foregoing pathway is a
classical representation of MAPK signaling, there is considerable
cross talk between the MAPK pathway and other signaling
cascades.
[0066] Aberrations in MAPK signaling have a significant role in
cancer biology. Altered expression of Ras is common in many
cancers, and activating mutations in Ras have also been identified.
Such mutations are found in up to 30% of all cancers, and are
especially common in pancreatic (90%) and colon (50%) carcinomas.
In addition, activating Raf mutations have been identified in
melanoma and ovarian cancer. The most common mutation, BRAF
600V>E, results in constitutive activation of the downstream MAP
kinase pathway and is required for melanoma cell proliferation,
soft agar growth, and tumor xenograft formation. Based on the
defined role of MAPK over-activation in human cancers, targeting
components of the MAPK pathway with specific inhibitors is a
promising approach to cancer therapy. MEK1 and MEK2 are attractive
targets for therapy because of the high degree of specificity they
display for their Erk1/2 substrates. The high degree of homology
between MEK1 and MEK2 make it likely that a small-molecule MEK
inhibitor would effectively inhibit both proteins. CI-1040 (also
known as PD184352) is a MEK inhibitor that has been tested in Phase
I and Phase II clinical trials. CI-1040 was found to inhibit MEK1
with an in vitro IC.sub.50 of 17 nmol/L (Friday et al. Clin. Cancer
Res. (2008) 14(2):342-346; incorporated herein by reference in its
entirety). CI-1040 additionally inhibited MEK in cell-based assays,
and reduced human colon cancer xenograft growth, indicating that
MEK inhibition is a viable method of cancer therapy.
[0067] As used interchangeably herein, the terms "MEK1 activity,"
"MEK1 biological activity" or "functional activity of MEK1,"
include activities exerted by a MEK1 protein on a MEK responsive
cell or tissue, e.g., a cancer cell, or on a MEK nucleic acid
molecule or protein target molecule, as determined in vivo or in
vitro, according to standard techniques. MEK1 activity can be a
direct activity, such as an association with a MEK-target molecule
e.g., ERK1/2, or phosphorylation of a substrate (e.g., ERK1/2).
Alternatively, a MEK1 activity is an indirect activity, such as a
downstream biological event mediated by interaction of the MEK1
protein with an MEK target molecule, e.g., ERK1/2. As MEK is in a
signal transduction pathway involving ERK1/2, modulation of MEK1
modulates a molecule in a signal transduction pathway involving
ERK1/2.
II. MEK Resistance Mutations
[0068] While treatment of cancer with RAF inhibitors, such as
PLX4032, or with MEK inhibitors, including AZD6244 and CI-1040, are
promising therapeutic approaches, patients receiving such therapies
frequently relapse or fail to respond, and as a result the
patients' disease progresses. As described herein, the present
invention relates to the discovery of a mutation in MEK1 that
confer resistance to RAF and MEK inhibitors currently in clinical
development. Acquisition of such a mutation in cancer cells makes
patients resistant to treatment with certain RAF and MEK
inhibitors. In exemplary embodiments, the invention regards
development of resistance to the RAF inhibitors PLX4032 and PLX4720
and the MEK inhibitor AZD6244.
[0069] The clinical emergence of a resistant MEK1 mutation in
metastatic BRAF.sup.v600E melanoma as described herein suggests
that the biological relevance of RAF/MEK-associated dependency is
maintained even in advanced stages of malignancy. Thus, the failure
of first-generation RAF or MEK inhibitors to elicit durable tumor
responses in many BRAF.sup.V600E melanomas may indicate suboptimal
drug potency or pharmacodynamics in the clinical setting. Based on
the findings described herein, treatment modalities involving
targeted agents in RAF- or MEK-driven tumors may benefit from more
potent drugs, altered dosing of existing drugs, or combined RAF and
MEK inhibition. These therapeutic innovations, together with robust
tumor genomic profiling to stratify patients, should speed the
advent of personalized cancer treatment in cancers with "druggable"
oncogene mutations.
(A) Identification of MEK1 Mutation Conferring Resistance to RAF
and MEK Inhibitors
[0070] In various embodiments, the present invention relates to
methods of identifying mutations in a MEK1 protein, or mutations in
a nucleic acid molecule encoding the MEK1 protein, that confer
resistance on cells expressing the MEK1 protein to drugs that
inhibit RAF or MEK activity. A "mutant MEK1 protein," as referenced
herein, includes a MEK1 protein containing one or more mutations
that confer resistance to one or more known RAF or MEK inhibitors.
Likewise, a "mutant MEK1 nucleic acid molecule," as referenced
herein, includes a nucleic acid molecule that encodes a mutant MEK1
protein. Nucleic acid molecules encoding MEK1 proteins that contain
one or more mutations can be created using any suitable method
known in the art, including, for example, random mutagenesis or
site-directed mutagenesis of a wild-type MEK1 nucleic acid
sequence, which can be conducted in E. coli. In exemplary
embodiments, the wild-type MEK1 nucleic acid sequence is a human
wild-type MEK1 nucleic acid sequence. In specific embodiments the
wild-type MEK1 nucleic acid sequence is wild-type human MEK1 (SEQ
ID NO:1), shown in FIG. 3. The mutant MEK1 nucleic acid molecules
can then be screened in cells otherwise sensitive to treatment with
a RAF or MEK inhibitor to identify a nucleic acid that encodes a
mutant MEK1 protein that is resistant to treatment with the RAF or
MEK inhibitor.
[0071] Any suitable method can be used to screen mutant MEK1
nucleic acids and mutant MEK1 proteins for resistance to treatment
with a RAF or MEK inhibitor. For example, a nucleic acid molecule
encoding a mutant MEK1 protein can be expressed in cells otherwise
sensitive to treatment with a RAF or MEK inhibitor. An exemplary
cell line useful for this purpose is the melanoma cell line A375.
Following expression of the mutant MEK1 protein, the cells can be
treated with a RAF or MEK inhibitor. The activity of the mutant
MEK1 protein can then be measured and compared to the activity of a
wild-type MEK1 protein similarly expressed and treated with the RAF
or MEK inhibitor. Activity of a MEK1 protein can be determined by,
for example, measuring proliferation or viability of cells
following treatment with the RAF or MEK inhibitor, wherein
proliferation or viability are positively correlated with MEK1
activity. Cell growth, proliferation, or viability can be
determined using any suitable method known in the art. In one
embodiment, cell growth can be determined using well-based cell
proliferation/viability assays such as MTS or Cell Titer GLo, in
which cell growth in the presence of a RAF or MEK inhibitor is
expressed as a percentage of that observed in untreated cells
cultured in the absence of the RAF or MEK inhibitor. In certain
embodiments, resistance is defined as a shift in the GI50 value of
at least 2 fold, more preferably at least 3 fold, most preferably
at least 4-5 fold, with respect to a suitable control. In other
embodiments, resistance is defined as a GI50 value of .about.1 uM).
Activity of a MEK1 protein can also be measured by, for example,
determining the relative amount of phosphorylated ERK1/2 present in
the cell following treatment with the RAF or MEK inhibitor.
Activity of a wild-type or mutant MEK1 protein can also be
determined using an in vitro phosphorylation assay, in which MEK1
activity is determined by measuring the proportion of
phosphorylated ERK 1/2 substrate in the assay following treatment
with the RAF or MEK inhibitor. A mutant MEK1 protein having greater
activity than a wild-type MEK1 protein following treatment with a
RAF or MEK inhibitor is identified as containing a mutation that
confers resistance to a RAF or MEK inhibitor. The mutation
conferring resistance to a RAF or MEK inhibitor can then be
identified by sequencing the nucleic acid encoding the mutant MEK1
protein, or by sequencing the mutant MEK1 protein directly.
[0072] In this manner, as well as using massively parallel sequence
methods, as described in Example 1, an amino acid substitution at
position 121 of the human MEK1 protein was identified that, when
mutated, confers resistance to the RAF inhibitors PLX4032 and
PLX4720, as well as the MEK inhibitor AZD6244. In particular, a
substitution of the wild-type cysteine at position 121 with serine
(referred to herein as C121S or 121C>S) was identified as
conferring resistance to RAF and MEK inhibitors. Moreover, the
invention encompasses other substitutions at amino acid position
121 of MEK1 that confer resistance to one or more RAF or MEK
inhibitors. For example, substitutions of the cysteine at position
121 with other amino acid structurally similar to serine, such as
alanine, threonine or glycine, are encompassed by the invention.
Thus, in various embodiments, the mutant MEK1 protein of the
invention can comprise a mutation such as C121A, C121T or
C121G.
[0073] As described herein, identification of mutations in MEK
conferring resistance to RAF or MEK inhibitors allows the design
and screening of "second generation MEK inhibitors," which are
effective at inhibiting a mutant MEK1 protein having a resistance
mutation at amino acid position 121. Such second-generation MEK
inhibitors are useful in many clinical and therapeutic
applications, for example, in the treatment of cancer.
Identification of resistance mutations in the MEK1 protein also
allows the screening of patients having a cancer in order to
determine the presence or absence of a MEK1 resistance mutations at
position 121 in the cancer. Determining the presence or absence of
a MEK1 resistance mutation at position 121 in a cancer allows
alteration of the treatment strategy of a cancer patient. For
example, identification of a MEK1 resistance mutation described
herein in a cancer cell-containing sample from a patient having a
cancer can be used to stratify the patient to treatment with a
second-generation MEK1 inhibitor. Identification of MEK1 resistance
mutations also allows the screening and identification of patients
having a high risk of relapse or lack of response to treatment with
certain RAF or MEK inhibitors.
[0074] The foregoing MEK1 resistance mutations also confer
resistance to inhibitors of RAF kinase, e.g., the B-RAF inhibitor
PLX4720. Resistance to inhibitors of a RAF kinase can be
determined, for example, by measuring the activity a mutant MEK1
protein in the presence of a RAF inhibitor, and comparing the
activity to that of a wild-type MEK1 protein similarly treated with
the RAF inhibitor. Activity of a MEK1 protein can be determined
using the methods set forth herein.
III. Methods for Identifying Second-Generation MEK Inhibitors
[0075] Identification of MEK1 resistance mutations allows the
development and/or identification of "second-generation MEK1
inhibitors." As used herein, a second-generation MEK1 inhibitor is
an agent that effectively inhibits the activity of a mutant MEK1
protein containing a mutation at amino acid position 121 as
described herein. A second-generation MEK1 inhibitor may or may not
inhibit the activity of a wild-type MEK1 protein in addition to a
mutant MEK1 protein. In a preferred embodiment, a second-generation
MEK1 inhibitor inhibits the activity of both a wild-type MEK1
protein and a mutant MEK1 protein. In an exemplary embodiment, a
second-generation MEK1 inhibitor inhibits the activity of a MEK1
protein containing a mutation at amino acid position 121,
preferably a C121S mutation.
[0076] Accordingly, the present invention provides methods for
identifying a test compound as a second-generation MEK1 inhibitor.
In one embodiment, a compound can be identified as a
second-generation MEK1 inhibitor by determining the relative MEK1
activity of a mutant MEK1 protein (having a substitution at
position 121) in the presence or absence of the compound, with
respect to a wild-type MEK1 protein. When in the presence of a
compound that is a second-generation MEK1 inhibitor, a mutant MEK1
protein has a lower level of MEK1 activity than in the absence of
the compound. When in the presence of a compound that is not a
second-generation MEK1 inhibitor, a mutant MEK1 protein has an
equivalent or higher level of MEK1 activity than in the absence of
the compound. In certain embodiments, MEK1 activity can be measured
in an in vitro assay using recombinant MEK1 proteins. In other
embodiments, MEK1 activity can be measured in an in vivo assay
using cultured cells or experimental animals.
[0077] Any indicator of MEK1 activity is suitable for determining
whether or not a compound is a second-generation MEK1 inhibitor. In
an exemplary embodiment, MEK1 activity is determined by measuring
phosphorylation of the MEK substrate ERK1/2, wherein a decrease in
ERK1/2 phosphorylation indicates a decrease in MEK1 activity. In
one embodiment, ERK1/2 phosphorylation is measured in a cell or
cell extract. In an alternate embodiment, ERK1/2 phosphorylation is
measured in an in vitro phosphorylation assay using purified or
recombinant proteins. Methods of detecting ERK1/2 phosphorylation
known in the art are suitable for measuring ERK1/2 phosphorylation
as an indication of the activity of a MEK1 protein or a mutant MEK1
protein. Such methods include, but are not limited to, Western blot
and mass spectroscopy. In certain embodiments, an ERK1/2
phosphorylation assay can be performed in vitro using recombinant
proteins. In other embodiments, an ERK1/2 phosphorylation assay can
be performed in vivo using cultured cells or experimental
animals.
[0078] In one embodiment, a host cell expressing a mutant MEK1
protein is used in the identification of a second-generation MEK1
inhibitor, wherein the sensitivity of the host cell to a test
compound identifies the test compound as a second-generation MEK1
inhibitor. As used herein, the term "sensitivity of the host cell
to a test compound" is intended to mean that the test compound has
a measurable effect on one or more parameters including cell
growth, cell proliferation, cell viability and/or intracellular
signal transduction (e.g., signal transduction mediated by MEK1 as
evidenced by, for example, phosphorylation of one or more MEK
substrates, such as ERK1/2).
[0079] A compound can be identified as a second-generation MEK1
inhibitor by determining the viability or proliferation rate of
cells expressing a mutant MEK1 protein in the presence or absence
of the compound. The cell line used in such an assay should be
sensitive to a MEK1 inhibitor when the cell line expresses a
wild-type MEK1 protein, and should be resistant to the MEK1
inhibitor (i.e., a first-generation MEK inhibitor) when the cell
line expresses a mutant MEK1 protein. An exemplary cell line useful
for identification of a second-generation MEK1 inhibitor is the
melanoma cell line A375. A375 cells are sensitive to the MEK1
inhibitor AZD6244 when expressing a wild-type MEK1 protein, but are
resistant to AZD6244 when expressing a mutant MEK1 protein, for
example, a MEK1 protein comprising a C121S mutation.
[0080] When in the presence of a compound that is a
second-generation MEK1 inhibitor, a cell line expressing a mutant
MEK1 protein has a lower viability or proliferation rate than in
the absence of the compound, and/or a lower viability or
proliferation rate than a cell line expressing a wild type MEK1
protein in the presence of the compound. When in the presence of a
compound that is not a second-generation MEK1 inhibitor, a cell
line expressing a mutant MEK1 protein has an equivalent or higher
viability or proliferation rate than in the absence of the
compound, and/or an equivalent or higher viability or proliferation
rate than a cell line expressing a wild type MEK1 protein in the
presence of the compound. Methods of measuring cell viability
and/or proliferation rate known in the art are suitable for
determining the sensitivity of a cell line expressing a MEK1
protein or a mutant MEK1 protein to a test compound. Such methods
include, but are not limited to, measurement of Trypan blue
exclusion, metabolism of tetrazolium compounds, tritiated thymidine
incorporation, BrdU incorporation, glucose uptake, ATP
concentration, and level of apoptosis. In one embodiment, cell
proliferation can be determined using well-based cell
proliferation/viability assays such as MTS or Cell Titer GLo. In
certain embodiments, sensitivity is defined as a shift in the GI50
value of at least 2 fold, more preferably at least 3 fold, most
preferably at lease 4-5 fold, with respect to a suitable
control.
[0081] Accordingly, in one embodiment, the invention provides a
method of identifying a compound that is a second generation MEK1
inhibitor, comprising providing an assay composition comprising a
MEK substrate and a MEK1 protein having a mutation at position 121
with respect to a wild-type MEK1 protein (e.g., a C121S mutation),
contacting the assay composition with a test compound under
conditions that permit phosphorylation of the MEK substrate in the
absence of the test compound, and determining the effect of the
compound of phosphorylation of the MEK substrate, wherein
downmodulation of phosphorylation of the MEK substrate as compared
to a suitable control identifies the compound as a second
generation MEK1 inhibitor. A compound identified in this way is a
compound useful for treating a cancer, e.g., a cancer in which a
mutant MEK1 protein has been detected. A MEK1 protein useful in the
foregoing methods is a MEK1 protein containing a mutation which
confers resistance to one or more MEK, e.g., a MEK1 protein
containing one or more of the mutations described herein. A MEK
substrate useful in the foregoing methods is ERK1/2. A decrease,
reduction, or downmodulation of ERK1/2 phosphorylation is an
indication that the compound is a MEK1 inhibitor. The foregoing
methods can be performed in vitro wherein the MEK1 protein and the
MEK substrate are isolated or purified proteins. The foregoing
methods can also be performed in vitro wherein the MEK1 protein and
the MEK substrate are components of a cell extract. In this
embodiment, the assay composition is a cell extract. A suitable
control is any control that would be apparent to a skilled person
performing the method, and includes, for example, a similar or
identical assay composition not treated with a test compound or
treated with a control compound, or an analogous assay composition
or cell extract comprising a "wild-type" MEK1 protein.
[0082] In another embodiment, the invention provides a method of
identifying a compound that is a second generation MEK1 inhibitor,
comprising providing a cell comprising a mutant MEK1 protein
comprising a mutation at position 121 (e.g., a C121S mutation),
contacting the cell with a test compound, and determining the
effect of the compound on ERK1/2 phosphorylation or cell
proliferation, wherein a decrease, reduction, or downmodulation of
ERK1/2 phosphorylation or cell proliferation as compared to an
appropriate control identifies the compound as a second generation
MEK1 inhibitor. A compound identified in this way is a compound
useful for treating a cancer, e.g., a cancer in which a mutant MEK1
protein has been detected. A suitable control is any control that
would be apparent to a skilled person performing the method, and
includes, for example, a similar or identical cell not treated with
a test compound or treated with a control compound, or an analogous
cell or cell extract in which recombinant, "wild-type" MEK1 was
expressed.
[0083] In one embodiment, the test compound used in the foregoing
methods is a MEK inhibitor that inhibits a biological activity of a
wild type MEK1 protein. MEK inhibitors that are second generation
MEK1 inhibitors are described herein.
[0084] In another embodiment, the test compound is a member of a
library of test compounds. A "library of test compounds" refers to
a panel comprising a multiplicity of test compounds. An approach
for the synthesis of molecular libraries of small organic molecules
has been described (Carell et al. (1994). Angew. Chem. Int. Ed.
Engl. 33:2059; Carell et al. (1994) Angew. Chem. Int. Ed. Engl.
33:2061). The compounds of the present invention can be obtained
using any of the numerous approaches in combinatorial library
methods known in the art, including: biological libraries;
spatially addressable parallel solid phase or solution phase
libraries, synthetic library methods requiring deconvolution, the
`one-bead one-compound` library method, and synthetic library
methods using affinity chromatography selection. The biological
library approach is limited to peptide libraries, while the other
four approaches are applicable to peptide, non-peptide oligomer or
small molecule libraries of compounds (Lam, K. S. (1997) Anticancer
Drug Des. 12:145). Other exemplary methods for the synthesis of
molecular libraries can be found in the art, for example in: Erb et
al. (1994). Proc. Natl. Acad. Sci. USA 91:11422; Horwell et al.
(1996) Immunopharmacology 33:68-; and in Gallop et al. (1994); J.
Med. Chem. 37:1233-. Libraries of compounds can be presented in
solution (e.g., Houghten (1992) Biotechniques 13:412-421), or on
beads (Lam (1991) Nature 354:82-84), chips (Fodor (1993) Nature
364:555-556), bacteria (Ladner U.S. Pat. No. 5,223,409), spores
(Ladner USP '409), plasmids (Cull et al. (1992) Proc Natl Acad Sci
USA 89:1865-1869) or on phage (Scott and Smith (1990) Science
249:386-390); (Devlin (1990) Science 249:404-406); (Cwirla et al.
(1990) Proc. Natl. Acad. Sci. 87:6378-6382); (Felici (1991) J. Mol.
Biol. 222:301-310). In still another embodiment, the combinatorial
proteins are produced from a cDNA library. Exemplary compounds that
can be screened for activity include, but are not limited to,
peptides, nucleic acids, carbohydrates, small organic molecules,
and natural product extract libraries.
