5-Azacytidine Induces NOXA and PUMA Expression to Prime AML Cells for Venetoclax-Mediated Apoptosis
Authors: Cojocari Dan , Jin Sha , Purkal Julie J , Popovic Relja , Talaty Nari N , Xiao Yu , Solomon Larry R , Boghaert Erwin , Leverson Joel D. , Phillips Darren C. 
Published: October 2019

Abstract
Acute myeloid leukemia (AML) is a clonal hematologic malignancy characterized by genomic heterogeneity and epigenetic changes, including aberrant DNA hypermethylation. Phase-Ib clinical data in relapsed/refractory AML patients indicate that combining venetoclax with the hypomethylating agents (HMAs) 5-azacitidine (5-Aza) or decitabine results in an overall response (OR) of 62% (DiNardo et al. 2018) compared to the historical OR of 28-29% with HMAs treatment alone (Kantarjian et al. 2013; Dombret et al. 2015). Subsequently, a randomized phase-III clinical trial was initiated to evaluate venetoclax activity in combination with 5-Aza in treatment-naïve AML patients ineligible for standard induction therapy (M15-656, NCT02993523). However, the underlying mechanism for the combinational activity observed between venetoclax and 5-Aza is unknown.
In this study, we demonstrate that both chronic low-dose 5-Aza treatment, which induced global DNA demethylation, and acute treatment (24 hours, non-epigenetic effects) can drive combinational activity with venetoclax in AML through distinct mechanisms. Chronic culture with a low-dose 5-Aza for one week sensitized AML cell lines to venetoclax in vitro. In contrast, acute treatment with 5-Aza, activated the integrated stress response (ISR) pathway to induce expression of the BH3-only proteins NOXA (PMAIP1) and PUMA (BBC3) in human AML cell lines, independent of DNA methylation. This resulted in an increase in the amount of NOXA and/or PUMA in complex with anti-apoptotic proteins like BCL-2, BCL-XL and MCL-1, thereby "priming" AML cells for induction of apoptosis by venetoclax treatment. Priming for apoptosis resulted in significant synergistic cell death in a panel of AML cell lines treated with venetoclax and 5-Aza in vitro. In this panel of cell lines, the level of the PMAIP1, BBC3, and DDIT3 gene induction correlated with the synergy observed between venetoclax and 5-Aza. Importantly, subsequent PMAIP1 deletion significantly impacted the kinetics and depth of apoptosis induced by 5-Aza or venetoclax alone or in combination. In accordance with the in vitro combinational activity, the venetoclax/5-Aza combination provided added benefit over either agent alone in two xenograft models of AML. Together, these data provide a rationale for an ongoing randomized phase-III clinical trial evaluating venetoclax activity in combination with 5-Aza (M15-656, NCT02993523).
Disclosures:
DC, SJ, JP, RP, NT, YX, EB, JL, and DP are employees of AbbVie. LS is a former employee of AbbVie and was employed during the duration of this study. The design, study conduct, and financial support for this research were provided by AbbVie and Genentech. AbbVie participated in the interpretation of data, review, and approval of the publication.

Disclosures
Cojocari: AbbVie Inc: Employment. Jin:AbbVie Inc: Employment, Equity Ownership. Purkal:AbbVie Inc: Employment, Equity Ownership. Popovic:AbbVie Inc: Employment, Equity Ownership. Talaty:AbbVie Inc: Employment, Equity Ownership. Xiao:AbbVie Inc: Employment, Equity Ownership. Solomon:AbbVie Inc: Equity Ownership. Boghaert:AbbVie Inc: Employment, Equity Ownership. Leverson:AbbVie Inc: Employment, Equity Ownership, Patents & Royalties. Phillips:AbbVie Inc: Employment, Equity Ownership, Patents & Royalties.