Co-Administration of the mTORC1/TORC2 Inhibitor INK128 and the Bcl-2/Bcl-Xl Antagonist ABT-737 Kills Human Myeloid Leukemia Cells through Mcl-1 Down-Regulation and AKT Inactivation
Authors: Rahmani Mohamed , Aust Mandy Mayo , Hawkins Elisa , Parker Rebecca E , Reshko Leonid , Ross Masey , Kmieciak Maciej , Rizzo Kathryn Ann , Dumur Catherine I , Ferreira-Gonzalez Andrea , Grant Steven 
Published: October 2019

Acute myelogenous leukemia (AML) is characterized by frequent aberrations of the PI3K/AKT/mTOR axis steming from diverse mechanisms, including FLT3, Ras, and c-KIT mutations, among multiple others. In a previous report, we demonstrated that dual inhibition of the PI3K/mTOR axis and the BH3-mimetic ABT-737, which inhibits both Bcl-2 and Bcl-xL, but not Mcl-1, exhibits potent anti-leukemia activity both in vitro and in vivo (Cancer Res. 2013;73(4):1340-51). It has also been shown that while TORC1 inhibitors such as rapamycin can elicit rebound activation of AKT in various tumor cells, dual inhibition of TORC1/2 potently inhibits AKT activation at both Thr308 and Ser473 sites. In the present studies, we examined whether concurrent inhibition of mTORC1/2 and Bcl-2/Bcl-xL would lead to enhanced anti-leukemia activity in AML cells. Notably, dual tetracycline-inducible Bcl-2/Bcl-xL knockdown markedly sensitized acute myeloid leukemia (AML) cells to the dual TORC1/2 inhibitor INK128 (ChemieTek) in vitro as well as in vivo. Moreover, INK128 co-administered with the Bcl-2/Bcl-xL antagonist ABT-737 (AbbVie) sharply induced cell death in multiple AML cell lines, including TKI-resistant FLT3-ITD mutants as well as primary AML blasts carrying various genetic aberrations e.g., FLT3, IDH2, NPM1, and Kras among others, while exerting minimal toxicity toward normal hematopoietic CD34+ cells. Combined treatment was particularly active against CD34+/CD38-/CD123+ primitive leukemia progenitor cells. The INK128/ABT-737 regimen was also effective in the presence of a protective stromal micro-environment (i.e., co-culture with HS-5 cells), suggesting that this strategy may circumvent the protective effects of bone marrow stromal cells, which are known to play an important role in leukemia cell survival as well as drug resistance. Notably, INK128 was very effective in down-regulating Mcl-1, diminishing AKT and 4EBP1 phosphorylation, and potentiating ABT-737-mediated apoptosis. Furthermore, ectopic expression of Mcl-1 dramatically attenuated INK128/ABT737 lethality, indicating an important functional role for Mcl-1 down-regulation in INK128/ABT-737 interactions. In contrast to INK128, rapamycin increased AML cell AKT phosphorylation, and was largely ineffective in down-regulating Mcl-1, decreasing 4EBP1 phosphorylation, or enhancing ABT-737 lethality. Interestingly, addition of a specific AKT inhibitor to the rapamycin/ABT-737 regimen sharply increased apoptosis in association with pronounced AKT inactivation and Mcl-1 down-regulation, analogous to effects observed with INK128/ABT-737. These findings argue that AKT activation may oppose rapamycin/ABT-737 lethality in AML cells. Immunoprecipitation analysis revealed that combined treatment markedly diminished Bax, Bak, and Bim binding to all major anti-apoptotic Bcl-2 members (Bcl-2/Bcl-xL/Mcl-1), while Bax/Bak shRNA knock-down reduced cell death. Finally, INK128/ABT-737 co-administration sharply attenuated leukemia growth and significantly prolonged survival in a systemic AML xenograft model (P < 0.0001; log-rank test for combined treatment vs either agent alone). In addition, analysis of subcutaneous AML-derived tumors showed significant declines in 4EBP1 phosphorylation and Mcl-1 protein level, consistent with in vitro results. Collectively, these findings demonstrate that co-administration of dual mTORC1/mTORC2 inhibitors and BH3-mimetics exhibits potent anti-leukemic activity in vitro and in vivo, arguing that this strategy warrants attention in AML. They also raise the possibility that dual mTORC1/mTORC2 inhibitors may offer advantages over pure mTORC1 inhibitors in this setting.

Disclosures

No relevant conflicts of interest to declare.