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.

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. 
     
     
         2 . A host cell comprising the expression vector of  claim 1 . 
     
     
         3 . The mutant C-RAF polypeptide according to  claim 1 , wherein the at least one amino acid substitution occurs at one or more amino acid positions selected from the group consisting of 104E, 257S, 261P, 356G, 361G, 427S, 447D, 469M, 478E and 554R. 
     
     
         4 . The mutant C-RAF polypeptide according to  claim 1 , wherein the at least one substitution is selected from the group consisting of 104E>K, 257S>P, 261P>T, 356G>E, 361G>A, 427S>T, 447D>N, 469M>I, 478E>K and 554R>K. 
     
     
         5 . The mutant C-RAF polypeptide according to  claim 1 , wherein the at least one substitution is selected from the group consisting of 257S, 261P and 361G. 
     
     
         6 . The mutant C-RAF polypeptide according to  claim 1 , wherein the RAF inhibitor is selected from the group consisting of 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-pyrazol-4-yl)pyrimidin-2-ylamino)propan-2-ylcarbamate. 
     
     
         7 . The mutant C-RAF polypeptide according to  claim 1 , wherein the RAF inhibitor is PLX4032. 
     
     
         8 . An antibody preparation which specifically binds to a polypeptide encoded by the expression vector of  claim 1 . 
     
     
         9 . A method of treating a subject having cancer, the method comprising:
 (a) extracting nucleic acid from cells of a cancer of the patient;   (b) 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; and   (c) 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 reside at one or more amino acids of the encoded C-RAF polypeptide as compared to a wild type C-RAF polypeptide.   
     
     
         10 . The method according to  claim 9 , wherein the second inhibitor is a MEK inhibitor. 
     
     
         11 . The method according to  claim 9 , wherein the RAF inhibitor is selected from the group consisting of 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-pyrazol-4-yl)pyrimidin-2-ylamino)propan-2-ylcarbamate. 
     
     
         12 . The method according to  claim 9 , wherein the MEK inhibitor is selected from the group consisting of CI-1040/PD184352, AZD6244, 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. 
     
     
         13 . The method according to  claim 9 , wherein the cancer is selected from the group consisting of melanoma, breast cancer, colorectal cancers, glioma, lung cancer, ovarian cancer, sarcoma and thyroid cancer. 
     
     
         14 . The method according to  claim 9 , wherein the cancer is a RAF dependent cancer. 
     
     
         15 . The method according to  claim 9 , wherein the cancer is melanoma. 
     
     
         16 . 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, the method comprising:
 (a) extracting nucleic acid from cells of a cancer of the patient; and   (b) assaying at least a portion of a nucleic acid molecule encoding a C-RAF polypeptide;   wherein 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.   
     
     
         17 . The method according to  claim 16 , further comprising administering a RAF inhibitor and a second inhibitor to the subject. 
     
     
         18 . The method according to  claim 16 , wherein 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 occurs at one or more amino acid positions selected from the group consisting of 104E>K, 257S>P, 261P>T, 356G>E, 361G>A, 427S>T, 447D>N, 469M>, 478E>K and 554R>K. 
     
     
         19 . The method according to any one of  claim 16 , wherein 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 occurs at one or more amino acid positions selected from the group consisting of 257S, 261P and 361G. 
     
     
         20 . The method according to  claim 16 , wherein the second inhibitor is a MEK inhibitor.