DIPG-52. Activators of the integrated stress response synergize to kill DIPG

DIPG has elevated baseline activation of the integrated stress response (ISR), an evolutionarily conserved system that allows cells to tolerate various forms of stress. Increased expression of activating transcription factor 4 (ATF4) indicates activation of the ISR. Intermediate levels of ATF4 protect cells from stress, while sustained high levels result in cell death. The imipridone drug ONC201 binds to and activates the mitochondrial protease ClpP, leading to increased mitochondrial stress and persistent ATF4 activation. Because DIPG has a high baseline level of ATF4, we hypothesized that the ISR activators Sal003, ONC201, and fenretinide would synergize to kill DIPG. Sal003 inhibits dephosphorylation of ATF4 upstream regulator, eIF2α. The retinoic acid derivative fenretinide induces ATF4, increases reactive oxygen species, and has clinical activity in pediatric patients with neuroblastoma. After determining the IC25 of Sal003, fenretinide, and ONC201, we treated patient-derived DIPG cell lines with low micromolar doses. The combination of Sal003 and ONC201 significantly increased apoptosis as measured by CC3 immunofluorescence in comparison to DMSO (p<0.0001, ANOVA). Combination therapy also significantly increased CC3 positivity compared to single treatment. Western blots for cleaved PARP expression detected induction of apoptosis in DIPG treated with both Sal003 and ONC201 over DMSO and monotherapy treated cells. In some cell lines, the combination increased ATF4 expression. Since Sal003 is not yet available for clinical testing in humans, we treated DIPG cells with ONC201 and fenretinide. CC3 immunofluorescence indicated synergistically elevated apoptosis in the combination of ONC201 and fenretinide vs. DMSO (p<0.0001, ANOVA). Western blots showed increased cleaved PARP, ATF4, and CHOP expression in DIPG treated with ONC201 and fenretinide. We are currently testing the efficacy of this combination in orthotopic DIPG xenografts. Our results suggest the combination of ONC201 with fenretinide could potentially serve as a therapy for DIPG.