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Ferroptosis is an iron dependent form of regulatory cell death caused by the accumulation of lipid peroxides that can be exploited as a therapeutic for carcinomas and possibly other cancers. However, some populations of carcinoma cells resist ferroptosis, especially those with an epithelial phenotype but the mechanisms that promote resistance are poorly understood. We discovered that ferroptosis resistance in triple-negative breast cancer cells (TNBC) with an epithelial phenotype is dependent on expression of the integrin β4 and its ligand laminin 332 (LM332). LM332 and the integrin β4 maintain an epithelial cell state by downregulation of the EMT factor ZEB1. We further demonstrate that repression of ZEB1 by integrin β4 and LM332 promotes YAP/TAZ-mediated transcription of the C2 subunit of LM332, activating a positive feedback loop. Lastly, we observed that disrupting the β4/LM332 signaling axis sensitizes epithelial TNBC cells to ferroptosis by inducing ZEB1 expression. Our findings reveal a novel role for the extracellular matrix in promoting ferroptosis resistance by sustaining an epithelial phenotype and they suggest potential therapeutic strategies for overcoming this resistance. Citation Format: Emmet R. Karner, Hira Lal Goel, Arthur M. Mercurio. Ferroptosis resistance in epithelial populations of triple negative breast cancer is mediated by laminin 332 and integrin beta4 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6097.

Introduction: Triple negative breast cancer (TNBC) represents 15% of all breast cancers, and is a notably aggressive subtype that is prone to early recurrence following initial diagnosis with a median survival of only 9 months following recurrence. One of the few treatment options for patients with advanced and metastatic TNBC is platinum-based chemotherapy, however not all tumors respond, and those that do often become refractory during the course of treatment. The cell surface receptor Neuropilin-2 (NRP2), which acts as a co-receptor for vascular endothelial growth factors (VEGFs), has been shown to be both highly expressed and associated with therapeutic resistance in TNBC. NRP2 also serves as a co-receptor for semaphorins, which may play tumor-suppressor functions in breast cancer. We have previously demonstrated that ATYR2810, a highly specific humanized monoclonal antibody which effectively blocks NRP2/VEGF signaling without disrupting NRP2/Semaphorin 3F signaling, down-regulated epithelial-mesenchymal transition (EMT) genes such as ZEB1 and sensitizes TNBC to chemotherapy both in vitro and in vivo. Results: In this study we have further characterized the breast cancer subtypes that are most responsive to ATYR2810 treatment. Using in vitro 3D colony formation assays (CFAs), we have interrogated ~15 breast cancer cell lines covering luminal, HER2+ and various TNBC subtypes, for responsiveness to ATYR2810 treatment in combination with chemotherapy. All responsive cell lines were associated with the more aggressive subtypes of TNBC such as BL2 (basal-like 2), ML (mesenchymal-like) and MSL (mesenchymal-stem like) subtypes. Additionally, we screened five patient derived xenografts (PDXs) and again found that the responsive PDXs were associated with the more aggressive and chemoresistant subtypes of TNBC. To gain insight into the potential mechanism for enhanced chemosensitivity by ATYR2810 treatment, we performed RNAseq on the PDX tumors. Interestingly, the chemokine receptor CXCR4, which is known to promote drug resistance and tumorigenic potential, was found to be significantly downregulated in responder PDX tumors treated with ATYR2810 in combination with cisplatin as compared to those treated with cisplatin alone. Further, gene set enrichment analysis (GSEA) showed that CXCR4 was found in numerous downregulated pathways enriched in the PDX responder tumors treated with ATYR2810/cisplatin, suggesting that CXCR4 may be driving the responder phenotype of the PDXs. Flow cytometry analysis of TNBC cells treated with ATYR2810 monotherapy in vitro resulted in a reduced frequency of cells expressing CXCR4. Furthermore, ATYR2810 monotherapy inhibited spontaneous lung metastasis in experimental models of mesenchymal TNBC. Conclusion: Taken together, our data suggest that ATYR2810 enhances chemosensitivity in highly aggressive TNBC subtypes, and that this response may be mediated through the downregulation of genes known to be associated with aggressive cancer states such as ZEB1 and CXCR4. ATYR2810 may therefore serve as a novel therapeutic agent for the treatment of advanced and metastatic TNBC and potentially other aggressive cancer types. Zhiwen Xu and Alison Barber are co-first authors of this abstract. Citation Format: Zhiwen Xu, Alison Barber, Christoph Burkart, Hira Lal Goel, Justin Rahman, Kristina Hamel, Zachary Fogassy, Lisa Eide, Clara Polizzi, Jasmine Stamps, Sofia Klopp Savino, Luke Burman, Esther Chong, Suzanne Paz, Arthur M. Mercurio, Leslie A. Nangle. ATYR2810, a fully humanized monoclonal antibody targeting the VEGF-NRP2 pathway sensitizes highly aggressive and chemoresistant TNBC subtypes to chemotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB085.

