ERK signaling plays an essential role in the IFNg-mediated apoptosis of melanoma cells. [RNAseq]

Interferon-gamma (IFNg) exerts potent growth inhibitory (antiproliferative/pro-apoptotic) effects on a wide range of tumors, including melanoma. While several terminal effectors of cell cycle arrest and apoptosis have been identified, a definitive account of the signaling pathway leading to these events is lacking. We set up a chemical genomics screen and a whole genome targeting CRISPR/Cas9 screen in a growth inhibition (GI)-sensitive patient-derived melanoma (PDM) cell line. Drug screening revealed that treatment with RAF and ERK inhibitors disrupted IFNg GI in PDM cells. For the CRISPR screen, the gene-level analysis showed that the magnitude of enrichment for guide RNAs targeting ERK2 in IFNg-treated cells was comparable to core IFNg signaling proteins like IFNGR2, JAK1, JAK2, and STAT1. We validated the involvement of ERK by detecting a sustained increase in phospho-ERK1/2 (p-ERK) levels following IFNg treatment. In live imaging experiments, we found that an ERK inhibitor (ERKi) blocks the induction of cell death in 17 of 23 (~74%) IFNg-sensitive PDM lines covering all the MAPK mutant and triple wildtype molecular subtypes. Through gene expression analysis, we found that IFNg-ERK signaling triggered the expression of stress response genes and subsequent upregulation of DR5 and NOXA, which were both critical for the induction of cell death. In summary, the IFNg-ERK signaling axis mediates cell death downstream of IFNg treatment in melanoma cells. Our results provide a new understanding of the IFNg-mediated GI pathway that will also be crucial to define mechanisms of GI resistance in tumor cells. Overall design: We performed gene expression analysis in order to understand which pathways downstream of ERK activation were involved in inducing cell death. To do this, we stimulated M230 and M238 cells with IFNg in the presence or absence of ERKi for 24 hours resulting in 4 treatments per line: DMSO, DMSO + IFNg, ERKi, and ERKi + IFNg. Ulixertinib was used as the ERKi at 6 µM and IFNg treatment was at 100 U/ml. RNAseq data was generated from 2 experiments per line.