Synergistic lethality of combination treatment with Trop2-directed antibody-drug conjugate (IMMU-132) and Apo2L/TRAIL in triple negative breast cancer (TNBC)

Background: Sacituzumab govitecan (IMMU-132) is an antibody drug conjugate targeting trophoblastic cell surface antigen 2 (Trop2) that's approved for treatment of patients with metastatic TNBC who have received two or more prior systemic therapies. The cytotoxic payload of IMMU-132 is a topoisomerase I inhibitor (SN-38) that kills cancer cells by causing DNA damage. Efforts to enhance the efficacy of IMMU-132 treatment include combination therapy with other agents. Apo2L/TRAIL interacts with death receptors on the cell surface to induce apoptosis in cancer cells, sparing normal cells. We previously have shown that TNBC is most sensitive to Apo2L/TRAIL. We investigated whether combination treatment with IMMU-132 and Apo2L/TRAIL synergistically inhibit TNBC cell growth. Methods: Human TNBC cell lines treated in vitro with IMMU-132 and Apo2L were assessed for cell viability via a propidium iodide-based cell death assay and ATP cell viability assay. The mode of cell death elicited by combination treatment was also investigated by using inhibitors of apoptosis, necroptosis and ferroptosis. Effects of treatment on cell cycle arrest were explored using flow cytometry. In vivo, NCr athymic nude (nu/nu) female mice with HCC1806 TNBC xenografts were treated with IMMU-132 and Apo2L after which tumor growth and survival were monitored. Results: Combination treatment with IMMU-132 and Apo2L synergistically induced cell death in triple negative breast cancer cell lines with high Trop2 expression (e.g. HCC1806, MDA-MB-468) but did not in the Trop2 low expressing MDA-MB-231 cell line. In some Trop2 low TNBC cell lines (e.g. BT-549, SUM-159), combination treatment also showed synergistic induction of cell death, suggesting extracellular deconjugation of the SN-38 cytotoxic payload and diffusion into cells. The synergy seen in Trop2 low TNBC cell lines was abrogated by limiting drug incubation periods - which minimizes the extracellular deconjugation. In contrast, the synergy seen in Trop2 high cells was maintained in these short incubation assays. Caspase activation assays and viability assays with caspase inhibitors showed that the primary mechanism of cell death for Apo2L and combination treatment with IMMU-132 is through apoptosis. Furthermore, flow cytometry-based cell cycle assays showed that IMMU-132 and combination treatment with Apo2L induce cell cycle arrest at the G2/M stage. Our xenograft model also showed significant antitumor effects with SG and TRAIL agonist combination treatment including decrease in tumor growth and improved survival in HCC1806 bearing mice. Conclusions: The data suggest a synergistic effect of combination treatment with Trop2 antibody drug conjugate IMMU-132 and TRAIL agonists in killing TNBC cells.