[0085] Second generation MEK inhibitors can also be rationally
designed based on the structure of MEK1 alleles containing a
resistance mutation described herein. As described herein, a
substitution at position 121 of MEK1 that confers resistance to RAF
or MEK inhibitors is located in or near the C-helix of the MEK1
protein. Identification of MEK1 mutant alleles conferring
resistance to RAF and MEK inhibitors allows comparison between the
structure of the C-helix of the mutant alleles and the wild-type
protein. Knowledge of the altered structural features that confer
resistance to RAF and MEK inhibitors allows rational design and
construction of ligands, including inhibitors, that will bind the
C-helix, activation loop, and/or the ATP binding pocket of the
mutant alleles. Such inhibitors can be designed such that they bind
both the mutant and wild-type MEK1 alleles. Inhibitors designed to
bind the C-helix, activation loop, and/or ATP binding pocket of
MEK1 alleles containing mutations described herein are
second-generation MEK1 inhibitors. The ability of such rationally
designed inhibitors to inhibit a biological activity of a mutant
MEK1 protein can be confirmed using the in vitro and/or in vivo
assays described herein.
[0086] The structure of a MEK protein containing a resistance
mutation described herein can be determined by computer-assisted
modeling, or by determining the crystal or solution structure of
the mutant MEK1 protein. Any suitable method known in the art can
be used to determine the structure of a mutant MEK1 protein.
[0087] Exemplary computer-assisted modeling methods include the use
of software programs such as PYMOL, CAVITY (described in J. Comp.
Aided. Mol. Des. (1990) 4:337-354 (incorporated herein by
reference)) and Discovery Studio.RTM. (Accelrys, San Diego,
Calif.). Additional techniques useful for computer-assisted
molecular modeling are described in J BUON. (2007) 12 Suppl
1:S101-18 (incorporated herein by reference). Computer-based
analysis of a protein with a known structure can also be used to
identify molecules which will bind to the protein. Such methods
rank molecules based on their shape complementary to a receptor
site. For example, using a 3-D database, a program such as DOCK can
be used to identify molecules which will bind to XBP-1, IRE-1
alpha, and/or EDEM. See DesJarlias et al. (1988) J. Med. Chem.
31:722; Meng et al. (1992) J. Computer Chem. 13:505; Meng et al.
(1993) Proteins 17:266; Shoichet et al. (1993) Science 259:1445. In
addition, the electronic complementarity of a molecule to a
targeted protein can also be analyzed to identify molecules which
bind to the target. This can be determined using, for example, a
molecular mechanics force field as described in Meng et al. (1992)
J. Computer Chem. 13:505 and Meng et al. (1993) Proteins 17:266.
Other programs which can be used include CLIX which uses a GRID
force field in docking of putative ligands (see, for example,
Lawrence et al. (1992) Proteins 12:31; Goodford et al. (1985) J.
Med. Chem. 28:849; and Boobbyer et al. (1989) J. Med. Chem.
32:1083, incorporated by reference in their entirety).
[0088] Crystallization can be performed by any crystallization
method including, but not limited to batch, dialysis and vapor
diffusion (e.g., sitting drop and hanging drop) methods. Micro,
macro and/or streak seeding of crystals can also be performed to
facilitate crystallization. Crystals comprising MEK1 mutant alleles
can be formed by a variety of different methods known in the art.
For example, crystallizations can be performed by batch, dialysis,
and vapor diffusion (sitting drop and hanging drop) methods. A
detailed description of basic protein crystallization setups can be
found in McRee, D., Practical Protein Crystallography, 2.sup.nd Ed.
(1999), Academic Press Inc. Further descriptions regarding
performing crystallization experiments are provided in Stevens et
al. (2000) Curr. Opin. Struct. Biol.: 10(5):558-63, and U.S. Pat.
Nos. 6,296,673; 5,419,278; and 5,096,676, the entire contents of
which are incorporated herein by reference. Such crystals can be
used to perform X-ray or neutron diffraction analysis in order to
determine the three-dimensional structure of MEK1 mutant alleles. A
solution structure of a MEK1 protein or mutant MEK1 protein can be
identified using nuclear magnetic resonance spectroscopy using
techniques known in the art. Suitable methods for protein structure
determination by X-Ray crystallography or NMR spectroscopy are
described in Brunger et al., (1998) "Crystallography & NMR
system (CNS): A new software system for macromolecular structure
determination," Acta Crystallogr D54, 905-921; Brunger et al.
(1987) "Solution of a Protein Crystal Structure With a Model
Obtained From NMR Interproton Distance Restraints," Science 235,
1049-1053; Drenth, "Principles of Protein X-ray Crystallography,"
(1994), Springer-Verlag. pp. 1-19; and Narula et al. (1995)
"Solution structure of the C-terminal SH2 domain of the human
tyrosine kinase Syk complexed with a phosphotyrosine pentapeptide,"
Structure 3, 1061-1073, incorporated herein by reference in their
entirety. Upon identification of the crystal or solution structure
of a mutant MEK1 protein, inhibitors of the mutant MEK1 protein can
be identified using the computer assisted modeling approaches
described above.
[0089] Second-generation MEK1 inhibitors identified by the
foregoing methods are useful for treating a disease or condition
associated with expression of a wild-type and/or mutant MEK1
protein. For example, second-generation MEK1 inhibitors are useful
for treating a cancer in a subject, particularly a cancer in which
a mutant MEK1 protein comprising a substitution at position 121 has
been identified. In an exemplary embodiment, second-generation MEK1
inhibitors are useful for treating a cancer containing a MEK1
protein having a C121S mutation.
IV. Isolated Nucleic Acid Molecules
[0090] The present invention concerns polynucleotides or nucleic
acid molecules relating to the MEK1 gene and its respective gene
product. These polynucleotides or nucleic acid molecules are
isolatable and purifiable from mammalian cells. In particular
aspects of the invention, the isolated MEK1 nucleic acid molecules
described herein comprise a mutation conferring resistance to one
or more RAF or MEK inhibitors. A "mutant MEK1 nucleic acid
molecule," as referenced herein, includes a MEK1 nucleic acid
molecule that encodes a mutant MEK1 protein, i.e., a MEK protein
containing one or more mutations that confer resistance to one or
more known RAF or MEK inhibitors.
[0091] In a preferred embodiment, the isolated nucleic acid of the
invention encoding a mutant MEK1 protein comprises the nucleotide
sequence set forth in SEQ ID NO: 3. In yet another embodiment, the
isolated nucleic acid of the invention encodes a mutant MEK1
protein comprising the amino acid sequence set forth in SEQ ID NO:
4.
[0092] It is contemplated that an isolated and purified MEK1
nucleic acid molecule, e.g., a mutant MEK1 nucleic acid molecule,
can take the form of RNA or DNA. As used herein, the term "RNA
transcript" refers to an RNA molecule that is the product of
transcription from a DNA nucleic acid molecule. Such a transcript
can encode for one or more proteins.
[0093] As used in this application, the term "polynucleotide"
refers to a nucleic acid molecule, RNA or DNA, that has been
isolated, such as being free of total genomic nucleic acid.
Therefore, a "polynucleotide encoding MEK1" refers to a nucleic
acid segment that contains MEK1 coding sequences, yet is isolated
away from, or purified and free of, total genomic DNA and proteins.
When the present application refers to the function or activity of
a MEK1-encoding polynucleotide or nucleic acid, it is meant that
the polynucleotide encodes a molecule that is capable of performing
an activity of a wild-type MEK1 protein, for example,
phosphorylation of the ERK1/2 substrate.
[0094] The term "cDNA" is intended to refer to DNA prepared using
RNA as a template. The advantage of using a cDNA, as opposed to
genomic DNA or an RNA transcript is stability and the ability to
manipulate the sequence using recombinant DNA technology (See
Sambrook, 1989; Ausubel, 1996). There can be times when the full or
partial genomic sequence is preferred. Alternatively, cDNA can be
advantageous because it represents coding regions of a protein and
eliminates introns and other regulatory regions.
[0095] It also is contemplated that a given MEK1-encoding nucleic
acid or MEK1 gene from a given cell may be represented by natural
variants or strains that have slightly different nucleic acid
sequences but, nonetheless, encode an active MEK1 protein. In a
preferred embodiment, the active MEK1 protein is an active human
MEK1 protein. In particularly preferred embodiments, the active
MEK1 protein is a mutant MEK1 protein that has an activity of a
wild-type MEK1 protein, but which is resistant to one or more known
RAF or MEK inhibitors. Consequently, certain aspects of the present
invention encompass derivatives of MEK1 with minimal amino acid
changes, but that possess the same biological function.
[0096] The term "gene" is used for simplicity to refer to a
functional protein, protein, or peptide-encoding unit. As will be
understood by those in the art, this functional term includes
genomic sequences, cDNA sequences, and smaller engineered gene
segments that express, or can be adapted to express, proteins,
proteins, domains, fusion proteins, and mutant proteins. The
nucleic acid molecule encoding MEK1 can comprise a contiguous
nucleic acid sequence of the following lengths: at least about 6,
7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130,
140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260,
270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390,
400, 410, 420, 430, 440, 441, 450, 460, 470, 480, 490, 500, 510,
520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640,
650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770,
780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900,
910, 920, 930, 940, 950, 960, 970, 980, 990, 1000, 1010, 1020,
1030, 1040, 1050, 1060, 1070, 1080, 1090, 1100, 1200, 1300, 1400,
1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500,
2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600,
3700, 3800, 3900, 4000, 4100, 4200, 4300, 4400, 4500, 4600, 4700,
4800, 4900, 5000, 5100, 5200, 5300, 5400, 5500, 5600, 5700, 5800,
5900, 6000, 6100, 6200, 6300, 6400, 6500, 6600, 6700, 6800, 6900,
7000, 7100, 7200, 7300, 7400, 7500, 7600, 7700, 7800, 7900, 8000,
8100, 8200, 8300, 8400, 8500, 8600, 8700, 8800, 8900, 9000, 9100,
9200, 9300, 9400, 9500, 9600, 9700, 9800, 9900, 10000, 10100,
10200, 10300, 10400, 10500, 10600, 10700, 10800, 10900, 11000,
11100, 11200, 11300, 11400, 11500, 11600, 11700, 11800, 11900,
12000 or more nucleotides, nucleosides, or base pairs. Such
sequences can be identical or complementary to, for example, SEQ ID
NO:1, or a fragment thereof.
[0097] Various embodiments of the invention relate to genetic
mutations in MEK1. As used herein, a mutation refers to an
addition, deletion, or substitution of a single nucleotide at a
site in a MEK1 nucleic acid molecule. In an exemplary embodiment, a
mutant MEK1 nucleic acid molecule contains one or more mutations
that confer resistance to a particular therapy, such as one or more
RAF or MEK inhibitors. In a related embodiment, a mutant MEK1
nucleic acid molecule contains one or more mutations such that the
mutant MEK1 nucleic acid molecule encodes a mutant MEK1 protein,
wherein the mutant MEK1 protein contains one or more mutations that
confer resistance to a particular therapy, such as one or more RAF
or MEK inhibitors. Thus, in particular aspects of the invention, an
alteration in a sequence results in a change that affects the
properties of a protein encoded by the sequence such that at least
some resistance to therapy, such as therapy with a RAF or MEK
inhibitor, occurs as a result.
[0098] "Isolated substantially away from other coding sequences"
means that the gene of interest forms part of the coding region of
the nucleic acid segment, and that the segment does not contain
large portions of naturally-occurring coding nucleic acid, such as
large chromosomal fragments or other functional genes or cDNA
coding regions. Of course, this refers to the nucleic acid segment
as originally isolated, and does not exclude genes or coding
regions later added to the segment by human manipulation.
[0099] In particular embodiments, the invention concerns isolated
nucleic acid segments and recombinant vectors incorporating DNA
sequences that encode mutant MEK1 proteins or peptides that include
within its amino acid sequence a contiguous amino acid sequence in
accordance with, or essentially corresponding to mutant MEK1
proteins. In exemplary embodiments, the invention concerns isolated
DNA segments and recombinant vectors incorporating DNA sequences
that encode a MEK1 protein, polypeptide or peptide that includes
within its amino acid sequence a contiguous amino acid sequence of
a MEK1 protein comprising one or more mutations that confer
resistance to one or more RAF or MEK inhibitors. In an exemplary
embodiment, the mutations occurs at position 121, and preferably is
a C121S substitution.
[0100] The nucleic acid segments used in the present invention,
regardless of the length of the coding sequence itself, can be
combined with other DNA or RNA sequences, such as promoters,
polyadenylation signals, additional restriction enzyme sites,
multiple cloning sites, other coding segments, and the like, such
that their overall length can vary considerably. It is therefore
contemplated that a nucleic acid fragment of almost any length can
be employed, with the total length preferably being limited by the
ease of preparation and use in the intended recombinant DNA
protocol.
[0101] It is contemplated that the nucleic acid constructs of the
present invention encode a MEK1 protein or a mutant MEK1 protein. A
"heterologous" sequence refers to a sequence that is foreign or
exogenous to the remaining sequence. A heterologous gene refers to
a gene that is not found in nature adjacent to the sequences with
which it is now placed.
[0102] In a non-limiting example, one or more nucleic acid
constructs can be prepared that include a contiguous stretch of
nucleotides identical to or complementary to all or part of a MEK1
gene. A nucleic acid construct can comprise at least 50, 60, 70,
80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1,000, 2,000,
3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 11,000,
12,000, 13,000, 14,000, 15,000, 20,000, 30,000, 50,000, 100, 000,
250,000, about 500,000, 750,000, to about 1,000,000 nucleotides in
length, as well as constructs of greater size, up to and including
chromosomal sizes (including all intermediate lengths and
intermediate ranges), given the advent of nucleic acids constructs
such as a yeast artificial chromosome are known to those of
ordinary skill in the art. It will be readily understood that
"intermediate lengths" and "intermediate ranges," as used herein,
means any length or range including or between the quoted values
(i.e., all integers including and between such values).
Non-limiting examples of intermediate lengths include about 11,
about 12, about 13, about 16, about 17, about 18, about 19, etc.;
about 21, about 22, about 23, etc.; about 31, about 32, etc.; about
51, about 52, about 53, etc.; about 101, about 102, about 103,
etc.; about 151, about 152, about 153, about 97001, about 1,001,
about 1002, about 50,001, about 50,002, about 750,001, about
750,002, about 1,000,001, about 1,000,002, etc. Non-limiting
examples of intermediate ranges include about 3 to about 32, about
150 to about 500,001, about 3,032 to about 7,145, about 5,000 to
about 15,000, about 20,007 to about 1,000,003, etc.
[0103] Certain embodiments of the present invention concern various
nucleic acids, including vectors, promoters, therapeutic nucleic
acids, and other nucleic acid elements involved in transformation
and expression in cells. In certain aspects, a nucleic acid
comprises a wild-type or a mutant nucleic acid. In particular
aspects, a nucleic acid encodes for or comprises a transcribed
nucleic acid.
[0104] The term "nucleic acid" is well known in the art. A "nucleic
acid" as used herein will generally refer to a molecule (i.e., a
strand) of DNA, RNA or a derivative or analog thereof, comprising a
nucleobase. A nucleobase includes, for example, a naturally
occurring purine or pyrimidine base found in DNA (e.g., an adenine
"A," a guanine "G," a thymine "T" or a cytosine "C") or RNA (e.g.,
an A, a G, an uracil "U" or a C). The term "nucleic acid"
encompasses the terms "oligonucleotide" and "polynucleotide," each
as a subgenus of the term "nucleic acid." The term
"oligonucleotide" refers to a molecule of between about 3 and about
100 nucleobases in length. The term "polynucleotide" refers to at
least one molecule of greater than about 100 nucleobases in length.
A "gene" refers to coding sequence of a gene product, as well as
introns and the promoter of the gene product. In addition to the
MEK1 gene, other regulatory regions such as enhancers for MEK1 are
contemplated as nucleic acids for use with compositions and methods
of the claimed invention.
[0105] These definitions generally refer to a single-stranded
molecule, but in specific embodiments will also encompass an
additional strand that is partially, substantially or fully
complementary to the single-stranded molecule. Thus, a nucleic acid
can encompass a double-stranded molecule or a triple-stranded
molecule that comprises one or more complementary strand(s) or
"complement(s)" of a particular sequence comprising a molecule. As
used herein, a single stranded nucleic acid can be denoted by the
prefix "ss", a double stranded nucleic acid by the prefix "ds", and
a triple stranded nucleic acid by the prefix "ts."
[0106] A nucleic acid can be made by any technique known to one of
ordinary skill in the art, for example, by chemical synthesis, or
by enzymatic production or biological production. Non-limiting
examples of a synthetic nucleic acid (e.g., a synthetic MEK1 primer
that facilitates identification of a mutation conferring resistance
to one or more RAF or MEK inhibitors), include a nucleic acid made
by in vitro chemically synthesis using phosphotriester, phosphite
or phosphoramidite chemistry and solid phase techniques such as
described in EP 266,032, incorporated herein by reference, or via
deoxynucleoside H-phosphonate intermediates as described by
Froehler et al., 1986 and U.S. Pat. No. 5,705,629, each
incorporated herein by reference. In the methods of the present
invention, one or more oligonucleotides can be used. Various
different mechanisms of oligonucleotide synthesis have been
disclosed in for example, U.S. Pat. Nos. 4,659,774, 4,816,571,
5,141,813, 5,264,566, 4,959,463, 5,428,148, 5,554,744, 5,574,146,
5,602,244, each of which is incorporated herein by reference.
[0107] A non-limiting example of an enzymatically produced nucleic
acid includes one produced by enzymes in an amplification reactions
such as PCR (see for example, U.S. Pat. Nos. 4,683,202 and
4,682,195, each incorporated herein by reference), or one produced
by synthesis of oligonucleotides, as described in U.S. Pat. No.
5,645,897, incorporated herein by reference. A non-limiting example
of a biologically produced nucleic acid includes a recombinant
nucleic acid produced (i.e., replicated) in a living cell, such as
a recombinant DNA vector replicated in bacteria.
[0108] A nucleic acid can be purified on polyacrylamide gels,
cesium chloride centrifugation gradients, or by any other means
known to one of ordinary skill in the art as part of assessment for
a mutation that confers resistance to RAF and MEK inhibitors. In
preferred aspects, a nucleic acid is a pharmacologically acceptable
nucleic acid. Pharmacologically acceptable compositions are known
to those of skill in the art, and are described herein.
[0109] In certain aspects, the present invention concerns a nucleic
acid that is an isolated nucleic acid. As used herein, the term
"isolated nucleic acid" refers to a nucleic acid molecule (e.g., an
RNA or DNA molecule) that has been isolated free of, or is
otherwise free of, the bulk of the total genomic and transcribed
nucleic acids of one or more cells. In certain embodiments,
"isolated nucleic acid" refers to a nucleic acid that has been
isolated free of, or is otherwise free of, the bulk of cellular
components or in vitro reaction components, including, for example,
macromolecules such as lipids or proteins, small biological
molecules, and the like.
V. Expression Vectors and Host Cells
[0110] The present invention encompasses expression vector
compositions and the use of such vectors to encode for a MEK1
protein, e.g., a mutant MEK1 protein, as well as host cell
compositions into which such expression vectors have been
introduced. The term "vector" is used to refer to a carrier nucleic
acid molecule into which a nucleic acid sequence can be inserted
for introduction into a cell where it can be replicated. A nucleic
acid sequence can be "exogenous," which means that it is foreign to
the cell into which the vector is being introduced or that the
sequence is homologous to a sequence in the cell but in a position
within the host cell nucleic acid in which the sequence is
ordinarily not found. Vectors include plasmids, cosmids, viruses
(bacteriophage, animal viruses, and plant viruses), and artificial
chromosomes (e.g., YACs). One of skill in the art would be well
equipped to construct a vector through standard recombinant
techniques.
[0111] The term "expression vector" or "expression construct"
refers to a vector containing a nucleic acid sequence coding for at
least part of a gene product capable of being transcribed. In some
cases, RNA molecules are then translated into a protein, protein,
or peptide. In other cases, these sequences are not translated, for
example, in the production of antisense molecules or ribozymes.
