Abstract 680: Topoisomerase 2 beta facilitates chromatin reorganization during Androgen Receptor induced transcription and contributes to chromoplexy in prostate cancer

Abstract

Abstract Prostate cancer (PCa) is the most common malignancy and second leading cause of cancer death in American men. Androgen Receptor (AR) mediated transcriptional program is central to normal prostate homeostasis and drives PCa growth and survival. Chromoplexy, a highly complex genomic architecture with several intra- and inter-chromosomal segments joined in a chain, is among the most prominent genetic alterations that drive both prostate cancer initiation and progression, and often involves sites of AR transcription. Previous studies have shown that AR induced, topoisomerase 2 beta (TOP2B) mediated double strand breaks were recombinogenic and led to de novo formation of TMPRSS2-ERG fusion gene, shedding light on the potential role of TOP2B in chromoplexy formation. However, the precise role of TOP2B in AR transcription was not well understood. Here, we hypothesize that TOP2B is recruited to resolve topological constraints arising during induction of AR transcriptional programs, and its catalytic activity is required to facilitate or maintain chromosomal interactions optimal for transcriptional induction. We performed Chromosomal Conformation Capture related techniques (3C and HiC) on LNCaP cells before and after androgen stimulation and observed an increase in chromatin interactions within 15kb from promoters of AR target genes upon androgen induction. These interactions depended on TOP2B, as TOP2B catalytic inhibition or knockdown reduced them significantly. Furthermore, TOP2B Hi-CHIP revealed that TOP2B is involved in key enhancer-promoter looping and in several interactions among gene body, enhancers, promoters of AR target genes, and nearby topological associated domain borders. We went on to isolate which steps during AR transcription induction required TOP2B by examining chromatin localization of the key factors, including AR, cohesin (SMC1A), CTCF, histone 3 lysine 27 acetylation (H3K27ac), and total and phosphorylated RNA Polymerase II (RNAPII) using ChIP-seq. These experiments revealed that TOP2B was not required for AR binding nor for localization of H3K27ac marks. However, it was required for recruitment of cohesin to AR binding sites as well as to AR target gene promoters and gene bodies, for displacement of CTCF near AR target genes, and for localization and phosphorylation of RNAPII at AR target genes. These data nominate TOP2B as a key AR coactivator, assisting in the proper assembly of cohesin during transcription induction, and maintaining chromosomal interactions optimal for binding and activation of RNAPII. Intriguingly, sites of binding of TOP2B, as well as of cohesin, were highly associated with sites of chromoplexy complex rearrangements in human prostate cancers. Taken together, this work elucidates the role of TOP2B in AR-induced transcription, and implicates its involvement in chromoplexy formation in PCa. Citation Format: Minh-Tam N. Pham, Michael C. Haffner, Heather C. Wick, Jonathan B. Coulter, Anuj Gupta, Roshan V. Chikarmane, Harshath Gupta, Sarah Wheelan, William G. Nelson, Srinivasan Yegnasubramanian. Topoisomerase 2 beta facilitates chromatin reorganization during Androgen Receptor induced transcription and contributes to chromoplexy in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 680.