Abstract 2757: The dual PI3K/mTOR inhibitor NVP-BEZ235 enhances dexamethasone induced apoptosis in models of T-cell ALL with PTEN dysfunction and hyperactivated PI3K/Akt pathway.

Abstract

Abstract Introduction: Glucocorticoid (GC) resistance is a negative prognostic factor in pediatric acute lymphoblastic leukemia (ALL) treatment. The tumor suppressor PTEN, when inactivated, may lead to increased GC resistance through aberrant activation of the PI3K/Akt pathway and inhibition of apoptosis. We investigated the effects of inhibiting the PI3K/Akt pathway with the dual PI3K/mTOR inhibitor NVP-BEZ235 on the enhancement of dexamethasone apoptosis activity in a panel of T-cell and B-cell ALL cell lines and the role PTEN plays in resistance to GCs. Methods: Cytotoxicity of NVP-BEZ235, dexamethasone, and the combination were evaluated using the fluorescence based microscopy system, DIMSCAN, in in vitro models of T- and B-cell ALL and combination indices (CI) were calculated using CalcuSyn. Apoptotic activity for each single agent and the combination was determined using the Annexin-V/PI staining method and measured by flow cytometry. Protein and mRNA expression were measured by immunoblotting and RT-PCR, respectively. Knockdown of PTEN was accomplished using siRNA. Results: The combination of dexamethasone plus NVP-BEZ235 showed strong synergistic activity (CI<0.3) in 6/6 T-cell ALL models tested, while only 1/6 B-cell ALL models, were characterized by such activity. In COG-LL-317h, a T-cell ALL cell line, the percentage of apoptotic cells were significantly greater in cells treated with the combination (17.3±1.0%) compared to vehicle control (4.0±1.7%, p=0.01), dexamethasone (4.5±0.4%, p=0.004), and NVP-BEZ235 (2.3±0.3%, p=0.002). In COG-LL-317h and another T-cell ALL cell line CCRF-CEM, the pro-apoptotic Bcl-2 family protein Bim was substantially increased at both the mRNA and protein levels when treated with the combination compared to either single agent alone, while the B-cell ALL models COG-LL-319h and RS4;11 did not show any enhancement with the combination. Knockdown of PTEN in dexamethasone-sensitive RS4;11 showed a significant reduction in dexamethasone cytotoxicity (non-targeting siRNA: 88.3±1.5% vs. PTEN siRNA: 82.6±2.6%; p<0.001), and activity was restored when dexamethasone was in combination with NVP-BEZ235 (non-targeting siRNA: 91.9±1.8% vs. PTEN siRNA: 92.1±1.6%, p=0.77). Conclusion: These data suggest PTEN dysfunction and subsequent PI3K/Akt activation play a significant role in altering GC responses and use of the combination may offset the negative consequences of PTEN loss by increasing Bim expression and apoptosis. Our results show that NVP-BEZ235 enhances dexamethasone apoptotic activity in models of T-cell ALL which have loss of PTEN function, and may provide a new adjunct to increase response in current T-cell ALL treatment protocols. Citation Format: Connor Hall, Min Kang. The dual PI3K/mTOR inhibitor NVP-BEZ235 enhances dexamethasone induced apoptosis in models of T-cell ALL with PTEN dysfunction and hyperactivated PI3K/Akt pathway. [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 2757. doi:10.1158/1538-7445.AM2013-2757