tak-632 and Melanoma

The BRAF inhibitors dabrafenib and vemurafenib induce remarkable clinical responses in patients with BRAF-mutated melanomas. However, adverse events, including the emergence of secondary tumors and drug resistance, have been reported. Studies have revealed that undesirable RAF dimerization induced by inhibitors promotes these adverse effects. Here, we developed highly sensitive biosensors of RAF dimerization in cells utilizing the split enhanced click beetle luciferase (Emerald Luc, ELuc) complementation technique. We demonstrated that our biosensor system works effectively for high-throughput screens in the microplate format. A comprehensive analysis of commercially available RAF inhibitors performed using this assay system revealed that the inhibitors exhibit various potencies in inducing the dimerization of RAF isoforms, and their dimerization potencies do not always correlate with the RAF enzyme inhibition. This sensitive assay system will become a powerful tool to discover next-generation BRAF inhibitors with safer profiles.

Topics: Benzothiazoles; Biosensing Techniques; Cell Line, Tumor; Dimerization; Heterocyclic Compounds, 2-Ring; Humans; Imidazoles; Melanoma; Nitriles; Oximes; Phenylurea Compounds; Protein Kinase Inhibitors; Protein Multimerization; Proto-Oncogene Proteins B-raf; Pyrimidines; Sulfonamides; Vemurafenib

The mitogen-activated protein kinase (MAPK) pathway is particularly important for the survival and proliferation of melanoma cells. Somatic mutations in BRAF and NRAS are frequently observed in melanoma. Recently, the BRAF inhibitors vemurafenib and dabrafenib have emerged as promising agents for the treatment of melanoma patients with BRAF-activating mutations. However, as BRAF inhibitors induce RAF paradoxical activation via RAF dimerization in BRAF wild-type cells, rapid emergence of acquired resistance and secondary skin tumors as well as presence of few effective treatment options for melanoma bearing wild-type BRAF (including NRAS-mutant melanoma) are clinical concerns. Here, we demonstrate that the selective pan-RAF inhibitor TAK-632 suppresses RAF activity in BRAF wild-type cells with minimal RAF paradoxical activation. Our analysis using RNAi and TAK-632 in preclinical models reveals that the MAPK pathway of NRAS-mutated melanoma cells is highly dependent on RAF. We also show that TAK-632 induces RAF dimerization but inhibits the kinase activity of the RAF dimer, probably because of its slow dissociation from RAF. As a result, TAK-632 demonstrates potent antiproliferative effects both on NRAS-mutated melanoma cells and BRAF-mutated melanoma cells with acquired resistance to BRAF inhibitors through NRAS mutation or BRAF truncation. Furthermore, we demonstrate that the combination of TAK-632 and the MAPK kinase (MEK) inhibitor TAK-733 exhibits synergistic antiproliferative effects on these cells. Our findings characterize the unique features of TAK-632 as a pan-RAF inhibitor and provide rationale for its further investigation in NRAS-mutated melanoma and a subset of BRAF-mutated melanomas refractory to BRAF inhibitors.

Topics: Animals; Antineoplastic Agents; Benzothiazoles; Cells, Cultured; Drug Resistance, Neoplasm; Humans; MAP Kinase Signaling System; Melanoma; Mice; Mice, Nude; Nitriles; Protein Kinase Inhibitors; Proto-Oncogene Proteins B-raf; raf Kinases; Skin Neoplasms; Xenograft Model Antitumor Assays