Abstract B36: FOXM1 induces DNA replication stress, and its bidirectional gene partner RHNO1 participates in the DNA replication stress response, in high-grade serous ovarian cancer

Abstract

Abstract Purpose: FOXM1 is located at 12p13.33, which shows copy number gains in the majority of high-grade serous ovarian cancer (HGSC) cases. The 12p13.33 amplicon contains 33 genes, some of which may cooperate with FOXM1 to exert oncogenic function. Although FOXM1 overexpression is associated with genomic instability in cancer, whether FOXM1 contributes directly to this phenotype is unclear. We hypothesized that FOXM1 induces DNA replication stress (DRS), resulting in ATR-CHK1 activation. Intriguingly, we noted that RHNO1, a component of the 9-1-1 complex required for ATR-CHK1 activation, is contained within the 12p13.33 amplicon and is arranged in a head-to-head orientation with FOXM1. Here, we characterized the FOXM1/RHNO1 bidirectional promoter, FOXM1/RHNO1 expression, and determined the role of FOXM1/RHNO1 in DRS, the DRS response, and clonogenic growth of HGSC cells. Methods: We used reporter assays to characterize the FOXM1/RHNO1 bidirectional promoter and FOXM1 transcriptional activity. We analyzed copy number, mRNA, and protein expression datasets in TCGA, CCLE, and GTEX. We measured FOXM1/RHNO1 expression in HGSC cell lines and tumors. We overexpressed FOXM1 in human immortalized fallopian tube epithelial (FTE) cells to determine its contribution to DRS and DNA damage. We knocked down RHNO1 in HGSC cells to determine its contribution to the DRS response and clonogenic growth. We performed Western blot analyses to measure markers of DRS and DNA damage. We characterized FOXM1-induced DNA damage at single-cell resolution using Comet assays and flow cytometry measurement of γ-H2AX. Results: FOXM1/RHNO1 showed copy number gains or amplifications in 58% of TCGA HGSC tumors, and FOXM1/RHNO1 mRNA expression significantly correlated in TCGA HGSC tumors, CCLE HGSC lines, and GTEX normal tissues. The FOXM1/RHNO1 bidirectional promoter showed balanced activity in each direction in FTE and HGSC cells, and bidirectional promoter activity correlated with endogenous mRNA expression. TCGA HGSC data indicated that FOXM1 expression associates with markers of DNA replication, global copy number alterations, and CHK1-Ser345-P, a canonical marker of DRS. In agreement, FOXM1 overexpression in FTE cells induced phosphorylation of ATR-Thr1989, CHK1-Ser317/345, H2AX-Ser139, and the proportion of DNA in Comet tails. In contrast, a DNA-binding domain mutant of FOXM1 did not induce DRS or DNA damage. RHNO1 knockdown attenuated CHK1-Ser345-P in response to DRS and RHNO1 shRNA knockdown or CRISPR-Cas9 knockout severely reduced HGSC cell clonogenic growth. Importantly, the effects of RHNO1 disruption were rescued by WT RHNO1 expression, but not by a RHNO1 mutant defective for interaction with the 9-1-1 complex. Conclusion: Our data reveal that FOXM1/RHNO1 share a bidirectional promoter and that this may account for their frequent coexpression in HGSC. FOXM1 induces DRS, and this is dependent on its ability to bind DNA. Conversely, RHNO1 contributes to ATR-CHK1 signaling and promotes the clonogenic growth of HGSC cells. We hypothesize that balanced FOXM1/RHNO1 expression promotes HGSC development and progression. Ongoing studies are characterizing the impact of FOXM1/RHNO1 on DRS and genomic instability in model systems. Finally, our data suggest that the ATR-CHK1 pathway is a synthetic lethal vulnerability in HGSC tumors that overexpress FOXM1. Citation Format: Carter J. Barger, Linda Chee, Connor Branick, Kunle Odunsi, Ronny Drapkin, Adam R. Karpf. FOXM1 induces DNA replication stress, and its bidirectional gene partner RHNO1 participates in the DNA replication stress response, in high-grade serous ovarian cancer. [abstract]. In: Proceedings of the AACR Conference: Addressing Critical Questions in Ovarian Cancer Research and Treatment; Oct 1-4, 2017; Pittsburgh, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(15_Suppl):Abstract nr B36.