U.S. patent application number 14/735973 was filed with the patent office on 2015-12-03 for mek mutations conferring resistance to mek inhibitors.
The applicant listed for this patent is Caroline Emery, Carlos Garcia-Echeverria, Levi A. Garraway. Invention is credited to Caroline Emery, Carlos Garcia-Echeverria, Levi A. Garraway.
Application Number | 20150346204 14/735973 |
Document ID | / |
Family ID | 41647153 |
Filed Date | 2015-12-03 |
United States Patent
Application |
20150346204 |
Kind Code |
A1 |
Garraway; Levi A. ; et
al. |
December 3, 2015 |
MEK Mutations Conferring Resistance to MEK Inhibitors
Abstract
The present invention relates to methods, compositions and kits
concerning resistance to treatment with an anti-cancer agent,
specifically an inhibitor of MEK. In particular embodiments, the
invention concerns mutations in a MEK sequence that confer
resistance to a MEK inhibitor. Identification of such mutations in
a MEK sequence allows the identification and design of
second-generation MEK inhibitors. Methods and kits for detecting
the presence of a mutant MEK sequence in a sample are also
provided.
Inventors: |
Garraway; Levi A.; (Newton,
MA) ; Emery; Caroline; (Cambridge, MA) ;
Garcia-Echeverria; Carlos; (Saint-Cloud, FR) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Garraway; Levi A.
Emery; Caroline
Garcia-Echeverria; Carlos |
Newton
Cambridge
Saint-Cloud |
MA
MA |
US
US
FR |
|
|
Family ID: |
41647153 |
Appl. No.: |
14/735973 |
Filed: |
June 10, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13139060 |
Aug 31, 2011 |
9084781 |
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PCT/US2009/067472 |
Dec 10, 2009 |
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14735973 |
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61121467 |
Dec 10, 2008 |
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61180739 |
May 22, 2009 |
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Current U.S.
Class: |
506/11 ;
435/252.33; 435/320.1; 435/366; 435/7.2; 435/7.6 |
Current CPC
Class: |
A61P 35/00 20180101;
G01N 2333/912 20130101; A61K 38/00 20130101; C12N 9/1205 20130101;
G01N 33/573 20130101; A61K 31/4184 20130101; A61P 43/00 20180101;
G01N 2500/04 20130101; A61P 35/02 20180101 |
International
Class: |
G01N 33/573 20060101
G01N033/573 |
Claims
1. A method of identifying a compound that is useful as a MEK
inhibitor that inhibits a mutant MEK polypeptide having MEK
activity, comprising: a) providing a cell comprising a mutant MEK
polypeptide having MEK activity, the mutant MEK polypeptide
comprising an amino acid sequence having at least one amino acid
substitution as compared to a wild type MEK1 polypeptide (SEQ ID
NO: 2) or a MEK2 polypeptide (SEQ ID NO:4), the at least one amino
acid substitution occurring at one or more amino acid positions
selected from the group consisting of Q56, I99, I103, K104, I111,
R113, L115, H119, E120, P124, G128, F129, F133, E203, V211, L215,
G237, V258, G276, G286, M302, P326, and H399 of SEQ ID NO: 2 or
Q60, I103, I107, K108, I115, R117, L119, H123, E124, P128, G132,
F133, F137, E207, V215, L219, G241, V262, G280, M310, and P334 of
SEQ ID NO: 4; b) contacting the cell with a test compound; and c)
determining the effect of the compound on MEK activity or
determining the effect of the compound on cell proliferation or
cell viability; wherein downmodulation of the MEK activity or
reduction in cell proliferation or a reduction in cell viability as
compared to a suitable control identifies the compound as a
compound that is useful as a MEK inhibitor that inhibits the mutant
MEK polypeptide having MEK activity.
2. The method according to claim 1, wherein the at least one amino
acid substitution occurs at one or more amino acid positions
selected from the group consisting of Q56P, I99T, I103, K104N,
I111N, R113S, L115P, L115P, H119P, E120D, P124L, P124S, G128D,
F129L, F133L, E203, V211D, L215P, G237V, V258G, G276E, G286E,
M302R, P326L, and H399D of SEQ ID NO: 2 or Q60P, I103T, I107N,
K108N, I115N, R117S, L119R, L119P, H123P, E124D, P128L, P128S,
G132D, F133L, F137L, E207K, V215D, L219P, G241V, V262G, G280E,
M310R, and P334L of SEQ ID NO: 4.
3. The method according to claim 1, wherein the at least one amino
acid substitution occurs at one or more amino acid positions
selected from the group consisting of Q56, I99, I103, K104, I111,
R113, L115, H119, E120, P124, G128, F129, F133, E203, V211, L215,
G237, V258, G276, G286, M302, P326, and H399 of SEQ ID NO: 2.
4. The method according to claim 1, wherein the at least one amino
acid substitution occurs at one or more amino acid positions
selected from the group consisting of Q56P, I99T, I103, K104N,
I111N, R113S, L115P, L115P, H119P, E120D, P124L, P124S, G128D,
F129L, F133L, E203, V211D, L215P, G237V, V258G, G276E, G286E,
M302R, P326L, and H399D of SEQ ID NO: 2.
5. The method of claim 1, wherein the downmodulation of MEK
activity comprises downmodulation of phosphorylation of an ERK1 or
ERK2 substrate.
6. The method of claim 1, comprising determining the effect of the
compound on cell proliferation.
7. The method of claim 6, wherein a shift in the GI50 value of at
least 2 fold identifies the compound as a compound that is useful
as a MEK inhibitor that inhibits the mutant MEK polypeptide having
MEK activity.
8. An expression vector comprising an isolated nucleic acid
molecule encoding a mutant MEK polypeptide having MEK activity, the
mutant MEK polypeptide comprising an amino acid sequence having at
least one amino acid substitution as compared to a wild type MEK1
polypeptide (SEQ ID NO: 2) or a MEK2 polypeptide (SEQ ID NO:4), the
at least one amino acid substitution occurring at one or more amino
acid positions selected from the group consisting of Q56, I99,
I103, K104, I111, R113, L115, H119, E120, P124, G128, F129, F133,
E203, V211, L215, G237, V258, G276, G286, M302, P326, and H399 of
SEQ ID NO: 2 or Q60, I103, I107, K108, I115, R117, L119, H123,
E124, P128, G132, F133, F137, E207, V215, L219, G241, V262, G280,
M310, and P334 of SEQ ID NO: 4.
9. The expression vector according to claim 8, wherein the at least
one amino acid substitution occurs at one or more amino acid
positions selected from the group consisting of Q56P, I99T, I103,
K104N, I111N, R113S, L115P, L115P, H119P, E120D, P124L, P124S,
G128D, F129L, F133L, E203, V211D, L215P, G237V, V258G, G276E,
G286E, M302R, P326L, and H399D of SEQ ID NO: 2 or Q60P, I103T,
I107N, K108N, I115N, R117S, L119R, L119P, H123P, E124D, P128L,
P128S, G132D, F133L, F137L, E207K, V215D, L219P, G241V, V262G,
G280E, M310R, and P334L of SEQ ID NO: 4.
10. The expression vector according to claim 8, wherein the at
least one amino acid substitution occurs at one or more amino acid
positions selected from the group consisting of Q56P, I99T, I103,
K104N, I111N, R113S, L115P, L115P, H119P, E120D, P124L, P124S,
G128D, F129L, F133L, E203, V211D, L215P, G237V, V258G, G276E,
G286E, M302R, P326L, and H399D of SEQ ID NO: 2.
11. A host cell comprising the expression vector of claim 8.
12. A cell-based screening method for identifying a test compound
as a MEK inhibitor that inhibits a mutant MEK polypeptide having
MEK activity, the method comprising: contacting a host cell
comprising the expression vector of claim 8 with a test compound,
measuring sensitivity of the host cell to the test compound using
an assay selected from the group consisting of a cell proliferation
assay, a cell viability assay, and MEK activity assay, wherein a
reduction in cell proliferation, cell viability, or MEK activity in
the presence of the test compound identifies the host cell as
having sensitivity to the test compound and wherein sensitivity of
the host cell to the test compound identifies the compound as a MEK
inhibitor that inhibits the mutant MEK polypeptide having MEK
activity.
13. The method of claim 12, wherein sensitivity of the host cell to
the test compound is measured using the MEK activity assay
comprising a an ERK phosphorylation assay, wherein a reduction in
ERK phosphorylation in the presence of the test compound identifies
the compound as a MEK inhibitor that inhibits the mutant MEK
polypeptide having MEK activity.
14. A method of identifying a compound as a MEK inhibitor that
inhibits a mutant MEK polypeptide having MEK activity, comprising:
(a) providing an assay composition comprising a mutant MEK
polypeptide having MEK activity, the mutant MEK polypeptide
comprising an amino acid sequence having at least one amino acid
substitution as compared to a wild type MEK1 polypeptide (SEQ ID
NO: 2) or a MEK2 polypeptide (SEQ ID NO:4), the at least one amino
acid substitution occurring at one or more amino acid positions
selected from the group consisting of Q56, I99, I103, K104, I111,
R113, L1115, H119, E120, P124, G128, F129, F133, E203, V211, L215,
G237, V258, G276, G286, M302, P326, and H399 of SEQ ID NO: 2 or
Q60, I103, I107, K108, I115, R117, L119, H123, E124, P128, G132,
F133, F137, E207, V215, L219, G241, V262, G280, M310, and P334 of
SEQ ID NO: 4 and a MEK substrate; (b) 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 (c) determining the effect of the compound on
phosphorylation of the MEK substrate; wherein downmodulation of
phosphorylation of the MEK substrate as compared to a suitable
control identifies the compound as a compound that inhibits the
mutant MEK polypeptide having MEK activity.
15. The method according to claim 14, wherein the at least one
amino acid substitution occurs at one or more amino acid positions
selected from the group consisting of Q56P, I99T, I103, K104N,
I111N, R113S, L115P, L115P, H119P, E120D, P124L, P124S, G128D,
F129L, F133L, E203, V211D, L215P, G237V, V258G, G276E, G286E,
M302R, P326L, and H399D of SEQ ID NO: 2 or Q60P, I103T, I107N,
K108N, I115N, R117S, L119R, L119P, H123P, E124D, P128L, P128S,
G132D, F133L, F137L, E207K, V215D, L219P, G241V, V262G, G280E,
M310R, and P334L of SEQ ID NO: 4.
16. The method according to claim 14, wherein the at least one
amino acid substitution occurs at one or more amino acid positions
selected from the group consisting of Q56P, I99T, I103, K104N,
I111N, R113S, L115P, L115P, H119P, E120D, P124L, P124S, G128D,
F129L, F133L, E203, V211D, L215P, G237V, V258G, G276E, G286E,
M302R, P326L, and H399D of SEQ ID NO: 2.
17. The method of claim 14, wherein the MEK substrate is ERK1 or
ERK2.
18. A method of identifying a compound as a MEK inhibitor that
inhibits a mutant MEK polypeptide having MEK activity, comprising:
a) selecting a potential drug using computer-assisted modeling with
a three-dimensional crystal or solution structure of a mutant MEK
polypeptide, wherein the mutant MEK polypeptide comprises an amino
acid sequence having at least one amino acid substitution as
compared to a wild type MEK1 polypeptide (SEQ ID NO: 2) or a MEK2
polypeptide (SEQ ID NO:4), the at least one amino acid substitution
conferring resistance to one or more MEK inhibitors on the mutant
MEK polypeptide; b) contacting the potential drug with the mutant
MEK polypeptide; and c) detecting the interaction of the potential
drug with the mutant MEK polypeptide; wherein a compound that is
capable of interacting with the mutant MEK polypeptide is
identified as a MEK inhibitor that inhibits the mutant MEK
polypeptide having MEK activity.
19. The method of claim 18, wherein the at least one amino acid
substitution occurring at one or more amino acid positions selected
from the group consisting of Q56, I99, I103, K104, I111, R113,
L115, H119, E120, P124, G128, F129, F133, E203, V21, L215, G237,
V258, G276, G286, M302, P326, and H399 of SEQ ID NO: 2 or Q60,
I103, I107, K108, I115, R117, L119, H123, E124, P128, G132, F133,
F137, E207, V215, L219, G241, V262, G280, M310, and P334 of SEQ ID
NO: 4.
20. The method of claim 18, wherein the test compound is a member
of a library of compounds.
Description
RELATED APPLICATIONS
[0001] This application is a divisional application of U.S. patent
application Ser. No. 13/139,060 filed Jun. 10, 2011, which claims
the benefit under 35 U.S.C. .sctn.371 of International Application
No. PCT/US2009/067472, filed Dec. 10, 2009, which claims the
benefit of U.S. Provisional Patent Application No. 61/121,467,
filed on Dec. 10, 2008, and U.S. Provisional Patent Application No.
61/180,739, filed on May 22, 2009. The entire contents of the
foregoing patent applications are incorporated herein by
reference.
SEQUENCE LISTING
[0002] The instant application contains a Sequence Listing which
has been submitted electronically in ASCII format and is hereby
incorporated by reference in its entirety. The ASCII copy, created
on Jun. 7, 2015, is named 14293-430 SEQ listing_ST25.txt and is
approximately 14.1 KB in size.
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.
SUMMARY OF THE INVENTION
[0004] The present invention pertains to mutation-mediated
resistance to chemotherapeutic treatment of cancer. In specific
embodiments, the present invention is directed to mutations
identified in MEK polypeptides, and in nucleic acid molecules
encoding the MEK polypeptides. These mutations confer resistance to
MEK inhibitors currently in therapeutic use. The identification of
these mutations allows for the development of second-generation MEK
inhibitors that exhibit activity against a MEK polypeptide
containing one or more mutations, such as the mutations described
herein. Such second-generation MEK inhibitors are useful in many
clinical and therapeutic applications, including the treatment of
cancer.
[0005] Accordingly, the invention features, in a first aspect, an
isolated nucleic acid molecule encoding a mutant MEK polypeptide
having a MEK activity, wherein said mutant MEK polypeptide
comprises an amino acid sequence having at least one amino acid
substitution as compared to a wild type MEK polypeptide selected
from the group consisting of MEK1 (SEQ ID NO:2) and MEK2 (SEQ ID
NO:4), the at least one amino acid substitution conferring
resistance to one or more MEK inhibitors on the mutant MEK protein.
In one embodiment, the wild type MEK polypeptide is MEK1 and the at
least one substitution occurs at one or more amino acid positions
selected from the group consisting of 67D, 74L, 103I, 106A, 107I,
111I, 115L, 119H, 124P, 128G, 129F, 167G, 203E, 211V, 247W, 258V,
260R, 276G, 286G, 302M, 303D, 308M, 328G and 399H. In another
embodiment, the wild type MEK polypeptide is MEK1 and the at least
one substitution occurs at one or more amino acid positions
selected from the group consisting of 56Q, 99I, 104K, 113R, 120E,
128G, 133F, 215L, 237G, 326P and 368E. In one embodiment, the wild
type MEK polypeptide is MEK2 and the at least one substitution
occurs at one or more amino acid positions selected from the group
consisting of 71D, 78L, 107I, 110A, 111I, 115I, 119L, 123I, 128P,
132G, 133F, 171G, 207E, 215V, 251W, 262V, 264R, 280G, 310M, 311D,
316M, and 336G. In another embodiment, the wild type MEK
polypeptide is MEK2, and the at least one substitution occurs at
one or more amino acid positions selected from the group consisting
of 60Q, 103I, 108K, 117R, 124E, 132G, 137F, 219L, 241G, 334P and
376E. In an exemplary embodiment, the wild type MEK polypeptide is
MEK1 and the at least one substitution is selected from the group
consisting of 67D>N, 74L>Q, 74L>R, 103I>N, 106A>T,
107I>M, 111I>N, 115L>R, 115L>P, 119H>P, 124P>L,
124P>S, 128G>D, 129F>L, 167G>R, 203E>K, 211V>D,
247W>G, 258V>G, 260R>G, 276G>E, 286G>E, 302M>R,
303D>A, 308M>L, 328G>L, 328G>R, 379I>L and
399H>D. In another exemplary embodiment, the wild type MEK
polypeptide is MEK1 and the at least one substitution is selected
from the group consisting of 56Q>P, 99I>T, 104K>N,
113R>S, 120E>D, 128G>D, 133F>L, 215L>P, 237G>V,
326P>L, 368E>K. In another exemplary embodiment, the wild
type MEK polypeptide is MEK2 and the at least one substitution is
selected from the group consisting of 71D>N, 78L>Q, 78L>R,
107I>N, 110A>T, 111I>M, 115I>N, 19L>R, 19L>P,
123H>P, 128P>L, 128P>S, 132G>D, 133F>L, 171G>R,
207E>K, 215V>D, 251 W>G, 262V>G, 264R>G, 280G>E,
310M>R, 311D>A, 316M>L, 336G>L, and 336G>R. In
another exemplary embodiment, the wild type MEK polypeptide is MEK2
and the at least one substitution is selected from the group
consisting of 60Q>P, 103I>T, 108K>N, 117R>S, 124E>D,
132G>D, 137F>L, 219L>P, 241G>V, 334P>L,
376E>K.
[0006] In one embodiment of the foregoing aspect, the mutant MEK
polypeptide has one to five amino acid substitutions as compared to
the wild type MEK polypeptide. In another embodiment, the mutant
MEK polypeptide has one amino acid substitution as compared to the
wild type MEK polypeptide. In an exemplary embodiment, the mutant
MEK polypeptide is a MEK1 polypeptide comprising a 111I>N
substitution, a 115L>R substitution, a 124P>L substitution
and/or a 129F>L substitution. In another exemplary embodiment,
mutant MEK polypeptide is a MEK2 polypeptide comprising a 115I>N
substitution, a 119L>R substitution, a 128P>L substitution or
a 133F>L substitution. In various embodiments of the foregoing
aspect, the MEK inhibitor is selected from the group consisting of
CI-1040, AZD6244, PD318088, PD98059, PD334581, RDEA119,
6-Methoxy-7-(3-morpholin-4-yl-propoxy)-4-(4-phenoxy-phenylamino)-quinolin-
e-3-carbonitrile (referenced herein as "Compound A"), and
4-[3-Chloro-4-(1-methyl-1H-imidazol-2-ylsulfanyl)-phenylamino]-6-methoxy--
7-(3-morpholin-4-yl-propoxy)-quinoline-3-carbonitrile (referenced
herein as "Compound B").
[0007] The isolated nucleic acid molecules encoding mutant MEK
polypeptides 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 MEK polypeptide, comprising culturing a host cell containing
an expression vector encoding a mutant MEK polypeptide, such that a
mutant MEK polypeptide is produced by the cell.
