U.S. patent application number 15/472934 was filed with the patent office on 2017-07-20 for c-raf mutants that confer resistance to raf inhibitors.
The applicant listed for this patent is Rajee Antony, Caroline Emery, Levi A. Garraway. Invention is credited to Rajee Antony, Caroline Emery, Levi A. Garraway.
Application Number | 20170204383 15/472934 |
Document ID | / |
Family ID | 47892065 |
Filed Date | 2017-07-20 |
United States Patent
Application |
20170204383 |
Kind Code |
A1 |
Emery; Caroline ; et
al. |
July 20, 2017 |
C-RAF Mutants that Confer Resistance to RAF Inhibitors
Abstract
Nucleic acids and proteins having a mutant C-RAF sequence, and
methods of identifying patients having cancer who are likely to
benefit from a combination therapy and methods of treatment are
provided.
Inventors: |
Emery; Caroline; (Kansas
City, MO) ; Antony; Rajee; (Norwood, MA) ;
Garraway; Levi A.; (Newton, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Emery; Caroline
Antony; Rajee
Garraway; Levi A. |
Kansas City
Norwood
Newton |
MO
MA
MA |
US
US
US |
|
|
Family ID: |
47892065 |
Appl. No.: |
15/472934 |
Filed: |
March 29, 2017 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
14387735 |
Sep 24, 2014 |
9629839 |
|
|
PCT/US2013/029513 |
Mar 7, 2013 |
|
|
|
15472934 |
|
|
|
|
61616999 |
Mar 28, 2012 |
|
|
|
61708372 |
Oct 1, 2012 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12Q 1/6886 20130101;
C12Y 207/11001 20130101; A61K 31/4184 20130101; G01N 33/5743
20130101; A61P 35/00 20180101; A61K 31/506 20130101; A61P 43/00
20180101; A61K 31/437 20130101; G01N 33/57484 20130101; C12Q
2600/156 20130101; C12Q 2600/106 20130101; C12N 9/12 20130101 |
International
Class: |
C12N 9/12 20060101
C12N009/12 |
Goverment Interests
FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
[0002] This invention was made with government support under
federal grant number DP2 OD002750 awarded by the National
Institutes of Health. The government has certain rights in the
invention.
Claims
1. An expression vector comprising an isolated nucleic acid
molecule encoding a mutant C-RAF polypeptide having C-RAF activity,
wherein said mutant C-RAF polypeptide comprises at least one amino
acid substitution as compared to a wild type C-RAF polypeptide
comprising SEQ. ID. NO. 2, the at least one amino acid substitution
conferring resistance to one or more RAF inhibitors on a cell
expressing the mutant C-RAF polypeptide.
Description
RELATED APPLICATIONS
[0001] This application is a division of U.S. application Ser. No.
14/387,735, filed Sep. 24, 2014, which claims the benefit under 35
U.S.C. .sctn.371 of International Application No.
PCT/US2013/029513, filed Mar. 7, 2013, which claims the benefit of
U.S. Provisional Application Nos. 61/616,999, filed Mar. 28, 2012,
and 61/708,372, filed Oct. 1, 2012, which are incorporated by
reference herein in their entirety.
BACKGROUND
[0003] Oncogenic mutations in the serine/threonine kinase B-RAF
(also known as BRAF) are found in 50-70% of malignant melanomas.
(Davies, H. et al., Nature 417, 949-954 (2002).) Melanoma is
considered to be the deadliest form of skin cancer and for the year
2012, the National Cancer Institute has estimated 72,250 new cases
which could lead to the death of approximately 9,180 people in the
United States. Pre-clinical studies have demonstrated that the
B-RAF(V600E) mutation predicts a dependency on the
mitogen-activated protein kinase (MAPK) signaling cascade in
melanoma (Hoeflich, K. P. et al., Cancer Res. 69, 3042-3051 (2009);
McDermott, U. et al., Proc. Natl Acad. Sci. USA 104, 19936-19941
(2007); Solit, D. B. et al. BRAF mutation predicts sensitivity to
MEK inhibition. Nature 439, 358-362 (2006); Wan, P. T. et al., Cell
116, 855-867 (2004); Wellbrock, C. et al., Cancer Res. 64,
2338-2342 (2004))--an observation that has been validated by the
success of RAF or MEK inhibitors in clinical trials (Flaherty, K.
T. et al., N. Engl. J. Med. 363, 809-819 (2010); Infante, J. R. et
al., J. Clin. Oncol. 28 (suppl.), 2503 (2010); Schwartz, G. K. et
al., J. Clin. Oncol. 27 (suppl.), 3513 (2009).)
[0004] Recently, the FDA approved Raf inhibitor Vemurafenib
(PLX4032). (Dummer et al., 2008; Infante et al., 2010; Joseph et
al., 2010; Flaherty et al., 2010) However, clinical responses to
targeted anticancer therapeutics are frequently confounded by de
novo or acquired resistance. (Engelman, J. A. et al., Science 316,
1039-1043 (2007); Gorre, M. E. et al., Science 293, 876-880 (2001);
Heinrich, M. C. et al., J. Clin. Oncol. 24, 4764-4774 (2006); Daub,
H., Specht, K. & Ullrich, A. Nature Rev. Drug Discov. 3,
1001-1010 (2004).) In the clinical and in-vitro settings, recently
this phenomenon has been shown to be governed either by
overexpression of a parallel signaling module (CRAF, COT) (Montagut
et al., Cancer Res 68:4853-4861 (2008); Johannessen et al., Nature
468:968-972 (2010), activation of a parallel signaling pathway
(PDGFRb, IGF-1R) (Nazarian et al., Nature 468: 973-977 (2010);
Villanueva et al., Cancer Cell 18: 683-695 (2010), amplification of
an upstream target (BRAF) (Shi et al., Nat Commun 3:724 (2012),
deletion in the target (p61BRAF) (Poulikakos et al., Nature
480:387-390 (2011) or by activating mutations in the downstream
target or the target protein itself (Mek) (Emery et al., Proc Natl
Acad Sci USA 106: 20411-20416 (2009); Wagle et al., JCO 29:
3085-3096 (2011). Accordingly, there remains a need for new methods
for identification of resistance mechanisms in a manner that
elucidates "druggable" targets for effective long-term treatment
strategies, for new methods of identifying patients that are likely
to benefit from the treatment strategies, and for methods of
treating patients with the effective long-term treatment
strategies.
BRIEF SUMMARY
[0005] The present invention relates to the development of
resistance to therapeutic agents in the treatment of cancer and
identification of targets that confer resistance to treatment of
cancer. The present invention also relates to identification of
parallel drug targets for facilitating an effective long-term
treatment strategy and to identifying patients that would benefit
from such treatment.
[0006] In one aspect, an isolated nucleic acid molecule encoding a
mutant C-RAF polypeptide having C-RAF activity is provided. The
mutant C-RAF polypeptide includes at least one amino acid
substitution as compared to a wild type C-RAF polypeptide
comprising SEQ. ID. NO. 2, the at least one amino acid substitution
confers resistance to one or more RAF inhibitors on a cell
expressing the mutant RAF polypeptide.
[0007] In another aspect, an expression vector in provided. The
expression vector includes the nucleic acid molecule encoding a
mutant C-RAF polypeptide having C-RAF activity where the mutant
C-RAF polypeptide includes at least one amino acid substitution as
compared to a wild type C-RAF polypeptide comprising SEQ. ID. NO. 2
and the at least one amino acid substitution confers resistance to
one or more RAF inhibitors on a cell expressing the mutant RAF
polypeptide.
[0008] In another aspect, a host cell is provided. The host cell
includes the expression vector.
[0009] In another aspect, an isolated mutant C-RAF polypeptide
having C-RAF activity is provided. The mutant C-RAF polypeptide
includes at least one amino acid substitution as compared to a wild
type C-RAF polypeptide comprising SEQ. ID. NO. 2 and the at least
one amino acid substitution confers resistance to one or more RAF
inhibitors on a cell expressing the mutant C-RAF polypeptide.
[0010] In another aspect, an antibody preparation is provided. The
antibody preparation specifically binds to an isolated mutant C-RAF
polypeptide of the present invention.
[0011] In yet another aspect, a method of treating a subject having
cancer is provided. The method includes extracting nucleic acid
from cells of a cancer of the patient and assaying at least a
portion of a nucleic acid molecule encoding a C-RAF polypeptide for
the presence of one or more mutations in a nucleic acid molecule
encoding a C-RAF polypeptide that alter the identity of an amino
acid residue at one or more amino acids of the encoded C-RAF
polypeptide as compared to a wild type C-RAF polypeptide at one or
more positions selected from the group consisting of 104E, 257S,
261P, 356G, 361G, 427S, 447D, 469M, 478E and 554R. The method also
includes administering an effective amount of a RAF inhibitor and
an effective amount of a second inhibitor to the subject when the
nucleic acid molecule includes nucleotides that alter the amino
acid residue at one or more amino acids of the encoded C-RAF
polypeptide as compared to a wild type C-RAF polypeptide.
[0012] In another aspect, a method of identifying a subject having
cancer who is likely to benefit from treatment with a combination
therapy with a RAF inhibitor and a second inhibitor is provided.
The method includes extracting nucleic acid from cells of a cancer
of the patient and assaying at least a portion of a nucleic acid
molecule encoding a C-RAF polypeptide. The presence of one or more
nucleotides that alter the identity of an amino acid residue at one
or more amino acids of the encoded mutant C-RAF polypeptide
relative to the amino acid at one or more positions of the wild
type C-RAF polypeptide at one or more of amino acid positions
selected from the group consisting of 104E, 257S, 261P, 356G, 361G,
427S, 447D, 469M, 478E and 554R indicates a need to treat the
subject with a RAF inhibitor and a second inhibitor.
BRIEF DESCRIPTION OF THE DRAWINGS
[0013] FIG. 1A-C illustrates C-RAF mutant alleles in A375 cells
(BRAFV600E) resistant to the RAF inhibitor PLX4720.
[0014] (A) The average variant score of candidate mutations across
the C-RAF coding sequence from the PLX4720 mutagenesis screen is
shown. The corresponding amino acid substitutions from high scoring
mutations (>2%) are indicated. (B) Left: crystal structure of
C-RAF kinase domain (residues 340-618; grey) (PDB code: 3OMV) is
shown, including representative C-RAF resistance mutants (sticks)
along with a space-filling model of bound PLX4720. The DFG motif
and P-loop are also indicated. Right: C-RAF structure rotated
90.degree. to expose the dimer interface residue R401 (Structures
are rendered with PyMOL). (C) C-RAF domain structure representing
(CR, conserved region; RBD, Ras binding domain and CRD, cysteine
rich domain) depicts the localization of C-RAF resistance mutants
(asterisks) and three serine residues important for C-RAF
regulation (circles).
[0015] FIG. 2A-G illustrates functional characterization of C-RAF
resistance mutants.
[0016] (A) A375 cells expressing C-RAF (WT) and C-RAF (alleles
identified) were treated with RAF inhibitor PLX4720 in a dose
dependent manner (0.08 .mu.M, 0.4 .mu.M, 2 .mu.M, 5 .mu.M and 10
.mu.M) for 90 min. Immunoblot showing pErk1/2, pMek1/2, C-RAF.
.alpha.-tubulin was used as a loading control. (B) A375 cells
expressing highly resistant C-RAF mutants were treated with 2 .mu.M
of PLX4720 for 16 h. Shown are the levels of pMek1/2, pErk1/2, Mek,
S259 C-RAF, S338 C-RAF, S621 C-RAF and actin. (C) Growth inhibition
curves of A375 and C-RAF resistance alleles in response to PLX4720
and (D) vemurafenib. (E) A375 cells expressing highly resistant
C-RAF mutants were treated with 1 .mu.M of MEK inhibitor AZD6244
for 16 h. The levels of pMek1/2, pErk1/2, Mek, S259 C-RAF, S338
C-RAF, S621 C-RAF and actin are shown. (F) Growth inhibition curves
of A375 and C-RAF resistance alleles in response to AZD6244 and (G)
Mek-GSK 1120212.
[0017] FIG. 3A-B illustrates that C-RAF resistance mutants exhibit
increased association with B-RAF.
[0018] (A) 293/T cells expressing C-RAF resistance alleles and (B)
A375 cells expressing C-RAF resistance alleles were
immunoprecipitated with total C-RAF. Levels of bound protein (B-RAF
and 14-3-3) were assessed by immunoblotting. Input lysate (lower
panels) show 14-3-3, B-RAF, C-RAF, pMek1/2, pErk1/2, Mek, actin and
S259 C-RAF, S338 C-RAF and S621 C-RAF (upper right panels). Results
are representative of more than two independent experiments.
[0019] FIG. 4A-D illustrates the biochemical characterization of
C-RAF resistance alleles using (S)-methyl
1-(4-(3-(5-chloro-2-fluoro-3-(methylsulfonamido)phenyl)-1-isopropyl-1H-py-
razol-4-yl)pyrimidin-2-ylamino)propan-2-ylcarbamate.
[0020] (A) Immunoblot represents pMek1/2, pErk1/2, Mek, Erk and
C-RAF levels in A375 cells expressing C-RAF resistance alleles in
response to 16 h treatment with 1 .mu.M of PLX4720, (S)-methyl
1-(4-(3-(5-chloro-2-fluoro-3-(methylsulfonamido)phenyl)-1-isopropyl-1H-py-
razol-4-yl)pyrimidin-2-ylamino)propan-2-ylcarbamate and AZD6244.
Actin was used as a loading control. (B) Growth inhibition curves
of A375 and C-RAF resistance alleles in response to PLX4720, (C)
(S)-methyl
1-(4-(3-(5-chloro-2-fluoro-3-(methylsulfonamido)phenyl)-1-isopropyl-1H-py-
razol-4-yl)pyrimidin-2-ylamino)propan-2-ylcarbamate and (D)
AZD6244.
[0021] FIG. 5A-E illustrates that C-RAF resistance mutants confer
resistance to Vemurafenib (PLX4032).
