Targeting PI3K, mTOR, ERK, and Bcl-2 Signaling Network Shows Superior Antileukemic Activity Against AML

Standard induction therapy for the majority of acute myeloid leukemia (AML) patients has not changed over the past 40 years in spite of unacceptably low overall survival rates and high relapse rates, emphasizing the shortfall of current chemotherapy and stressing the need for new therapies. The PI3K/mTOR pathway, which is involved in proliferation, differentiation, and survival, has been shown to be constitutively active in 50-80% of AML patients. Promising preclinical data of PI3K inhibitors have been overshadowed by disappointing clinical results. PI3K/mTOR inhibition has been shown to cause compensatory ERK activation, resulting in cell survival. Thus, both signaling pathways must be targeted to induce AML cell death. However, other mechanisms of survival can remain intact after PI3K/mTOR and ERK inhibition. AML cells can escape cell death through upregulation of the anti-apoptotic Bcl-2 family proteins. Although results from a Phase II AML clinical trial using the Bcl-2-selective inhibitor ABT-199 (Venetoclax) showed promising results, all the ABT-199 treated patients relapsed in a short period of time (median time to relapse was 2.5 months). Therefore, we hypothesize that targeting the PI3K, mTOR, ERK, and Bcl-2 signaling network would result in superior antileukemic activity against AML. To begin our investigation, we first considered inhibition of PI3K and mTOR in AML cells using a novel dual PI3K/mTOR inhibitor VS-5584. VS-5584 sensitivity, as measured by MTT assays, varied among the AML cell lines (n=7) and primary AML patient samples (n=43), with IC₅₀s ranging from 303 nM to 1.4 µM in the AML cell lines and 7 nM to 5.3 µM in the primary AML patient samples (median IC₅₀ was 1.1 µM). Annexin V/propidium iodide staining and flow cytometry analysis revealed that VS-5584 induced cell death including caspase-independent apoptosis due to the lack of PARP and caspase-3 cleavage and change on the mitochondrial outer membrane potential, and the inability of the pan-caspase inhibitor Z-VAD-FMK to prevent cell death. Western blot analysis showed that VS-5584 treatment resulted in decreased phosphorylation of AKT (S473 and T308) and S6 (S240/244), indicating inhibition of both PI3K and mTOR. However, increased phosphorylation of ERK (T202/204) was detected, which could be abolished by the ERK-selective inhibitor SCH772984. Although the combination of VS-5584 and SCH772984 indeed resulted in synergistic induction of cell death, the levels of cell death were modest in some of the AML cell lines and primary patient samples tested. Although Z-VAD-FMK could partially prevent cell death of AML cells exposed to VS-5584 and SCH772984 for a short time (12 h), it could not save the cells post longer drug exposure (48 h). Western blot analysis showed downregulation of Mcl-1 and upregulation of Bim following inhibition of PI3K/mTOR, which were further enhanced by the combined inhibition of PI3K/mTOR and ERK. shRNA knockdown of Bim and overexpression of Mcl-1 at least partially prevented the combination of VS-5584 and SCH772984 induced cell death at an early time point, showing that Bim and Mcl-1 play an important role in cell death induced by the combination. Interestingly, immunoprecipitation of Bcl-2 revealed increased binding of Bim to Bcl-2 in AML cells post combined VS-5584 and SCH772984 treatment, which was abolished by the addition of ABT-199, suggesting that the increased Bim was bound to Bcl-2 which prevented its enhancement of cell death. The three drug combination induced significantly more cell death compared to control, single drug treatment or any of the two drug combinations, and was at least partially mediated through the intrinsic apoptotic pathway, as demonstrated by the ability of Z-VAD-FMK to partially prevent cell death induced by the three drug combination. Treatment of normal human CD34+ cells with the VS-5584, SCH772984, and ABT-199 combination showed no significant effect on colony formation, suggesting that the three drug combination spares normal hematopoietic stem/progenitor cells. Taken together, our findings show that inhibition of PI3K/mTOR and ERK synergistically induce cell death in AML cells, and addition of ABT-199 can improve the efficacy. Thus, our data support targeting the PI3K, mTOR, ERK, and Bcl-2 signaling network for the treatment of AML.