[0008] In other aspects, the invention features an isolated mutant
MEK polypeptide having a MEK activity, wherein said mutant MEK
polypeptide comprises an amino acid sequence having at least one
amino acid substitution as compared to a wild type MEK polypeptide
selected from the group consisting of MEK1 (SEQ ID NO: 2) and MEK2
(SEQ ID NO:4), the at least one amino acid substitution conferring
resistance to one or more MEK inhibitors on the mutant MEK
polypeptide. In one embodiment of this aspect, the wild type MEK
polypeptide is MEK1 and the at least one substitution occurs at one
or more amino acid positions selected from the group consisting of
67D, 74L, 103I, 106A, 107I, 111I, 115L, 119H, 124P, 128G, 129F,
167G, 203E, 211V, 247W, 258V, 260R, 276G, 286G, 302M, 303D, 308M,
328G and 399H. In another embodiment, the wild type MEK polypeptide
is MEK1 and the at least one substitution occurs at one or more
amino acid positions selected from the group consisting of 56Q,
99I, 104K, 113R, 120E, 128G, 133F, 215L, 237G, 326P and 368E. In
one embodiment, the wild type MEK polypeptide is MEK2 and the at
least one substitution occurs at one or more amino acid positions
selected from the group consisting of 71D, 78L, 107I, 110A, 111I,
115I, 119L, 123H, 128P, 132G, 133F, 171G, 207E, 215V, 251W, 262V,
264R, 280G, 310M, 311D, 316M, and 336G. In another embodiment, the
wild type MEK polypeptide is MEK2, and the at least one
substitution occurs at one or more amino acid positions selected
from the group consisting of 60Q, 103I, 108K, 117R, 124E, 132G,
137F, 219L, 241G, 334P and 376E. In an exemplary embodiment, the
wild type MEK polypeptide is MEK1 and the at least one substitution
is selected from the group consisting of 67D>N, 74L>Q,
74L>R, 103I>N, 106A>T, 107I>M, 111I>N, 115L>R,
115L>P, 119H>P, 124P>L, 124P>S, 128G>D, 129F>L,
167G>R, 203E>K, 211V>D, 247W>G, 258V>G, 260R>G,
276G>E, 286G>E, 302M>R, 303D>A, 308M>L, 328G>L,
328G>R, 379I>L and 399H>D. In another exemplary
embodiment, the wild type MEK polypeptide is MEK1 and the at least
one substitution is selected from the group consisting of 56Q>P,
99I>T, 104K>N, 113R>S, 120E>D, 128G>D, 133F>L,
215L>P, 237G>V, 326P>L and 368E>K. In another exemplary
embodiment, the wild type MEK polypeptide is MEK2 and the at least
one substitution is selected from the group consisting of 71D>N,
78L>Q, 78L>R, 107I>N, 110A>T, 111I>M, 115I>N,
119L>R, 119L>P, 123H>P, 128P>L, 128P>S, 132G>D,
133F>L, 171G>R, 207E>K, 215V>D, 251W>G, 262V>G,
264R>G, 280G>E, 310M>R, 311 D>A, 316M>L, 336G>L,
and 336G>R. In another exemplary embodiment, the wild type MEK
polypeptide is MEK2 and the at least one substitution is selected
from the group consisting of 60Q>P, 103I>T, 108K>N,
117R>S, 124E>D, 132G>D, 137F>L, 219L>P, 241G>V,
334P>L and 376E>K.
[0009] In one embodiment of the foregoing aspect, the mutant MEK
polypeptide has one to five amino acid substitutions as compared to
the wild type MEK polypeptide. In another embodiment, the mutant
MEK polypeptide has one amino acid substitution as compared to the
wild type MEK polypeptide. In an exemplary embodiment, the mutant
MEK polypeptide is a MEK1 polypeptide that comprises a 111I>N
substitution, a 115L>R substitution, a 124P>L substitution
and/or a 129F>L substitution. In another exemplary embodiment,
mutant MEK polypeptide is a MEK2 polypeptide that comprises a
115I>N substitution, a 119L>R substitution, a 128P>L
substitution or a 133F>L substitution. In various embodiments of
the foregoing aspect, the MEK inhibitor is selected from the group
consisting of CI-1040, AZD6244, PD318088, PD98059, PD334581,
RDEA119, Compound A, and Compound B.
[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 MEK
polypeptide and a MEK substrate; 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 on phosphorylation of the
MEK substrate; wherein downmodulation of phosphorylation of the MEK
substrate as compared to a suitable control identifies the compound
as a compound that is useful in treating cancer.
[0011] In a related aspect, the invention features a method of
identifying a compound as a second generation MEK inhibitor,
comprising: providing an assay composition comprising a mutant MEK
polypeptide and a MEK substrate; 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 on 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 MEK inhibitor.
[0012] In exemplary embodiments of the foregoing aspects, the MEK
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 MEK polypeptide;
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 MEK inhibitor, comprising: providing a cell comprising a
mutant MEK polypeptide; 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 MEK inhibitor.
[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 MEK polypeptide;
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 MEK inhibitor, comprising:
providing a cell comprising a mutant MEK polypeptide; 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 MEK inhibitor.
[0015] In another aspect, the invention features a cell-based
screening method for identifying a test compound as a second
generation MEK inhibitor, the method comprising contacting the host
cell comprising a mutant MEK polypeptide with a test compound,
wherein sensitivity of the host cell to the test compound
identifies the compound as a second-generation MEK 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 MEK inhibitor.
[0016] In another aspect, the invention features a method of
identifying a second-generation MEK inhibitor, comprising:
selecting a potential drug using computer-assisted modeling with a
three-dimensional crystal or solution structure of a mutant MEK
polypeptide, wherein said mutant MEK polypeptide comprises an amino
acid sequence having at least one amino acid substitution as
compared to a wild type MEK polypeptide selected from the group
consisting of MEK1 (SEQ ID NO: 2) and MEK2 (SEQ ID NO:4), the at
least one amino acid substitution conferring resistance to one or
more MEK inhibitors on the mutant MEK polypeptide; contacting said
potential drug with the mutant MEK polypeptide; and detecting the
interaction of said potential drug with the mutant MEK polypeptide;
wherein a compound that is capable of interacting with the mutant
MEK polypeptide is identified as a second-generation MEK
inhibitor.
[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 MEK
polypeptide. Accordingly, in another aspect, the invention features
a method of inhibiting the activity of a mutant MEK polypeptide,
comprising contacting the mutant MEK polypeptide with a compound
identified according to one ofthe foregoing methods. In an
exemplary embodiment, the compound inhibits the activity of a
mutant MEK polypeptide and a wild-type MEK polypeptide. 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
first generation MEK inhibitor selected from the group consisting
of CI-1040, AZD6244, PD318088, PD98059, PD334581. RDEA119, Compound
A, and Compound B. 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 MEK polypeptide and a wild-type MEK
polypeptide. In one embodiment, the subject has relapsed from
treatment with a first generation MEK inhibitor selected from the
group consisting of CI-1040, AZD6244, PD318088, PD98059, PD334581,
RDEA119, Compound A, and Compound B. In a particular embodiment,
the cancer comprises a MEK polypeptide having one or more mutations
with respect to a wild type MEK polypeptide, and/or a MEK nucleic
acid molecule encoding a MEK polypeptide having one or more
mutations with respect to a wild type MEK polypeptide, wherein the
one or more mutations confer resistance to one or more MEK
inhibitors on the mutant MEK polypeptide. 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 one or more amino acid substitutions in
a wild type MEK polypeptide selected from the group consisting of
MEK1 (SEQ ID NO: 2) and MEK2 (SEQ ID NO:4) in the cancer
cell-containing sample. In one embodiment of this aspect, the wild
type MEK polypeptide is MEK1 and the one or more amino acid
substitutions occur at one or more residues selected from the group
consisting of 67D, 74L, 103I, 106A, 107I, 111I, 115L, 119H, 124P,
128G, 129F, 167G, 203E, 211V, 247W, 258V, 260R, 276G, 286G, 302M,
303D, 308M, 3280 and 399H. In another embodiment, the wild type MEK
polypeptide is MEK1 and the one or more amino acid substitutions
occur at one or more residues selected from the group consisting of
56Q, 99I, 104K, 113R, 120E, 128G, 133F, 215L, 237G, 326P and 368E.
In one embodiment, the wild type MEK polypeptide is MEK2 and the
one or more amino acid substitutions occur at one or more residues
selected from the group consisting of 71D, 78L, 107I, 110A, 111I,
115I, 119L, 123H, 128P, 132G, 133F, 171G, 207E, 215V, 251W, 262V,
264R, 280G, 310M, 311D, 316M, and 336G. In another embodiment, the
wild type MEK polypeptide is MEK2 and the one or more amino acid
substitutions occur at one or more residues selected from the group
consisting of 60Q, 103I, 108K, 117R, 124E, 132G, 137F, 219L, 241G,
334P and 376E.
[0021] In one embodiment of the foregoing aspect, detection of one
or more mutations in the MEK1 or MEK2 polypeptides identifies the
subject as having a relatively high risk of relapse during
treatment with a first generation MEK inhibitor. In another
embodiment, detection of one or more mutations in the MEK1 or MEK2
polypeptides identifies the subject as being unresponsive to
treatment with a first generation MEK inhibitor. In exemplary
embodiments, the first generation MEK inhibitor is selected from
the group consisting of CI-1040, AZD6244, PD318088, PD98059,
PD334581, RDEA119, Compound A, and Compound B. In another
embodiment of the foregoing aspect, the presence of one or more
mutations in the MEK1 or MEK2 polypeptides stratifies the subject
to treatment with a second generation MEK inhibitor. In another
embodiment, the presence of one or more mutations in the MEK
polypeptide stratifies the subject to treatment with a combination
therapy that includes administration of both a MEK inhibitor (e.g.,
a first generation MEK inhibitor or a second generation MEK
inhibitor) and a RAF inhibitor.
[0022] 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 and a RAF inhibitor, comprising (a)
assaying a nucleic acid sample obtained from the cancer for the
presence of one or more mutations in a nucleic acid molecule
encoding a MEK polypeptide that alter the identity of one or more
amino acid residues of the encoded MEK polypeptide relative to the
amino acid at one or more positions selected from the group
consisting of 56Q, 67D, 74L, 99I, 103I, 104K, 106A, 107I, 111I,
113R, 115L, 119H, 120E, 124P, 128G, 129F, 133F, 167G, 203E, 211V,
215L, 237G, 247W, 258V, 260R, 276G, 286G, 302M, 303D, 308M, 326P,
328G, 368E and 399H of SEQ ID NO:2 and/or assaying a nucleic acid
sample obtained from the cancer for the presence of one or more
mutations in a nucleic acid molecule encoding a MEK polypeptide
that alter the identity of one or more amino acid residues of the
encoded MEK polypeptide relative to the amino acid at one or more
positions selected from the group consisting of 60Q, 71D, 78L,
103I, 107I, 108K, 110A, 111I, 115I, 117R, 119L, 123H, 124E, 128P,
132G, 133F, 137F, 171G, 207E, 215V, 219L, 241G, 251W, 262V, 264R,
280G, 310M, 311D, 316M, 334P, 336G, and 376E of SEQ ID NO:4; and
(b) correlating the presence of the one or more mutations in a
nucleic acid molecule encoding a MEK polypeptide with a subject who
is likely to benefit from treatment with a MEK inhibitor and a RAF
inhibitor.
[0023] In another aspect, the invention provides a method of
optimizing treatment of a subject having cancer, comprising: (a)
extracting nucleic acid from cells of the cancer, and (b)
sequencing a nucleic acid molecule encoding a MEK polypeptide;
wherein the presence of nucleotides that alter the identity of an
amino acid residue at one or more amino acids of the encoded MEK
polypeptide relative to the amino acid at one or more positions
selected from the group consisting of 56Q, 67D, 74L, 99I, 103I,
104K, 106A, 107I, 111I, 113R, 115L, 119H, 120E, 124P, 128G, 129F,
133F, 167G, 203E, 211V, 215L, 237G, 247W, 258V, 260R, 276G, 286G,
302M, 303D, 308M, 326P, 328G, 368E and 399H of SEQ ID NO:2, or the
presence of nucleotides that alter the identity of an amino acid
residue at one or more amino acids of the encoded MEK polypeptide
relative to the amino acid at one or more positions selected from
the group consisting of 60Q, 71D, 78L, 103I, 107I, 108K, 110A,
111I, 115I, 117R, 119L, 123H, 124E, 128P, 132G, 133F, 137F, 171G,
207E, 215V, 219L, 241G, 251W, 262V, 264R, 280G, 310M, 311 D, 316M,
334P, 336G, and 376E of SEQ ID NO:4, indicates a need to treat the
subject with a MEK inhibitor and a RAF inhibitor.
[0024] In another aspect, the invention provides a method of
optimizing treatment of a subject having cancer, comprising: (a)
extracting nucleic acid from cells of the cancer, and (b)
subjecting the sample to PCR and identifying the nucleotide
sequence of a nucleic acid molecule encoding a MEK polypeptide;
wherein the presence of nucleotides that alter the identity of an
amino acid residue at one or more amino acid of the encoded MEK
polypeptide relative to the amino acid at one or more positions
selected from the group consisting of 56Q, 67D, 74L, 99I, 103I,
104K, 106A, 107I, 111I, 113R, 115L, 119H, 120E, 124P, 128G, 129F,
133F, 167G, 203E, 211V, 215L, 237G, 247W, 258V, 260R, 276G, 286G,
302M, 303D, 308M, 326P, 328G, 368E and 399H of SEQ ID NO:2, or the
presence of nucleotides that alter the identity of an amino acid
residue at one or more amino acid of the encoded MEK polypeptide
relative to the amino acid at one or more positions selected from
the group consisting of 60Q, 71D, 78L, 103I, 107I, 108K, 110A,
111I, 115I, 117R, 119L, 123H, 124E, 128P, 132G, 133F, 137F, 171G,
207E, 215V, 219L, 241G, 251W, 262V, 264R, 280G, 310M, 311D, 316M,
334P, 336G, and 376E of SEQ ID NO:4, indicates a need to treat the
subject with a MEK inhibitor and a RAF inhibitor.
[0025] In another aspect, the invention provides 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 MEK polypeptide; and (c) administering a
MEK inhibitor and a RAF inhibitor to the subject when the nucleic
acid molecule contains nucleotides that alter the identity of an
amino acid residue at one or more amino acid of the encoded MEK
polypeptide relative to the amino acid at one or more positions
selected from the group consisting of 56Q, 67D, 74L, 99I, 103I,
104K, 106A, 107I, 111I, 113R, 115L, 119H, 120E, 124P, 128G, 129F,
133F, 167G, 203E, 211V, 215L, 237G, 247W, 258V, 260R, 276G, 286G,
302M, 303D, 308M, 326P, 328G, 368E and 399H of SEQ ID NO:2, or when
the nucleic acid molecule contains nucleotides that alter the
identity of an amino acid residue at one or more amino acid of the
encoded MEK polypeptide relative to the amino acid at one or more
positions selected from the group consisting of 60Q, 71D, 78L,
103I, 107I, 108K, 110A, 111I, 115I, 117R, 119L, 123H, 124E, 128P,
132G, 133F, 137F, 171G, 207E, 215V, 219L, 241G, 251W, 262V, 264R,
280G, 310M, 311D, 316M, 334P, 336G, and 376E of SEQ ID NO:4.
[0026] In another aspect, the invention provides a method of
treating a subject having cancer, comprising: (a) extracting
nucleic acid from cells of the cancer; (b) subjecting the sample to
PCR and identifying the nucleotide sequence of a nucleic acid
molecule encoding a MEK polypeptide; and (c) administering a MEK
inhibitor and a RAF inhibitor to the subject when the nucleic acid
molecule contains nucleotides that alter the identity of an amino
acid residue at one or more amino acids of the encoded MEK
polypeptide relative to the amino acid at one or more positions
selected from the group consisting of 56Q, 67D, 74L, 99I, 103I,
104K, 106A, 107I, 111I, 113R, 115L, 119H, 120E, 124P, 128G, 129F,
133F, 167G, 203E, 211V, 215L, 237G, 247W, 258V, 260R, 276G, 286G,
302M, 303D, 308M, 326P, 328G, 368E and 399H of SEQ ID NO:2, or when
the nucleic acid molecule contains nucleotides that alter the
identity of an amino acid residue at one or more amino acid of the
encoded MEK polypeptide relative to the amino acid at one or more
positions selected from the group consisting of 60Q, 71 D, 78L,
103I, 107I, 108K, 110A, 111I, 115I, 117R, 119L, 123H, 124E, 128P,
132G, 133F, 137F, 171G, 207E, 215V, 219L, 241G, 251W, 262V, 264R,
280G, 310M, 311D, 316M, 334P, 336G, and 376E of SEQ ID NO:4.
[0027] In exemplary embodiments of the foregoing aspects, the RAF
inhibitor is PLX4720, PLX4032, BAY 43-9006 (Sorafenib). ZM 336372,
RAF 265, AAL-881, LBT-613, or CJS352.
[0028] 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 first-generation MEK inhibitor,
comprising: (a) extracting nucleic acid from cells of the cancer,
and (b) sequencing a nucleic acid molecule encoding a MEK
polypeptide; wherein the presence of nucleotides that alter the
identity of an amino acid residue at one or more amino acids of the
encoded MEK polypeptide relative to the amino acid at one or more
positions selected from the group consisting of 56Q, 67D, 74L, 99I,
103I, 104K, 106A, 107I, 111I, 113R, 115L, 119H, 120E, 124P, 128G,
129F, 133F, 167G, 203E, 211V, 215L, 237G, 247W, 258V, 260R, 276G,
286G, 302M, 303D, 308M, 326P, 328G, 368E and 399H of SEQ ID NO:2,
or the presence of nucleotides that alter the identity of an amino
acid residue at one or more amino acids of the encoded MEK
polypeptide relative to the amino acid at one or more positions
selected from the group consisting of 60Q, 71 D, 78L, 103I, 107I,
108K, 110A, 111I, 115I, 117R, 119L, 123H, 124E, 128P, 132G, 133F,
137F, 171G, 207E, 215V, 219L, 241G, 251W, 262V, 264R, 280G, 310M,
311D, 316M, 334P, 336G, and 376E of SEQ ID NO:4, identifies the
subject as having a high risk of relapse during treatment with a
first-generation MEK inhibitor.
[0029] 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 first generation MEK inhibitor,
comprising: (a) assaying a nucleic acid sample obtained from the
cancer for the presence of one or more mutations in a nucleic acid
molecule encoding a MEK polypeptide that alter the identity of one
or more amino acid residues of the encoded MEK polypeptide relative
to the amino acid at one or more positions selected from the group
consisting of 56Q, 67D, 74L, 99I, 103I, 104K, 106A, 107I, 111I,
113R, 115L, 119H, 120E, 124P, 128G, 129F, 133F, 167G, 203E, 211V,
215L, 237G, 247W, 258V, 260R, 276G, 286G, 302M, 303D, 308M, 326P,
328G, 368E and 399H of SEQ ID NO:2 and/or assaying a nucleic acid
sample obtained from the cancer for the presence of one or more
mutations in a nucleic acid molecule encoding a MEK polypeptide
that alter the identity of one or more amino acid residues of the
encoded MEK polypeptide relative to the amino acid at one or more
positions selected from the group consisting of 60Q, 71D, 78L,
103I, 107I, 108K, 110A, 111I, 115I, 117R, 119L, 123H, 124E, 128P,
132G, 133F, 137F, 171G, 207E, 215V, 219L, 241G, 251W, 262V, 264R,
280G, 310M, 311D, 316M, 334P, 336G, and 376E of SEQ ID NO:4; and
(b) correlating the presence of the one or more mutations in a
nucleic acid molecule encoding a MEK polypeptide with a subject
having a high risk of relapse during treatment with a first
generation MEK inhibitor.