[0022] (A) Growth inhibition curves of A375 and C-RAF resistance
alleles in response to Vemurafenib (PLX4032). (B) C-RAF kinase
activity in extracts from A375 cells expressing WT, S257P, P261T
and G361A in the presence and absence of Vemurafenib for 16 h.
Immunoblot represents pMek1/2, pErk1/2, Mek, Erk and actin. Results
are representative of three independent experiments. (C) Growth
inhibition curves of A375 and C-RAF resistance alleles in response
to PLX4720, (D) AZD6244 and (E) PLX4720 and AZD6244.
[0023] FIG. 6A-D illustrates homodimerization and kinase activity
of C-RAF resistance mutants.
[0024] (A) 293T cells co-expressing His/V5-tagged and Flag-tagged
C-RAF resistance mutants for 48 h were immunoprecipitated with
Nickel (see Methods) to pull down His-tagged C-RAF, and Flag tagged
C-RAF was assessed by immunoblotting. Input lysate was also
assessed using antibodies that detected Flag-C-RAF, V5-C-RAF, total
C-RAF, p-MEK, p-ERK and total ERK. (B) 293T cells co-expressing
His/V5-tagged and Flag-tagged C-RAF resistance mutants were treated
with either vehicle (DMSO) or 2 .mu.M of vemurafenib for 1 h, and
His/V5-tagged C-RAF was immunoprecipitated as in (A) above. Input
lysates were immunoblotted using antibodies recognizing C-RAF
(S338), p-MEK, p-ERK, and actin (loading control). (C) In vitro
C-Raf kinase activity was measured in cell extracts derived from
293T cells transiently expressing Flag-tagged empty vector ("C"),
wild type C-RAF ("WT"), and C-RAF harboring the resistance mutants
S257P, P261T, G361A and E478K. Assays were performed in the
presence or absence of 2 .mu.M vemurafenib (see Methods). Input
lysate was also immunoblotted using antibodies that detect
p-MEK1/2, p-ERK1/2, total MEK, total ERK and actin. (D) In vitro
C-Raf kinase activity was measured in cell extracts derived from
A375 cells (B-RAF.sup.V600E) ("C") and A375 stably expressing wild
type C-RAF ("WT"), and C-RAF harboring the resistance mutants
S257P, P261T, and G361A in the presence and absence of 2 .mu.M
vemurafenib. Immunoblotting studies were performed on input lysate
using antibodies recognizing p-MEK1/2, p-ERK1/2, total MEK, total
ERK, and actin. All results are representative of three independent
experiments.
[0025] FIG. 7A-B illustrates heterodimerization and 14-3-3 binding
properties of C-RAF resistance mutants.
[0026] (A) 293/T cells transiently expressing the indicated C-RAF
resistance mutants in the absence (-) or presence (+) of 2 .mu.M
vemurafenib for 1 h were immunoprecipitated with C-RAF and levels
of bound protein (B-RAF and 14-3-3 .zeta.) (upper panels) was
assessed by immunoblotting. Input lysate (lower panels) show 14-3-3
.zeta., B-RAF, C-RAF, pMek1/2, pErk1/2, Mek, S338 C-RAF and actin.
Results are representative of more than two independent
experiments. (B) A375 cells stably expressing the indicated C-RAF
resistance mutants in the presence and absence of 2 .mu.M
vemurafenib for 16 h were immunoprecipitated with C-RAF and levels
of bound protein (B-RAF and 14-3-3 .zeta.) (upper panels) was
assessed by immunoblotting. Input lysate was blotted for the same
as in FIG. 7A. Results are representative of more than two
independent experiments.
[0027] FIG. 8A-B illustrates that C-RAF resistance mutants require
dimerization for MEK/ERK signaling.
[0028] (A) Constructs expressing either Flag-tagged, wild-type
C-RAF or the indicated C-RAF resistance mutants in the absence
(left) or presence (right) of the dimerization deficient mutant
R401H (pink) were expressed in 293T cells. Lysates were blotted
using antibodies recognizing p-MEK, P-ERK, total MEK, or total
C-RAF. (B) 293/T cells coexpressing His-tagged C-Raf resistance
mutants by themselves and in the dimerization deficient (pink)
context were cultured in the absence or presence of vemurafenib (2
.mu.M, 1 hr). Immunoprecipitations were performed using Nickel
beads and levels of Flag-tagged C-RAF were assessed by
immunoblotting. Input lysates blotted with antibodies recognizing
Flag-C-RAF, V5-C-RAF, total C-RAF, p-MEK, p-ERK, S338-C-RAF and
actin are also shown.
[0029] FIG. 9A-B illustrates biochemical characterization of C-RAF
resistance alleles
[0030] (A) Comparison of pMek/pErk levels using A375 cells
expressing C-RAF containing various resistance alleles that emerged
from the random mutagenesis screens was expressed in A375 cells.
Tubulin was included as a positive control. (B) A375 cells
expressing either wild-type C-RAF or C-RAF resistance alleles were
treated with the Raf inhibitor PLX4720 for 90 minutes at the doses
indicated. Immunoblotting studies were performed with antibodies
against p-ERK, p-MEK, and total C-RAF. Tubulin was used as a
loading control.
[0031] FIG. 10A-B illustrates homodimerization and kinase
activity.
[0032] (A) 293/T cells coexpressing His/V5 tagged C-RAF resistance
mutants with Flag tagged WT-C-RAF were immunoprecipitated after 48
h with His and levels of Flag tagged C-RAF interaction was assessed
by immunoblotting. Input lysate represents Flag-C-RAF, V5-C-RAF,
C-RAF, pMek, pErk and Erk. (B) 293/T cells were transfected with
the dimerization deficient C-RAF mutant R401H and gatekeeper mutant
T421N and Mek/Erk sensitivity was detected in the presence of
vemurafenib (2 .mu.M) for 1 h. T421N was used as a negative control
and G361A was used as a positive control.
DETAILED DESCRIPTION OF THE INVENTION
[0033] The present invention relates to the development of
resistance to therapeutic agents in the treatment of cancer and
identification of targets that confer resistance to treatment of
cancer. The present invention also relates to identification of
parallel drug targets for facilitating an effective long-term
treatment strategy and to identifying patients that would benefit
from such treatment.
[0034] The practice of the present invention will employ, unless
otherwise indicated, conventional techniques of molecular biology,
immunology, microbiology, cell biology and recombinant DNA, which
are within the skill of the art. See e.g., Sambrook, Fritsch and
Maniatis, MOLECULAR CLONING: A LABORATORY MANUAL, (Current
Edition); CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (F. M. Ausubel et
al. eds., (Current Edition)); the series METHODS IN ENZYMOLOGY
(Academic Press, Inc.); PCR 2: A PRACTICAL APPROACH (Current
Edition); ANTIBODIES, A LABORATORY MANUAL and ANIMAL CELL CULTURE
(R. I. Freshney, ed. (1987)). DNA Cloning: A Practical Approach,
vol. I & II (D. Glover, ed.); Oligonucleotide Synthesis (N.
Gait, ed., Current Edition); Nucleic Acid Hybridization (B. Hames
& S. Higgins, eds., Current Edition); Transcription and
Translation (B. Hames & S. Higgins, eds., Current Edition);
Fundamental Virology, 2nd Edition, vol. I & II (B. N. Fields
and D. M. Knipe, eds.)
[0035] 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.
[0036] 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-I 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, C-Raf or Raf-1) is recruited to the cell membrane
through interaction with Ras or in a Ras independent manner in the
cytosol where it undergoes conformational changes and binding to
scaffold proteins such as 14-3-3 (King et al., Nature 396; 180-183
(1998); Chaudhary et al., Curr Bio/10: 551-554 (2000); Avruch et
al., Endo Rev 56: 127-156 (2001), Wellbrock et al., Nat Rev Mol
Cell Biol 5: 875-885 (2004). 14-3-3 binding and
stabilization/activation of CRAF is governed by phosphorylation of
activating residues such as S338, Y341 in the negative charge
regulatory region (N-region) and S621 in the C-terminus, outside
the kinase domain and dephosphorylation of negative regulatory
residues such as S259 in the CR2 domain (FIG. 1C) and numerous
other phosphorylation sites distributed throughout the protein
which further reflects its complex regulation (Avruch et al., Id.
(2001); Wellbrock et al., Id. (2004); Garnett et al., Mol. Cell 20:
963-969 (2005). CRAF activation is also induced by artificial
homodimer formation (Avruch et al., Id. (2001); Wellbrock et al.,
Id., (2004).)
[0037] Raf is then phosphorylated. Raf directly activates MEKI and
MEK2 by phosphorylation of two serine residues at positions 217 and
221. Following activation, MEKI and MEK2 phosphorylate tyrosine
(Tyr-185) and threonine (Thr-183) residues in serine/threonine
kinases ErkI 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-I, c-EtsI, c-Ets2, p90RSKI, MNKI, MNK2, MSKI, 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.
[0038] 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 B-Raf mutations have been identified in
melanoma and ovarian cancer. The most common mutation,
BRAF.sup.V600E, results in constitutive activation of the
downstream MAP kinase pathway and is required for melanoma cell
proliferation, soft agar growth, and tumor xenograft formation.
CRAF amplification have been implicated in prostate cancer and
bladder cancer (Edwards et al., 2003; Simon et al., 2001), besides
chromosomal translocations in stomach cancer and pilocytic
astrocytomas (Shimizu et al., 1986; Jones et al., 2009). However,
the occurrence rate of CRAF mutations in human cancers is 1%
(COSMIC) which is attributable to its low basal kinase activity
when compared to BRAF (Marais et al., Science 280: 109-112 (1997);
Emuss et al., Cancer Res 65: 9719-9726 (2005); Garnett et al., Mol.
Cell 20: 963-969 (2005)). 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. However, patients may have innate resistance or acquire
resistance to these promising therapies. Identification of
resistance conferring mutations in target kinases, diagnostic
and/or prognostic markers and treatment therapies for these
patients with innate or acquired resistance are described
below.
C-RAF Mutations
[0039] While treatment of cancer with RAF inhibitors, such as
PLX4032, 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 C-RAF
that confer resistance to RAF inhibitors, some of which are
currently in clinical development. Acquisition of such a mutation
in cancer cells makes cells of the patient resistant to treatment
with certain RAF inhibitors. In exemplary embodiments, the
invention regards development of resistance to RAF inhibitors, that
may include but are not limited to RAF265, sorafenib, SB590885, PLX
4720, PLX4032, GDC-0879, ZM 336372 and (S)-methyl
1-(4-(3-(5-chloro-2-fluoro-3-(methylsulfonamido)phenyl)-1-isopropyl-1H-py-
razol-4-yl)pyrimidin-2-ylamino)propan-2-ylcarbamate. By way of
non-limiting example, exemplary RAF inhibitors are shown in Table
1. The RAF inhibitor (S)-methyl
1-(4-(3-(5-chloro-2-fluoro-3-(methylsulfonamido)phenyl)-1-isopropyl-1H-py-
razol-4-yl)pyrimidin-2-ylamino)propan-2-ylcarbamate is generically
and specifically covered (compound 9, table 1, page 50 of the
provisional; Compound 9 on pg. 72 of PCT publication
WO2011/025927.
[0040] The clinical emergence of a C-RAF mutation conferring
resistance to a RAF inhibitor 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 RAF
inhibitors to elicit durable tumor responses in many malignancies,
including 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. Exemplary RAF inhibitors include, but are not limited
to the inhibitors listed in Table I. Non-limiting examples of MEK
inhibitors include, AZD6244; CI-1040; PD184352; PD318088, PD98059,
PD334581, RDEA119,
6-Methoxy-7-(3-morpholin-4-yl-propoxy)-4-(4-phenoxy-phenylamino)-
-quinoline-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. Exemplary
MEK inhibitors are shown in Table 2. These therapeutic innovations,
together with robust tumor genomic profiling to stratify patients,
should speed the advent of personalized cancer treatment in cancers
with "druggable" oncogene mutations.
TABLE-US-00001 TABLE 1 Exemplary RAF Inhibitors CAS Name No.
Structure 1 RAF265 927880-90- ##STR00001## 2 Sorafenib Tosylate
Nexavar Bay 43-9006 475207-59-1 ##STR00002## Sorafenib
4-[4-[[4-chloro-3- (trifluoromethyl)phenyl] carbamoylamino]
phenoxy]-N-methyl- pyridine-2-carboxamide 284461-73-0 ##STR00003##
3 SB590885 ##STR00004## 4 PLX4720 918505-84-7 ##STR00005## 5
PLX4032 1029872-54-5 ##STR00006## 6 GDC-0879 905281-76-7
##STR00007## 7 ZM 336372 208260-29-1 ##STR00008## 8 (S)-methyl
1-(4-(3-(5- chloro-2-fluoro-3- (methylsulfonamido)
phenyl)-1-isopropyl-1H- pyrazol-4-yl)pyrimidin-2- ylamino)propan-2-
ylcarbamate ##STR00009##
TABLE-US-00002 TABLE 2 Exemplary MEK Inhibitors Name CAS No.
