Abstract Diffuse Midline Glioma (DMG) is a highly aggressive brainstem tumour, with a dismal median survival of less than one year despite extensive clinical trials. Over 80% of DMGs harbour histone-altering mutations, notably H3K27M, which results in global hypomethylation, dysregulate gene expression, and contributes to the tumour’s aggressiveness. We and others have shown that DMGs harbouring TP53 mutations exhibit resistance to radiation therapy (RT); however, the precise role of mutant TP53 and other transcription factors (TFs) in mediating radioresistance remains unclear. To address this, we developed an advanced CRISPR activation (CRISPRa) platform to systematically screen and validate TFs involved in DMG fitness and response to RT. Utilizing DMG-patient-derived cells expressing dCas9-VP64, we performed an arrayed CRISPRa screen targeting 1,889 TFs and appropriate non-targeting controls, in combination with fractionated radiotherapy (2 Gy/day for four days). Hits were identified based on a Log2FC threshold of 0.6, revealing 30 TFs implicated in DMG survival and mechanisms of radiosensitization or radioresistance. In support of our primary screen, we observed that 15 hits have already been associated in mechanisms of radioresistance and/or tumorigenesis. To further investigate the roles of our candidate TFs, a secondary CRISPRa screen was conducted to distinguish factors deriving general cellular fitness from those directly modulating RT resistance. We are actively targeting the most promising hits with pharmacological inhibitors in combination with radiation therapy. Concurrently, we are mapping the TF mechanisms of tumorigenicity within the context of cellular addiction to H3K27M. Our study aims to uncover the mechanisms driving radioresistance in DMG cells to identify novel therapeutic strategies that can be combined with radiotherapy to overcome this resistance.
Bimbati et al. (Fri,) studied this question.