[0030] In another aspect, the invention provides a method of
identifying a subject having cancer as being unresponsive to
treatment with a first-generation MEK inhibitor, comprising: (a)
extracting nucleic acid from cells of the cancer, and (b)
sequencing a nucleic acid molecule encoding a MEK polypeptide;
wherein the presence of nucleotides that alter the identity of an
amino acid residue at one or more amino acids of the encoded MEK
polypeptide relative to the amino acid at one or more positions
selected from the group consisting of 56Q, 67D, 74L, 99I, 103I,
104K, 106A, 107I, 111I, 113R, 115L, 119H, 120E, 124P, 128G, 129F,
133F, 167G, 203E, 211V, 215L, 237G, 247W, 258V, 260R, 276G, 286G,
302M, 303D, 308M, 326P, 328G, 368E and 399H of SEQ ID NO:2, or the
presence of nucleotides that alter the identity of an amino acid
residue at one or more amino acids of the encoded MEK polypeptide
relative to the amino acid at one or more positions selected from
the group consisting of 60Q, 71D, 78L, 103I, 107I, 108K, 10A, 111I,
115I, 117R, 119L, 123H, 124E, 128P, 132G, 133F, 137F, 171G, 207E,
215V, 219L, 241G, 251W, 262V, 264R, 280G, 310M, 311D, 316M, 334P,
336G, and 376E of SEQ ID NO:4, identifies the subject as being
unresponsive to treatment with a first-generation MEK
inhibitor.
[0031] In another aspect, the invention provides a method of
identifying a subject having cancer as being unresponsive to
treatment with a first-generation MEK inhibitor, comprising: (a)
assaying a nucleic acid sample obtained from the cancer for the
presence of one or more mutations in a nucleic acid molecule
encoding a MEK polypeptide that alter the identity of one or more
amino acid residues of the encoded MEK polypeptide relative to the
amino acid at one or more positions selected from the group
consisting of 56Q, 67D, 74L, 99I, 103I, 104K, 106A, 107I, 111I,
113R, 115L, 119H, 120E, 124P, 128G, 129F, 133F, 167G, 203E, 211V,
215L, 237G, 247W, 258V, 260R, 276G, 286G, 302M, 303D, 308M, 326P,
328G, 368E and 399H of SEQ ID NO:2 and/or assaying a nucleic acid
sample obtained from the cancer for the presence of one or more
mutations in a nucleic acid molecule encoding a MEK polypeptide
that alter the identity of one or more amino acid residues of the
encoded MEK polypeptide relative to the amino acid at one or more
positions selected from the group consisting of 60Q, 71 D, 78L,
103I, 107I, 108K, 110A, 111I, 115I, 117R, 119L, 123H, 124E, 128P,
132G, 133F, 137F, 171G, 207E, 215V, 219L, 241G, 251W, 262V, 264R,
280G, 310M, 311D, 316M, 334P, 336G, and 376E of SEQ ID NO:4; and
(b) correlating the presence of the one or more mutations in a
nucleic acid molecule encoding a MEK polypeptide with a subject who
is unresponsive to treatment with a first-generation MEK
inhibitor.
[0032] In exemplary embodiments of the foregoing aspects, the MEK
inhibitor is CI-1040, AZD6244, PD318088, PD98059, PD334581,
RDEA119, Compound A, or Compound B.
[0033] In another aspect, the invention provides a method of
identifying a subject having cancer who is likely to benefit from
treatment with a second-generation MEK inhibitor, comprising: (a)
extracting nucleic acid from cells of the cancer; and (b)
sequencing a nucleic acid molecule encoding a MEK polypeptide;
wherein the presence of nucleotides that alter the identity of an
amino acid residue at one or more amino acids of the encoded MEK
polypeptide relative to the amino acid at one or more positions
selected from the group consisting of 56Q, 67D, 74L, 99I, 103I,
104K, 106A, 107I, 111I, 113R, 115I, 119H, 120E, 124P, 128G, 129F,
133F, 167G, 203E, 211V, 215L, 2370, 247W, 258V, 260R, 276G, 286G,
302M, 303D, 308M, 326P, 328G, 368E and 399H of SEQ ID NO:2, or the
presence of nucleotides that alter the identity of an amino acid
residue at one or more amino acids of the encoded MEK polypeptide
relative to the amino acid at one or more positions selected from
the group consisting of 60Q, 71 D, 78L, 103I, 107I, 108K, 110A,
111I, 115I, 117R, 119L, 123H, 124E, 128P, 132G, 133F, 137F, 171G,
207E, 215V, 219L, 241G, 251W, 262V, 264R, 280G, 310M, 311D, 316M,
334P, 336G, and 376E of SEQ ID NO:4, identifies the subject as
being likely to benefit from treatment with a second-generation MEK
inhibitor.
[0034] In another aspect, the invention provides a method of
identifying a subject having cancer who is likely to benefit from
treatment with a second-generation MEK inhibitor, comprising: (a)
assaying a nucleic acid sample obtained from the cancer for the
presence of one or more mutations in a nucleic acid molecule
encoding a MEK polypeptide that alter the identity of one or more
amino acid residues of the encoded MEK polypeptide relative to the
amino acid at one or more positions selected from the group
consisting of 56Q, 67 D, 74L, 99I, 103I, 104K, 106A, 107I, 111I,
113R, 115L, 119H, 120E, 124P, 128G, 129F, 133F, 167G, 203E, 211V,
215L, 237G, 247W, 258V, 260R, 276G, 286G, 302M, 303D, 308M, 326P,
328G, 368E and 399H of SEQ ID NO:2 and/or assaying a nucleic acid
sample obtained from the cancer for the presence of one or more
mutations in a nucleic acid molecule encoding a MEK polypeptide
that alter the identity of one or more amino acid residues of the
encoded MEK polypeptide relative to the amino acid at one or more
positions selected from the group consisting of 60Q, 71D, 78L,
103I, 107I, 108K, 110A, 111I, 115I, 117R, 119L, 123H, 124E, 128P,
132G, 133F, 137F, 171G, 207E, 215V, 219L, 241G, 251W, 262V, 264R,
280G, 310M, 311D, 316M, 334P, 336G, and 376E of SEQ ID NO:4; and
(b) correlating the presence of the one or more mutations in a
nucleic acid molecule encoding a MEK polypeptide with a subject who
is likely to benefit from treatment with a second-generation MEK
inhibitor.
[0035] In one embodiment of the foregoing aspects, the second
generation MEK inhibitor is the Roche compound RG7420.
[0036] 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
genitourinary cancer. In an exemplary embodiment, the cancer is a
melanoma.
[0037] In one aspect, the invention provides kits for optimizing
treatment of a subject having cancer, identifying a subject having
cancer who is likely to benefit from treatment with a MEK inhibitor
and a RAF inhibitor, identifying a subject having cancer who has a
high risk of relapse during treatment with a first-generation MEK
inhibitor, identifying a subject having cancer who is unresponsive
to treatment with a first-generation MEK inhibitor, and/or for
identifying a subject having cancer who is likely to benefit from
treatment with a second-generation MEK inhibitor, comprising: (a) a
detection reagent useful for identifying the presence or absence of
one or more mutations in a MEK polypeptide 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 MEK polypeptide.
[0038] In various embodiments of the foregoing aspects, the
presence of one or more mutations in a MEK polypeptide, e.g., a MEK
polypeptide in a cancer cell-containing sample, is determined by a
method comprising determining the sequence of a nucleic acid
molecule encoding the MEK polypeptide. In other embodiments, the
presence of one or more mutations in a MEK polypeptide is
determined using an antibody that recognizes a MEK polypeptide
comprising the one or more mutations. 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 MEK polypeptide was
detected.
[0039] In another aspect, the invention features a method of
inhibiting a MEK polypeptide in a subject, comprising administering
a compound identified by one of the foregoing methods to a subject
in whom the presence of one or more mutations in a MEK polypeptide
was detected.
BRIEF DESCRIPTION OF THE DRAWINGS
[0040] FIG. I presents a schematic diagram depicting the design of
a study to identify "on-target" resistance mutations in vitro and
in vivo. A random mutagenesis screen was performed using a class of
first-generation MEK inhibitors in clinical development.
High-frequency mutations in MEK1 were identified, and the
corresponding exons were probed by deep sequencing of patient
samples. The resistance phenotype of representative MEK1 alleles
was confirmed by experimental studies.
[0041] FIGS. 2a-2c depict MEK1 resistance mutations identified in a
mutagenesis screen. (A): The average variant score (based on a lane
of Illumina sequencing) of candidate mutations across the MEK1
coding sequence from the AZD6244 mutagenesis screen is shown. The
corresponding amino acid substitutions from high scoring mutations
(>15%) are indicated in bold. (B): The locations of putative
MEK1 inhibitor resistance alleles are indicated within the crystal
structure of MEK1 (red). Both ATP and an aryl amine MEK inhibitor
(PD318088) are shown to bind MEK1; this inhibitor chemotype binds a
hydrophobic pocket adjacent to the ATP binding site. Candidate
resistance alleles cluster within Helix C, the hydrophobic drug
binding pocket, and the activation loop. (C): The majority of
high-frequency mutations cluster in two distinct regions encoded by
exons 3 and 6 of MEK1. The locations of the C helix and activation
loop are indicated.
[0042] FIGS. 3a-3b show the frequency of mutations across the MEK
coding sequence from a mutagenesis screen using the
first-generation MEK inhibitor CI-1040. The corresponding amino
acids are shown for high frequency alleles (Panel A). Panel B shows
the relative levels of pERK1/2, pMEK1/2, MEK1/2 and .alpha.-Tubulin
in A375 cells expressing resistant MEK1 alleles.
[0043] FIGS. 4a-4c depict the identification of an acquired MEK1
mutation in AZD6244-resistant metastatic melanoma. PCR products
corresponding to MEK1 exons 3 and 6 were generated from relapsed
melanomas treated with AZD6244; products were pooled and
interrogated by massively-parallel sequencing (a single Illumina
lane). (A): A single mutation, MEK.sup.P/24-.sup.L, was recovered
by this analysis. (B): Sanger sequencing chromatograms from MEK1
exon 3 corresponding to pre-treatment and post-relapse melanoma DNA
from patient #7. A C>T transition, indicative of MEK.sup.PI24L,
is evident only in the relapsed sample. (C): Changes in RECIST
total count (blue graph) and serum S100 levels (red graph) over the
clinical course of patient #7. The times of pre-treatment and
post-relapse biopsies are indicated. The intervals of temozolomide
treatment and AZD6244 treatment are shown.
[0044] FIGS. 5a-5g depict the functional characterization of MEK
resistance mutations. (A): AZD6244-mediated growth inhibition ex
vivo of treatment-naive BRAFV600E melanoma cells (black) or cells
cultured from an AZD6244-resistant metastatic focus (red). (B): ERK
phosphorylation (p-ERK) is shown following treatment with
increasing concentrations of AZD6244 in treatment-naive or
AZD6244-resistant melanoma cells cultured ex vivo. Phosphorylated
and total MEK (p-MEK1/2 and MEK1/2) are also shown, together with
the tubulin loading control (.alpha.-tubulin). (C): Growth
inhibition curves are shown for A375 cells expressing MEK1
resistance mutations derived from in vitro screens (blue), the
AZD6244-resistant metastasis (MEK.sup.PI24L; red), a constitutive
active MEK variant (MEK-DD; green), wild-type MEK1 (hatched black)
and parental A375 (solid black). (D): PLX4720 growth inhibition
curves are shown for the cultured melanoma cells from panel A,
above. (E): Immunoblot studies of p-ERK, p-MEK1/2 and MEK1/2 levels
following treatment with increasing concentrations of PLX4720 in
treatment-naive or AZD6244-resistant melanoma cells. (F): PLX4720
growth inhibition curves of A375 cells expressing MEK1 resistance
mutations or controls as outlined in panel C, above. (G): Colony
formation of parental A375 cells or cells expressing a mutagenized
MEK1 cDNA library. Cells were treated with vehicle, AZD6244 alone
(1.5 .mu.M), PLX4720 alone (1.5 .mu.M), or AZD6244+PLX4720 (1.5
.mu.M each).
[0045] FIGS. 6a-6c depict growth inhibition curves and alterations
in signaling in A375 cells containing resistance mutations. (A)
shows growth inhibition curves with CI-1040 for A375 cells
expressing MEK1 resistance mutations derived from in vitro screens
(blue), the AZD6244-resistant metastasis (MEK.sup.PI24L; red), a
constitutive active MEK variant (MEK-DD; green), wild-type MEK1
(hatched black) and parental A375 (solid black). (B) shows the
levels of pERK1/2, pMEK1/2, MEK1/2 and .alpha.-Tubulin in A375
cells expressing resistant MEK1 alleles following a 16 hour
incubation with CI-1040 at 10 .mu.M, 5 .mu.M, 2 .mu.M, 0.4 .mu.M,
0.08 .mu.M and 0 .mu.M. (C) shows protein levels following
treatment with ADZ6244 in A375 cells compared to A375 expressing
MEK1 wild type (MEK1.sup.WT), constitutive activated MEK
(MEK.sup.DD) and MEK.sup.PI24L.
[0046] FIG. 7 depicts the level of pERK1/2, pMEK1/2, MEK1/2 and
.alpha.-tubulin in A375 cells expressing resistant MEK1 alleles
following a 16-hour incubation with PLX4720 at 10 .mu.M, 5 .mu.M, 2
.mu.M, 0.4 .mu.M, 0.08 .mu.M and 0 .mu.M.
[0047] FIG. 8 depicts the wild-type human MEK nucleic acid sequence
(SEQ ID NO:1) (Accession No. NM.sub.--002755; gi:169790828).
[0048] FIG. 9 depicts the wild-type human MEK1 polypeptide sequence
(SEQ ID NO:2) (Accession No. NP.sub.--002746; gi:5579478).
[0049] FIG. 10 depicts the wild-type human MEK2 nucleic acid
sequence (SEQ ID NO:3) (Accession No. NM.sub.--030662;
gi:187171274).
[0050] FIG. 11 depicts the wild-type human MEK2 polypeptide
sequence (SEQ ID NO:4) (Accession No. NP.sub.--109587;
gi:13489054).
[0051] FIG. 12 depicts a sequence alignment between wild-type human
MEK1 (SEQ ID NO:2) and wild-type human MEK2 (SEQ ID NO:4).
DETAILED DESCRIPTION OF THE INVENTION
[0052] The present invention relates to the development of
resistance to chemotherapeutic therapy for cancer, particularly in
cancers having aberrant activation of MAP kinase signaling
pathways. Aberrant activation of MAP kinase signaling can result
from mutation or overexpression of one or more MAP kinase signaling
components. Mutation or overexpression of a component of a MAP
kinase signaling pathway may allow a cancer cell to become
resistant to one or more chemotherapeutic agents. In particular
embodiments, the present invention pertains to development of
resistance to a drug that targets a MEK polypeptide. The MEK
polypeptide is a key component of MAP kinase signaling pathways. In
specific embodiments, the invention concerns mutations in a MEK
polypeptide that confer resistance to a drug that targets the MEK
polypeptide, but that do not destroy the endogenous enzymatic
activity of the MEK polypeptide. In an exemplary embodiment, the
mutations in the MEK polypeptide confer resistance to the MEK
inhibitors CI-1040, AZD6244, PD318088, PD98059, PD334581, RDEA119,
Compound A, and Compound B. Accordingly, a "resistance mutation,"
as used herein, is a mutation in a MEK polypeptide which confers
resistance to one or more MEK inhibitors, e.g., CI-1040, AZD6244,
PD318088, PD98059, PD334581, RDEA119, Compound A, and Compound B.
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. Jun.; 3(6):755-62 (2004), the
entire contents of which are incorporated herein by reference.
[0053] The identification of mutations in the MEK polypeptide that
confer resistance to a drug that targets the MEK polypeptide allows
the development of "second-generation MEK inhibitors." As used
herein, the term "second-generation MEK inhibitor" refers to an
agent that inhibits a biological activity of a MEK polypeptide
containing one or more resistance mutations, such as the mutations
described herein. In a preferred embodiment, a second-generation
MEK inhibitor also inhibits a biological activity of a wild-type
MEK polypeptide. Accordingly, a second generation MEK inhibitor of
the invention can inhibit a biological activity of a MEK
polypeptide containing one or more resistance mutations and can
inhibit a biological activity of a wild-type MEK polypeptide. 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 polypeptide, but does not inhibit a biological
activity of a MEK polypeptide containing one or more resistance
mutations, such as the mutations described herein. Exemplary
first-generation MEK inhibitors include CI-1040, AZD6244, PD318088,
PD98059, PD334581, RDEA119, Compound A, and Compound B. In one
embodiment, a first-generation MEK inhibitor binds to a MEK
polypeptide at or near the ATP binding site of the MEK polypeptide.
An exemplary second-generation MEK inhibitor is the Roche compound
RG7420.
[0054] Additional MEK inhibitors include the compounds described in
the following patent publications, the entire contents of which are
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and U.S. Pat. No. 7,019,033. Compounds described in the foregoing
publications can be tested using the methods described herein to
determine if they are "first-generation MEK inhibitors" or
"second-generation MEK inhibitors."
[0056] Identification of resistance mutations in the MEK
polypeptide also allows for the screening of patients having a
cancer in order to determine the presence or absence of one or more
MEK resistance mutations 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 MEK inhibitor, or starting or
stopping treatment with a second generation MEK inhibitor.
[0057] The present invention further describes methods of
suppressing the emergence of MEK resistance mutations in a subject
having cancer. Such methods involve the combined administration of
a MEK inhibitor (e.g., a first generation MEK inhibitor or a second
generation MEK inhibitor) and a RAF inhibitor. Accordingly, the
present invention provides a combination therapy for treating or
preventing the symptoms of a cancer, comprising administration of a
MEK inhibitor and a RAF inhibitor. The MEK 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 MEK inhibitor and a RAF inhibitor
suppresses the emergence of MEK resistance mutations to a greater
extent than administration of either a MEK inhibitor or a RAF
inhibitor alone.
[0058] 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
[0059] As used herein, the terms "MEK protein" and "MEK
polypeptide" are intended to encompass MEK isoforms, such as MEK1
and MEK2. MEK1 and MEK2 (also known as MKK1 (MAP kinase kinase 1)
and MKK2 (MAP kinase kinase 2)) are dual-specific tyrosine and
serine/theronine kinases that play a key role in mitogen-activated
protein kinase (MAPK) intracellular signaling. Both MEK isoforms
are approximately 45 kDa in size, and are expressed ubiquitously in
mammalian cells. MEK1 and MEK2 are highly homologous, with MEK2
being 80% identical and 90% similar to MEK1. MEK1 and MEK2 each
contain an activation loop that includes two serine residues at
positions 217 and 221. Phosphorylation of these residues by the
protein kinase Raf results in MEK activation during MAPK signaling.
MEK 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 MEK activity.
[0060] 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.
[0061] 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.
[0062] 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.
[0063] As used interchangeably herein, the terms "MEK activity,"
"MEK biological activity" or "functional activity of MEK," include
activities exerted by a MEK protein, e.g., MEK1 or MEK2, 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. MEK 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 MEK activity is an indirect activity, such as a
downstream biological event mediated by interaction of the MEK
protein with an MEK target molecule, e.g., ERK1/2. As MEK is in a
signal transduction pathway involving ERK1/2, modulation of MEK
modulates a molecule in a signal transduction pathway involving
ERK1/2.
