Activation of the PP2A-B56α heterocomplex synergizes with venetoclax therapies in AML through BCL2 and MCL1 modulation

•PP2A activators enhance the efficacy of venetoclax and venetoclax-azacitidine combination in primary samples and preclinical models of AML.•PP2A-B56α holoenzyme drives this synergy by modulating BCL2 and MCL1, and PPP2R5A expression is a potential biomarker to predict responses. PP2A activators enhance the efficacy of venetoclax and venetoclax-azacitidine combination in primary samples and preclinical models of AML. The PP2A-B56α holoenzyme drives the synergistic effects through simultaneous inhibition of BCL2 and ERK signaling, with the latter decreasing MCL1 protein stability. [Display omitted] Venetoclax combination therapies are becoming the standard of care in acute myeloid leukemia (AML). However, the therapeutic benefit of these drugs in older/unfit patients is limited to only a few months, highlighting the need for more effective therapies. Protein phosphatase 2A (PP2A) is a tumor suppressor phosphatase with pleiotropic functions that becomes inactivated in ∼70% of AML cases. PP2A promotes cancer cell death by modulating the phosphorylation state in a variety of proteins along the mitochondrial apoptotic pathway. We therefore hypothesized that pharmacological PP2A reactivation could increase BCL2 dependency in AML cells and, thus, potentiate venetoclax–induced cell death. Here, by using 3 structurally distinct PP2A-activating drugs, we show that PP2A reactivation synergistically enhances venetoclax activity in AML cell lines, primary cells, and xenograft models. Through the use of gene editing tools and pharmacological approaches, we demonstrate that the observed therapeutic synergy relies on PP2A complexes containing the B56α regulatory subunit, of which expression dictates response to the combination therapy. Mechanistically, PP2A reactivation enhances venetoclax-driven apoptosis through simultaneous inhibition of antiapoptotic BCL2 and extracellular signal-regulated kinase signaling, with the latter decreasing MCL1 protein stability. Finally, PP2A targeting increases the efficacy of the clinically approved venetoclax and azacitidine combination in vitro, in primary cells, and in an AML patient-derived xenograft model. These preclinical results provide a scientific rationale for testing PP2A-activating drugs with venetoclax combinations in AML.