Abstract Cancer cells are continually challenged by both intrinsic and extrinsic stresses that interfere with DNA replication, causing DNA replication stress (DRS). DRS can fuel cancer progression by increasing genomic instability, but if left unregulated, it can also cause widespread DNA damage and cell death. Consequently, cancer cells are highly dependent on the ATR/Chk1 signaling pathway to regulate DRS and ensure survival. Rad9-Hus1-Rad1 interacting nuclear orphan 1 (RHNO1) is a novel component of the ATR/Chk1 pathway and is often overexpressed in cancer cells with high DRS, where it contributes to chemotherapy resistance. However, the precise function of RHNO1 in the DRS response is still largely unknown. Here, we investigated the roles of RHNO1 in cancer progression and DRS response using an ovarian cancer cell (OVC) model in both in vitro and in vivo systems. Furthermore, we generated an endogenously HiBiT-tagged and degron-tagged RHNO1 cell line to precisely track RHNO1 levels and temporally deplete RHNO1 during different phases of the DRS response in HEK293T cells. Depletion of RHNO1 significantly reduces OVC proliferation rate, clonogenicity, and in vivo tumor growth, while extending host mouse survival. RHNO1 knockdown sensitized cells to hydroxyurea (HU), increasing cell death and micronuclei formation. Mechanistically, RHNO1 was upregulated and stabilized following DRS. Notably, we show that RHNO1 stabilization is mediated by ATR/Chk1 phosphorylation, which is also required for RHNO1 translocation to stressed replication forks. Interestingly, chromatin recruitment of RHNO1 appears to be independent of 9-1-1, a DNA clamp complex previously known to bind RHNO1. Furthermore, cells with stable RHNO1 knockdown demonstrated normal initial ATR/Chk1 signaling activation in response to HU treatment, however, at later time points, the absence of RHNO1 caused premature termination of ATR/Chk1 signaling despite the persistence of DRS. These findings are consistent with a dispensable role of RHNO1 in the initial activation of the ATR/Chk1 pathway but a key role in sustaining the ATR/Chk1 response. In summary, we demonstrated the RHNO1 promote critical OVC phenotypes and uncovered a novel positive feedback loop between RHNO1 and ATR/Chk1 signaling. This work identifies RHNO1 as a key component in maintaining the DRS response, highlighting its potential as a therapeutic target for cancers reliant on ATR/Chk1 signaling. Citation Format: Niphat Jirapongwattana, Carley M. Conover, Catalina Trujillo Jaramillo, Adam R. Karpf. A RHNO1-ATR/Chk1 positive feedback loop sustains the cellular DNA replication stress response abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 4684.
Jirapongwattana et al. (Fri,) studied this question.