II. MEK Resistance Mutations
[0064] While treatment of cancer with MEK inhibitors, including
CI-1040, is a promising therapeutic approach, 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 mutations in MEK that
confer resistance to MEK inhibitors currently in clinical
development. Acquisition of such mutations in cancer cells makes
patients resistant to treatment with certain MEK inhibitors. In
exemplary embodiments, the invention regards development of
resistance to the MEK inhibitors CI-1040, AZD6244, PD318088,
PD98059, PD334581, RDEA119, Compound A, and Compound B.
[0065] As described herein, tumors harboring a druggable oncogene
dependency tend to develop on-target clinical resistance through
point mutations. Elaboration of on-target resistance mechanisms has
enabled the design of second-generation inhibitors with enhanced
potency against a range of resistant variants. Toward this end,
most resistance mutations identified herein populate the MEK
allosteric drug binding pocket adjacent to the ATP binding motif.
Some first-generation MEK inhibitors, e.g., aryl amine inhibitors,
function by locking MEK into a "closed" but inactive conformation,
in which the activation loop causes the C helix to become
externally rotated and displaced. Without wishing to be bound by
theory, MEK mutations may introduce resistance through direct
interference of drug binding or by forcing the C helix towards a
closed conformation that disrupts the binding pocket. The design of
second-generation MEK inhibitors, e.g., ATP-competitive inhibitors
or novel allosteric modulators, may override resistance to this
mode of non-competitive kinase inhibition.
[0066] 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 "druggablc"
oncogene mutations.
(A) Identification of MEK Mutations Conferring Resistance to MEK1
inhibitors
[0067] In various embodiments, the present invention relates to
methods of identifying mutations in a MEK polypeptide, or mutations
in a nucleic acid molecule encoding the MEK polypeptide, that
confer resistance of the MEK polypeptide to drugs that inhibit MEK
activity. A "mutant MEK polypeptide," as referenced herein,
includes a MEK polypeptide containing one or more mutations that
confer resistance to one or more known MEK inhibitors. Likewise, a
"mutant MEK nucleic acid molecule," as referenced herein, includes
a nucleic acid molecule that encodes a mutant MEK polypeptide.
Nucleic acid molecules encoding MEK polypeptides 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 MEK nucleic acid sequence,
which can be conducted in E. coli. In exemplary embodiments, the
wild-type MEK nucleic acid sequence is a human wild-type MEK
nucleic acid sequence. In specific embodiments the wild-type MEK
nucleic acid sequence is wild-type human MEK1 (SEQ ID NO:1) or
wild-type human MEK2 (SEQ ID NO:3). The mutant MEK nucleic acid
molecules can then be screened in cells otherwise sensitive to
treatment with a MEK inhibitor to identify a nucleic acid that
encodes a mutant MEK polypeptide that is resistant to treatment
with the MEK inhibitor.
[0068] Any suitable method can be used to screen mutant MEK nucleic
acids and mutant MEK polypeptides for resistance to treatment with
a MEK inhibitor. For example, a nucleic acid molecule encoding a
mutant MEK polypeptide can be expressed in cells otherwise
sensitive to treatment with a MEK inhibitor. An exemplary cell line
useful for this purpose is the melanoma cell line A375. Following
expression of the mutant MEK polypeptide, the cells can be treated
with a MEK inhibitor. The activity of the mutant MEK polypeptide
can then be measured and compared to the activity of a wild-type
MEK polypeptide similarly expressed and treated with the MEK
inhibitor. Activity of a MEK polypeptide can be determined by, for
example, measuring proliferation or viability of cells following
treatment with the MEK inhibitor, wherein proliferation or
viability are positively correlated with MEK 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 MEK inhibitor is expressed as a percentage of that observed in
untreated cells cultured in the absence of the 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 MEK polypeptide can also be measured
by, for example, determining the relative amount of phosphorylated
ERK1/2 present in the cell following treatment with the MEK
inhibitor. Activity of a wild-type or mutant MEK polypeptide can
also be determined using an in vitro phosphorylation assay, in
which MEK activity is determined by measuring the proportion of
phosphorylated ERK1/2 substrate in the assay following treatment
with the MEK inhibitor. A mutant MEK polypeptide having greater
activity than a wild-type MEK polypeptide following treatment with
a MEK inhibitor is identified as containing a mutation that confers
resistance to a MEK inhibitor. The mutation conferring resistance
to a MEK inhibitor can then be identified by sequencing the nucleic
acid encoding the mutant MEK polypeptide, or by sequencing the
mutant MEK polypeptide directly.
[0069] In this manner, several amino acid residues of the human
MEK1 polypeptide were identified that, when mutated, confer
resistance to the MEK inhibitor CI-1040. These amino acid residues
include one or more of the following: 67D, 74L, 103I, 106A, 107I,
111I, 115I, 119H, 124P, 128G, 129F, 167G, 203E, 211V, 247W, 258V,
260R, 276G, 286G, 302M, 303D, 308M, 328G, 399H, 56Q, 99I, 104K,
113R, 120E, 128G, 133F, 215L, 237G, 326P, and 368E. In certain
embodiments, the mutant MEK polypeptides of the invention contain a
mutation with respect to the wild-type human MEK1 polypeptide
sequence at one or more of these amino acid residues. In related
embodiments, the mutant MEK nucleic acid molecules of the invention
contain a mutation with respect to the wild-type human MEK1 nucleic
acid sequence at one or more nucleotides encoding one or more of
the these amino acid residues. In exemplary embodiments, the mutant
MEK polypeptides of the invention contain one of more of the
following resistance mutations: 67D>N, 74L>Q, 74L>R,
103I>N, 106A>T, 107I>M, 111I>N, 115L>R, 115L>P,
119H>P, 124P>L, 124P>S, 128G>D, 129F>L, 167G>R,
203E>K, 211V>D, 247W>G, 258V>G, 260R>G, 276G>E,
286G>E, 302M>R, 303D>A, 308M>L, 328G>L, 328G>R,
379I>L, 399H>D, 56Q>P, 99I>T, 104K>N, 113R>S,
120E>D, 128G>D, 133F>L, 215L>P, 237G>V, 326P>L
and 368E>K. In other exemplary embodiments, the mutant MEK
nucleic acid molecules of invention encode a mutant MEK polypeptide
containing one or more of the following resistance mutations:
67D>N, 74L>Q, 74L>R, 103I>N, 106A>T, 107I>M,
111>N, 115L>R, 115L>P, 119H>P, 124P>L, 124P>S,
128G>D, 129F>L, 167G>R, 203E>K, 211V>D, 247W>G,
258V>G, 260R>G, 276G>E, 286G>E, 302M>R, 303D>A,
308M>L, 328G>L, 328G>R, 379I>L, 399H>D, 56Q>P,
99I>T, 104K>N, 113R>S, 120E>D, 128G>D, 133F>L,
215L>P, 237G>V, 326P>L, and 368E>K.
[0070] The MEK resistance mutations identified above also confer
resistance to a MEK inhibitor when introduced at corresponding
residues in MEK2. Accordingly, amino acid residues of the human
MEK2 polypeptide that, when mutated, confer resistance to the MEK
inhibitor CI-1040 include one or more of the following: 71D, 78L,
107I, 110A, 111I, 115I, 119L, 123H, 128P, 132G, 133F, 171G, 207E,
215V, 251W, 262V, 264R, 280G, 310M, 311D, 316M, 3360, 60Q, 103I,
108K, 117R, 124E, 132G, 137F, 219L, 241G, 334P and 376E. In certain
embodiments, the mutant MEK polypeptides of the invention contain a
mutation with respect to the wild-type human MEK2 polypeptide
sequence at one or more of these amino acid residues. In related
embodiments, the mutant MEK nucleic acid molecules of the invention
contain a mutation with respect to the wild-type human MEK2 nucleic
acid sequence at one or more nucleotides encoding one or more of
the these amino acid residues. In exemplary embodiments, the mutant
MEK polypeptides of the invention contain one of more of the
following resistance mutations: 71D>N, 78L>Q, 78L>R,
107I>N, 110A>T, 111I>M, 115I>N, 119L>R, 119L>P,
123H>P, 128P>L, 128P>S, 132G>D, 133F>L, 171G>R,
207E>K, 215V>D, 251W>G, 262V>G, 264R>G, 280G>E,
310M>R, 311D>A, 316M>L, 336G>L, 336G>R, 60Q>P,
103I>T, 108K>N, 117R>S, 124E>D, 132G>D, 137F>L,
219L>P, 241G>V, 334P>L and 376E>K. In all embodiments
of the invention, a mutant MEK molecule or a mutant MEK polypeptide
refers to either a MEK1 or MEK2 polypeptide having one or more
mutations that confer resistance to a MEK inhibitor.
[0071] As described herein, identification of mutations in MEK
conferring resistance to MEK inhibitors allows the design and
screening of "second generation MEK inhibitors," which are
effective at inhibiting a MEK protein having one or more resistance
mutations. 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
MEK polypeptide also allows the screening of patients having a
cancer in order to determine the presence or absence of one or more
MEK resistance mutations in the cancer. Determining the presence or
absence of one or more MEK resistance mutations in a cancer allows
alteration of the treatment strategy of a cancer patient. For
example, identification of one or more of the MEK resistance
mutations 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 MEK inhibitor. Identification of
MEK resistance mutations also allows the screening and
identification of patients having a high risk of relapse or lack of
response to treatment with certain MEK inhibitors.
[0072] The foregoing MEK 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 MEK
polypeptide in the presence of a RAF inhibitor, and comparing the
activity to that of a wild-type MEK polypeptide similarly treated
with the RAF inhibitor. Activity of a MEK polypeptide can be
determined using the methods set forth herein.
III. Methods for Identifying Second-Generatio MEK1 Inhibitors
[0073] Identification of MEK resistance mutations allows the
development and/or identification of "second-generation MEK
inhibitors." As used herein, a second-generation MEK inhibitor is
an agent that effectively inhibits the activity of a MEK
polypeptide containing one or more mutations described herein. A
second-generation MEK inhibitor may or may not inhibit the activity
of a wild-type MEK polypeptide in addition to a mutant MEK
polypeptide. In a preferred embodiment, a second-generation MEK
inhibitor inhibits the activity of both a wild-type MEK polypeptide
and a mutant MEK polypeptide. In an exemplary embodiment, a
second-generation MEK inhibitor inhibits the activity of a MEK1
polypeptide containing mutations at one or more of the following
amino acid residues: 67D, 74L, 103I, 106A, 107I, 111I, 115L, 119H,
124P, 128G, 129F, 167G, 203E, 211V, 247W, 258V, 260R, 276G, 286G,
302M, 303D, 308M, 328G and 399H; and/or a MEK2 polypeptide
containing mutations at one or more of the following residues 71D,
78L, 107I, 110A, 111I, 115I, 119L, 123H, 128P, 132G, 133F, 171G,
207E, 215V, 251W, 262V, 264R, 280G, 310M, 311 D, 316M, and 336G. In
another exemplary embodiment, a second-generation MEK inhibitor
inhibits the activity of a MEK1 polypeptide containing mutations at
one or more of the following amino acid residues: 56Q, 99I, 104K,
113R, 120E, 128G, 133F, 215L, 237G, 326P and 368E; and/or a MEK2
polypeptide containing mutations at one or more of the following
residues: 60Q, 103I, 108K, 117R, 124E, 132G, 137F, 219L, 241G, 334P
and 376E.
[0074] Accordingly, the present invention provides methods for
identifying a test compound as a second-generation MEK inhibitor.
In one embodiment, a compound can be identified as a
second-generation MEK inhibitor by determining the relative MEK
activity of a mutant MEK polypeptide in the presence or absence of
the compound, with respect to a wild-type MEK polypeptide. When in
the presence of a compound that is a second-generation MEK
inhibitor, a mutant MEK polypeptide has a lower level of MEK
activity than in the absence of the compound. When in the presence
of a compound that is not a second-generation MEK inhibitor, a
mutant MEK polypeptide has an equivalent or higher level of MEK
activity than in the absence of the compound. In certain
embodiments, MEK activity can be measured in an in vitro assay
using recombinant MEK polypeptides. In other embodiments, MEK
activity can be measured in an in vi assay using cultured cells or
experimental animals.
[0075] Any indicator of MEK activity is suitable for determining
whether or not a compound is a second-generation MEK inhibitor. In
an exemplary embodiment, MEK activity is determined by measuring
phosphorylation of the MEK substrate ERK1/2, wherein a decrease in
ERK1/2 phosphorylation indicates a decrease in MEK 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 MEK polypeptide or a mutant
MEK polypeptide. 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 vive using cultured cells
or experimental animals.
[0076] In one embodiment, a host cell expressing a mutant MEK
polypeptide is used in the identification of a second-generation
MEK inhibitor, wherein the sensitivity of the host cell to a test
compound identifies the test compound as a second-generation MEK
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 MEK as
evidenced by, for example, phosphorylation of one or more MEK
substrates, such as ERK1/2).
[0077] A compound can be identified as a second-generation MEK
inhibitor by determining the viability or proliferation rate of
cells expressing a mutant MEK polypeptide in the presence or
absence of the compound. The cell line used in such an assay should
be sensitive to a MEK inhibitor when the cell line expresses a
wild-type MEK polypeptide, and should be resistant to the MEK
inhibitor (i.e., a first-generation MEK inhibitor) when the cell
line expresses a mutant MEK polypeptide. An exemplary cell line
useful for identification of a second-generation MEK inhibitor is
the melanoma cell line A375. A375 cells are sensitive to the MEK
inhibitor CI-1040 when expressing a wild-type MEK polypeptide, but
are resistant to CI-1040 when expressing a mutant MEK polypeptide,
for example, a MEK1 polypeptide containing one or more of the
following resistance mutations: 67D>N, 74L>Q, 74L>R,
103I>N, 106A>T, 107I>M, 111I>N, 115L>R, 115L>P,
19H>P, 124P>L, 124P>S, 128G>D, 129F>L, 167G>R,
203E>K, 211V>D, 247W>G, 258V>G, 260R>G, 276G>E,
286G>E, 302M>R, 303D>A, 308M>L, 328G>L, 328G>R,
379I>L, 399H>D, 56Q>P, 99I>T, 104K>N, 113R>S,
120E>D, 128G>D, 133F>L, 215L>P, 237G>V, 326P>L
and 368E>K; or a MEK2 polypeptide containing one or more of the
following resistance mutations: 71D>N, 78L>Q, 78L>R,
107I>N, 10A>T, 111I>M, 115I>N, 119L>R, 119L>P,
123H>P, 128P>L, 128P>S, 132G>D, 133F>L, 171G>R,
207E>K, 215V>D, 251W>G, 262V>G, 264R>G, 280G>E,
310M>R, 311D>A, 316M>L, 336G>L, 336G>R, 60Q>P,
103I>T, 108K>N, 117R>S, 124E>D, 132G>D, 137F>L,
219L>P, 241G>V, 334P>L and 376E>K.
[0078] When in the presence of a compound that is a
second-generation MEK inhibitor, a cell line expressing a mutant
MEK polypeptide has a lower viability or proliferation rate than in
the absence of the compound, and/or a lower viability or
prolideration rate than a cell line expressing a wild type MEK
polypeptide in the presence of the compound. When in the presence
of a compound that is not a second-generation MEK inhibitor, a cell
line expressing a mutant MEK polypeptide has an equivalent or
higher viability or proliferation rate than in the absence of the
compound, and/or an equivalent or higher viability or prolideration
rate than a cell line expressing a wild type MEK polypeptide 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 MEK
polypeptide or a mutant MEK polypeptide 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.
[0079] Accordingly, in one embodiment, the invention provides a
method of identifying a compound that is a second generation MEK
inhibitor, comprising providing an assay composition comprising a
MEK substrate and a MEK polypeptide having one or more mutations
with respect to a wild-type MEK polypeptide, 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 MEK
inhibitor. A compound identified in this way is a compound useful
for treating a cancer, e.g., a cancer in which a mutant MEK
polypeptide has been detected. A MEK polypeptide useful in the
foregoing methods is a MEK polypeptide containing one or more
mutations which confer resistance to the MEK inhibitors CI-1040,
AZD6244, PD318088, PD98059, PD334581, RDEA119, Compound A, and
Compound B, e.g., a MEK polypeptide 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 MEK
inhibitor. The foregoing methods can be performed in vitro wherein
the MEK polypeptide and the MEK substrate are isolated or purified
proteins. The foregoing methods can also be performed in vitro
wherein the MEK polypeptide 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 or
MEK2 polypeptide.
[0080] In another embodiment, the invention provides a method of
identifying a compound that is a second generation MEK inhibitor,
comprising providing a cell comprising a mutant MEK polypeptide,
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
MEK inhibitor. A compound identified in this way is a compound
useful for treating a cancer, e.g., a cancer in which a mutant MEK
polypeptide 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 or MEK2 was expressed.
[0081] In one embodiment, the test compound used in the foregoing
methods is a MEK inhibitor that inhibits a biological activity of a
wild type MEK polypeptide. MEK inhibitors that are second
generation MEK inhibitors are described herein.
[0082] 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 (Lamin, 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) Imnuunopharmacology 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 polypeptides 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.
[0083] Second generation MEK inhibitors can also be rationally
designed based on the structure of MEK1 or MEK2 alleles containing
one or more of the resistance mutations described herein. As
described herein, residues of MEK that confer resistance to MEK
inhibitors are located in or near the C-helix, the activation loop,
and the ATP binding pocket of the MEK polypeptide. This region of
the MEK polypeptide is the same region bound by the
first-generation MEK inhibitors CI-1040 and AZD6244. Identification
of MEK1 and MEK2 mutant alleles conferring resistance to MEK
inhibitors allows comparison between the structure of the C-helix,
activation loop and ATP binding pocket of the mutant alleles and
the wild-type protein. Knowledge of the altered structural features
that confer resistance to first-generation 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 MEK alleles.
Inhibitors designed to bind the C-helix, activation loop, and/or
ATP binding pocket of MEK alleles containing one or mutations
described herein are second-generation MEK inhibitors. The ability
of such rationally designed inhibitors to inhibit a biological
activity of a mutant MEK polypeptide can be confirmed using the in
vitro and/or in vivo assays described herein.
[0084] The structure of a MEK polypeptide containing one or more of
the resistance mutations described herein can be determined by
computer-assisted modeling, or by determining the crystal or
solution structure of the mutant MEK polypeptide. Any suitable
method known in the art can be used to determine the structure of a
mutant MEK polypeptide.
[0085] 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).
[0086] 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 or MEK2 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 MEK mutant alleles. A solution structure of a MEK
polypeptide or mutant MEK polypeptide can be identified using
nuclear magnetic resonance spectroscopy using techniques known in
the art. Suitable methods for polypeptide 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 MEK
polypeptide, inhibitors of the mutant MEK polypeptide can be
identified using the computer assisted modeling approaches
described above.
