Activation of the Ras-Raf-MEK-ERK pathway plays an integral role in tumor cell proliferation and survival. Mutations in Ras and B-Raf have been reported in many human cancers, making these proteins attractive targets for cancer therapies. The pathway can also be activated by overexpression or constitutive activation through mutations of upstream modulators, such as receptor tyrosine kinases (RTK). The recent successes of RTK inhibitors as targeted cancer therapeutics provides proof of concept for the inhibition of signaling through Ras-Raf-MEK-ERK as a means of providing clinical benefit to cancer patients. We have identified CHIR-265 as a novel, orally bioavailable, potent inhibitor of mutant and wild-type B-Raf as well as c-Raf. In biochemical and cell-based assays, CHIR-265 also exhibits activity against VEGFR2. To further define CHIR-265 mechanism of action, we characterized its activity in cell lines where the MAPK pathway is activated by different mechanisms. We measured phospho-MEK and phospho-ERK as endpoints of Raf inhibition in cell lines harboring a B-Raf, an N-Ras or a K-Ras mutation, an upstream receptor tyrosine kinase activation (RTK), or no known activation. CHIR-265 showed the most potent activity in cells lines carrying a B-Raf V600E mutation, and significant inhibition was also seen in cells expressing mutant Ras. In contrast, target modulation was not detected in cells where neither Ras nor B-Raf is mutated. These observations were extended to xenograft tumor models of differing genotypes. CHIR-265 exhibited potent anti-tumor activity in a colorectal xenograft models harboring a B-Raf (HT29) or a K-Ras (HCT116) mutation. This was accompanied by down-modulation of phospho-MEK and phospho-ERK as well as changes in downstream markers associated with induction of apoptosis and cell cycle arrest. Anti-tumor activity was also seen in a tumor model driven by RTK activation (MV4;11 AML with FLT3 ITD mutation). However, this activity was not associated with a decrease in Raf activity as measured by phospho-MEK, suggesting that this anti-tumor effect may have been mediated through another target of CHIR-265. The activity of CHIR-265 against VEGFR has been demonstrated in vitro and in vivo, supporting the hypothesis that part of the anti-tumor activity is through inhibition of angiogenesis. In summary, CHIR-265 exhibited efficacy in all tumor models evaluated. It was most potent in models expressing either mutant B-Raf or mutant Ras, where target inhibition was associated with tumor regressions. Efficacy was also observed against tumors that are wild type for B-Raf and Ras; the anti-tumor activity in these models appears to be due to the anti-angiogenic activity. These results provide a rationale for the clinical evaluation of CHIR-265 in cancers where Ras or B-Raf are mutated, as well as those with a strong angiogenic component.

[Proc Amer Assoc Cancer Res, Volume 47, 2006]