Abstract 1838: Accumulation of DNA:RNA hybrids (R-loops) and cell death after YM155 treatment of anaplastic thyroid cancer hints at a novel therapeutic approach

Abstract

Abstract Introduction: Anaplastic thyroid cancer (ATC) is a rapidly fatal disease with a median survival of less than 6 months. Although rare, ATC is responsible for a disproportionate number of thyroid cancer deaths and is highly resistant to common cancer therapies. YM155 is a small molecule drug identified in a screen by the National Cancer Institute that potently kills ATC cells in vitro and in vivo. We have previously shown the ability of YM155 to inhibit ATC cell proliferation, however, the mechanism is still unclear. This study investigates the DNA damage and accumulation of R-loops that is seen with YM155 treatment of ATC cells. R-loops are structures characterized by a DNA:RNA hybrid with a displaced single strand of DNA and can be formed by transcription-replication collision, nucleotide depletion, and other situations where transcription is stalled. Materials & Methods: Clonogenic assays were performed in four ATC cell lines (ACT1, THJ11T, THJ16T, and THJ29T) to study the effect of YM155 on cell survival. DNA damage was measured by COMET assay and by counting foci of phosphorylated histone H2AX (γH2AX). R-loops were measured via immunofluorescent microscopy with S9.6 anti-R-loop antibody and anti-nucleolin antibody in conjunction with an AI algorithm using inFormTM cell segmentation software to quantify non-nucleolar nuclear R-loop signal. Results: We show in multiple ATC cell lines that YM155 potently kills ATC cells at low nanomolar concentrations. COMET assay found significant increases in DNA damage after treatment with 10 nM YM155, with large increases in tail moment seen by 24 hours in three ATC cell lines (p &lt 0.001 for all) with no change in primary benign thyroid (BT) cells. Similar trends were seen when measuring formation of γH2AX foci, a marker for double strand DNA breaks. R-loop immunofluorescence more than doubled with 10 nM YM155 treatment (p &lt 0.001) similar to hydroxyurea, a positive control. This finding was confirmed when addition of RNAseH, which is specific for RNA in DNA:RNA hybrids, reduced R-loop signal down to baseline levels. Conclusion: YM155's ability to kill ATC cells at low concentrations while sparing BT cells coincides with increases in DNA damage and accumulation of R-loops. A variety of factors aid in the resolution of R-loops, and we have previously shown patient data exhibiting overexpression of DNA topoisomerase genes TOP1 and TOP2A. Combined with published information describing the ability of YM155 to inhibit Topoisomerase Iiα (Top2α) activity, these results suggest a newly revealed, targetable aspect of ATC pathophysiology and a potential novel role for the involvement of Top2α in the resolution of R-loops. Citation Format: Ryan Mackay, Qinqin Xu, Paul Weinberger. Accumulation of DNA:RNA hybrids (R-loops) and cell death after YM155 treatment of anaplastic thyroid cancer hints at a novel therapeutic approach [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1838.