Expression vectors can contain a variety of "control sequences,"
which refer to nucleic acid sequences necessary for the
transcription and possibly translation of an operably linked coding
sequence in a particular host organism. In addition to control
sequences that govern transcription and translation, vectors and
expression vectors can contain nucleic acid sequences that serve
other functions as well and are described infra.
1. Promoters and Enhancers
[0112] A "promoter" is a control sequence that is a region of a
nucleic acid sequence at which initiation and rate of transcription
are controlled. It can contain genetic elements at which regulatory
proteins and molecules can bind such as RNA polymerase and other
transcription factors. The phrases "operatively positioned,"
"operatively linked," "under control," and "under transcriptional
control" mean that a promoter is in a correct functional location
and/or orientation in relation to a nucleic acid sequence to
control transcriptional initiation and/or expression of that
sequence. A promoter may or may not be used in conjunction with an
"enhancer," which refers to a cis-acting regulatory sequence
involved in the transcriptional activation of a nucleic acid
sequence.
[0113] A promoter can be one naturally associated with a gene or
sequence, as can be obtained by isolating the 5' non-coding
sequences located upstream of the coding segment and/or exon. Such
a promoter can be referred to as "endogenous." Similarly, an
enhancer can be one naturally associated with a nucleic acid
sequence, located either downstream or upstream of that sequence.
Alternatively, certain advantages will be gained by positioning the
coding nucleic acid segment under the control of a recombinant or
heterologous promoter, which refers to a promoter that is not
normally associated with a nucleic acid sequence in its natural
environment. A recombinant or heterologous enhancer refers also to
an enhancer not normally associated with a nucleic acid sequence in
its natural environment. Such promoters or enhancers can include
promoters or enhancers of other genes, and promoters or enhancers
isolated from any other prokaryotic, viral, or eukaryotic cell, and
promoters or enhancers not "naturally occurring," i.e., containing
different elements of different transcriptional regulatory regions,
and/or mutations that alter expression.
[0114] Naturally, it will be important to employ a promoter and/or
enhancer that effectively directs the expression of the nucleic
acid segment in the cell type, organelle, and organism chosen for
expression. Those of skill in the art of molecular biology
generally know the use of promoters, enhancers, and cell type
combinations for protein expression. The promoters employed can be
constitutive, tissue-specific, inducible, and/or useful under the
appropriate conditions to direct high level expression of the
introduced DNA segment. The promoter can be heterologous or
exogenous, for example, a non-MEK1 promoter with respect to MEK1
encoding sequence. In some examples, a prokaryotic promoter is
employed for use with in vitro transcription of a desired sequence.
Prokaryotic promoters for use with many commercially available
systems include T7, T3, and Sp6.
2. Initiation Signals and Internal Ribosome Binding Sites
[0115] A specific initiation signal also may be required for
efficient translation of coding sequences. These signals include
the ATG initiation codon or adjacent sequences. Exogenous
translational control signals, including the ATG initiation codon,
may need to be provided. One of ordinary skill in the art would
readily be capable of determining this and providing the necessary
signals. It is well known that the initiation codon must be
"in-frame" with the reading frame of the desired coding sequence to
ensure translation of the entire insert. The exogenous
translational control signals and initiation codons can be either
natural or synthetic. The efficiency of expression may be enhanced
by the inclusion of appropriate transcription enhancer elements. In
certain embodiments of the invention, the use of internal ribosome
entry sites (IRES) elements are used to create multigene, or
polycistronic, messages.
3. Multiple Cloning Sites
[0116] Vectors can include a multiple cloning site (MCS), which is
a nucleic acid region that contains multiple restriction enzyme
sites, any of which can be used in conjunction with standard
recombinant technology to digest the vector (see, for example,
Carbonelli et al., 1999, Levenson et al., 1998, and Cocea, 1997,
incorporated herein by reference). "Restriction enzyme digestion"
refers to catalytic cleavage of a nucleic acid molecule with an
enzyme that functions only at specific locations in a nucleic acid
molecule. Many of these restriction enzymes are commercially
available. Use of such enzymes is widely understood by those of
skill in the art. Frequently, a vector is linearized or fragmented
using a restriction enzyme that cuts within the MCS to enable
exogenous sequences to be ligated to the vector. "Ligation" refers
to the process of forming phosphodiester bonds between two nucleic
acid fragments, which may or may not be contiguous with each other.
Techniques involving restriction enzymes and ligation reactions are
well known to those of skill in the art of recombinant
technology.
4. Splicing Sites
[0117] Most transcribed eukaryotic RNA molecules will undergo RNA
splicing to remove introns from the primary transcripts. Vectors
containing genomic eukaryotic sequences can require donor and/or
acceptor splicing sites to ensure proper processing of the
transcript for protein expression (see, for example, Chandler et
al., 1997, herein incorporated by reference).
5. Termination Signals
[0118] The vectors or constructs of the present invention will
generally comprise at least one termination signal. A "termination
signal" or "terminator" is comprised of the DNA sequences involved
in specific termination of an RNA transcript by an RNA polymerase.
Thus, in certain embodiments a termination signal that ends the
production of an RNA transcript is contemplated. A terminator may
be necessary in vivo to achieve desirable message levels.
6. Polyadenylation Signals
[0119] For expression, particularly eukaryotic expression, one will
typically include a polyadenylation signal to effect proper
polyadenylation of the transcript. The nature of the
polyadenylation signal is not believed to be crucial to the
successful practice of the invention, and/or any such sequence can
be employed. Preferred embodiments include the SV40 polyadenylation
signal and/or the bovine growth hormone polyadenylation signal,
convenient and/or known to function well in various target cells.
Polyadenylation can increase the stability of the transcript or may
facilitate cytoplasmic transport.
7. Origins of Replication
[0120] In order to propagate a vector in a host cell, it may
contain one or more origins of replication sites (often termed
"ori"), which is a specific nucleic acid sequence at which
replication is initiated. Alternatively an autonomously replicating
sequence (ARS) can be employed if the host cell is yeast.
8. Selectable and Screenable Markers
[0121] In certain embodiments of the invention, the cells
containing a nucleic acid construct of the present invention can be
identified in vitro or in vivo by including a marker in the
expression vector. Such markers would confer an identifiable change
to the cell permitting easy identification of cells containing the
expression vector. Generally, a selectable marker is one that
confers a property that allows for selection. A positive selectable
marker is one in which the presence of the marker allows for its
selection, while a negative selectable marker is one in which its
presence prevents its selection. An example of a positive
selectable marker is a drug resistance marker.
[0122] Usually the inclusion of a drug selection marker aids in the
cloning and identification of transformants, for example, genes
that confer resistance to neomycin, puromycin, hygromycin, DHFR,
GPT, zeocin and histidinol are useful selectable markers. In
addition to markers conferring a phenotype that allows for the
discrimination of transformants based on the implementation of
conditions, other types of markers including screenable markers
such as GFP, whose basis is calorimetric analysis, are also
contemplated. Alternatively, screenable enzymes such as herpes
simplex virus thymidine kinase (tk) or chloramphenicol
acetyltransferase (CAT) can be utilized. One of skill in the art
would also know how to employ immunologic markers, possibly in
conjunction with FACS analysis. The marker used is not believed to
be important, so long as it is capable of being expressed
simultaneously with the nucleic acid encoding a gene product.
Further examples of selectable and screenable markers are well
known to one of skill in the art.
9. Host Cells
[0123] As used herein, the terms "cell," "cell line," and "cell
culture" may be used interchangeably. A cell comprising a MEK1
polynucleotide, either mutated or wild-type, can be employed in the
invention. All of these terms also include their progeny, which
refers to any and all subsequent generations. It is understood that
all progeny may not be identical due to deliberate or inadvertent
mutations. In the context of expressing a heterologous nucleic acid
sequence, "host cell" refers to a prokaryotic or eukaryotic cell,
and it includes any transformable organisms that is capable of
replicating a vector and/or expressing a heterologous gene encoded
by a vector. A host cell can, and has been, used as a recipient for
vectors. A host cell may be "transfected" or "transformed," which
refers to a process by which exogenous nucleic acid is transferred
or introduced into the host cell. A transformed cell includes the
primary subject cell and its progeny. A "recombinant host cell"
refers to a host cell that carries a recombinant nucleic acid, i.e.
a nucleic acid that has been manipulated in vitro or that is a
replicated copy of a nucleic acid that has been so manipulated.
[0124] A host cell can be derived from prokaryotes or eukaryotes,
depending upon whether the desired result is replication of the
vector, expression of part or all of the vector-encoded nucleic
acid sequences, or production of infectious viral particles.
Numerous cell lines and cultures are available for use as a host
cell, and they can be obtained through the American Type Culture
Collection (ATCC), which is an organization that serves as an
archive for living cultures and genetic materials. An appropriate
host can be determined by one of skill in the art based on the
vector backbone and the desired result. A plasmid or cosmid, for
example, can be introduced into a prokaryote host cell for
replication of many vectors. Bacterial cells used as host cells for
vector replication and/or expression include DH5.alpha., JM109, and
KCB, as well as a number of commercially available bacterial hosts
such as SURE.TM.. Competent Cells and Solopack.TM. Gold Cells
(Strategene.RTM., La Jolla). Alternatively, bacterial cells such as
E. coli LE392 could be used as host cells for phage viruses.
[0125] A preferred eukaryotic host cell of the invention is the
melanoma cell line A375, wherein the cell has been transformed with
an expression vector encoding a MEK1 protein, e.g., a mutant MEK1
protein of the invention.
10. Expression Systems
[0126] Numerous expression systems exist that comprise at least a
part or all of the compositions discussed above. Prokaryote- and/or
eukaryote-based systems can be employed for use with the present
invention to produce MEK1 nucleic acid sequences, or their cognate
proteins, proteins and peptides. Many such systems are commercially
and widely available.
[0127] The insect cell/baculovirus system can produce a high level
of protein expression of a heterologous nucleic acid segment, such
as described in U.S. Pat. Nos. 5,871,986, 4,879,236, both herein
incorporated by reference, and which can be bought, for example,
under the name MaxBac.TM. 2.0 from Invitrogen.TM. and BacPack.TM.
Baculovirus Expression System from Clontech.TM..
[0128] Other examples of expression systems include Stratagene's
Complete Control.TM. Inducible Mammalian Expression System, which
involves a synthetic ecdysone-inducible receptor, or its pET
Expression System, an E. coli expression system. Another example of
an inducible expression system is available from Invitrogen, which
carries the T-Rex.TM. (tetracycline-regulated expression) System,
an inducible mammalian expression system that uses the full-length
CMV promoter. The Tet-On.TM. and Tet-Off.TM. systems from
Clontech.TM. can be used to regulate expression in a mammalian host
using tetracycline or its derivatives. The implementation of these
systems is described in Gossen et al., 1992 and Gossen et al.,
1995, and U.S. Pat. No. 5,650,298, all of which are incorporated by
reference.
[0129] Invitrogen also provides a yeast expression system called
the Pichia methanolica Expression System, which is designed for
high-level production of recombinant proteins in the methylotrophic
yeast Pichia methanolica. One of skill in the art would know how to
express a vector, such as an expression construct, to produce a
nucleic acid sequence or its cognate protein, protein, or
peptide.
VI. Isolated Protein Molecules
[0130] Another aspect of the invention pertains to isolated and/or
purified MEK1 proteins, and biologically active portions thereof.
In particular aspects of the invention, the MEK1 proteins described
herein comprise a mutation at amino acid position 121 conferring
resistance to one or more RAF and/or MEK inhibitors. A "mutant MEK1
protein", as referenced herein, includes a MEK1 protein containing
a mutation at amino acid position 121 that confers resistance to
one or more known RAF and/or MEK inhibitors. Preferably, the
isolated mutant MEK1 protein comprises a C121S mutation. In a
preferred embodiment, the isolated mutant MEK1 protein comprises
the amino acid sequence set forth in SEQ ID NO: 4.
[0131] An "isolated" or "purified" protein or biologically active
portion thereof is substantially free of cellular material when
produced by recombinant DNA techniques, or chemical precursors or
other chemicals when chemically synthesized. The language
"substantially free of cellular material" includes preparations of
MEK1 proteins in which the protein is separated from cellular
components of the cells in which it is naturally or recombinantly
produced. In one embodiment, the language "substantially free of
cellular material" includes preparations of MEK1 protein having
less than about 30% (by dry weight) of non-MEK1 protein (also
referred to herein as a "contaminating protein"), more preferably
less than about 20% of non-MEK1 protein, still more preferably less
than about 10% of non-MEK1 protein, and most preferably less than
about 5% of non-MEK1 protein. When the MEK1 protein or biologically
active portion thereof is recombinantly produced, it is also
preferably substantially free of culture medium, i.e., culture
medium represents less than about 20%, more preferably less than
about 10%, and most preferably less than about 5% of the volume of
the protein preparation. The language "substantially free of
chemical precursors or other chemicals" includes preparations of
MEK1 protein in which the protein is separated from chemical
precursors or other chemicals which are involved in the synthesis
of the protein. In one embodiment, the language "substantially free
of chemical precursors or other chemicals" includes preparations of
MEK1 protein having less than about 30% (by dry weight) of chemical
precursors or non-MEK1 chemicals, more preferably less than about
20% chemical precursors or non-MEK1 chemicals, still more
preferably less than about 10% chemical precursors or non-MEK1
chemicals, and most preferably less than about 5% chemical
precursors or non-MEK1 chemicals.
[0132] Biologically active portions of a MEK1 protein include
peptides comprising amino acid sequences derived from the amino
acid sequence of a MEK1 protein, e.g., the amino acid sequence
shown in SEQ ID NOs:2 and 4, which include fewer amino acids than a
full length MEK1 protein, and exhibit at least one activity of a
MEK1 protein. Typically, biologically active portions (peptides,
e.g., peptides which are, for example, 5, 10, 15, 20, 30, 35, 36,
37, 38, 39, 40, 50, 100 or more amino acids in length) comprise a
domain or motif with at least one activity of a MEK1 protein.
Moreover, other biologically active portions, in which other
regions of the protein are deleted, can be prepared by recombinant
techniques and evaluated for one or more of the activities
described herein. Preferably, the biologically active portions of a
MEK1 protein include one or more selected domains/motifs or
portions thereof having biological activity. In preferred
embodiments, biologically active portions of a MEK1 protein
comprise a mutation at amino acid position 121 with respect to a
wild-type MEK1 sequence, wherein said mutation, when present in a
full-length mutant MEK1 protein, confer resistance of the mutant
MEK1 protein to known RAF and MEK inhibitors. In an exemplary
embodiment, the mutation occurs at amino acid 121, preferably a
C121S mutation.
[0133] MEK1 proteins are preferably produced by recombinant DNA
techniques. For example, a nucleic acid molecule encoding the
protein is cloned into an expression vector (as described above),
the expression vector is introduced into a host cell (as described
above) and the MEK1 protein is expressed in the host cell. The MEK1
protein can then be isolated from the cells by an appropriate
purification scheme using standard protein purification techniques.
Alternative to recombinant expression, a MEK1 protein, protein, or
peptide can be synthesized chemically using standard peptide
synthesis techniques. Moreover, a native MEK1 protein and/or a
mutant MEK1 protein can be isolated from cells (e.g., cancer
cells), for example using an anti-MEK1 antibody, which can be
produced by standard techniques utilizing a MEK1 protein or
fragment thereof of this invention.
[0134] The invention also provides MEK1 chimeric or fusion
proteins. As used herein, a MEK1 "chimeric protein" or "fusion
protein" comprises a MEK1 protein operatively linked to a non-MEK1
protein. A "MEK1 protein" refers to a protein having an amino acid
sequence corresponding to a MEK1 protein, whereas a "non-MEK1
protein" refers to a protein having an amino acid sequence
corresponding to a protein which is not substantially homologous to
the MEK1 protein, e.g., a protein which is substantially different
from the MEK1 protein, which does not display a MEK1 activity and
which is derived from the same or a different organism. Within the
fusion protein, the term "operatively linked" is intended to
indicate that the MEK1 protein and the non-MEK1 protein are fused
in-frame to each other. The non-MEK1 protein can be fused to the
N-terminus or C-terminus of the MEK1 protein. For example, in one
embodiment the fusion protein is a GST-MEK1 fusion protein in which
the MEK1 sequences are fused to the C-terminus of the GST
sequences. Such fusion proteins can facilitate the purification of
recombinant MEK1 proteins. In another embodiment, the fusion
protein is a MEK1 protein containing a heterologous signal sequence
at its N-terminus. In certain host cells (e.g., mammalian host
cells), expression and/or secretion of a MEK1 protein can be
increased through use of a heterologous signal sequence.
[0135] Preferably, a MEK1 chimeric or fusion protein of the
invention is produced by standard recombinant DNA techniques. For
example, DNA fragments coding for the different protein sequences
are ligated together in-frame in accordance with conventional
techniques, for example by employing blunt-ended or stagger-ended
termini for ligation, restriction enzyme digestion to provide for
appropriate termini, filling-in of cohesive ends as appropriate,
alkaline phosphatase treatment to avoid undesirable joining, and
enzymatic ligation. In another embodiment, the fusion gene can be
synthesized by conventional techniques including automated DNA
synthesizers. Alternatively, PCR amplification of gene fragments
can be carried out using anchor primers which give rise to
complementary overhangs between two consecutive gene fragments
which can subsequently be annealed and reamplified to generate a
chimeric gene sequence (see, for example, Current Protocols in
Molecular Biology, eds. Ausubel et al. John Wiley & Sons:
1992). Moreover, many expression vectors are commercially available
that already encode a fusion moiety (e.g., a GST protein). A
MEK1-encoding nucleic acid can be cloned into such an expression
vector such that the fusion moiety is linked in-frame to the MEK1
protein.
[0136] In a preferred embodiment, the isolated MEK1 proteins of the
invention contain one or more mutations (e.g., substitutions or
deletions) with respect to a wild-type MEK1 protein sequence. In
one embodiment, the mutant MEK1 proteins contain one or more
mutations with respect to a human wild-type MEK1 protein sequence
(SEQ ID NO:2). In a particularly preferred embodiment, the one or
more mutations confer resistance to one or more RAF and/or MEK
inhibitors. In an exemplary embodiment, the RAF inhibitor is
PLX4032 and/or PLX4720 and the MEK inhibitor is AZD6244. A mutant
MEK1 protein of the invention exhibits a biological activity
characteristic of a wild-type MEK1 protein. Such a biological
activity can include, for example, phosphorylation of ERK1/2.
Exemplary mutant MEK1 proteins of the invention include MEK1
proteins comprising a mutation at amino acid position 121,
preferably a C121s mutation.
[0137] Mutant MEK1 proteins can be generated by mutagenesis of a
wild-type MEK1 protein, or of the nucleic acid molecule encoding a
wild-type MEK1 protein. Mutant MEK1 proteins can also be identified
by screening combinatorial libraries of MEK1 mutants for a mutant
MEK1 protein having a desired activity, e.g., resistance to one or
more RAF and/or MEK inhibitors. Several techniques are known in the
art for screening gene products of combinatorial libraries made by
point mutations or truncation, and for screening cDNA libraries for
gene products having a selected property. Such techniques are
adaptable for rapid screening of the gene libraries generated by
combinatorial mutagenesis. The most widely used techniques, which
are amenable to high through-put analysis, for screening large gene
libraries typically include cloning the gene library into
replicable expression vectors, transforming appropriate cells with
the resulting library of vectors, and expressing the combinatorial
genes under conditions in which detection of a desired activity
facilitates isolation of the vector encoding the gene whose product
was detected.
VIII. Detection of Mutations
[0138] In another aspect, the invention pertains to methods of
detecting the presence of a mutant MEK1 protein in a sample (e.g.,
a biological sample from a cancer patient). A variety of screening
methods can be used to detect the presence of a mutant MEK1 protein
of the invention in a sample, e.g., a nucleic acid and/or a protein
sample. In specific embodiments, the sample contains a cell or cell
extract. In exemplary embodiments, the sample is obtained from a
subject, e.g., a subject having cancer.
