Resistance to chemotherapy and PARP inhibitors remains a major challenge in treating tubo-ovarian high-grade serous carcinoma (HGSC), primarily due to defective apoptosis in cancer cells. To overcome this, we investigated the potential of Second Mitochondria-derived Activator of Caspases (SMAC) mimetics in restoring chemotherapy sensitivity.

We performed high-throughput screening of 528 single agents and six combination treatments in genetically characterized ovarian cancer cell lines (n=6) to identify synergistic interactions. Promising combinations, including the SMAC mimetic-birinapant with carboplatin, were validated in ex vivo patient-derived HGSC cultures and syngeneic mouse models, with tumor-associated macrophages (TAMs) dependence assessed via macrophage depletion. Mechanistic studies included caspase-3 and 8 activation assays, TNFα supplementation, and NF-κB pathway modeling. Multi-omic analyses, scRNA-seq, bulk RNA-seq, sequential immunohistochemistry, and imaging mass cytometry, across seven independent HGSC cohorts encompassing chemo-naïve, post-NACT, and relapsed tumors, were used to further study potential predictive biomarkers and examine microenvironment-driven mechanisms.

Dose-response profiling revealed significant synergy between birinapant and carboplatin, confirmed in ex vivo patient-derived HGSC cultures and in vivo mouse models, where sequential birinapant–chemotherapy treatment extended overall survival. Mechanistically, chemotherapy induced TNFα expression specifically in TAMs, which sensitized cancer cells to birinapant-induced, caspase-8–dependent apoptosis. Macrophage depletion in mouse models abolished the survival benefit, demonstrating TAM-derived TNFα dependent cancer cell killing.

Chemotherapy primes the tumor microenvironment by increasing TNFα expression primarily in TAMs, enabling birinapant to trigger apoptosis in HGSC cells. Sequential birinapant administration following chemotherapy may enhance treatment responses in chemoresistant tumors. Furthermore, elevated TNFα levels and higher TAM abundance could serve as surrogates for treatment stratification. These findings provide a strong rationale for clinical testing of sequential birinapant–chemotherapy regimens in HGSC, incorporating TNFα and TAM-associated biomarkers for patient selection.