Abstract PD1-01: Estrogen therapy induces R-loop-dependent DNA damage that can be enhanced by PARP inhibition to improve response in ER+ breast cancer
Authors: Traphagen Nicole A. , Tau Steven , Jiang Amanda , Wells Jason D. , Hosford Sarah R. , Goen Abigail E. , Demidenko Eugene 
Published: March 2022

Abstract
Treatment with estrogens can elicit anti-cancer effects in a subset of patients with anti-estrogen-resistant ER+ breast cancer. Clinical studies suggest that ~30% of postmenopausal patients with advanced ER+ breast cancer are likely to respond to estrogen, translating into thousands of patients who could benefit from this treatment each year. Despite the proven efficacy of estrogen therapy, it is under-utilized in the clinic due in large part to the unknown mechanism of action. We found that overexpression of ER confers resistance to estrogen deprivation through upregulation of estrogen-independent ER transcriptional activity. Further, this overexpression of ER confers sensitivity to treatment with the natural estrogen 17β-estradiol (E2) through stimulation of high levels of ER transcriptional activity. Using cell line- and patient-derived xenograft (PDX) models, including the novel PDX model CTG-3346, we aimed to determine the mechanism through which high levels of ER transcriptional activation elicit anti-cancer effects. RNA sequencing revealed a DNA damage response and G2/M checkpoint activation upon treatment with estrogen therapy. We further determined that treatment with E2 induces DNA damage specifically in ER-overexpressing cells, and knockdown of ER prevents E2-induced DNA damage. We performed a genome-wide CRISPR/Cas9 knockout screen to determine gene essentiality in the context of estrogen therapy. This revealed that knockout of CDK2/4/7/9, as well as MYC target genes, rescues from the cytotoxic effects of E2. This suggests that proliferation is required prior to apoptosis from E2 treatment. In agreement, E2 therapy initially induces a proliferative burst which precedes apoptosis. Blocking proliferation through pharmacological inhibition of CDK4/6 or knockdown of MYC prevented induction of DNA damage upon E2 treatment. MYC is an ER target gene, so high levels of ER transcriptional activity induce overexpression of MYC. Both ER and MYC are transcription factors that stimulate increased proliferation and transcription, so we hypothesized that E2/ER-induced DNA damage is a result of replication stress in ER-overexpressing cells. We found that E2 induces the formation of DNA:RNA hybrids called R-loops. R-loops occur when transcribed RNA anneals to the DNA template, leaving a displaced single-stranded DNA prone to breakage and replication stress. Reversal of R-loops through overexpression of the endonuclease RNase H1, which specifically degrades RNA in DNA:RNA hybrids, prevented the accumulation of DNA damage upon E2 treatment. Poly (ADP-ribose) polymerase-1 (PARP) has been implicated in the repair of R-loop-associated DNA damage. We therefore hypothesized that inhibition of PARP with FDA-approved drugs would enhance the effects of E2 therapy. Indeed, treatment with olaparib enhanced E2-induced DNA damage and growth-inhibition in vitro. In two PDX models, olaparib had minimal effects as a single agent but synergized with E2 to enhance anti-cancer effects. Collectively, this work implicates estrogen-induced DNA damage through R-loop formation and replication stress as the mechanism of action of estrogen therapy. Importantly, these results suggest that estrogen therapy should not be used in combination with CDK4/6 inhibitors. Our finding that E2 therapy synergizes with olaparib even in models that do not respond to olaparib monotherapy presents a novel use for PARP inhibitors outside of the setting of germline BRCA1/2 mutations.
Citation Format: Nicole A. Traphagen, Steven Tau, Amanda Jiang, Jason D. Wells, Sarah R. Hosford, Abigail E. Goen, Eugene Demidenko. Estrogen therapy induces R-loop-dependent DNA damage that can be enhanced by PARP inhibition to improve response in ER+ breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr PD1-01.