PURPOSE:Triple-negative breast cancer (TNBC) has the worst prognosis among breast cancers. Immunotherapy is a therapeutic option, but there is no biomarker to guide promising combination treatments. Mucin 4 (MUC4) favors metastasis in preclinical cancer models. This study evaluates the efficacy of soluble TNF (sTNF) neutralization to tackle MUC4 expression preventing metastasis in combination with immunotherapy, and the potential use of MUC4 as a prognostic and predictive biomarker in TNBC patients. EXPERIMENTAL DESIGN:To explore TNF modulation of MUC4 expression, a panel of TNBC cell lines was used. To assess the effect of sTNF blockade with a dominant negative molecule in combination with anti-PD-1 antibody on lung metastasis and overall survival (OS), 4T1 and LMM3 tumors were used. MUC4, PD-L1 and Ki-67 expression was evaluated by immunohistochemistry, and tumor infiltrating lymphocytes (TILs) were assessed by H&E staining, in a cohort of 49 early TNBC patients treated with chemotherapy. RESULTS:TNF neutralization reduces MUC4 expression in TNBC cell lines. Only the combination of sTNF blockade with anti-PD-1 antibody prevents metastasis and increases mice survival. In early TNBC patients MUC4 expression is inversely associated with TILs presence and PD-L1 and Ki-67 expression. Finally, MUC4 is associated with metastasis and is an independent biomarker of poor OS. CONCLUSIONS:We proved the existence of a sTNF/MUC4 axis in TNBC that can be actionable by sTNF neutralization, preventing metastasis. We suggest that MUC4 is a suitable biomarker to guide immunotherapy in TNBC, together with the administration of sTNF blocking drugs to improve outcome.

Abstract Background Trastuzumab deruxtecan (T-DXd) administration improves response for patients with HER2-positive metastatic breast cancer (HER2+ BC). Unfortunately, 50% of patients relapse after 2 years. T-DXd resistance mechanisms are being explored. For trastuzumab we have shown that mucin 4 (MUC4) expression is an independent predictor of poor response in HER2+BC patients. MUC4 is upregulated by soluble TNF (sTNF) secreted by the tumor, confers resistance to trastuzumab by hiding its epitope on the HER2, reducing its binding and decreasing anti-tumor phagocytic function. In preclinical models of de novo trastuzumab-resistant tumors, combination of a sTNF blocking agent INB03, (DN), with T-DXd decreases tumor growth compared to T-DXd alone. To disclose the underlying mechanism, we studied whether DN improved internalization of T-DXd in tumor cells and modified the innate immune response to enhance T-DXd antitumor effects in a multiple HER2-targeted therapy-resistant model. Methods 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 figures/field (mean) were analyzed in H&E tumor sections. Tumor-infiltrating macrophages were studied by flow cytometry. IFNƔ was determined in tumor extracts by ELISA. Internalization of T-DXd in JIMT-1 cells was studied in a S1 Incucyte along 18h by Fab-rhodo red labeling. Results T-DXd dose-response curves showed inhibition in tumor growth of 83%, 61% and 37% for T-DXd 5, 2.5 and 1.25 mg/kg treatment respectively compared with IgG-treated tumors. DN alone exhibited no antitumor activity. T-DXd+DN increased the antitumor effect by 10%, 33% and 97% for T-DXd dose of 5, 2.5 and 1.25 mg/kg respectively. Tumor growth inhibition of T-DXd 5 was similar to combination of T-DXd1.25+DN. Addition of DN did not have toxicity. DN increases IFNƔ production in the TME of T-DXd treated tumors vs T-DXd alone and promoted macrophage recruitment to the tumor bed and polarization to the antitumor M1-like phenotype. Histopathological analysis of the tumor showed a significant decrease in proliferation in all the combined and in the T-DXd 5mg/kg (2.8-3.7 mitotic figures/field) vs IgG (6.0 mitotic figures/field). In vitro, JIMT-1 cells treated with DN internalized 40% more T-DXd than its vehicle-treated counterparts. Conclusions Neutralizing sTNF with DN enhances T-DXd effect in a multiple HER2 targeted therapy resistant model of MUC4 expressing HER2+ BC. Combination of DN with T-DXd increases tumor response at all dose levels tested. The largest effect was seen at lower doses (T-DXd 1.25+DN), which mimicked the effect of 4 times the dose of T-DXd alone, suggesting significant synergy with DN as dose of T-DXd decreases. Combination of DN with T-DXd in MUC4 expressing HER2+ BC improves response to T-DXd alone by increasing T-DXd internalization and improving anti-tumor innate immune responses in the TME without increasing toxicity. The results suggest this combination should be investigated in clinical trials in patients who have MUC4 expressing tumors when T-DXd is started or become resistant to T-DXd therapy. Citation Format: Sofia Bruni, Florencia Mauro, Sofia Naveiro, Rosalia Cordo-Russo, Agustina Dupont, Mercogliano María Florencia, Roxana Schillaci. Blocking soluble TNF to Improve potency of trastuzumab deruxtecan by increasing internalization and antitumor innate immune response in a resistant HER2-positive breast cancer model [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO2-18-05.

