Thesis (Ph.D.)--Boston UniversityMetastatic 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. Previous reports have demonstrated the sensitivity of cancer cell lines carrying RAS mutations to apoptosis initiated by inhibition of protein kinase C delta (PKCδ), suggesting the possible association between RAS mutational status and sensitivity to PKCδ inhibition. I therefore hypothesized that PKCδ inhibitors might also be cytotoxic in melanomas with primary or acquired NRAS mutations. In this project, the effect of PKCδ inhibition, and the efficacy of a new PKCcS inhibitor, BJE6-106 (B106), in melanoma were investigated. Inhibition of PKCδ inhibited the growth of multiple human melanoma cell lines carrying NRAS mutations, and induced apoptosis mediated by terminal caspase activation. Analysis of the molecular mechanisms demonstrated activation of the JNK pathway after PKCδ 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 PKCδ inhibition. Consistent with recent reports on the apoptotic role of phospho-H2AX, knockdown of H2AX prior to PKCδ inhibition mitigated the induction of caspase-dependent apoptosis. To explore the potential of B106 further, melanoma cell lines harboring BRAF mutations that had evolved resistance to a BRAF inhibitor, PLX4032 (vemurafenib), were developed. B106 effectively induced cytotoxicity in these cells, suggesting the potential clinical application of targeting PKCδ in patients who have relapsed following treatment with PLX4032. Taken together, this work suggests that inhibition of PKCδ 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
