Inhibition of AKT/mTOR Signaling Pathway Induces Cell Cycle Arrest and Apoptosis in Acute Myelogenous Leukemia.

Acute myelogeneous leukemia (AML) is a heterogeneous disease and includes a subset of neoplasms that harbor activating mutations of the fms-like tyrosine kinase-3 (FLT3) gene. Mutated FLT3 has recently been shown to activate downstream oncogenic pathways including the PI3K/AKT pathway (Scheijen, et al. Oncogene. 23:3338–3349, 2004; Choudhary, et al. Blood. 106:265–273, 2005). It is known that activated AKT mediates its effects, at least in part, through activation of mammalian target of rapamycin (mTOR). However, the potential role of PI3K/AKT/mTOR signaling pathway in tumor cell survival in AML remains largely unknown. We hypothesized that the PI3K/AKT signaling pathway is activated in AML and contributes to tumor cell survival through activation (phosphorylation) of mTOR and its downstream effectors 4EBP1, p70S6K, ribosomal protein S6 (rpS6), and eIF-4E. We used 3 AML cell lines, including MV4-11 and MOLM-13, that are homozygous and heterozygous for mutated FLT3, respectively, as well as U937 (wild-type FLT3). All 3 cell lines expressed activated (serine 473-phosphorylated) AKT (Ser473pAKT), and phosphorylated 4EBP1, p70S6K and rpS6 shown by Western Blot analysis. Treatment of AML cell lines with LY294002, an inhibitor of PI3K, resulted in a dose-dependent decrease of phosphorylation of AKT, mTOR, 4EBP1, p70S6K, and rpS6. This was associated with decreased cell viability as assessed by trypan-blue exclusion assay. Cell death following inhibition of the PI3K/AKT pathway was predominantly attributed to apoptosis as shown by increased annexin V staining assessed by flow cytometry. These changes were associated with downregulation of the anti-apoptotic proteins cFLIP, Mcl-1, and Bcl-XL that are involved in the extrinsic and intrinsic apoptosis. Cell cycle analysis using flow cytometry also showed that inhibition of PI3K resulted in decreased S-phase and increased G1-phase fraction. These cell cycle changes were associated with increased levels of the cyclin-dependent kinase inhibitor p27 and underphosphorylated Rb in a dose-dependent manner. Similar biologic effects, although to a lesser degree, were found after treatment of AML cells with rapamycin, an inhibitor of mTOR. In addition, expression of activated AKT, mTOR, 4EBP1, p70S6K and rpS6 was assessed in AML tumors (n=19) using tissue microarrays of bone marrow samples and immunohistochemical methods. These included tumors with (n=14) and without (n=5) FLT3 mutations. Using a 10% cutoff to define positivity, 13/19 (68%) expressed Ser473pAKT, 16/18 (89%) mTOR, 15/19 (79%) p4E-BP1, 18/19 (95%) p-p70S6K, and 15/18 (83%) p-rpS6. However, no association between expression of activated AKT, or mTOR signaling proteins and FLT3 mutational status was observed. Our study provides first evidence that the AKT/mTOR signaling pathway is activated in AML cell lines and tumors regardless of FLT3 mutational status. The AKT/mTOR signaling pathway may contribute to cell cycle progression and tumor cell survival in AML. Inhibition of this oncogenic pathway represents a potential target for therapy in patients with AML.