Structure 1 Cl-1040/PD184352 212631-79-3 ##STR00010## 2 AZD6244
606143-52-6 ##STR00011## 3 PD318088 391210-00-7 ##STR00012## 4
PD98059 167869-21-8 ##STR00013## 5 PD334581 ##STR00014## 6 RDEA119
N-[3,4-difluoro-2-[(2- fluoro-4- iodophenyl)amino]-6-
methoxyphenyl]-1-[(2R)- 2,3-dihydroxypropyl]-
Cyclopropanesulfonamide 923032-38-6 ##STR00015## 7 6-Methoxy-7-(3-
morpholin-4-yl-propoxy)- 4-(4-phenoxy- phenylamino)-quinoline-3-
carbonitrile ##STR00016## 8 4-[3-Chloro-4-(1-methyl-
1H-imidazol-2-ylsulfanyl)- phenylamino]-6-methoxy-
7-(3-morpholin-4-yl- propoxy)-quinoline-3- carbonitrile
##STR00017##
[0041] In various embodiments, the present invention relates to
methods of identifying mutations in a C-RAF polypeptide, or
mutations in a nucleic acid molecule encoding the C-RAF
polypeptide, that confer resistance on cells expressing the C-RAF
polypeptide to drugs that inhibit RAF activity. A "mutant C-RAF
polypeptide," as referenced herein, includes a C-RAF polypeptide
including one or more mutations that confer resistance to one or
more known RAF inhibitors. Likewise, a "mutant C-RAF nucleic acid
molecule," as referenced herein, includes a nucleic acid molecule
that encodes a mutant C-RAF polypeptide. Nucleic acid molecules
encoding C-RAF polypeptides that include 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 C-RAF nucleic acid sequence, which can be conducted in E.
coli. In exemplary embodiments, the wild-type C-RAF nucleic acid
sequence is a human wild-type MEK1 nucleic acid sequence. In
specific embodiments, the wild-type C-RAF nucleic acid sequence is
wild-type human C-RAF (SEQ ID NO: 1) (Accession Number
BC018119.2.). The mutant C-RAF nucleic acid molecules can then be
screened in cells otherwise sensitive to treatment with a RAF
inhibitor to identify a nucleic acid that encodes a mutant C-RAF
polypeptide compared to a wild-type C-RAF polypeptide that is
resistant to treatment with the RAF inhibitor. In some embodiments,
the C-RAF polypeptide is the wild-type human C-RAF (SEQ ID NO: 2)
(Swiss-Prot ID # is P04049-10).
[0042] Any suitable method can be used to screen mutant C-RAF
nucleic acids and mutant C-RAF polypeptides for resistance to
treatment with a RAF inhibitor. For example, a nucleic acid
molecule encoding a mutant C-RAF polypeptide can be expressed in
cells otherwise sensitive to treatment with a RAF. An exemplary
cell line useful for this purpose is the melanoma cell line A375.
Following expression of the mutant C-RAF polypeptide, the cells can
be treated with a RAF. The activity of the mutant C-RAF polypeptide
can then be measured and compared to the activity of a wild-type
C-RAF polypeptide similarly expressed and treated with the RAF
inhibitor. Activity of a C-RAF polypeptide can be determined by,
for example, measuring proliferation or viability of cells
following treatment with the RAF inhibitor, wherein proliferation
or viability are positively correlated with C-RAF activity. Cell
growth, proliferation, or viability can be determined using any
suitable method known in the art. In one embodiment, cell growth
can be determined using well-based cell proliferation/viability
assays such as MTS or Cell Titer GLo, in which cell growth in the
presence of a RAF inhibitor is expressed as a percentage of that
observed in untreated cells cultured in the absence of the RAF
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 C-RAF polypeptide can also be
measured by, for example, determining the relative amount of
phosphorylated ERK1/2 present in the cell following treatment with
the RAF inhibitor. Activity of a wild-type or mutant C-RAF
polypeptide can also be determined using an in vitro
phosphorylation assay, in which MEK1 activity is determined by
measuring the proportion of phosphorylated ERK 1/2 substrate in the
assay following treatment with the RAF or MEK inhibitor. A mutant
C-RAF polypeptide having greater activity than a wild-type C-RAF
polypeptide following treatment with a RAF inhibitor is identified
as containing a mutation that confers resistance to a RAF
inhibitor. The mutation conferring resistance to a RAF inhibitor
can then be identified by sequencing the nucleic acid encoding the
mutant C-RAF polypeptide, or by sequencing the mutant C-RAF
polypeptide directly.
[0043] In this manner, as well as using massively parallel sequence
methods, as described in Example 1, amino acid substitutions were
identified in the C-RAF polypeptide that when mutated confer
resistance to the RAF inhibitors PLX4032, PLX4720 and (S)-methyl
1-(4-(3-(5-chloro-2-fluoro-3-(methylsulfonamido)phenyl)-1-isopropyl-1H-py-
razol-4-yl)pyrimidin-2-ylamino)propan-2-ylcarbamate. In particular,
substitutions at one or more of the following amino acids of the
human C-RAF polypeptide confer resistance to RAF inhibitors
including 104E, 257S, 261P, 356G, 361G, 427S, 447D, 469M, 478E and
554R. In certain embodiments, the mutant C-RAF polypeptide includes
a mutation with respect to the wild-type human C-RAF polypeptide at
one or more of these amino acid residues. In exemplary embodiments,
the mutant C-RAF polypeptide includes one or more of the following
resistance mutations: 104E>K, 257S>P, 261P>T, 356G>E,
361G>A, 427S>T, 447D>N, 469M>l, 478E>K and
554R>K.
Isolated Nucleic Acid Molecules
[0044] The present invention concerns polynucleotides or nucleic
acid molecules relating to the C-RAF 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 C-RAF nucleic acid molecules
described herein comprise a mutation conferring resistance to one
or more RAF inhibitors. A "mutant C-RAF nucleic acid molecule," as
referenced herein, includes a C-RAF nucleic acid molecule that
encodes a mutant C-RAF polypeptide, i.e., a C-RAF polypeptide
including one or more mutations that confer resistance to one or
more RAF inhibitors.
[0045] It is contemplated that an isolated and purified C-RAF
nucleic acid molecule, e.g., a mutant C-RAF nucleic acid molecule,
can take the form of RNA or DNA. As used herein, the term "RNA
transcript" refers to an RNA molecule that is the product of
transcription from a DNA nucleic acid molecule. Such a transcript
can encode for one or more proteins.
[0046] 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 C-RAF" refers to a nucleic
acid segment that includes C-RAF 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 C-RAF-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 C-RAF polypeptide, for example,
phosphorylation of the ERK1/2 substrate.
[0047] The term "cDNA" is intended to refer to DNA prepared using
RNA as a template. It also is contemplated that a given
C-RAF-encoding nucleic acid or C-RAF 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
C-RAF polypeptide. In a preferred embodiment, the active C-RAF
polypeptide is an active human C-RAF polypeptide. In particularly
preferred embodiments, the active C-RAF polypeptide is a mutant
C-RAF polypeptide that has an activity of a wild-type C-RAF
polypeptide, but which is resistant to one or more known RAF
inhibitors. Consequently, certain aspects of the present invention
encompass derivatives of C-RAF nucleic acids or polypeptides with
minimal nucleic acid or amino acid changes, but that possess the
same biological function.
[0048] In some embodiments, the invention relates to recombinant
vectors incorporating DNA sequences that encode mutant C-RAF
polypeptides or peptides that include within its amino acid
sequence a contiguous amino acid sequence in accordance with, or
essentially corresponding to mutant C-RAF polypeptides. In
exemplary embodiments, the invention relates to isolated DNA
segments and recombinant vectors incorporating DNA sequences that
encode a C-RAF polypeptide that includes within its amino acid
sequence a contiguous amino acid sequence of a C-RAF polypeptide
comprising one or more mutations that confer resistance to one or
more RAF inhibitors.
[0049] 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. 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.
[0050] In some embodiments, the nucleic acid sequence may encode a
mutant C-RAF polypeptide having C-RAF activity where at least one
amino acid substitution occurs at one or more amino acid positions
including the following: 104E, 257S, 261P, 356G, 361G, 427S, 447D,
469M, 478E and 554R. In other embodiments, the mutant C-RAF
polypeptide includes one or more of the following resistance
mutations: 104E>K, 257S>P, 261P>T, 356G>E, 361G>A,
427S>T, 447D>N, 469M>l, 478E>K and 554R>K.
Expression Vectors and Host Cells
[0051] The present invention encompasses expression vector
compositions and the use of such vectors to encode for a C-RAF
polypeptide, e.g., a mutant C-RAF 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.
[0052] The term "expression vector" or "expression construct"
refers to a vector containing a nucleic acid sequence coding for at
least part of a gene product capable of being transcribed. In some
cases, RNA molecules are then translated into a protein, protein,
or peptide. In other cases, these sequences are not translated, for
example, in the production of antisense molecules or ribozymes.
Expression vectors can contain a variety of "control sequences,"
which refer to nucleic acid sequences necessary for the
transcription and possibly translation of an operably linked coding
sequence in a particular host organism. In addition to control
sequences that govern transcription and translation, vectors and
expression vectors can contain nucleic acid sequences that serve
other functions as well.
[0053] As used herein, the terms "cell," "cell line," and "cell
culture" may be used interchangeably. A cell comprising a C-RAF
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. 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.
Isolated Polypeptide Molecules
[0054] Another aspect of the invention pertains to isolated and/or
purified C-RAF polypeptides, and biologically active portions
thereof. In particular aspects of the invention, the C-RAF
polypeptides described herein comprise a mutation at one or more
amino acids conferring resistance to one or more RAF inhibitors. A
"mutant C-RAF polypeptide", as referenced herein, includes a C-RAF
polypeptide including a mutation at one or more amino acids
positions that confer resistance to one or more RAF inhibitors.
[0055] Biologically active portions of a C-RAF polypeptide include
peptides comprising amino acid sequences derived from the amino
acid sequence of a C-RAF polypeptide, e.g., the amino acid sequence
shown in SEQ ID NO: 2, which include fewer amino acids than a full
length C-RAF polypeptide, and exhibit at least one activity of a
C-RAF polypeptide. 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 C-RAF
polypeptide. 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 C-RAF polypeptide include one or more selected
domains/motifs or portions thereof having biological activity.
[0056] C-RAF polypeptides may be 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 C-RAF polypeptide is expressed in the host cell. The
C-RAF polypeptide can then be isolated from the cells by an
appropriate purification scheme using standard protein purification
techniques. Alternative to recombinant expression, a C-RAF
polypeptide can be synthesized chemically using standard peptide
synthesis techniques. Moreover, a native C-RAF polypeptide and/or a
mutant C-RAF polypeptide can be isolated from cells (e.g., cancer
cells), for example using an anti-C-RAF antibody, which can be
produced by standard techniques utilizing a C-RAF polypeptide or
fragment thereof of this invention.
[0057] C-RAF chimeric or fusion proteins may also be used. As used
herein, a MEK1 "chimeric protein" or "fusion protein" comprises a
C-RAF polypeptide operatively linked to a non-C-RAF polypeptide. A
"C-RAF polypeptide" refers to a protein having an amino acid
sequence corresponding to a C-RAF polypeptide, whereas a "non-C-RAF
polypeptide" refers to a protein having an amino acid sequence
corresponding to a protein which is not substantially homologous to
the C-RAF polypeptide, e.g., a protein which is substantially
different from the C-RAF polypeptide, which does not display a
C-RAF 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 C-RAF polypeptide and the
non-C-RAF polypeptide are fused in-frame to each other. The
non-C-RAF polypeptide can be fused to the N-terminus or C-terminus
of the C-RAF polypeptide. For example, in one embodiment the fusion
protein is a GST-C-RAF fusion protein in which the C-RAF amino
acids are fused to the C-terminus of the GST polypeptide. Such
fusion proteins can facilitate the purification of recombinant
C-RAF polypeptide. In another embodiment, the fusion protein is a
C-RAF polypeptide containing a heterologous signal sequence at its
N-terminus. In certain host cells (e.g., mammalian host cells),
expression and/or secretion of a MEK1 protein can be increased
through use of a heterologous signal sequence.
[0058] Mutant C-RAF polypeptide can be generated by mutagenesis of
a wild-type C-RAF polypeptide, or of the nucleic acid molecule
encoding a wild-type C-RAF polypeptide. Mutant C-RAF polypeptide
can also be identified by screening combinatorial libraries of
C-RAF mutants for a mutant C-RAF polypeptide having a desired
activity, e.g., resistance to one or more RAF inhibitors. Several
techniques are known in the art for screening gene products of
combinatorial libraries made by point mutations or truncation, and
for screening cDNA libraries for gene products having a selected
property. Such techniques are adaptable for rapid screening of the
gene libraries generated by combinatorial mutagenesis. The most
widely used techniques, which are amenable to high through-put
analysis, for screening large gene libraries typically include
cloning the gene library into replicable expression vectors,
transforming appropriate cells with the resulting library of
vectors, and expressing the combinatorial genes under conditions in
which detection of a desired activity facilitates isolation of the
vector encoding the gene whose product was detected.
Antibodies
[0059] The polypeptides expressed from the polynucleotides of the
invention can be used for generating antibodies. In some
embodiments, the antibodies can be used to detect and quantitate
expression of the mutant C-RAF polypeptides. In some embodiments,
the antibodies can be used to alter the activity of a mutant C-RAF
polypeptide. Polypeptides expressed from the polynucleotides of the
invention comprising at least six, eight, ten, twelve, fifteen,
twenty or thirty consecutive amino acids can be used as immunogens.
The polypeptides can be used to obtain a preparation of antibodies
which specifically bind to a mutant C-Raf polypeptide of the
invention having one or more amino acid substitutions at one or
more of the following amino acids of the human C-RAF polypeptide
that confer resistance to RAF inhibitors including 104E, 257S,
261P, 356G, 361G, 427S, 447D, 469M, 478E and 554R. In exemplary
embodiments, the mutant C-RAF polypeptide includes one or more of
the following resistance mutations: 104E>K, 257S>P,
261P>T, 356G>E, 361G>A, 427S>T, 447D>N, 469M>l,
478E>K and 554R>K.
[0060] The antibodies can be monoclonal and polyclonal antibodies,
single chain antibodies, chimeric antibodies,
bifunctional/bispecific antibodies, humanized antibodies, human
antibodies, and complementary determining region (CDR)-grafted
antibodies, that are specific for the target protein or fragments
thereof; and also include antibody fragments, including Fab, Fab',
F(ab')2, scFv, Fv, camelbodies, or microantibodies. An antibody can
also refer to an anti-idiotype antibody, i.e., an antibody directed
against the antigen specific part of the sequence of an antibody
and thus recognizes the binding sites of other antibodies; or an
anti-anti-idiotype antibody, i.e., an antibody with a combining
site that mimics the epitope on the antigen that was used to
generate the original antibody. Techniques for raising antibodies
are well known in the art.
