Background: Novel strategies aimed to overcome trastuzumab (Tz) resistance of HER2+ breast cancer (BC) are needed. Recently, we demonstrated a novel immune evasion strategy used by BC where tumor necrosis factor alpha (TNF) induces upregulation of the transmembrane glycoprotein mucin 4 (MUC4) via NF-kB activation to impair Tz binding that prevents antibody mediated killing of BC cells. Etanercept, a non-selective inhibitor of soluble and transmembrane TNF (sTNF, tmTNF), downregulated MUC4 expression and sensitized de novo Tz-resistant BC xenografts to Tz. Moreover, we showed that MUC4 expression is an independent predictor of poor disease-free survival in patients treated with Tz in the adjuvant setting (Clin Cancer Res 2017, 23:636). Etanercept is immunosuppressive due to off-target effects on tmTNF while selective inhibition of sTNF improves the immune response to the tumor (Cancer Immunol Res 2016, 4:441). Because of the immunosuppressive properties of etanercept, we wanted study if the dominant negative-TNF protein XPro1595 (DN-TNF; also known as INB03) that neutralizes sTNF without affecting tmTNF is able to downregulate MUC4 to inhibit Tz-resistant tumor growth and improve innate antitumor immune response.

Methods: To assess the effect of DN-TNF on Tz-resistant HER2+ tumor growth, JIMT-1 cells were s.c. injected in nude mice. When tumors were established, animals were treated with IgG, DN-TNF, Tz or DN-TNF+Tz, i.p. twice a week for one month. Innate immune response was determined by flow cytometry analysis of NK cells activation and degranulation and myeloid-derived suppressor cells (MDSC) subtypes in tumor microenvironment (TME) and in spleen. Tz-dependent NK cells degranulation was assessed in splenocytes using HER2+, Tz-sensitive cell line BT-474 as the target. MUC4 and phospho NF-kB expression was determined by Western blot.

Results: Treatment with Tz or DN-TNF had no impact on JIMT-1 tumor growth. However, co-treatment with DN-TNF and Tz resulted in significantly less growth. At day 21st, tumor volume was 75mm3 in DN-TNF+Tz vs 300mm3 control groups. DN-TNF+Tz treatment showed a decrease in myeloid cell infiltration and MDSC phenotype was enriched in the granulocytic-MDSC vs monocytic-MDSC suggesting a less immunosuppressive TME. DN-TNF+Tz administration significantly increased activation and degranulation of tumor infiltrating NK cells. In addition, spleen NK cells from these animals exhibited enhanced Tz-dependent degranulation vs control groups. MUC4 expression was downregulated in tumors treated with DN-TNF and NF-kB phosphorylation was inhibited (all comparisons p<0.05).

Conclusion: These results suggest that targeting sTNF together with Tz treatment improves antitumor immune response reducing tumor burden. Activated NK cells can more effectively attack the tumor due to a less suppressive TME and decreased MUC4 expression enhancing Tz binding in Tz-resistant HER2+ BC. Patients with increased levels of TNF expressing MUC4 in their tumors could be eligible for a combined therapy with DN-TNF and Tz to overcome/avoid resistance to therapy. These results can be translated quickly into the clinic.

Citation Format: Schillaci R, Bruni S, De Martino M, Mercogliano MF, Inurrigarro G, Frahm I, Proietti CJ, Elizalde PV. Neutralizing soluble tumor necrosis factor alpha overcomes trastuzumab-resistant breast cancer immune evasion by downregulating mucin 4, improving NK cell function and decreasing myeloid-derived suppressor cells in tumor microenvironment [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P6-20-14.