Acid ceramidase inhibition enhances BCL-2 targeting in venetoclax-resistant acute myeloid leukemia

Resistance to combination regimens containing the B-cell lymphoma 2 (BCL-2) inhibitor and BH3 mimetic venetoclax in acute myeloid leukemia (AML) is a growing clinical challenge for this extensively used agent. We previously established the antileukemic properties of ceramide, a tumor-suppressive sphingolipid, in AML, and demonstrated that upregulated expression of acid ceramidase (AC), a ceramide-neutralizing enzyme, supports leukemic survival and resistance to BH3 mimetics. Here, we report the antileukemic efficacy and mechanisms of cotargeting AC and BCL-2 in venetoclax-resistant AML. Analysis of the BeatAML data set revealed a positive relationship between increased AC gene expression and venetoclax resistance. Pharmacologic AC inhibition with the ceramide analog SACLAC enhanced single-agent venetoclax cytotoxicity and the venetoclax + cytarabine combination in AML cell lines with primary or acquired venetoclax resistance. SACLAC + venetoclax was synergistically lethal when evaluated ex vivo across a cohort of venetoclax-resistant (n = 21) and venetoclax-sensitive (n = 46) primary samples from patients with AML. Moreover, the SACLAC + venetoclax combination was equipotent to the combination of venetoclax + cytarabine at reducing cell viability across primary patient samples. Mechanistically, cotargeting AC and BCL-2 increased ceramide to levels that trigger a cytotoxic integrated stress response (ISR), ISR-mediated NOXA protein upregulation, mitochondrial dysregulation, and caspase-dependent cell death. Importantly, AC knockdown sensitized AML cells to venetoclax and induced NOXA protein accumulation, whereas NOXA knockdown protected against AC and BCL-2 cotargeting. Collectively, these findings demonstrate the efficacy of cotargeting AC and BCL-2, and rationalize targeting AC as a therapeutic approach for venetoclax-sensitive and -resistant AML.