BRAF mutations conferring resistance to BRAF inhibitors
The present invention relates to methods, compositions and kits concerning resistance to treatment with an anti-cancer agent, specifically an inhibitor of BRAF. In particular embodiments, the invention concerns mutations in a BRAF sequence that confer resistance to a BRAF inhibitor. Identification of such mutations in a BRAF sequence allows the identification and design of second-generation BRAF inhibitors. Methods and kits for detecting the presence of a mutant BRAF sequence in a sample are also provided.
1. A method of detecting a BRAF mutation in a subject having a cancer selected from: melanoma, colon carcinoma or thyroid carcinoma, the method comprising
(a) obtaining a cancer cell-containing sample from the subject; and
(b) performing a nucleic acid-based assay to analyze a nucleic acid molecule encoding a BRAF polypeptide for one or more mutations with respect to a nucleic acid molecule encoding a wild-type BRAF polypeptide (SEQ ID NO: 1) or a BRAF V600E polypeptide (SEQ ID NO: 3); and
(c) detecting in the assay the one or more mutations occurring at positions encoding one or more amino acids in the BRAF polypeptide selected from the group consisting of A29, H72, SI 13, S124, P162, C194, L227, P231, C251, V291, Q329, V483, L485, T521, V528, D587, P655, S657, S683, P686, C696, L697, P722, F738, and C748.
2. The method of claim 1 , wherein the nucleic acid molecule encodes a BRAF polypeptide having one or more amino acid substitutions with respect to a wild type BRAF polypeptide (SEQ ID NO:2) or a BRAF V600E polypeptide (SEQ ID NO:4) selected from the group consisting of A29V, H72N, SI 131, S124F, P162H, C194*, L227F, P231T, C251F, V291F, Q329K, V483E, L485F, T521K, V528F, D587E, P655T, S657*, S683R, P686Q, P686T, C696*, L697I, P722T, F738L, and C748F.
3. The method of claim 1 , wherein the mutant BRAF polypeptide is a BRAF polypeptide comprising a substitution at one or more of amino acid positions T521, V528, or P686.
4. The method of claim 1 , wherein the mutant BRAF polypeptide is a BRAF polypeptide comprising a substitution at one or more of amino acid positions T521K, V528F, or P686Q.
5. The method of claim 1 , wherein the presence of the one or more mutations in the nucleic acid molecule in the cancer cell-containing sample confers resistance to a RAF inhibitor selected from the group consisting of RAF-265 and PLX4720.
6. The method of claim 5 , wherein the BRAF inhibitor is RAF-265.
7. The method of claim 5 , wherein the RAF inhibitor is PLX4720.
8. The method of claim 1 , wherein the cancer is melanoma.