Patterns and Prognostic Impact of PI3K-AKT Pathway Activation, Regulation and Downstream Activity in AML Using Reverse Phase Proteins Arrays (RPPA).

Functional activation of the PI3K/AKT signaling pathway provides survival signals to leukemic cells and pathway blockade may facilitate cell death. We have previously demonstrated that activation of AKT (phosphorylated on threonine 308 and serine 473 [pAKT308 & pAKT473]) pathway is a frequent event with adverse prognostic consequences in AML. To further explore the upstream regulation and downstream consequences of activation of this pathway in AML we assessed the level of expression and activation of PTEN/phosphoPTEN (pPTEN) (which controls AKT activation), P53 and DJ1 (which regulates PTEN), along with targets of activated AKT including proteins regulating proliferation (S6RP, Cyclin D1, GSK3, MTOR), and apoptosis (MCL, BAD, XIAP, BCL2). Total and phospho-protein levels from 258 leukemia-enriched samples of newly diagnosed, untreated AML patients were measured using high-throughput reverse phase protein array technology (RPPA). High P53 expression negatively correlated with PTEN levels (p=0.001). Almost all cases with very high P53 had unfavorable cytogenetics with complex karyotypes, and P53 mutations were found in 54% cases (see accompanying abstract). AKT activation, determined as high ratios of phospho to total (P/T) pAKT473 and pAKT308 were strongly correlated with loss of PTEN expression or increased PTEN phosphorylation,(p=0.0006 & 0.006) but not with increased levels of PTEN suppressor DJ1 (P=0.33 & 0.34). AKT activation (pAKT473 & pAKT308 P/T ratios) was associated with increased signaling through mTOR kinase (increased phosphorylation of S6RP on p236, p240, p244, p<0.0001) and inactivation (via phosphorylation) of GSK3 (p<0.004). Furthermore, AKT-mTOR activation strongly correlated with high levels of Cyclin D1 (p<0.008), inactivated BAD(p136) and increased expression of the antiapoptotic protein MCL1 (p=0.04 for S6RP-p236). In turn, GSK3 inactivation in samples with active AKT correlated strongly with increased expression levels of β-Catenin, Survivin and cyclin D1 (all p<0.005). Phosphorylated AKT ratios were significantly correlated with high levels of anti-apoptotic proteins XIAP and BCL2 (p=0.011 and p=0.005) but did not correlate with P27, pBAD or P70SK6. Confirming our prior findings, patients with higher P/T AKT ratios (473, 308 or both), had significantly shorter survival (p=0.02, 0.07 & 0.03 respectively). These results demonstrate that in newly diagnosed AML, inactivation of PTEN via phosphorylation or downregulation, in part via p53 pathway, results in robust activation of PI3K/AKT signaling. In summary this is the first analysis in a large AML dataset of AKT targets and regulators. Combined this demonstrates that the PI3K-AKT/mTOR pathway is commonly activated in AML, and functions canonically as a proximal regulator of critical components of the apoptotic machinery by maintaining mitochondrial integrity (Bcl-2 family proteins) and inhibiting caspase activation (IAPs). Since mTOR can be activated in PI3K-independent fashion, we tested the concept of intra-pathway inhibition of PI3K pathway at multiple sites. Indeed, combining rapamycin analogues with PI3K inhibitors synergistically enhances apoptosis induction in AML cell lines and in primary AML samples, via complete blockade of AKT phosphorylation. These results provide rationale for targeting the PI3K/AKT and mTOR pathways in the therapy of AML.