Abstract A046: MAPK pathway mediates resistance to p53 reactivation therapies in Diffuse Midline Glioma

Every year, up to 300 children are diagnosed with H3K27-altered Diffuse Midline Gliomas (DMGs), a type of high-grade glioma (HGG) that originates in the midline structures of the brain. Its location renders surgical removal challenging, and current treatment options, namely chemotherapy and radiation, are ineffective. This highlights the urgency for a better understanding of the disease and more effective therapies. While DMGs are aggressive tumors, a subset retains wild-type TP53, presenting p53 reactivation therapy as a potential therapeutic strategy. However, the p53 pathway may be functionally suppressed by alteration of negative regulators such as PPM1D and MDM2/4. PPM1D, a phosphatase with several targets in the DNA Damage Response pathway including p53, is frequently altered in DMGs, largely mutually exclusively with TP53 mutations. Whole Genome Sequencing of approximately 180 pediatric HGGs found that 10% of DMGs harbor nonsense mutations in PPM1D, leading to a truncated and more stable phosphatase (PPM1Dtr). PPM1Dtr DMGs therefore present a relevant model for exploring the therapeutic potential of p53 reactivation therapy in DMGs. To investigate potential mechanisms of resistance to p53 reactivation in DMGs, a CRISPR activation (CRISPRa) screen was performed in a p53 wild-type, PPM1Dtr patient derived cell line (PDCL) under treatment with inhibitors of PPM1D and MDM2. The screen identified MAPK pathway genes as significantly enriched, suggesting the pathway’s involvement in resisting p53 reactivation. To validate that the hits are responsible for the phenotype, the PDCL constitutively expressing dCas9 fused to transcriptional activator VP64 was transduced with sgRNAs targeting those hits to activate their endogenous transcription. CRISPRa-mediated upregulation was confirmed in the transduced cells via western blotting. To assess their sensitivity to p53 reactivation, dose-response curves were performed using CellTiter-Glo assay to quantify cell viability. Cells transduced with CRISPRa sgRNAs targeting candidate genes displayed a rightward shift in their curves and higher IC50 values compared to cells transduced with non-targeting control sgRNAs. As a follow-up experiment, the cells were monitored for proliferation in drug treatment using a live-cell imaging system. Cells transduced with CRISPRa sgRNAs targeting candidate genes increased proliferation compared to the controls, further supporting the role of the MAPK pathway in conferring resistance to p53 reactivation therapy. Citation Format: Adam F Kebede, Eric Morin, Ipsita Kundu, Apichaya Sethaudom, Nicolas Poux, Daren Zhang, Leslie Lupien, Veronica Rendo, Timothy N Phoenix, Pratiti Bandopadhayay. MAPK pathway mediates resistance to p53 reactivation therapies in Diffuse Midline Glioma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Discovery and Innovation in Pediatric Cancer— From Biology to Breakthrough Therapies; 2025 Sep 25-28; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2025;85(18_Suppl_2):Abstract nr A046.