Targeting midkine with HBS-101 enhances chemotherapy and immunotherapy response in TNBC via immune activation and oncogenic pathway suppression

Background: Triple-negative breast cancer (TNBC) is an aggressive subtype with high recurrence and resistance to standard therapies. Midkine (MDK), a heparin-binding growth factor that functions as a cytokine, is overexpressed in TNBC and promotes tumor progression, immune evasion, and chemoresistance. We recently developed HBS-101, as a first-in-class small-molecule MDK inhibitor and demonstrated its therapeutic efficacy in preclinical TNBC models. This study evaluates whether MDK inhibition enhances chemo and immunotherapy efficacy as well as the mechanistic understanding of combination therapy effects. Methods: TNBC cell lines were treated with HBS-101 alone or in combination with chemotherapy (doxorubicin and paclitaxel), and assessed for viability, apoptosis, and synergy. 3D organoids derived from patient-derived xenografts (PDX) were employed to examine the ex vivo effects of combination therapy. The efficacy of HBS-101 with chemotherapy and immunotherapy (PD-L1 inhibitor) was evaluated using human and murine cell line-derived xenograft (CDX) models of TNBC respectively. In vivo, orthotopic TNBC tumors were established in immunocompetent mice and treated with combination treatments. Tumor growth, and immune cell infiltration were analyzed using xenograft models. RNA-seq was performed on HBS-101 treated cells to identify changes in immune and apoptotic pathways. Molecular and immunological effects were examined using RT-qPCR, Western blotting, flow cytometry, and immunohistochemistry. Results: Combination treatment with HBS-101 and chemotherapy produced synergistic anti-tumor activity in TNBC models, significantly reducing 2D and 3D cell viability, stemness, and tumor growth relative to monotherapies. Similarly, combining HBS-101 with immunotherapy also yielded strong synergistic effects in syngeneic TNBC models, outperforming individual treatments. Mechanistic studies revealed that HBS-101 disrupted MDK-mediated signaling pathways, including STAT3, thereby sensitizing tumor cells to treatment and enhancing immune activation. Combination therapy also increased infiltration of CD8 positive T cells and macrophages, accompanied by elevated levels of IFN-gamma and granzyme B. RNA-seq analysis showed upregulation of immune and apoptotic pathways and suppression of immunosuppressive signals. These molecular changes correlated with reduced tumor burden, and increased apoptosis, supporting the mechanistic synergy of MDK inhibition in combination therapy. Conclusion: MDK inhibition with HBS-101 enhances the efficacy of chemo-immunotherapy in TNBC by disrupting oncogenic signaling and promoting immune activation. These findings support the therapeutic potential of HBS-101 as part of a combination strategy to overcome resistance and improve outcomes in TNBC.