MECHANISMS OF POL II ELONGATION IN DIFFUSE MIDLINE GLIOMA

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Project Summary/Abstract Diffuse midline gliomas (DMGs) are aggressive brain tumors in both children and adults with no effective therapies. Over 80% of pediatric DMG and a majority of adult DMG harbor non-synonymous mutations in histone H3 (H3.3K27M and H3.1K27M), which dysregulate chromatin and result in aberrant Pol II transcription in brain tumors compared to normal tissue. Aberrant transcription is not only a cause of tumorigenesis, but also a druggable vulnerability as there are multiple studies investigating the utility of Pol II and epigenome-targeting drugs for the treatment of cancer. Despite the initial promise of compounds targeting transcriptional dysregulation as potential cancer therapies, none of the drugs under investigation in clinical trials have improved DMG patient survival thus far. A critical barrier to the discovery of more effective and less toxic therapies for DMG is our lack of functional knowledge regarding the molecular pathways connecting H3K27M mutant histones to tumor initiation and progression. We have recently conducted CRISPR screens identifying multiple regulators of Pol II transcriptional elongation as genetic dependencies in H3K27M mutant cells, including Elongin B (ELOB), and its binding partner, the Von Hippel-Lindau disease protein and tumor suppressor, VHL. Further preliminary studies reveal that genetic ablation of ELOB alters the chromatin binding patterns of H3K27M mutant histones, and disrupts DMG gene expression. These data support our overall hypothesis that H3K27M mutant histones enlist the activity of specific gene regulatory complexes to induce Pol II transcriptional deregulation and trigger DMG tumorigenesis. Our broad, long-term goal is to identity genes and molecular pathways driving glioma-specific transcriptional programs and to determine their roles in brain cancer development and progression. We propose the following specific aims: 1) Establish roles for Pol II elongation regulators in pre-clinical mouse models of DMG and investigate genetic interactions between the elongation machinery and H3K27M; 2) Apply cutting edge next generation sequencing analyses to dissect mechanisms of crosstalk between Pol II regulatory proteins and H3K27M and determine their roles in promoting abnormal gene regulation in DMG; 3) Investigate an unexpected oncogenic role for Elongin/VHL complexes in promoting malignant gene expression patterns and DMG tumor growth. The significance of this work is that these studies will reveal new insights into how H3K27M oncogenic histones co-opt specific Pol II regulators to promote tumorigenesis, and will uncover molecular mechanisms driving aberrant chromatin and gene regulation in DMG and other cancers.