[0061] Single chain antibodies can also be constructed. Single
chain antibodies which specifically bind to a polypeptide expressed
from the polynucleotides of the invention can be isolated, for
example, from single-chain immunoglobulin display libraries, as are
known in the art. The library is "panned" against a polypeptide,
and a number of single chain antibodies which bind different
epitopes of the polypeptide with high-affinity can be isolated.
Hayashi et al., 1995, Gene 160: 129-30. Such libraries are known
and available to those in the art. The antibodies can also be
constructed using the polymerase chain reaction (PCR), using
hybridoma cDNA as a template. Thirion et al., 1996, Eur. J. Cancer
Prey. 5: 507-11.
[0062] The single chain antibody can be mono- or bi-specific, and
can be bivalent or tetravalent. Construction of tetravalent
bispecific single chain antibodies is taught in Coloma and
Morrison, 1997, Nat. Biotechnol. 15: 159-63 Construction of
bivalent bispecific single chain antibodies is taught in Mallender
and Voss, 1994, J. Biol. Chem. 269: 199-206.
[0063] A nucleotide sequence encoding the single chain antibody can
then be constructed using manual or automated nucleotide synthesis,
cloned into DNA expression vectors using standard recombinant DNA
methodologies, and introduced into cells which express the selected
gene, as described below. Alternatively, the antibodies can be
produced directly using filamentous phage technology Verhaar et
al., 1995, Int. J. Cancer 61:497-501; Nicholls et al., 1993. J.
Immunol. Meth. 165:81-91.
[0064] The antibodies bind specifically to the epitopes of the
polypeptides expressed from the polynucleotides of the invention.
In a preferred embodiment, the epitopes are not present on other
human proteins. Typically a minimum number of contiguous amino
acids to encode an epitope is 6, 8, or 10. However, more can be
used, for example, at least 15, 25, or 50, especially to form
epitopes which involve non-contiguous residues or particular
conformations.
[0065] Antibodies that bind specifically to the polypeptides
include those that bind to full-length polypeptides. Specific
binding antibodies do not detect other proteins on Western blots of
human cells, or provide a signal at least ten-fold lower than the
signal provided by the target protein of the invention. Antibodies
which have such specificity can be obtained by routine screening.
In a preferred embodiment of the invention, the antibodies
immunoprecipitate the polypeptides expressed from the
polynucleotides of the invention from cell extracts or solution.
Additionally, the antibodies can react with polypeptides expressed
from the polynucleotides of the invention in tissue sections or on
Western blots of polyacrylamide gels. Preferably the antibodies do
not exhibit nonspecific cross-reactivity with other human proteins
on Western blots or in immunocytochemical assays.
[0066] Techniques for purifying antibodies to the polypeptides
expressed from the polynucleotides of the invention are available
in the art. In a preferred embodiment, the antibodies are passed
over a column to which a particular protein or polypeptide
expressed from the polynucleotides of the invention is bound. The
bound antibodies are then eluted, for example, with a buffer having
a high salt concentration.
Detection of Mutations
[0067] In another aspect, the invention pertains to methods of
detecting the presence of a mutant C-RAF polypeptide 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
C-RAF polypeptide of the invention in a sample, e.g., a nucleic
acid and/or a protein sample. In specific embodiments, the sample
includes a cell or cell extract. In exemplary embodiments, the
sample is obtained from a subject, e.g., a subject having
cancer.
[0068] Methods for detecting the presence of resistance mutations
in genomic DNA, cDNA, and RNA (i.e., mRNA) containing a sequence
encoding a C-RAF polypeptide, 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 C-RAF polypeptide, 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 C-RAF
polypeptide, or a nucleic acid molecule encoding C-RAF polypeptide,
having a mutation at one or more amino acid positions as compared
to the wild-type C-RAF polypeptide (SEQ ID NO: 2). In some
embodiments, antibodies directed to a mutant C-RAF polypeptide may
be used to detect the presence of the mutant polypeptide.
[0069] 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.
Other methods for detecting mutations known to one skilled in the
art may also be used.
[0070] 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 C-RAF nucleic acid or a C-RAF 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.
[0071] The presence or absence of one or more mutations described
herein determines the likelihood of the screened individuals to
resist therapy with a RAF inhibitor. According to methods provided
by the invention, these results will be used to adjust and/or alter
the dose of the RAF inhibitor, or to select a course of treatment
using a second inhibitor. In some embodiments, the second inhibitor
may be a 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.
[0072] The resistance mutations in C-RAF polypeptide, or in nucleic
acid molecules encoding C-RAF polypeptide, 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 C-RAF polypeptide, or any
other biochemical interpretation.
Diagnostic/Prognostic Markers for Resistance to Targeted
Therapies
[0073] In some aspects, the present invention relates to methods of
detecting the presence of one or more diagnostic or prognostic
markers in a sample (e.g. a biological sample from a cancer
patient). A variety of screening methods known to one of skill in
the art may be used to detect the presence of the marker in the
sample including DNA, RNA and protein detection. The techniques can
be used to determine the presence or absence of a mutation in a
sample obtained from a patient. In some embodiments, the patient
may have innate or acquired resistance to kinase targeted
therapies, including B-RAF inhibitors or pan-RAF inhibitors. For
example, the patient may have an innate or acquired resistance to
RAF inhibitors PLX4720 and/or PLX4032 and/or (S)-methyl
1-(4-(3-(5-chloro-2-fluoro-3-(methylsulfonamido)phenyl)-1-isopropyl-1H-py-
razol-4-yl)pyrimidin-2-ylamino)propan-2-ylcarbamate. In one
embodiment, identification of a C-RAF nucleic acid or polypeptide
including one or more mutations described herein in a cancer-cell
containing sample obtained from a patient indicates that the
patient is at a relatively high risk of relapse or lack of response
to treatment with a RAF inhibitor. Identification of one or more
C-RAF mutations described above in a patient assists the physician
in determining and implementing a treatment protocol for the
patient. For example, in a patient having one or more mutations in
the C-RAF polypeptide identified above, the physician may treat the
patient with a combination therapy as described in more detail
below.
[0074] Identification of resistance mutations in the C-RAF
polypeptide also allows for the screening of patients having a
cancer in order to determine the presence or absence of a C-RAF
resistance mutation at one or more amino acid positions in the
cancer. Determining the presence or absence of one or more C-RAF
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 RAF
inhibitor or a MEK inhibitor, giving a combination therapy,
providing sequential dosing of a RAF inhibitor and a second
inhibitor and the like.
[0075] In some embodiments, the RAF resistance mutations may be
identified in a nucleic acid encoding a mutant C-RAF polypeptide
having C-RAF activity, where the mutant C-RAF polypeptide includes
at least one amino acid substitution as compared to a wild type
C-RAF polypeptide shown in SEQ ID NO: 2 and where the at least one
amino acid substitution confers resistance to one or more RAF
inhibitors on a cell expressing the mutant RAF polypeptide. In some
embodiments, the RAF resistance mutations may be identified in a
mutant C-RAF polypeptide having C-RAF activity, where the mutant
C-RAF polypeptide includes at least one amino acid substitution as
compared to a wild type C-RAF polypeptide shown in SEQ. ID. NO. 2,
and where the at least one amino acid substitution confers
resistance to one or more RAF inhibitors on a cell expressing the
mutant C-RAF polypeptide. In some embodiments, the substitution at
one or more of the following amino acids of the wild-type C-RAF
polypeptide confer resistance to RAF inhibitors including 104E,
257S, 261P, 356G, 361G, 427S, 447D, 469M, 478E and 554R. In some
embodiments, the substitution of one or more amino acids of the
wild-type C-RAF polypeptide is selected from the group consisting
of 104E>K, 257S>P, 261P>T, 356G>E, 361G>A,
427S>T, 447D>N, 469M>l, 478E>K and 554R>K. In some
embodiments, the substitution of one or more amino acids of the
wild-type C-RAF polypeptide is selected from the group consisting
of 257S, 261P and 361G.
Methods of Treatment
[0076] In various embodiments, the invention provides methods for
treatment of a patient having cancer. The methods generally
comprise administration of a first inhibitor and a second
inhibitor. One inhibitor may be a RAF inhibitor. Exemplary RAF
inhibitors are shown in Table 1 above. One inhibitor may be a MEK
inhibitor (see Table 2 illustrating exemplary MEK inhibitors). In
some embodiments, a combination therapy for cancer is provided,
comprising an effective amount of a RAF inhibitor and an effective
amount of a second inhibitor. In some embodiments the second
inhibitor is a MEK inhibitor.
[0077] In exemplary embodiments of the foregoing aspects, the RAF
inhibitor provided herein is PLX4720, PLX4032, BAY 43-9006
(Sorafenib), ZM 336372, RAF 265, AAL-881, LBT-613, (S)-methyl
1-(4-(3-(5-chloro-2-fluoro-3-(methylsulfonamido)phenyl)-1-isopropyl-1H-py-
razol-4-yl)pyrimidin-2-ylamino)propan-2-ylcarbamate or CJS352.
Additional exemplary RAF inhibitors useful for combination therapy
include pan-RAF inhibitors, inhibitors of B-RAF, inhibitors of
A-RAF, and inhibitors of RAF-1. Additional RAF inhibitors known in
the art may also be used.
[0078] As a non-limiting example, the MEK inhibitor provided herein
can be CI-1040, AZD6244, PD318088, PD98059, PD334581, RDEA119,
6-Methoxy-7-(3-morpholin-4-yl-propoxy)-4-(4-phenoxy-phenylamino)-quinolin-
e-3-carbonitrile or
4-[3-Chloro-4-(1-methyl-1H-imidazol-2-ylsulfanyl)-phenylamino]-6-methoxy--
7-(3-morpholin-4-yl-propoxy)-quinoline-3-carbonitrile, Roche
compound RG7420, or combinations thereof. Additional MEK inhibitors
known in the art may also be used.
[0079] Administration of the combination includes administration of
the combination in a single formulation or unit dosage form,
administration of the individual agents of the combination
concurrently but separately, or administration of the individual
agents of the combination sequentially by any suitable route. The
dosage of the individual agents of the combination may require more
frequent administration of one of the agents as compared to the
other agent in the combination. Therefore, to permit appropriate
dosing, packaged pharmaceutical products may contain one or more
dosage forms that contain the combination of agents, and one or
more dosage forms that contain one of the combinations of agents,
but not the other agent(s) of the combination.
[0080] Agents may contain one or more asymmetric elements such as
stereogenic centers or stereogenic axes, e.g., asymmetric carbon
atoms, so that the compounds can exist in different stereoisomeric
forms. These compounds can be, for example, racemates or optically
active forms. For compounds with two or more asymmetric elements,
these compounds can additionally be mixtures of diastereomers. For
compounds having asymmetric centers, it should be understood that
all of the optical isomers and mixtures thereof are encompassed. In
addition, compounds with carbon-carbon double bonds may occur in Z-
and E-forms; all isomeric forms of the compounds are included in
the present invention. In these situations, the single enantiomers
(optically active forms) can be obtained by asymmetric synthesis,
synthesis from optically pure precursors, or by resolution of the
racemates. Resolution of the racemates can also be accomplished,
for example, by conventional methods such as crystallization in the
presence of a resolving agent, or chromatography, using, for
example a chiral HPLC column.
[0081] Unless otherwise specified, or clearly indicated by the
text, reference to compounds useful in the combination therapy of
the invention includes both the free base of the compounds, and all
pharmaceutically acceptable salts of the compounds. A preferred
salt is the hydrochloride salt.
[0082] The term "pharmaceutically acceptable salts" includes
derivatives of the disclosed compounds, wherein the parent compound
is modified by making non-toxic acid or base addition salts
thereof, and further refers to pharmaceutically acceptable
solvates, including hydrates, of such compounds and such salts.
Examples of pharmaceutically acceptable salts include, but are not
limited to, mineral or organic acid addition salts of basic
residues such as amines; alkali or organic addition salts of acidic
residues such as carboxylic acids; and the like, and combinations
comprising one or more of the foregoing salts. The pharmaceutically
acceptable salts include non-toxic salts and the quaternary
ammonium salts of the parent compound formed, for example, from
non-toxic inorganic or organic acids. For example, non-toxic acid
salts include those derived from inorganic acids such as
hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, and
nitric; other acceptable inorganic salts include metal salts such
as sodium salt, potassium salt, and cesium salt; and alkaline earth
metal salts, such as calcium salt and magnesium salt; and
combinations comprising one or more of the foregoing salts.
[0083] Pharmaceutically acceptable organic salts include salts
prepared from organic acids such as acetic, trifluoroacetic,
propionic, succinic, glycolic, stearic, lactic, malic, tartaric,
citric, ascorbic, pamoic, maleic, hydroxymaleic, phenylacetic,
glutamic, benzoic, salicylic, mesylic, esylic, besylic, sulfanilic,
2-acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic, ethane
disulfonic, oxalic, isethionic, HOOC(CH.sub.2).sub.nCOOH where n is
0-4; organic amine salts such as triethylamine salt, pyridine salt,
picoline salt, ethanolamine salt, triethanolamine salt,
dicyclohexylamine salt, N,N'-dibenzylethylenediamine salt; and
amino acid salts such as arginate, asparginate, and glutamate, and
combinations comprising one or more of the foregoing salts.
[0084] An "effective amount" of a combination of agents is an
amount sufficient to provide an observable improvement over the
baseline clinically observable signs and symptoms of the disorder
treated with the combination.
[0085] The pharmaceutical products can be administrated by oral or
other forms, e.g., rectally or by parenteral injection. "Oral
dosage form" is meant to include a unit dosage form prescribed or
intended for oral administration. An oral dosage form may or may
not comprise a plurality of subunits such as, for example,
microcapsules or microtablets, packaged for administration in a
single dose.
[0086] The pharmaceutical products can be released in various
forms. "Releasable form" is meant to include instant release,
immediate-release, controlled-release, and sustained-release
forms.
[0087] "Instant-release" is meant to include a dosage form designed
to ensure rapid dissolution of the active agent by modifying the
normal crystal form of the active agent to obtain a more rapid
dissolution.
[0088] "Immediate-release" is meant to include a conventional or
non-modified release form in which greater than or equal to about
50% or more preferably about 75% of the active agents is released
within two hours of administration, preferably within one hour of
administration.
