5-Azacitidine Induces NOXA to Prime AML Cells for Venetoclax-mediated Apoptosis.

Purpose: Patients with acute myeloid leukemia (AML) frequently do not respond to conventional therapies. Leukemic cell survival and treatment resistance have been attributed to the overexpression of B-cell lymphoma 2 (BCL-2) and aberrant DNA hypermethylation. In a phase Ib study in elderly patients with AML, combining the BCL-2 selective inhibitor venetoclax with hypomethylating agents 5-azacitidine (5-Aza) or decitabine resulted in 67% overall response rate; however, the underlying mechanism for this activity is unknown. Experimental Design: We studied the consequences of combining two therapeutic agents, venetoclax and 5-Aza, in AML preclinical models and primary patient samples. We measured expression changes in the integrated stress response (ISR) and the BCL-2 family by Western blot and qPCR Subsequently, we engineered PMAIP1 (NOXA)- and BBC3 (PUMA)-deficient AML cell lines using CRISPR-Cas9 methods to understand their respective roles in driving the venetoclax/5-Aza combinatorial activity. Results: In this study, we demonstrate that venetoclax and 5-Aza act synergistically to kill AML cells in vitro and display combinatorial antitumor activity in vivo. We uncover a novel nonepigenetic mechanism for 5-Aza-induced apoptosis in AML cells through transcriptional induction of the proapoptotic BH3-only protein NOXA. This induction occurred within hours of treatment and was mediated by the ISR pathway. NOXA was detected in complex with antiapoptotic proteins, suggesting that 5-Aza may be "priming" the AML cells for venetoclax-induced apoptosis. PMAIP1 knockout confirmed its major role in driving venetoclax and 5-Aza synergy. Conclusions: These data provide a novel nonepigenetic mechanism of action for 5-Aza and its combinatorial activity with venetoclax through the ISR-mediated induction of PMAIP1.