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

Abstract

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 (PKCδ). Here, we report that PKCδ inhibition is cytotoxic in melanomas with primary NRAS mutations. Novel small-molecule inhibitors of PKCδ were designed as chimeric hybrids of two naturally-occurring PKCδ inhibitors, staurosporine and rottlerin. The specific hypothesis interrogated and validated is that combining two domains of two naturally-occurring PKCδ inhibitors into a chimeric or hybrid structure retains biochemical and biological activity, and improves PKCδ isozyme selectivity. We have devised a potentially general synthetic protocol to make these chimeric species using Molander trifluorborate coupling chemistry. Inhibition of PKCδ, by siRNA or small molecule inhibitors, suppressed the growth of multiple melanoma cell lines carrying NRAS mutations, mediated via caspase-dependent apoptosis. Following PKCδ 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 PKCδ inhibition mitigated the induction of caspase-dependent apoptosis. Furthermore, PKCδ 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 PKCδ in patients who have relapsed following treatment with BRAF inhibitors. Taken together, the present work demonstrates that inhibition of PKCδ by novel small molecule inhibitors causes caspase-dependent apoptosis mediated via the JNK-H2AX pathway in melanomas with NRAS mutations or BRAF inhibitor-resistance.

Mechanisms of Acquired BRAF Inhibitor Resistance in Melanoma: A Systematic Review

“…A considerable number of preclinical studies are investigating other novel targets for overcoming BRAF inhibitor resistance. These include combining BRAF and/or MEK inhibitors with inhibitors of pre-mRNA splicing (to counteract resistance caused by BRAF splicing) [ 251 ], BH3-mimetics [ 252 , 253 ], BCL2 inhibitors [ 254 ], mitochondrial-targeted agents [ 255 , 256 ], inhibitors of p90 ribosomal S6 kinases [ 257 , 258 ], pro-caspase activating compounds [ 259 ], Rho kinase 1 (ROCK1) inhibitors [ 260 ], protein kinase Cδ inhibitors [ 261 ], tubulin inhibitors [ 262 ], ErbB2 or ErbB3 inhibitors [ 222 , 263 , 264 ], activators of the liver-X nuclear hormone receptor [ 265 ], an antibody conjugate targeting the endothelin B receptor [ 266 ], monoclonal antibodies against chondroitin sulfate proteoglycan 4 [ 267 ], inhibitors of sterol regulator element binding protein I (SREBP-1) [ 268 ], copper chelators [ 269 ], polo-like 3 kinase inhibitors (including in models of BRAF + MEK inhibitor resistance) [ 270 , 271 ], anti-nodal antibodies [ 272 ], PAK1 inhibitors [ 273 ], GLI1/2 inhibitors [ 274 ], inhibitors of IQ motif-containing GTPase activating protein 1 (IQGAP1) [ 275 ], serotonin agonists [ 276 ], CK2 inhibitors [ 277 ], p53 activators [ 278 ], metformin [ 279 ], statins [ 280 ], non-steroidal anti-inflammatory drugs [ 281 ], mibefradil [ 282 ], hydroxychloroquine (an autophagy inhibitor) [ 83 ], and A100 (a reactive oxygen species-activated prodrug) [ 283 ].…”

Multifunctional roles of PKCδ: Opportunities for targeted therapy in human disease

“…However, it was ultimately demonstrated that rottlerin could inhibit multiple kinases and was not selective for PKCδ (Soltoff, 2007). Recently, Faller and Williams et al used computational pharmacophore modeling of the interactions of rottlerin and staurosporine with the kinase domains of PKC to develop novel PKCδ selective inhibitors (Chen et al, 2011, 2014; Takashima et al, 2014). These chimeric hybrid inhibitors were designed to retain the features of rottlerin that give it the apparent specificity over other PKC isozymes, while retaining features of staurosporine (and other general inhibitors) that make it a potent inhibitor.…”

