Characterization of a novel Raf kinase inhibitor that causes target dependent tumor regression in human melanoma xenografts expressing mutant B-Raf

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The Ras/Raf/MEK/ERK pathway plays a central role in mediating proliferation and survival signals from the cell membrane to the nucleus and cytoplasm. Activation of this pathway often occurs in cancer cells, the importance of which is underscored by the observation that activating mutations in Ras and B-Raf frequently occur in human cancer. Thus a potent Raf inhibitor could have a significant impact in treating cancers that are dependent on activated Ras or Raf for survival and proliferation signaling. Malignant melanoma represents a promising indication for a Raf inhibitor given the high percentage of B-Raf (60-70%) and N-Ras (10-20%) mutations and the huge unmet medical need. CHIR-265 is a novel, orally bioavailable small molecule inhibitor of c-Raf, B-Raf and mutant B-Raf (V600E) that also inhibits VEGFR-2. This target profile provides two potential mechanisms for inhibiting tumor growth: direct anti-proliferative/pro-apoptotic effects on tumor cells through inhibition of Raf, and anti-angiogenic activity through inhibition of VEGFR-2. In melanoma cells expressing mutant B-Raf (V600E), CHIR-265 inhibits MEK phosphorylation, causes cell-cycle arrest and induces apoptosis. In B-Raf mutant human melanoma tumor xenografts grown in nude mice, CHIR-265 administration caused dose-dependent inhibition of tumor growth, with tumor regression at higher doses. Western blot analysis of tumor lysates indicate that CHIR-265 treatment decreased levels of phosphorylated MEK and markers for cell-cycle and apoptosis were modulated in a way that is consistent with inhibition of the Ras/Raf/MEK/ERK pathway. In human melanoma tumor xenografts that express wild-type B-Raf, CHIR-265 also caused significant tumor growth inhibition; however, without tumor regression or evidence of Raf inhibition. In these models, efficacy did not appear to be mediated by Raf inhibition, as decreases in phospho-MEK were not apparent. It appears that the efficacy in the wild-type B-Raf models may be through an anti-angiogenic mechanism mediated by VEGFR-2 inhibition. This hypothesis is supported by data indicating that CHIR-265 inhibited VEGF-induced vascular permeability, VEGF-induced angiogenesis in Matrigel^TM plugs and decreased microvessel density in tumor xenografts. Thus, the pre-clinical data suggest that CHIR-265 has both direct anti-proliferative/pro-apoptotic activity on tumor cells and indirect anti-angiogenic activity which contribute to the efficacy. Therefore, CHIR-265 is expected to have broad-based efficacy in human cancer with selectivity for cancers with a high frequency of B-Raf mutations such as malignant melanoma.