Adaptation to Metabolic Stress By Mondoα in Common B-Cell Acute Lymphoblastic Leukemia

Malignant cells evolve adaptive mechanisms to survive metabolic stress to drive progression. We previously described the stress sensor MondoA as promoting malignancy of common acute lymphoblastic leukemia (cALL). MondoA knockdown (MKD) in cALL cell lines xenografted into mice reduced the number of leukemic blasts compared to control cells (Sipol 2014). Next we investigated potential mechanisms of MondoA mediated malignancy. Microarray analyses of MKD cALL lines revealed an induction of glycolytic and hypoxia response associated gene sets. To validate and expand these results we generated cALL lines with MondoA knockout (MKO) via CRISPR/cas9 gene editing. Colony forming assays and direct cell counting demonstrated reduced proliferation of MKO cells under normoxic conditions compared to controls. However, under hypoxia, there was a diminished MondoA dependent growth advantage. This suggests that MondoA proficient cells may have a selective growth advantage in the presence of oxygen, and that MondoA might confer the ability to utilize oxidative phosphorylation and thus increase metabolic activity. We therefore measured mitochondrial respiration by determining oxygen consumption rates (OCR) in control and MKO cells using an extracellular flux analyzer. Surprisingly, MKO cells displayed a higher basal OCR as well as significantly increased extracellular acidification rate (ECAR) and glycolysis. This suggested that MondoA might actually limit oxidative phosphorylation and glycolysis in response to glucose. We therefore hypothesized that MondoA might remodel cALL metabolism towards an alternative energy source. Indeed, Kegg pathway analysis of microarray data showed upregulation of fatty acid synthesis (FAS) and fatty acid oxidation (FAO) genes by MondoA. This suggested that MondoA might facilitate sustained fatty acid metabolism to maintain high proliferative rates. Key regulators of citrate to oleate conversion, fatty acid elongation and cholesterol synthesis (ACLY, ELOVL5, ELOVL6, ELOVL1, FASN, HMGCR, HMGCS1) were stimulated by MondoA. Colorimetric assays demonstrated decreased NADPH and Acetyl-CoA levels corresponding to disregulated fatty acid metabolism by MKO. In addition, ROS measurements by electron paramagnetic resonance (EPR) using the spin trap CMH in the presence or absence of NADPH oxidase NOX2 inhibitory peptide gp91ds-Tat, a readout of NOX activity, demonstrated that MKO cells increase cytoplasmic ROS production, mediated by NOX2. Moreover, MondoA depleted leukemia cells generate more ROS in glutamine-restricted media. In summary, (1) MondoA increases leukemic cell proliferation under both normoxia and hypoxia; (2) MondoA increases metabolic activity, in particular fatty acid metabolism; and (3) MondoA decreases cytoplasmic ROS production in glutamine-deprived conditions. We conclude that MondoA has multiple functions in supporting malignancy of cALL, most likely by increasing fatty acid metabolism while simultaneously providing adaptation to oxidative stress.