Lapatinib (LAP), a dual EGFR/HER2 tyrosine kinase inhibitor, is used as second-line therapy in women with HER2+ breast cancer (BC), but less than 25% of the patients achieve an objective response. An alternative therapeutic approach is needed to overcome LAP resistance in women with metastatic HER2+ BC, particularly in patients with central nervous system (CNS) metastasis where large biological molecules are not effective. Previously, we reported that women with HER2+ tumors that express transmembrane glycoprotein mucin 4 (MUC4) have worse survival, and that HER2+/MUC4+ cell lines resistant to trastuzumab (T) express higher levels of tumor necrosis factor α (TNF) than T-sensitive cell lines. In addition, we proved that inhibition of soluble TNF (sTNF) decreases the expression of MUC4 and reverses T resistance. The aim of this work is to evaluate the participation of sTNF and transmembrane TNF (tmTNF) in LAP resistance in vivo and in the anti-tumor innate immune response. We used the LAP-resistant human BC cell line JIMT-1 and compared etanercept (E), a fusion protein that non-selectively blocks both sTNF and tmTNF, with the dominant negative-TNF protein INB03 (DN), that neutralizes sTNF without affecting tmTNF. Nude mice bearing JIMT-1 tumors (~50 mm3), received LAP (100 mg/kg) daily by oral gavage and IgG (5 mg/kg), E (5 mg/kg), DN (10 mg/kg), LAP+E or LAP+DN, twice a week i.p. Tumor volume was monitored routinely. At the end of the experiment, tumor-infiltrating immune cells were evaluated by immunofluorescence and analyzed by flow cytometry. DN or E treatments did not exhibit any anti-tumor effect alone, but in combination with LAP (LAP+DN and LAP+E) tumor growth decreased in a 54% and 34% vs. IgG, respectively (p<0.0001). LAP+DN combination was significantly more effective in decreasing JIMT-1 tumor growth than LAP+E (p<0.05). Analysis of tumor-infiltrating immune cells showed that tumor growth inhibition was accompanied by an increase in NK cell activation and degranulation, and a decrease in monocytic-myeloid-derived suppressor cells in the tumor bed of LAP+E and LAP+DN treated groups (p<0.01, vs. IgG). This is the first report to show that TNF blockade is able to overcome LAP resistance. In addition, TNF neutralization together with LAP treatment unleashes an anti-tumor innate immune response. These data, combined with previous results, suggest that MUC4 expression in patients with HER2+ BC could act not only as a biomarker of T resistance but also of LAP resistance. Women with HER2+/MUC4+ tumors undergoing treatment with LAP would benefit from the combined administration of LAP with the selective sTNF inhibitor DN to help overcome resistance; a hypothesis to be tested in a clinical trial. This therapeutic strategy may be particularly useful in patients with CNS metastasis because LAP and DN cross the blood-brain-barrier.

Citation Format: Sofia Bruni, Florencia L. Mauro, Mara De Martino, Agustina Roldán Deamicis, María F. Mercogliano, Patricia V. Elizalde, Roxana Schillaci. Soluble TNFα blockade overcomes lapatinib resistance and induces an innate immune response in HER2-positive breast cancer [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 1913.