Abstract Triple negative breast cancer (TNBC) accounts for about 10-15% of all breast cancers and has the worst survival rate. Immunotherapy is a new treatment option but resistance is frequent. We have demonstrated that TNF induces trastuzumab resistance through mucin 4 (MUC4) upregulation and it is an independent biomarker of poor response to therapy in HER2+ breast cancer. MUC4 is a transmembrane glycoprotein involved in metastasis (MTS) dissemination. Here, we evaluated the impact of the axis TNF/MUC4 on the invasive capacity of TNBC cell lines and in a preclinical model of spontaneous MTS. Finally, we assessed the clinical impact of MUC4 expression in TNBC patients. TNF blockade was achieved with etanercept (E), which blocks the soluble (sTNF) and transmembrane isoform of TNF, or with the dominant negative protein INB03 (DN) which neutralizes only sTNF. BT-549 and MDA-MB-231 human TNBC cell lines treated with E or DN exhibit a decrease in MUC4 expression. To assess the impact of TNF blockade on tumor cells and its effect on the tumor microenvironment, we collected conditioned media (CM) from 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 and MDA-MB-231 was impaired with CM-Eta (p≤0.01) and CM-DN (p≤0.01 and p≤0.05, respectively). Female BALB/c mice bearing the TNBC LMM3 tumors were treated with IgG, DN, anti-PD-1 antibodies or DN+anti-PD-1 for two weeks. After the treatments we performed surgery to remove the primary tumor and the animals were sacrificed 2 weeks later. No effect on tumor growth was observed. However, the DN+anti-PD-1 group was the only one with less of 3 MTS per lung (p≤0.05). We obtained tumor extracts and found that the tumors of the animals treated with DN or DN+anti-PD-1 had decreased MUC4 expression measured by Western blot. 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 56 early TNBC patients. MUC4 expression was inversely correlated with TILs presence (p=0.0003). Since TILs are associated with good prognosis, we evaluated the effect of BT-549-CM and MDA-MB-231-CM on T cell migration. CM-DN from both cell lines increased T cell migration compared to control CM (p≤0.01). MUC4 expression was inversely correlated with Ki67 (≥30%, p=0.036), 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 MTS risk (p=0.005). MUC4 is associated with poorly-infiltrated TNBC, and sTNF blockade downregulates its expression decreasing MTS when combined with anti-PD-1. We propose the TNF as a new target for the treatment of TNBC, and MUC4 as a predictive biomarker to guide a combined treatment of TNF blockers with immunotherapy. Citation Format: Florencia Luciana Mauro, Sofia Bruni, Agustina Dupont, Gloria Inurrigarro, Silvina Figurelli, Sabrina Barchuk, Daniel Lopez Della Vecchia, Rosalia Cordo Russo, Ernesto Gil Deza, Maria Florencia Mercogliano, Roxana Schillaci. Muc4 is a biomarker of metastasis in TNBC and its downregulation by blocking soluble TNF prevents metastasis in combination with immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2753.

