Abstract B086: Anchored in Resistance: Overcoming Nidogen-2 (NID2) - mediated Immunoresistance in Ovarian Cancer with Ras and Bcl-xl Inhibition

Abstract Background: Adoptive cell therapies (ACTs), such as chimeric antigen receptor (CAR) T cells and bispecific T cell engagers (BiTEDs), offer promising immunotherapeutic strategies for ovarian cancer (OC) due to their robust cytolytic capacity and ability to establish long-term immune surveillance. However, despite encouraging preclinical data, clinical responses remain limited. This discrepancy highlights an urgent need to uncover tumor-intrinsic resistance mechanisms that may impair ACT efficacy in OC. Methods: To investigate mechanisms of immunotherapy (IT) resistance in OC, we developed OVCAR3-derived cell models resistant to Ipilimumab/Nivolumab in vivo (OV3 IE ) and to BiTEDs in vitro (OV3 R ), and established patient-derived organoids (PDOs) from IT responders and non-responders. Candidate mediators of resistance were identified via RNA sequencing of OV3 IE cells and validated by Western blot. Tumor cell sensitivity to granzyme/perforin (Gr/PFN), Muc16/CA-125 (4h11-28ζ) - targeting CAR T cells, and Muc16/CA-125 (4H11) - targeting BiTEDs was assessed in cytotoxicity assays using Incucyte live-cell imaging. The functional role of the extracellular matrix protein Nidogen-2 (NID2) in resistance was evaluated through gene overexpression, knockdown, and knockout. Western blot analyses of Ras and Bcl-xl signalling pathways were performed, and pharmacologic inhibitors targeting these pathways were used to test their potential to reverse immune resistance. Results: IT-resistant cell lines and PDOs exhibited elevated NID2 expression. NID2 overexpression conferred resistance to Gr/PFN, 4h11-28ζ CAR T cells, and 4H11 BiTEDs, whereas knockdown or knockout restored tumor cell susceptibility. Mechanistically, NID2 upregulation enhanced Ras activation and Bcl-xl expression. Pharmacologic inhibition of either pathway - using the pan-Ras inhibitor ADT-007 or Bcl-xl inhibitors (Navitoclax, Venetoclax, A-1331852) - restored cytotoxicity in NID2-high models, including resistant PDOs. Conclusion: NID2 is a tumor-intrinsic driver of IT resistance in OC, acting through Ras activation and Bcl-xl upregulation. It may serve as a predictive biomarker for ACT response. Targeting this pathway, particularly via Bcl-xl inhibitors offers a mechanistically grounded and clinically actionable strategy to overcome immune resistance. Citation Format: Fabian BT. Kraus, Mengyao Xu, Raj Kumar, Irva Veillard, Will B. Manning, Timothy S. Bond, Eugene Kim, Yusuke Matoba, Maryam Azimi, Bastian Czogalla, Sven Mahner, Fabian Trillsch, Bo R. Rueda, David R. Spriggs, Oladapo O. Yeku. Anchored in Resistance: Overcoming Nidogen-2 (NID2) - mediated Immunoresistance in Ovarian Cancer with Ras and Bcl-xl Inhibition [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2025 Oct 22-26; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2025;24(10 Suppl):Abstract nr B086.