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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.

The mitotic kinase Aurora A is an important therapeutic target for cancer therapy. This study evaluated new mechanism-based pharmacodynamic biomarkers in cancer patients in two phase I studies of MLN8054, a small-molecule inhibitor of Aurora A kinase. Patients with advanced solid tumors received MLN8054 orally for 7 consecutive days in escalating dose cohorts, with skin and tumor biopsies obtained before and after dosing. Skin biopsies were evaluated for increased mitotic cells within the basal epithelium. Tumor biopsies were assessed for accumulation of mitotic cells within proliferative tumor regions. Several patients in the highest dose cohorts showed marked increases in the skin mitotic index after dosing. Although some tumors exhibited increases in mitotic cells after dosing, others displayed decreases, a variable outcome consistent with dual mechanisms of mitotic arrest and mitotic slippage induced by antimitotics in tumors. To provide a clearer picture, mitotic cell chromosome alignment and spindle bipolarity, new biomarkers of Aurora A inhibition that act independently of mitotic arrest or slippage, were assessed in the tumor biopsies. Several patients, primarily in the highest dose cohorts, had marked decreases in the percentage of mitotic cells with aligned chromosomes and bipolar spindles after dosing. Evidence existed for an exposure-effect relationship for mitotic cells with defects in chromosome alignment and spindle bipolarity that indicated a biologically active dose range. Outcomes of pharmacodynamic assays from skin and tumor biopsies were concordant in several patients. Together, these new pharmacodynamic assays provide evidence for Aurora A inhibition by MLN8054 in patient skin and tumor tissues.

Aurora A is a serine/threonine protein kinase essential for normal mitotic progression. Aberrant increased expression of Aurora A, which occurs frequently in human cancers, results in abnormal mitoses leading to chromosome instability and possibly tumorigenesis. Consequently, Aurora A has received considerable attention as a potential target for anticancer therapeutic intervention. Aurora A coordinates several essential mitotic activities through phosphorylation of a variety of proteins, including TACC3, which modulates microtubule stabilization of the mitotic spindle. Recent studies identified a conserved serine in Xenopus (Ser(626)) and Drosophila (Ser(863)) TACC3 orthologues that is phosphorylated by Aurora A. We show that this conserved serine on human TACC3 (Ser(558)) is also phosphorylated by Aurora A. Moreover, phosphorylation of TACC3 by Aurora A in human cells is essential for its proper localization to centrosomes and proximal mitotic spindles. Inhibition of Aurora A with the selective small molecule inhibitor MLN8054 in cultured human tumor cells resulted in mislocalization of TACC3 away from mitotic spindles in a concentration-dependent manner. Furthermore, oral administration of MLN8054 to nude mice bearing HCT-116 human tumor xenografts caused a dose-dependent mislocalization of TACC3 away from spindle poles that correlated with tumor growth inhibition. As TACC3 localization to mitotic spindles depends on Aurora A-mediated phosphorylation, quantifying TACC3 mislocalization represents a novel pharmacodynamic approach for measuring Aurora A activity in cancer patients treated with inhibitors of Aurora A kinase.