Abstract 2226: Mechanisms for cross-talk between Notch1 and AKT signaling in T-cell acute lymphoblastic leukemia
Authors: Hales Eric Christopher , Orr Steven , Gedman Amanda Larson , Taub Jeffrey , Matherly Larry 
Published: August 2012

Abstract
Pediatric T-cell acute lymphoblastic leukemia (T-ALL) has a variable prognosis, with many patients exhibiting high-risk features at diagnosis, including older age and high presenting white blood cell counts. Activating mutations involving the Notch1 heterodimerizeration and PEST domains occur in more than 50% of T-ALL cases. Increased Notch1 activity promotes survival and proliferation of leukemic cells, in part by transcriptionally repressing PTEN (via HES1). This provides compelling rationale for the use of gamma secretase inhibitors (GSIs) to block Notch1 activation and to restore PTEN levels, which antagonize AKT signaling. Further, PTEN mutations are implicated in GSI resistance. PTEN activity is also modulated by an assortment of posttranslational modifications and Notch1 may regulate PTEN at the posttranslational level. To investigate the impact of Notch1 on AKT signaling, independent of its transcriptional regulation of PTEN, we used PTEN-null Jurkat cells with constitutively active Notch1 and conditional ectopic PTEN expression under control of a tetracycline (tet)-inducible promoter. Induction of PTEN was accompanied by increased phosphorylation of AKTT308, increased insulin-like growth factor 1 receptor (IGF1R) protein and transcripts, and increased Notch1. Increased AKTT308 phosphorylation was blocked with an IGF1R inhibitor (GSK1904259A) and was abolished in Jurkat cells expressing a tet-inducible G129R PTEN phosphatase-inactive mutant. This suggests that PTEN can activate AKT signaling by modulating IGF1R levels. In Jurkat cells, inhibition of Notch1, either through GSI treatment or shRNA knockdown (KD), resulted in increased phosphorylation of AKTT308. Although this result could involve Notch1-dependent decreased cMyc, reflecting indirect effects of cMyc on mTOR1 and negative feedback on AKT, this was not impacted by treatment with a cMyc inhibitor (10058-F4) or by ectopic overexpression of cMyc, suggesting that other effectors and mechanisms are involved. Microarray analysis was performed on wild-type and KD Jurkat cells. Using a 1.5-fold cutoff, 1208 differentially regulated genes were identified. These include PH domain leucine-rich repeat protein phosphatase-1 (PHLPP1) and prokineticin-2 (PROK2), both of which could significantly impact AKT phosphorylation in response to Notch1. By real-time RT-PCR, PHLPP1 was significantly decreased (∼4-fold), and PROK2 was significantly increased (∼6-fold) in Notch1 KD cells compared to wild-type cells. Studies are underway to characterize the potential roles of PHLPP1 and PROK2 in mediating the inhibitory effects of Notch1 on AKT activation in T-ALL. Better understanding of the cross-talk between Notch1 and AKT in T-ALL may identify rational therapeutic strategies for combining GSIs with inhibitors of AKT, mTOR, or IGF1R, and/or for designing therapies based on capacities for constitutive Notch1 signaling.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2226. doi:1538-7445.AM2012-2226