Abstract 2362: Long non-coding RNA NORAD interaction with miR-346 impacts DNA damage response and anti-tumor immunity in prostate cancer
Authors: Fletcher Claire , Deng Lin , Orafidiya Folake , Yuan Wei , Figueiredo Ines , Gurel Bora , Leach Damien , Issa Fadi , Neeb Antje , Bogdan Denisa , Dobbs Felix , Philippou Yiannis , Murphy Emma A. , Zhao Shuang G. , Hester Joanna , Bryant Richard J. , Reed Simon H. , Knudsen Karen E. , Mills Ian G. , de Bono Johann , Bevan Charlotte L. 
Published: July 2021

Abstract
NORAD (NOn-Coding RNA Activated by DNA Damage) is a highly-abundant, evolutionarily-conserved lncRNA. It maintains mitosis, DNA damage repair (DDR), and chromosomal integrity through PUM1/2 sequestration (PUM1/2 activity increases turnover of DDR factors), and through formation of a TOPO2-containing complex critical for genome integrity. We show that NORAD activity is regulated by microRNA-346 (miR-346), which disrupts NORAD:PUM2, interaction, leading to PUM2 destabilization and derepression of PUM1/2 DDR targets in prostate cancer (PCa) cells. RNA-seq reveals widespread miR-346 dysregulation of DNA damage, DNA replication and cell cycle transcripts. A novel method for high resolution, amplification-free genome-wide mapping of double strand DNA breaks (DSBs) (INDUCE-seq) reveals miR-346 induces DSBs specifically at transcription start sites characterized by phospho-PolII/CTCF/ZFX binding - a phenomenon not previously described for any microRNA. Mechanistically, DSBs result from miR-346 activation of transcription, R-loop formation and replicative catastrophe. This results in rapid dose-dependent induction of DNA damage, leading to checkpoint activation and cytosolic DNA accumulation, rescuable by NORAD. This cytosolic DNA activates cytokine-inducing cGAS-STING/RIG-1 innate immune pathways. Indeed, RNA-seq analysis reveals the top NORAD-enriched pathway as interferon signaling, while cytokine arrays reveal secretion of pro-Treg, MDSC and TAM factors by NORAD-overexpressing cells. NORAD inversely correlates with tumor immune response in gene expression data sets, and expression-based immune infiltration scoring predicts increased M2 macrophages, and reduced NK, CD8+ve, Th1 and cytotoxic T cells in NORAD-high vs NORAD-low PCa, indicating an ‘immune-cold' microenvironment. Excitingly, NORAD silencing results in several thousand-fold increase in mature miR-346 without affecting pri-miR levels, supporting NORAD's ability to drive target-directed microRNA decay (TDMD) of miR-346 as a critical novel genome protection mechanism. However, miR-346-induced DNA damage is in part NORAD-independent, since miR-346 induces DSBs within 1h, and in contrast to most miRs is predominantly chromatin-bound (NORAD is cytoplasmic). Critically, miR-346 sensitizes PCa cells to DNA-damaging chemotherapy and PARP inhibition. MiR-346 expression is associated with improved PCa survival, and reduced in high vs low Gleason grade PCa. Notably, NORAD strongly correlates with DDR signatures in early-stage, but not advanced metastatic PCa. Despite its DDR-promoting activity, and in contrast to miR-346, it is associated with worse survival across multiple patient cohorts. In conclusion, these data demonstrate that the NORAD:miR-346 interaction determines DNA damage response and innate immune pathway activity to regulate tumor immune response in PCa.
Citation Format: Claire Fletcher, Lin Deng, Folake Orafidiya, Wei Yuan, Ines Figueiredo, Bora Gurel, Damien Leach, Fadi Issa, Antje Neeb, Denisa Bogdan, Felix Dobbs, Yiannis Philippou, Emma A. Murphy, Shuang G. Zhao, Joanna Hester, Richard J. Bryant, Simon H. Reed, Karen E. Knudsen, Ian G. Mills, Johann de Bono, Charlotte L. Bevan. Long non-coding RNA NORAD interaction with miR-346 impacts DNA damage response and anti-tumor immunity in prostate cancer [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 2362.