Protein Kinase Cδ Is a Therapeutic Target in Malignant Melanoma with NRAS Mutation

NRAS is the second most frequently mutated gene in melanoma. Previous reports have demonstrated the sensitivity of cancer cell lines carrying KRAS mutations to apoptosis initiated by inhibition of protein kinase C delta (PKC delta). Here, we report 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 interrogated and validated is that combining two domains of two naturally occurring PKC delta inhibitors into a chimeric or hybrid structure retains biochemical and biological activity and improves PKC delta isozyme selectivity. 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. Taken together, the present work demonstrates that inhibition of PKC delta by novel small Molecule inhibitors causes caspase-dependent apoptosis mediated via the JNK-H2AX pathway in melanomas with NRAS mutations or BRAF inhibitor resistance.