Tumor necrosis factor alpha (TNFα) is a pleiotropic cytokine known to have contradictory roles in oncoimmunology. Indeed, TNFα has a central role in the onset of the immune response, inducing both activation and the effector function of macrophages, dendritic cells, natural killer (NK) cells, and B and T lymphocytes. Within the tumor microenvironment, however, TNFα is one of the main mediators of cancer-related inflammation. It is involved in the recruitment and differentiation of immune suppressor cells, leading to evasion of tumor immune surveillance. These characteristics turn TNFα into an attractive target to overcome therapy resistance and tackle cancer. This review focuses on the diverse molecular mechanisms that place TNFα as a source of resistance to immunotherapy such as monoclonal antibodies against cancer cells or immune checkpoints and adoptive cell therapy. We also expose the benefits of TNFα blocking strategies in combination with immunotherapy to improve the antitumor effect and prevent or treat adverse immune-related effects.

BackgroundThe success of HER2-positive (HER2+) breast cancer treatment with trastuzumab, an antibody that targets HER2, relies on immune response. We demonstrated that TNFα induces mucin 4 (MUC4) expression, which shields the trastuzumab epitope on the HER2 molecule decreasing its therapeutic effect. Here, we used mouse models and samples from HER2+ breast cancer patients to unravel MUC4 participation in hindering trastuzumab effect by fostering immune evasion.MethodsWe used a dominant negative TNFα inhibitor (DN) selective for soluble TNFα (sTNFα) together with trastuzumab. Preclinical experiments were performed using two models of conditionally MUC4-silenced tumors to characterize the immune cell infiltration. A cohort of 91 patients treated with trastuzumab was used to correlate tumor MUC4 with tumor-infiltrating lymphocytes.ResultsIn mice bearing de novo trastuzumab-resistant HER2+ breast tumors, neutralizing sTNFα with DN induced MUC4 downregulation. Using the conditionally MUC4-silenced tumor models, the antitumor effect of trastuzumab was reinstated and the addition of TNFα-blocking agents did not further decrease tumor burden. DN administration with trastuzumab modifies the immunosuppressive tumor milieu through M1-like phenotype macrophage polarization and NK cells degranulation. Depletion experiments revealed a cross-talk between macrophages and NK cells necessary for trastuzumab antitumor effect. In addition, tumor cells treated with DN are more susceptible to trastuzumab-dependent cellular phagocytosis. Finally, MUC4 expression in HER2+ breast cancer is associated with immune desert tumors.ConclusionsThese findings provide rationale to pursue sTNFα blockade combined with trastuzumab or trastuzumab drug conjugates for MUC4+ and HER2+ breast cancer patients to overcome trastuzumab resistance.

Breast cancer is the most common cancer in women and the leading cause of death. HER2 overexpression is found in approximately 20% of breast cancers and is associated with a poor prognosis and a shorter overall survival. Tratuzumab, a monoclonal antibody directed against the HER2 receptor, is the standard of care treatment. However, a third of the patients do not respond to therapy. Given the high rate of resistance, other HER2-targeted strategies have been developed, including monoclonal antibodies such as pertuzumab and margetuximab, trastuzumab-based antibody drug conjugates such as trastuzumab-emtansine (T-DM1) and trastuzumab-deruxtecan (T-DXd), and tyrosine kinase inhibitors like lapatinib and tucatinib, among others. Moreover, T-DXd has proven to be of use in the HER2-low subtype, which suggests that other HER2-targeted therapies could be successful in this recently defined new breast cancer subclassification. When patients progress to multiple strategies, there are several HER2-targeted therapies available; however, treatment options are limited, and the potential combination with other drugs, immune checkpoint inhibitors, CAR-T cells, CAR-NK, CAR-M, and vaccines is an interesting and appealing field that is still in development. In this review, we will discuss the highlights and pitfalls of the different HER2-targeted therapies and potential combinations to overcome metastatic disease and resistance to therapy.