[0139] Methods for detecting the presence of resistance mutations
in genomic DNA, cDNA, and RNA (i.e., mRNA) containing a sequence
encoding a MEK1 protein, or biologically active portion thereof,
can be used within the scope of the present invention. Likewise,
methods for detecting the presence of resistance mutations in MEK1
proteins, or biologically active portions thereof, can be used
within the scope of the present invention. In particular
embodiments, methods including, but not limited to, the following
can be used to detect the presence of a MEK1 protein, or a nucleic
acid molecule encoding a MEK1 protein, having a mutation at amino
acid position 121 as compared to the wild-type MEK1 (SEQ ID NO:
2).
[0140] Point mutations can be detected using any suitable method
known in the art, including, for example, denaturing gradient gel
electrophoresis ("DGGE"), restriction fragment length polymorphism
analysis ("RFLP"), chemical or enzymatic cleavage methods, direct
sequencing of target regions amplified by PCR (see above),
single-strand conformation polymorphism analysis ("SSCP"),
polymerase chain reaction, sequencing, hybridization, or "hybrid
capture" followed by pyrosequencing or single-molecule sequencing.
One method of screening for point mutations is based on RNase
cleavage of base pair mismatches in RNA/DNA or RNA/RNA
heteroduplexes. As used herein, the term "mismatch" is defined as a
region of one or more unpaired or mispaired nucleotides in a
double-stranded RNA/RNA, RNA/DNA or DNA/DNA molecule. This
definition thus includes mismatches due to insertion/deletion
mutations, as well as single or multiple base point mutations. U.S.
Pat. No. 4,946,773 describes an RNase A mismatch cleavage assay
that involves annealing single-stranded DNA or RNA test samples to
an RNA probe, and subsequent treatment of the nucleic acid duplexes
with RNase A. For the detection of mismatches, the single-stranded
products of the RNase A treatment, electrophoretically separated
according to size, are compared to similarly treated control
duplexes. Samples containing smaller fragments (cleavage products)
not seen in the control duplex are scored as positive.
[0141] Other investigators have described the use of RNase I in
mismatch assays. For example, Promega markets a kit containing
RNase I that is reported to cleave three out of four known
mismatches. Others have described using the MutS protein or other
DNA-repair enzymes for detection of single-base mismatches.
[0142] Alternative methods for detection of deletion, insertion or
substitution mutations that can be used in the practice of the
present invention are disclosed in U.S. Pat. Nos. 5,849,483,
5,851,770, 5,866,337, 5,925,525 and 5,928,870, each of which is
incorporated herein by reference in its entirety.
[0143] Screening methods can be performed to screen an individual
for the occurrence of the mutations identified above. For example,
in one embodiment, a sample (such as blood or other bodily fluid or
cell or tissue sample) is taken from a patient for analysis. In an
exemplary embodiment, the patient is a cancer patient. Methods
suitable for processing such samples for detection of a mutation in
a MEK1 nucleic acid or a MEK1 protein are known in the art, and the
skilled artisan may adapt the processing of such samples in
accordance with the chosen method of detection. For example, when
detecting the presence of mutations in a nucleic acid encoding a
MEK1 protein, biological samples (e.g., cell samples or tissue
samples) may be processed such that the nucleic acid material in
the sample is accessible to reagents used to detect mutations
(e.g., nucleic acid probes, primers, etc.). Accordingly, in certain
embodiments, the nucleic acid material in a biological sample is
extracted from cells within the sample. "Extraction" of nucleic
acid material as used herein refers to the process of making the
nucleic acid material in a biological sample accessible to contact
by reagents used for detection. For example, in a cell sample, the
nucleic acid material (e.g., mRNA encoding a MEK1 protein, DNA
encoding a MEK1 protein, etc.) is located inside the cell membrane,
making it inaccessible by detection reagents added to the sample.
Accordingly, extraction of the nucleic acid material by disrupting
the integrity of the cell membrane is commonly employed. Exemplary
means of extracting nucleic acid material from a cell are known in
the art and include, but are not limited to, mechanical disruption
(e.g., sonication, French press, vortexing, Dounce homogenization,
etc.), and/or detergent-mediated disruption (e.g., disruption in
lysis buffer containing detergents such as Triton-X-100, CHAPS,
SDS, etc.). Depending on the method of detection, the nucleic acid
material may or may not be isolated from other components of the
sample during or following extraction. For example, for detection
methods employing PCR amplification of a nucleic acid molecule
encoding a MEK1 protein, nucleic acid material that has been
extracted from cells may be used without further isolation (e.g.,
as a component of a crude lysate). In one embodiment, a biological
sample may be isolated on filter paper that is then processed using
standard extraction techniques prior to PCR amplification of a
nucleic acid molecule encoding a MEK1 protein. Such filter paper
can contain chemical reagents that lyse cells, and can additionally
contain reagents that stabilize and protect nucleic acid material
(e.g., Whatman FTA.TM., GE Healthcare). PCR amplification of a MEK1
nucleic acid molecule may be performed from samples stored on
filter paper without isolation from other cellular components. In
other embodiments, nucleic acid material may be isolated from other
non-nucleic acid components in the sample using techniques known in
the art. As described therein, modifications to the methods of
extraction and/or isolation may be used depending on the type of
nucleic acid material to be assayed (e.g. DNA or RNA). Methods of
detecting mutations in a MEK1 protein also may employ extraction
and/or isolation of proteins from a biological sample. Techniques
suitable for extracting and/or isolating proteins from a biological
sample are well known in the art.
[0144] The presence or absence of one or more mutations described
herein determines the ability of the screened individuals to resist
therapy with a RAF inhibitor and/or a first-generation MEK1
inhibitor. According to methods provided by the invention, these
results will be used to adjust and/or alter the dose of the RAF
inhibitor and/or the first-generation MEK1 inhibitor, or to select
a course of treatment using a second-generation MEK1 inhibitor.
Effective treatment of a subject having cancer can comprise the
eradication of a cancer cell, the cessation or reduction of cancer
(such as solid tumor) growth rate, or the amelioration of at least
one cancer symptom.
[0145] The resistance mutations in MEK1 proteins, or in nucleic
acid molecules encoding MEK1 proteins, can be detected using any
suitable methods known in the art, or modifications thereof,
including the methods described below. Such methods include the use
of allele-specific polymerase chain reaction, direct or indirect
sequencing of the site, the use of restriction enzymes where the
respective alleles of the site create or destroy a restriction
site, the use of allele-specific hybridization probes, the use of
antibodies that are specific for mutant MEK1 proteins, or any other
biochemical interpretation.
1. DNA Sequencing
[0146] The most commonly used method of characterizing a mutation
is direct DNA sequencing of the genetic locus that flanks and
includes the polymorphism. Such analysis can be accomplished using
either the "dideoxy-mediated chain termination method," also known
as the "Sanger Method" (Sanger, F., et al., 1975) or the "chemical
degradation method," also known as the "Maxam-Gilbert method"
(Maxam, A. M., et al., 1977). Sequencing in combination with
genomic sequence-specific amplification technologies, such as the
polymerase chain reaction, can be utilized to facilitate the
recovery of the desired genes (Mullis, K. et al., 1986; European
Patent Appln. 50,424; European Patent Appln. 84,796, European
Patent Application 258,017, European Patent Appln. 237,362;
European Patent Appln. 201,184; U.S. Pat. Nos. 4,683,202;
4,582,788; and 4,683,194), all of which are incorporated herein by
reference. Sequencing of pooled samples can also be accomplished
using Solexa/Illumina sequencing (Illumina,.RTM. San Diego,
Calif.), pyrosequencing, or other single-molecule sequencing
approaches. In one embodiment, mutations in the MEK1 gene can be
detected by cloning and sequencing a MEK1 allele present in a
sample obtained from the subject. If desired, MEK1 mRNA can be
sequenced directly, or the polymerase chain reaction technique
("PCR") can be used to amplify MEK1 DNA or mRNA to produce encoding
DNA ("cDNA"), and the resultant cDNA can be sequenced. PCR can also
be used to selectively amplify a region of the MEK1 allele.
2. Exonuclease Resistance
[0147] Other methods that can be employed to determine the identity
of a nucleotide present at a mutated site utilize a specialized
exonuclease-resistant nucleotide derivative (U.S. Pat. No.
4,656,127). A primer complementary to an allelic sequence
immediately 3'- to the polymorphic site is hybridized to the DNA
under investigation. If the polymorphic site on the DNA contains a
nucleotide that is complementary to the particular
exonucleotide-resistant nucleotide derivative present, then that
derivative will be incorporated by a polymerase onto the end of the
hybridized primer. Such incorporation makes the primer resistant to
exonuclease cleavage and thereby permits its detection. As the
identity of the exonucleotide-resistant derivative is known, one
can determine the specific nucleotide present in the polymorphic
site of the DNA.
3. Microsequencing Methods
[0148] Several other primer-guided nucleotide incorporation
procedures for assaying mutated sites in DNA have been described
(Komher, J. S. et al., 1989; Sokolov, B. P., 1990; Syvanen 1990;
Kuppuswamy et al., 1991; Prezant et al., 1992; Ugozzoll, L. et al.,
1992; Nyren et al., 1993). These methods rely on the incorporation
of labeled deoxynucleotides to discriminate between bases at a
mutated site. As the signal is proportional to the number of
deoxynucleotides incorporated, mutations that occur in runs of the
same nucleotide result in a signal that is proportional to the
length of the run (Syvanen et al., 1993).
4. Extension in Solution
[0149] French Patent 2,650,840 and PCT Application No. WO91/02087
discuss a solution-based method for determining the identity of the
nucleotide of a mutated site. According to these methods, a primer
complementary to allelic sequences immediately 3'- to a polymorphic
site is used. The identity of the nucleotide of that site is
determined using labeled dideoxynucleotide derivatives which are
incorporated at the end of the primer if complementary to the
nucleotide of the polymorphic site.
5. Genetic Bit.TM. Analysis or Solid-Phase Extension
[0150] PCT Appln. No. 92/15712 describes a method that uses
mixtures of labeled terminators and a primer that is complementary
to the sequence 3' to a polymorphic site. The labeled terminator
that is incorporated is complementary to the nucleotide present in
the polymorphic site of the target molecule being evaluated and is
thus identified. Here, the primer or the target molecule is
immobilized to a solid phase.
6. Oligonucleotide Ligation Assay (OLA)
[0151] Oligonucleotide Ligation Assay is a solid phase method that
uses different methodology than that described above (Landegren et
al., 1988). Two oligonucleotides capable of hybridizing to abutting
sequences of a single strand of a target DNA are utilized. One of
these oligonucleotides is biotinylated while the other is
detectably labeled. If the precise complementary sequence is found
in a target molecule, the oligonucleotides will hybridize such that
their termini abut, and create a ligation substrate. Ligation
permits the recovery of the labeled oligonucleotide using avidin.
Other nucleic acid detection assays, based on this method, combined
with PCR are also described (Nickerson et al., 1990). Here, PCR is
used to achieve the exponential amplification of target DNA, which
is then detected using the OLA.
7. Ligase/Polymerase-Mediated Genetic Bit Analysis
[0152] U.S. Pat. No. 5,952,174 describes a method that also
involves two primers capable of hybridizing to abutting sequences
of a target molecule. The hybridized product is formed on a solid
support to which the target is immobilized. Here the hybridization
occurs such that the primers are separated from one another by a
space of a single nucleotide. Incubating this hybridized product in
the presence of a polymerase, a ligase, and a nucleoside
triphosphate mixture containing at least one deoxynucleoside
triphosphate allows the ligation of any pair of abutting hybridized
oligonucleotides. Addition of a ligase results in two events
required to generate a signal, extension and ligation. This
provides a higher specificity and lower "noise" than methods using
either extension or ligation alone, and unlike the polymerase-based
assays, this method enhances the specificity of the polymerase step
by combining it with a second hybridization and a ligation step for
a signal to be attached to the solid phase.
8. Methods of Nucleic Acid Transfer
[0153] For some methods of the present invention, methods of
nucleic acid transfer can be employed. Suitable methods for nucleic
acid delivery to effect expression of compositions of the present
invention are believed to include virtually any method by which a
nucleic acid (e.g., DNA, including viral and nonviral vectors) can
be introduced into an organelle, a cell, a tissue or an organism,
as described herein or as would be known to one of ordinary skill
in the art. Such methods include, but are not limited to, direct
delivery of DNA such as by injection (U.S. Pat. Nos. 5,994,624,
5,981,274, 5,945,100, 5,780,448, 5,736,524, 5,702,932, 5,656,610,
5,589,466 and 5,580,859, each incorporated herein by reference),
including microinjection (Harlan and Weintraub, 1985; U.S. Pat. No.
5,789,215, incorporated herein by reference); by electroporation
(U.S. Pat. No. 5,384,253, incorporated herein by reference); by
calcium phosphate precipitation (Graham and Van Der Eb, 1973; Chen
and Okayama, 1987; Rippe et al., 1990); by using DEAE-dextran
followed by polyethylene glycol (Gopal, 1985); by direct sonic
loading (Fechheimer et al., 1987); by liposome mediated
transfection (Nicolau and Sene, 1982; Fraley et al., 1979; Nicolau
et al., 1987; Wong et al., 1980; Kaneda et al., 1989; Kato et al.,
1991); by microprojectile bombardment (PCT Application Nos. WO
94/09699 and 95/06128; U.S. Pat. Nos. 5,610,042; 5,322,783
5,563,055, 5,550,318, 5,538,877 and 5,538,880, and each
incorporated herein by reference); by agitation with silicon
carbide fibers (Kaeppler et al., 1990; U.S. Pat. Nos. 5,302,523 and
5,464,765, each incorporated herein by reference); by
Agrobacterium-mediated transformation (U.S. Pat. Nos. 5,591,616 and
5,563,055, each incorporated herein by reference); by PEG-mediated
transformation of protoplasts (Omirulleh et al., 1993; U.S. Pat.
Nos. 4,684,611 and 4,952,500, each incorporated herein by
reference); or by desiccation/inhibition-mediated DNA uptake
(Potrykus et al., 1985). Through the application of techniques such
as these, organelle(s), cell(s), tissue(s) or organism(s) can be
stably or transiently transformed.
9. Allele-Specific Antibodies
[0154] MEK1 proteins having a resistance mutation described herein
can be detected using antibodies that specifically recognize the
mutant MEK1 protein, but do not recognize wild-type MEK1 protein.
Antibodies can be raised against one or more allelic forms of the
MEK1 protein having one or more resistance mutations. Techniques
for using a specific protein or an oligopeptide as an antigen to
elicit antibodies that specifically recognize epitopes on the
peptide or protein are well known. In one embodiment, the DNA
sequence of the desired allelic form of the target gene can be
cloned by insertion into an appropriate expression vector and
translated into protein in a prokaryotic or eukaryotic host cell.
The protein can be recovered and used as an antigen to elicit the
production of specific antibodies. In another embodiment, the DNA
of the desired allelic form of the target gene is amplified by PCR
technology and is subsequently translated in vitro into protein to
be used as the antigen to elicit the production of specific
antibodies. A third embodiment is to use the DNA sequence of the
alternative alleles as a basis for the generation of synthetic
peptides representing the amino acid sequence of the alleles for
use as antigen to elicit the production of specific antibodies.
[0155] Antibodies can be generated either by standard monoclonal
antibody techniques or generated through recombinant based
expression systems. The term "antibodies" is meant to include
intact antibody molecules as well as antibody fragments or
derivatives, such as Fab and F(ab')2, which are capable of
specifically binding to antigen. The antibodies so produced will
preferentially bind only the mutant protein produced in the allelic
form which was used as an antigen to create the antibody. Methods
of generating allele-specific antibodies are also described in U.S.
Pat. No. 6,200,754 and U.S. Pat. No. 6,054,273, the entire contents
of which are incorporated herein by reference.
[0156] Such antibodies specific for mutant MEK1 proteins can be
used to detect the presence of a MEK1 protein having one or more
resistance mutations in a sample, e.g., an assay sample, a cell
sample, a cell extract, a biological sample, or a patient sample,
using techniques known in the art. These techniques include, for
example, Western blot, immunohistochemistry, indirect
immunofluorescence, and antibody microarray. Antibodies which
specifically recognize mutant MEK1 proteins can also be
second-generation MEK1 inhibitors. The ability of an antibody which
specifically recognizes a mutant MEK1 protein to inhibit the
biological activity of the mutant MEK1 protein can be determined
using the methods described herein for identifying
second-generation MEK1 inhibitors.
IX. Diagnostic and Screening Applications
[0157] The foregoing techniques can be used to determine the
presence or absence of a previously identified resistance mutation
in a MEK1 nucleic acid or protein molecule in a sample obtained
from a patient. Identification of a mutant MEK1 nucleic acid or
protein molecule in a patient sample can be useful for
characterizing a disease or condition in the patient. For example,
in a patient having a disease associated with aberrant expression
or activation of MEK1 (e.g., a cancer), identification of a mutant
MEK1 nucleic acid or protein molecule in sample (e.g., a
cancer-cell containing sample) obtained from the patient indicates
that the patient is at a relatively high risk of relapse or lack of
response to treatment with a RAF or MEK inhibitor. In one
embodiment, identification of a MEK1 nucleic acid or protein
molecule containing one or more mutations described herein in a
cancer-cell containing sample obtained from a patient indicates
that the patient is at a relatively high risk of relapse or lack of
response to treatment with a RAF inhibitor, such as PLX4032 or
PLX4720, or with a first generation MEK inhibitor, e.g., CI-1040,
AZD6244, PD318088, PD98059, PD334581, RDEA119, Compound A, or
Compound B.
[0158] A patient who has a MEK1 resistance mutation described
herein has a higher risk of relapse from treatment with a RAF
inhibitor or a first-generation MEK inhibitor than a patient in
whom a MEK1 resistance mutation cannot be detected. Accordingly, as
used herein, the term "relatively high risk of relapse" is in
relation to a patient in whom a mutant MEK1 nucleic acid or protein
molecule cannot be detected. That is, a patient who has a
"relatively high risk of relapse" is a patient who has a greater
risk of relapse as compared to a patient in whom a mutant MEK1
nucleic acid or protein molecule cannot be detected.
[0159] In certain embodiments, identification of a MEK1 protein, or
a nucleic acid encoding a MEK1 protein, having a mutation at amino
acid position 121 of wild-type MEK1 shown in SEQ ID NO: 2,
indicates that the patient is at relatively high risk of relapse or
lack of response to treatment with a RAF inhibitor and/or a
first-generation MEK inhibitor.
[0160] Determining the presence or absence of a mutant MEK1 nucleic
acid or protein molecule in a patient sample also allows for the
selection of an optimized treatment regimen for the patient, or
stratification of the patient to a certain treatment group. In one
embodiment, a treatment regimen comprising treatment with a RAF
inhibitor and/or a first-generation MEK inhibitor is selected for a
patient wherein a cancer cell-containing sample from the patient
does not contain a mutant MEK1 nucleic acid or protein molecule.
Such a patient can be stratified to a treatment regimen comprising
a RAF inhibitor and/or a first-generation MEK inhibitor. The RAF
and/or MEK inhibitor can be given to such a patient at a standard
dosage, at standard dosing intervals.
[0161] In another embodiment, a treatment regimen comprising a
combination therapy with a MEK inhibitor and a RAF inhibitor is
selected for a patient wherein a cancer cell-containing sample from
the patient does not contain a mutant MEK1 nucleic acid or protein
molecule. Such a patient can be stratified to a treatment regimen
comprising a first generation MEK inhibitor or a second generation
MEK inhibitor, in combination with a RAF inhibitor. A treatment
regimen comprising a combination therapy with a RAF inhibitor and a
MEK inhibitor that targets the C121S MEK1 mutation advantageously
suppresses the emergence of MEK resistant alleles in a patient
whose cancer does not contain a mutant MEK1 molecule.
[0162] In another embodiment, a treatment regimen comprising
treatment without a MEK inhibitor is selected for a patient wherein
a cancer cell-containing sample from the patient contains a mutant
MEK1 nucleic acid or protein molecule, e.g., a MEK1 nucleic acid or
protein molecule containing one or more of the mutations described
herein. Such a patient can be stratified to a treatment regimen
that does not comprise a MEK inhibitor.