“…These chimeric hybrid inhibitors were designed to retain the features of rottlerin that give it the apparent specificity over other PKC isozymes, while retaining features of staurosporine (and other general inhibitors) that make it a potent inhibitor. The third-generation compound (BJE106) targets PKCδ with an IC 50 of 50 nM and shows a 1000-fold selectivity for PKCδ over PKCα (IC 50 50 μM) (Takashima et al, 2014). These inhibitors were effective in suppressing growth of NRAS mutant melanoma cell lines, and this was mediated by caspase-dependent apoptosis.…”

“…These inhibitors were effective in suppressing growth of NRAS mutant melanoma cell lines, and this was mediated by caspase-dependent apoptosis. Furthermore, they showed that these inhibitors were also effective in inducing cell death in BRAF-mutant melanomas that had evolved resistance to BRAF inhibitors, suggesting a potential clinical application (Takashima et al, 2014). …”

NRAS Mutant Melanoma: An Overview for the Clinician for Melanoma Management

“…Clinical benefit was seen in 8 out of 11 patients (73%) with NRAS mutant melanoma treated with PD-1/PD-1L therapy compared with 13 out of 37 (35%) in the non-NRAS mutant group (WT and BRAF mutant). The proportion of PD-L1-positive cells was also higher in NRAS mutant melanoma samples (from PI3K/mTOR + BRAF + MEK Omipalisib (GSK2126458) PI3K/mTOR (with dabrafenib + trametinib) [111] PKC delta B106 Novel PKC delta inhibitors demonstrated activity in NRAS melanoma cell lines and triggers apoptosis (B106, most selective and potent) [112] c-MET PHA665752 c-Met phosphorylation elevated in NRAS melanoma primary tumors [113] RAF PRi Enhanced efficacy of combination pan-RAF inhibitor (Amgen) with MEK inhibitor (trametinib) in NRAS melanoma cell lines [114] RAF PLX7904 Novel Raf inhibitors -PLX7904 showed activity in BRAF mutant melanoma with a secondary NRAS Q61 resistance mutation [115] ERK SCH772984 ERK 1/2 inhibitor, SCH772984, has single agent activity in both BRAF and NRAS mutant melanoma [116] Multitarget Amuvatinib (MP-470) Multitarget kinase inhibitor (activity against c-Kit, Axl, PDGFRA, Rad61) inhibited growth of NRAS (but not BRAF) mutant melanoma cell lines [117] HSP90 XL888 HSP90 inhibitor XL888 downregulated Wee1, AKT, and CDK4 in NRAS mutant melanoma in vitro and in vivo [118] GRM1 + mTOR Riluzole (GRM1 -metabotropic glutamate receptor 1 inhibitor) with mTOR inhibitor slowed melanoma growth regardless of BRAF status [119] MEK + TBK1 Overexpression of TBK1 (an effector of the RAS-RAL pathway) promoted invasive behavior and inhibition/depletion of TBK1 decreased invasive features, which was enhanced with a MEK inhibitor [120] mTOR + Wee1 MK-1775 + Torin 1 Wee1 inhibition potentiated inhibition of mTOR in NRAS mutant leukemia and melanoma [121] MEK/ERK + ROCK GSK269962A + trametinib MEK inhibitor (or ERK inhibitor) + ROCK inhibitor resulted in decreased proliferation and cell death in vitro [122] MEK + Plk1 JTP-74057 + BI 6727 Plk1 overexpressed in NRAS melanoma. MEK and Plk1 inhibition demonstrated activity in vitro and in xenograft model of NRAS melanoma (MEK inhibitor -JTP-74057 and Plk1 inhibitor -BI 6727 [123] MEK PD325901 GAB2 induces angiogenesis in NRAS mutant melanoma (and is sensitive to MEK inhibition) [124] Akt/NF-kB BI-69A11 BI-69A11 (inhibitor of Akt/sphingosine kinase-NF-kB) Inhibited tumor growth in a murine model of NRAS melanoma [125] MEK Trametinib + metformin Combination metformin and trametinib decreased NRAS melanoma growth in vitro and in xenograft model [126] an independent cohort of archived samples), although this result was not statistically significant.…”