[0087] Second-generation MEK inhibitors identified by the foregoing
methods are useful for treating a disease or condition associated
with expression of a wild-type and/or mutant MEK polypeptide. For
example, second-generation MEK inhibitors are useful for treating a
cancer in a subject, particularly a cancer in which a mutant MEK
polypeptide has been identified. In an exemplary embodiment,
second-generation MEK inhibitors are useful for treating a cancer
containing a MEK1 polypeptide having a mutation at one or more of
the following amino acid residues: 67D, 74L, 103I, 106A, 107I,
111I, 115L, 119H, 124P, 128G, 129F, 167G, 203E, 211V, 247W, 258V,
260R, 276G, 286G, 302M, 303D, 308M, 328G and 399H, and/or a MEK2
polypeptide having a mutation at one or more of the following amino
acid residues: 71D, 78L, 107I, 110A, 111I, 115I, 119L, 123H, 128P,
132G, 133F, 171G, 207E, 215V, 251W, 262V, 264R, 280G, 310M, 311 D,
316M, and 336G. In another exemplary embodiment, second-generation
MEK inhibitors are useful for treating a cancer containing a MEK1
polypeptide having a mutation at one or more of the following amino
acid residues: 56Q, 99I, 104K, 113R, 120E, 128G, 133F, 215L, 237G,
326P, and 368E; and/or a MEK2 polypeptide having a mutation at one
or more of the following amino acid residues: 60Q, 103I, 108K,
117R, 124E, 132G, 137F, 219L, 241G, 334P and 376E. In a related
embodiment, second-generation MEK inhibitors are useful for
treating a cancer containing a MEK1 polypeptide having one or more
of the following mutations: 67D>N, 74L>Q, 741,>R,
103I>N, 106A>T, 107I>M, 111I>N, 115L>R, 115L>P,
119H>P, 124P>L, 124P>S, 128G>D, 129F>L, 167G>R,
203E>K, 211V>D, 247W>G, 258V>G, 260R>G, 276G>E,
286G>E, 302M>R, 303D>A, 308M>L, 328G>L, 328G>R,
379I>L and 399H>D, and/or a MEK2 polypeptide having one or
more of the following mutations: 71D>N, 78L>Q, 78L>R,
107I>N, 110A>T, 111I>M, 115I>N, 119L>R, 119L>P,
123H>P, 128P>L, 128P>S, 132G>D, 133F>L, 171G>R,
207E>K, 215V>D, 251W>G, 262V>G, 264R>G, 280G>E,
310M>R, 311D>A, 316M>L, 336G>L, and 336G>R. In
another exemplary embodiment, second-generation MEK inhibitors are
useful for treating a cancer containing a MEK1 polypeptide having
one or more of the following mutations: 56Q>P, 99I>T,
104K>N, 113R>S, 120E>D, 128G>D, 133F>L, 215L>P,
237G>V, 326P>L, and 368E>K, and/or a MEK2 polypeptide
having one or more of the following mutations: 60Q>P, 103I>T,
108K>N, 117R>S, 124E>D, 132G>D, 137F>L, 219L>P,
241G>V, 334P>L, and 376E>K.
IV. Isolated Nucleic Acid Molecules
[0088] The present invention concerns polynucleotides or nucleic
acid molecules relating to the MEK 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 MEK nucleic acid molecules
described herein comprise one or more mutations conferring
resistance to a MEK inhibitor. A "mutant MEK nucleic acid
molecule," as referenced herein, includes a MEK nucleic acid
molecule that encodes a mutant MEK polypeptide, i.e., a MEK
polypeptide containing one or more mutations that confer resistance
to one or more known MEK inhibitors.
[0089] It is contemplated that an isolated and purified MEK nucleic
acid molecule, e.g., a mutant MEK 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 polypeptides.
[0090] 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 MEK" refers to a nucleic acid
segment that contains MEK 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
MEK-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 MEK polypeptide, for example,
phosphorylation of the ERK1/2 substrate.
[0091] 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 polypeptide
and eliminates introns and other regulatory regions.
[0092] It also is contemplated that a given MEK-encoding nucleic
acid or MEK 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 MEK polypeptide. In a
preferred embodiment, the active MEK polypeptide is an active human
MEK polypeptide. In particularly preferred embodiments, the active
MEK polypeptide is a mutant MEK polypeptide that has an activity of
a wild-type MEK polypeptide, but which is resistant to one or more
known MEK inhibitors. Consequently, certain aspects of the present
invention encompass derivatives of MEK with minimal amino acid
changes, but that possess the same biological function.
[0093] The term "gene" is used for simplicity to refer to a
functional protein, polypeptide, 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,
polypeptides, domains, fusion proteins, and mutant proteins. The
nucleic acid molecule encoding MEK 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.
[0094] Various embodiments of the invention relate to genetic
mutations in MEK. As used herein, a mutation refers to an addition,
deletion, or substitution of a single nucleotide at a site in a MEK
nucleic acid molecule. In an exemplary embodiment, a mutant MEK
nucleic acid molecule contains one or more mutations that confer
resistance to a particular therapy, such as a MEK inhibitor, e.g.,
CI-1040. In a related embodiment, a mutant MEK nucleic acid
molecule contains one or more mutations such that the mutant MEK
nucleic acid molecule encodes a mutant MEK polypeptide, wherein the
mutant MEK polypeptide contains one or more mutations that confer
resistance to a particular therapy, such as a MEK inhibitor, e.g.,
CI-1040. Thus, in particular aspects of the invention, an
alteration in a sequence results in a change that affects the
properties of a polypeptide encoded by the sequence such that at
least some resistance to therapy, such as therapy with a MEK
inhibitor, occurs as a result.
[0095] "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.
[0096] In particular embodiments, the invention concerns isolated
nucleic acid segments and recombinant vectors incorporating DNA
sequences that encode mutant MEK polypeptides or peptides that
include within its amino acid sequence a contiguous amino acid
sequence in accordance with, or essentially corresponding to mutant
MEK polypeptides. In exemplary embodiments, the invention concerns
isolated DNA segments and recombinant vectors incorporating DNA
sequences that encode a MEK protein, polypeptide or peptide that
includes within its amino acid sequence a contiguous amino acid
sequence of a MEK polypeptide comprising one or more mutations that
confer resistance to a MEK inhibitor. In certain embodiments, the
one or more mutations occur at positions 67D, 74L, 103I, 106A,
107I, 11I, 115L, 119H, 124P, 128G, 129F, 167G, 203E, 211V, 247W,
258V, 260R, 276G, 286G, 302M, 303D, 308M, 328G or 399H of a MEK1
polypeptide. In other embodiments, the one or more mutations occur
at positions 56Q, 99I, 104K, 113R, 120E, 128G, 133F, 215L, 237G,
326P or 368E of a MEK1 polypeptide. In other embodiments, the one
or more MEK1 mutations include the following: 67D>N, 74L>Q,
74L>R, 103I>N, 106A>T, 107I>M, 111I>N, 115L>R,
115L>P, 119H>P, 124P>L, 124P>S, 128G>D, 129F>L,
167G>R, 203E>K, 21I V>D, 247W>G, 258V>G, 260R>G,
276G>E, 286G>E, 302M>R, 303D>A, 308M>L, 328G>L,
328G>R, 379I>L or 399H>D. In other embodiments, the one or
more MEK1 mutations include the following: 56Q>P, 99I>T,
104K>N, 113R>S, 120E>D, 128G>D, 133F>L, 215L>P,
237G>V, 326P>L or 368E>K. In certain embodiments, the one
or more mutations occur at positions 71D, 78L, 107I, 10A, 111I,
115I, 119L, 123H, 128P, 132G, 133F, 171G, 207E, 215V, 251W, 262V,
264R, 280G, 310M, 311D, 316M or 336G of a MEK2 polypeptide. In
other embodiments, the one or more mutations occur at positions
60Q, 103I, 108K, 117R, 124E, 132G, 137F, 219L, 241G, 334P or 376E
of a MEK2 polypeptide. In other embodiments, the one or more MEK2
mutations include the following: 71D>N, 78L>Q, 78L>R,
107I>N, 110A>T, 111I>M, 115I>N, 119L>R, 119L>P,
123H>P, 128P>L, 128P>S, 132G>D, 133F>L, 171G>R,
207E>K, 215V>D, 251 W>G, 262V>G, 264R>G, 280G>E,
310M>R, 311 D>A, 316M>L, 336G>L, and 336G>R. In
other embodiments, the one or more MEK2 mutations include the
following: 60Q>P, 103I>T, 108K>N, 117R>S, 124E>D,
132G>D, 137F>L, 219L>P, 241G>V, 334P>L,
376E>K.
[0097] 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.
[0098] It is contemplated that the nucleic acid constructs of the
present invention encode a MEK polypeptide or a mutant MEK
polypeptide. 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.
[0099] 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 MEK
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.
[0100] 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.
[0101] 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
MEK gene, other regulatory regions such as enhancers for MEK are
contemplated as nucleic acids for use with compositions and methods
of the claimed invention.
[0102] 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."
[0103] 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 MEK primer
that facilitates identification of a mutation conferring resistance
to a MEK inhibitor), 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.
[0104] 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 (see for example,
Sambrook et al, 1989, incorporated herein by reference).
[0105] 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 MEK (see for example,
Sambrook et al., 1989, incorporated herein by reference). 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.
[0106] 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
[0107] The present invention encompasses expression vector
compositions and the use of such vectors to encode for a MEK
polypeptide, e.g., a mutant MEK polypeptide, 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, which are described in Sambrook et al., 1989 and
Ausubel et al., 1996, both incorporated herein by reference.
[0108] 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,
polypeptide, 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
[0109] 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.
[0110] 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.
[0111] 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, for example, see Sambrook et
al. (1989), incorporated herein by reference. 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-MEK promoter with
respect to MEK 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
[0112] 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
[0113] 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
[0114] 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
[0115] 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
[0116] 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
[0117] 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
[0118] 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.
[0119] 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
[0120] As used herein, the terms "cell," "cell line," and "cell
culture" may be used interchangeably. A cell comprising a MEK
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.
[0121] 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
KC8, 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.
[0122] 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 MEK polypeptide, e.g., a mutant MEK
polypeptide of the invention.
10. Expression Systems
[0123] 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 MEK nucleic acid sequences, or their cognate
polypeptides, proteins and peptides. Many such systems are
commercially and widely available.
[0124] 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..
[0125] 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.
[0126] 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 polypeptide, protein, or
peptide.
VI. Isolated Polypeptide Molecules
[0127] Another aspect of the invention pertains to isolated and/or
purified MEK proteins, and biologically active portions thereof. In
particular aspects of the invention, the MEK polypeptides described
herein comprise one or more mutations conferring resistance to a
MEK inhibitor. A "mutant MEK polypeptide", as referenced herein,
includes a MEK polypeptide containing one or more mutations that
confer resistance to one or more known MEK inhibitors.
[0128] 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
MEK 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 MEK protein having less
than about 30% (by dry weight) of non-MEK protein (also referred to
herein as a "contaminating protein"), more preferably less than
about 20% of non-MEK protein, still more preferably less than about
10% of non-MEK protein, and most preferably less than about 5% of
non-MEK protein. When the MEK 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 MEK 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 MEK protein
having less than about 30% (by dry weight) of chemical precursors
or non-MEK chemicals, more preferably less than about 20% chemical
precursors or non-MEK chemicals, still more preferably less than
about 10% chemical precursors or non-MEK chemicals, and most
preferably less than about 5% chemical precursors or non-MEK
chemicals.
[0129] Biologically active portions of a MEK protein include
peptides comprising amino acid sequences derived from the amino
acid sequence of a MEK protein, e.g., the amino acid sequence shown
in SEQ ID NOs:2 and 4, or the amino acid sequence of a protein
homologous to a MEK protein, which include fewer amino acids than a
full length MEK protein or the full length protein which is
homologous to a MEK protein, and exhibit at least one activity of a
MEK 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 MEK 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
MEK protein include one or more selected domains/motifs or portions
thereof having biological activity. In preferred embodiments,
biologically active portions of a MEK protein comprise one or more
mutations with respect to a wild-type MEK sequence, wherein said
one or more mutations, when present in a full-length mutant MEK
polypeptide, confer resistance of the mutant MEK polypeptide to
known MEK inhibitors. In exemplary embodiments, the mutations occur
at amino acids corresponding to one or more of the following
positions in a wild-type MEK1 protein: 67D, 74L, 103I, 106A, 107I,
111I, 115L, 119H, 124P, 128G, 129F, 167G, 203E, 211V, 247W, 258V,
260R, 2760, 286G, 302M, 303D, 308M, 328G and 399H; or in one or
more of the following positions in a wild-type MEK2 protein: 71D,
78L, 107I, 110A, 111I, 115I, 119L, 123H, 128P, 132G, 133F, 171G,
207E, 215V, 251W, 262V, 264R, 280G, 310M, 311D, 316M, and 336G. In
other embodiments, the mutations occur at amino acids corresponding
to one or more of the following positions in a wild-type MEK1
protein: 56Q, 99I, 104K, 113R, 120E, 128G, 133F, 215L, 237G, 326P
and 368E; or in one or more of the following positions in a
wild-type MEK2 protein: 60Q, 103I, 108K, 117R, 124E, 132G, 137F,
219L, 241G, 334P and 376E. In a related embodiment, the mutations
include one or more of the following MEK1 residues: 67D>N,
74L>Q, 74L>R, 103I>N, 106A>T, 107I>M, 111I>N,
115L>R, 115L>P, 119H>P, 124P>L, 124P>S, 128G>D,
129F>L, 167G>R, 203E>K, 211V>D, 247W>G, 258V>G,
260R>G, 276G>E, 286G>E, 302M>R, 303D>A, 308M>L,
328G>L, 328G>R, 379I>L and 399H>D; or one or more of
the following MEK2 residues: 71D>N, 78L>Q, 78L>R,
107I>N, 110A>T, 111I>M, 115I>N, 119L>R, 119L>P,
123H>P, 128P>L, 128P>S, 132G>D, 133F>L, 171G>R,
207E>K, 215V>D, 251 W>G, 262V>G, 264R>G, 280G>E,
310M>R, 311D>A, 316M>L, 336G>L, and 336G>R. In
another embodiment, the mutations include one or more of the
following MEK1 residues: 56Q>P, 99I>T, 104K>N, 113R>S,
120E>D, 128G>D, 133F>L, 215L>P, 237G>V, 326P>L
and 368E>K; or one or more of the following MEK2 residues:
60Q>P, 103I>T, 108K>N, 117R>S, 124E>D, 132G>D,
137F>L, 219L>P, 241G>V, 334P>L, 376E>K.
[0130] MEK 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 MEK protein is expressed in the host cell. The MEK
protein can then be isolated from the cells by an appropriate
purification scheme using standard protein purification techniques.
Alternative to recombinant expression, a MEK protein, polypeptide,
or peptide can be synthesized chemically using standard peptide
synthesis techniques. Moreover, a native MEK protein and/or a
mutant MEK protein can be isolated from cells (e.g., cancer cells),
for example using an anti-MEK antibody, which can be produced by
standard techniques utilizing a MEK protein or fragment thereof of
this invention.
[0131] The invention also provides MEK chimeric or fusion proteins.
As used herein, a MEK "chimeric protein" or "fusion protein"
comprises a MEK polypeptide operatively linked to a non-MEK
polypeptide. A "MEK polypeptide" refers to a polypeptide having an
amino acid sequence corresponding to a MEK protein, whereas a
"non-MEK polypeptide" refers to a polypeptide having an amino acid
sequence corresponding to a protein which is not substantially
homologous to the MEK protein, e.g., a protein which is
substantially different from the MEK protein, which does not
display a MEK 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 MEK
polypeptide and the non-MEK polypeptide are fused in-frame to each
other. The non-MEK polypeptide can be fused to the N-terminus or
C-terminus of the MEK polypeptide. For example, in one embodiment
the fusion protein is a GST-MEK fusion protein in which the MEK
sequences are fused to the C-terminus of the GST sequences. Such
fusion proteins can facilitate the purification of recombinant MEK
proteins. In another embodiment, the fusion protein is a MEK
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 MEK protein can be increased
through use of a heterologous signal sequence.
[0132] Preferably, a MEK chimeric or fusion protein of the
invention is produced by standard recombinant DNA techniques. For
example, DNA fragments coding for the different polypeptide
sequences are ligated together in-frame in accordance with
conventional techniques, for example by employing blunt-ended or
stagger-ended termini for ligation, restriction enzyme digestion to
provide for appropriate termini, filling-in of cohesive ends as
appropriate, alkaline phosphatase treatment to avoid undesirable
joining, and enzymatic ligation. In 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 polypeptide). A MEK-encoding nucleic acid can be cloned into
such an expression vector such that the fusion moiety is linked
in-frame to the MEK protein.
[0133] In a preferred embodiment, the isolated MEK polypeptides of
the invention contain one or more mutations (e.g., substitutions or
deletions) with respect to a wild-type MEK polypeptide sequence. In
one embodiment, the mutant MEK polypeptides contain one or more
mutations with respect to a human wild-type MEK polypeptide
sequence (SEQ ID NO:2). In a particularly preferred embodiment, the
one or more mutations confer resistance to a MEK inhibitor. In an
exemplary embodiment, the MEK inhibitor is CI-1040. A mutant MEK
protein of the invention exhibits a biological activity
characteristic of a wild-type MEK protein. Such a biological
activity can include, for example, phosphorylation of ERK1/2.
Exemplary mutant MEK polypeptides of the invention include MEK
polypeptides comprising a mutation at one or more of the following
amino acid residues with respect to the wild type human MEK1
polypeptide: 67D, 74L, 103I, 106A, 107I, 111I, 115L, 119H, 124P,
128G, 129F, 167G, 203E, 211V, 247W, 258V, 260R, 276G, 286G, 302M,
303D, 308M, 3280 and 399H, or at one or more of the following amino
acid residues with respect to the wild-type human MEK2 polypeptide:
71D, 78L, 107I, 110A, 111I, 115I, 119L, 123H, 128P, 132G, 133F,
171G, 207E, 215V, 251W, 262V, 264R, 280G, 310M, 311D, 316M, and
336G. In another embodiment, exemplary mutant MEK polypeptides of
the invention include MEK polypeptides comprising a mutation at one
or more of the following amino acid residues with respect to the
wild type human MEK1 polypeptide: 56Q, 99I, 104K, 113R, 120E, 128G,
133F, 215L, 237G, 326P, and 368E, or at one or more of the
following amino acid residues with respect to the wild-type human
MEK2 polypeptide: 60Q, 103I, 108K, 117R, 124E, 132G, 137F, 219L,
241G, 334P, and 376E. Exemplary mutant MEK polypeptides of the
invention also include MEK polypeptides comprising one or more of
the following amino acid substitutions with respect to the wild
type human MEK1 protein: 67D>N, 74L>Q, 74L>R, 103I>N,
106A>T, 107I>M, 111I>N, 115L>R, 115L>P, 119H>P,
124P>L, 124P>S, 128G>D, 129F>L, 167G>R, 203E>K,
211V>D, 247W>G, 258V>G, 260R>G, 276G>E, 286G>E,
302M>R, 303D>A, 308M>L, 328G>L, 328G>R, 379I>L
and 399H>D; or the wild-type MEK2 protein: 71D>N, 78L>Q,
78L>R, 107I>N, 110A>T, 111I>M, 115I>N, 119L>R,
119L>P, 123H>P, 128P>L, 128P>S, 132G>D, 133F>L,
171G>R, 207E>K, 215V>D, 251W>G, 262V>G, 264R>G,
280G>E, 310M>R, 311D>A, 316M>L, 336G>L, and
336G>R. In another embodiment, exemplary mutant MEK polypeptides
of the invention also include MEK polypeptides comprising one or
more of the following amino acid substitutions with respect to the
wild type human MEK1 protein: 56Q>P, 99I>T, 104K>N,
113R>S, 120E>D, 128G>D, 133F>L, 215L>P, 237G>V,
326P>L and 368E>K; or the wild-type MEK2 protein: 60Q>P,
103I>T, 108K>N, 117R>S, 124E>D, 132G>D, 137F>L,
219L>P, 241G>V, 334P>L, 376E>K.
