Ung, J.; Tan, S.-F.; Shaw, J. J.; Taori, M.; Deddens, T. M.; Venancio, G. d. C.; Montgomery, M. M.; Hagen, J. T.; Aruleba, R. T.; Golla, U.; Sharma, A.; Paudel, B. B.; Lee, I. S.-A.; Ramamoorthy, B.; Janes, K. A.; Garrett-Bakelman, F. E.; Cabot, M. C.; Fisher-Wellman, K. H.; Fox, T. E.; Claxton, D. F.; Chalfant, C. E.; Feith, D. J.; Loughran, T. P.

Resistance to combination regimens containing the BCL-2 inhibitor venetoclax in acute myeloid leukemia (AML) is a growing clinical challenge for this extensively utilized agent. We previously established the anti-leukemic properties of ceramide, a tumor-suppressive sphingolipid, in AML and demonstrated that upregulated expression of acid ceramidase (AC), a ceramide-neutralizing enzyme, supported leukemic survival and resistance to BH3 mimetics. Here, we report the anti-leukemic efficacy and mechanisms of co-targeting AC and BCL-2 in venetoclax-resistant AML. Analysis of the BeatAML dataset revealed a positive relationship between increased AC gene expression and venetoclax resistance. Targeting AC enhanced single-agent venetoclax cytotoxicity and the venetoclax + cytarabine combination in AML cell lines with primary or acquired venetoclax resistance. SACLAC + venetoclax was equipotent to the combination of venetoclax + cytarabine at reducing cell viability when evaluated ex vivo across a cohort of 71 primary AML patient samples. Mechanistically, SACLAC + venetoclax increased ceramide to levels that trigger a cytotoxic integrated stress response (ISR), ISR-mediated NOXA protein upregulation, mitochondrial dysregulation, and caspase-dependent cell death. Collectively, these data demonstrate the efficacy of co-targeting AC and BCL-2 in AML and rationalize targeting AC as a therapeutic approach to overcome venetoclax resistance.