Abstract 4116: Notch1 regulates AKT-T308 dephosphorylation through modulation of the PP2A phosphatase in GSI-resistant T-cell acute lymphoblastic leukemia (T-ALL) cells.

Abstract

Abstract Pediatric T-ALL typically has a poorer prognosis than B-precursor ALL, as T-ALL patients exhibit 5-event free survival rates approximating 70-75% compared to > 90% for B-precursor-ALL. Notch1 activating mutations occur in more than 50% of T-ALL cases. Notch1 mutations cluster within the heterodimerization (HD) and/or PEST domains and liberate activated intracellular Notch 1 (ICN1) by increasing susceptibility to HD domain cleavage by gamma-secretase (GS) and/or by increasing the half-life of ICN1, respectively. This results in increased expression of ICN1 target genes including hes1, deltex1, cmyc, igf1r, calcineurin, e2a, and il7r, which affect cell proliferation and survival at least in part by activating AKT. Notch1 increases Hes1 expression and transcriptional silencing of PTEN which antagonizes AKT activation. Mutational loss of PTEN, which occurs frequently in T-ALL, renders cells resistant to Notch1 inhibition with GS inhibitors (GSIs). This suggests that Notch1 favors AKT activation. However, Notch1 can exert inhibitory control over AKT signaling even in GSI-resistant, PTEN-null Jurkat cells, which have mutant activated Notch1. We found that both GSI treatment and Notch1 knockdown (N1KD) in Jurkat cells increased AKT activation loop (T308) phosphorylation and signaling, and protected cells from induction of apoptosis. This was not due to increased AKT phosphorylation by PI3K. Rather this was due to decreased dephosphorylation of AKT-T308 in the N1KD cells. PP2A is the major Ser/Thr phosphatase that dephosphorylates AKT at T308. Treatment of cells with the PP2A inhibitor okadaic acid increased AKT-T308 phosphorylation in non-targeted control (NTC) cells but had no overt effect in the N1KD cells, suggesting decreased PP2A activity. Notch1 primarily functions as a transcriptional regulator and could affect genes encoding the PP2A catalytic or regulatory subunits. However, neither the levels of the individual subunits nor PP2A catalytic activity were changed in N1KD cells. By immunoprecipitation, N1KD cells showed a decreased interaction between PP2A and AKT. This was accompanied by increased phosphorylation of AKT-T308 but also of other PP2A targets including AMPK and p70S6K. Increased phosphorylation of these targets also resulted from transient transfection with a dominant-negative MAML which interferes with ICN1 transcriptional effects. This was not mediated by cMyc, as established by cMyc transfections and treatment with a cMyc inhibitor. Conversely, transfection with Hes1 decreased phosphorylation of these PP2A targets in the N1KD cells. This suggests a causal role for Hes1, at least in part, in the Notch1 effects on PP2A and AKT-T308 phosphorylation. To our knowledge, these effects of Notch1 and Hes1 on PP2A and their impact on AKT and AMPK signaling have not been previously described. Citation Format: Eric Christopher Hales, Steven M. Orr, Amanda Larson Gedman, Jeffrey W. Taub, Larry H. Matherly. Notch1 regulates AKT-T308 dephosphorylation through modulation of the PP2A phosphatase in GSI-resistant T-cell acute lymphoblastic leukemia (T-ALL) cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4116. doi:10.1158/1538-7445.AM2013-4116