OTHR-14. Integrated stress response activators ixazomib and gemcitabine synergize to induce apoptosis in diffuse midline glioma and atypical teratoid/rhabdoid tumor

The integrated stress response (ISR) is an evolutionarily conserved signaling pathway that helps cells adapt to various stress conditions and restore homeostasis. Transient or low-level activation of the ISR provides protection from stress, while persistent or high-level activation results in cell death. H3K27-altered diffuse midline glioma (DMG) and atypical teratoid/rhabdoid tumor (ATRT) are aggressive pediatric brain tumors with dismal survival outcomes. According to the Children’s Brain Tumor Network Pediatric Brain Tumor Atlas, DMG and ATRT have significantly increased ATF4 RNA expression compared to low grade glioma, indicating high baseline activation of the ISR (DMG p < 0.0001, ATRT p = 0.0472 by ANOVA). Ixazomib is an orally bioavailable proteasome inhibitor that triggers the ISR via endoplasmic reticulum stress. We have previously shown that ixazomib significantly induced apoptosis and suppressed proliferation in DMG and ATRT cell lines, as demonstrated by western blots for cleaved PARP and phospho-Rb expression as well as cleaved caspase-3 (CC3) and BrdU immunofluorescence (DMSO vs. 25nM p < 0.0001 by t-test). Additionally, western blots detected elevated ATF4 expression in ixazomib treated cells. Gemcitabine is a brain-penetrant nucleoside analog that inhibits DNA synthesis and activates the ISR. We hypothesized that ixazomib would combine with gemcitabine to hyper-activate the ISR in DMG and ATRT, leading to tumor cell death. Synergy testing of ixazomib and gemcitabine showed an overall Bliss synergy score of 16.17 in CHLA06 (scores of 10 or above indicate synergy). Combination treatment significantly increased apoptosis in both DMG and ATRT as measured by CC3 immunofluorescence (HSJD007 p = 0.0001 and CHLA06 p < 0.0001 by ANOVA and Tukey’s multiple comparison’s test compared to DMSO control). Similarly, western blots measuring cleaved PARP expression displayed a synergistic effect on apoptosis in cells treated with both ixazomib and gemcitabine. We are currently investigating the efficacy of this combination in vivo. Our studies suggest that the combination of ixazomib and gemcitabine may serve as a promising therapy for DMG and ATRT.