Background HER2-positive (+) and triple negative breast cancer (TNBC) have the worst survival among BC. BC patients are treated with chemotherapy (CT) and/or radiotherapy (RT), and HER2+ BC patients also receive targeted therapies, such as trastuzumab (Tz). The abundance of tumor infiltrating lymphocytes (TILs), in both HER2+ and TNBC, has a major good prognostic value. Thus, indicating that immunological evasion mechanisms are present in the tumor microenvironment (TME) hampering the efficacy of the treatments. We previously showed that soluble tumor necrosis factor α (sTNF) induces upregulation of mucin 4 (MUC4), which shields Tz epitope on HER2 impairing Tz binding and its effects. In preclinical models of de no5vo Tz-resistant tumors, administration of the sTNF blocking agent INB03 (DN) together with Tz inhibited tumor growth. We proved that MUC4 expression is an independent predictor of poor DFS in patients treated with adjuvant Tz. Our goal is to study whether MUC4 plays a role in tumor immune evasion in HER2+ and TNBC. Methods Untreated primary BC samples were assessed for TILs density (H&E) and MUC4 expression by immunohistochemistry. Tumors with TILs ≥30% and >50%, for TNBC and HER2+ BC respectively, and MUC4 scores 2 and 3 (0-3) were deemed positive. A cohort of 56 TNBC and 90 HER2+BC, stage I-III were retrospectively retrieved from Hospital Fernández and Instituto Henry Moore from 2013-2017, and clinicopathological and treatment characteristics were obtained from electronic records. TNBC were treated with adjuvant (41) or neoadjuvant CT +/- RT (15). HER2+BC patients received adjuvant Tz + CT. The association between MUC4 and OS was assessed by Kaplan Meier and log rank test and between MUC4 and TILs using Chi2. JIMT-1 HER2+ BC, de novo resistant tumors to Tz, containing a doxycycline (Dox)-inducible shRNA MUC4 plasmid (JIMT-1shMUC4) growing in nude mice were treated with IgG, Tz, DN or Tz + DN. Tumor growth was measured and macrophages and NK cells were determined in the TME by flow cytometry. Anti-asialo GM1 and clodronate-encapsulated liposomes were used to deplete NK cells and macrophages, respectively. Results We found an inverse relationship between TILs and MUC4 expression in HER2+ and TNBC (P=0.02 and P= 5 x10-5, respectively). Patients with MUC4+ TNBC have a shorter OS (P=0.03) and MUC4 was an independent predictor of OS [P=0.01; HR 4.9 (95%CI 1.4-17.0)]. To study MUC4 involvement in macrophage and NK cells recruitment in a Tz resistant model, nude mice bearing JIMT-1-shMUC4 tumors were treated or not with Dox to abolish MUC4 expression. Both groups received IgG, Tz, DN or DN + Tz. In control groups (without Dox), only Tz + DN administration was able to inhibit tumor growth (75% inhibition, P<0.0001 vs. IgG), in line with our previous results, and DN treatment reduced MUC4 expression. Knockdown of MUC4 expression by Dox, showed that Tz alone was effective in inhibiting JIMT-shMUC4 tumor growth at similar levels than Tz + DN group. Tumor growth inhibition was accompanied by an increase in NK cells activation and degranulation, and a rise in M1/M2 macrophage ratio. Depletion of macrophages or NK cells totally blunted antitumor effect of Tz + DN in control tumors. In MUC4-silenced tumors only macrophage depletion was able to abolish Tz antitumor effect. Conclusion Our results suggest that i) MUC4 expression is associated with immunologically “cold” HER2+ and TNBC, inducing an immunosuppressive TME that reflects in poor DFS/OS, and it confers resistance to Tz in HER2+ BC; ii) elimination of MUC4 expression reverses resistance to Tz; iii) tumor infiltrating macrophages are critical to the anti-tumor response in HER2+ BC. Patients with MUC4+ HER2+ or MUC4+ TNBC should benefit from sTNF blockade treatment leading to MUC4 downregulation and higher TILs, which would result in a better response to Tz and probably to immune checkpoint inhibitors. Citation Format: Roxana Schillaci, Sofia Bruni, Florencia Mauro, María F Mercogliano, Agustina Roldan-Deamicis, Cecilia J Proietti, Rosalía Cordo-Russo, Gloria Inurrigarro, Agustina Dupont, Carla Adami, Daniel Lopez Della Vecchia, Sabrina Barchuck, Silvina Figurelli, Ernesto Gil Deza, Sandra Ares, Felipe G Gercovich, Patricia V Elizalde. Mucin 4 expression in high risk breast cancer: Predicting and overcoming resistance to immunotherapy [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-13-32.

