OTHR-16. HYPERACTIVATION OF THE INTEGRATED STRESS RESPONSE VIA PROTEASOME INHIBITION KILLS DMG AND ATRT WHILE SPARING NORMAL IPSC BRAIN ORGANOIDS

Abstract

Abstract BACKGROUND H3K27-altered diffuse midline glioma (DMG) and atypical teratoid/rhabdoid tumor (ATRT) have increased baseline activation of the integrated stress response (ISR), an evolutionarily conserved system that enables cells to tolerate stress. This activation is manifested by elevated levels of ATF4. Transient or low-level expression protects cells from stress, while persistent, 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 ATRT would make these tumors susceptible to ISR activators such as ixazomib. METHODS After determining the IC50, DMG and ATRT cell lines were treated with increasing concentrations of ixazomib. To evaluate if ixazomib selectively kills DMG and ATRT, we treated iPSC brain organoids in parallel with tumor cells. Combination treatment of ixazomib and idarubicin, a brain-penetrant anthracycline, was conducted to assess synergy. RESULTS Ixazomib significantly induced apoptosis and suppressed proliferation in multiple cell lines, as shown by western blots for cleaved PARP and phospho-Rb expression as well as cleaved caspase-3 and BrdU immunofluorescence (DMSO vs. 25nM p<0.0001 by ANOVA). Additionally, western blots detected elevated ATF4 and CHOP expression in ixazomib treated cells, indicating activation of the ISR. CC3 immunofluorescence and western blot for cleaved PARP showed significantly increased apoptosis in DMG and ATRT cell lines treated with ixazomib (JHHDIPG1 and CHLA06 – DMSO vs. 25nM p<0.0001 by ANOVA), while iPSC brain organoids were not affected. Synergy testing of ixazomib and idarubicin showed an overall zero-interaction-potency (ZIP) synergy score of 23.464 in CHLA06 and 19.084 in HSJD007 (scores of 10 or above indicate synergy). CONCLUSIONS By over-activating the ISR, ixazomib kills DMG and ATRT cells without impacting normal iPSC brain organoids. This effect may potentially be enhanced with the addition of idarubicin.