Multiple signaling pathways converge onto the regulation of HAD-like phosphatases to modulate cellular resistance to the metabolic inhibitor 2-deoxyglucose

Cancer cells display an altered metabolism with an increased glycolysis and a lower respiration rate, leading to an increased glucose uptake. These particularities provide therapeutic opportunities and anti-cancer strategies targeting glycolysis through metabolic inhibitors have been put forth. One of these inhibitors, the glucose analogue 2-deoxyglucose (2DG), is imported into cells and phosphorylated into 2DG-6-phosphate, a toxic by-product that accumulates in cells and inhibits glycolysis. Recent data suggest that 2DG has additional effects in the cell, and resistance to 2DG has also been observed. It appears crucial to better understand the mechanisms leading to this resistance. Using budding yeast as a model system, we engaged an unbiased, mass-spectrometry-based approach to probe the cellular effects of 2DG exposure on the total proteome and reveal the molecular basis of 2DG resistance. This led to the identification of two 2DG-6-Phosphate phosphatases, Dog1 and Dog2, that are induced upon exposure to 2DG and participate in 2DG detoxification. We reveal that 2DG induces Dog2 by upregulating several signaling pathways, such as the MAPK (Hog1/p38)-based stress-responsive pathway, the Unfolded Protein Response (UPR) pathway triggered by 2DG-induced ER stress, and the MAPK (Slt2)-based Cell Wall Integrity pathway. Consequently, loss of the UPR or CWI pathways leads to hypersensitivity to 2DG. Moreover, we show that DOG2 is additionally regulated by glucose availability in a Snf1/AMPK-dependent manner through the transcriptional repressors Mig1/Mig2 and Cyc8, explaining why several mutants impaired in this pathway were found as 2DG-resistant. The isolation and characterization of spontaneous 2DG-resistant mutants revealed that DOG2 overexpression is a common strategy for 2DG resistance. Thus, 2DG-induced interference with cellular signaling rewires the expression of these endogenous phosphatases to promote 2DG resistance. Importantly, a human orthologue of Dog1/Dog2, named HDHD1, displays an in vitro 2DG-6-phosphate phosphatase activity, and its overexpression confers 2DG resistance in HeLa cells, which has important implications for potential future chemotherapies involving 2DG.