CRISPR/Cas9 screen identifies oxidative phosphorylation as essential for cancer cell survival at low extracellular pH

Unlike most cell types, many cancer cells survive at low extracellular pH (pHe), a chemical signature of tumors. Genes that facilitate survival under acid stress are therefore potential targets for cancer therapies. We performed a genome-wide CRISPR/Cas9 cell viability screen at physiological and acidic conditions to systematically identify gene knockouts associated with pH-related fitness defects in colorectal cancer cells. Knockouts of genes involved in oxidative phosphorylation (NDUFS1) and iron-sulfur cluster biogenesis (IBA57, NFU1) grew well at physiological pHe, but underwent profound cell death under acidic conditions. We identified several small-molecule inhibitors of mitochondrial metabolism that can kill cancer cells at low pHe only. Xenografts established from NDUFS1-/- cells grew considerably slower than their wild-type controls, but growth could be stimulated with systemic bicarbonate therapy which lessens the tumoral acid stress. These findings raise the possibility of therapeutically targeting mitochondrial metabolism in combination with acid stress as a cancer treatment option. Overall design: CRISPR screen. SW480 cells were infected with TKO v3 virus library with a target MOI of 0.3 After puromycin selection, 3 million cells per 15 cm-dish were seeded and were incubated with 32 mL medium (NaHCO3-free DMEM D7777) containing either 22 mM, 5.5 mM or 2.75 mM sodium bicarbonate (equilibrated in 5% CO2 to pHe 7.4, 6.9 and 6.63) supplemented with various concentrations of NaCl to maintain constant osmolarity. The cells were exposed to different pH levels for 11 days. The screen was carried out in duplicate, independent experimental setups.