Efficacy and Mechanisms of Apoptosis Induction by Simultaneous Inhibition of PI3K with GDC-0941 and Blockade of Bcl-2 (ABT-737) or FLT3 (Sorafenib) In AML Cells In the Hypoxic Bone Marrow Microenvironment

Abstract 777

Hypoxia and the interactions with bone marrow (BM) stromal cells have emerged as important mechanisms of leukemia cell survival and chemoresistance. The PI3K-Akt-mTOR-HIF1a signaling pathway has been shown to be activated in response to hypoxia in several cancer models including AML. Therefore, effective targeting of PI3K/Akt signaling pathway could suppress AML cell survival in the hypoxic BM microenvironment. Furthermore, concomitant intra-pathway blockade, or inhibition of the key pro-survival pathways may be more effective. In this study, we investigated the anti-leukemia effects and molecular mechanisms of apoptosis induction in the context of hypoxic bone marrow microenvironment by simultaneous use of a selective Class I PI3K inhibitor GDC-0941 (Genentech) with BH3 mimetic ABT-737 (Abbott) or FLT3 inhibitor sorafenib in AML cells. For hypoxia experiments, AML cells with FLT3-ITD mutation and wild-type FLT3 (FLT3-ITD: MOLM13, MV4;11, wt-FLT3: HL60) were cultured under 1.0% O₂ for at least 14 days to assure their continuous proliferation and survival. Under hypoxia, more cells accumulated in G₀/G₁ phase, indicating that hypoxic conditions promote cell cycle quiescence in leukemic cells.

GDC-0941 treatment in normoxia resulted in a reduction of cell proliferation with G₀/G₁ cell cycle arrest and minimal apoptosis induction, in a time and concentration-dependent manner (IC₅₀ at 48 hrs; 0.7 μM for HL-60, 0.9 μM for MOLM13, 0.7 μM for MV4;11). In hypoxia, GDC-0941 further enhanced cell growth inhibition and G₀/G₁ cell cycle arrest. Importantly, co-culture with BM mesenchymal stroma cells (MSC), which protected AML cells from cytarabine induced apoptosis, did not affect cell growth inhibition by GDC-0941 both in normoxia and hypoxia. Combined treatment of GDC-0941 synergistically enhanced the ABT-737- or sorafenib induced apoptosis under both normoxic- and hypoxic conditions (p<0.05). Combination Index of GDC-0941/ABT-737 was 0.25 for HL-60 and 0.86 for MOLM13 cells; GDC-0941/sorafenib 0.58 for MV4;11 and 0.65 for MOLM13. In FLT3/ITD harboring MOLM13 and MV4;11 cells, ABT-737 and GDC-0941/ABT-737, or sorafenib and GDC-0941/sorafenib-induced apoptosis was partially reversed by MSC co-culture in normoxia, but not in hypoxia.

We next examined the molecular mechanisms of apoptosis induction by simultaneous blockade of PI3K and mutant FLT3 in MOLM13 cells. GDC-0941 inhibited phospho-Akt^Ser473, irrespective of oxygen tension. Hypoxia increased/induced phosphorylation of Akt^Ser473, and of the pro-survival serine/threonine-protein kinase Pim-1, known to promote hypoxia-induced chemoresistance. Pim-1 is a downstream target of Akt and FLT-3 signaling that promotes cell survival and inhibits apoptosis through Bcl-2-dependent mechanisms. While GDC-0941 or ABT-737 alone did not affect hypoxia-induced Pim-1 levels, the GDC-0941/ABT-737 combination down-regulated Pim-1 by 86% (24 hrs). Treatment with sorafenib induced Pim-1 down-regulation by itself by 70%. Under hypoxia, expression levels of Bcl-2 family proteins Bcl-2 and BIM were not significantly changed by any treatment and cell culture conditions. In turn, Mcl-1 expression was upregulated by MSC co-cultures. Further, sorafenib alone but not GDC-0941 or ABT-737 decreased Mcl-1 protein levels, which was reversed by MSC co-culture in hypoxia; in turn, the GDC-0941/sorafenib or GDC-0941/ABT-737 combination potently suppressed Mcl-1 expression under MSC/hypoxia conditions. These changes were associated with the observed apoptotic responses.

In summary, our findings indicate that hypoxic conditions of BM microenvironment result in stimulation of the pro-survival Akt and PIM kinase pathways and increase anti-apoptotic Mcl-1 protein in AML cells with mutated FLT3. In turn, simultaneous blockade of PI3K and FLT3 pathways, or of PI3K and Bcl-2 results in inhibition of these signaling modules and synergistic induction of apoptosis in AML. This data suggest that combined inhibition of Bcl-2 with PI3K or FLT3-ITD may constitute a targeted approach to eradicate chemoresistant AML cells sequestered in the hypoxic BM niches.