Prednisolone Resistance in Pediatric Acute Lymphoblastic Leukemia Can Be Synergistically Overcome by Inhibition of Anti-Apoptotic MCL1 and Glycolysis

Abstract 3528 Background: Unsuccessful treatment of pediatric precursor B acute lymphoblastic leukemia (ALL) can be ascribed to cellular resistance to antileukemic drugs. In particular, resistance towards prednisolone is associated with poor prognosis in pediatric ALL. For three reasons, we hypothesized that anti-apoptosis sustained by the BCL2 family member MCL1 and glycolysis are linked processes and concomitantly induce resistance to prednisolone: 1) Glycolysis and apoptosis are closely related survival pathways both associated with prednisolone resistance, 2) Increased glucose metabolism has been directly linked to MCL1 stabilization and attenuation of apoptosis, and 3) BCL2 family members can adjust oxidative phosphorylation, a process that together with anaerobic glycolysis, is responsible for cellular respiration and ATP production. In this study, we functionally determined the synergistic contribution of MCL1 and glycolysis to prednisolone resistance in childhood ALL. Methods: Leukemic cells of pediatric ALL patients, >90% blasts, were treated in vitro with prednisolone for 48 hours. Changes in MCL1 protein levels were measured by reverse phase protein array. MCL1 knockdown was achieved by locked nucleic acid oligonucleotides (LNAs) and lentiviral silencing in two different prednisolone resistant leukemic cell lines, and the effect was assessed with RTQ-PCR and Western blot. Cell viability and cell count were analyzed by MACSQuant. Glucose consumption was measured using the GAGO20 glucose assay, in which glucose is oxidized to form the spectrophotometric end-product Oxidized o-Dianisidine. 2-deoxyglucose (2DG) was used to inhibit glycolysis. Cytotoxicity of prednisolone in leukemic cells was determined by the in vitro 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) drug-resistance assay. Sensitivity and resistance to prednisolone was assessed using previously established LC50 cut-off values, shown to be linked to the prognosis of patients. Results: MCL1 protein expression decreased by 2.9-fold after in vitro prednisolone treatment in prednisolone sensitive patients' leukemic cells (p<0.001). In contrast, MCL1 protein expression increased in prednisolone resistant ALL patient cells by maximum 2.3-fold (p<0.01). Three MCL1 LNA oligonucleotides efficiently diminished MCL1 protein levels by 82±16% compared to MCL1 levels measured in non-silencing control cells (p<0.05). This decrease was similar to the reduction by 72±12% seen for 2 lentivirally delivered shMCL1 (p<0.05). Silencing of MCL1 decreased leukemic cell proliferation by 9-fold and sensitized leukemic cells to prednisolone by maximum 80-fold (p<0.05). MCL1 silencing by either MCL1 LNA or shMCL1 upregulated the glucose consumption of leukemic cells by 2.5-fold (p<0.05), indicating a rescue mechanism mediated by anaerobic glycolysis. Inhibition of the anaerobic glycolysis by 2-DG diminished the proliferation rate of MCL1-silenced cells by 3.9-fold compared to MCL1-silenced cells alone (p<0.05). Most importantly, the combination of 2DG and silencing of MCL1 synergistically sensitized to prednisolone by 33±16% compared to the prednisolone response of leukemic cells treated with 2DG or MCL1 LNA alone (p<0.05, n=3). Conclusion: MCL1 is a potent target to sensitize to prednisolone in pediatric ALL. However, MCL1-silenced cells increase anaerobic glycolysis to avoid prednisolone-induced apoptosis. MCL1 and glycolysis should therefore be targeted simultaneously to effectively and synergistically reverse prednisolone resistance in ALL. Disclosures: No relevant conflicts of interest to declare.