[0163] In an alternative embodiment, identification of a mutant
MEK1 nucleic acid or protein molecule, wherein the mutant MEK1
comprises a C121S mutation, in a cancer-cell containing sample
obtained from a patient indicates that the patient is likely to
respond to treatment with a second-generation MEK inhibitor that
targets a MEK1 mutant comprising a C121S mutation. Accordingly, in
one embodiment, a treatment regimen comprising treatment with a
second-generation MEK inhibitor that targets a C121S mutant MEK1
protein is selected for a patient wherein a cancer cell-containing
sample from the patient contains a mutant MEK1 nucleic acid or
protein molecule comprising the C121S mutation. Such a patient can
be stratified to a treatment regimen comprising such a
second-generation MEK inhibitor.
[0164] The practice of the present invention will employ, unless
otherwise indicated, conventional techniques of molecular biology,
immunology, microbiology, cell biology and recombinant DNA, which
are within the skill of the art. See e.g., Sambrook, Fritsch and
Maniatis, MOLECULAR CLONING: A LABORATORY MANUAL, (Current
Edition); CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (F. M. Ausubel et
al. eds., (Current Edition)); the series METHODS IN ENZYMOLOGY
(Academic Press, Inc.): PCR 2: A PRACTICAL APPROACH (Current
Edition) ANTIBODIES, A LABORATORY MANUAL and ANIMAL CELL CULTURE
(R. I. Freshney, ed. (1987)). DNA Cloning: A Practical Approach,
vol. I & II (D. Glover, ed.); Oligonucleotide Synthesis (N.
Gait, ed., Current Edition); Nucleic Acid Hybridization (B. Hames
& S. Higgins, eds., Current Edition); Transcription and
Translation (B. Hames & S. Higgins, eds., Current Edition);
Fundamental Virology, 2nd Edition, vol. I & II (B. N. Fields
and D. M. Knipe, eds.)
X. Methods of Treatment
[0165] In various embodiments, the invention provides methods of
treating a subject having a cancer. Said methods generally comprise
administration of a MEK1 inhibitor, e.g., a second-generation MEK1
inhibitor, as described herein, that targets a mutant MEK1 having a
substitution at amino acid position 121 as compared to the
wild-type MEK1 of SEQ ID NO: 2 (.e.g., a C121S mutation). In an
exemplary embodiment, the subject has a cancer containing a mutant
MEK1 protein having a resistance mutation as described herein. In
related embodiments, the subject has a cancer containing a nucleic
acid molecule encoding a MEK1 protein having a resistance mutation
as described herein. The term "treated," "treating" or "treatment"
includes the diminishment or alleviation of at least one symptom
associated with the condition being treated. For example, treatment
can be diminishment of one or several symptoms of a disorder or
complete eradication of a disorder, such as cancer.
[0166] In an exemplary embodiment, the cancer is a melanoma, such
as a metastatic melanoma. In other exemplary embodiments, the
cancer is selected from the group consisting of leukemias,
lymphomas, myelomas, carcinomas, metastatic carcinomas, sarcomas,
adenomas, nervous system cancers and geritourinary cancers.
However, because mutations in MEK1 broadly enhance resistance in a
number of types of cancer, the methods and second-generation MEK1
inhibitors described herein are useful in treating a broad spectrum
of tumors, including all solid tumors and hematological
malignancies. Examples of such tumors include but are not limited
to leukemias, lymphomas, myelomas, carcinomas, metastatic
carcinomas, sarcomas, adenomas, nervous system cancers and
geritourinary cancers. In exemplary embodiments, the foregoing
methods are useful in treating adult and pediatric acute
lymphoblastic leukemia, acute myeloid leukemia, adrenocortical
carcinoma, AIDS-related cancers, anal cancer, cancer of the
appendix, astrocytoma, basal cell carcinoma, bile duct cancer,
bladder cancer, bone cancer, osteosarcoma, fibrous histiocytoma,
brain cancer, brain stem glioma, cerebellar astrocytoma, malignant
glioma, ependymoma, medulloblastoma, supratentorial primitive
neuroectodermal tumors, hypothalamic glioma, breast cancer, male
breast cancer, bronchial adenomas, Burkitt lymphoma, carcinoid
tumor, carcinoma of unknown origin, central nervous system
lymphoma, cerebellar astrocytoma, malignant glioma, cervical
cancer, childhood cancers, chronic lymphocytic leukemia, chronic
myelogenous leukemia, chronic myeloproliferative disorders,
colorectal cancer, cutaneous T-cell lymphoma, endometrial cancer,
ependymoma, esophageal cancer, Ewing family tumors, extracranial
germ cell tumor, extragonadal germ cell tumor, extrahepatic bile
duct cancer, intraocular melanoma, retinoblastoma, gallbladder
cancer, gastric cancer, gastrointestinal stromal tumor,
extracranial germ cell tumor, extragonadal germ cell tumor, ovarian
germ cell tumor, gestational trophoblastic tumor, glioma, hairy
cell leukemia, head and neck cancer, hepatocellular cancer, Hodgkin
lymphoma, non-Hodgkin lymphoma, hypopharyngeal cancer, hypothalamic
and visual pathway glioma, intraocular melanoma, islet cell tumors,
Kaposi sarcoma, kidney cancer, renal cell cancer, laryngeal cancer,
lip and oral cavity cancer, small cell lung cancer, non-small cell
lung cancer, primary central nervous system lymphoma, Waldenstrom
macroglobulinema, malignant fibrous histiocytoma, medulloblastoma,
melanoma, Merkel cell carcinoma, malignant mesothelioma, squamous
neck cancer, multiple endocrine neoplasia syndrome, multiple
myeloma, mycosis fungoides, myelodysplastic syndromes,
myeloproliferative disorders, chronic myeloproliferative disorders,
nasal cavity and paranasal sinus cancer, nasopharyngeal cancer,
neuroblastoma, oropharyngeal cancer, ovarian cancer, pancreatic
cancer, parathyroid cancer, penile cancer, pharyngeal cancer,
pheochromocytoma, pineoblastoma and supratentorial primitive
neuroectodermal tumors, pituitary cancer, plasma cell neoplasms,
pleuropulmonary blastoma, prostate cancer, rectal cancer,
rhabdomyosarcoma, salivary gland cancer, soft tissue sarcoma,
uterine sarcoma, Sezary syndrome, non-melanoma skin cancer, small
intestine cancer, squamous cell carcinoma, squamous neck cancer,
supratentorial primitive neuroectodermal tumors, testicular cancer,
throat cancer, thymoma and thymic carcinoma, thyroid cancer,
transitional cell cancer, trophoblastic tumors, urethral cancer,
uterine cancer, uterine sarcoma, vaginal cancer, vulvar cancer, and
Wilms tumor.
[0167] In some embodiments, pharmaceutical compositions of the
compounds (or combinations) of the invention can be in unitary
dosage form suitable for administration orally, rectally or by
parenteral injection. For example, in preparing compositions in
oral dosage form, any of the usual pharmaceutical media may be
employed, such as, for example, water, glycols, oils, alcohols and
the like, as in the case of oral liquid preparations such as
suspensions, syrups, elixirs and solutions; or solid carriers such
as starches, sugars, kaolin, lubricants, binders, disintegrating
agents and the like in the case of powders, pills, capsules and
tablets. Because of their ease in administration, tablets and
capsules represent the most advantageous oral dosage unit form, in
which case solid pharmaceutical carriers are employed. For
parenteral compositions, carriers usually comprise sterile water,
at least in large part, though other ingredients, for example, to
aid solubility, may be included. Injectable solutions, for example,
are prepared using a carrier which comprises saline solution,
glucose solution or a mixture of saline and glucose solution.
Injectable suspensions may also be prepared in which case
appropriate liquid carriers, suspending agents and the like may be
employed. In case of compositions suitable for percutaneous
administration, carrier optionally comprises a penetration
enhancing agent and/or a suitable wetting agent, which may be
combined with suitable additives of any nature in minor
proportions, which additives do not cause a significant deleterious
effect to the skin. Additives may facilitate the administration to
the skin and/or may be helpful for preparing desired compositions.
These compositions may be administered in various ways, e.g., as a
transdermal patch, as a spot-on, as an ointment.
[0168] It is especially advantageous to formulate the
pharmaceutical compositions described herein in dosage unit form
for ease of administration and uniformity of dosage. Dosage unit
form, as used herein, refers to physically discrete units suitable
as unitary dosages, each unit containing a predetermined quantity
of active ingredient calculated to produce the desired therapeutic
effect in association with the required pharmaceutical carrier.
Examples of such dosage unit forms are tablets (including scored or
coated tablets), capsules, pills, powder packets, wafers,
injectable solutions or suspensions, teaspoonfuls, tablespoonfuls
and the like, and segregated multiples thereof.
[0169] In general it is contemplated that a therapeutically
effective amount of a first or a second compound would be from
about 0.0001 mg/kg to 0.001 mg/kg; 0.001 mg/kg to about 10 mg/kg
body weight or from about 0.02 mg/kg to about 5 mg/kg body weight.
In some embodiments, a therapeutically effective amount of a first
or a second compound is from about 0.007 mg to about 0.07 mg, about
0.07 mg to about 700 mg, or from about 1.4 mg to about 350 mg. A
method of prophylactic or curative treatment may also include
administering the composition in a regimen of between one to five
intakes per day.
[0170] In some embodiments, a therapeutically effective amount of a
first compound or a second compound includes, but is not limited
to, the amount less than about 0.01 mg/dose, or less than about 0.5
mg/dose, or less than about 1 mg/dose, or less than about 2
mg/dose, or less than about 5 mg/dose, or less than about 10
mg/dose, or less than about 20 mg/dose, or less than about 25
mg/dose, or less than about 50 mg/dose, or less than about 100
mg/dose. The number of times a day a first or a second compound is
administrated to a subject can be determined based on various
criteria commonly used in the art and/or those described
herein.
[0171] Wetting agents, emulsifiers and lubricants, such as sodium
lauryl sulfate and magnesium stearate, as well as coloring agents,
release agents, coating agents, sweetening, flavoring and perfuming
agents, preservatives and antioxidants can also be present in the
compositions.
[0172] Examples of pharmaceutically acceptable antioxidants
include: water soluble antioxidants, such as ascorbic acid,
cysteine hydrochloride, sodium bisulfate, sodium metabisulfite,
sodium sulfite and the like; oil-soluble antioxidants, such as
ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated
hydroxytoluene (BHT), lecithin, propyl gallate, .alpha.-tocopherol,
and the like; and metal chelating agents, such as citric acid,
ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid,
phosphoric acid, and the like.
[0173] Formulations of the present invention include those suitable
for oral, nasal, topical, buccal, sublingual, rectal, vaginal
and/or parenteral administration. The formulations may conveniently
be presented in unit dosage form and may be prepared by any methods
well known in the art of pharmacy. The amount of active ingredient
that can be combined with a carrier material to produce a single
dosage form will generally be that amount of the composition that
produces a therapeutic effect. Generally, out of one hundred
percent, this amount will range from about 1 percent to about
ninety-nine percent of active ingredient, preferably from about 5
percent to about 70 percent, most preferably from about 10 percent
to about 30 percent.
[0174] Methods of preparing these formulations or compositions
include the step of bringing into association a composition of the
present invention with the carrier and, optionally, one or more
accessory ingredients. In general, the formulations are prepared by
uniformly and intimately bringing into association a composition of
the present invention with liquid carriers, or finely divided solid
carriers, or both, and then, if necessary, shaping the product.
[0175] Formulations of the invention suitable for oral
administration may be in the form of capsules, cachets, pills,
tablets, lozenges (using a flavored basis, usually sucrose and
acacia or tragacanth), powders, granules, or as a solution or a
suspension in an aqueous or non-aqueous liquid, or as an
oil-in-water or water-in-oil liquid emulsion, or as an elixir or
syrup, or as pastilles (using an inert base, such as gelatin and
glycerin, or sucrose and acacia) and/or as mouth washes and the
like, each containing a predetermined amount of a composition of
the present invention as an active ingredient. A composition of the
present invention may also be administered as a bolus, electuary or
paste.
[0176] In solid dosage forms of the invention for oral
administration (capsules, tablets, pills, dragees, powders,
granules and the like), the active ingredient is mixed with one or
more pharmaceutically acceptable carriers, such as sodium citrate
or dicalcium phosphate, and/or any of the following: fillers or
extenders, such as starches, lactose, sucrose, glucose, mannitol,
and/or silicic acid; binders, such as, for example,
carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone,
sucrose and/or acacia; humectants, such as glycerol; disintegrating
agents, such as agar-agar, calcium carbonate, potato or tapioca
starch, alginic acid, certain silicates, and sodium carbonate;
solution retarding agents, such as paraffin; absorption
accelerators, such as quaternary ammonium compounds; wetting
agents, such as, for example, cetyl alcohol and glycerol
monostearate; absorbents, such as kaolin and bentonite clay;
lubricants, such a talc, calcium stearate, magnesium stearate,
solid polyethylene glycols, sodium lauryl sulfate, and mixtures
thereof; and coloring agents. In the case of capsules, tablets and
pills, the pharmaceutical compositions may also comprise buffering
agents. Solid compositions of a similar type may also be employed
as fillers in soft and hard-filled gelatin capsules using such
excipients as lactose or milk sugars, as well as high molecular
weight polyethylene glycols and the like.
[0177] A tablet may be made by compression or molding, optionally
with one or more accessory ingredients. Compressed tablets may be
prepared using binder (for example, gelatin or hydroxypropylmethyl
cellulose), lubricant, inert diluent, preservative, disintegrant
(for example, sodium starch glycolate or cross-linked sodium
carboxymethyl cellulose), surface-active or dispersing agent.
Molded tablets may be made by molding in a suitable machine a
mixture of the powdered composition moistened with an inert liquid
diluent.
[0178] The tablets, and other solid dosage forms of the
pharmaceutical compositions of the present invention, such as
dragees, capsules, pills and granules, may optionally be scored or
prepared with coatings and shells, such as enteric coatings and
other coatings well known in the pharmaceutical-formulating art.
They may also be formulated so as to provide slow or controlled
release of the active ingredient therein using, for example,
hydroxypropylmethyl cellulose in varying proportions to provide the
desired release profile, other polymer matrices, liposomes and/or
microspheres. They may be sterilized by, for example, filtration
through a bacteria-retaining filter, or by incorporating
sterilizing agents in the form of sterile solid compositions that
can be dissolved in sterile water, or some other sterile injectable
medium immediately before use. These compositions may also
optionally contain opacifying agents and may be of a composition
that they release the active ingredient(s) only, or preferentially,
in a certain portion of the gastrointestinal tract, optionally, in
a delayed manner. Examples of embedding compositions that can be
used include polymeric substances and waxes. The active ingredient
can also be in micro-encapsulated form, if appropriate, with one or
more of the above-described excipients.
[0179] Liquid dosage forms for oral administration of the
compositions of the invention include pharmaceutically acceptable
emulsions, microemulsions, solutions, suspensions, syrups and
elixirs. In addition to the active ingredient, the liquid dosage
forms may contain inert diluent commonly used in the art, such as,
for example, water or other solvents, solubilizing agents and
emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl
carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate,
propylene glycol, 1,3-butylene glycol, oils (in particular,
cottonseed, groundnut, corn, germ, olive, castor and sesame oils),
glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty
acid esters of sorbitan, and mixtures thereof.
[0180] Besides inert diluents, the oral compositions can also
include adjuvants such as wetting agents, emulsifying and
suspending agents, sweetening, flavoring, coloring, perfuming and
preservative agents.
[0181] Suspensions, in addition to the active compositions, may
contain suspending agents as, for example, ethoxylated isostearyl
alcohols, polyoxyethylene sorbitol and sorbitan esters,
microcrystalline cellulose, aluminum metahydroxide, bentonite,
agar-agar and tragacanth, and mixtures thereof.
[0182] Formulations of the pharmaceutical compositions of the
invention for rectal or vaginal administration may be presented as
a suppository, which may be prepared by mixing one or more
compositions of the invention with one or more suitable
nonirritating excipients or carriers comprising, for example, cocoa
butter, polyethylene glycol, a suppository wax or a salicylate, and
which is solid at room temperature, but liquid at body temperature
and, therefore, will melt in the rectum or vaginal cavity and
release the active composition.
[0183] Formulations of the present invention which are suitable for
vaginal administration also include pessaries, tampons, creams,
gels, pastes, foams or spray formulations containing such carriers
as are known in the art to be appropriate.
[0184] Dosage forms for the topical or transdermal administration
of a composition of this invention include powders, sprays,
ointments, pastes, creams, lotions, gels, solutions, patches and
inhalants. The active composition may be mixed under sterile
conditions with a pharmaceutically acceptable carrier, and with any
preservatives, buffers, or propellants that may be required.
[0185] The ointments, pastes, creams and gels may contain, in
addition to an active composition of this invention, excipients,
such as animal and vegetable fats, oils, waxes, paraffins, starch,
tragacanth, cellulose derivatives, polyethylene glycols, silicones,
bentonites, silicic acid, talc and zinc oxide, or mixtures
thereof.
[0186] Powders and sprays can contain, in addition to a composition
of this invention, excipients such as lactose, talc, silicic acid,
aluminum hydroxide, calcium silicates and polyamide powder, or
mixtures of these substances. Sprays can additionally contain
customary propellants, such as chlorofluorohydrocarbons and
volatile unsubstituted hydrocarbons, such as butane and
propane.
[0187] Transdermal patches have the added advantage of providing
controlled delivery of a composition of the present invention to
the body. Such dosage forms can be made by dissolving or dispersing
the composition in the proper medium. Absorption enhancers can also
be used to increase the flux of the composition across the skin.
The rate of such flux can be controlled by either providing a rate
controlling membrane or dispersing the active composition in a
polymer matrix or gel.
[0188] Ophthalmic formulations, eye ointments, powders, solutions
and the like, are also contemplated as being within the scope of
this invention.
[0189] Pharmaceutical compositions of this invention suitable for
parenteral administration comprise one or more compositions of the
invention in combination with one or more pharmaceutically
acceptable sterile isotonic aqueous or nonaqueous solutions,
dispersions, suspensions or emulsions, or sterile powders which may
be reconstituted into sterile injectable solutions or dispersions
just prior to use, which may contain antioxidants, buffers,
bacteriostats, solutes which render the formulation isotonic with
the blood of the intended recipient or suspending or thickening
agents.
[0190] Examples of suitable aqueous and nonaqueous carriers that
may be employed in the pharmaceutical compositions of the invention
include water, ethanol, polyols (such as glycerol, propylene
glycol, polyethylene glycol, and the like), and suitable mixtures
thereof, vegetable oils, such as olive oil, and injectable organic
esters, such as ethyl oleate. Proper fluidity can be maintained,
for example, by the use of coating materials, such as lecithin, by
the maintenance of the required particle size in the case of
dispersions, and by the use of surfactants.
[0191] These compositions may also contain adjuvants such as
preservatives, wetting agents, emulsifying agents and dispersing
agents. Prevention of the action of microorganisms may be ensured
by the inclusion of various antibacterial and antifungal agents,
for example, paraben, chlorobutanol, phenol sorbic acid, and the
like. It may also be desirable to include isotonic agents, such as
sugars, sodium chloride, and the like into the compositions. In
addition, prolonged absorption of the injectable pharmaceutical
form may be brought about by the inclusion of agents that delay
absorption such as aluminum monostearate and gelatin.
[0192] In some cases, in order to prolong the effect of a drug, it
is desirable to slow the absorption of the drug from subcutaneous
or intramuscular injection. This may be accomplished by the use of
a liquid suspension of crystalline or amorphous material having
poor water solubility. The rate of absorption of the drug then
depends upon its rate of dissolution which, in turn, may depend
upon crystal size and crystalline form. Alternatively, delayed
absorption of a parenterally-administered drug form is accomplished
by dissolving or suspending the drug in an oil vehicle.
