Abstract 4038: Anti-ROR2 therapies target cancer stem cells in triple-negative breast cancer

Abstract ROR1 and ROR2 are developmentally restricted, structurally related cell surface proteins expressed during embryogenesis but absent in adult tissues. Many cancers, including breast cancer, aberrantly express ROR1/ROR2. We hypothesize that ROR1 and ROR2 mark triple-negative breast cancer (TNBC) stem cells (CSCs) and mediate cancer stemness, epithelial-mesenchymal transition (EMT), metastasis, and resistance to reactive oxygen species (ROS) triggered by radiation or chemotherapy via ROR1/ROR2 signaling. Patient-derived xenografts (PDX) in immunodeficient NOD-scid gamma (NSG) mice (N=7) enabled direct comparison of TNBC tissue and early-passage (T1) PDX using oligonucleotide-barcoded antibodies and single-cell RNA-seq. PDX retained high proportions of ROR2+ TNBC cells with variable or absent ROR1. ROR2+ cell abundance in T1 PDX matched biopsy and showed enriched stemness, EMT, and upregulated ERK1/2, NF-κB, NRF2 target genes (FDR<0.0001). Extreme limiting dilution assays showed 1,000 or 50,000 ROR2+, but not ROR2-negative, cells, formed secondary PDX tumors (p<0.0001). APR-246, an investigational anti-cancer agent, promotes cell death via p53 restoration/ROS generation. We generated a high-affinity, humanized monoclonal antibody (mAb) specific for human ROR2 (h6E6) that could block ROR2 signaling, analogous to the capacity of our previous anti-ROR1 mAb (zilovertamab) to block ROR1 signaling. Observing that the vast majority of ROR1+ TNBC co-express ROR2, we hypothesized that targeting ROR2+ TNBC cells with h6E6 mAb could synergize with APR-246 to reverse CSC properties, inhibit PDX engraftment, and overcome resistance to oxidative stress induced by therapy. In vitro, h6E6 treatment enhanced TNBC PDX cell sensitivity to APR-246 versus isotype control (hIgG1) (p<0.005). In NSG mice with ROR2+ TNBC PDX, intravenous h6E6, compared to control hIgG1, reduced expression of cancer stemness and EMT genes, as well as ERK1/2, NF-κB, and NRF2 pathway targets (FDR<0.0001), and caused a 5-fold reduction in NQO1, the main NRF2 downstream target, and increased sensitivity to APR-246 (p<0.001). By integrating rigorous experimental controls in both in vitro and in vivo studies using early-passage PDX, our work demonstrates that anti-ROR2 antibody therapy, combined with redox-modulating agents like APR-246, can effectively target CSC-driven disease persistence and therapy resistance in TNBC, supporting future clinical trials of h6E6 targeting ROR2+ TNBC. Citation Format: Emanuela M. Ghia, Can Huang, Rebecca A. Shatsky, George F. Widhopf, Dan Elson, Jie Xian, Somaye Zare, Anne M. Wallace, Barbara A. Parker, Thomas J. Kipps. Anti-ROR2 therapies target cancer stem cells in triple-negative breast cancer [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 4038.