HER2+ is a breast cancer (BC) subtype characterized by the overexpression/amplification of HER2. Patients receive trastuzumab (Tz) but many (27-42%) do not achieve an objective response. We demonstrated that the overexpression of TNFɑ induces Tz resistance in tumors by upregulating the membrane glycoprotein MUC4, which masks Tz epitope on HER2, impairing its binding and reducing its therapeutic effects. We have also proved that blocking the soluble TNFɑ isoform with INB03 (DN) reduces MUC4 expression, overcomes Tz resistance and unleashes an antitumor innate immune response characterized by an increase in NK cell-activation and degranulation and a macrophage (Mφ) polarization to the M1 subtype. This study aims to determine the impact of MUC4 expression on Mφ and NK cells antitumor activity in the presence of Tz or Tz+DN, and on human T-lymphocyte recruitment and differentiation. Tz-resistant HER2+ BC human cell lines, JIMT-1 and KPL-4, were genetically modified to stably express a doxycycline-inducible (Dox) MUC4 shRNA (shMUC4) or a scramble one (shControl), and injected s.c. into female nude mice. After tumor establishment, the animals were randomly divided into a control group (-Dox) or an induced group (+Dox), and treated twice a week i.p. with IgG (5 mg/kg) or Tz+DN (5 and 10 mg/kg each, respectively) (-Dox) and with IgG or Tz (+Dox). Chlodronate or anti-asialo GM1 was used to deplete Mφ or NK cells, respectively. Tumor volume was measured routinely and, at the end point, tumors were processed and infiltrating immune cells were analyzed by flow cytometry. In -Dox tumors, both Mφ and NK cells are needed to achieve Tz+DN antitumor effect (p<0.01 and p<0.05, respectively). However, upon MUC4 silencing, only Mφ are required to mediate Tz antitumor effect (p<0.01). To address MUC4 role on T-lymphocyte differentiation, we isolated human T-cells from peripheral blood, activated them with CD3/CD28 beads and exposed them to conditioned mediums obtained from JIMT-1 and KPL-4 cells with or without MUC4 expression. The absence of MUC4 generated a secretome that promoted the differentiation of naïve T-cells to an effector subtype. Finally, we studied the tumor infiltrating lymphocytes (TILs) in a cohort of 90 patients with HER2+ BC and found a negative correlation between the presence of TILs and MUC4 expression (p=0.01). We conclude that Mφ are key players in the Tz-mediated antitumor innate immune response. Moreover, MUC4 expression impairs T-cell effector function and promotes immunologically cold HER2+ tumors, an additional resistance mechanism of this glycoprotein. We propose that women with HER2+MUC4+ BC could benefit from the combined treatment of Tz+DN to enhance innate and adaptive antitumor immune responses and prevent or overcome Tz resistance. Because MUC4 expression can be easily determined from biopsy tissue, early determination of MUC4 status in HER2+ BC could guide therapeutic choices to improve treatment outcomes. Citation Format: Sofia Bruni, Florencia L. Mauro, Maria F. Mercogliano, Cecilia J. Proietti, Carla Adami, Agustina Dupont, Gloria Inurrigarro, Rosalia Cordo Russo, Patricia V. Elizalde, Roxana Schillaci. MUC4 enables immune tumor evasion in HER2+ breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2047.

