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 (FDR0.0001). Extreme limiting dilution assays showed 1,000 or 50,000 ROR2+, but not ROR2-negative, cells, formed secondary PDX tumors (p0.0001). 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) (p0.005). 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 (FDR0.0001), and caused a 5-fold reduction in NQO1, the main NRF2 downstream target, and increased sensitivity to APR-246 (p0.001). 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.
Ghia et al. (Fri,) studied this question.