Methods of Diagnosing and Treating Cancer in Patients Having or Developing Resistance to a First Cancer Therapy

Abstract

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. A method of treating cancer in a subject in need thereof is also provided and includes administering to the subject an effective amount of a RAF inhibitor and an effective amount of a second inhibitor, wherein the second inhibitor is a MEK inhibitor, a CRAF inhibitor, a CrkL inhibitor or a TPL2/COT inhibitor. A method of identifying a kinase target that confers resistance to a first inhibitor is also provided.

1 . 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, comprising:
 (a) assaying a gene copy number, a mRNA or a protein level or phosphorylation of one or more kinase targets selected from the group consisting of MAP3K8 (TPL2/COT), RAF1 (CRAF), CRKL (CrkL), FGR (Fgr), PRKCE (Prkce), PRKCH (Prkch), ERBB2 (ErbB2), AXL (Axl), or PAK3 (Pak3) in cancer cells obtained from the subject and comparing the gene copy number, the mRNA or the protein level or the phoshorylation with a gene copy number, a mRNA or a protein level or phosphorylation of the target kinase in cells obtained from a subject without the cancer;   (b) correlating increased gene copy number or an alteration in mRNA expression or protein overexpression or phosphorylation of the target kinase in the cancer cells relative to the cells from the subject without the cancer with the subject having the cancer who is likely to benefit from treatment with the combination therapy; and   (c) selecting the subject having increased gene copy number or an alteration in mRNA expression or protein overexpression or phosphorylation of the target kinase in the cancer cells for treatment with the combination therapy.   
     
     
         2 . The method of  claim 1 , further comprising (c) assaying a nucleic acid sample obtained from the cancer cells for the presence of a mutation in a nucleic acid molecule encoding a B-RAF polypeptide with a mutation at about amino acid position 600 and (d) correlating the presence of the mutation in the nucleic acid molecule encoding the B-RAF polypeptide with a subject who is likely to benefit from treatment with the combination therapy. 
     
     
         3 . The method of  claim 1 , comprising correlating the presence of a Valine to Glutamic Acid mutation at about amino acid position 600 (V600E) in a nucleic acid molecule encoding B-RAF with a subject who is likely to benefit from treatment with the combination therapy. 
     
     
         4 . The method of  claim 1 , comprising assaying the gene copy number, the mRNA or the protein level of MAP3K8 (TPL/COT). 
     
     
         5 . The method of  claim 1 , comprising assaying phosphorylation of C-RAF at amino acid S338. 
     
     
         6 . The method of  claim 1 , wherein the second inhibitor is a MEK inhibitor, a CRAF inhibitor, a CrkL inhibitor or a TPL2/COT inhibitor. 
     
     
         7 . The method of  claim 1 , wherein the RAF inhibitor is a B-RAF inhibitor or a pan-RAF inhibitor. 
     
     
         8 . The method of  claim 1 , wherein the RAF inhibitor is selected from the group consisting of RAF265, sorafenib, SB590885, PLX 4720, PLX4032, GDC-0879 and ZM 336372. 
     
     
         9 . The method of  claim 1 , wherein the subject has innate resistance to the RAF inhibitor or is likely to develop resistance to the RAF inhibitor. 
     
     
         10 . The method of  claim 1 , wherein the cancer is selected from the group consisting of melanoma, breast cancer, colorectal cancers, glioma, lung cancer, ovarian cancer, sarcoma and thyroid cancer. 
     
     
         11 . The method of  claim 10 , wherein the cancer is melanoma. 
     
     
         12 . A method of treating cancer in a subject in need thereof, comprising administering to the subject an effective amount of a RAF inhibitor and an effective amount of a second inhibitor, wherein the second inhibitor is a MEK inhibitor, or a MAP3K8 (TPL2/COT) inhibitor. 
     
     
         13 . The method of  claim 12 , wherein the subject has cancer cells comprising a BRAF mutation or a B-RAF V600E  mutation. 
     
     
         14 . (canceled) 
     
     
         15 . The method of  claim 12 , further comprising assaying a gene copy number, a mRNA or a protein level or phosphorylation of one or more kinase targets selected from the group consisting of MAP3K8 (TPL2/COT), RAF1 (CRAF), CRKL (CrkL), FGR (Fgr), PRKCE (Prkce), PRKCH (Prkch), ERBB2 (ErbB2), AXL (Axl), or PAK3 (Pak3) in cancer cells obtained from the subject and comparing the gene copy number, the mRNA or the protein level or the phoshorylation with a gene copy number, a mRNA or a protein level or phosphorylation of the target kinase in cells obtained from a subject without the cancer; and
 administering to the subject the effective amount of a RAF inhibitor and the effective amount of a second inhibitor in subjects having an increased gene copy number or an alteration in mRNA expression or protein overexpression or phosphorylation of the target kinase in the cancer cells.   
     
     
         16 . The method of  claim 12 , wherein the RAF inhibitor is selected from the group consisting of RAF265, sorafenib, SB590885, PLX 4720, PLX4032, GDC-0879 and ZM 336372. 
     
     
         17 . The method of  claim 12 , 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. 
     
     
         18 . The method of  claim 12 , wherein the subject has innate resistance to the RAF inhibitor or the MEK inhibitor or is likely to develop resistance to the RAF inhibitor or the MEK inhibitor. 
     
     
         19 . The method of  claim 12 , wherein the cancer is selected from the group consisting of melanoma, breast cancer, colorectal cancers, glioma, lung cancer, ovarian cancer, sarcoma and thyroid cancer. 
     
     
         20 . The method of  claim 12 , wherein the cancer is melanoma. 
     
     
         21 . (canceled) 
     
     
         22 . The method of  claim 12 , wherein the second inhibitor is a MAP3K8 (TPL2/COT) inhibitor. 
     
     
         23 . A method of identifying a kinase target that confers resistance to a first inhibitor, the method comprising:
 culturing cells having sensitivity to the first inhibitor;   expressing a plurality of kinase ORF clones in the cell cultures, each cell culture expressing a different kinase ORF clone;   exposing each cell culture to the inhibitor;   identifying cell cultures having greater viability than a control cell culture after exposure to the inhibitor to identify one or more kinase ORF clones that confers resistance to the first inhibitor.   
     
     
         24 . The method of  claim 23 , wherein the cultured cells have sensitivity to a RAF inhibitor or a MEK inhibitor. 
     
     
         25 . (canceled) 
     
     
         26 . The method of  claim 23 , wherein the cultured cells comprise a B-RAF mutation or a B-RAF V600E  mutation. 
     
     
         27 . (canceled) 
     
     
         28 . The method of  claim 23 , wherein the cultured cells comprise a melanoma cell line. 
     
     
         29 . The method of  claim 23 , comprising providing the plurality of kinase ORF clones in a lentiviral vector or a retroviral vector.