Abstract Trastuzumab resistance is an important clinical issue in HER2+ breast cancer (BC), and few actionable targets are available. We demonstrated that soluble TNF (sTNF) upregulates mucin 4 (MUC4) expression, which shields trastuzumab epitope on HER2 hindering its therapeutic effect, and that sTNF blockade with INB03 decreases MUC4 expression which, together with trastuzumab, triggers an effective antitumor immune response based on antitumoral macrophage-NK cell collaboration. Because immune checkpoint molecules (ICP) and T cell exhaustion foster tumor immune escape, we addressed whether INB03 could modulate macrophage polarization, T cell exhaustion, and ICP expression in both populations to boost an antitumor immune response. For in vitro assays, human monocytes and T cells were isolated from buffy coats and enriched with specific RosetteSep. Macrophages (Mϕ) were differentiated for 6 days with M-CSF (10 ng/ml) to M0, and polarized for 48h to the M1-like subtype with LPS (50 ng/ml)+IFNγ (20 ng/ml), or to the M2-like subtype with IL-4 (20 ng/ml)+IL-10 (20 ng/ml). Then, INB03 was added (10 μg/ml) for 96h with corresponding cytokines to already-polarized Mϕ to test its ability to revert polarization. Also, Mϕ antibody-dependent cellular phagocytosis (ADCP) against human HER2+ breast cancer cell line JIMT-1 was assessed. T cells were activated with CD3/CD28 beads and cultured for 7 or 14 days. INB03 was added at those times for 48h to test its ability to modulate ICP and exhaustion markers. For in vivo assays, tumor-free mice or bearing the HER2+ C4HD tumor were treated with vehicle or INB03 (10 mg/kg, i.p.) for 21 days. Polarization, exhaustion and ICP markers were studied in Mϕ and T cells from spleen and tumor. Adding INB03 to already-polarized human Mϕ enhanced polarization towards M1-like (CD86+CD206−). Polarized Mϕ exposed to INB03 showed decreased PD1 and PDL-1 expression. Also, the expression of ADCP-inhibitory markers B7H4 and SIRPα was diminished when INB03 was present. These Mϕ exhibited increased ADCP against JIMT-1 cells treated with INB03, which showed downregulation of the inhibitory signal CD47. Murine Mϕ from tumor-bearing mice treated with INB03 showed increased polarization to the M1-like phenotype. Human CD8+ T cells showed decreased expression of PD1, PD-L1, LAG3, TIM3 and TIGIT. However, only TIM3 and PD1 were downregulated in murine CD8+ T cells of tumor-bearing mice treated with INB03. In all, sTNF blockade skews Mϕ to the M1-like phenotype and reprograms already-polarized pro-tumoral M2-like Mϕ to antitumoral ones. INB03 can tailor the tumor microenvironment by promoting Mϕ ADCP against tumor cells and by acting as an ICP inhibitor for CD8+ T cells, possibly relieving their exhaustion. HER2+MUC4+ BC patients could benefit from the administration of INB03 to boost targeted therapy efficacy and overcome tumor-induced macrophage and T-cell immune escape. Citation Format: Sofia Bruni, Maria F. Mercogliano, Roxana Schillaci. INB03: a new immune checkpoint inhibitor that reprograms macrophage polarization, boosts ADCP and reverts T-cell exhaustion markers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1365.

