EVALUATION OF 15dPMJ2 CYTOTOXICITY AND GASDERMIN E-MEDIATED PYROPTOTIC CELL DEATH IN BRAF MUTANT MELANOMA CELLS

Abstract

Melanoma is the leading cause of death from skin disease in the United States. The American Cancer Society estimates nearly 234,680 new melanomas will be diagnosed in 2026. Immune checkpoint inhibitors and targeted therapies against oncogenic BRAF mutations are commonly used to manage unresectable metastatic melanoma tumors; however, approximately half of patients are primary non-responders, or develop resistance to these therapies and survive less than 5 years after diagnosis. Thus, additional options for melanoma treatment are needed. 15-deoxy-Δ12,14-prostamide J2 (15dPMJ2) is a novel small molecule therapeutic shown to reduce the viability of melanoma cells in vitro, and to reduce tumor burden in vivo. However, BRAF mutant cell lines were not previously evaluated against 15dPMJ2. Here, we report that 15dPMJ2 significantly reduces viability at low micromolar concentrations in this cell type and was found to induce markers of apoptosis and endoplasmic reticulum stress. Emerging evidence suggests that caspase-3 activation can promote pyroptosis, a secondary lytic cell death pathway. Consistent with pyroptotic signaling, our results demonstrated enriched GSDME cleavage, membrane permeabilization, and HMGB1 release following treatment with 15dPMJ2. We also evaluated the combination of 15dPMJ2 with clinically approved MAPK inhibitors dabrafenib and trametinib, however, the triple combination did not yield synergistic effects. Furthermore, we identified molecular targets of 15dPMJ2 by conducting discovery proteomic analysis in B16F10 mitochondrial extracts. We report that 15dPMJ2 forms an adduct with PGC-1α, a transcriptional co-activator that stimulates gene expression, mitochondrial biogenesis and respiration. Altogether, these results demonstrate that 15dPMJ2 exerts anti-melanoma activity through reduction of cell viability and prompting markers of apoptotic and pyroptotic signaling pathways in BRAF-mutant melanoma cells. Additionally, data identify PGC-1α to form a drug- protein adducts in B16F10 mitochondrial samples following treatment with 15dPMJ2.