Abstract 4986: Single-cell multimodal characterization of ovarian cancer microenvironment under neoadjuvant niraparib treatment informs an effective therapeutic strategy of suppressing HRD-dependent eTreg hyper-expansion

Abstract Homologous recombination deficiency (HRD) is a widespread hallmark in cancer, rendering tumors susceptible to PARP inhibition. Despite this well-established tumor-intrinsic vulnerability, the effects of HRD and related therapies on the tumor microenvironment (TME) remain poorly understood. In this study, utilizing single-cell RNA and T cell receptor profiling (scRNA/TCR-seq), we characterized 79 high-grade serous ovarian cancer (HGSOC) samples, both pre- and post-treatment, from 46 patients treated with neoadjuvant niraparib or chemotherapy in a phase II clinical trial (NCT04507841). This effort established the most comprehensive HGSOC TME atlas to date, encompassing over 700,000 cells. By integrating multiplex immunofluorescence imaging, flow cytometry, and bulk TCR-seq data from additional tumors and matched peripheral blood samples, we systematically assessed HRD-induced and treatment-perturbed dynamics of tumor-infiltrating and circulating immune cells. Surprisingly, among over 120 cell states across 10 cell types, effector regulatory CD4+ T cells (eTreg) exhibited the strongest enrichment in treatment-naïve HRD tumors and a marked treatment-induced reduction. Further analyses of transcriptional states and clonal kinetics revealed HRD-driven functional coordination between eTregs and exhausted CD8+ T cells (Tex), both displaying uniquely heightened tumor reactivity signatures and clonal co-expansions. The intra-population heterogeneity analysis of Tregs and Texs further demonstrated an effectiveness gradient, wherein the terminal subsets had a greater presence in HRD and were reversed by both niraparib and chemotherapy treatments. Delineating the clonal evolution of Tregs and Texs, we identified their notable crosstalk with other tumor-infiltrating T cell populations while remaining remarkably isolated from the peripheral blood repertoire, in line with their terminally differentiated status. Furthermore, we observed widespread, chronic activation of interferon signaling across TME components, downstream of HRD. This orchestrated CD4+ T cell anergy induction into Tregs, mainly through direct engagement by cancer cells that upregulated MHC-II molecules but lacked essential co-stimulatory ligands. Finally, we experimentally validated a therapeutic strategy to conjunctively and effectively break down these factors in HGSOC by combining PARP inhibition and eTreg deletion through a humanized therapeutic anti-CCR8 antibody in multiple syngeneic HGSOC mouse models. Our study unravels a key immunoregulatory role of eTregs in the HGSOC TME amplified by HRD. These findings underscore the underappreciated value of eTreg-focused therapeutics as novel immunotherapies for HGSOC and other genomically unstable cancers. Citation Format: Yikai Luo, Yu Xia, Dan Liu, Xiong Li, Huayi Li, Yi Zhao, Ding Ma, Yong Fang, Han Liang, Qinglei Gao. Single-cell multimodal characterization of ovarian cancer microenvironment under neoadjuvant niraparib treatment informs an effective therapeutic strategy of suppressing HRD-dependent eTreg hyper-expansion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4986.