Background. Clinical trials have demonstrated that trastuzumab deruxtecan (T-DXd) provides a durable responses for patients with HER2-positive and HER2 low metastatic breast cancer (BC). With T-DXd treatment, approximately 50% of patients with HER2+ metastatic BC were still alive and progression-free at 24 months (DESTINY-Breast03). We previously shown that mucin 4 (MUC4) expression is an independent predictor of poor response to trastuzumab in HER2-positive BC patients. We also showed that MUC4 is upregulated by soluble TNFα (sTNFα) secreted by the tumor, which confers primary trastuzumab resistance since it hides trastuzumab epitope on the HER2 molecule, reducing its binding and diminishing its therapeutic effects. In preclinical models of de novo trastuzumab-resistant tumors, we proved that administration of the sTNFα blocking agent INB03 (DN) together with trastuzumab inhibited tumor growth and induced an innate immune response in the tumor microenvironment (TME). DN is a dominant-negative inhibitor of sTNFα that is not immunosuppressive because it does not affect transmembrane TNFα. Our goal is to study whether neutralizing sTNFα can improve T-DXd effects in a multiple HER2-targeted therapy-resistant model. Methods JIMT-1 is a HER2-positive BC cell line resistant to trastuzumab, pertuzumab and lapatinib, which expresses MUC4. JIMT-1 tumor-bearing nude mice were treated with (1) IgG 5 mg/kg, (2) T-DXd 5 mg/kg, (3) T-DXd 2.5 mg/kg, (4) T-DXd 1.25 mg/kg, (5) DN 10 mg/kg, (6) T-DXd 5 mg/kg +DN, (7) T-DXd 2.5 mg/kg +DN and (8) T-DXd 1.25 mg/kg +DN. 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 regularly. Tumor-infiltrating macrophages, NK cells and myeloid-derived suppressor cells (MDSCs) were evaluated by immunofluorescence and flow cytometry. Results The dose-response curve of T-DXd exhibited tumor growth inhibitions of 83% (5 mg/kg), 61% (2.5 mg/kg) and 37% (1.25 mg/kg) vs IgG-treated tumors. DN alone had no antitumor effect. Combination of T-DXd with DN resulted in a reinforced antitumor effect, as the tumor growth inhibition escalated to 98% (T-DXd 5 mg/kg+ DN), 81% (T-DXd 2.5 mg/kg+DN) and 73% (T-DXd 1.25 mg/kg+DN). Moreover, we observed that addition of DN to T-DXd 1.25 and 5 mg/kg enhances the infiltration of resident macrophages (p < 0.05 and p<0.01, respectively) and promotes polarization to the M1-like phenotype (p < 0.05 and p<0.001, respectively). While T-DXd 2.5 and 5 mg/kg alone achieved a decrease in M2-like macrophages (p < 0.05), combination of T-DXd 1.25mg/kg+DN impaired M2-like polarization at similar levels of that observed with 2.5 and 5 mg/kg T-DXd (p < 0.05). Notably, the M1/M2 ratio escalated from 10.9% to 51.5% when DN was added to the lowest T-DXd dose (p < 0.01). In addition, T-DXd 2.5 and 5 mg/kg induce an increase in the proportion of NK cells in TME (p < 0.001 and p<0.05, respectively), which T-DXd 1.25 mg/kg+ DN treatment mimicked (p < 0.05). Although an increase in NK cell activation was observed with 1.25 and 2.5 mg/kg treatments (p < 0.05 and p<0.001, respectively), adding DN did not further improved this effect. Only the highest dose of T-DXd in combination with DN was able to increase NK cell degranulation (p < 0.05), compared to T-DXd alone. Finally, the percentage of MDSCs population decreases with the addition of DN to the T-DXd 2.5 and 5 mg/kg. Conclusions Our results suggest that sTNFα blockade is able to enhance T-DXd effect in a multiple HER2-targeted therapy resistant model. Notably, the administration of T-DXd 1.25 mg/kg+DN achieved a similar antitumor effect to T-DXd 5 mg/kg alone and also transforms the TME to an antitumor one with a reinforced immune response. This finding highlights that sTNFα and MUC4 expression are important variables in the response to T-DXd. Neutralization of sTNFα may open new therapeutic strategies for treatment of patients who present with MUC4 expression or have progression on T-DXd therapy. Citation Format: Sofia Bruni, Florencia Mauro, Cecilia Proietti, Rosalia Cordo-Russo, Mercogliano María Florencia, Roxana Schillaci. Soluble TNFα blockade enhances trastuzumab deruxtecan antitumor effect in HER2-positive breast cancer model [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P1-11-12.

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.

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.

