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

Abstract

Resistance to BRAFV600E 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 BRAFV600E 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 BRAFV600E 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 BRAFV600E- or MEK-inhibitors. Here, CDK2/4 inhibition statistically significantly augmented the effects of BRAFV600E- 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 BRAFV600E inhibitor vs BRAFV600E 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 = 4–8 mice per group).