Combination of the PI3k inhibitor Paxalisib with the nucleoside analog Gemcitabine over-activates the integrated stress response and induces atypical teratoid/rhabdoid tumor cell death

Abstract We have previously identified high activation of both mTORC1 and mTORC2 in the aggressive infantile brain tumor, atypical teratoid/rhabdoid tumor (AT/RT). Paxalisib is a highly brain penetrant PI3k inhibitor which acts upstream of mTOR to simultaneously inhibit mTORC1/2. Paxalisib is well-tolerated in ongoing pediatric clinical trials. In murine orthotopic xenograft models of AT/RT, Paxalisib significantly extended survival (CHLA-06: 40 to 54 days, p=0.001; BT12: 21 to 35 days, p=0.02). However, to improve the durability of this single agent therapy, we evaluated the efficacy of rational therapeutic partners. Analysis of RNAseq following mTOR inhibition (mTORi) of 4 cell lines representative of AT/RT showed an upregulation of genes associated with the integrated stress response (ISR), (ATF4: CHLA-06, t-test p<0.05; CHOP: CHLA-02, CHLA-06 t-test p<0.001). The activation of the ISR is further suggested through induction of PPP1R15A, a gene whose activation is closely associated with stress events and leads to downstream cell growth arrest and apoptosis (BT37 t-test p<0.001, CHLA-02, CHLA-06, CHLA-266, t-test p<0.01). Gemcitabine is a pyrimidine nucleoside prodrug, which inhibits DNA synthesis and induces DNA damage. As a result, Gemcitabine also induces phosphorylation of eIF2α and the activation of the ISR. While the ISR can be protective, intense or prolonged activation leads to cell death. We therefore hypothesized that Paxalisib would combine with Gemcitabine to hyper-activate the ISR and drive AT/RT cell death. Through Western blot analysis, we demonstrate that combination therapy increases phospho-eIF2α, ATF4, and CHOP compared to DMSO control. Furthermore, we demonstrate that combination therapy increases apoptosis (Western blot cPARP, MUSE Annexin V Assay) and synergizes to decrease AT/RT cell growth (SynergyFinder BLISS score CHLA-06: 16.8, BT12: 14.3, CHLA-266: 13.9, BT37: 11.4, CHLA-05: 10.4). Ongoing treatment of mice bearing CHLA06 orthotopic tumors demonstrate that combination therapy further extends median survival. Our studies suggest that pharmacologically activating the ISR may be an effective strategy to target AT/RT and combination of Paxalisib and Gemcitabine may be an effective treatment to help extend survival in this deadly disease. Citation Format: Tyler Findlay, Kristen Malebranche, Anupa Geethadevi, Eric Raabe, Charles Eberhart, Jeffrey Rubens. Combination of the PI3k inhibitor Paxalisib with the nucleoside analog Gemcitabine over-activates the integrated stress response and induces atypical teratoid/rhabdoid tumor cell death. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5490.