Among all breast cancers, triple negative (TNBC) subtype has the worst survival rate. We have demonstrated that the proinflammatory cytokine TNFα induces trastuzumab resistance through Mucin4 (MUC4) upregulation and it is an independent biomarker of poor response to therapy in HER2+ breast cancer. MUC4 is a transmembrane glycoprotein expressed in several tumors and it is involved in metastasis dissemination. Here, we evaluated the role of TNFα and MUC4 in metastasis in TNBC cell lines and we assessed the clinical impact of MUC4 expression in TNBC patients. TNFα blockade was achieved using etanercept (E), which blocks the soluble (sTNFα) and transmembrane isoforms of TNFα, or dominant negative protein INB03 (DN) which only neutralizes sTNFα. BT-549 and MDA-MB-231 TNBC cell lines treated with E or DN exhibit a decrease in MUC4 expression. TNFα blockade decreases the expression of the mesenchymal markers vimentin and snail in both cell lines. To assess the impact of TNFα blockade on tumor cells and its effect on the tumor microenvironment (TME), we collected conditioned media (CM) of MDA-MB-231 and BT-549 cells treated with E or DN which were used to evaluate the invasive capacity of the TNBC cell lines. The invasion of BT-549 was impaired with CM-Eta (p<0.01) and CM-DN (p<0.01), and MDA-MB-231 invasive capacity was reduced only with CM-DN (p<0.01). We evaluated MUC4 participation on invasion in MDA-MB-231 cell line transfected with a siRNA targeting MUC4 with or without DN treatment, and an invasion assay was performed. MUC4 knockdown impaired cell invasion (p=0.0001 vs siRNA control transfected cells). The treatment with DN of the MUC4-silenced cells did not further enhanced the effect, demonstrating that the impairment of the invasion is due to the decrease in MUC4. We determined the presence of TILs by H&E and the expression of MUC4, Ki67, PD-L1 (SP142), androgen receptor (AR) and cytokeratin 5 by immunohistochemistry in a cohort of 55 TNBC patients. MUC4 expression inversely correlated with TILs (p=0.00013). Since TILs are associated with good prognosis, we evaluated the effect of BT-549-CM on T cell migration. CM-DN increased T cell migration compared to control CM (p<0.01). MUC4 expression inversely correlated with Ki67 (p=0.016), PD-L1 (p=0.001) and AR (p=0.047) in our cohort. Moreover, MUC4 proved to be an independent predictor of poor overall survival (p=0.02), and is associated with a higher metastasis risk (p=0.005).In conclusion, TNFα blocking agents decrease MUC4, mesenchymal markers and the invasive capacity of TNBC cells, and in turn increase human T cell migration. MUC4 is an independent biomarker of poor overall survival, it is associated with an increased risk of metastasis and immune desert tumors. We propose TNFα as a new target for the treatment of TNBC, and MUC4 as a predictive marker to guide a combined treatment of TNFα blockers with chemotherapy or immunotherapy. Citation Format: Florencia Mauro, Sofia Bruni, Agustina Dupont, Gloria Inurrigarro, Silvina Figurelli, Sabrina Barchuk, Daniel Lopez Della Vecchia, Rosalia Cordo Russo, Ernesto Gil Deza, María Florencia Mercogliano, Roxana Schillaci. New therapeutic approach for triple negative breast cancer: soluble TNFα blockade and MUC4 expression as a prognostic biomarker [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1306.

Enhancer of Zeste homolog 2 (EZH2) is a histone methyltransferase which catalyzes the trimethylation of lysine 27 of histone H3 (H3K27me3), an epigenetic mark associated with chromatin compaction and transcriptional repression of target genes. This constitutes its canonical mechanism of action. Several studies have shown that EZH2 is able to activate gene transcription by forming transcriptional complexes through mechanisms that do not involve histone methylation. Indeed, EZH2 can also act in a non-canonical function by regulating transcription independently of its enzymatic activity. We have previously described that progestins induce the interaction between Progesterone Receptor (PR) and EZH2 in breast cancer (BC) cells, which results in the downregulation of tumor suppressor GATA3 and in the increase in cell proliferation. Since EZH2 has been implicated in the progression of several types of cancer, including those of the breast, and our own previous results indicate that EZH2 is required for progestin-induced breast cancer growth, we hypothesized that EZH2 could function as a mediator in the pro-tumorigenic effects of progestins, targeting specific tumor