CO-REGULATION AND FUNCTIONAL COOPERATIVITY BETWEEN FOXM1 AND RHNO1 BIDIRECTIONAL GENES IN HIGH-GRADE SEROUS OVARIAN CANCER

PURPOSE: The FOXM1 transcription factor is canonically involved in cell cycle progression, but also promotes DNA repair and drug resistance, via its transcriptional activity. We previously reported that FOXM1 is located within the Ch.12p13.33 amplicon, which sustains copy number gains in ~60% of high-grade serous ovarian cancer (HGSC). More recently, we discovered that FOXM1 is arranged in a head-to-head configuration with RHNO1, a gene involved in the ATR/CHK1-dependent DNA replication stress (DRS) response. By virtue of its role in the DRS response, RHNO1 promotes homologous recombination (HR) DNA repair. We hypothesized that FOXM1 and RHNO1 are co-expressed in HGSC due to their bidirectional configuration, and that they functionally cooperate to promote HGSC cell survival, HR repair, and PARPi resistance. METHODS: We used 59 RACE, reporter constructs, and CRISPR-Cas9 (CRISPR) to characterize the FOXM1-RHNO1 bidirectional promoter. We correlated FOXM1 and RHNO1 mRNA expression and copy number in normal tissue and cancer data, including single cell RNA sequencing (scRNA-seq) of immortalized fallopian tube epithelial (FTE) cells and HGSC cells. We used RNAi and CRISPR to disrupt FOXM1 and/or RHNO1 in HGSC cells, and determined the resulting impact on gene expression, cell cycle, cell survival, HR repair, and olaparib (PARPi) sensitivity. RESULTS: FOXM1 and RHNO1 mRNA expression significantly correlated in normal tissues, including FTE, and in HGSC tumors and cell lines, both in bulk cells/tissues and at the single cell level. Co-expression appeared to result from the FOXM1-RHNO1 bidirectional promoter, which showed similar activity in each direction in cell models and correlated with endogenous mRNA expression. FOXM1 knockdown in HGSC cells led to enriched gene expression signatures for the G2/M checkpoint and reduced clonogenic survival. RHNO1 knockdown attenuated ATR/CHK1 signaling, reduced HR repair efficiency, and reduced HGSC clonogenic survival. Dual targeting of FOXM1 and RHNO1 using shRNA caused an additive effect on HR repair and clonogenic survival and caused synergistic sensitization of HGSC cells to olaparib treatment. Critically, bidirectional promoter targeting via a CRISPR-KRAB repressor recapitulated the effects of shRNA knockdown. Finally, FOXM1/RHNO1 bidirectional promoter repression led to olaparib sensitization in an in vitro HGSC model of acquired olaparib resistance. CONCLUSIONS: We demonstrate that FOXM1 and RHNO1 are frequently co-expressed in HGSC, most likely due to coordinated activity from their bidirectional promoter. Importantly, the FOXM1/RHNO1 bidirectional unit exhibits functional cooperativity for HR DNA repair and cell survival, and synergistically promotes olaparib resistance. Based on our data, we suggest that functionally interactive bidirectional gene modules are an under-appreciated oncogenic mechanism. Finally, we suggest that FOXM1 and RHNO1, or their associated pathways, are promising combinatorial therapeutic targets in HGSC. Citation Format: Carter J Barger, Linda Chee, Connor Branick, Kunle Odunsi, Lee Zou, Adam R. Karpf. CO-REGULATION AND FUNCTIONAL COOPERATIVITY BETWEEN FOXM1 AND RHNO1 BIDIRECTIONAL GENES IN HIGH-GRADE SEROUS OVARIAN CANCER [abstract]. In: Proceedings of the 12th Biennial Ovarian Cancer Research Symposium; Sep 13-15, 2018; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2019;25(22 Suppl):Abstract nr GMM-034.