[0134] Mutant MEK proteins can be generated by mutagenesis of a
wild-type MEK protein, or of the nucleic acid molecule encoding a
wild-type MEK protein. Mutant MEK proteins can also be identified
by screening combinatorial libraries of MEK mutants for a mutant
MEK protein having a desired activity, e.g., resistance to a MEK
inhibitor. 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.
VII. Detection of Mutations
[0135] In another aspect, the invention pertains to methods of
detecting the presence of a mutant MEK 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 MEK protein
of the invention in a sample, e.g., a nucleic acid and/or a
polypeptide 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.
[0136] Methods for detecting the presence of resistance mutations
in genomic DNA, cDNA, and RNA (i.e., mRNA) containing a sequence
encoding a MEK 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 MEK
polypeptides, 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 polypeptide, or a
nucleic acid molecule encoding a MEK1 polypeptide, having a
mutation at one or more of the following amino acid residues: 67D,
74L, 103I, 106A, 107I, 111I, 115L, 119H, 124P, 128G, 129F, 167G,
203E, 211V, 247W, 258V, 260R, 276G, 286G, 302M, 303D, 308M, 328G,
399H, 56Q, 99I, 104K, 113R, 120E, 128G, 133F, 215L, 237G, 326P, and
368E; or to detect the presence of a MEK2 polypeptide, or a nucleic
acid molecule encoding a MEK2 polypeptide, having a mutation at one
or more of the following amino acid residues: 71D, 78L, 107I, 110A,
111I, 115I, 119L, 123H, 128P, 132G, 133F, 171G, 207E, 215V, 251W,
262V, 264R, 280G, 310M, 311D, 316M, 336G, 60Q, 103I, 108K, 117R,
124E, 132G, 137F, 219L, 241G, 334P, and 376E. In exemplary
embodiments, methods including, but not limited to, the following
can be used to detect the presence of a MEK1 polypeptide, or a
nucleic acid molecule encoding a MEK1 polypeptide, having one or
more of the following mutations: 67D>N, 74L>Q, 74L>R,
103I>N, 106A>T, 107I>M, 111I>N, 115L>R, 115L>P,
19H>P, 124P>L, 124P>S, 128G>D, 129F>L, 167G>R,
203E>K, 211V>D, 247W>G, 258V>G, 260R>G, 276G>E,
286G>E, 302M>R, 303D>A, 308M>L, 328G>L, 328G>R,
379I>L, 399H>D, 56Q>P, 99I>T, 104K>N, 113R>S,
120E>D, 128G>D, 133F>L, 215L>P, 237G>V, 326P>L,
and 368E>K; or a MEK2 polypeptide, or a nucleic acid molecule
encoding a MEK2 polypeptide, having one or more of the following
mutations: 71D>N, 78L>Q, 78L>R, 107I>N, 110A>T,
111I>M, 115I>N, 119L>R, 119L>P, 123H>P, 128P>L,
128P>S, 132G>D, 133F>L, 171G>R, 207E>K, 215V>D,
251W>G, 262V>G, 264R>G, 280G>E, 310M>R, 311D>A,
316M>L, 336G>L, 336G>R, 60Q>P, 103I>T, 108K>N,
117R>S, 124E>D, 132G>D, 137F>L, 219L>P, 241G>V,
334P>L, and 376E>K.
[0137] 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.
[0138] 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.
[0139] 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.
[0140] 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 MEK nucleic acid or a MEK polypeptide 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
MEK polypeptide, 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 MEK polypeptide, DNA
encoding a MEK polypeptide, 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 MEK polypeptide, 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 MEK
polypeptide. 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 MEK 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, including, for
example, those described by Sambrook, Fritsch and Maniatis,
Molecular Cloning: Cold Spring Harbor Laboratory Press (1989), and
by Current Protocols in Molecular Biology, eds. Ausubel et al.,
John Wiley & Sons (1992). 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 MEK polypeptide also may
employ extraction and/or isolation of polypeptides from a
biological sample. Techniques suitable for extracting and/or
isolating polypeptides from a biological sample are well known in
the art and are further described in, for example, Current
Protocols in Molecular Biology, eds. Ausubel et al., John Wiley
& Sons (1992).
[0141] The presence or absence of one or more mutations described
herein determines the ability of the screened individuals to resist
therapy with a first-generation MEK inhibitor. According to methods
provided by the invention, these results will be used to adjust
and/or alter the dose of the first-generation MEK inhibitor, or to
select a course of treatment using a second-generation MEK
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.
[0142] The resistance mutations in MEK polypeptides, or in nucleic
acid molecules encoding MEK polypeptides, 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 MEK polypeptides, or any
other biochemical interpretation.
1. DNA Sequencing
[0143] 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 MEK gene can be
detected by cloning and sequencing a MEK allele present in a sample
obtained from the subject. If desired, MEK mRNA can be sequenced
directly, or the polymerase chain reaction technique ("PCR") can be
used to amplify MEK 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 MEK allele. Techniques that may
be used to detect one or more mutations in a MEK nucleic acid
include, but are not limited to, those described in Sambrook,
Fritsch and Maniatis, Molecular Cloning. Cold Spring Harbor
Laboratory Press (1989); Current Protocols in Molecular Biology,
Ausubel et al., eds., John Wiley & Sons (1992); PCR Protocols,
Current Methods and Applications, White, B. A., ed., Humana Press
Inc. (1993), PCR Sequencing Protocols, Rapley R. et al., eds.,
Humana Press Inc. (1996)
2. Exonuclease Resistance
[0144] 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
[0145] 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
[0146] 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
[0147] 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)
[0148] 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
[0149] 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 polymerasc, 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
[0150] 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;
5,322,7835,563,055, 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
[0151] MEK1 or MEK2 polypeptides having one or more resistance
mutations described herein can be detected using antibodies that
specifically recognize the mutant MEK1 or MEK2 polypeptides, but do
not recognize wild-type MEK1 or MEK2 polypeptides. Antibodies can
be raised against one or more allelic forms of the MEK1 or MEK2
polypeptides 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.
[0152] Antibodies can be generated either by standard monoclonal
antibody techniques or generated through recombinant based
expression systems. See generally. Abbas. Lichtman, and Pober,
Cellular and Molecular Immunology, W. B. Saunders Co. (1991). The
term "antibodies" is meant to include intact antibody molecules as
well as antibody fragments or derivatives, such as Fab and F(ab')2,
which are capable of specifically binding to antigen. The
antibodies so produced 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.
[0153] Such antibodies specific for mutant MEK polypeptides can be
used to detect the presence of a MEK polypeptide 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 MEK polypeptides can also be
second-generation MEK inhibitors. The ability of an antibody which
specifically recognizes a mutant MEK polypeptide to inhibit the
biological activity of the mutant MEK polypeptide can be determined
using the methods described herein for identifying
second-generation MEK inhibitors.
VIII. Diagnostic and Screening Applications
[0154] The foregoing techniques can be used to determine the
presence or absence of a previously identified resistance mutation
in a MEK nucleic acid or polypeptide molecule in a sample obtained
from a patient. Identification of a mutant MEK nucleic acid or
polypeptide 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 MEK (e.g., a cancer), identification of a mutant
MEK nucleic acid or polypeptide 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 MEK inhibitor. In one embodiment,
identification of a MEK nucleic acid or polypeptide 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 first generation MEK inhibitor. e.g., CI-1040,
AZD6244, PD318088, PD98059, PD334581, RDEA119, Compound A, or
Compound B.
[0155] A patient who has a MEK resistance mutation described herein
has a higher risk of relapse from treatment with a first-generation
MEK inhibitor than a patient in whom a MEK resistance mutation can
not be detected. Accordingly, as used herein, the term "relatively
high risk of relapse" is in relation to a patient in whom a mutant
MEK nucleic acid or polypeptide 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 MEK nucleic acid or polypeptide molecule cannot be
detected.
[0156] In certain embodiments, identification of a MEK polypeptide,
or a nucleic acid encoding a MEK polypeptide, having a mutation at
one or more of the following residues indicates that the patient is
at relatively high risk of relapse or lack of response to treatment
with a first-generation MEK inhibitor, e.g., CI-1040: 67D, 74L,
103I, 106A, 107I, 111I, 115L, 119H, 124P, 128G, 129F, 167G, 203E,
211V, 247W, 258V, 260R, 276G, 286G, 302M, 303D, 308M, 328G and 399H
in MEK1; and 71D, 78L, 107I, 110A, 111 I, 115I, 119L, 123H, 128P,
132G, 133F, 171G, 207E, 215V, 251W, 262V, 264R, 280G, 310M, 311D,
316M, and 336G in MEK2. In other embodiments, identification of a
MEK polypeptide, or a nucleic acid encoding a MEK polypeptide,
having a mutation at one or more of the following residues
indicates that the patient is at relatively high risk of relapse or
lack of response to treatment with a first-generation MEK
inhibitor, e.g., CI-1040: 56Q, 99I, 104K, 113R, 120E, 128G, 133F,
215L, 237G, 326P and 368E in MEK1; and 60Q, 103I, 108K, 117R, 124E,
132G, 137F, 219L, 241G, 334P and 376E in MEK2. In exemplary
embodiments, identification of a MEK polypeptide, or a nucleic acid
encoding a MEK polypeptide, having one or more of the following
resistance mutations indicates that the patient is at relatively
high risk of relapse or lack of response to treatment with a MEK
inhibitor, e.g., CI-1040: 67D>N, 74L>Q, 74L>R, 103I>N,
106A>T, 107I>M, 111I>N, 115L>R, 115L>P, 119H>P,
124P>L, 124P>S, 128G>D, 129F>L, 167G>R, 203E>K,
211V>D, 247W>G, 258V>G, 260R>G, 276G>E, 286G>E,
302M>R, 303D>A, 308M>L, 328G>L, 328G>R, 379I>L
and 399H>D in MEK1; and 71D>N, 78L>Q, 78L>R, 107I>N,
110A>T, 111I>M, 115I>N, 119L>R, 119L>P, 123H>P,
128P>L, 128P>S, 132G>D, 133F>L, 171G>R, 207E>K,
215V>D, 251W>G, 262V>G, 264R>G, 280G>E, 310M>R,
311D>A, 316M>L, 336G>L, and 336G>R in MEK2. In other
embodiments, identification of a MEK polypeptide, or a nucleic acid
encoding a MEK polypeptide, having one or more of the following
resistance mutations indicates that the patient is at relatively
high risk of relapse or lack of response to treatment with a MEK
inhibitor, e.g., CI-1040: 56Q>P, 99I>T, 104K>N, 113R>S,
120E>D, 128G>D, 133F>L, 215L>P, 237G>V, 326P>L,
and 368E>K in MEK1; and 60Q>P, 103I>T, 108K>N,
117R>S, 124E>D, 132G>D, 137F>L, 219L>P, 241G>V,
334P>L, and 376E>K in MEK2.
[0157] Determining the presence or absence of a mutant MEK nucleic
acid or polypeptide 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
first-generation MEK inhibitor is selected for a patient wherein a
cancer cell-containing sample from the patient does not contain a
mutant MEK nucleic acid or polypeptide molecule. Such a patient can
be stratified to a treatment regimen comprising a first-generation
MEK inhibitor. The MEK inhibitor can be given to such a patient at
a standard dosage, at standard dosing intervals.
[0158] 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 MEK nucleic acid or
polypeptide 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 MEK inhibitor and a RAF inhibitor advantageously suppresses
the emergence of MEK resistant alleles in a patient whose cancer
does not contain a mutant MEK molecule.
[0159] 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
MEK nucleic acid or polypeptide molecule, e.g., a MEK nucleic acid
or polypeptide 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.
[0160] In another embodiment, a treatment regimen comprising
treatment with an elevated dosage of a first-generation MEK
inhibitor is selected for a patient wherein a cancer
cell-containing sample from the patient contains a mutant MEK
nucleic acid or polypeptide molecule. Such a patient can be
stratified to a treatment regimen comprising an elevated dosage of
a first-generation MEK inhibitor. In an exemplary aspect of the
foregoing embodiments, the first-generation MEK inhibitor is
CI-1040, AZD6244, PD318088, PD98059, PD334581, RDEA119, Compound A,
or Compound B.
[0161] In an alternative embodiment, identification of a mutant MEK
nucleic acid or polypeptide molecule 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.
Accordingly, in one embodiment, a treatment regimen comprising
treatment with a second-generation MEK inhibitor is selected for a
patient wherein a cancer cell-containing sample from the patient
contains a mutant MEK nucleic acid or polypeptide molecule. Such a
patient can be stratified to a treatment regimen comprising a
second-generation MEK inhibitor.
[0162] In particular aspects of the foregoing embodiments, the
mutant MEK polypeptide is a mutant MEK1 polypeptide that has a
mutation at one or more of the following residues: 67D, 74L, 103I,
106A, 107I, 111I, 115L, 119H, 124P, 128G, 129F, 167G, 203E, 211V,
247W, 258V, 260R, 276G, 286G, 302M, 303D, 308M, 328G and 399H. In
another embodiment, the mutant MEK polypeptide is a mutant MEK1
polypeptide that has a mutation at one or more of the following
residues: 56Q, 99I, 104K, 113R, 120E, 128G, 133F, 215L, 237G, 326P,
and 368E. In exemplary embodiments, the mutant MEK1 polypeptide has
one or more of the following mutations: 67D>N, 74L>Q,
74L>R, 103I>N, 106A>T, 107I>M, 111I>N, 115L>R,
15L>P, 19H>P, 124P>L, 124P>S, 128G>D, 129F>L,
167G>R, 203E>K, 211V>D, 247W>G, 258V>G, 260R>G,
276G>E, 286G>E, 302M>R, 303D>A, 308M>L, 328G>L,
328G>R, 379I>L and 399H>D. In other embodiments, the
mutant MEK1 polypeptide has one or more of the following mutations:
56Q>P, 99I>T, 104K>N, 113R>S, 120E>D, 128G>D,
133F>L, 215L>P, 237G>V, 326P>L, and 368E>K. In
related embodiments, the mutant MEK polypeptide is a mutant MEK2
polypeptide that has a mutation at one or more of the following
residues: 71D, 78L, 107I, 110A, 111I, 115I, 119L, 123H, 128P, 132G,
133F, 171G, 207E, 215V, 251W, 262V, 264R, 280G, 310M, 311D, 316M,
and 336G. In another embodiment, the mutant MEK polypeptide is a
mutant MEK2 polypeptide that has a mutation at one or more of the
following residues: 60Q, 103I, 108K, 117R, 124E, 132G, 137F, 219L,
241G, 334P, and 376E. In exemplary embodiments, the mutant MEK2
polypeptide has one or more of the following mutations: 71D>N,
78L>Q, 78L>R, 107I>N, 110A>T, 111I>M, 115I>N,
119L>R, 119L>P, 123H>P, 128P>L, 128P>S, 132G>D,
133F>L, 171G>R, 207E>K, 215V>D, 251W>G, 262V>G,
264R>G, 280G>E, 310M>R, 311 D>A, 316M>L, 336G>L,
and 336G>R. In other embodiments, the mutant MEK2 polypeptide
has one or more of the following mutations: 60Q>P, 103I>T,
108K>N, 117R>S, 124E>D, 132G>D, 137F>L, 219L>P,
241G>V, 334P>L, and 376E>K. In a preferred embodiment, a
mutant MEK nucleic acid molecule encodes a mutant MEK polypeptide
containing a mutation at one or more of the foregoing amino acid
residues.
IX. Methods of Treatment
[0163] In various embodiments, the invention provides methods of
treating a subject having a cancer. Said methods generally comprise
administration of a MEK inhibitor, e.g., a second-generation MEK
inhibitor, as described herein. In exemplary embodiments, the
subject has a cancer containing a MEK polypeptide having one or
more mutations, e.g., one or more of the resistance mutations
described herein. In related embodiments, the subject has a cancer
containing a nucleic acid molecule encoding a MEK polypeptide
having one or more mutations, e.g., one or more of the resistance
mutations 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.
[0164] Because mutations in MEK broadly enhance resistance in a
number of types of cancer, the methods and second-generation MEK
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
macroglobulinemia, 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.
[0165] 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 case 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.
[0166] 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.
[0167] 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 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.
[0168] 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.
[0169] 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.
[0170] 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.
[0171] 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.
[0172] 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.
[0173] 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.
[0174] 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.
[0175] 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.
[0176] 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.
[0177] 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.
[0178] 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.
[0179] 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.
[0180] 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.
[0181] 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.
[0182] 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.
[0183] 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.
[0184] 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.
[0185] 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.
[0186] Ophthalmic formulations, eye ointments, powders, solutions
and the like, are also contemplated as being within the scope of
this invention.
[0187] 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.
[0188] 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.
[0189] 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.
[0190] 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.
[0191] 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.
[0192] 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.
[0193] 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 intrastemal injection and
infusion.
[0194] 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.
[0195] 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.
[0196] 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.
[0197] 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.
[0198] 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.
[0199] 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.
[0200] 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 is that amount
treats a viral infection.
[0201] 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.
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.
X. Methods of Treatment Using a Combination Therapy
[0202] The present invention further describes methods of
suppressing the emergence of MEK resistance mutations in a subject
having cancer. Such methods involve the combined administration of
a MEK inhibitor (e.g., a first generation MEK inhibitor or a second
generation MEK inhibitor) and a RAF inhibitor. Accordingly, the
present invention provides a combination therapy for treating or
preventing the symptoms of a cancer, comprising administration of a
MEK inhibitor and a RAF inhibitor. The term "combination therapy",
as used herein, refers to the administration of two or more
therapeutic substances, e.g., a MEK inhibitor or a RAF inhibitor.
The MEK 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 MEK inhibitor and a RAF
inhibitor suppresses the emergence of MEK resistance mutations to a
greater extent than administration of either a MEK inhibitor or a
RAF inhibitor alone. Standard dosages of the MEK 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, 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.
[0203] Exemplary MEK inhibitors include first-generation and second
generation MEK inhibitors. In particular embodiments, MEK
inhibitors useful for combination therapy include CI-1040. AZD6244,
PD318088, PD98059, PD334581, RDEA119, Compound A, and Compound B.
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.
[0204] Accordingly, the invention provides methods of treating
cancer in a subject, comprising administering a combination therapy
comprising a MEK 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 MEK. In a preferred embodiment, a combination therapy
comprising a MEK inhibitor and a RAF inhibitor is selected for a
subject in whom one or more mutations in MEK are not detected. In
such a subject, the combination therapy advantageously suppresses
the emergence of MEK resistance mutations. The invention further
provides methods of suppressing the emergence of MEK resistance
mutations in a subject having cancer, comprising administering to
the subject a combination therapy comprising a MEK inhibitor and a
RAF inhibitor, as described herein. Accordingly, a combination
therapy comprising a MEK inhibitor and a RAF inhibitor may be
administered to a subject in whom one or more MEK resistance
mutations are detected. Such a combination therapy suppresses the
growth of cells containing MEK resistance mutations. In some
embodiments, the method further comprises screening a subject to
detect the presence or absence of one or more mutations in MEK. In
these embodiments, a combination therapy comprising a MEK inhibitor
and a RAF inhibitor can be given, for example, to a subject in whom
a MEK resistance mutation was not detected, or, for example, to a
subject in whom a MEK resistance mutation was detected.
