Dual Suppression of the Cyclin-Dependent Kinase Inhibitors CDKN2C and CDKN1A in Human Melanoma

Resistance to BRAF(V600E) inhibitors is associated with reactivation of mitogen-activated protein kinase (MAPK) signaling at different levels in melanoma. To identify downstream effectors of MAPK signaling that could be used as potential additional therapeutic targets for BRAF(V600E) inhibitors, we used hTERT/CDK4R24C/p53DD-immortalized primary human melanocytes genetically modified to ectopically express BRAF(V600E) or NRAS(G12D) and observed induction of the AP-1 transcription factor family member c-Jun. Using a dominant negative approach, in vitro cell proliferation assays, western blots, and flow cytometry showed that MAPK signaling via BRAF(V600E) promotes melanoma cell proliferation at G1 through AP-1-mediated negative regulation of the INK4 family member, cyclin-dependent kinase inhibitor 2C (CDKN2C), and the CIP/KIP family member, cyclin-dependent kinase inhibitor 1A (CDKN1A). These effects were antagonized by pharmacological inhibition of CDKN2C and CDKN1A targets CDK2 and CDK4 in vitro. In contrast to BRAF(V600E) or NRAS(G12D)-expressing melanocytes, melanoma cells have an inherent resistance to suppression of AP-1 activity by BRAF(V600E)- or MEK-inhibitors. Here, CDK2/4 inhibition statistically significantly augmented the effects of BRAF(V600E)- or MEK-inhibitors on melanoma cell viability in vitro and growth in athymic nude Foxn1(nu) mice (P .03 when mean tumor volume at day 13 was compared for BRAF(V600E) inhibitor vs BRAF(V600E) inhibitor plus CDK2/4 inhibition; P .02 when mean tumor volume was compared for MEK inhibitor vs MEK inhibitor plus CDK2/4 inhibition; P values were calculated by a two-sided Welch t test; n 48 mice per group).