Abstract 605: A novel role for AMPK as a molecular switch regulating the activation of Akt and RAS signaling in acute lymphoblastic leukemia

Abstract

Abstract Acute Lymphoblastic Leukemia (ALL) is the most common hematological malignancy and the main cause of cancer-related death in children. In search for novel treatment strategies, we identified AMP activated protein kinase (AMPK) as a potential target for ALL therapy due to its effects on cell proliferation and cell cycle regulation, as well as its crosstalk with critical metabolic and oncogenic pathways. We demonstrated that treatment of NALM6 and CCRF-CEM cells with the AMPK activators AICAR and metformin induced significant cell growth inhibition and apoptosis. Rescue experiments using the AMPK inhibitor compound-C (CC) failed to completely abrogate the cytotoxic effects induced by AICAR, attenuating AICAR's apoptosis by 42% in CCRF-CEM and 45% in NALM6. Whereas when used alone, both CC and Ara-A induced significant apoptosis in ALL cells. Using the caspase inhibitors Z-VAD and Z-IETD, we demonstrated that activation of AMPK by AICAR induced cell death via both caspase-dependent and independent mechanisms, whereas inhibition of AMPK by CC induced apoptosis mainly via the intrinsic caspase-dependent pathway. To examine the effects of AMPK activation vs. inhibition on downstream signaling, we used Western blot analysis of key signaling factors associated with the PI3K/Akt/mTOR and RAS/RAF/ERK pathways in ALL cells. We found that AICAR and CC exerted opposite effects on PI3K/Akt and RAS pathway signaling. CC decreased P-Akt (Ser473) and activated the RAS pathway, whereas AICAR increased P-Akt, suggesting that AMPK may play a role as regulator of Akt and RAS activity. We showed that activation of AMPK and Akt by AICAR down-regulated the RAS pathway via phosphorylation of cRAF at Ser621 and Ser259, respectively. Inhibition of the RAS pathway with farnesylthiosalicylic acid (FTS) and U-0126 induced significant cell growth arrest and apoptosis in ALL cells (p<0.0001) and resulted in up-regulation of P-AMPK (Thr172) and P-Akt (Thr308). The phosphorylation of Akt at Thr308 was mediated by AMPK-dependent activation of IGF-1R/IRS-1, since down-regulation of AMPK using shRNA abolished the activation of both P-Akt (Thr308) and IGF-1R (Tyr1131). Inhibition of AMPK activity with either CC or shRNA sensitized ALL cells to RAS inhibition (p<0.001). In addition, co-targeting RAS pathway signaling plus either IGF-1R or Akt pathways resulted in significant cell growth inhibition and apoptosis in ALL cells (p<0.001), confirming the functional relevance of this feedback loop mechanism. Taken together, our results indicate that activation and inhibition of AMPK induce cell death in ALL cells through alternative signaling mechanisms impinging on the expression of critical cell proliferation pathways. The data presented here support a new role for AMPK as a molecular regulatory switch coordinating cellular survival responses by activating PI3K/Akt/mTOR and RAS/RAF/ERK oncogenic pathways in ALL. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 605. doi:10.1158/1538-7445.AM2011-605