[0205] In another embodiment of the invention, a pharmaceutical
composition can comprise (a) a MEK inhibitor and also (b) a RAF
inhibitor. In some embodiments, the MEK inhibitor can be a first
generation MEK inhibitor or a second generation MEK 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 exemplary embodiments, the MEK inhibitor can be CI-1040,
AZD6244, PD318088, PD98059, PD334581, RDEA119, Compound A, or
Compound B, and 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 pharmaceutical composition
comprises (a) AZD6244 and also (b) PLX4720.
[0206] 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 MEK
inhibitor, and administering to the subject a pharmaceutical
composition comprising a RAF inhibitor. The MEK inhibitor and the
RAF inhibitor can be administered to the subject sequentially or
simultaneously. A sequential administration includes (a) first
administering the MEK inhibitor followed by (b) administering the
RAF inhibitor. An alternative sequential administration includes
(a) first administering the RAF inhibitor followed by (b)
administering the MEK inhibitor. A simultaneous administration
includes administering the MEK inhibitor and the RAF inhibitor at
the same time; or at substantially the same time.
[0207] When administration involves the separate administration
(e.g., sequential administration) of the first compound (e.g., a
MEK 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.
[0208] When the MEK inhibitor and the RAF inhibitor are
administered sequentially, they are separately formulated and can
be provided in any order. When the MEK 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 MEK 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 MEK inhibitor and the RAF
inhibitor includes the following: [0209] A) release and
bioavailability of the MEK inhibitor followed by release and
bioavailability of the RAF inhibitor, [0210] B) release and
bioavailability of the RAF inhibitor followed by release and
bioavailability of the MEK inhibitor; [0211] C) release and
bioavailability of the MEK inhibitor at the same time (or
substantially at the same time as) release and bioavailability of
the RAF inhibitor.
[0212] 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 MEK
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 MEK inhibitor and a RAF inhibitor, wherein the cancer
is selected from the group consisting of 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.
[0213] A combination of compounds described herein (e.g., a MEK
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 MEK inhibitor and
a RAF inhibitor suppress the emergence of MEK resistance alleles to
a greater extent than when either compound is used alone.
[0214] 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.
[0215] 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.
[0216] 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.
[0217] In another embodiment of the invention, the MEK 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. The CDA substrate and the
compound of any one of formulae I-VIII can be formulated together
with or separately from such other pharmaceutical agents.
XI. Kits
[0218] Various kits may be assembled as part of the present
invention. A kit can contain components to assay for mutations in
MEK to evaluate a particular patient for the risk of developing
resistance to therapy using a 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 MEK inhibitor (e.g., CI-1040). It is
contemplated that primers (or pairs of primers) that are
complementary to or identical to all or part of SEQ ID NO:1 or SEQ
ID NO:3, 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 polypeptide
containing a mutation at one or more of the following amino acid
residues: 67D, 74L, 103I, 106A, 107I, 111I, 115L, 119H, 124P, 128G,
129F, 167G, 203E, 21I V, 247W, 258V, 260R, 276G, 286G, 302M, 303D,
308M, 328G and 399H, or a mutant MEK2 polypeptide containing a
mutation at one or more of the following residues: 71D, 78L, 107I,
10A, 111I, 115I, 119L, 123H, 128P, 132G, 133F, 171G, 207E, 215V,
251W, 262V, 264R, 280G, 310M, 311D, 316M, and 336G. In other
embodiments, the primers can be used to specifically detect or
amplify a nucleic acid molecule encoding a mutant MEK1 polypeptide
containing a mutation at one or more of the following amino acid
residues: 56Q, 99I, 104K, 113R, 120E, 128G, 133F, 215L, 237G, 326P,
and 368E, or a mutant MEK2 polypeptide containing a mutation at one
or more of the following residues: 60Q, 103I, 108K, 117R, 124E,
132G, 137F, 219L, 241G, 334P, and 376E. In exemplary embodiments,
the primers specifically detect or amplify a nucleic acid molecule
encoding a MEK1 polypeptide containing one or more of the following
mutations: 67D>N, 74L>Q, 74L>R, 103I>N, 106A>T,
107I>M, 111I>N, 115L>R, 115L>P, 119H>P, 124P>L,
124P>S, 128G>D, 129F>L, 167G>R, 203E>K, 211V>D,
247W>G, 258V>G, 260R>G, 276G>E, 286G>E, 302M>R,
303D>A, 308M>L, 328G>L, 328G>R, 379I>L and
399H>D, or a MEK2 polypeptide containing one or more of the
following mutations: 71D>N, 78L>Q, 78L>R, 107I>N,
110A>T, 111I>M, 115I>N, 19L>R, 19L>P, 123H>P,
128P>L, 128P>S, 132G>D, 133F>L, 171G>R, 207E>K,
215V>D, 251 W>G, 262V>G, 264R>G, 280G>E, 310M>R,
311D>A, 316M>L, 336G>L, and 336G>R. In other
embodiments, the primers specifically detect or amplify a nucleic
acid molecule encoding a MEK1 polypeptide containing one or more of
the following mutations: 56Q>P, 99I>T, 104K>N, 113R>S,
120E>D, 128G>D, 133F>L, 215L>P, 237G>V, 326P>L,
and 368E>K, or a MEK2 polypeptide containing one or more of the
following mutations: 60Q>P, 103I>T, 108K>N, 117R>S,
124E>D, 132G>D, 137F>L, 219L>P, 241G>V, 334P>L,
and 376E>K. In other embodiments, the kits contain instructions
for using primers that are complementary or identical to all or
part of SEQ ID NO:1 or SEQ ID NO:3 to amplify a nucleotide sequence
encoding a MEK polypeptide.
[0219] In other embodiments, the kits comprise compositions for
detecting a mutation comprising a MEK polypeptide, such as an
antibody which specifically recognizes a mutant MEK polypeptide
containing one or more resistance mutations. Exemplary polypeptides
include a mutant MEK1 polypeptide containing a mutation at one or
more of the following amino acid residues: 67D, 74L, 103I, 106A,
107I, 111I, 115L, 119H, 124P, 1280, I29F, 167G, 203E, 211V, 247W,
258V, 260R, 276G, 286G, 302M, 303D, 308M, 328G and 399H, or a
mutant MEK2 polypeptide containing a mutation at one or more of the
following amino acid residues: 71D, 78L, 107I, 110A, 111I, 115I,
119L, 123H, 128P, 132G, 133F, 171G, 207E, 215V, 251W, 262V, 264R,
280G, 310M, 311D, 316M, and 336G. Other exemplary polypeptides
include a mutant MEK1 polypeptide containing a mutation at one or
more of the following amino acid residues: 56Q, 99I, 104K, 113R,
120E, 128G, 133F, 215L, 237G, 326P, and 368E, or a mutant MEK2
polypeptide containing a mutation at one or more of the following
amino acid residues: 60Q, 103I, 108K, 117R, 124E, 132G, 137F, 219L,
241G, 334P, and 376E. In other embodiments, the mutant MEK
polypeptide contains one or more of the following mutations:
67D>N, 74L>Q, 74L>R, 103I>N, 106A>T, 107I>M, 11I
I>N, 115L>R, 115L>P, 119H>P, 124P>L, 124P>S,
128G>D, 129F>L, 167G>R, 203E>K, 211V>D, 247W>G,
258V>G, 260R>G, 276G>E, 286G>E, 302M>R, 303D>A,
308M>L, 328G>L, 328G>R, 379I>L and 399H>D in MEK1;
and 71D>N, 78L>Q, 78L>R, 107I>N, 110A>T, 111I>M,
115I>N, 119L>R, 119L>P, 123H>P, 128P>L, 128P>S,
132G>D, 133F>L, 171G>R, 207E>K, 215V>D, 251W>G,
262V>G, 264R>G, 280G>E, 310M>R, 311D>A, 316M>L,
336G>L, and 336G>R in MEK2. In other embodiments, the mutant
MEK polypeptide contains one or more of the following mutations:
56Q>P, 99I>T, 104K>N, 113R>S, 120E>D, 128G>D,
133F>L, 215L>P, 237G>V, 326P>L, and 368E>K in MEK1;
and 60Q>P, 103I>T, 108K>N, 17R>S, 124E>D, 132G>D,
137F>L, 219L>P, 241G>V, 334P>L, and 376E>K in
MEK2.
[0220] All of the essential materials and reagents required for
assaying for MEK 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.
[0221] The invention further provides kits comprising compositions,
(e.g., pharmaceutical compositions), comprising a second generation
MEK inhibitor. 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.
[0222] The invention likewise provides kits comprising compositions
suitable for use in a combination therapy involving a MEK inhibitor
and a RAF inhibitor. Such kits may include, for example, a
composition comprising a MEK inhibitor and a composition comprising
a RAF inhibitor. Such kits may alternatively include a composition
comprising both a MEK 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 MEK resistance alleles in a subject having cancer. The
instructions may describe the administration of a composition
comprising a MEK inhibitor simultaneously, prior to, or following
administration of a RAF inhibitor.
[0223] 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.
[0224] 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.
[0225] 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 MEK
polypeptide has been identified, discontinue treatment with a
first-generation MEK inhibitor, be monitored for relapse during
treatment with a first-generation MEK inhibitor, continue treatment
with a first-generation MEK inhibitor at an elevated dosage, or
initiate treatment with a second-generation MEK inhibitor. An
instruction sheet included with the kits of the invention can
likewise recommend that a patient having a disorder in which a
mutant MEK polypeptide is not detectable continue treatment with a
first-generation MEK 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.
[0226] 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 MEK Mutations that Confer Resistance to
First-Generation MEK Inhibitors
[0227] To investigate the importance of RAF/MEK dependency and
"on-target" resistance to MEK inhibition in melanoma, random
mutagenesis screens together with massively-parallel sequencing
were employed to discover the spectrum of variants associated with
resistance to MEK inhibition (outlined in FIG. 1). Here, a
saturating cDNA library of MEK1 mutations was introduced into A375
melanoma cells, which harbor the BRAF.sup.v600E mutation and are
highly sensitive to MEK inhibition. After culturing these cells for
4 weeks in the presence of a diaryl amine MEK inhibitor (e.g.,
AZD6244 or CI-1040) resistant clones emerged, .about.1000 of which
were pooled and characterized en masse by massively-parallel
sequencing. An additional 100 clones were sequenced by the Sanger
method. This combined analysis of .about.1100 resistant clones
vastly exceeded the scope of prior mutagenesis studies and offered
a comprehensive framework for unbiased characterization of
MEK/-mediated drug resistance.
[0228] The distribution of candidate resistance alleles across each
MEK1 nucleotide position, and the resulting amino acid
substitutions within the MEK three-dimensional structure, were then
investigated. Nearly all non-synonymous nucleotide mutations
clustered within exons 3 and 6 of MEK (FIG. 2a), which encode "C
helix" and flanking hydrophobic residues, plus the majority of the
kinase activation loop. Notably, these alleles corresponded closely
to the known binding pocket of diaryl amine-based MEK inhibitors,
which engage a novel allosteric site adjacent to the ATP binding
motif (FIG. 2b). Furthermore, several high-frequency MEK amino acid
substitutions converted a hydrophobic residue to a charged residue
or introduced a proline into the proximal aspect of the C helix
(FIG. 2c). Such alterations may disrupt drug binding either through
steric hindrance or altered C helical conformation. A few mutations
localized elsewhere within MEK1, but preliminary functional studies
suggested that these were less likely to exert a prominent
resistance phenotype. The foregoing data indicate that MEK1 exons 3
and 6 represent promising candidate loci for the clinical emergence
of resistance to MEK inhibition. FIG. 3a depicts the frequency of
mutations across the MEK1 coding sequence. Corresponding amino
acids are shown for high frequency alleles. FIG. 3b depicts the
relative levels of pERK1/2, pMEK1/2 and .alpha.-tubulin in A375
cells expressing resistant MEK1 alleles.
Example 2
Acquired MEK1 Mutations in AZD6244-Resistant Metastatic
Melanoma
[0229] To test the hypothesis that MEK exons 3 and 6 are likely
candidates for emergence of resistance to MEK inhibition, the MEK1
locus in melanoma genomic DNA from patients enrolled in a phase II
clinical trial of the MEK inhibitor AZD6244.sup.5 was studied.
Table 1 summarizes pertinent clinical characteristics of patients
examined. In this trial, AZD6244 treatment did not improve clinical
outcomes when compared to temozolomide; however, we identified
seven patients who experienced transient disease stabilization
followed by relapse on AZD6244 (mean stable disease
duration=55+/-18 days in patients 1-6). Five of these patients
contained dermal metastases amenable to both pre-treatment and
post-relapse biopsy, and three harbored BRAF.sup.V600E melanomas.
One patient (patient 7) with BRAF.sup.V600E melanoma experienced
prolonged disease stabilization on AZD6244.
[0230] To investigate the clinical relevance of on-target
resistance to MEK inhibitors, MEK1 exons 3 and 6 were PCR-amplified
in tumor DNA from relapsed patients. PCR products from all patients
were pooled and subjected to massively parallel sequencing. This
analysis identified a C>T transition in MEK1 exon 3 that encodes
a P124L substitution (FIG. 4a). MEK1 codon 124 was also implicated
by Sanger sequencing in random mutagenesis screens using the MEK
inhibitor CI-1040, although an alternative amino acid was selected
(P124S; Table 2). Sequencing of individual pre-treatment and
post-relapse melanoma samples revealed that MEK1.sup.PI24L occurred
in the post-relapse tumor DNA from patient #7, but this mutation
was absent in the corresponding pre-treatment tumor sample (FIG.
4b).
[0231] The MEK1.sup.PI24L allele was identified in a 55 year old
male with metastatic melanoma, including pulmonary and skeletal
involvement at the time of clinical trial enrollment. The patient
was treated with oral temozolomide but developed progressive
disease characterized by mediastinal lymph node enlargement and
pulmonary infiltration (FIG. 4c). Switching to the AZD6244
treatment arm resulted in regression or stabilization of all
disease sites except for a left axillary lymph note metastasis,
which expanded over 16 weeks with a concomitant rise in the S100
tumor marker. Surgical removal of this metastasis led to a marked
decrease in S100 levels; MEK1P124L was detected in this specimen.
Following resection, the patient continued on AZD6244 and showed
prolonged disease stabilization (>44 weeks), including a
steadily declining RECIST score and a 28% overall reduction in
tumor burden (FIG. 4c).
[0232] To confirm the functional effects of MEK1.sup.PI24L and the
putative MEK1 resistance alleles identified by random mutagenesis,
biochemical and pharmacologic studies were performed on several
representative variants. First, ex vivo melanoma cultures were
derived from the AZD6244-resistant metastasis of patient #7 (M307),
and the effects of MEK inhibition in these cells was examined. The
GI.sub.50 for AZD6244 was 10-50 nM in treatment-naive
BRAF.sup.V600E melanoma cultures (WM3482 and WM3457; Table 3) but
exceeded 2 .mu.M in cells derived from the AZD6244-resistant
metastasis (M307; FIG. 5a). Likewise, marked biochemical inhibition
of ERK phosphorylation (p-ERK) was achieved following 16-hour
exposure to .about.400 nM AZD6244 in treatment-naive lines, but
p-ERK remained robust in the resistant cells even at 10 .mu.M
AZD6244 (FIG. 5b). Similar results were observed in studies of the
related MEK inhibitor C1-1040 (Table 3).
TABLE-US-00001 TABLE 1 Patient Characteristics Patient Age Breslow
Staging at Pre-treatment Post-treatment Durations of stable BRAF #
(years) Gender (mm) trial entry biopsy location biopsy location
disease (days) status 1 48 Female 6 T4b Nx Mx Back, left Skin,
thoracal, 77 V600E left 2 66 Male 3.1 T3b N3 M1c Skin, axillary,
Skin, shoulder, 48 wt thoracal left 3 72 Female 5.5 T4b N2b M1b
Lung, upper lobe n/a 40 -- 4 48 Female 0.3 T1a N1b M1c Axillary
lymph Skin, retro- 39 V600E node, left auricular, right 5 63 Male
unknown Tx Nx M1c Skin, axillary, left n/a 78 wt 6 49 Female 0.8
T1a N3 M1c n/a Skin, neck, left 51 wt 7 58 Male 7 T4 N1 M0 Axillary
lymph Axillary lymph Ongoing V600E node, left node, left
TABLE-US-00002 TABLE 2 Summary of inhibitor-resistant MEK1 alleles
MEK1 allele Method of identification Experimental Validation Q56P
Massively-parallel Not done D67N Massively-parallel and Sanger
Failed L74R Massively-parallel and Sanger Failed I99T
Massively-parallel Not done I103N Sanger Validated K104N
Massively-parallel Not done A106T Sanger Failed I107M
Massively-parallel Failed I111N Massively-parallel and Sanger
Validated L115P Massively-parallel Validated L115R Sanger Validated
H119P Massively-parallel Validated E120D Massively-parallel Not
done P124S Sanger Validated G128D Massively-parallel Not done F129L
Massively-parallel and Sanger Validated F133L Massively-parallel
Not done G167R Massively-parallel and Sanger Failed V211D
Massively-parallel and Sanger Validated L215P Massively-parallel
Not done W247G Massively-parallel and Sanger Failed V258G
Massively-parallel Not done R260G Massively-parallel Failed M302R
Massively-parallel Not done D303A Massively-parallel Failed M308L
Massively-parallel Failed P326L Massively-parallel Not done G328R
Massively-parallel Failed E368K Massively-parallel Failed I379L
Massively-parallel Failed
TABLE-US-00003 TABLE 3 GI.sub.50 values for cell lines and MEK1
alleles Cell culture AZD6244 (.mu.M) PLX4720 (.mu.M) CI-1040
(.mu.M) A375 0.04 0.91 0.07 A375-MEK-wt 0.04* 1.29* 0.11**
A375-MEK-DD 0.05* 16.80* 0.11* A375-MEK-I103N 1.89** 2.94***
6.14*** A375-MEK-I111N 5.96*** 2.94*** 7.59*** A375-MEK-L115P
55.05** 1.97** 11.34*** A375-MEK-L115R 43.65*** 1.17*** 6.80**
A375-MEK-H119P 9.08*** 1.42*** 6.91** A375-MEK-P124S 0.17***
3.44*** 0.62*** A375-MEK-P124L 0.19** 2.84*** 0.56***
A375-MEK-F129L 7.32*** 2.79*** 6.05** A375-MEK-V211D 113.90**
2.73** 8.47*** M307 7.18*** 20.28*** -- WM3457 0.05 0.07 -- WM3482
0.01 0.05 --
Example 3
Pharmacological and Biochemical Characterization of Mutant MEK
Alles
[0233] Next, we introduced P124L and several additional candidate
resistance alleles into the sequence of wild-type MEK1 and
expressed these variants in A375 melanoma cells. All variant
constructs were overexpressed at comparable levels. A375 cells
expressing wild-type MEK1 or a constitutively active control
(MEK-DD) showed AZD6244 GI50 values comparable to wild-type A375
(.about.50 nM; FIG. 5c). In contrast, P124L and P124S alleles
increased the G150 of AZD6244 and CI-1040 by 5-10-fold; several
additional alleles located in the MEK1 C helix (I103N, I111N,
L115P/R, H119P), drug binding pocket (F129L) or activation loop
(V211D) increased the GI.sub.50 by .about.100-fold (FIG. 5c).