[0193] Injectable depot forms are made by forming microencapsule
matrices of the subject compositions in biodegradable polymers such
as polylactide-polyglycolide. Depending on the ratio of drug to
polymer, and the nature of the particular polymer employed, the
rate of drug release can be controlled. Examples of other
biodegradable polymers include poly(orthoesters) and
poly(anhydrides). Depot injectable formulations are also prepared
by entrapping the drug in liposomes or microemulsions that are
compatible with body tissue.
[0194] The preparations of the present invention may be given
orally, parenterally, topically, or rectally. They are of course
given by forms suitable for each administration route. For example,
they are administered in tablets or capsule form, by injection,
inhalation, eye lotion, ointment, suppository, etc., administration
by injection, infusion or inhalation; topical by lotion or
ointment; and rectal by suppositories. Oral and/or IV
administration is preferred.
[0195] The phrases "parenteral administration" and "administered
parenterally" as used herein means modes of administration other
than enteral and topical administration, usually by injection, and
includes, without limitation, intravenous, intramuscular,
intraarterial, intrathecal, intracapsular, intraorbital,
intracardiac, intradermal, intraperitoneal, transtracheal,
subcutaneous, subcuticular, intraarticular, subcapsular,
subarachnoid, intraspinal and intrasternal injection and
infusion.
[0196] The phrases "systemic administration," "administered
systemically," "peripheral administration" and "administered
peripherally" as used herein mean the administration of a compound,
drug or other material other than directly into the central nervous
system, such that it enters the patient's system and, thus, is
subject to metabolism and other like processes, for example,
subcutaneous administration.
[0197] These compounds may be administered to humans and other
animals for therapy by any suitable route of administration,
including orally, nasally, as by, for example, a spray, rectally,
intravaginally, parenterally, intracisternally and topically, as by
powders, ointments or drops, including buccally and
sublingually.
[0198] Regardless of the route of administration selected, the
compounds of the present invention, which may be used in a suitable
hydrated form, and/or the pharmaceutical compositions of the
present invention, are formulated into pharmaceutically acceptable
dosage forms by conventional methods known to those of skill in the
art.
[0199] Actual dosage levels of the active ingredients in the
pharmaceutical compositions of this invention may be varied so as
to obtain an amount of the active ingredient which is effective to
achieve the desired therapeutic response for a particular patient,
composition, and mode of administration, without being toxic to the
patient.
[0200] The selected dosage level will depend upon a variety of
factors including the activity of the particular compound of the
present invention employed, or the ester, salt or amide thereof,
the route of administration, the time of administration, the rate
of excretion of the particular compound being employed, the
duration of the treatment, other drugs, compounds and/or materials
used in combination with the particular compound employed, the age,
sex, weight, condition, general health and prior medical history of
the patient being treated, and like factors well known in the
medical arts.
[0201] A physician or veterinarian having ordinary skill in the art
can readily determine and prescribe the effective amount of the
pharmaceutical composition required. For example, the physician or
veterinarian could start doses of the compounds of the invention
employed in the pharmaceutical composition at levels lower than
that required in order to achieve the desired therapeutic effect
and gradually increase the dosage until the desired effect is
achieved.
[0202] In general, a suitable daily dose of a compound of the
invention will be that amount of the compound that is the lowest
dose effective to produce a therapeutic effect. Such an effective
dose will generally depend upon the factors described above.
Generally, intravenous and subcutaneous doses of the compounds of
this invention for a patient, when used for the indicated analgesic
effects, will range from about 0.0001 to about 100 mg per kilogram
of body weight per day, more preferably from about 0.01 to about 50
mg per kg per day, and still more preferably from about 1.0 to
about 100 mg per kg per day. An effective amount can be, for
example, an amount that inhibits growth of tumor cells in the
subject.
[0203] If desired, the effective daily dose of the active compound
may be administered as two, three, four, five, six or more
sub-doses administered separately at appropriate intervals
throughout the day, optionally, in unit dosage forms.
[0204] While it is possible for a compound of the present invention
to be administered alone, it is preferable to administer the
compound as a pharmaceutical composition.
XI. Methods of Treatment using a Combination Therapy
[0205] The present invention further describes methods of
suppressing the emergence of MEK1 resistance mutations in a subject
having cancer. Such methods involve the combined administration of
a RAF inhibitor and a second generation MEK1 inhibitor that targets
a mutant MEK1 protein having a substitution at amino acid position
121 as compared to wild-type MEK1 of SEQ ID NO: 1. Preferably, the
second generation MEK1 inhibitor targets a mutant MEK1 protein
having a C121S substitution. Accordingly, the present invention
provides a combination therapy for treating or preventing the
symptoms of a cancer, comprising administration of a RAF inhibitor
and a MEK1 inhibitor that targets a mutant MEK1 protein having a
substitution at amino acid position 121 as compared to wild-type
MEK1 of SEQ ID NO: 1 (preferably a C121S mutation). The term
"combination therapy", as used herein, refers to the administration
of two or more therapeutic substances, e.g., a MEK1 inhibitor or a
RAF inhibitor. The MEK1 inhibitor may be administered concomitant
with, prior to, or following the administration of a RAF inhibitor.
As set forth herein, a combination therapy involving a second
generation MEK1 inhibitor and a RAF inhibitor suppresses the
emergence of MEK resistance mutations to a greater extent than
administration of either a MEK1 inhibitor or a RAF inhibitor alone.
Standard dosages of the second generation MEK1 inhibitor and RAF
inhibitors are also suitable for the combination therapies
described herein. Exemplary cancers that can be treated with a
combination therapy include those described herein, for example,
melanoma, leukemias, lymphomas, myelomas, carcinomas, metastatic
carcinomas, sarcomas, adenomas, nervous system cancers and
geritourinary cancers, e.g., melanoma, myelodysplastic syndrome,
leukemia, pancreatic cancer, ovarian cancer, peritoneal cancer, non
small cell lung cancer, and breast cancer.
[0206] Second generation MEK1 inhibitors that target a mutant MEK1
protein having a substitution at amino acid position 121 as
compared to wild-type MEK1 of SEQ ID NO: 1 (preferably a C121S
mutation) can be identified as described herein. Exemplary RAF
inhibitors useful for combination therapy include pan-RAF
inhibitors, inhibitors of B-RAF, inhibitors of A-RAF, and
inhibitors of RAF-1. In exemplary embodiments RAF inhibitors useful
for combination therapy include PLX4720, PLX4032, BAY 43-9006
(Sorafenib), ZM 336372, RAF 265, AAL-881, LBT-613, and CJS352.
Exemplary RAF inhibitors further include the compounds set forth in
PCT Publication No. WO/2008/028141, the entire contents of which
are incorporated herein by reference. Exemplary RAF inhibitors
additionally include the quinazolinone derivatives described in PCT
Publication No. WO/2006/024836, and the pyridinylquinazolinamine
derivatives described in PCT Publication No. WO/2008/020203, the
entire contents of which are incorporated herein by reference.
[0207] Accordingly, the invention provides methods of treating
cancer in a subject, comprising administering a combination therapy
comprising a second generation MEK1 inhibitor and a RAF inhibitor,
as described herein. In some embodiments, the method further
comprises screening a subject to detect the presence or absence of
one or more mutations in MEK1. In a preferred embodiment, a
combination therapy comprising a second generation MEK inhibitor
and a RAF inhibitor is selected for a subject in whom one or more
mutations in MEK1 are not detected. In such a subject, the
combination therapy advantageously suppresses the emergence of MEK1
resistance mutations. The invention further provides methods of
suppressing the emergence of MEK1 resistance mutations in a subject
having cancer, comprising administering to the subject a
combination therapy comprising a second generation MEK1 inhibitor
and a RAF inhibitor, as described herein. Accordingly, a
combination therapy comprising a second generation MEK1 inhibitor
and a RAF inhibitor may be administered to a subject in whom one or
more MEK1 resistance mutations are detected. Such a combination
therapy suppresses the growth of cells containing MEK1 resistance
mutations. In some embodiments, the method further comprises
screening a subject to detect the presence or absence of one or
more mutations in MEK1. In these embodiments, a combination therapy
comprising a second generation MEK1 inhibitor and a RAF inhibitor
can be given, for example, to a subject in whom a MEK1 resistance
mutation was not detected, or, for example, to a subject in whom a
MEK1 resistance mutation was detected.
[0208] In another embodiment of the invention, a pharmaceutical
composition can comprise (a) a second generation MEK1 inhibitor
that targets a mutant MEK1 protein having a substitution at amino
acid position 121 as compared to wild-type MEK1 of SEQ ID NO: 1
(preferably a C121S mutation) and also (b) a RAF inhibitor. In some
embodiments, the RAF inhibitor can be a pan-RAF inhibitor, an
inhibitor of B-RAF, an inhibitor of A-RAF, and an inhibitor of
RAF-1. In preferred embodiments, the RAF inhibitor can be PLX4720,
PLX4032, BAY 43-9006 (Sorafenib), ZM 336372, RAF 265, AAL-881,
LBT-613, and CJS352. In a particular embodiment, the RAF inhibitor
is (a) PLX4720 or PLX4032.
[0209] Another embodiment of the invention is directed to methods
of administering the pharmaceutical compositions described herein.
Hence, the present invention is directed to a method of treating a
subject for cancer comprising administering to the subject a second
generation MEK1 inhibitor that targets a mutant MEK1 protein having
a substitution at amino acid position 121 as compared to wild-type
MEK1 of SEQ ID NO: 1 (preferably a C121S mutation); and
administering to the subject a pharmaceutical composition
comprising a RAF inhibitor. The second generation MEK1 inhibitor
and the RAF inhibitor can be administered to the subject
sequentially or simultaneously. A sequential administration
includes (a) first administering the second generation MEK1
inhibitor followed by (b) administering the RAF inhibitor. An
alternative sequential administration includes (a) first
administering the RAF inhibitor followed by (b) administering the
second generation MEK1 inhibitor. A simultaneous administration
includes administering the second generation MEK1 inhibitor and the
RAF inhibitor at the same time; or at substantially the same
time.
[0210] When administration involves the separate administration
(e.g., sequential administration) of the first compound (e.g., a
second generation MEK1 inhibitor) and a second compound (e.g., a
RAF inhibitor), as described herein, the compounds are administered
sufficiently close in time to have the desired therapeutic effect.
For example, the period of time between each administration, which
can result in the desired therapeutic effect, can range from
minutes to hours and can be determined based on the properties of
each compound such as potency, solubility, bioavailability, plasma
half-life and kinetic profile. For example, the compounds can be
administered in any order within about 24 hours of each other or
within any time less than 24 hours of each other.
[0211] When the second generation MEK1 inhibitor and the RAF
inhibitor are administered sequentially, they are separately
formulated and can be provided in any order. When the second
generation MEK1 inhibitor and the RAF inhibitor are administered
simultaneously, however, they may be either separately formulated
or combined in the same formulation. When combined in the same
formulation, the second generation MEK1 inhibitor and the RAF
inhibitor can be formulated so as to be released into the subject
at the same time or at different times. The release profile of a
formulation comprising both the second generation MEK1 inhibitor
and the RAF inhibitor includes the following: [0212] A) release and
bioavailability of the second generation MEK1 inhibitor followed by
release and bioavailability of the RAF inhibitor; [0213] B) release
and bioavailability of the RAF inhibitor followed by release and
bioavailability of the second generation MEK1 inhibitor; [0214] C)
release and bioavailability of the second generation MEK1 inhibitor
at the same time (or substantially at the same time as) release and
bioavailability of the RAF inhibitor.
[0215] In another embodiment, provided herein is a method of
treating cancer in a subject in need thereof comprising
administering to the subject a composition comprising a second
generation MEK1 inhibitor and a RAF inhibitor. In still another
embodiment, provided herein is a method of treating cancer in a
subject in need thereof comprising administering to the subject a
composition comprising a second generation MEK1 inhibitor and a RAF
inhibitor, wherein the cancer is selected from the group consisting
of melanoma, leukemias, lymphomas, myelomas, carcinomas, metastatic
carcinomas, sarcomas, adenomas, nervous system cancers and
geritourinary cancers, e.g., melanoma, myelodysplastic syndrome,
leukemia, pancreatic cancer, ovarian cancer, peritoneal cancer, non
small cell lung cancer, and breast cancer.
[0216] A combination of compounds described herein (e.g., a second
generation MEK1 inhibitor and a RAF inhibitor) can either result in
synergistic increase in anti-cancer activity, or such an increase
can be additive. Compositions described herein typically include
lower dosages of each compound in a composition, thereby avoiding
adverse interactions between compounds and/or harmful side effects,
such as ones which have been reported for similar compounds.
Furthermore, normal amounts of each compound when given in
combination could provide for greater efficacy in subjects who are
either unresponsive or minimally responsive to each compound when
used alone. For example, when given in combination, a second
generation MEK1 inhibitor and a RAF inhibitor suppress the
emergence of MEK1 resistance alleles to a greater extent than when
either compound is used alone.
[0217] A synergistic effect can be calculated, for example, using
suitable methods such as the Sigmoid-Emax equation (Holford, N. H.
G. and Scheiner, L. B., Clin. Pharmacokinet. 6: 429-453 (1981)),
the equation of Loewe additivity (Loewe, S, and Muischnek, H.,
Arch. Exp. Pathol Pharmacol. 114: 313-326 (1926)) and the
median-effect equation (Chou, T. C. and Talalay, P., Adv. Enzyme
Regul. 22: 27-55 (1984)). Each equation referred to above can be
applied to experimental data to generate a corresponding graph to
aid in assessing the effects of the drug combination. The
corresponding graphs associated with the equations referred to
above are the concentration-effect curve, isobologram curve and
combination index curve, respectively.
[0218] In certain embodiments, the invention provides a
pharmaceutical composition of any of the compositions of the
present invention. In a related embodiment, the invention provides
a pharmaceutical composition of any of the compositions of the
present invention and a pharmaceutically acceptable carrier or
excipient of any of these compositions.
[0219] In one embodiment, the invention includes a packaged cancer
treatment. The packaged treatment includes a composition of the
invention packaged with instructions for using an effective amount
of the composition of the invention for an intended use. In other
embodiments, the present invention provides a use of any of the
compositions of the invention for manufacture of a medicament to
treat a cancer in a subject.
[0220] In another embodiment of the invention, the second
generation MEK1 inhibitor and the RAF inhibitor can be administered
sequentially (in any order) or simultaneously with other
pharmaceutical agents typically administered to subjects being
treated for cancer. Such other pharmaceutical agents include
without limitation anti-emetics, agents that increase appetite,
other cytotoxic or chemotherapeutic agents, and agents that relieve
pain.
XII. Kits
[0221] Various kits may be assembled as part of the present
invention. A kit can contain components to assay for mutations in
MEK1 to evaluate a particular patient for the risk of developing
resistance to therapy using one or more RAF inhibitors and/or MEK
inhibitor, and thus allow a clinician to determine whether an
alternative treatment for the patient is needed. Such kits can
contain reagents that allow for mutations to be evaluated, such as
primer sets to evaluate mutations correlated with relevant
phenotypic manifestations concerning resistance to a RAF inhibitor
and/or a MEK inhibitor. It is contemplated that primers (or pairs
of primers) that are complementary to or identical to all or part
of SEQ ID NO:1, encoding wild-type human MEK1, for example, can be
part of a kit. In preferred embodiments, the primers can be used to
specifically detect or amplify a nucleic acid molecule encoding a
mutant MEK1 protein containing a mutation at amino acid position
121, such as a C121S mutation. In other embodiments, the kits
contain instructions for using primers that are complementary or
identical to all or part of SEQ ID NO:1 to amplify a nucleotide
sequence encoding a MEK1 protein.
[0222] In other embodiments, the kits comprise compositions for
detecting a mutation comprising a MEK1 protein, such as an antibody
which specifically recognizes a mutant MEK1 protein containing a
resistance mutation. Exemplary proteins include a mutant MEK1
protein containing a mutation at amino acid position 121, such as a
C121S mutation.
[0223] All of the essential materials and reagents required for
assaying for MEK1 mutations by a particular method discussed above
can also be assembled together in a kit. When the components of the
kit are provided in one or more liquid solutions, the liquid
solution preferably is an aqueous solution, with a sterile aqueous
solution being particularly preferred.
[0224] The invention further provides kits comprising compositions,
(e.g., pharmaceutical compositions), comprising a second generation
MEK1 inhibitor that targets a mutant MEK1 protein having a mutation
at amino acid position 121 as compared to the wild-type MEK1 of SEQ
ID NO: 2, such as the C121S mutation. Such kits can be used in the
methods set forth herein, for example, for treating a cancer in a
subject. Accordingly, the kits may further contain instructions
that describe the use of the composition for the treatment of
cancer.
[0225] The invention likewise provides kits comprising compositions
suitable for use in a combination therapy involving a second
generation MEK1 inhibitor that targets a mutant MEK1 protein having
a mutation at amino acid position 121 as compared to the wild-type
MEK1 of SEQ ID NO: 2, such as the C121S mutation, and a RAF
inhibitor. Such kits may include, for example, a composition
comprising a second generation MEK1 inhibitor and a composition
comprising a RAF inhibitor. Such kits may alternatively include a
composition comprising both a second generation MEK1 inhibitor and
a RAF inhibitor. The kits may further contain instructions that
describe the use of the composition for the treatment of cancer,
and/or for suppressing the emergence of MEK1 resistance alleles in
a subject having cancer. The instructions may describe the
administration of a composition comprising a second generation MEK1
inhibitor simultaneously, prior to, or following administration of
a RAF inhibitor.
[0226] The components of the kit can also be provided in dried or
lyophilized forms. When reagents or components are provided as a
dried form, reconstitution generally is by the addition of a
suitable solvent. It is envisioned that the solvent also can be
provided in another container means.
[0227] The kits of the present invention also will typically
include a means for containing the vials in close confinement for
commercial sale such as, e.g., injection or blow-molded plastic
containers into which the desired vials are retained. Irrespective
of the number or type of containers, the kits of the invention also
can comprise, or be packaged with, an instrument for assisting with
sample collection and evaluation. Such an instrument can be an
inhalant, syringe, pipette, forceps, measured spoon, eye dropper or
any such medically approved delivery vehicle, for example.
[0228] The kits of the invention can also include an instruction
sheet outlining suggested alternative therapies when particular
mutations are identified in a patient. For example, an instruction
sheet included with the kits of the invention can recommend that a
patient having a disorder, e.g., a cancer, in which a mutant MEK1
protein has been identified, discontinue treatment with a
first-generation MEK1 inhibitor and/or a RAF inhibitor, be
monitored for relapse during treatment with a first-generation MEK1
inhibitor and/or a RAF inhibitor, continue treatment with a
first-generation MEK1 inhibitor and/or a RAF inhibitor at an
elevated dosage, or initiate treatment with a second-generation
MEK1 inhibitor, alone or in combination with a RAF inhibitor. An
instruction sheet included with the kits of the invention can
likewise recommend that a patient having a disorder in which a
mutant MEK1 protein is not detectable continue treatment with a
first-generation MEK1 inhibitor and/or a RAF inhibitor at a
standard dosage. In exemplary embodiments, the first-generation MEK
inhibitor is CI-1040, AZD6244, PD318088, PD98059, PD334581,
RDEA119, Compound A, or Compound B.
[0229] This invention is further illustrated by the following
examples, which should not be construed as limiting. The contents
of all references, patents and published patent applications cited
throughout this application are incorporated herein by
reference.
EXAMPLES
Example 1
Identification of Activating MEK1 C121S Mutation
[0230] In this example, DNA samples from a patient with metastatic
melanoma who developed resistance to the BRAF inhibitor PLX4032
were used to identify an activating MEK1 mutation that conferred
resistance to both BRAF and MEK1 inhibitors. More specifically,
massively parallel sequencing was used to conduct a comparative
genomic analysis of 3 different DNA samples from the patient: (i)
tumor that was sensitive to PLX4032, (ii) tumor that was resistant
to PLX4032, and (iii) normal skin, to thereby allow for the
identification of the activating MEK1 mutation.
