Intra-Pathway Inhibition of Upstream (PI3K) and Downstream (mTOR) Kinases Synergistically Induces Apoptosis in AML.

Constitutive activation of the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) pathway regulates the growth and survival of acute myeloid leukemia (AML). We hypothesized that targeting this pathway with both PI3K and mTOR inhibitors may greatly enhance the effectiveness of these two inhibitors in the treatment of AML. PI3KI1 is a novel PI3K inhibitor that induced apoptosis in AML cell lines and primary AML cells at an IC₅₀ of 5μM. It directly inhibited AKT at Ser473, however had limited effects on pGSK3b and on the mTOR downstream target p70S6K at Thr389. Colony-forming assays demonstrated that PI3KI1 decreased the viability of primary AML samples but spared normal bone marrow progenitor cells. mTOR inhibitor CCI779 inhibited phosphorylation of downstream mTOR targets p70S6K and 4EBP, however showed only minor cytotoxicity to AML cell lines and primary samples, suggesting that inhibition of mTOR signaling is not sufficient to cause growth inhibition in the majority of AML. Combined use of PI3KI1 and CCI779 synergistically induced apoptosis in U937 cells, with a combination index of 0.061±0.02. Western blot analysis demonstrated enhanced suppression of pP70S6K, pAKT and p4EBP1(Thr70) when PI3KI1 and CCI779 were used in combination. In primary AML samples, combined inhibition of PI3K and mTOR pathways enhanced apoptosis induction in 8/12 samples, with true synergistic responses in 3 samples. Importantly, the combination, but not PI3KI1 or CCI779 alone, was able to overcome the growth advantage conferred to AML cell lines or primary AML samples by adherence to bone marrow stromal cells. Taken together, our results indicate that PI3K and mTOR are relevant molecular targets in AML and that intra-pathway inhibition of both, upstream and downstream proteins may be required for maximal inhibition of leukemia cell growth.