Imipridone treatment activates canonical NFkB and inflammatory signaling in THP1 differentiated macrophages 2258348

Abstract

Abstract Introduction Diffuse intrinsic pontine glioma (DIPG) is a devastating pediatric high-grade glioma and is the leading cause of brain cancer related mortality in children. Modeyso (ONC201/dordaviprone), the first in class imipridone, has been recently FDA approved for use in progressive H3K27M mutated diffuse midline gliomas (DMG) such as DIPG. We aim to better understand the effects of ONC201 on the predominantly-macrophage immune microenvironment of DIPG by characterizing their direct impact on bone marrow derived macrophages. Methods We conducted gene set enrichment analysis on RNA sequencing data derived from ONC201 DMG clinical trials. We next aimed to better understand NFκB signaling and cytokine production following imipridone treatment. We utilized the THP1 human-monocyte cancer cell line as a model for bone marrow derived macrophages by differentiating them using 200ng/mL PMA for 48 hours. Following differentiation, we treated the cells with 5µM or 2µM ONC201, 1.5µM or 0.75µM ONC206, or 0.1µM or 0.05µM ONC212 and collected lysates for western blot. Results Using the hallmarks gene set, we found that increased expression of genes associated with a baseline inflammatory, IFNα and IFNγ response were associated with patient radiographic response. By western blot, we found a dose dependent increase in protein levels of canonical NFκB subunits and increased phosphorylation of p65. At later time-points, we found an increase in ATF4 levels and subsequent decrease in inflammatory protein expression. We also identified an increase in pro-IL1β suggesting an activation of the NLRP3 inflammasome. However, we did not find an increase in the active form through western blotting. Conclusion We have identified a link between a more immunologically active TME and response to ONC201 in patients with DMG, and that changes in NFkB signaling occurred following imipridone treatment of the THP1 cell line. We will continue our analysis of changes in cytokine production through ELISA and luciferase reporter assays. Funding Source n/a Topic Categories Tumor Immunology: Cellular Responses and Tumor Microevironment (TIME)