[0089] "Sustained-release" or "extended-release" includes the
release of active agents at such a rate that blood (e.g., plasma)
levels are maintained within a therapeutic range but below toxic
levels for at least about 8 hours, preferably at least about 12
hours, more preferably about 24 hours after administration at
steady-state. The term "steady-state" means that a plasma level for
a given active agent or combination of active agents, has been
achieved and which is maintained with subsequent doses of the
active agent(s) at a level which is at or above the minimum
effective therapeutic level and is below the minimum toxic plasma
level for a given active agent(s).
[0090] The term "treat", "treated," "treating" or "treatment" is
used herein to mean to relieve, reduce or alleviate at least one
symptom of a disease in a subject. For example, treatment can be
diminishment of one or several symptoms of a disorder or complete
eradication of a disorder, such as cancer. Within the meaning of
the present invention, the term "treat" also denote to arrest,
delay the onset (i.e., the period prior to clinical manifestation
of a disease) and/or reduce the risk of developing or worsening a
disease. The term "protect" is used herein to mean prevent delay or
treat, or all, as appropriate, development or continuance or
aggravation of a disease in a subject. Within the meaning of the
present invention, the disease is associated with a cancer.
[0091] The term "subject" or "patient" is intended to include
animals, which are capable of suffering from or afflicted with a
cancer or any disorder involving, directly or indirectly, a cancer.
Examples of subjects include mammals, e.g., humans, dogs, cows,
horses, pigs, sheep, goats, cats, mice, rabbits, rats, and
transgenic non-human animals. In certain embodiments, the subject
is a human, e.g., a human suffering from, at risk of suffering
from, or potentially capable of suffering from cancers.
[0092] The term "about" or "approximately" usually means within
20%, more preferably within 10%, and most preferably still within
5% of a given value or range. Alternatively, especially in
biological systems, the term "about" means within about a log
(i.e., an order of magnitude) preferably within a factor of two of
a given value.
[0093] The use of the terms "a" and "an" and "the" and similar
referents in the context of describing the invention (especially in
the context of the following claims) are to be construed to cover
both the singular and the plural, unless otherwise indicated herein
or clearly contradicted by context. The terms "comprising,
"having," "including," and "containing" are to be construed as
open-ended terms (i.e., meaning "including, but not limited to")
unless otherwise noted. Recitation of ranges of values herein are
merely intended to serve as a shorthand method of referring
individually to each separate value falling within the range,
unless otherwise indicated herein, and each separate value is
incorporated into the specification as if it were individually
recited herein.
[0094] As specified above, in one aspect, the instant invention
provides a drug combination useful for treating, preventing,
arresting, delaying the onset of and/or reducing the risk of
developing, or reversing at least one symptom of cancer, in a
subject comprising administering to the subject a combination
therapy, comprising an effective amount of a RAF inhibitor and a
second inhibitor. In some embodiments, the second inhibitor is a
MEK inhibitor. Preferably, these inhibitors are administered at
therapeutically effective dosages which, when combined, provide a
beneficial effect. The administration may be simultaneous or
sequential.
[0095] The term "cancer" is used herein to mean a broad spectrum of
tumors, including all solid tumors and hematological malignancies.
Examples of such tumors include but are not limited to leukemias,
lymphomas, myelomas, carcinomas, metastatic carcinomas, sarcomas,
adenomas, nervous system cancers and geritourinary cancers. In
exemplary embodiments, the foregoing methods are useful in treating
adult and pediatric acute lymphoblastic leukemia, acute myeloid
leukemia, adrenocortical carcinoma, AIDS-related cancers, anal
cancer, cancer of the appendix, astrocytoma, basal cell carcinoma,
bile duct cancer, bladder cancer, bone cancer, osteosarcoma,
fibrous histiocytoma, brain cancer, brain stem glioma, cerebellar
astrocytoma, malignant glioma, ependymoma, medulloblastoma,
supratentorial primitive neuroectodermal tumors, hypothalamic
glioma, breast cancer, male breast cancer, bronchial adenomas,
Burkitt lymphoma, carcinoid tumor, carcinoma of unknown origin,
central nervous system lymphoma, cerebellar astrocytoma, malignant
glioma, cervical cancer, childhood cancers, chronic lymphocytic
leukemia, chronic myelogenous leukemia, chronic myeloproliferative
disorders, colorectal cancer, cutaneous T-cell lymphoma,
endometrial cancer, ependymoma, esophageal cancer, Ewing family
tumors, extracranial germ cell tumor, extragonadal germ cell tumor,
extrahepatic bile duct cancer, intraocular melanoma,
retinoblastoma, gallbladder cancer, gastric cancer,
gastrointestinal stromal tumor, extracranial germ cell tumor,
extragonadal germ cell tumor, ovarian germ cell tumor, gestational
trophoblastic tumor, glioma, hairy cell leukemia, head and neck
cancer, hepatocellular cancer, Hodgkin lymphoma, non-Hodgkin
lymphoma, hypopharyngeal cancer, hypothalamic and visual pathway
glioma, intraocular melanoma, islet cell tumors, Kaposi sarcoma,
kidney cancer, renal cell cancer, laryngeal cancer, lip and oral
cavity cancer, small cell lung cancer, non-small cell lung cancer,
primary central nervous system lymphoma, Waldenstrom
macroglobulinema, malignant fibrous histiocytoma, medulloblastoma,
melanoma, Merkel cell carcinoma, malignant mesothelioma, squamous
neck cancer, multiple endocrine neoplasia syndrome, multiple
myeloma, mycosis fungoides, myelodysplastic syndromes,
myeloproliferative disorders, chronic myeloproliferative disorders,
nasal cavity and paranasal sinus cancer, nasopharyngeal cancer,
neuroblastoma, oropharyngeal cancer, ovarian cancer, pancreatic
cancer, parathyroid cancer, penile cancer, pharyngeal cancer,
pheochromocytoma, pineoblastoma and supratentorial primitive
neuroectodermal tumors, pituitary cancer, plasma cell neoplasms,
pleuropulmonary blastoma, prostate cancer, rectal cancer,
rhabdomyosarcoma, salivary gland cancer, soft tissue sarcoma,
uterine sarcoma, Sezary syndrome, non-melanoma skin cancer, small
intestine cancer, squamous cell carcinoma, squamous neck cancer,
supratentorial primitive neuroectodermal tumors, testicular cancer,
throat cancer, thymoma and thymic carcinoma, thyroid cancer,
transitional cell cancer, trophoblastic tumors, urethral cancer,
uterine cancer, uterine sarcoma, vaginal cancer, vulvar cancer, and
Wilms tumor.
[0096] In particular, the cancer may be associated with a mutation
in the B-RAF gene. In some embodiments, the cancer may be a RAF
dependent cancer. These cancers include but are not limited to
melanoma, breast cancer, colorectal cancers, glioma, lung cancer,
ovarian cancer, sarcoma and thyroid cancer.
[0097] In a particular embodiment, the therapeutic combination
provided herein is effective for the treatment of moderate to
severe cancer in a subject.
Dosages
[0098] The optimal dose of the combination of agents for treatment
of cancer can be determined empirically for each subject using
known methods and will depend upon a variety of factors, including
the activity of the agents; the age, body weight, general health,
gender and diet of the subject; the time and route of
administration; and other medications the subject is taking.
Optimal dosages may be established using routine testing and
procedures that are well known in the art.
[0099] The amount of combination of agents that may be combined
with the carrier materials to produce a single dosage form will
vary depending upon the individual treated and the particular mode
of administration. In some embodiments the unit dosage forms
containing the combination of agents as described herein will
contain the amounts of each agent of the combination that are
typically administered when the agents are administered alone.
[0100] 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.
[0101] 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 and is
readily determined by one having skill in the art.
[0102] Generally, therapeutically effective 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 1000 mg
per kilogram of body weight per day, more preferably from about
0.01 to about 50 mg per kg per day.
[0103] 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.
Pharmaceutical Formulations and Routes of Administration
[0104] Provided herein are pharmaceutical formulations comprising a
combination of agents for the treatment of cancer, e.g., melanoma.
The pharmaceutical formulations may additionally comprise a carrier
or excipient, stabilizer, flavoring agent, and/or coloring
agent.
[0105] Provided herein are pharmaceutical formulations comprising
combination of agents which can be, for example, a combination of
two types of agents: (1) a RAF inhibitor and/or pharmacologically
active metabolites, salts, solvates and racemates of the inhibitor
and (2) a MEK inhibitor and/or pharmacologically active
metabolites, salts, solvates and racemates of the MEK
inhibitor.
[0106] The combination of agents may be administered using a
variety of routes of administration known to those skilled in the
art. The combination of agents may be administered to humans and
other animals orally, parenterally, sublingually, by aerosolization
or inhalation spray, rectally, intracisternally, intravaginally,
intraperitoneally, bucally, or topically in dosage unit
formulations containing conventional nontoxic pharmaceutically
acceptable carriers, adjuvants, and vehicles as desired. Topical
administration may also involve the use of transdermal
administration such as transdermal patches or ionophoresis devices.
The term parenteral as used herein includes subcutaneous
injections, intravenous, intramuscular, intrasternal injection, or
infusion techniques.
[0107] Methods of formulation are well known in the art and are
disclosed, for example, in Remington: The Science and Practice of
Pharmacy, Mack Publishing Company, Easton, Pa., 19th Edition
(1995). Pharmaceutical compositions for use in the present
invention can be in the form of sterile, non-pyrogenic liquid
solutions or suspensions, coated capsules, suppositories,
lyophilized powders, transdermal patches or other forms known in
the art.
[0108] Injectable preparations, for example, sterile injectable
aqueous or oleaginous suspensions may be formulated according to
the known art using suitable dispersing or wetting agents and
suspending agents. The sterile injectable preparation may also be a
sterile injectable solution, suspension or emulsion in a nontoxic
parenterally acceptable diluent or solvent, for example, as a
solution in 1,3 propanediol or 1,3 butanediol. Among the acceptable
vehicles and solvents that may be employed are water, Ringer's
solution, U.S.P. and isotonic sodium chloride solution. In
addition, sterile, fixed oils are conventionally employed as a
solvent or suspending medium. For this purpose any bland fixed oil
may be employed including synthetic mono or di glycerides. In
addition, fatty acids such as oleic acid find use in the
preparation of injectables. The injectable formulations can be
sterilized, for example, by filtration through a
bacterial-retaining filter, or by incorporating sterilizing agents
in the form of sterile solid compositions which can be dissolved or
dispersed in sterile water or other sterile injectable medium prior
to use.
[0109] In order to prolong the effect of a drug, it is often
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 with 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 may be
accomplished by dissolving or suspending the drug in an oil
vehicle. Injectable depot forms are made by forming microencapsule
matrices of the drug in biodegradable polymers such as polylactide
polyglycolide. Depending upon 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 may also be prepared by entrapping the drug in
liposomes or microemulsions, which are compatible with body
tissues.
[0110] Compositions for rectal or vaginal administration are
preferably suppositories which can be prepared by mixing the
compounds of this invention with suitable non irritating excipients
or carriers such as cocoa butter, polyethylene glycol or a
suppository wax which are solid at ambient temperature but liquid
at body temperature and therefore melt in the rectum or vaginal
cavity and release the active compound.
[0111] Solid dosage forms for oral administration include capsules,
tablets, pills, powders, and granules. In such solid dosage forms,
the active compound is mixed with at least one inert,
pharmaceutically acceptable excipient or carrier such as sodium
citrate or dicalcium phosphate and/or a) fillers or extenders such
as starches, lactose, sucrose, glucose, mannitol, and silicic acid,
b) binders such as, for example, carboxymethylcellulose, alginates,
gelatin, polyvinylpyrrolidinone, sucrose, and acacia, c) humectants
such as glycerol, d) disintegrating agents such as agar-agar,
calcium carbonate, potato or tapioca starch, alginic acid, certain
silicates, and sodium carbonate, e) solution retarding agents such
as paraffin, f) absorption accelerators such as quaternary ammonium
compounds, g) wetting agents such as, for example, acetyl alcohol
and glycerol monostearate, h) absorbents such as kaolin and
bentonite clay, and i) lubricants such as talc, calcium stearate,
magnesium stearate, solid polyethylene glycols, sodium lauryl
sulfate, and mixtures thereof. In the case of capsules, tablets and
pills, the dosage form may also comprise buffering agents.
[0112] 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 sugar as well as high molecular
weight polyethylene glycols and the like.
[0113] The solid dosage forms of tablets, dragees, capsules, pills,
and granules can be prepared with coatings and shells such as
enteric coatings and other coatings well known in the
pharmaceutical formulating art. They may optionally contain
opacifying agents and can also be of a composition that they
release the active ingredient(s) only, or preferentially, in a
certain part of the intestinal tract, optionally, in a delayed
manner. Examples of embedding compositions that can be used include
polymeric substances and waxes.
[0114] The active compounds can also be in micro-encapsulated form
with one or more excipients as noted above. The solid dosage forms
of tablets, dragees, capsules, pills, and granules can be prepared
with coatings and shells such as enteric coatings, release
controlling coatings and other coatings well known in the
pharmaceutical formulating art. In such solid dosage forms the
active compound may be admixed with at least one inert diluent such
as sucrose, lactose or starch. Such dosage forms may also comprise,
as is normal practice, additional substances other than inert
diluents, e.g., tableting lubricants and other tableting aids such
a magnesium stearate and microcrystalline cellulose. In the case of
capsules, tablets and pills, the dosage forms may also comprise
buffering agents. They may optionally contain opacifying agents and
can also be of a composition that they release the active
ingredient(s) only, or preferentially, in a certain part of the
intestinal tract, optionally, in a delayed manner. Examples of
embedding compositions that can be used include polymeric
substances and waxes.