[0231] The patient was a 38 year old man who was diagnosed with
metastatic melanoma in February 2009. He initially presented with a
mass in his right axilla, and a staging PET-CT demonstrated a large
lesion in the right latissimus dorsi as well as multiple
hypermetabolic foci in the lungs, liver, bones and several
subcutaneous sites. Biopsy of the latissimus dorsi mass showed
malignant melanoma. He was treated with several therapeutic
regimens with minimal clinical response. By October 2009, the
patient had developed numerous subcutaneous metastatic deposits.
Genotyping of the BRAF oncogene at this time demonstrated a V600E
mutation, and he enrolled in a clinical trial of the specific BRAF
inhibitor PLX4032. The patient had a profound response to therapy,
with a visible partial response within 2 weeks of starting PLX4032
and near complete regression of all visible tumor nodules by
January 2010, after 15 weeks of therapy.
[0232] In late February 2010, after 4 months of therapy with
PLX4032, the patient rapidly developed new subcutaneous nodules all
over his body and PLX4032 was stopped. At this point, he provided
written informed consent and a percutaneous core biopsy of a
recurrent subcutaneous nodule on the chest was obtained. In order
to determine possible mechanisms of resistance to PLX4032 in this
tumor, the exons from 138 cancer genes from this sample were
sequenced using massively parallel sequencing. The patient
continued to have rapid progression of his disease and died on
hospice in March 2010.
[0233] All exons from 138 cancer genes were sequenced from DNA
isolated from both normal skin and the PLX4032-resistant tumor.
Illumina sequencing libraries were generated from genomic DNA
extracted from the patient's normal skin and resistant tumor
specimen. Targeted hybrid capture of the genomic regions of
interest were performed as previously described (Gnirke, A. et al.
(2009) Nature Biotechnol. 27:182-189). Briefly, approximately 7000
biotinylated RNA baits corresponding to the coding sequence of 138
genes known to undergo somatic genomic alterations in cancer were
designed and synthesized. Genomic DNA libraries were subjected to
solution-phase hybrid capture with the biotinylated RNA baits
followed by massively parallel sequencing. 36 bases from both ends
of the samples were sequenced using an Illumina GAIIx. The
sequencing data were deconvoluted to match all high-quality reads
with the corresponding tumor samples and call base mutations,
insertions, deletions, and copy number alterations.
[0234] A total of 14 somatic base substitutions were seen. Of
these, 9 caused amino acid changes (non-synonymous), all of which
were missense mutations. There were 5 silent (synonymous)
substitutions. There were no insertions, deletions, or significant
copy number alterations. The previously seen BRAF V600E mutation
was again detected. In addition, amino acid changes in ERBB4, FLT1,
MEK1, PTPRD, RET, RUNX1T1, and TERT were seen. To validate these 9
missense mutations, mass spectrometric genotypic assays to each
somatic mutation seen in the tumor sample were designed. All 9 of
the missense mutations were confirmed by mass spectrometric
genotyping. In particular, mutations detected by massively parallel
sequencing were tested for using multi-base hME extension chemistry
by methods known in the art. To further confirm the sequencing
results, the tumor DNA and normal DNA were also sequenced using a
larger independent set targeting exons from approximately 2000
genes, and all 9 mutations were again seen.
[0235] Next, to determine if any of these mutations were de novo
mutations that developed after the patient started PLX4032, the
mass spectrometric genotypic assays were used to query the original
pre-PLX4032 biopsy sample for the presence of these mutations. Two
of the 9 missense mutations detected in the PLX4032-resistant tumor
were not detected in the original PLX4032-sensitive tumor,
suggesting that they were de novo somatic mutations. The first was
a missense mutation (C121S) in MEK1, the kinase immediately
downstream from BRAF, with a mutant allele frequency of 14%. The
other was a missense mutation in RET with a mutant allele frequency
of 28%.
[0236] It has previously been shown that mutations proximal to the
C-helix of MEK1 can confer resistance to BRAF inhibition by PLX4032
by upregulating intrinsic MEK1 kinase activity (Emery, C. M. et
al., (2009) Proc. Natl. Acad. Sci. USA 106:20411-20416). Therefore,
it seemed plausible that the MEK1 C121 mutation, which is also
located proximal to the C-helix, might have caused resistance to
PLX4032 in the patient's recurrent tumor. To test this, the C121S
was introduced mutation into the sequence of wild-type MEK1 and the
mutant cDNA was expressed in the A375 melanoma cell line, which
harbors a BRAF V600E mutation. To introduce the C1212S mutation
into the wild-type MEK1 sequence, MEK1 site-directed mutagenesis
was performed as previously described (Emery, C. M. et al., (2009)
Proc. Natl. Acad. Sci. USA 106:20411-20416). Briefly, MEK1 cDNA was
amplified by PCR and site-directed mutagenesis was conducted using
Quick-Change II (Stratagene) as per the manufacturer's
instructions. As controls, the wild-type MEK1 sequence or a
constitutively active variant of MEK1, referred to as MEK1-DD, was
expressed in the A375 melanoma cells.
[0237] To examine the effect of the MEK1 C121S mutation on
responsiveness of the A375 melanoma cells to inhibitors, A375 cells
(either untransfected cells, or cells transfected with either the
wild-type MEK1, the C121S mutant MEK1, or the constitutively active
variant MEK1-DD) were treated with increasing amounts of either the
BRAF inhibitor PLX4720 (purchased from Symansis, Inc.), which is a
compound closely related to PLX4032, or the MEK1 inhibitor AZD6244
(purchased from Selleck Chemical Co., Ltd.). The results are shown
in the graphs of FIGS. 1A (PLX4720 treatment) and 1B (AZD6244
treatment).
[0238] As shown in FIG. 1A, cells expressing the MEK1 C121S
mutation were strongly resistant to PLX4720, with 100-fold greater
GI-50 values than wild-type A375 cells or A375 cells expressing
wild-type MEK1 (MEK-WT). A375 cells expressing a constitutively
active variant of MEK1 (MEK-DD) showed similar resistance to
PLX4720 as MEK1 C121S. Similarly, as shown in FIG. 1B, cells
expressing the MEK1 C121S mutation were strongly resistant to
AZD6244, with nearly a 1000-fold greater GI-50 value than wild-type
A375 cells, A375 cells expressing MEK-WT, and A375 cells expressing
MEK-DD.
[0239] Biochemical evidence suggests that this resistance is indeed
due to increased kinase activity of MEK1 C121S. To examine this,
immunoblot studies were performed using standard procedures.
Briefly, A375 melanoma cells were lysed with TNN buffer containing
protease inhibitor (Roche), NaF and NaV0.sub.3 (1 mM each). Lysates
were quantified (Bradford assay), denatured (95.degree. C.), and
resolved by SDS gel electrophoresis. Protein was transferred to
nitrocellulose membranes and probed with primary antibodies
recognizing p-ERK1/2, p-MEK1/2 (Ser217/221), MEK1/2 and
.alpha.-tubulin (Cell Signaling Technology; 1:1000 dilution). After
incubation with the appropriate secondary antibody (anti-rabbit or
anti-mouse IgG, HRP-linked; 1:1000 dilution) (Cell Signaling
Technology), proteins were detected using chemiluminescence
(Pierce). The results are summarized in FIGS. 2A and 2B, wherein
FIG. 2A shows the immunoblot results and FIG. 2B is a graph
quantitating the relative percentage of phosphorylated ERK1
(pERK1). As shown in FIGS. 2A and 2B, A375 cells expressing MEK1
C121S had higher levels of phosphorylated ERK1/2 as compared to
A375 cells expressing MEK-WT. A375 cells expressing the
constitutively active MEK1 variant MEK1-DD exhibited even higher
levels of phosphorylated ERK1/2.
[0240] An in vitro kinase assay also demonstrated increased kinase
activity of MEK1 C121S as compared to MEK1-WT. 293T cells at 70%
confluency were transfected with 15 .mu.g pc-DNA-DEST40 containing
MEK-WT, MEK-DD, or MEK C121S. 48 hours post infection, lysate was
generated and pull down with 40 ul of cobalt beads was performed
for 30 mins at 4.degree. C. on 1 mg of whole cell extract.
Following pull down, in vitro kinase assays were performed as
described previously (Emery, C. M. et al., (2009) Proc. Natl. Acad.
Sci. USA 106:20411-20416).
[0241] In summary, a novel activating mutation in MEK1 has been
identified, which arose de novo in a patient with BRAF-mutant
metastatic melanoma who had developed resistance to the selective
BRAF inhibitor PLX4032. The patient was initially highly responsive
PLX4032, but after a dramatic 4-month response he developed rapid
progression of disease. Analysis of a biopsy of the
PLX4032-resistant tumor with massively parallel sequencing revealed
a mutation in MEK1 (C121S) that was not detected in the
pre-treatment, PLX4032-sensitive biopsy sample. This mutation
results in increased kinase activity and confers resistance to
selective BRAF inhibition, as well as MEK1 inhibition, in vitro.
Taken together, this suggests a mechanism by which this patient's
tumor became resistant to BRAF inhibition.
[0242] A MEK1 mutation (P124L) that had arisen in a patient with
metastatic melanoma who had developed resistance to the MEK1
inhibitor AZD6244 was previously described (Emery et al. (2009)
supra). Like MEK1 C121S, MEK1 P124L mutation was proximal to the
C-helix and conferred resistance to both the MEK1 inhibitor AZD6244
as well as the BRAF inhibitor PLX4720. A notable difference,
however, is that the MEK1 C121S mutation seen here arose in the
setting of BRAF inhibition rather than direct MEK1 inhibition.
[0243] Several mechanisms of acquired resistance to targeted kinase
inhibition in patients have been described, the majority of which
can be grouped into two categories. In the first category,
secondary genomic alterations in the target kinase prevent access
of the drug to the mutant kinase through various mechanisms while
maintaining the oncogenic catalytic activity. These alterations are
among the most common mechanisms of acquired resistance to kinase
inhibitors and have been described in BCR-ABL, EGFR, and FLT3. The
development of MEK1 P124L in response to MEK1 inhibition with
AZD6244, as described in Emery et al. (2009) supra, is an example
of this type of resistance mechanism. Notably, we did not detect
any secondary mutations in BRAF in the PLX4032-resistant
sample.
[0244] The second category involves genomic alterations in the
target signaling pathway in genes other than the target of
inhibition. These alterations compensate for the signals lost due
to target inhibition, thereby "bypassing" inhibition of the target
kinase. The best described example of this in patients is
amplification of the MET oncogene, which has been observed in 20%
of EGFR-mutant lung cancers with acquired resistance to gefitinib
MET amplification leads to persistent activation of both PI3K/AKT
and ERK signaling in the presence of EGFR inhibition. Other similar
type mechanisms, including activation of IGF-1R.beta./IRS-1
signaling and signaling via the MET ligand HGF have also been
described in cell lines with acquired resistance to targeted kinase
inhibition.
[0245] The development of a MEK1 mutation in response to
BRAF-inhibition represents the first reported example in a patient
of an acquired resistance mechanism in which the tumor develops an
activating mutation downstream of the target kinase. MEK1 C121S, in
particular, adds an additional layer of complexity, as treatment
with the BRAF inhibitor leads to acquisition of a genetic
alteration which confers resistance to both the BRAF inhibitor as
well as a MEK1 inhibitor. This has clinical significance when
considering allosteric MEK1 inhibitors in patients who progress on
PLX4032 or other BRAF inhibitors. Indeed, combination trials of
combined PLX4032 and AZD6244 are now underway; the development of a
MEK C121S or similar mutation might render a patient simultaneously
resistant to both therapies. Overcoming resistance due to MEK1
C121S thus likely requires inhibition downstream of MEK1 or an
alternative mechanism of inhibiting MEK1.
[0246] Reversion of the target kinase mutation to wildtype has not
been described in patients treated with targeted kinase inhibitors,
suggesting that cancers remain dependent on the original oncogenic
mutations. Consistent with this, we detected continued presence of
the V600E mutation. However, the BRAF mutation was detected at an
allele frequency of 37%, which is lower than the expected 50%.
Although this is likely this is due to "contamination" by normal
DNA from surrounding stroma, we cannot rule out the possibility
that a subset of the tumor cells lost the BRAF V600E mutation. One
intriguing possibility is that tumor cells with the MEK1 C121S
mutation (which was present at an allele frequency of 14%) no
longer required BRAF V600E to activate the MAPK pathway. Supporting
this idea, we have observed a reversion to BRAF wildtype in short
term cultures derived from the AZD6244-resistant patient with
metastatic melanoma with MEK1 P124L. Nevertheless, the fact that
BRAF remains mutated at least in some subset of the cancer cells
indicates that a preferred treatment approach is continued
treatment with a BRAF inhibitor plus a second targeted therapy to
overcome the resistance mutation.
EQUIVALENTS
[0247] The invention has been described herein with reference to
certain examples and embodiments only. No effort has been made to
exhaustively describe all possible examples and embodiments of the
invention. Indeed, those of skill in the art will appreciate that
various additions, deletions, modifications and other changes can
be made to the above-described examples and embodiments, without
departing from the intended spirit and scope of the invention as
recited in the following claims. It is intended that all such
additions, deletions, modifications and other changes be included
within the scope of the following claims.
TABLE-US-00003 SUMMARY OF SEQUENCE LISTING SEQ ID NO: 1 - Wild-type
Human MEK1 Nucleic Acid Sequence (NM_002755; gi:169790828) [The
start codon and the codon encoding amino acid position 121 are in
bold and underlined] aggcgaggct tccccttccc cgcccctccc ccggcctcca
gtccctccca gggccgcttc gcagagcggc taggagcacg gcggcggcgg cactttcccc
ggcaggagct ggagctgggc tctggtgcgc gcgcggctgt gccgcccgag ccggagggac
tggttggttg agagagagag aggaagggaa tcccgggctg ccgaaccgca cgttcagccc
gctccgctcc tgcagggcag cctttcggct ctctgcgcgc gaagccgagt cccgggcggg
tggggcgggg gtccactgag accgctaccg gcccctcggc gctgacggga ccgcgcgggg
cgcacccgct gaaggcagcc ccggggcccg cggcccggac ttggtcctgc gcagcgggcg
cggggcagcg cagcgggagg aagcgagagg tgctgccctc cccccggagt tggaagcgcg
ttacccgggt ccaaaatgcc caagaagaag ccgacgccca tccagctgaa cccggccccc
gacggctctg cagttaacgg gaccagctct gcggagacca acttggaggc cttgcagaag
aagctggagg agctagagct tgatgagcag cagcgaaagc gccttgaggc ctttcttacc
cagaagcaga aggtgggaga actgaaggat gacgactttg agaagatcag tgagctgggg
gctggcaatg gcggtgtggt gttcaaggtc tcccacaagc cttctggcct ggtcatggcc
agaaagctaa ttcatctgga gatcaaaccc gcaatccgga accagatcat aagggagctg
caggttctgc atgagtgcaa ctctccgtac atcgtgggct tctatggtgc gttctacagc
gatggcgaga tcagtatctg catggagcac atggatggag gttctctgga tcaagtcctg
aagaaagctg gaagaattcc tgaacaaatt ttaggaaaag ttagcattgc tgtaataaaa
ggcctgacat atctgaggga gaagcacaag atcatgcaca gagatgtcaa gccctccaac
atcctagtca actcccgtgg ggagatcaag ctctgtgact ttggggtcag cgggcagctc
atcgactcca tggccaactc cttcgtgggc acaaggtcct acatgtcgcc agaaagactc
caggggactc attactctgt gcagtcagac atctggagca tgggactgtc tctggtagag
