Poster Presentation 32nd Lorne Cancer 2020

Recurrent and metastatic cancer often undergo a period of dormancy, which is closely associated with cancer cell quiescence, a state whereby cancer cells exit the cell cycle and are reversibly arrested in G0 phase. Quiescent cancer cells are inherently resistant to cell death and refractory to therapeutic drugs. Nevertheless, the mechanisms responsible for this resistance remain largely undefined. This is related to technical difficulties associated with purifying and characterizing quiescent cancer cells. Here we show that dual genome-editing of endogenous p27 and Ki67 with green and red fluorescent proteins, respectively, allows for visualization, isolation and analysis of viable p27^highKi67^low quiescent cancer cells. By use of CRISPR-Cas9 genome editing, we introduced sequences encoding EGFP at the endogenous locus of CDKN1B, the gene encoding p27, and sequences encoding mCherry at the endogenous locus of MKI67 into Mel-RM melanoma cells. Purified EGFP-p27^highmCherry-Ki67^low cells were diploid, contained low RNA content, exhibited minimal levels of phosphorylated retinoblastoma protein, cyclin D1 and phosphorylated cyclin-dependent kinase 2, corroborating their quiescent state. Moreover, gene set enrichment analysis (GSEA) of transcriptomes showed that the E2F1 and G2/M progression pathways were strongly decreased in purified EGFP-p27^highmCherry-Ki67^low cells. Strikingly, the oxidative phosphorylation (OXPHOS) pathway was markedly increased in purified EGFP-p27^highmCherry-Ki67^low cells. In accordance, the oxygen consumption rate (OCR) and mitochondrial oxidative stress were increased, whereas the cellular lactate levels were reduced in quiescent compared with proliferating cells, indicating that metabolic switching towards OXPHOS is a biological trait of melanoma cells in quiescence. Demonstrating the functional importance of the increased OXPHOS activity, purified EGFP-p27^highmCherry-Ki67^low quiescent cells were markedly more sensitive to killing by treatment with the complex I inhibitor, ICAS-010759, and the proton ionophore, 2,4-dinitrophenol (DNP). Thus, targeting OXPHOS to kill quiescent melanoma cells may constitute a useful strategy in combination with other therapeutic agents that predominantly affect proliferating cells towards curative treatment of the disease.