INTRODUCTION: A growing body of evidence suggests that increased expression of the vascular endothelial growth factor (VEGF) co-receptor Neuropilin-2 (NRP2) is associated with aggressive breast cancers and that VEGF/NRP2 signaling contributes to clinical resistance to chemotherapy and tumor recurrence, making NRP2 a promising therapeutic target. A major limitation that has hampered the development of such a therapy, however, has been the lack of availability of high-quality anti-human NRP2 monoclonal antibodies (mAbs) that specifically block VEGF/NRP2 signaling. aTyr has generated a panel of high-quality, anti-human NRP2 mAbs that have the potential to be developed for the clinical management of diseases that involve NRP2 signaling. Among them, ATYR2810 has been characterized to bind to the b1 domain of NRP2 that encompasses the VEGF binding sites. It completely blocks the binding of VEGF to NRP2, and VEGF-induced NRP2/VEGFR dimerization. Importantly, ATYR2810 had no effect on Semaphorin 3F (Sema3F) induced NRP2/PlexinA1 dimerization, demonstrating its specificity for blocking the VEGF/NRP2 pathway. We have shown that ATYR2810, but not a Sema3F-blocking mAb has tumor-inhibitory effects on triple negative breast cancer (TNBC) cell lines or patient-derived organoids. RESULTS: We further evaluated the efficacy of ATYR2810 in combination with standard-of-care therapeutics including Cisplatin and Bevacizumab (anti-VEGF-A blocking antibody). In in vitro 3D methylcellulose colony formation assays, ATYR2810 sensitized TNBC cells to Cisplatin or Bevacizumab and considerably reduced colony formation in combination with these treatments. In an in vivo TNBC xenograft cancer model (MDA-MB-231), ATYR2810 augmented the anti-tumor effects of Cisplatin or Bevacizumab. To explore the underlying molecular mechanism, we performed gene expression profiling with samples treated by ATYR2810 alone and the combo therapy with Cisplatin or Bevacizumab. A number of gene markers of cancer stem cells (CSC) and/or epithelial-mesenchymal transition (EMT) were found to be down-regulated by ATYR2810 treatment in TNBC patient-derived organoids, including a key EMT transcription factor ZEB1. We also confirmed the reduction of ZEB1 protein expression by ATYR2810 treatment in TNBC cells. CONCLUSIONS: These results demonstrate the efficacy of ATYR2810 in combination with standard-of-care therapeutics in in vitro and in vivo TNBC models, and suggest its activity is mediated through inhibiting both EMT and cellular dedifferentiation that renders tumors more sensitive to the treatment regimes. The targeting of VEGF/NRP2 signaling by ATYR2810 may provide a new therapeutic option, and lead to the identification of new treatment biomarkers, which could offer improved efficacy and reduced toxicity in aggressive breast cancers. Citation Format: Zhiwen Xu, Christoph Burkart, Hira L. Goel, Justin Rahman, Clara Polizzi, Matt Seikkula, Luke Burman, Arthur M. Mercurio, Leslie A. Nangle. A domain-specific antibody to NRP2 down-regulated epithelial-mesenchymal transition genes and enhanced efficacy of standard-of-care therapeutics for aggressive breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB095.

Lung cancer remains one of the leading cancer types and is the foremost cause of cancer-related deaths, yet treatment options for advanced lung cancer remain limited. Novel therapies targeting factors critical to the progression of lung cancer are essential. Neuropilin-2 (NRP2) acts as a co-receptor for VEGF and has been correlated with aggressive cancer growth and metastasis. The expression of NRP2 has been shown to be upregulated in several cancers, including non-small cell lung cancer (NSCLC), and the expression of NRP2 in NSCLC is associated with increased invasive tumor growth in vitro as well as a significant decrease in patient survival. As shown previously, aTyr has developed ATYR2810, a domain-specific anti-human monoclonal antibody to NRP2 which blocks its binding to VEGF. Importantly, ATYR2810 blocks VEGF binding in a highly specific manner and does not affect the binding of Semaphorin-3F or subsequent Semaphorin-3F-induced dimerization of NRP2 and PlexinA1. In this study, we have used ATYR2810 to examine the effects of blocking VEGF-mediated NRP2 signaling both as monotherapy and in combination therapy. Using in vitro 3D colony formation assays, we found that ATYR2810 used as monotherapy led to a significant reduction in colony formation in the A549 NSCLC cell line. The effects of ATYR2810 were also examined in NSCLC xenografts, where the use of ATYR2810 as a single agent was found to result in the inhibition of tumor growth. Interestingly, the tumor growth inhibition observed with ATYR2810 as monotherapy was comparable to that of cisplatin monotherapy, suggesting that ATYR2810 may be a potent inhibitor of tumor growth in the A549 xenograft model. The effects of ATYR2810 combination therapy in NSCLC xenografts was also examined, and we found that the use of ATYR2810 in combination with either 5-FU or cisplatin enhanced tumor growth inhibition as compared to the use of either chemotherapeutic agent alone. Taken together, these results suggest that NRP2 may be an important target in aggressive NSCLC and that the use of our novel ATYR2810 antibody to block VEGF-mediated NRP2 signaling may serve as a valuable therapeutic agent both as monotherapy as well as in combination therapy. Citation Format: Alison G. Barber, Zhiwen Xu, Justin Rahman, Hira Lal Goel, Arthur M. Mercurio, Christoph Burkart, Leslie A. Nangle. The Neuropilin-2 targeting antibody ATYR2810 inhibits non-small cell lung cancer tumor growth in monotherapy and combination therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB234.

