Abstract 3849: Pygo2 deletion stimulates tertiary lymphoid structure formation and forms the cornerstone for a quadruple combination immunotherapy of prostate cancer

Abstract Introduction: Advanced prostate cancer (PCa) shows overwhelming de novo resistance to immune checkpoint blockade (ICB). We previously identified Pygopus 2 (Pygo2) as a critical epigenetic regulator in PCa to orchestrate the Pygo2/p53/Sp1/Kit/Ido1 pathway to foster a microenvironment hostile to cytotoxic T lymphocytes (CTLs), and inhibiting Pygo2 genetically or pharmacologically sensitized PCa mouse models to ICB and adoptive CTL transfer therapy (Sci Immunol. 2023; PMID: 36897957). Here, we continued this line of research and examined the effect of Pygo2 targeting on castration (androgen deprivation therapy), anti-MDSC therapy and ICB in genetically engineered mouse models. Methods: We crossed Pygo2 conditional null allele to the PB-Cre/Pten/Smad4 (DKO) mice and generated PB-Cre/Pten/Smad4/Pygo2 (TKO) PCa mouse model. Using this model, we performed a series of combination treatments including ICB, surgical castration, and anti-MDSC therapy SX682 (Cxcr1/2 inhibitor). Various immune-phenotyping techniques (single-cell RNA-seq, bulk RNA-seq, flow cytometry and immunohistochemical staining) were performed on treated tumors. Results: The best tumor protection phenotype came from the combination of Pygo2 deletion, castration, ICB and SX682 treatment. This quadruple combination regimen led to nearly curative outcome, rendering the mouse tumors extensively necrotic. scRNA-seq and IHC revealed that Pygo2 deletion promoted the formation of tertiary lymphoid structures (TLS) within the tumor microenvironment. The quadruple combination demonstrated the highest level of infiltration of B cells and CTLs. Emerging data suggest that Pygo2 loss instigates cancer-cell-intrinsic signaling distinct from the previously identified Kit/Ido1-dependent pathway and connects an antiviral mimicry state of the cancer cells to adaptive tumor immunity that culminates in TLS formation and maturation. Conclusions: Our study further consolidates Pygo2 as a valid and promising therapeutic target in combination immunotherapy of prostate cancer. The quadruple combination immunotherapy composed of Pygo2 inhibition, androgen deprivation therapy, SX682 and ICB demonstrates superior preclinical efficacy and should be further investigated for its clinical implications. Citation Format: Yan Liu, Ziyu Zeng, Yini Zhu, Xin Lu. Pygo2 deletion stimulates tertiary lymphoid structure formation and forms the cornerstone for a quadruple combination immunotherapy of prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 3849.