[0115] Liquid dosage forms for oral administration include
pharmaceutically acceptable emulsions, microemulsions, solutions,
suspensions, syrups and elixirs. In addition to the active
compounds, the liquid dosage forms may contain inert diluents
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, EtOAc, benzyl alcohol,
benzyl benzoate, propylene glycol, 1,3 butylene glycol,
dimethylformamide, oils (in particular, cottonseed, groundnut,
corn, germ, olive, castor, and sesame oils), glycerol,
tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid
esters of sorbitan, and mixtures thereof. Besides inert diluents,
the oral compositions can also include adjuvants such as wetting
agents, emulsifying and suspending agents, sweetening, flavoring,
and perfuming agents.
[0116] Dosage forms for topical or transdermal administration of a
compound of this invention include ointments, pastes, creams,
lotions, gels, powders, solutions, sprays, inhalants or patches.
The active component is admixed under sterile conditions with a
pharmaceutically acceptable carrier and any needed preservatives or
buffers as may be required. Ophthalmic formulations, ear drops, and
the like are also contemplated as being within the scope of this
invention.
[0117] The ointments, pastes, creams and gels may contain, in
addition to an active compound 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.
[0118] Compositions of the invention may also be formulated for
delivery as a liquid aerosol or inhalable dry powder. Liquid
aerosol formulations may be nebulized predominantly into particle
sizes that can be delivered to the terminal and respiratory
bronchioles.
[0119] Aerosolized formulations of the invention may be delivered
using an aerosol forming device, such as a jet, vibrating porous
plate or ultrasonic nebulizer, preferably selected to allow the
formation of an aerosol particles having with a mass medium average
diameter predominantly between 1 to 5 .mu.m..quadrature. Further,
the formulation preferably has balanced osmolarity ionic strength
and chloride concentration, and the smallest aerosolizable volume
able to deliver effective dose of the compounds of the invention to
the site of the infection. Additionally, the aerosolized
formulation preferably does not impair negatively the functionality
of the airways and does not cause undesirable side effects.
[0120] Aerosolization devices suitable for administration of
aerosol formulations of the invention include, for example, jet,
vibrating porous plate, ultrasonic nebulizers and energized dry
powder inhalers, that are able to nebulize the formulation of the
invention into aerosol particle size predominantly in the size
range from 1 to 5 .mu.m..quadrature. Predominantly in this
application means that at least 70% but preferably more than 90% of
all generated aerosol particles are within 1 to 5 .mu.m
range..quadrature. A jet nebulizer works by air pressure to break a
liquid solution into aerosol droplets. Vibrating porous plate
nebulizers work by using a sonic vacuum produced by a rapidly
vibrating porous plate to extrude a solvent droplet through a
porous plate. An ultrasonic nebulizer works by a piezoelectric
crystal that shears a liquid into small aerosol droplets. A variety
of suitable devices are available, including, for example, AERONEB
and AERODOSE vibrating porous plate nebulizers (AeroGen, Inc.,
Sunnyvale, Calif.), SIDESTREAM nebulizers (Medic Aid Ltd., West
Sussex, England), PARI LC and PARI LC STAR jet nebulizers (Pari
Respiratory Equipment, Inc., Richmond, Va.), and AEROSONIC
(DeVilbiss Medizinische Produkte (Deutschland) GmbH, Heiden,
Germany) and ULTRAAIRE (Omron Healthcare, Inc., Vernon Hills, Ill.)
ultrasonic nebulizers.
[0121] Compounds of the invention may also be formulated for use as
topical powders and sprays that can contain, in addition to the
compounds 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.
[0122] Transdermal patches have the added advantage of providing
controlled delivery of a compound to the body. Such dosage forms
can be made by dissolving or dispensing the compound in the proper
medium. Absorption enhancers can also be used to increase the flux
of the compound across the skin. The rate can be controlled by
either providing a rate controlling membrane or by dispersing the
compound in a polymer matrix or gel. The compounds of the present
invention can also be administered in the form of liposomes. As is
known in the art, liposomes are generally derived from
phospholipids or other lipid substances. Liposomes are formed by
mono or multi lamellar hydrated liquid crystals that are dispersed
in an aqueous medium. Any nontoxic, physiologically acceptable and
metabolizable lipid capable of forming liposomes can be used. The
present compositions in liposome form can contain, in addition to a
compound of the present invention, stabilizers, preservatives,
excipients, and the like. The preferred lipids are the
phospholipids and phosphatidyl cholines (lecithins), both natural
and synthetic. Methods to form liposomes are known in the art. See,
for example, Prescott (ed.), "Methods in Cell Biology," Volume XIV,
Academic Press, New York, 1976, p. 33 et seq.
EXAMPLES
Example 1: Identification and Characterization of CRAF Resistance
Mutations In Vitro
[0123] To identify somatic RAF mutations that confer resistance to
RAF inhibitors, saturating random mutagenesis screens were
performed using the XL1-Red bacterial system and retroviral vectors
expressing C-RAF cDNAs (Emery et al., Id. 2009, Wagle et al., Id.
2011). The resulting randomly mutagenized saturating cDNA library
of CRAF mutations was expressed in A375 melanoma cells harboring
the BRAF.sup.V600E mutation which is highly responsive to the RAF
inhibitors (Li et al., 2007; Tsai et al., 2008, Emery et al., Id.
2009). These cells were cultured for 4 weeks in the presence of
fully inhibitory concentrations of PLX4720 (1.5 .mu.M) and the
resistant clones emerged were pooled and characterized by massive
parallel sequencing (Emery et al., Id. 2009, Wagle et al., Id.
2011). All C-RAF mutations examined (FIG. 1A) could be stably
expressed in A375 cells. (FIG. 9A). Furthermore, 8 of the 10 most
prominent C-RAF mutations conferred biochemical resistance to the
RAF inhibitor PLX4720 in A375 cells, as evidenced by p-MEK and
p-ERK levels (FIG. 9B). One of these alleles (CRAF.sup.G361A)
conferred substantial "paradoxical" activation of p-MEK at high
PLX4720 concentrations (FIG. 9B).
[0124] The CRAF mutations attained through the screen were mapped
on to the crystal structure of the CRAF kinase domain (340-618)
(Hatzivassiliou et al., 2010) (PDB code: 30MV) (FIG. 1B). Three of
the mutations were not mapped since the full length structure of
CRAF has not been solved to date. The CRAF resistance alleles
clustered towards two distinct regions, outside or within the
regulatory region (N-terminus) and in the kinase domain
(C-terminus) (FIG. 1C). CRAF consists of two 14-3-3 consensus
binding sites and two of the resistance alleles S257P and P261T
occupied one of the 14-3-3 consensus binding sites in the CR2
except the E104K allele in CR1 region (FIG. 1B).
[0125] The second category of resistance alleles encompasses the
kinase domain including the glycine rich loop. Mutations such as
G356E and G361A were found in the glycine residue of the
ATP-binding P-loop, GxGxxG motif. The P-loop mutations has been
found in several protein kinases (Christopher et al., 2007)
including BRAF which possess cellular transformation capacity by
activating Mek (Wan et al., Cell 116: 855-867, 2004; Garnett et
al., Id. 2005). The other subset of mutations (S427T, D447N, M469I,
E478K and R554K) populated outside the activation segment (FIG.
1B).
Example 2: Functional Characterization of C-RAF Resistance
Mutants
[0126] To study the functional consequences of the identified C-RAF
resistance alleles, the representative mutations (FIG. 1A) were
introduced into and expressed in A375 melanoma cells. Biochemical
analysis followed by treatment with Raf inhibitor, PLX4720
attenuated Mek phosphorylation (pMek) and Erk phosphorylation
(pErk) at a concentration of 2 .mu.M (FIG. 2A) in A375 cells and
WT-C-RAF expressing cells. However, the resistance alleles showed
sustained pMek and pErk at the same (FIG. 2A) concentration. Also,
the resistance alleles clustered towards the 14-3-3 binding region
(S257P and P261T) and one in the ATP binding region, particularly
G361A conferred profound pharmacological resistance to PLX4720.
These mutants increased the PLX4720 GI.sub.50 values by .about.100
fold (S257P and P261T) and 30 fold (G361A) when compared to the WT
(0.4 .mu.M) (FIG. 2C). Moreover, stability of C-Raf has been
correlated to S259 and S621 phosphorylation (FIG. 1C) and
subsequent 14-3-3 binding and activation of C-RAF has been
correlated with phosphorylation of S338 and S621 residues (FIG. 1C)
(Wellbrock et al., Id. 2004). PLX4720 induced the phosphorylation
of S338 and a modest increase of S621 particularly in S257P, P261T
and G361A resistance mutants which is consistent with paradoxical
activation of pMek in these variants (FIG. 2B). This C-RAF
activation of MEK/ERK signaling was suppressed by pharmacologic MEK
inhibition (FIG. 2G). The WT expressing cells exhibited a modest
increase in S338 but not in S621. On the contrary, the S259 site
was phosphorylated under basal conditions and ectopic expression of
WT-C-RAF exacerbated the effect in the absence of PLX4720 (FIG.
2B). However, PLX4720 also induced S259 phosphorylation in the
resistance mutants but comparatively these mutants exhibited lower
S259 phosphorylation levels (FIG. 2B), but this effect was
attenuated in the presence of AZD6244 with diminishing levels of
pErk (FIG. 2E). As C-RAF is highly modulated and activated by
phosphorylation, these data suggest that there is a feedback
activation and inhibition loop which is constantly at work
maintaining robustness and stringency to the MAPK signaling output.
Hence, the C-RAF resistance allele's activity might be modulated by
a balance between the amount of phosphorylation attained by sites
which render activity (S338, S621) and sites that render inhibition
(S259). Only one allele (G361A) conferred evidence of pharmacologic
resistance to MEK inhibition (FIGS. 2F and 2G).
Example 3: C-RAF Resistance Mutants Exhibit Increased Association
with B-RAF
[0127] The cumulative data above show that the resistance alleles
encompassing the 14-3-3 consensus binding site (S257P and P261T)
and the ATP binding region (G361A) (FIG. 1C) confer resistance due
to decreased inhibition of phosphorylated MEK and ERK. Also, it has
been shown that a double mutant of S259/S621A completely abrogates
the interaction between C-RAF and 14-3-3 without affecting the
interaction with RAS or MEK (Tzivion et al., Nature 394: 88-92,
1998). To investigate the underlying mechanism, we
immunoprecipitated C-RAF from 293/T cells ectopically expressing
all the resistance alleles identified during the initial screen.
Mutations that decreased interaction with 14-3-3 and increased the
interaction with B-RAF maintained a higher MEK and ERK
phosphorylation status when compared to the WT (FIG. 3A) and also
increased C-RAF kinase activity in vitro (data not shown). These
results corroborated the interaction status of 14-3-3 and B-RAF in
A375 cells expressing C-RAF alleles (FIG. 3B). Hence, these
resistance mutants possess higher activity towards its substrate
even in the absence of an oncogenic driver such as Ras (Weber et
al., 2001).
Example 4: Biochemical Characterization of C-RAF Resistance Alleles
Using (S)-methyl
1-(4-(3-(5-chloro-2-fluoro-3-(methylsulfonamido)phenyl)-1-isopropyl-1H-py-
razol-4-yl)pyrimidin-2-ylamino)propan-2-ylcarbamate
[0128] To exclude the possibility of inefficient binding of PLX4720
to the resistance mutants, mutant C-RAF responses to (S)-methyl
1-(4-(3-(5-chloro-2-fluoro-3-(methylsulfonamido)phenyl)-1-isopropyl-1H-py-
razol-4-yl)pyrimidin-2-ylamino)propan-2-ylcarbamate (Novartis), in
A375 cells and cells expressing WT-C-RAF (FIG. 3C) were examined.
The C-RAF resistance alleles (S257P, P261T) increased (S)-methyl
1-(4-(3-(5-chloro-2-fluoro-3-(methylsulfonamido)phenyl)-1-isopropyl-1H-py-
razol-4-yl)pyrimidin-2-ylamino)propan-2-ylcarbamate GI50 values by
.about.10,000 and .about.30,000 fold respectively and G361A by 20
fold (FIG. 3C). As observed in FIGS. 2C and E, the response to
PLX4720 and AZD6244 remained same (FIGS. 3B and D). Furthermore,
biochemically the C-RAF resistance mutants conferred resistance to
(S)-methyl
1-(4-(3-(5-chloro-2-fluoro-3-(methylsulfonamido)phenyl)-1-isopropyl-1H-py-
razol-4-yl)pyrimidin-2-ylamino)propan-2-ylcarbamate even at a
concentration of 1 .mu.M (FIG. 3A).
Example 5: C-RAF Resistance Mutants Confer Resistance to
Vemurafenib (PLX4032)
[0129] Vemurafenib is highly responsive to BRAF.sup.V600E mutations
but causes a paradoxical activation of Mek and Erk in cells
expressing oncogenic Ras (Hatzivassiliou et al., 2010; Heidorn et
al., 2010; Poulikakos et al., 2010). C-RAF resistance alleles were
tested to determine whether the C-RAF resistance alleles exhibited
similar response to Vemurafenib in the presence of oncogenic B-RAF.
A375 cells expressing WT-C-RAF (0.4 .mu.M) (FIG. 5A) showed
comparable GI.sub.50 values to that of PLX4720 treated WT-C-RAF
cells (FIG. 2C). The resistance conferred by G361A was 50 fold
higher than the WT, whereas surprisingly, the S257P and P261T
displayed an increased resistance towards Vemurafenib. As, these
mutants conferred resistance even in the absence of oncogenic Ras,
it was further determined, if resistance to the Raf inhibitor was
due to an increased activity. Total C-RAF was immunoprecipitated
from extracts of A375 cells expressing the C-RAF variants in the
presence and absence of Vemurafenib. Mutations that displayed high
resistance (S257P and P261T) during pharmacological inhibition
(FIG. 5A) had increased kinase activity in vitro compared to the WT
(FIG. 5B); however G361A resistance mutant displayed even higher
kinase activity in presence of the drug (FIG. 5B). Moreover, it is
shown that C-RAF resistance alleles remain sensitive to
combinatorial treatment with PLX4720 and AZD6244 (FIGS. 5C, D and
E).
[0130] Together, these data are consistent with the notion that
just an optimal amount of Erk signaling is required for the cells
to confer resistance.
