Mutations in BRAF a serine/threonine protein kinase are frequently observed in human cancers. A valine to glutamic acid mutation at amino acid position 600 accounts for nearly 80% of all the activating mutations found for this kinase. This mutation (V600E) forces BRAF to become constitutively active and phosphorylate its substrates regardless of upstream signaling. Numerous cancers in tissues such as skin, colon, thyroid, and ovary have been attributed to uncontrolled proliferation driven by this mutation. TAK-733 represents a novel and distinct chemotype which is capable of allosteric inhibition of the BRAF substrates MEK-1 and MEK-2 at low nanomolar concentrations. Viability assays in established human cancer cell lines which bear the BRAF V600E mutation demonstrate this compound was also capable of lowering NADH levels with single digit nanomolar EC50s. Loss of NADH production can be attributed to the induction of programmed cell death pathways involving Caspases 3 and 7. Induction of these apoptosis markers was confirmed in vitro by a Caspase-GLO assay. TAK-733 induces apoptosis more potently in COLO 205 (BRAF V600E) human colo-rectal carcinoma cells when compared to other MEK inhibitors. Pharmacodynamic analysis of COLO 205 tumor bearing mice confirmed TAK-733, unlike other MEK inhibitors tested, is capable of inducing apoptosis 36 hours after a single oral administration. To determine whether the anti-tumor effect of TAK-733 is associated with apoptosis, A375 (BRAF V600E) human melanoma tumors were harvested at sacrifice and protein extracted to determine cleavage products of PARP. A single oral treatment with 1, 10, and 30 mg/kg TAK-733 increased protein expression of cleaved PARP in a dose and time dependent manner. Elevated cleaved PARP protein expression was detected as early as 0.5-hours in tumors following 30 mg/kg dose and detectable at all groups by 4-hours. The increase cleaved PARP protein levels persisted for at least 24-hours and returned to control expression levels by 48-hours. Daily oral administration of 1, 10, and 30 mg/kg/day TAK-733 for 14 days potently inhibited tumor growth resulting in tumor growth delay of 3.4, 10.8, and 26.6 days in A375 cell implanted mice. A 60% partial regression response rate (tumor volume reduced by more than 50% of initial volume) was observed in mice that received 30 mg/kg/day TAK-733. These data suggest a possible explanation as to why TAK-733 is capable of inducing regressions in COLO 205 and A375 tumor growth inhibition studies, and may represent a differentiation point from other MEK inhibitors currently in clinical trials.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2518.