Biochemical analyses of both drugs corroborated these findings
(FIG. 6). These results validated the functional relevance of
resistance-associated MEK1 alleles.
Example 4
MEK Mutations Confer Cross-Resistance to RAF Inhibition
[0234] To determine if MEK1 mutations might confer cross-resistance
to RAF inhibition, we examined the effects of the selective B-RAF
inhibitor PLX4720 in the cultured melanoma lines described above.
AZD6244-resistant primary melanoma cells (M307) demonstrated
profound resistance to PLX4720, with a GI.sub.50 value of >20
.mu.M compared to 50-70 nM in treatment-naive lines (FIG. 5d).
These findings were reflected in biochemical studies of MEK
phosphorylation (pMEK) following B-RAF inhibition (FIG. 5c).
Expression of several MEK1 resistance alleles in A375 cells
modestly increased PLX4720 GI.sub.50 levels by 3-4 fold, despite
comparable levels of p-MEK reduction by this compound (FIG. 5f and
Table 3). Interestingly, the constitutively active MEK-DD variant
conferred robust resistance to PLX4720, indicating a specific
dependency on RAF-activated MEK activity in BRAF.sup.V600E melanoma
(FIG. 7). Thus, MEK resistance alleles may confer some
cross-resistance to RAF inhibition in MAP kinase-dependent cancer
cells.
Example 5
Suppression of the emergence of drug-resistant variants
[0235] Whether the Stringency of MAP Kinase Pathway Inhibition
Might Influence the emergence of on-target resistance variants in
cells harboring a MAP kinase oncogene dependency was next examined.
A375 melanoma cells expressing either wild-type MEK1 or a library
of MEK1-mutant cDNAs (described above) were cultured for 4 weeks in
the presence of AZD6244 or PLX4720 (1.5 PM) singly or in
combination. As expected, exposure to AZD6244 completely suppressed
the growth of A375 cells expressing wild-type MEK1, whereas
AZD6244-resistant clones emerged on plates containing mutagenized
MEK1-expressing cells (FIG. 5g). PLX4720-resistant clones emerged
from both wild-type and mutant MEK1-expressing cells under these
conditions. Notably, simultaneous exposure to both AZD6244 and
PLX4720 completely suppressed the emergence of resistant variants
(FIG. 5g). Thus, combined RAF and MEK inhibition can help
circumvent acquired resistance in MAP kinase-dependent cancers.
Methodology
[0236] The following materials and methods were used in connection
with the examples. These materials and methods are illustrative
only and are not intended to be limiting.
MEK1 Expression Constructs
[0237] MEK1 cDNA was cloned into the pWZL-Blast vector by
recombinational cloning (Invitrogen) to generate pWZL-Blast-MEK1.
Specific mutations were introduced into MEK1 cDNA using QuikChange
II Site Directed Mutagenesis (Stratagene).
MEK1 Random Mutagenesis Screen
[0238] MEK1 cDNA was cloned in pWZL (Blast) via Gateway cloning
technology (Invitrogen). To generate random mutant libraries the
plasmid was propagated in E. coli deficient for DNA repair genes
MutS, MutDS- and MutT-(XL1-Red, Stratagene), thus introducing
random mutations into the plasmid. DNA extracted from the bacteria
was subsequently transformed into XL1-Blue (Stratagene) to amplify
the library. The mutagenized MEK1 plasmid or non-mutagenized
control was used to infect A375 melanoma cells. After selection
with blasticidin, cells were plated on 15-cm dishes and cultured in
the presence of MEK inhibitors (AZD6244 or CI-1040; 1.5 .mu.M or 2
.mu.M respectively) for 4 weeks until resistant clones emerged.
[0239] To determine the approximate coverage of mutations within
the library a parallel experiment was performed using the
pDNR-1r-SacB vector. Expression of SacB is lethal in sucrose
containing media. The vector was replicated in XL1-red following
manufacturers protocol. Following transformation bacteria was
plated in triplicate +/- sucrose at different dilutions. Colonies
were then quantified to infer a frequency of mutation (number of
colonies--sucrose/number of colonies+sucrose). The frequency
determined by this method is an underestimate: stop codons are most
scored, and silent and non-deleterious mutations cannot be
detected. One can assume the actual mutation rate would approach a
value 4 times greater than scored with this assay. The number of
MEK1 containing E. coli was quantified per 15 cm plate, then the
number of plates to generate the library was calculated to generate
coverage of approximately 60.times., meaning each base had the
potential to be mutated 60 individual occasions within the library
as a whole. This was achieved by harvesting bacteria from
40.times.15 cm plates (.about.54 000 colonies per plate).
MEK1 Polymerase Chan Reaction
[0240] MEK1 exon 3 was amplified using forward primer
5'-CTTTCATCCCTTCCTCCCTC-3 (SEQ ID NO: 5) and reverse primer
5'-CACCTCCCAGACCAAAGATTAG-3' (SEQ ID NO: 6). MEK1 exon 6 was
amplified using 10-20 ng of genomic DNA, forward primer
5'-CTTCTCTTCCCCAATCTACCTGTG-3 (SEQ ID NO: 7)' and reverse primer
5'-CCTACCCAGCACAAGACTCTG-3 (SEQ ID NO: 8). For each exon,
thermocycling reactions were as follows; 95.degree. C./5 minutes,
followed by 30 cycles of (95.degree. C./15 seconds, 52.degree.
C./20 seconds, 68.degree. C./30 seconds), followed by 68.degree.
C./10 minutes. MEK1 cDNA was amplified using forward primer
5'-CGATCCTCCCTTATCCAGCCCTCACTCCTTCTCTAGG-3 (SEQ ID NO: 9) and
reverse primer 5'-GAGGCCAGCATCGGTTGGTGTG-3' (SEQ ID NO: 10).
Thermocycling reactions were 95.degree. C./5 minutes,
35.times.(95.degree. C./15 seconds, 66.degree. C./60 seconds,
68.degree. C./90 seconds), 68.degree. C./10 minutes. All reactions
were performed using pfx polymerase (Invitrogen).
Sequencing of MEK1 DNA
[0241] AZD6244 or CI-1040-resistant cells emerging from the random
mutagenesis screens were pooled and genomic DNA was prepared
(Qiagen DNeasy). MEK1 cDNA was amplified from genomic DNA using
primers specific to flanking vector sequence at the 5' and 3' end.
In separate experiments, genomic DNA was prepared from melanoma
tumor samples, and PCR was performed using intronic primers that
amplified exons 3 and 6 from MEK1. PCR products were gel purified,
pooled and subjected to single molecule sequencing using an
Illumina 3G instrument according to the manufacturer's
instructions. Sequence reads (2-3 million 36-base pair sequences
per lane) were filtered based on quality metrics and assigned a
variant score according to the manufacturer's instructions.
Analysis of Massively-Parallel Sequencing
[0242] Raw data from massively-parallel sequencing lanes (Illumina)
were analyzed using a "next-generation" sequencing analysis
pipeline. Output from data files representing the nucleotide
sequence, per-base quality measure, variants detected and alignment
to cDNA reference sequence (as determined by alignment with the
ELAND algorithm) were integrated and processed for each run.
Coverage (i.e., the number of fragments including each base of the
cDNA reference) was determined for all bases, and variant alleles
were mapped from individual DNA fragments onto the reference
sequence. Frequency of variation for each non-wild type allele was
determined, and an `average variant score` was calculated as the
average of all quality scores for the position and variant allele
in question. All coding mutations were translated to determine
amino acid variation, if any, and data for high-frequency
(>0.5%), high-quality (AVS>7) mutations were loaded into the
CCGD results database.
Patients and Samples
[0243] Pre-treatment and post-relapse metastatic melanoma samples
were obtained from patients enrolled on a Phase II clinical trial
of AZD6244.sup.5. All patients were consented according to the
approved biobanking IRB protocol (University of Zurich, no, 647)
prior to biopsy.
Cell Lines and Primary Melanoma Cultures
[0244] A375 melanoma cells and 293T retroviral producer cells
(ATCC) were cultured in DMEM (MediaTech) with 10% fetal bovine
serum (Gemini). A primary culture (M307) derived from an
AZD6244-resistant metastatic tumor was generated by treating fresh
biopsy material with Dispase for 4 hours and collagenase for 3
hours at 37.degree. C. Cells were washed (phosphate-buffered
saline) and seeded into RPMI medium (MediaTech) supplemented with
10% fetal calf serum. Treatment-naive BRAFV600E melanoma primary
cultures (WM3482 and WM3457) were kindly provided by Dr. M. Herlyn
(Wistar Institute) and cultured in RPMI, 10% fetal bovine serum and
1% penicillin plus streptomycin.
Retroviral Infections
[0245] 293T cells (70% confluent) were transfected with
pWZL-Blast-MEK1 and pCL-Ampho packaging vector using Lipofectamine
2000 (Invitrogen). Supernatants containing virus were passed
through a 0.45 .mu.m syringe. A375 cells were infected for 16 hr
with virus together with polybrene (4 .mu.g/ml, Sigma). The
selective marker blasticidin (3 .mu.g/ml) was introduced 48 hours
post infection.
Pharmacologic Studies
[0246] CI-1040 was purchased from Shanghai Lechen International
Trading Company, AZD6244 was purchased from Selleck Chemicals Co.,
Ltd., and PLX4720 was purchased from Symansis, Inc. For growth
inhibition analysis, cultured melanoma cells were seeded in 96-well
plates (5000 cells/well, or 3000 cells/well for A375 cells) and
allowed to adhere for 16 hr. Afterwards, media containing serial
dilutions of MEK or RAF inhibitor were added, ensuring that the
final volume of DMSO did not exceed 1%. Cells were incubated for 96
hours in the presence of drug, and viability was measured by the
CellTiter-Glo assay (Promega). Curves and GI.sub.50 values were
generated from the composite of three independent experiments, 6
replicates per experiment.
Pharmacological Growth Inhibition Assays
[0247] Cultured cells were seeded in 96-well plates at a density of
5000 cells/well for all cell lines, with the exception of A375,
where 3000 cells were seeded. Following adherence of the cells,
usually an overnight period was allowed for this, serial dilutions
of a compound were performed 100 fold in DMSO, then transferred to
cells accordingly to yield dilutions ranging from 100 .mu.M to
1.times.10.sup.6 .mu.M, ensuring the final volume of DMSO did not
exceed 1%. Compounds used included the MEK inhibitors CI-1040
(purchased from Shanghai Lechen International Trading Co), and
AZD6244 (purchased from Selleck Chemicals Co., Ltd.) and the BRAF
inhibitor PLX4720 (purchased from Symansis). Following addition of
a drug, cells were incubated for 96 hours. Subsequently cell
viability was measured using the CellTiter-Glo viability assay
(Promega). Viability was calculated as a percentage of the control
(untreated cells) after background subtraction. A minimal number of
three replicates were made for each cell line and drug combination
experiment, and the entire experiment repeated 3 times. The data
from the phaunacologic growth-inhibition assays were modeled using
a nonlinear regression curve fit with a sigmoidal dose response.
These curves were displayed using GraphPad Prism 5 for Windows
(GraphPad Software). The GI.sub.50 was calculated by determining
the slope of the line connecting the data points which flanked the
50% point. The GI.sub.50 of each MEK allele was compared to A375
using two-sample Student's T-test, and M302 was compared to
treatment naive short term cultures. H.sub.A (alternative
hypothesis) was used; mean GI.sub.50 greater than mean GI.sub.50 of
A375 or naive short term culture, .alpha.=0.05, one-sided test. All
P-value calculations were performed using MATLAB Statistics Toolbox
(MATLAB Version 7.1), the MathWorks, Inc.
Western Blot Analysis
[0248] Immunoblot studies were performed using standard procedures.
Briefly, 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). Biochemical effects of MEK or RAF inhibition were
assessed by Western blot analysis of cell lysates collected
following 16 hr exposure to drug at varying concentrations.
EQUIVALENTS
[0249] 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.
Sequence CWU 1
1
1012603DNAHomo 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 31759DNAHomo sapiens 3cccctgcctc tcggactcgg gctgcggcgt
cagccttctt cgggcctcgg cagcggtagc 60ggctcgctcg cctcagcccc agcgcccctc
ggctaccctc ggcccaggcc cgcagcgccg 120cccgccctcg gccgccccga
cgccggcctg ggccgcggcc gcagccccgg gctcgcgtag 180gcgccgaccg
ctcccggccc gccccctatg ggccccggct agaggcgccg ccgccgccgg
240cccgcggagc cccgatgctg gcccggagga agccggtgct gccggcgctc
accatcaacc 300ctaccatcgc cgagggccca tcccctacca gcgagggcgc
ctccgaggca aacctggtgg 360acctgcagaa gaagctggag gagctggaac
ttgacgagca gcagaagaag cggctggaag 420cctttctcac ccagaaagcc
aaggtcggcg aactcaaaga cgatgacttc gaaaggatct 480cagagctggg
cgcgggcaac ggcggggtgg tcaccaaagt ccagcacaga ccctcgggcc
540tcatcatggc caggaagctg atccaccttg agatcaagcc ggccatccgg
aaccagatca 600tccgcgagct gcaggtcctg cacgaatgca actcgccgta
catcgtgggc ttctacgggg 660ccttctacag tgacggggag atcagcattt
gcatggaaca catggacggc ggctccctgg 720accaggtgct gaaagaggcc
aagaggattc ccgaggagat cctggggaaa gtcagcatcg 780cggttctccg
gggcttggcg tacctccgag agaagcacca gatcatgcac cgagatgtga
840agccctccaa catcctcgtg aactctagag gggagatcaa gctgtgtgac
ttcggggtga 900gcggccagct catcgactcc atggccaact ccttcgtggg
cacgcgctcc tacatggctc 960cggagcggtt gcagggcaca cattactcgg
tgcagtcgga catctggagc atgggcctgt 1020ccctggtgga gctggccgtc
ggaaggtacc ccatcccccc gcccgacgcc aaagagctgg 1080aggccatctt
tggccggccc gtggtcgacg gggaagaagg agagcctcac agcatctcgc
1140ctcggccgag gccccccggg cgccccgtca gcggtcacgg gatggatagc
cggcctgcca 1200tggccatctt tgaactcctg gactatattg tgaacgagcc
acctcctaag ctgcccaacg 1260gtgtgttcac ccccgacttc caggagtttg
tcaataaatg cctcatcaag aacccagcgg 1320agcgggcgga cctgaagatg
ctcacaaacc acaccttcat caagcggtcc gaggtggaag 1380aagtggattt
tgccggctgg ttgtgtaaaa ccctgcggct gaaccagccc ggcacaccca
1440cgcgcaccgc cgtgtgacag tggccgggct ccctgcgtcc cgctggtgac
ctgcccaccg 1500tccctgtcca tgccccgccc ttccagctga ggacaggctg
gcgcctccac ccaccctcct 1560gcctcacccc tgcggagagc accgtggcgg
ggcgacagcg catgcaggaa cgggggtctc 1620ctctcctgcc cgtcctggcc
ggggtgcctc tggggacggg cgacgctgct gtgtgtggtc 1680tcagaggctc
tgcttcctta ggttacaaaa caaaacaggg agagaaaaag caaaaaaaaa
1740aaaaaaaaaa aaaaaaaaa 17594400PRTHomo sapiens 4Met Leu Ala Arg
Arg Lys Pro Val Leu Pro Ala Leu Thr Ile Asn Pro 1 5 10 15 Thr Ile
Ala Glu Gly Pro Ser Pro Thr Ser Glu Gly Ala Ser Glu Ala 20 25 30
Asn Leu Val Asp Leu Gln Lys Lys Leu Glu Glu Leu Glu Leu Asp Glu 35
40 45 Gln Gln Lys Lys Arg Leu Glu Ala Phe Leu Thr Gln Lys Ala Lys
Val 50 55 60 Gly Glu Leu Lys Asp Asp Asp Phe Glu Arg Ile Ser Glu
Leu Gly Ala 65 70 75 80 Gly Asn Gly Gly Val Val Thr Lys Val Gln His
Arg Pro Ser Gly Leu 85 90 95 Ile Met Ala Arg Lys Leu Ile His Leu
Glu Ile Lys Pro Ala Ile Arg 100 105 110 Asn Gln Ile Ile Arg Glu Leu
Gln Val Leu His Glu Cys Asn Ser Pro 115 120 125 Tyr Ile Val Gly Phe
Tyr Gly Ala Phe Tyr Ser Asp Gly Glu Ile Ser 130 135 140 Ile Cys Met
Glu His Met Asp Gly Gly Ser Leu Asp Gln Val Leu Lys 145 150 155 160
Glu Ala Lys Arg Ile Pro Glu Glu Ile Leu Gly Lys Val Ser Ile Ala 165
170 175 Val Leu Arg Gly Leu Ala Tyr Leu Arg Glu Lys His Gln Ile Met
His 180 185 190 Arg Asp Val Lys Pro Ser Asn Ile Leu Val Asn Ser Arg
Gly Glu Ile 195 200 205 Lys Leu Cys Asp Phe Gly Val Ser Gly Gln Leu
Ile Asp Ser Met Ala 210 215 220 Asn Ser Phe Val Gly Thr Arg Ser Tyr
Met Ala Pro Glu Arg Leu Gln 225 230 235 240 Gly Thr His Tyr Ser Val
Gln Ser Asp Ile Trp Ser Met Gly Leu Ser 245 250 255 Leu Val Glu Leu
Ala Val Gly Arg Tyr Pro Ile Pro Pro Pro Asp Ala 260 265 270 Lys Glu
Leu Glu Ala Ile Phe Gly Arg Pro Val Val Asp Gly Glu Glu 275 280 285
Gly Glu Pro His Ser Ile Ser Pro Arg Pro Arg Pro Pro Gly Arg Pro 290
295 300 Val Ser Gly His Gly Met Asp Ser Arg Pro Ala Met Ala Ile Phe
Glu 305 310 315 320 Leu Leu Asp Tyr Ile Val Asn Glu Pro Pro Pro Lys
Leu Pro Asn Gly 325 330 335 Val Phe Thr Pro Asp Phe Gln Glu Phe Val
Asn Lys Cys Leu Ile Lys 340 345 350 Asn Pro Ala Glu Arg Ala Asp Leu
Lys Met Leu Thr Asn His Thr Phe 355 360 365 Ile Lys Arg Ser Glu Val
Glu Glu Val Asp Phe Ala Gly Trp Leu Cys 370 375 380 Lys Thr Leu Arg
Leu Asn Gln Pro Gly Thr Pro Thr Arg Thr Ala Val 385 390 395 400
520DNAArtificial SequenceSynthetic forward primer for MEK 1 exon 3
5ctttcatccc ttcctccctc 20622DNAArtificial SequenceSynthetic reverse
primer for MEK 1 exon 3 6cacctcccag accaaagatt ag
22724DNAArtificial SequenceSynthetic forward primer for MEK1 exon 6
7cttctcttcc ccaatctacc tgtg 24821DNAArtificial SequenceSynthetic
reverse primer for MEK1 exon 6 8cctacccagc acaagactct g
21938DNAArtificial SequenceSynthetic forward primer for MEK1 cDNA
9cgatcctccc tttatccagc cctcactcct tctctagg 381022DNAArtificial
SequenceSynthetic reverse primer for MEK1 cDNA 10gaggccagca
tcggttggtg tg 22
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