Transcriptomics-Guided High-Throughput Drug Screening Identifies Potent Therapies for P53 Pathway Alterated DIPG/DMG

Abstract Background Diffuse midline gliomas (DMGs), particularly diffuse intrinsic pontine gliomas (DIPGs), are aggressive pediatric brain tumors with a median survival of less than 12 months. Notably, approximately 70% of these tumors harbor P53 pathway alterations, including TP53 or PPM1D mutations. Identifying precision therapies for this subset is crucial. This study aims to employ transcriptomics-guided high-throughput drug screening to identify effective treatments for DIPG/DMG with P53 pathway alterations. Methods Transcriptomic profiling of 98 patient samples containing 31 DIPGs revealed key activated pathways. Patient-derived cell lines were subjected to high-throughput screening using a cell cycle-targeting drug library. Lead candidates were validated both in vitro and in orthotopic xenograft models, while combination therapies were assessed for their ability to overcome TP53-mutant resistance. Results Transcriptomic analysis revealed activation of P53 and cell cycle pathways in DIPGs. High-throughput drug screening identified SN-38, a topoisomerase I inhibitor, as selectively targeting TP53 wild-type tumor cells by inducing G2 arrest and apoptosis. TP53 knockdown abolished SN-38’s efficacy, while PPM1D knockdown enhanced sensitivity, confirming a TP53-dependent mechanism. Conversely, TP53-mutated cells exhibited SN-38 resistance via ATR pathway activation. Combining SN-38 with the ATR inhibitor AZ20 restored apoptosis and suppressed TP53-mutant tumor growth in vitro and in vivo. Conclusions Guided by transcriptomic profiling, this study utilized high-throughput drug screening to identify SN-38 as a potential therapy for TP53 wild-type DIPG/DMG, while the SN-38 and AZ20 combination was effective against TP53-mutant tumors. This approach provides a promising treatment strategy for this malignancy and establishes a novel paradigm for drug screening in DIPG/DMG.