Abstract 2342: Inhibition of protein kinase C delta induces apoptosis through JNK-H2AX pathway in melanoma.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Metastatic melanoma is the major cause of skin cancer death, and the annual incidence of melanoma continues to increase. Despite the impressively high rates of response to BRAF inhibitors in patients with melanomas harboring BRAF mutations, most of these patients eventually relapse after developing resistance to the drug, due in part to secondary mutations in NRAS. Although NRAS mutation is the second most common genetic mutation in melanoma patients (after BRAF mutation), there is currently no treatment option that targets NRAS-mutated melanomas. New targeted therapies are urgently needed. Our previous reports have demonstrated the sensitivity of pancreatic cancer cell lines carrying oncogenic KRAS mutations to apoptosis initiated by inhibition of protein kinase C delta (PKCd), suggesting that PKCd inhibitors might be effective in melanomas with primary or acquired NRAS mutations. PKCd is not required for normal mammalian development, making it a potential tumor-specific target. In this study, we investigated the effect of PKCd inhibition, and the efficacy of a new PKCd inhibitor BJE6-106 (B106), in melanoma. Inhibition of PKCd by B106 (at nano-molar concentrations), or by siRNA, inhibited the growth of multiple melanoma cell lines carrying NRAS mutations, and induced apoptosis mediated by terminal caspase activation. Analysis of the molecular mechanisms involved in this NRAS signaling-targeted approach demonstrated activation of the JNK pathway after PKCd inhibition, leading to the activation (phosphorylation) of H2AX, a histone H2A variant. Activation of H2AX was attenuated when JNK1/2 levels were repressed, indicating that H2AX activation is mediated by the JNK pathway in response to PKCd inhibition. Although the phosphorylation of H2AX has in the past been primarily known for its role in repair of DNA double strand breakage, more recent studies have proposed an active role for phospho-H2AX in the induction of apoptosis. Consistent with these reports, knockdown of H2AX prior to inhibition of PKCd mitigated the induction of caspase-dependent apoptosis in NRAS mutant melanoma cell lines. To explore the potential activity of new PKCd inhibitors in inhibitor-resistant BRAF-mutant tumors, we derived melanoma cell lines harboring BRAF mutations that evolved resistance to a BRAF inhibitor PLX4032 (vemurafenib). B106 effectively induced cytotoxicity in these cells, suggesting the potential clinical application of targeting PKCd in patients who have relapsed following treatment with PLX4032. Taken together, our present study suggests that inhibition of PKCd causes caspase-dependent apoptosis in melanomas with NRAS mutations and in PLX4032-resistant BRAF mutant melanomas. This apoptosis is mediated via activation of the JNK-H2AX pathway, which involves a novel role for phospho-H2AX in the execution of apoptosis. Citation Format: Asami Takashima, Douglas V. Faller. Inhibition of protein kinase C delta induces apoptosis through JNK-H2AX pathway in melanoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2342. doi:10.1158/1538-7445.AM2013-2342