Abstract B36: Targeting oncogenic RAS with small molecule PKC-delta inhibitors

Somatic point mutations of RAS genes at codons 12, 13, and 61 are the most common dominant oncogenic lesions in human cancer, making aberrant RAS signaling an important therapeutic target. Unfortunately, the oncogenic RAS/GAP switch is an exceedingly difficult target for rational drug discovery. We have developed a novel, alternative strategy to circumvent this limitation of targeting RAS directly. Mutated, constitutively-activated RAS is lethal to the tumor cell unless a survival pathway, mediated by a novel PKC isozyme, PKC-delta, is also active. This ”non-oncogene” dependency of RAS-mutant cells on PKC-delta activity can be exploited therapeutically. We have demonstrated the selective susceptibility of human pancreatic, lung, colon, ovarian and neuroendocrine tumor cells with mutant K- or HRAS alleles to PKC-delta inhibition. Importantly, unlike the classical PKC isozymes, PKC-delta is not required for the survival of normal cells, and its inhibition or down-regulation in normal cells and organisms has no adverse effects. Furthermore, we demonstrate that cancer stem-like cells derived from RAS-mutant tumor specimens and cell lines are equally or more susceptible to PKC-delta inhibition as the non-stem-like population, both in culture and in xenograft studies. There is currently no treatment option that targets melanoma with NRAS mutations, which is the second most frequently mutated gene in melanoma. We report here that PKC-delta inhibition is cytotoxic in melanomas with primary NRAS mutations. Novel small-molecule inhibitors of PKC-delta were designed as chimeric hybrids of two naturally-occurring PKC-delta inhibitors, staurosporine and rottlerin. The specific hypothesis we have interrogated and validated is the concept that combining two domains of two naturally-occurring PKC-delta inhibitors into a chimeric or hybrid structure retains biochemical and biological activity, and improves selectivity for the specific PKC-delta isozyme. We have devised a potentially general synthetic protocol to make these chimeric species using Molander trifluorborate coupling chemistry. Inhibition of PKC-delta, by siRNA or small molecule inhibitors, suppressed the growth of multiple melanoma cell lines carrying NRAS mutations, mediated via caspase-dependent apoptosis. Following PKC-delta inhibition, the stress-responsive JNK pathway was activated, leading to the activation of H2AX. Consistent with recent reports on the apoptotic role of phospho-H2AX, knockdown of H2AX prior to PKC-delta inhibition mitigated the induction of caspase-dependent apoptosis. Furthermore, PKC-delta inhibition effectively induced cytotoxicity in BRAF-mutant melanoma cell lines that had evolved resistance to a BRAF inhibitor, suggesting the potential clinical application of targeting PKC-delta in patients who have relapsed following treatment with BRAF inhibitors. Collectively, these findings demonstrate that inhibition of PKC-delta by novel small molecule inhibitors represents a novel and targeted therapeutic approach to tumors bearing mutant RAS alleles. Citation Format: Asami Takashima, Zhihong Chen, Brandon English, Robert M. Williams, Douglas V. Faller. Targeting oncogenic RAS with small molecule PKC-delta inhibitors. [abstract]. In: Proceedings of the AACR Special Conference on RAS Oncogenes: From Biology to Therapy; Feb 24-27, 2014; Lake Buena Vista, FL. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(12 Suppl):Abstract nr B36. doi: 10.1158/1557-3125.RASONC14-B36