Purpose: Acute myeloid leukemia (AML) is an aggressive disease with a poor outcome. We investigated mechanisms by which the anti-AML activity of ABT-199 (venetoclax) could be potentiated by dual mTORC1/TORC2 inhibition. Methods: Venetoclax/INK128 synergism was assessed in various AML cell lines and primary patient AML samples in vitro. AML cells over-expressing MCL-1, constitutively active AKT, BAK and/or BAX knock-out, and acquired venetoclax resistance were investigated to define mechanisms underlying interactions. The antileukemic efficacy of this regimen was also examined in xenograft and patient-derived xenograft (PDX) models. Results: Combination treatment with venetoclax and INK128 (but not the mTORC1 inhibitor Rapamycin) dramatically enhanced cell death in AML cell lines. Synergism was associated with p-AKT and p-4EBP1 down-regulation and dependent upon MCL-1 downregulation and BAK/BAX up-regulation as MCL-1 overexpression and BAX/BAK knock out abrogated cell death. Constitutive AKT activation opposed synergism between venetoclax and PI3K or AKT inhibitors, but not INK128. Combination treatment also synergistically induced cell death in venetoclax- resistant AML cells. Similar events occurred in primary patient-derived leukemia samples but not normal CD34+ cells. Finally, venetoclax and INK128 co-treatment displayed increased anti-leukemia effects in in vivo xenograft and PDX models. Conclusions: The venetoclax/INK128 regimen exerts significant anti-leukemic activity in various preclinical models through mechanisms involving MCL-1 down-regulation and BAK/BAX activation, and offers potential advantages over PI3K or AKT inhibitors in cells with constitutive AKT activation. This regimen is active against primary and venetoclax resistant AML cells, and in in vivoAML models. Further investigation of this strategy appears warranted.

<p>Supplementary Figures S1-S14</p>

<p>Supplementary Figures S1-S14</p>

<p>Clinical, molecular, and cytogenetic characteristics of patient samples (CD34+CD38-CD123+ population) exposed to ABT-199 ± INK-128</p>

<p>Clinical, molecular, and cytogenetic characteristics of patient samples (CD45dim SS low population) exposed to ABT-199 ± INK-128</p>