suppressor and differentiating genes to allow ER/PR-positive BC growth. In the present work we found that progestin treatment of T47D cells induced EZH2 mRNA and protein expression, which was abolished by the use of the progestin antagonist RU486 and when PR expression was blocked by siRNAs. We also studied the participation of EZH2 in the regulation of PR-regulated genes TNFalpha (TNFα), cyclin D1 and TIMP2. By ELISA assays, we observed that progestin treatment of T47D cells for 24 h induced TNFα secretion. This effect was abolished when EZH2 expression was inhibited in the presence of EZH2 siRNAs and when EZH2 enzymatic activity was blocked by the use of GSK126. Regarding TIMP2, we observed that EZH2 canonical activity participates in TIMP2 downregulation exerted by progestin treatment. We showed that EZH2 non-canonical function is also involved in progestin modulation of TIMP2 given that blockage of EZH2 activity did not completely revert progestin effect. Finally, we demonstrated that progestin upregulation of cyclin D1 did not require EZH2 activity. Our results prove that EZH2 engagement in PR target genes may involve its canonical or non-canonical function and therefore support further studies on other progestin-regulated cancer genes implicated in tumor growth. Presentation: No date and time listed

Clinical trials showed that trastuzumab deruxtecan (T-DXd) provides durable responses for patients with HER2+ and HER2 low metastatic breast cancer (BC), determined by immunohistochemistry. Approximately 50% of patients with HER2+ metastatic BC were still alive and progression-free at 24 months (DESTINY-Breast03). We proved that mucin 4 (MUC4) expression is an independent predictor of poor response to trastuzumab in HER2+ BC patients. In JIMT-1 tumors we proved that soluble TNFα (sTNFα) upregulates MUC4, conferring trastuzumab resistance by hiding its epitope on the HER2 molecule and reducing its binding. Here, we study whether sTNFα blockade with INB03 (DN) plays a role in regulation of innate immunity to enhance T-DXd antitumor effects in a multiple HER2-targeted therapy-resistant model. Nude mice bearing HER2+MUC4+ JIMT-1 tumor, primary resistant to trastuzumab, pertuzumab and lapatinib, were treated with IgG 5 mg/kg, T-DXd 5 mg/kg (T-DXd 5), 2.5 mg/kg (T-DXd 2.5) or 1.25 mg/kg (T-DXd 1.25), DN 10 mg/kg or the combined therapies. T-DXd and IgG were administered i.v. on days 0, 7 and 14. DN was administered i.p. twice a week for 3 weeks. Tumor growth was monitored. Mitotic index was analyzed in H&E tumor sections. The tumor-infiltrating innate cells, macrophages, NK cells and myeloid-derived suppressor cells (MDSCs), were studied by flow cytometry. T-DXd dose-response curves exhibited tumor growth inhibitions of 83% (T-DXd 5), 61% (T-DXd 2.5) and 37% (T-DXd 1.25) vs IgG-treated tumors. DN alone had no antitumor effect. T-DXd+DN reinforced the antitumor effect, as tumor growth inhibition escalated to 98% (T-DXd 5+DN), 81% (T-DXd 2.5+DN) and 73% (T-DXd 1.25+DN). A reduced number of mitotic figures were observed in T-DXd 5, T-DXd 1.25+DN and T-DXd 5+DN. Combining DN with T-DXd 1.25 and 5 enhanced the infiltration of resident macrophages and promoted polarization to the M1-like phenotype The tumor associated macrophages (TAMs) were similar among treatments. However, the combination T-DXd 1.25+DN showed an increase in M1-like tumor associated macrophages (TAMs) and a decrease in M2-like TAMs vs T-DXd 1.25 alone. T-DXd 1.25+DN treatment mimics the increase of infiltrating NK cells observed in the T-DXd 2.5 and 5 doses Finally, adding DN to T-DXd 2.5 and 5 diminishes MDSCs infiltration. Combination therapies were well tolerated without evidence of toxicity. Our results suggest that sTNFα blockade enhances T-DXd effect in a multiple HER2-targeted therapy resistant model. Adding DN allows to lower T-DXd doses to induce a reinforced antitumor innate immune response, reduced tumor cell mitosis and achieve similar tumor inhibition. Since sTNFα and MUC4 expression proved to be important variables in the response to T-DXd, neutralizing this cytokine may open new therapeutic strategies to treat patients with MUC4 expressing tumors or have progression on T-DXd therapy. Citation Format: Sofia Bruni, Florencia L. Mauro, Sofia Naveiro, Maria F. Mercogliano, Roxana Schillaci. Soluble TNFα blockade improves effectiveness of trastuzumab deruxtecan and boosts antitumor potential of macrophages in a HER2+ tumor model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2273.