Abstract #4405: Cytotoxic effects of simultaneous inhibition of AMPK/mTOR and IGF-1R signaling pathways on acute lymphoblastic leukemia
Authors: Gilles Leclerc, Guilian Fu, Guy Leclerc, Julio Barredo
Journal: Cancer Research (2009)

Children 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 energy homeostasis regulator AMP activated protein kinase (AMPK) based on its effects on cell growth and cell cycle. We found that treatment with the AMPK activator AICAR not only increased phosphorylation of AMPK but also resulted in a dose-dependent decrease of P-mTOR. We reported that treatment of NALM6 (Bp-ALL) and CEM (T-ALL) cells with AICAR plus the mTOR inhibitor rapamycin synergistically increased cytotoxicity compared to each agent alone. As a compensatory survival mechanism AICAR induced phosphorylation of Akt (P-Akt) at Ser473 and Thr308. To further understand the signaling cross-talk leading to AICAR-induced apoptosis, we examined the effects of simultaneously targeting AMPK and factors within the IGF-1R and mTOR signaling cascades using Western blots, cell proliferation and apoptosis assays. We detected that IGF-1R signaling, via phosphorylation of the insulin receptor substrate-1 (IRS-1), was higher in NALM6 than CEM, suggesting a greater requirement for activated IGF-1R signaling in B-lineage lymphoblasts. Consequently, inhibition of IGF-1R with HNMPA resulted in significantly higher cell death in NALM6 compared to CEM cells. We then determined that treatment with AICAR increased IRS-1 phosphorylation at Ser789 by 3-5 fold, indicating a direct interaction between AMPK and IRS-1 in ALL cells. To further delineate the signaling cross-talk between IGF-1R and AMPK, we simultaneously targeted these two pathways with HNMPA and AICAR, respectively. As expected, HNMPA decreased the levels of P-IRS-1 (Ser789) and P-Akt (Thr308 & Ser473), but more important AICAR plus HNMPA abrogated the phosphorylation of Akt at Thr308 dependent on PDK1 downstream of IGF-1R. Targeting IGF-1R and AMPK resulted in highly synergistic inhibition of cell viability (NALM6, CI = 0.55; CEM, CI = 0.47). A similar cytotoxic effect was observed when AICAR was combined with the Akt inhibitor X (NALM6, CI = 0.85; CEM, CI = 0.90). Finally, since mTOR inhibits IGF-1R signaling via feedback phosphorylation of IRS-1 at Ser312, we evaluated the effect of simultaneously inhibiting mTOR and IGF-1R using rapamycin and HNMPA, respectively. Cells treated with both agents exhibited synergistic cytotoxicity (NALM6, CI = 0.88; CEM, CI = 0.41), reflecting blockade of both mTOR-induced phosphorylation of IRS-1 (Ser312) and downstream activation of Akt (Thr308). We conclude that rationally designed simultaneous targeting of key factors within the AMPK, IGF-1R, and mTOR pathways leads to synergistic induction of cell death by blocking compensatory survival responses triggered by treatment with single agents. These combinations warrant future testing as novel targeted strategies for childhood ALL.  Citation Information: In: Proc Am Assoc Cancer Res; 2009 Apr 18-22; Denver, CO. Philadelphia (PA): AACR; 2009. Abstract nr 4405.