Feedbacks and adaptive capabilities of the PI3K/Akt/mTOR axis in acute myeloid leukemia revealed by pathway selective inhibition and phosphoproteome analysis

Abstract

Acute myeloid leukemia (AML) primary cells express high levels of phosphorylated Akt, a master regulator of cellular functions regarded as a promising drug target. By means of reverse phase protein arrays, we examined the response of 80 samples of primary cells from AML patients to selective inhibitors of the phosphatidylinositol 3 kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) axis. We confirm that >60% of the samples analyzed are characterized by high pathway phosphorylation. Unexpectedly, however, we show here that targeting Akt and mTOR with the specific inhibitors Akti 1/2 and Torin1, alone or in combination, result in paradoxical Akt phosphorylation and activation of downstream signaling in 70% of the samples. Indeed, we demonstrate that cropping Akt or mTOR activity can stabilize the Akt/mTOR downstream effectors Forkhead box O and insulin receptor substrate-1, which in turn potentiate signaling through upregulation of the expression/phosphorylation of selected growth factor receptor tyrosine kinases (RTKs). Activation of RTKs in turn reactivates PI3K and downstream signaling, thus overruling the action of the drugs. We finally demonstrate that dual inhibition of Akt and RTKs displays strong synergistic cytotoxic effects in AML cells and downmodulates Akt signaling to a much greater extent than either drug alone, and should therefore be explored in AML clinical setting.

Current treatment strategies for inhibiting mTOR in cancer

“…These include cyclin-dependent kinase-2 (CDK-2), cyclin D1, c-Myc, signal activator and transducer of transcription 3 (STAT3), B-cell lymphoma 2 (Bcl-2), Bcl-xL, myeloid cell leukemia-1 (Mcl-1), survivin, and ornithine decarboxylase [43,44]. Third, the disappointing performance of rapamycin/rapalogs has also been ascribed to S6K1-dependent feedback loops that lead to reactivation of RTK, PI3K/Akt, and Ras/Raf/MEK/ERK signaling on mTORC1 inhibition [27,[45][46][47]. Moreover, mTORC1 directly phosphorylates and inhibits the RTK inhibitor growth factor receptor-bound protein 10 (Grb10) [48], leading to accumulation of Grb10 and PI3K/Akt activation in some cell types [1].…”

Dual targeting of eIF4E by blocking MNK and mTOR pathways in leukemia

“…In fact, PI3K/AKT/mTOR pathways were shown to promote leukemic cell proliferation and survival and have been implicated in resistance to antileukemic drugs [54, 55]. It has been reported that in 50-80% of AML cases there is constitutive activation of the PI3K and mTOR kinases [56].…”

Targeting mTOR signaling pathways and related negative feedback loops for the treatment of acute myeloid leukemia

“…This feedback loop was successfully blocked by the combination of an AKT inhibitor with the PDGFR inhibitor sunitinib, the IGF-1R/IR inhibitor linsitinib, or the FLT3 inhibitor quizartinib. 10 Hence, these findings suggest that turning off the PI3K/AKT/mTOR axis goes beyond combining individual inhibitors of axis nodes and may require strategies accounting for the plasticity of feedback loops that emerge after exposure to certain drugs. 107 Altogether, these results epitomize the complexity of PI3K/AKT/mTOR pathway regulatory network and highlight the potential importance of incorporating receptor tyrosine kinase inhibitors into genomic-based treatment plans for AML patients.…”

“…[103][104][105][106] Recent results have demonstrated paradoxical AKT phosphorylation in AML cells treated with AKT or PI3K inhibitors for 24 hours compared to the shorter duration treatment of 4 hours. 10 These inhibitors also increased the expression of IGF-1R, PDGFR and insulin receptors and induced their autophosphorylation. These effects ultimately result in sustained activation of the insulin receptor substrate 1.…”

“…6 None of these agents is FDA-approved for the treatment of acute myeloid leukemia (AML), a disease in which there is very strong preclinical evidence implicating this pathway in its pathogenesis and pathophysiology. [7][8][9][10][11] In one study, more than 90% of primary AML blasts had evidence of activation of AKT, a critical substrate of PI3K, as determined by the presence of phosphorylation or a positive kinase assay. 12 Recher et al showed that S6K and 4E-BP1, both downstream effectors of mTOR, were constitutively phosphorylated among the majority of freshly isolated AML samples from 23 patients but were not phosphorylated in normal freshly isolated CD34 Downloaded by [University of Western Ontario] at 01: 13 13 April 2015 control cells.…”