Functional Genomics Pipeline Reveals Therapeutic Dependencies In Diffuse Intrinsic Pontine Glioma.

Abstract Diffuse intrinsic pontine glioma (DIPG) is a highly aggressive and infiltrative pediatric brainstem tumor with universal lethality. Current treatment is limited to palliative radiotherapy. Molecular characterization of DIPG has revealed recurrent heterozygous mutation (p.K27M) in the Histone H3 variant genes; H3.1 (HIST1H3B, HIS1H3C) or H3.3 (H3F3A) in >80% of cases. While H3K27M function is still under heavy investigation, accumulating molecular evidence now suggests its subsequent role in tumorigenesis is driven through epigenetic dysregulation of the chromatin via the loss of tri-methylation markers. Co-occurring mutations in known oncogenes and amplification in cancer prone proliferative pathways further contribute to the DIPG's diverse molecular landscape and may represent therapeutic vulnerabilities. The purpose of this present study is to utilize a functional genomics pipeline to investigate the intrinsic molecular mechanisms of H3K27M DIPG. Identification of novel molecular dependencies and pathways that contribute to overall disease progression serve as potential targets for therapeutic exploitation and development for clinical utility. Sixteen established and validated patient derived cell lines (10 H3.3K27M, 4 H3.1K27M and 2 Wt), obtained from external collaborators were used in this study. High throughput screening for DIPG drug sensitivity was carried out using a panel of 2048 molecular compounds. Cell viability data following 72 hours treatment was z- score transformed and potential compounds identified based on a z-score ≤-1.5 and no effect in a neural stem cell (NSC) control line. Highly prevalent targets identified from this primary screen include HDAC, Proteasome, Topoisomerase and Microtubule Associated inhibitors. In parallel, a 300 gene pooled CRISPR/Cas9 loss of function screen was used to determine key DIPG survival dependencies. Cas9 expressing DIPG cell lines (3 H3.1K27M and 3 H3.3K27M) were transduced with 1200 gRNAs and cultured for 21-28 day period. Next Generation Sequencing (NGS) of samples was used to determine gRNA depletion and a set z score threshold of ≤-1.5 to distinguish potential “hits”. Hierarchical clustering revealed genotype specific patterns suggesting differing tumorigenic mechanisms between H3.1 and H3.3 K27M DIPG. Comparison between CRISPR and drug screens revealed 24 molecular targets with therapeutic sensitivity and DIPG cell survival dependency. We are currently carrying out validation studies of these targets for therapeutic development, effectively demonstrating a functional genomics pipeline is able to identify therapeutic dependencies in DIPG. Citation Format: Sarah Parackal, Gabrielle Bradshaw, Claire Sun, Wai Chin Chong, Paul Daniel, Samantha Jayasekara, Duncan Crombie, Ron Firestein, Jason Cain, Department of Molecular Translational Science, Monash University. Functional genomics pipeline reveals therapeutic dependencies in diffuse intrinsic pontine glioma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3029.