Introduction: There is a strong body of evidence indicating that the expression of Neuropilin-2 (NRP2) is enriched in breast cancer stem cells (CSCs) and that NRP2 signaling is critical for breast CSC function and resistance development. For this reason, the rationale for targeting NRP2 as a therapeutic strategy is compelling and timely. A major limitation that has hampered the development of this therapy, however, has been the lack of availability of high-quality anti-human NRP2 monoclonal antibodies (mAbs) that block NRP2 signalling. aTyr Pharma has generated a panel of high-quality, anti-human NRP2 mAbs that have the potential to be developed for the clinical management of breast cancer. A significant advance made by aTyr is that through specific domain reactivity, they have demonstrated differential effects on ligand blocking, receptor homo and heterodimerization and functional activity. Importantly a subset of such antibodies show differential activity in the mammosphere assay of triple negative breast cancer. Results: Flow cytometry was used to assess the specificity of the in-house anti-NRP2 mAbs to NRP2 using A549 wild type versus NRP2 knockout clonal cells. The in-house anti-NRP2 mAbs bound to A549 wild-type cells while showing little or no binding to the NRP2 knockout clonal cells, and exhibited significantly superior specificity, and sensitivity compared to existing commercially available antibodies. Displacement studies demonstrated that the tested anti-NRP2 mAbs showed different capabilities in blocking of VEGF-C or SEMA-3F binding to Expi293-hNRP2 cells, and were categorized as blockers (>90% inhibition), partial blockers (30-90% inhibition), or non-blockers (no obvious inhibition). To further extend the assessment of the biological activity of the anti-NRP2 antibodies, their activity was assessed in a receptor dimerization assay. Vectors encoding a split luciferase pBiT1.1 and pBiT2.1, and a cell permeable substrate, were obtained from Promega corporation. The complete extracellular domain and transmembrane helices of NRP2, FLT4 (VEGFR3), KDR (VEGFR2) and plexin A1 (PLXNA1) were cloned into the vectors and screened for optimal orientation, following established methods. Tested antibodies were able to impair respective VEGF and SEMA3 induced dimerization of receptor pairs NRP2/FLT4, NRP2/KDR and NRP2/PLXNA1. Surprisingly select antibodies show extremely specific and non-obvious functional differentiation. Direct functional assessment of a subset of these antibodies on breast CSCs revealed that the VEGF blocking, but not the SEMA3 blocking anti-NRP2 amAbs had the ability to inhibit serial passage mammosphere formation, an indicator of self-renewal potential. Conclusions: A series of domain specific antibodies to NRP2 have been developed and characterized. These antibodies show differential binding to specific domains of NRP2, can inhibit either VEGF-C or Sema3F binding to NRP2, and differentially effect receptor dimerization. The use of these antibodies enabled us to implicate VEGF-C/NRP2 signaling but not SEMA-3F/NRP2 signaling in the function of breast CSCs. Citation Format: Leslie A. Nangle, Luke Burman, Hira L. Goel, Zhiwen Xu, Kristina Hamel, Nathaniel Bloom, Arthur M. Mercurio. Domain-specific antibodies to Neuropilin 2 implicate VEGF-C and not Semaphorin 3F in breast cancer stem cell function [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1785.

The interactions between cancer cells and the extracellular matrix (ECM) regulate cancer progression. The β1C and β1A integrins, two cytoplasmic variants of the β1 integrin subfamily, are differentially expressed in prostate cancer. Using gene expression analysis, we show here that the β1C variant, an inhibitor of cell proliferation, which is down-regulated in prostate cancer, up-regulates insulin-like growth factor-II (IGF-II) mRNA and protein levels. In contrast, β1A does not affect IGF-II levels. We provide evidence that β1C-mediated up-regulation of IGF-II levels increases adhesion to Laminin-1, a basement membrane protein down-regulated in prostate cancer, and that the β1C cytoplasmic domain contains the structural motif sufficient to increase cell adhesion to Laminin-1. This autocrine mechanism that locally supports cell adhesion to Laminin-1 via IGF-II is selectively regulated by the β1 cytoplasmic domain via activation of the growth factor receptor binding protein 2–associated binder-1/SH2-containing protein-tyrosine phosphatase 2/phosphatidylinositol 3-kinase pathway. Thus, the concurrent local loss of β1C integrin, of its ligand Laminin-1, and of IGF-II in the tumor microenvironment may promote prostate cancer cell invasion and metastasis by reducing cancer cell adhesive properties. It is, therefore, conceivable that reexpression of β1C will be sufficient to revert a neoplastic phenotype to a nonproliferative and highly adherent normal phenotype. (Cancer Res 2006; 66(1): 331-42)