LGG-57. Bh3 Mimetics Improve Response to Autophagy Inhibition in Brafv600e Gliomas

Abstract BACKGROUND Autophagy inhibition improves the effectiveness of RAF pathway inhibition (RAFi) and overcomes resistance mechanisms. Mechanistic links between autophagy and apoptotic cell death may explain this ability. BH3 mimetics with autophagy and RAFi further improves this response. METHODS RAFi sensitive (MAF 794, AM38) and resistant (MAF 794R, MAF 905-3, AM38R, B76) BRAFV600E tumor cell lines were analyzed at baseline, following RAFi (vemurafenib), autophagy inhibition (chloroquine or shRNAs), BH3 protein inhibition (navitoclax or shRNAs), and combination therapy. Growth assays and caspase activation were monitored by Incucyte Zoom. qRT-PCR evaluated key pro-apoptotic BH3-only members of the BCL-2 family. BH3 profiling was completed using the Letai JC-1 plate-based protocol. Western blot analysis assessed protein levels. Cell Titer Glo determined cell viability. RESULTS Combination therapy increased key pro-apoptotic BH3-only proteins including PUMA. shRNAs targeting ATG5 and ATG7 (proteins required for autophagosome formation) also increased PUMA protein and mRNA levels following RAFi. This suggests autophagy-mediated regulation of BH3 proteins helps determine cellular apoptotic thresholds. Combined autophagy and RAFi increased caspase activation and cell death compared to single drug treatment. BH3 profiling demonstrated BCL-2 dependence to inhibit apoptosis. Inhibition of pro-survival BCL-2 family members BCL-2 and BCL-XL (pharmacologically and genetically) with RAFi, MEKi, and autophagy inhibition further increased caspase activation and reduced cell viability. CONCLUSIONS BH3 mimetics competitively bind pro-survival BCL-2 family members, increasing apoptosis. Autophagy inhibition improves treatment response by overcoming the apoptotic threshold in RAFi resistant cells and magnifying the apoptotic response in sensitive cells. BH3 profiling reveals CNS BRAFV600E are BCL-2 and BCL-XL dependent, making them good candidates for additional treatment with BH3 mimetics. The addition of navitoclax increased caspase activation and reduced cell viability. Autophagy inhibition can magnify this response even more. This presents an attractive treatment for MAPK activated CNS tumors by enhancing apoptotic cell death by targeting the MAPK pathway, autophagy and BH3.