DIPG-03. IDENTIFICATION OF NEW THERAPEUTIC TARGETS BY ARRAYED CRISPRA-BASED APPROACH TO MAP RADIORESISTANCE IN DMG

Abstract

Abstract Pediatric Diffuse Midline Glioma (DMG) is amongst the most aggressive childhood brain tumors. The dismal prognosis is less than a year for children diagnosed. Radiotherapy (RT) remains the only standard treatment that provides only transient relief of clinical symptoms of DMG patients. Poor survival is associated with radio-resistance due in part to P53 mutational status and to invasive tumor properties related to epigenetic dysregulation. Over 80% of DMG patients harbor oncohistone 3 mutations (H3K27M) leading to global genome hypomethylation and transcriptional disruption. Transcriptional factors are known as master regulators of the genome but their implication in response to radiation treatment remains unclear. We developed an arrayed-CRISPR screen strategy to understand the role of transcription factors in radioresistance mechanisms in DMG-patient-derived cells. We performed arrayed-CRISPRa screening in multiwell plates with DMG cells expressing the dCas9-VP64, infected with gRNA lentiviral library to target each transcription factor (one transcription factor overexpressed per well). Each plate was irradiated fractionally (2Gy/day) and after irradiation stopped a comparison between cells overexpressing one targeted transcription to internal controls (non-targeted-gRNA infected cells) has been conducted. Transcription factor “hits” identification is based on Log2FC (logarithm 2 fold-change) with a value higher than 0.6, considering these targets as potentially involved in DMG cell radio-resistance and/or proliferation mechanisms. We identified 82 “hits” with more than 30% described as involved in radio-resistance mechanisms and/or tumorigenesis process, sustaining our screen results. We focus on “hits” with a Log2FC higher than 1 to identify the most robust targets playing a role in resistance to radiation. Currently, DMG cells are being treated with RT combination with pharmacological inhibitors to validate our candidate target involvement in DMG radio-resistance. Our goal is to achieve a better understanding of resistance mechanisms to identify therapeutic treatments to combine with radiotherapy to overcome radio-resistance developed by DMG cells.