atggcggttg ggaggtatcc catccctcct ccagatgcca aggagctgga gctgatgttt
gggtgccagg tggaaggaga tgcggctgag accccaccca ggccaaggac ccccgggagg
ccccttagct catacggaat ggacagccga cctcccatgg caatttttga gttgttggat
tacatagtca acgagcctcc tccaaaactg cccagtggag tgttcagtct ggaatttcaa
gattttgtga ataaatgctt aataaaaaac cccgcagaga gagcagattt gaagcaactc
atggttcatg cttttatcaa gagatctgat gctgaggaag tggattttgc aggttggctc
tgctccacca tcggccttaa ccagcccagc acaccaaccc atgctgctgg cgtctaagtg
tttgggaagc aacaaagagc gagtcccctg cccggtggtt tgccatgtcg cttttgggcc
tccttcccat gcctgtctct gttcagatgt gcatttcacc tgtgacaaag gatgaagaac
acagcatgtg ccaagattct actcttgtca tttttaatat tactgtcttt attcttatta
ctattattgt tcccctaagt ggattggctt tgtgcttggg gctatttgtg tgtatgctga
tgatcaaaac ctgtgccagg ctgaattaca gtgaaatttt ggtgaatgtg ggtagtcatt
cttacaattg cactgctgtt cctgctccat gactggctgt ctgcctgtat tttcgggatt
ctttgacatt tggtggtact ttattcttgc tgggcatact ttctctctag gagggagcct
tgtgagatcc ttcacaggca gtgcatgtga agcatgcttt gctgctatga aaatgagcat
cagagagtgt acatcatgtt attttattat tattatttgc ttttcatgta gaactcagca
gttgacatcc aaatctagcc agagcccttc actgccatga tagctggggc ttcaccagtc
tgtctactgt ggtgatctgt agacttctgg ttgtatttct atatttattt tcagtatact
gtgtgggata cttagtggta tgtctcttta agttttgatt aatgtttctt aaatggaatt
attttgaatg tcacaaattg atcaagatat taaaatgtcg gatttatctt tccccatatc
caagtaccaa tgctgttgta aacaacgtgt atagtgccta aaattgtatg aaaatccttt
taaccatttt aacctagatg tttaacaaat ctaatctctt attctaataa atatactatg
aaataaaaaa aaaaggatga aagctaaaaa aaaaaaaaaa aaa SEQ ID NO: 2 -
Wild-type Human MEK1 Amino Acid Sequence (NP_002746; gi:5579478)
[Cysteine at amino acid position 121 is in bold and underlined]
mpkkkptpiq lnpapdgsav ngtssaetnl ealqkkleel eldeqqrkrl eafltqkqkv
gelkdddfek iselgagngg vvfkvshkps glvmarklih leikpairnq iirelqvlhe
cnspyivgfy gafysdgeis icmehmdggs ldqvlkkagr ipeqilgkvs iavikgltyl
rekhkimhrd vkpsnilvns rgeiklcdfg vsgqlidsma nsfvgtrsym sperlqgthy
svqsdiwsmg lslvemavgr ypipppdake lelmfgcqve gdaaetpprp rtpgrplssy
gmdsrppmai felldyivne pppklpsgvf slefqdfvnk cliknpaera dlkqlmvhaf
ikrsdaeevd fagwlcstig lnqpstptha agv SEQ ID NO: 3 - Mutant MEK1
C121S Nucleic Acid Sequence start codon and the codon encoding
amino acid position 121 are in bold and underlined; n = a, c, g or
t] aggcgaggct tccccttccc cgcccctccc ccggcctcca gtccctccca
gggccgcttc gcagagcggc taggagcacg gcggcggcgg cactttcccc ggcaggagct
ggagctgggc tctggtgcgc gcgcggctgt gccgcccgag ccggagggac tggttggttg
agagagagag aggaagggaa tcccgggctg ccgaaccgca cgttcagccc gctccgctcc
tgcagggcag cctttcggct ctctgcgcgc gaagccgagt cccgggcggg tggggcgggg
gtccactgag accgctaccg gcccctcggc gctgacggga ccgcgcgggg cgcacccgct
gaaggcagcc ccggggcccg cggcccggac ttggtcctgc gcagcgggcg cggggcagcg
cagcgggagg aagcgagagg tgctgccctc cccccggagt tggaagcgcg ttacccgggt
ccaaaatgcc caagaagaag ccgacgccca tccagctgaa cccggccccc gacggctctg
cagttaacgg gaccagctct gcggagacca acttggaggc cttgcagaag aagctggagg
agctagagct tgatgagcag cagcgaaagc gccttgaggc ctttcttacc cagaagcaga
aggtgggaga actgaaggat gacgactttg agaagatcag tgagctgggg gctggcaatg
gcggtgtggt gttcaaggtc tcccacaagc cttctggcct ggtcatggcc agaaagctaa
ttcatctgga gatcaaaccc gcaatccgga accagatcat aagggagctg caggttctgc
atgagtcnaa ctctccgtac atcgtgggct tctatggtgc gttctacagc gatggcgaga
tcagtatctg catggagcac atggatggag gttctctgga tcaagtcctg aagaaagctg
gaagaattcc tgaacaaatt ttaggaaaag ttagcattgc tgtaataaaa ggcctgacat
atctgaggga gaagcacaag atcatgcaca gagatgtcaa gccctccaac atcctagtca
actcccgtgg ggagatcaag ctctgtgact ttggggtcag cgggcagctc atcgactcca
tggccaactc cttcgtgggc acaaggtcct acatgtcgcc agaaagactc caggggactc
attactctgt gcagtcagac atctggagca tgggactgtc tctggtagag atggcggttg
ggaggtatcc catccctcct ccagatgcca aggagctgga gctgatgttt gggtgccagg
tggaaggaga tgcggctgag accccaccca ggccaaggac ccccgggagg ccccttagct
catacggaat ggacagccga cctcccatgg caatttttga gttgttggat tacatagtca
acgagcctcc tccaaaactg cccagtggag tgttcagtct ggaatttcaa gattttgtga
ataaatgctt aataaaaaac cccgcagaga gagcagattt gaagcaactc atggttcatg
cttttatcaa gagatctgat gctgaggaag tggattttgc aggttggctc tgctccacca
tcggccttaa ccagcccagc acaccaaccc atgctgctgg cgtctaagtg tttgggaagc
aacaaagagc gagtcccctg cccggtggtt tgccatgtcg cttttgggcc tccttcccat
gcctgtctct gttcagatgt gcatttcacc tgtgacaaag gatgaagaac acagcatgtg
ccaagattct actcttgtca tttttaatat tactgtcttt attcttatta ctattattgt
tcccctaagt ggattggctt tgtgcttggg gctatttgtg tgtatgctga tgatcaaaac
ctgtgccagg ctgaattaca gtgaaatttt ggtgaatgtg ggtagtcatt cttacaattg
cactgctgtt cctgctccat gactggctgt ctgcctgtat tttcgggatt ctttgacatt
tggtggtact ttattcttgc tgggcatact ttctctctag gagggagcct tgtgagatcc
ttcacaggca gtgcatgtga agcatgcttt gctgctatga aaatgagcat cagagagtgt
acatcatgtt attttattat tattatttgc ttttcatgta gaactcagca gttgacatcc
aaatctagcc agagcccttc actgccatga tagctggggc ttcaccagtc tgtctactgt
ggtgatctgt agacttctgg ttgtatttct atatttattt tcagtatact gtgtgggata
cttagtggta tgtctcttta agttttgatt aatgtttctt aaatggaatt attttgaatg
tcacaaattg atcaagatat taaaatgtcg gatttatctt tccccatatc caagtaccaa
tgctgttgta aacaacgtgt atagtgccta aaattgtatg aaaatccttt taaccatttt
aacctagatg tttaacaaat ctaatctctt attctaataa atatactatg aaataaaaaa
aaaaggatga aagctaaaaa aaaaaaaaaa aaa SEQ ID NO: 4 - Mutant MEK1
C121S Amino Acid Sequence [Serine at amino acid position 121 is in
bold and underlined] mpkkkptpiq lnpapdgsav ngtssaetnl ealqkkleel
eldeqqrkrl eafltqkqkv gelkdddfek iselgagngg vvfkvshkps glvmarklih
leikpairnq iirelqvlhe snspyivgfy gafysdgeis icmehmdggs ldqvlkkagr
ipeqilgkvs iavikgltyl rekhkimhrd vkpsnilvns rgeiklcdfg vsgqlidsma
nsfvgtrsym sperlqgthy svqsdiwsmg lslvemavgr ypipppdake lelmfgcqve
gdaaetpprp rtpgrplssy gmdsrppmai felldyivne pppklpsgvf slefqdfvnk
cliknpaera dlkqlmvhaf ikrsdaeevd fagwlcstig lnqpstptha agv
Sequence CWU 1
1
412603DNAHomo sapiens 1aggcgaggct tccccttccc cgcccctccc ccggcctcca
gtccctccca gggccgcttc 60gcagagcggc taggagcacg gcggcggcgg cactttcccc
ggcaggagct ggagctgggc 120tctggtgcgc gcgcggctgt gccgcccgag
ccggagggac tggttggttg agagagagag 180aggaagggaa tcccgggctg
ccgaaccgca cgttcagccc gctccgctcc tgcagggcag 240cctttcggct
ctctgcgcgc gaagccgagt cccgggcggg tggggcgggg gtccactgag
300accgctaccg gcccctcggc gctgacggga ccgcgcgggg cgcacccgct
gaaggcagcc 360ccggggcccg cggcccggac ttggtcctgc gcagcgggcg
cggggcagcg cagcgggagg 420aagcgagagg tgctgccctc cccccggagt
tggaagcgcg ttacccgggt ccaaaatgcc 480caagaagaag ccgacgccca
tccagctgaa cccggccccc gacggctctg cagttaacgg 540gaccagctct
gcggagacca acttggaggc cttgcagaag aagctggagg agctagagct
600tgatgagcag cagcgaaagc gccttgaggc ctttcttacc cagaagcaga
aggtgggaga 660actgaaggat gacgactttg agaagatcag tgagctgggg
gctggcaatg gcggtgtggt 720gttcaaggtc tcccacaagc cttctggcct
ggtcatggcc agaaagctaa ttcatctgga 780gatcaaaccc gcaatccgga
accagatcat aagggagctg caggttctgc atgagtgcaa 840ctctccgtac
atcgtgggct tctatggtgc gttctacagc gatggcgaga tcagtatctg
900catggagcac atggatggag gttctctgga tcaagtcctg aagaaagctg
gaagaattcc 960tgaacaaatt ttaggaaaag ttagcattgc tgtaataaaa
ggcctgacat atctgaggga 1020gaagcacaag atcatgcaca gagatgtcaa
gccctccaac atcctagtca actcccgtgg 1080ggagatcaag ctctgtgact
ttggggtcag cgggcagctc atcgactcca tggccaactc 1140cttcgtgggc
acaaggtcct acatgtcgcc agaaagactc caggggactc attactctgt
1200gcagtcagac atctggagca tgggactgtc tctggtagag atggcggttg
ggaggtatcc 1260catccctcct ccagatgcca aggagctgga gctgatgttt
gggtgccagg tggaaggaga 1320tgcggctgag accccaccca ggccaaggac
ccccgggagg ccccttagct catacggaat 1380ggacagccga cctcccatgg
caatttttga gttgttggat tacatagtca acgagcctcc 1440tccaaaactg
cccagtggag tgttcagtct ggaatttcaa gattttgtga ataaatgctt
1500aataaaaaac cccgcagaga gagcagattt gaagcaactc atggttcatg
cttttatcaa 1560gagatctgat gctgaggaag tggattttgc aggttggctc
tgctccacca tcggccttaa 1620ccagcccagc acaccaaccc atgctgctgg
cgtctaagtg tttgggaagc aacaaagagc 1680gagtcccctg cccggtggtt
tgccatgtcg cttttgggcc tccttcccat gcctgtctct 1740gttcagatgt
gcatttcacc tgtgacaaag gatgaagaac acagcatgtg ccaagattct
1800actcttgtca tttttaatat tactgtcttt attcttatta ctattattgt
tcccctaagt 1860ggattggctt tgtgcttggg gctatttgtg tgtatgctga
tgatcaaaac ctgtgccagg 1920ctgaattaca gtgaaatttt ggtgaatgtg
ggtagtcatt cttacaattg cactgctgtt 1980cctgctccat gactggctgt
ctgcctgtat tttcgggatt ctttgacatt tggtggtact 2040ttattcttgc
tgggcatact ttctctctag gagggagcct tgtgagatcc ttcacaggca
2100gtgcatgtga agcatgcttt gctgctatga aaatgagcat cagagagtgt
acatcatgtt 2160attttattat tattatttgc ttttcatgta gaactcagca
gttgacatcc aaatctagcc 2220agagcccttc actgccatga tagctggggc
ttcaccagtc tgtctactgt ggtgatctgt 2280agacttctgg ttgtatttct
atatttattt tcagtatact gtgtgggata cttagtggta 2340tgtctcttta
agttttgatt aatgtttctt aaatggaatt attttgaatg tcacaaattg
2400atcaagatat taaaatgtcg gatttatctt tccccatatc caagtaccaa
tgctgttgta 2460aacaacgtgt atagtgccta aaattgtatg aaaatccttt
taaccatttt aacctagatg 2520tttaacaaat ctaatctctt attctaataa
atatactatg aaataaaaaa aaaaggatga 2580aagctaaaaa aaaaaaaaaa aaa
26032393PRTHomo sapiens 2Met Pro Lys Lys Lys Pro Thr Pro Ile Gln
Leu Asn Pro Ala Pro Asp 1 5 10 15 Gly Ser Ala Val Asn Gly Thr Ser
Ser Ala Glu Thr Asn Leu Glu Ala 20 25 30 Leu Gln Lys Lys Leu Glu
Glu Leu Glu Leu Asp Glu Gln Gln Arg Lys 35 40 45 Arg Leu Glu Ala
Phe Leu Thr Gln Lys Gln Lys Val Gly Glu Leu Lys 50 55 60 Asp Asp
Asp Phe Glu Lys Ile Ser Glu Leu Gly Ala Gly Asn Gly Gly 65 70 75 80
Val Val Phe Lys Val Ser His Lys Pro Ser Gly Leu Val Met Ala Arg 85
90 95 Lys Leu Ile His Leu Glu Ile Lys Pro Ala Ile Arg Asn Gln Ile
Ile 100 105 110 Arg Glu Leu Gln Val Leu His Glu Cys Asn Ser Pro Tyr
Ile Val Gly 115 120 125 Phe Tyr Gly Ala Phe Tyr Ser Asp Gly Glu Ile
Ser Ile Cys Met Glu 130 135 140 His Met Asp Gly Gly Ser Leu Asp Gln
Val Leu Lys Lys Ala Gly Arg 145 150 155 160 Ile Pro Glu Gln Ile Leu
Gly Lys Val Ser Ile Ala Val Ile Lys Gly 165 170 175 Leu Thr Tyr Leu
Arg Glu Lys His Lys Ile Met His Arg Asp Val Lys 180 185 190 Pro Ser
Asn Ile Leu Val Asn Ser Arg Gly Glu Ile Lys Leu Cys Asp 195 200 205
Phe Gly Val Ser Gly Gln Leu Ile Asp Ser Met Ala Asn Ser Phe Val 210
215 220 Gly Thr Arg Ser Tyr Met Ser Pro Glu Arg Leu Gln Gly Thr His
Tyr 225 230 235 240 Ser Val Gln Ser Asp Ile Trp Ser Met Gly Leu Ser
Leu Val Glu Met 245 250 255 Ala Val Gly Arg Tyr Pro Ile Pro Pro Pro
Asp Ala Lys Glu Leu Glu 260 265 270 Leu Met Phe Gly Cys Gln Val Glu
Gly Asp Ala Ala Glu Thr Pro Pro 275 280 285 Arg Pro Arg Thr Pro Gly
Arg Pro Leu Ser Ser Tyr Gly Met Asp Ser 290 295 300 Arg Pro Pro Met
Ala Ile Phe Glu Leu Leu Asp Tyr Ile Val Asn Glu 305 310 315 320 Pro
Pro Pro Lys Leu Pro Ser Gly Val Phe Ser Leu Glu Phe Gln Asp 325 330
335 Phe Val Asn Lys Cys Leu Ile Lys Asn Pro Ala Glu Arg Ala Asp Leu
340 345 350 Lys Gln Leu Met Val His Ala Phe Ile Lys Arg Ser Asp Ala
Glu Glu 355 360 365 Val Asp Phe Ala Gly Trp Leu Cys Ser Thr Ile Gly
Leu Asn Gln Pro 370 375 380 Ser Thr Pro Thr His Ala Ala Gly Val 385
390 32603DNAHomo sapiensmisc_feature(838)..(838)n is a, c, g, or t
3aggcgaggct tccccttccc cgcccctccc ccggcctcca gtccctccca gggccgcttc
60gcagagcggc taggagcacg gcggcggcgg cactttcccc ggcaggagct ggagctgggc
120tctggtgcgc gcgcggctgt gccgcccgag ccggagggac tggttggttg
agagagagag 180aggaagggaa tcccgggctg ccgaaccgca cgttcagccc
gctccgctcc tgcagggcag 240cctttcggct ctctgcgcgc gaagccgagt
cccgggcggg tggggcgggg gtccactgag 300accgctaccg gcccctcggc
gctgacggga ccgcgcgggg cgcacccgct gaaggcagcc 360ccggggcccg
cggcccggac ttggtcctgc gcagcgggcg cggggcagcg cagcgggagg
420aagcgagagg tgctgccctc cccccggagt tggaagcgcg ttacccgggt
ccaaaatgcc 480caagaagaag ccgacgccca tccagctgaa cccggccccc
gacggctctg cagttaacgg 540gaccagctct gcggagacca acttggaggc
cttgcagaag aagctggagg agctagagct 600tgatgagcag cagcgaaagc
gccttgaggc ctttcttacc cagaagcaga aggtgggaga 660actgaaggat
gacgactttg agaagatcag tgagctgggg gctggcaatg gcggtgtggt
720gttcaaggtc tcccacaagc cttctggcct ggtcatggcc agaaagctaa
ttcatctgga 780gatcaaaccc gcaatccgga accagatcat aagggagctg
caggttctgc atgagtcnaa 840ctctccgtac atcgtgggct tctatggtgc
gttctacagc gatggcgaga tcagtatctg 900catggagcac atggatggag
gttctctgga tcaagtcctg aagaaagctg gaagaattcc 960tgaacaaatt
ttaggaaaag ttagcattgc tgtaataaaa ggcctgacat atctgaggga
1020gaagcacaag atcatgcaca gagatgtcaa gccctccaac atcctagtca
actcccgtgg 1080ggagatcaag ctctgtgact ttggggtcag cgggcagctc
atcgactcca tggccaactc 1140cttcgtgggc acaaggtcct acatgtcgcc
agaaagactc caggggactc attactctgt 1200gcagtcagac atctggagca
tgggactgtc tctggtagag atggcggttg ggaggtatcc 1260catccctcct
ccagatgcca aggagctgga gctgatgttt gggtgccagg tggaaggaga
1320tgcggctgag accccaccca ggccaaggac ccccgggagg ccccttagct
catacggaat 1380ggacagccga cctcccatgg caatttttga gttgttggat
tacatagtca acgagcctcc 1440tccaaaactg cccagtggag tgttcagtct
ggaatttcaa gattttgtga ataaatgctt 1500aataaaaaac cccgcagaga
gagcagattt gaagcaactc atggttcatg cttttatcaa 1560gagatctgat
gctgaggaag tggattttgc aggttggctc tgctccacca tcggccttaa
1620ccagcccagc acaccaaccc atgctgctgg cgtctaagtg tttgggaagc
aacaaagagc 1680gagtcccctg cccggtggtt tgccatgtcg cttttgggcc
tccttcccat gcctgtctct 1740gttcagatgt gcatttcacc tgtgacaaag
gatgaagaac acagcatgtg ccaagattct 1800actcttgtca tttttaatat
tactgtcttt attcttatta ctattattgt tcccctaagt 1860ggattggctt
tgtgcttggg gctatttgtg tgtatgctga tgatcaaaac ctgtgccagg
1920ctgaattaca gtgaaatttt ggtgaatgtg ggtagtcatt cttacaattg
cactgctgtt 1980cctgctccat gactggctgt ctgcctgtat tttcgggatt
ctttgacatt tggtggtact 2040ttattcttgc tgggcatact ttctctctag
gagggagcct tgtgagatcc ttcacaggca 2100gtgcatgtga agcatgcttt
gctgctatga aaatgagcat cagagagtgt acatcatgtt 2160attttattat
tattatttgc ttttcatgta gaactcagca gttgacatcc aaatctagcc
2220agagcccttc actgccatga tagctggggc ttcaccagtc tgtctactgt
ggtgatctgt 2280agacttctgg ttgtatttct atatttattt tcagtatact
gtgtgggata cttagtggta 2340tgtctcttta agttttgatt aatgtttctt
aaatggaatt attttgaatg tcacaaattg 2400atcaagatat taaaatgtcg
gatttatctt tccccatatc caagtaccaa tgctgttgta 2460aacaacgtgt
atagtgccta aaattgtatg aaaatccttt taaccatttt aacctagatg
2520tttaacaaat ctaatctctt attctaataa atatactatg aaataaaaaa
aaaaggatga 2580aagctaaaaa aaaaaaaaaa aaa 26034393PRTHomo sapiens
4Met Pro Lys Lys Lys Pro Thr Pro Ile Gln Leu Asn Pro Ala Pro Asp 1
5 10 15 Gly Ser Ala Val Asn Gly Thr Ser Ser Ala Glu Thr Asn Leu Glu
Ala 20 25 30 Leu Gln Lys Lys Leu Glu Glu Leu Glu Leu Asp Glu Gln
Gln Arg Lys 35 40 45 Arg Leu Glu Ala Phe Leu Thr Gln Lys Gln Lys
Val Gly Glu Leu Lys 50 55 60 Asp Asp Asp Phe Glu Lys Ile Ser Glu
Leu Gly Ala Gly Asn Gly Gly 65 70 75 80 Val Val Phe Lys Val Ser His
Lys Pro Ser Gly Leu Val Met Ala Arg 85 90 95 Lys Leu Ile His Leu
Glu Ile Lys Pro Ala Ile Arg Asn Gln Ile Ile 100 105 110 Arg Glu Leu
Gln Val Leu His Glu Ser Asn Ser Pro Tyr Ile Val Gly 115 120 125 Phe
Tyr Gly Ala Phe Tyr Ser Asp Gly Glu Ile Ser Ile Cys Met Glu 130 135
140 His Met Asp Gly Gly Ser Leu Asp Gln Val Leu Lys Lys Ala Gly Arg
145 150 155 160 Ile Pro Glu Gln Ile Leu Gly Lys Val Ser Ile Ala Val
Ile Lys Gly 165 170 175 Leu Thr Tyr Leu Arg Glu Lys His Lys Ile Met
His Arg Asp Val Lys 180 185 190 Pro Ser Asn Ile Leu Val Asn Ser Arg
Gly Glu Ile Lys Leu Cys Asp 195 200 205 Phe Gly Val Ser Gly Gln Leu
Ile Asp Ser Met Ala Asn Ser Phe Val 210 215 220 Gly Thr Arg Ser Tyr
Met Ser Pro Glu Arg Leu Gln Gly Thr His Tyr 225 230 235 240 Ser Val
Gln Ser Asp Ile Trp Ser Met Gly Leu Ser Leu Val Glu Met 245 250 255
Ala Val Gly Arg Tyr Pro Ile Pro Pro Pro Asp Ala Lys Glu Leu Glu 260
265 270 Leu Met Phe Gly Cys Gln Val Glu Gly Asp Ala Ala Glu Thr Pro
Pro 275 280 285 Arg Pro Arg Thr Pro Gly Arg Pro Leu Ser Ser Tyr Gly
Met Asp Ser 290 295 300 Arg Pro Pro Met Ala Ile Phe Glu Leu Leu Asp
Tyr Ile Val Asn Glu 305 310 315 320 Pro Pro Pro Lys Leu Pro Ser Gly
Val Phe Ser Leu Glu Phe Gln Asp 325 330 335 Phe Val Asn Lys Cys Leu
Ile Lys Asn Pro Ala Glu Arg Ala Asp Leu 340 345 350 Lys Gln Leu Met
Val His Ala Phe Ile Lys Arg Ser Asp Ala Glu Glu 355 360 365 Val Asp
Phe Ala Gly Trp Leu Cys Ser Thr Ile Gly Leu Asn Gln Pro 370 375 380
Ser Thr Pro Thr His Ala Ala Gly Val 385 390
* * * * *