Example 6: C-RAF Resistance Alleles Enable Paradoxical C-RAF
Activation and Enhanced Dimerization
[0131] Paradoxical MEK/ERK activation induced by RAF inhibitors
involves dimerization of RAF proteins (Hatzivassiliou et al., 2010;
Poulikakos et al., 2010). Moreover, a truncated form of
BRAF.sup.V600E that shows enhanced dimerization confers resistance
to RAF inhibitors (Poulikakos et al., 2011). To determine whether
the C-RAF resistance mutations mediate resistance through increased
dimerization, co-transfections were performed in 293/T cells using
expression constructs in which several representative C-RAF
resistance alleles were differentially tagged with two distinct
epitopes (His/V5 or Flag). Immunoprecipitation reactions were
carried out using Ni.sup.2+ beads (to capture the His-tagged
protein) followed by immunoblotting using anti-Flag antibody. In
these experiments, all C-RAF mutations that conferred pharmacologic
resistance to RAF inhibitors also exhibited increased
homodimerization compared to wild type C-RAF (S257P, P261T, G361A,
and E478K;). As expected, the increased dimerization generally
correlated with increased p-MEK levels (FIG. 6A, input lysate).
Similar results were observed when His/V5-tagged C-RAF mutants were
co-transfected with Flag-tagged wild-type C-RAF (FIG. 10A),
although the magnitude of MEK/ERK activation seemed qualitatively
reduced (FIG. 10A, input lysate). The three C-RAF mutants that
conferred the most profound pharmacologic resistance to PLX4720 and
vemurafenib (S257P, P261T, and G361A) (FIGS. 2C and 2F) also showed
evidence of increased total protein accumulation (FIG. 6A, input
lysate). Thus, the resistance phenotype linked to C-RAF mutations
correlated strongly with RAF dimerization.
[0132] In 293T cells (which lack oncogenic BRAF mutations), the
increased C-RAF dimerization engendered by the presence of
resistance mutations was sustained but not further enhanced upon
exposure of transfected cells to RAF inhibitor (vemurafenib; FIG.
6B). However, both C-RAF activation (evidenced by S338
phosphorylation) and downstream MEK/ERK signaling were robustly
induced by the RAF inhibitor (FIG. 6B, input lysate). To determine
the effects of these resistance mutations on intrinsic C-RAF kinase
activity, in vitro kinase reactions were performed from 293T cells
cultured in the presence or absence of RAF inhibitor. In the
absence of drug, steady-state C-RAF kinase activity (p-MEK) was not
measurably increased by the resistance mutations in most cases
(FIG. 6C). CRAF.sup.G361A was the one exception to this; here,
modest steady-state kinase activity was detected that correlated
with robust intrinsic p-MEK levels in the corresponding whole cell
lysates (FIG. 6C). In contrast, treatment of 293T cells with 2
.mu.M vemurafenib prior to the in vitro kinase assays resulted in a
marked up-regulation of C-RAF kinase activity in all resistance
alleles examined (FIG. 6C). Similar experiments in BRAF.sup.V600E
melanoma cells (A375) revealed an increase in intrinsic kinase
activity in the three most potent C-RAF resistance mutants examined
(S257P, P261T, and G361A; FIG. 6D). This kinase activity was
further augmented upon exposure of these cells to vemurafenib (FIG.
6D), as observed in 293T cells. Together, these results suggested
that potent C-RAF resistance mutants enhanced both RAF dimerization
and RAF inhibitor-mediated C-RAF kinase activity.
Example 7: C-RAF Resistance Mutants Exhibit Reduced 14-3-3 Binding
and Increased B-RAF Heterodimerization
[0133] The cumulative data above suggested that C-RAF mutations
encompassing its 14-3-3 consensus binding site (S257P and P261T)
and the ATP binding region of the P loop (G361A) conferred
pharmacological and biochemical resistance to RAF inhibition,
enhanced RAF dimerization, and increased C-RAF kinase activation
upon treatment with RAF inhibitors. To investigate the role of
14-3-3 protein binding in relation to RAF dimerization,
immunoprecipitation experiments were performed from cells
engineered to ectopically express C-RAF resistance alleles. To
examine the effects of pharmacologic RAF inhibition on 14-3-3
binding and B-RAF heterodimerization, these experiments were
conducted in both the absence and presence of RAF inhibition (in
this case, vemurafenib). In the absence of RAF inhibitor, the C-RAF
resistance alleles S257P, P261T and G361A tended to demonstrate
reduced interactions with 14-3-3.zeta. and increased interactions
with B-RAF in both 293T cells (FIG. 7A) and, in particular, A375
(BRAF.sup.V600E) melanoma cells (FIG. 7B). In 293T cells, the
enhanced C-RAF/B-RAF heterodimerization triggered by C-RAF
mutations correlated with C-RAF protein stabilization and robust
MEK/ERK phosphorylation (FIG. 7A). On the other hand, the robust
MEK/ERK activation observed in BRAF.sup.V600E melanoma cells was
only marginally enhanced by the C-RAF resistance mutants (FIG. 7B);
this result was expected given the constitutive oncogenic B-RAF
signaling in these cells. Interestingly, one of the C-RAF mutants
(G356E) exhibited very low 14-3-3.zeta. binding in both cellular
contexts (FIGS. 7A and 7B); however, C-RAF.sup.G356E showed no
enrichment in B-RAF heterodimerization and no increase in MEK/ERK
signaling under steady-state conditions. These results suggest that
while reduced 14-3-3 binding may promote enhanced mutant C-RAF
dimerization, some degree of 14-3-3 binding (perhaps within the
C-terminal domain) is needed to promote maximal RAF-dependent
signaling.
[0134] As expected, the RAF inhibitor vemurafenib induced
B-RAF/C-RAF heterodimerization in 293/T cells ectopically
expressing wild-type C-RAF (FIG. 7A), but abrogated this
heterodimerization in A375 melanoma cells (FIG. 7B). In contrast,
ectopic expression of the most robust C-RAF resistance mutants
enabled sustained B-RAF heterodimerization even in the presence of
drug in A375 cells (FIG. 7B). These results suggest that
BRAF.sup.V600E assumes a dominant conformation that favors
heterodimerization with resistance-associated C-RAF variants.
[0135] Pharmacologic RAF inhibition had variable effects on the
14-3-3/C-RAF interaction depending on the cellular genetic context.
In A375 cells (BRAF.sup.V600E), vemurafenib modestly decreased
14-3-3.zeta. binding to wild-type C-RAF, but had no effect in the
setting of the C-RAF.sup.S257P, C-RAF.sup.P261T and C-RAF.sup.G361A
mutants (FIG. 7B). On the other hand, vemurafenib enhanced these
14-3-3/C-RAF interactions in 293/T cells (FIG. 7A). These findings
lent further support to the premise that the resistance phenotype
conferred by these C-RAF mutants in the BRAF.sup.V600E context
involved enhanced RAF dimerization, which correlated with
diminished 14-3-3/C-RAF interactions.
Example 8: Enhanced MEK/ERK Signaling by C-RAF Resistance Mutants
Requires Dimerization
[0136] To test whether C-RAF dimerization is necessary for the
enhanced MEK/ERK signaling conferred by C-RAF resistance mutants,
an arginine-histidine mutation was introduced at residue R401
(C-RAF.sup.R401H) (FIG. 10B). This mutant has previously been shown
to disrupt C-RAF homodimerization ((Hatzivassiliou et al., Nature
464: 431-435, 2010; Poulikakos et al., Nature 464:427-430, 2010).
The R401H dimerization deficient mutation was introduced into the
respective C-RAF resistance alleles. As expected, C-RAF double
mutants were rendered largely incapable of enhanced MEK/ERK
signaling (FIG. 8A). Next, co-transfections were performed using
differentially epitope-tagged C-RAF resistance/R401H double
mutants. As described earlier, the C-RAF resistance alleles
augmented C-RAF homodimerization in a manner unaffected by RAF
inhibitor (FIG. 8B). In contrast, introduction of the R401H allele
suppressed C-RAF homodimerization and abrogated MEK/ERK signaling
in most C-RAF mutant contexts examined. The exception to this was
the C-RAF.sup.G361A allele, which exhibited constitutive (albeit
markedly reduced) MEK/ERK activation that was further induced by
vemurafenib exposure, even when co-expressed with the
dimerization-deficient double mutant. Together with the in vitro
kinase activity results above, these data suggest that the
C-RAF.sup.G361A/R401H allele may also contain increased intrinsic
kinase activity. Overall, these results provide direct evidence
that the enhanced MEK/ERK signaling conferred by C-RAF resistance
mutants requires RAF dimerization. They may also provide a
rationale for the future development of allosteric RAF inhibitors
that disrupt the RAF dimerization interface.
Methods
Cell Culture
[0137] 293/T cells, A375 cells (ATCC) and Phoenix cells (Allele
Biotech) were cultured and maintained at 37.degree. C. in
Dulbecco's modified Eagle's medium supplemented with 10% fetal
bovine serum in a humidified atmosphere containing 5% CO.sub.2.
C-RAF Random Mutagenesis Screen
[0138] C-RAF cDNA was cloned into pWZL-Blast vector (gift from J.
Boehm and W. C. Hahn) by recombinational cloning (Invitrogen).
Specific mutations were introduced into c-raf cDNA using
QuickChange II Site Directed Mutagenesis (Stratagene). Random
mutagenesis was done based on established protocol (Emery et al.,
Id. 2009). The mutagenized C-RAF plasmid was used to infect A375
melanoma cells. Following selection with Blasticidin, cells were
plated on 15-cm dishes and cultured in the presence of RAF
inhibitor, PLX4720 (1.5 .mu.M) for 4 weeks until resistant clones
emerged.
Sequencing of c-RAF DNA
[0139] PLX4720 resistant cells emerging from the random mutagenesis
screens were pooled and genomic DNA was prepared (Qiagen DNeasy).
C-RAF cDNA was amplified from genomic DNA using primers specific to
flanking vector sequence at the 5' and 3' end and sequenced by the
Sanger method using established protocols.
Analysis of Massively Parallel Sequencing
[0140] Raw data from massively parallel sequencing lanes (IIlumina;
2-3 million 36-base-pair sequences per lane) were analyzed using a
"next-generation" sequencing analysis pipeline (Emery et al., PNAS,
2009.). 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. The frequency of variation for each
nonwild-type allele was determined, and an average variant score
(AVS) was calculated as the mean of all quality scores for the
position and variant allele in question. All coding mutations were
translated to determine the amino acid variation (if any) and data
for high-frequency (>0.5%) and high-quality (AVS>7) mutations
were loaded into the CCGD results database.
Retroviral Infections
[0141] Phoenix cells (70% confluent) were transfected with
pWZLBlast-C-RAF or the mutants using Fugene 6 (Roche). Supernatants
containing virus were passed through a 0.45-.mu.m syringe. The A375
cells were infected for 16 h with virus together with polybrene (4
.mu.g/mL, Sigma). The selective marker blasticidin (3 .mu.g/mL) was
introduced 48 h postinfection.
Western Blot Analysis
[0142] Samples were extracted after washing twice with PBS and
lysed with 150 mM NaCl, 50 mM Tris pH 7.5, 1 mM EDTA, PMSF, Sodium
Fluoride, Sodium Orthovanadate and protease inhibitor cocktail in
the presence of 1% NP-40. The protein content was estimated with
protein assay reagent (Bio-Rad) according to the manufacturer's
instructions. Equal amounts of whole cell lysates were loaded onto
and separated by 8-16% SDS-PAGE ready-made gels. Proteins were
transferred to polyvinylidene difluoride membranes in a Trans-Blot
apparatus. Membranes were blocked with 5% skim milk in TBS
containing 0.1% Tween 20 for 1 h at room temperature or overnight
at 4.degree. C. Membranes were then incubated with monoclonal or
polyclonal antibody raised against the protein of interest for 1 h
at room temperature or overnight at 4.degree. C. followed by three
washes with TBS containing 0.1% Tween 20. The immunoreactivity of
the primary antibodies C-RAF, S259C-RAF, S338C-RAF, S621C-RAF,
pERK, ERK, pMEK, MEK, 14-3-3.zeta. Flag (Cell Signaling), B-RAF
(Santa Cruz Biotechnology) and actin (Sigma) was visualized with a
secondary anti-rabbit (BD Transduction Laboratories) or anti-mouse
(Santa Cruz Biotechnology) antibodies conjugated with horseradish
peroxidase and subsequent development with ECL Plus (Amersham
Biosciences) and autoradiography on X-OMAR TAR films. The bands
were scanned and quantified by the Gel Doc system using the
Quantity One software.
Immunoprecipitation
[0143] For immunoprecipitation with C-RAF antibody (BD Biosciences)
protein G-Sepharose slurry (Thermo Scientific) was washed with
1.times.PBS and incubated with C-RAF antibody (BD Biosciences) or
normal mouse IgG (control) for 1 h at 4.degree. C. After three
washes with lysis buffer, the beads were incubated with whole cell
lysates (0.5 mg of total protein) for 2 h and then washed three
times with lysis buffer. The proteins were then eluted by boiling
in 1.times.SDS-sample buffer.
C-RAF Kinase Assay
[0144] The 293T cells (70% confluent) were transfected with 6 .mu.g
pc-DNA with His or V5 tag towards the C-terminal containing
C-RAF-WT and C-RAF variant alleles. Cells were treated with
vemurafenib (Allele Biotech) for 1 h and 48 h post-transfection,
lysates were extracted by general protocol. Immunoprecipitation
using cobalt beads was performed overnight for 1 hr at 4.degree. C.
The protein-bound cobalt beads where incubated with 20 .mu.L
ATP/magnesium mixture (20 mM Mops pH 7.2, 25 mM
.beta.-glycerophosphate, 5 mM EGTA, 1 mM Na3VO4, 1 mM DTT, 75 mM
MgCl2, and 0.5 mMATP), 20 .mu.L of dilution buffer (20 mM Mops, pH
7.2, 25 mM-glycerol phosphate, 5 mM EGTA, 1 mM sodium
orthovanadate, 1 mM DTT), and 1 .mu.g of inactive MEK (obtained
from Millipore) for 30 min at 30.degree. C. The phosphorylated MEK
product was detected by immunostaining using a p-MEK antibody (Cell
Signaling Technology), and relative p-MEK signals were quantified
using densitometry, normalized to the amount of input C-RAF, and
compared to C-RAF-WT as a reference.
