Abstract 5227: Combination therapy activating the integrated stress response synergistically suppresses proliferation and induces apoptosis in diffuse intrinsic pontine glioma

Abstract

Abstract Diffuse intrinsic pontine glioma (DIPG) is a lethal pediatric brain tumor. Radiation, the standard of care, extends life for 6-18 months but has never cured a patient. Therefore, the discovery of novel treatments is imperative. DIPG tumors have 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. Low levels of ATF4 protect cells from stress, while sustained high-levels of ATF4 result in cell death. Because DIPG has a high baseline level of ATF4, we hypothesized that the ISR activators Sal003 and ONC201 would synergize and kill DIPG cells. After determining the IC25 of Sal003 and ONC201, we treated three patient-derived cell lines: JHH-DIPG1, SF-7761, and JHH-DIPG16A with low micromolar doses. To measure proliferation, we performed immunofluorescence staining for bromodeoxyuridine (BrdU) incorporation. The combination treatment significantly reduced BrdU incorporation (JHH-DIPG1 p=0.0026, SF-7761 p=0.0002, JHH-DIPG16A p<0.0001 by ANOVA and Dunnett’s multiple comparisons test compared to DMSO control). In all three cell lines, the combination also significantly reduced proliferation compared to monotherapy. We next measured apoptosis by staining for cleaved caspase-3 (CC3) and performing western blots for cleaved PARP. The combination of Sal003 and ONC201 significantly increased apoptosis as measured by CC3 immunofluorescence in comparison to DMSO (JHH-DIPG1 p<0.0001, SF-7761 p<0.0001, JHH-DIPG16A p<0.0001 by ANOVA and Dunnett’s multiple comparisons test compared to DMSO control). In all cell lines, combination therapy significantly increased CC3 positivity compared to single treatment. Western blots for cleaved PARP expression detected induction of apoptosis in all three cell lines treated with the combination over DMSO and monotherapy treated cells. In JHH-DIPG1 and SF-7761, the combination increased ATF4 expression. Since Sal003 is not yet available for clinical testing in humans, we will next investigate treatment with ONC201 and the well-tolerated ATF4 inducer fenretinide. The combination of ONC201 with another ISR activating agent has the potential to serve as a therapy for DIPG. Citation Format: Orlandi V. Novak, Antje Arnold, Charles Eberhart, Eric H. Raabe. Combination therapy activating the integrated stress response synergistically suppresses proliferation and induces apoptosis in diffuse intrinsic pontine glioma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5227.