Abstract 6720: Using proteasome inhibition to hyperactivate the integrated stress response in aggressive pediatric brain tumors

Abstract

Abstract H3K27-altered diffuse midline glioma (DMG) and atypical teratoid/rhabdoid tumor (AT/RT) are aggressive pediatric brain tumors with dismal outcomes. DMG and AT/RT have increased baseline activation of the integrated stress response (ISR), an evolutionarily conserved system that enables cells to tolerate various forms of stress. This activation is manifested by elevated levels of ATF4 and NRF2 in DMG and AT/RT, respectively. Transient or low-level expression of these transcription factors protect cells from stress, while sustained, high-level activation leads to cell death. Ixazomib is an orally bioavailable proteasome inhibitor that causes endoplasmic reticulum stress, which is a major upstream activator of the ISR. We hypothesized that the high baseline level of ATF4 in DMG and NRF2 in AT/RT would make these tumors susceptible to ISR activators such as ixazomib. After determining the IC50 of ixazomib, DMG and AT/RT cell lines (JHH-DIPG1, JHH-DIPG16A, and CHLA06) were treated with increasing concentrations of the drug. Cleaved caspase 3 immunofluorescence showed a significant increase of apoptosis in all cell lines (JHH-DIPG16A and CHLA06 p<0.0001, JHH-DIPG1 p=0.0008 by ANOVA). Additionally, western blots for cleaved PARP and phospho-Rb expression detected induction of apoptosis and suppression of cell proliferation in JHH-DIPG1 treated with ixazomib. We are currently testing ixazomib in combination with idarubicin, a brain-penetrant anthracycline, and gemcitabine, a brain-penetrant nucleoside analog. Synergy testing in CHLA06 showed an overall zero-interaction-potency (ZIP) synergy score of 23.464 with inhibitory concentrations of ixazomib and idarubicin in the low nanomolar range (scores of 10 or above indicate synergy). Similarly, ixazomib and gemcitabine synergized to suppress CHLA06 growth and induce apoptosis (67% Annexin V+ cells in combination compared to 14 and 24% Annexin V+ with single agent treatment). In the future, we will determine if ixazomib selectively kills DMG and AT/RT while sparing normal cells in iPSC brain organoids. We will also test ixazomib singly and in combination with traditional chemotherapy in orthotopic xenografts of AT/RT. These results suggest that ixazomib, especially in combination with idarubicin, gemcitabine or other ISR activators, has the potential to serve as an effective therapy for aggressive pediatric brain tumors. Citation Format: Orlandi Valencia Novak, Tyler Findlay, Jeffrey Rubens, Charles Eberhart, Eric H. Raabe. Using proteasome inhibition to hyperactivate the integrated stress response in aggressive pediatric brain tumors [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 6720.