Simultaneous Inhibition of Ceramide Hydrolysis and Glycosylation Synergizes to Corrupt Mitochondrial Respiration and Signal Caspase Driven Cell Death in Drug-Resistant Acute Myeloid Leukemia.

Simple Summary: The sphingolipid ceramide is a key player in cytotoxic responses elicited by many anticancer drugs. Confounding this asset, however, are enzymes that promote ceramide clearance, thus limiting the propagation of ceramide-driven cancer cell death. Because several key ceramide-metabolizing enzymes have been shown to be upregulated in chemotherapy-resistant acute myeloid leukemia (AML) cells compared to chemotherapy-naïve counterparts, the current study was designed to determine the effects of blocking ceramide clearance in drug-resistant AML. For this, we employed simultaneous inhibition of ceramide hydrolysis and ceramide glycosylation and demonstrated that this dual blockade produced multi-fold elevations in intracellular ceramide levels, corrupted mitochondrial function, enhanced caspase activation, and elicited cell death in models of drug-resistant AML. We have herein identified sphingolipid metabolic junctures that can be targeted to enhance leukemia cell vulnerability in the drug-resistant setting, thus providing a novel therapeutic modality in difficult-to-treat cancers. Acute myelogenous leukemia (AML), the most prevalent acute and aggressive leukemia diagnosed in adults, often recurs as a difficult-to-treat, chemotherapy-resistant disease. Because chemotherapy resistance is a major obstacle to successful treatment, novel therapeutic intervention is needed. Upregulated ceramide clearance via accelerated hydrolysis and glycosylation has been shown to be an element in chemotherapy-resistant AML, a problem considering the crucial role ceramide plays in eliciting apoptosis. Herein we employed agents that block ceramide clearance to determine if such a "reset" would be of therapeutic benefit. SACLAC was utilized to limit ceramide hydrolysis, and D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (D-threo-PDMP) was used to block the glycosylation route. The SACLAC D-threo-PDMP inhibitor combination was synergistically cytotoxic in drug-resistant, P-glycoprotein-expressing (P-gp) AML but not in wt, P-gp-poor cells. Interestingly, P-gp antagonists that can limit ceramide glycosylation via depression of glucosylceramide transit also synergized with SACLAC, suggesting a paradoxical role for P-gp in the implementation of cell death. Mechanistically, cell death was accompanied by a complete drop in ceramide glycosylation, concomitant, striking increases in all molecular species of ceramide, diminished sphingosine 1-phosphate levels, resounding declines in mitochondrial respiratory kinetics, altered Akt, pGSK-3β, and Mcl-1 expression, and caspase activation. Although ceramide was generated in wt cells upon inhibitor exposure, mitochondrial respiration was not corrupted, suggestive of mitochondrial vulnerability in the drug-resistant phenotype, a potential therapeutic avenue. The inhibitor regimen showed efficacy in an in vivo model and in primary AML cells from patients. These results support the implementation of SL enzyme targeting to limit ceramide clearance as a therapeutic strategy in chemotherapy-resistant AML, inclusive of a novel indication for the use of P-gp antagonists. [ABSTRACT FROM AUTHOR]