Abstract 3958: Targeting HSP70 enhances immunotherapy efficacy in triple-negative breast cancer by modulating tumor intrinsic and immunosuppressive pathways

Triple-negative breast cancer (TNBC) is an aggressive subtype characterized by resistance to cytotoxic therapies and immunotherapies due ​to the overexpression of survival proteins such as A20 and HSP70, which sustain the epithelial-to-mesenchymal transition (EMT) and cancer stem cell (CSC) phenotypes. We previously demonstrated that TNF-α stimulation in TNBC cell lines induces A20 expression, which upregulates HSP70 and protects cells from apoptosis. Luminal cell lines (MCF7 and ZR75-1), by contrast, exhibit approximately 70% apoptosis upon TNF-α treatment, and overexpression of A20 confers resistance and promotes aggressive metastatic properties in xenograft models. In vitro studies revealed that HSP70 secretion into the conditioned media is TNF-α-dependent in the EMT6 TNBC cell line, but no secretion was observed in the 4T1 TNBC cell line. LIMBO chaperone-binding assays confirmed A20 as a potential client protein of HSP70, and inhibition of HSP70 with the specific inhibitor JG-231 suppressed A20 expression in TNF-α-treated TNBC cells, reducing EMT/CSC phenotypes. Moreover, the A20/HSP70 axis was found to attract tumor-infiltrating lymphocytes (TILs) while promoting immunosuppressive myeloid-derived suppressor cells (MDSCs), as shown by increased Ly6G-positive cells in syngeneic mouse models. In vivo experiments using the EMT6 syngeneic mouse model demonstrated that JG-231, a small molecule HSP70 inhibitor, alone did not reduce primary tumor weight or metastasis after mammary fat pad injection. However, when combined with anti-PD-L1 therapy, tumor weight was significantly reduced, accompanied by a decrease in Ly6G-positive MDSCs in the tumor microenvironment. These findings suggest that the combination of JG-231 and immune checkpoint inhibitors reprograms the immunosuppressive tumor microenvironment and enhances therapeutic efficacy. In the 4T1 syngeneic model, JG-231 in combination with anti-PD-L1 and cyclophosphamide (CTX) achieved a greater than 10-fold reduction in primary tumor growth and complete elimination of metastases, while single-agent treatments showed no significant effect. Our study highlights the role of the A20/HSP70 axis in driving resistance to immunotherapy and shaping an immunosuppressive TME. By targeting HSP70, we demonstrate dual benefits: direct suppression of tumor-intrinsic survival pathways and reprogramming of the TME to reduce MDSCs and enhance TIL activity. These findings establish a strong preclinical rationale for combining HSP70 inhibitors like JG-231 with immune checkpoint inhibitors and standard chemotherapies to achieve durable therapeutic responses in TNBC. Further investigations are warranted to evaluate the clinical potential of this combinatorial strategy in aggressive breast cancers. Citation Format: Ahmet B. Caglayan, Fulya Koksalar Alkan, Hilmi K. Alkan, Cordarryl Jones, Sukran Ozdatli Kurtulus, Elana Ishaya, Mahassen Elboraii, Hasan Korkaya. Targeting HSP70 enhances immunotherapy efficacy in triple-negative breast cancer by modulating tumor intrinsic and immunosuppressive pathways [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 3958.