C-RAF Kinase Assay (A375)
[0145] A375 cells infected with WT and mutant C-RAF alleles were
cultured in the absence and presence of PLX4032 (Allele Biotech)
for 16 h. Lysates were prepared with 150 mM NaCl, 50 mM Tris pH
7.5, 1 mM EDTA, PMSF, Sodium Fluoride, Sodium Orthovanadate and
protease inhibitor cocktail in the presence of 1% NP-40.
Immunoprecipitation with C-RAF antibody was performed overnight and
bound beads were washed three times with lysis buffer, followed by
kinase buffer (1.times.). The beads were incubated with 20 ul of
ATP/Magnesium mixture (Millipore) and 0.5 .mu.g of inactive MEK
(Millipore) for 30 min at 30.degree. C. The phosphorylated
substrate MEK was detected by immunoblotting.
Pharmacologic Growth Inhibition Assays
[0146] Cultured cells were seeded into 96-well plates at a density
of 3,000 cells per well for all melanoma short-term cultures
including A375. After 16 h, serial dilutions of the compound were
performed in DMSO and transferred to cells to yield drug
concentrations based on the potency of the drug, ensuring that the
final volume of DMSO did not exceed 1%. The B-RAF inhibitor PLX4720
(purchased from Symansis, PLX4032 (purchased from Allele Biotech),
AZD6244 (purchased from Selleck Chemicals) and GSK1120212
(Purchased from Active Biochem). Following addition of the drug,
cell viability was measured using the Cell-Titer-96 aqueous
non-radioactive proliferation assay (Promega) after 4 days.
Viability was calculated as a percentage of the control (untreated
cells) after background subtraction. A minimum of six replicates
was made for each cell line and the entire experiment was repeated
at least three times. The data from the pharmacologic
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). GI50
values were calculated by determining the slope of the line
connecting the data points that flanked the 50% point.
[0147] The definitions and disclosures provided herein govern and
supersede all others incorporated by reference. Although the
invention herein has been described in connection with preferred
embodiments thereof, it will be appreciated by those skilled in the
art that additions, modifications, substitutions, and deletions not
specifically described may be made without departing from the
spirit and scope of the invention as defined in the appended
claims. It is therefore intended that the foregoing detailed
description be regarded as illustrative rather than limiting, and
that it be understood that it is the following claims, including
all equivalents, that are intended to define the spirit and scope
of this invention.
Sequence CWU 1
1
213216DNAHuman 1tcgcgggcgc ttgggccgcc atcttagatg gcgggagtaa
gaggaaaacg attgtgaggc 60gggaacggct ttctgctgcc ttttttgggc cccgaaaagg
gtcagctggc cgggctttgg 120ggcgcgtgcc ctgaggcgcg gagcgcgttt
gctacgatgc gggggctgct cggggctccg 180tcccctgggc tggggacgcg
ccgaatgtga ccgcctcccg ctccctcacc cgccgcgggg 240aggaggagcg
ggcgagaagc tgccgccgaa cgacaggacg ttggggcggc ctggctccct
300caggtttaag aattgtttaa gctgcatcaa tggagcacat acagggagct
tggaagacga 360tcagcaatgg ttttggattc aaagatgccg tgtttgatgg
ctccagctgc atctctccta 420caatagttca gcagtttggc tatcagcgcc
gggcatcaga tgatggcaaa ctcacagatc 480cttctaagac aagcaacact
atccgtgttt tcttgccgaa caagcaaaga acagtggtca 540atgtgcgaaa
tggaatgagc ttgcatgact gccttatgaa agcactcaag gtgaggggcc
600tgcaaccaga gtgctgtgca gtgttcagac ttctccacga acacaaaggt
aaaaaagcac 660gcttagattg gaatactgat gctgcgtctt tgattggaga
agaacttcaa gtagatttcc 720tggatcatgt tcccctcaca acacacaact
ttgctcggaa gacgttcctg aagcttgcct 780tctgtgacat ctgtcagaaa
ttcctgctca atggatttcg atgtcagact tgtggctaca 840aatttcatga
gcactgtagc accaaagtac ctactatgtg tgtggactgg agtaacatca
900gacaactctt attgtttcca aattccacta ttggtgatag tggagtccca
gcactacctt 960ctttgactat gcgtcgtatg cgagagtctg tttccaggat
gcctgttagt tctcagcaca 1020gatattctac acctcacgcc ttcaccttta
acacctccag tccctcatct gaaggttccc 1080tctcccagag gcagaggtcg
acatccacac ctaatgtcca catggtcagc accaccctgc 1140ctgtggacag
caggatgatt gaggatgcaa ttcgaagtca cagcgaatca gcctcacctt
1200cagccctgtc cagtagcccc aacaatctga gcccaacagg ctggtcacag
ccgaaaaccc 1260ccgtgccagc acaaagagag cgggcaccag tatctgggac
ccaggagaaa aacaaaatta 1320ggcctcgtgg acagagagat tcaagctatt
attgggaaat agaagccagt gaagtgatgc 1380tgtccactcg gattgggtca
ggctcttttg gaactgttta taagggtaaa tggcacggag 1440atgttgcagt
aaagatccta aaggttgtcg acccaacccc agagcaattc caggccttca
1500ggaatgaggt ggctgttctg cgcaaaacac ggcatgtgaa cattctgctt
ttcatggggt 1560acatgacaaa ggacaacctg gcaattgtga cccagtggtg
cgagggcagc agcctctaca 1620aacacctgca tgtccaggag accaagtttc
agatgttcca gctaattgac attgcccggc 1680agacggctca gggaatggac
tatttgcatg caaagaacat catccataga gacatgaaat 1740ccaacaatat
atttctccat gaaggcttaa cagtgaaaat tggagatttt ggtttggcaa
1800cagtaaagtc acgctggagt ggttctcagc aggttgaaca acctactggc
tctgtcctct 1860ggatggcccc agaggtgatc cgaatgcagg ataacaaccc
attcagtttc cagtcggatg 1920tctactccta tggcatcgta ttgtatgaac
tgatgacggg ggagcttcct tattctcaca 1980tcaacaaccg agatcagatc
atcttcatgg tgggccgagg atatgcctcc ccagatctta 2040gtaagctata
taagaactgc cccaaagcaa tgaagaggct ggtagctgac tgtgtgaaga
2100aagtaaagga agagaggcct ctttttcccc agatcctgtc ttccattgag
ctgctccaac 2160actctctacc gaagatcaac cggagcgctt ccgagccatc
cttgcatcgg gcagcccaca 2220ctgaggatat caatgcttgc acgctgacca
cgtccccgag gctgcctgtc ttctagttga 2280ctttgcacct gtcttcaggc
tgccagggga ggaggagaag ccagcaggca ccacttttct 2340gctccctttc
tccagaggca gaacacatgt tttcagagaa gctgctgcta aggaccttct
2400agactgctca cagggcctta acttcatgtt gccttctttt ctatcccttt
gggccctggg 2460agaaggaagc catttgcagt gctggtgtgt cctgctccct
ccccacattc cccatgctca 2520aggcccagcc ttctgtagat gcgcaagtgg
atgttgatgg tagtacaaaa agcaggggcc 2580cagccccagc tgttggctac
atgagtattt agaggaagta aggtagcagg cagtccagcc 2640ctgatgtgga
gacacatggg attttggaaa tcagcttctg gaggaatgca tgtcacaggc
2700gggactttct tcagagagtg gtgcagcgcc agacattttg cacataaggc
accaaacagc 2760ccaggactgc cgagactctg gccgcccgaa ggagcctgct
ttggtactat ggaacttttc 2820ttaggggaca cgtcctcctt tcacagcttc
taaggtgtcc agtgcattgg gatggttttc 2880caggcaaggc actcggccaa
tccgcatctc agccctctca gggagcagtc ttccatcatg 2940ctgaattttg
tcttccagga gctgccccta tggggcgggg ccgcagggcc agccttgttt
3000ctctaacaaa caaacaaaca aacagccttg tttctctagt cacatcatgt
gtatacaagg 3060aagccaggaa tacaggtttt cttgatgatt tgggttttaa
ttttgttttt attgcacctg 3120acaaaataca gttatctgat ggtccctcaa
ttatgttatt ttaataaaat aaattaaatt 3180taaaaaaaaa aaaaaaaaaa
aaaaaaaaaa aaaaaa 32162648PRTHuman 2Met Glu His Ile Gln Gly Ala Trp
Lys Thr Ile Ser Asn Gly Phe Gly 1 5 10 15 Phe Lys Asp Ala Val Phe
Asp Gly Ser Ser Cys Ile Ser Pro Thr Ile 20 25 30 Val Gln Gln Phe
Gly Tyr Gln Arg Arg Ala Ser Asp Asp Gly Lys Leu 35 40 45 Thr Asp
Pro Ser Lys Thr Ser Asn Thr Ile Arg Val Phe Leu Pro Asn 50 55 60
Lys Gln Arg Thr Val Val Asn Val Arg Asn Gly Met Ser Leu His Asp 65
70 75 80 Cys Leu Met Lys Ala Leu Lys Val Arg Gly Leu Gln Pro Glu
Cys Cys 85 90 95 Ala Val Phe Arg Leu Leu His Glu His Lys Gly Lys
Lys Ala Arg Leu 100 105 110 Asp Trp Asn Thr Asp Ala Ala Ser Leu Ile
Gly Glu Glu Leu Gln Val 115 120 125 Asp Phe Leu Asp His Val Pro Leu
Thr Thr His Asn Phe Ala Arg Lys 130 135 140 Thr Phe Leu Lys Leu Ala
Phe Cys Asp Ile Cys Gln Lys Phe Leu Leu 145 150 155 160 Asn Gly Phe
Arg Cys Gln Thr Cys Gly Tyr Lys Phe His Glu His Cys 165 170 175 Ser
Thr Lys Val Pro Thr Met Cys Val Asp Trp Ser Asn Ile Arg Gln 180 185
190 Leu Leu Leu Phe Pro Asn Ser Thr Ile Gly Asp Ser Gly Val Pro Ala
195 200 205 Leu Pro Ser Leu Thr Met Arg Arg Met Arg Glu Ser Val Ser
Arg Met 210 215 220 Pro Val Ser Ser Gln His Arg Tyr Ser Thr Pro His
Ala Phe Thr Phe 225 230 235 240 Asn Thr Ser Ser Pro Ser Ser Glu Gly
Ser Leu Ser Gln Arg Gln Arg 245 250 255 Ser Thr Ser Thr Pro Asn Val
His Met Val Ser Thr Thr Leu Pro Val 260 265 270 Asp Ser Arg Met Ile
Glu Asp Ala Ile Arg Ser His Ser Glu Ser Ala 275 280 285 Ser Pro Ser
Ala Leu Ser Ser Ser Pro Asn Asn Leu Ser Pro Thr Gly 290 295 300 Trp
Ser Gln Pro Lys Thr Pro Val Pro Ala Gln Arg Glu Arg Ala Pro 305 310
315 320 Val Ser Gly Thr Gln Glu Lys Asn Lys Ile Arg Pro Arg Gly Gln
Arg 325 330 335 Asp Ser Ser Tyr Tyr Trp Glu Ile Glu Ala Ser Glu Val
Met Leu Ser 340 345 350 Thr Arg Ile Gly Ser Gly Ser Phe Gly Thr Val
Tyr Lys Gly Lys Trp 355 360 365 His Gly Asp Val Ala Val Lys Ile Leu
Lys Val Val Asp Pro Thr Pro 370 375 380 Glu Gln Phe Gln Ala Phe Arg
Asn Glu Val Ala Val Leu Arg Lys Thr 385 390 395 400 Arg His Val Asn
Ile Leu Leu Phe Met Gly Tyr Met Thr Lys Asp Asn 405 410 415 Leu Ala
Ile Val Thr Gln Trp Cys Glu Gly Ser Ser Leu Tyr Lys His 420 425 430
Leu His Val Gln Glu Thr Lys Phe Gln Met Phe Gln Leu Ile Asp Ile 435
440 445 Ala Arg Gln Thr Ala Gln Gly Met Asp Tyr Leu His Ala Lys Asn
Ile 450 455 460 Ile His Arg Asp Met Lys Ser Asn Asn Ile Phe Leu His
Glu Gly Leu 465 470 475 480 Thr Val Lys Ile Gly Asp Phe Gly Leu Ala
Thr Val Lys Ser Arg Trp 485 490 495 Ser Gly Ser Gln Gln Val Glu Gln
Pro Thr Gly Ser Val Leu Trp Met 500 505 510 Ala Pro Glu Val Ile Arg
Met Gln Asp Asn Asn Pro Phe Ser Phe Gln 515 520 525 Ser Asp Val Tyr
Ser Tyr Gly Ile Val Leu Tyr Glu Leu Met Thr Gly 530 535 540 Glu Leu
Pro Tyr Ser His Ile Asn Asn Arg Asp Gln Ile Ile Phe Met 545 550 555
560 Val Gly Arg Gly Tyr Ala Ser Pro Asp Leu Ser Lys Leu Tyr Lys Asn
565 570 575 Cys Pro Lys Ala Met Lys Arg Leu Val Ala Asp Cys Val Lys
Lys Val 580 585 590 Lys Glu Glu Arg Pro Leu Phe Pro Gln Ile Leu Ser
Ser Ile Glu Leu 595 600 605 Leu Gln His Ser Leu Pro Lys Ile Asn Arg
Ser Ala Ser Glu Pro Ser 610 615 620 Leu His Arg Ala Ala His Thr Glu
Asp Ile Asn Ala Cys Thr Leu Thr 625 630 635 640 Thr Ser Pro Arg Leu
Pro Val Phe 645
* * * * *