Abstract
BackgroundChildren with Acute Lymphoblastic Leukemia (ALL) diagnosed with resistant phenotypes and those who relapse have a dismal prognosis for cure. In search for novel treatment strategies, we identified the AMP activated protein kinase (AMPK) as a potential drug target based on its effects on cell growth and survival. We have shown previously that AICAR-induced AMPK activation also induced a compensatory survival mechanism via PI3K/Akt signaling.ResultsIn the present study, we further investigated the downstream signaling induced by AMPK activation in ALL cells. We found that AICAR-induced AMPK activation resulted in up-regulation of P-Akt (Ser473 and Thr308) and decrease of P-mTOR (Ser2448) expression and downstream signaling. We determined that activation of P-Akt (Thr308) was mediated by AMPK-induced IGF-1R activation via phosphorylation of the insulin receptor substrate-1 (IRS-1) at Ser794. Inhibition of IGF-1R signaling using the tyrosine kinase inhibitor HNMPA(AM)3 resulted in significant decrease in P-IRS-1 (Ser794) and P-Akt (Thr308). Co-treatment of AICAR plus HNMPA(AM)3 prevented AMPK-induced up-regulation of P-Akt (Thr308) but did not alter the activation of P-Akt (Ser473). Inhibition of AMPK using compound-C resulted in decreased P-Akt expression at both residues, suggesting a central role for AMPK in Akt activation. In addition, inhibition of IGF-1R signaling in ALL cells resulted in cell growth arrest and apoptosis. Additional Western blots revealed that P-IGF-1R (Tyr1131) and P-IRS-1 (Ser794) levels were higher in NALM6 (Bp-ALL) than CEM (T-ALL), and found differences in IGF-1R signaling within Bp-ALL cell line models NALM6, REH (TEL-AML1, [t(12;21)]), and SupB15 (BCR-ABL, [t(9;22)]). In these models, higher sensitivity to IGF-1R inhibitors correlated with increased levels of IGF-1R expression. Combined therapy simultaneously targeting IGF-1R, AMPK, Akt, and mTOR pathways resulted in synergistic growth inhibition and cell death.ConclusionsOur study demonstrates that AMPK activates Akt through IGF-1R dependent and independent mechanisms. Co-targeting IGF-1R and related downstream metabolic and oncogenic signaling pathways represent a potential strategy for future translation into novel ALL therapies.
Highlights
Children with Acute Lymphoblastic Leukemia (ALL) diagnosed with resistant phenotypes and those who relapse have a dismal prognosis for cure
AICAR-induced AMP activated protein kinase (AMPK) activation promotes phosphorylation of insulin receptor substrate-1 (IRS-1) at Ser794 Recently, we reported that treatment of ALL cell lines with AICAR induced growth inhibition and apoptosis, and resulted in increased expression of P-protein kinase B (Akt) (Ser473) [3]
To investigate the role of AMPK and mammalian target of rapamycin (mTOR) in this process, we examined the levels of P-mTOR (Ser2448) and P-IRS-1 (Ser794, a residue known to be phosphorylated by AMPK) [31] in CCRF-CEM (T-ALL) and NALM6 (Bp-ALL) cells treated with AICAR
Summary
Children with Acute Lymphoblastic Leukemia (ALL) diagnosed with resistant phenotypes and those who relapse have a dismal prognosis for cure. Phosphorylation of Akt at Ser473 involves rictor, a member of the TORC2 complex known to modulate the activity of mTOR [7,10,11,12], while phosphorylation of Thr308 is mediated by PDK1 and PIP3 following phosphorylation of PIP2 by PI3K [13,14] The latter mechanism is responsible for the described feedback loop inhibition of Akt phosphorylation mediated by mTORdependent phosphorylation of IRS-1 at Ser312, the immediate downstream effector protein of the insulinlike growth factor-1 receptor (IGF-1R) [15,16]. Inhibition of mTOR results in IRS-1 activation and increased phosphorylation of Akt at Thr308 [19]
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