203 RNA Polymerase II Meningioma Mutations Activate Forebrain Neurodevelopmental Pathways

Abstract

INTRODUCTION: RNA polymerase II, which transcribes DNA into RNA, is mutated in a subset of skull base meningiomas. Patient-derived Pol II mutant meningiomas are dysregulated in pathways directing neural crest differentiation and neurodevelopment. The molecular mechanism of the Pol II driver mutation has remained elusive, and may provide novel therapeutic insight. METHODS: The POLR2A Q403K meningioma driver mutation was modeled in the mouse embryonic stem cell system using CRISPR-Cas9. Metabolic labeling of nascent RNA was performed with 4-thiouridine (SLAMseq). Pol II ChIPseq was performed to calculate the travel ratio (proportion of initiated Pol II to elongating Pol II). POLR2A Q403K and wildtype mES cells differentiated over 12 days in permissive media, RNA was harvested over multiple days, and RNAseq was performed. RESULTS: POLR2A Q403K cells had a global increase in transcription per nascent RNA sequencing, as measured by T>C conversions (p = 5.71x10-66). For active and highly-paused genes, which were enriched for neurodevelopmental pathways (FDR = 3.52x10-25), a left shift in the mean travel ratio revealed defects in pause-release as the mechanism for increased levels of nascent transcription. Recapitulating human disease, POLR2A Q403K mES cells had significant activation of Hedgehog (Padj. = 2.4x10-3) and WNT (Padj. = 5.2x10-3) signaling pathways, and by day 12 of differentiation showed significant upregulation of pathways critical for rostral cortical development including forebrain neuronal differentiation (FDR = 4.86x10-4), telencephalon development (FDR = 4.30x10-4), and neuronal differentiation (FDR = 4.39x10-4). CONCLUSIONS: Meningioma Pol II mutations drive global increases in transcription via dysregulation of pause-release. Highly-paused genes, including master regulators of neural development, are disproportionately activated by mutant Pol II. This dysregulation leads to aberrant induction of neurodevelopmental signaling, including Hedgehog and WNT, and provides a rationale for pharmacologic targeting of these pathways.