Background Trastuzumab resistance is an important clinical issue and few actionable targets are available. We have demonstrated that soluble TNF (sTNF) upregulates mucin 4 (MUC4) expression, which shields trastuzumab epitope on HER2 hindering its therapeutic effect,1 2 and that sTNF blockade with INB03 decreases MUC4 expression which, together with trastuzumab, triggers an effective antitumor immune response that relies on M1-macrophage-NK cell collaboration. Because macrophages have a plastic phenotype and express immune checkpoint molecules (ICP) to foster immune escape, we addressed whether INB03 could modulate macrophage polarization and ICP expression to boost the antitumor immune response. Methods Human monocytes were isolated from buffy coats and enriched with RosetteSep enrichment cocktail. Macrophages (Mφ) were differentiated for 7 days with M-CSF (10 ng/ml) to M0 (day 0) and then for 72h to the M1 subtype with LPS (50 ng/ml)+IFNg (20 ng/ml), or to the M2 subtype with IL-4 (20 ng/ml)+IL-10 (20 ng/ml) (day 3). INB03 was added (10 μg/ml) for 72h with corresponding cytokines to M0 (day 0) or to already-polarized M1 or M2 (day 3) to test its ability to inhibit or revert polarization, respectively. M1, M2 and ICP markers were analyzed by flow cytometry. M1 were considered CD14+CD86+CD206- and M2 CD14+CD86-CD206+. For functional assays, Mφ antibody-dependent cellular phagocytosis (ADCP) against the human HER2+ breast cancer cells JIMT-1 was performed using IncuCyte live-cell imaging. Results At day 0, addition of INB03 showed no effect on Mφ polarization fate. However, adding it to already-polarized M1 enhanced polarization towards M1 (CD86+CD206-). M2 treated with INB03 lost CD206 and gained CD86, reverting their polarization commitment towards M1. Mφ exposed to INB03 showed a decreased in B7H4 and PD1 expression, in both unpolarized M0 and M2. This effect was stronger when INB03 was added 72h after polarization to M2 was induced. The expression of PD-L1 and of SIRPα, which inhibits ADCP, diminished when INB03 was added to M1. M2 exhibited a more powerful downregulation of these inhibitory molecules. Finally, Mφ exhibited an increased ADCP against JIMT-1 cells treated with INB03, which showed downregulation of the inhibitory signal CD47. Conclusions In all, sTNF blockade commits M0 Mφ to the M1 phenotype and reprograms already-polarized pro-tumoral M2 Mφ to antitumoral ones. INB03 can tailor the tumor microenvironment by acting as an ICP inhibitor and by promoting ADCP against tumor cells. HER2+MUC4+ BC patients could benefit from the administration of INB03 to boost targeted-therapy efficacy and overcome macrophage immune escape. References Mercogliano MF, De Martino M, Venturutti L, et al. TNFα-Induced Mucin 4 Expression Elicits Trastuzumab Resistance in HER2-Positive Breast Cancer. Clin Cancer Res. 2017;23(3):636–648. Bruni S, Mauro FL, Proietti CJ, et al. Blocking soluble TNFα sensitizes HER2-positive breast cancer to trastuzumab through MUC4 downregulation and subverts immunosuppression. J Immunother Cancer. 2023;11(3):e005325. Ethics Approval Healthy donor's blood was collected with the donor's informed consent from Fundación Hematologica Sarmiento under IRB approval from Instituto de Biologia y Medicina Experimental (IBYME-CONICET) in Buenos Aires, Argentina (CEI # 6217).

Immunotherapy has changed the course of cancer treatment. The initial steps were made through tumor-specific antibodies that guided the setup of an antitumor immune response. A new and successful generation of antibodies are designed to target immune checkpoint molecules aimed to reinvigorate the antitumor immune response. The cellular counterpart is the adoptive cell therapy, where specific immune cells are expanded or engineered to target cancer cells. In all cases, the key for achieving positive clinical resolutions rests upon the access of immune cells to the tumor. In this review, we focus on how the tumor microenvironment architecture, including stromal cells, immunosuppressive cells and extracellular matrix, protects tumor cells from an immune attack leading to immunotherapy resistance, and on the available strategies to tackle immune evasion.

Triple-negative breast cancer (TNBC) is clinically defined by the absence of estrogen and progesterone receptors and the lack of membrane overexpression or gene amplification of receptor tyrosine kinase ErbB-2/HER2. Due to TNBC heterogeneity, clinical biomarkers and targeted therapies for this disease remain elusive. We demonstrated that ErbB-2 is localized in the nucleus (NErbB-2) of TNBC cells and primary tumors, from where it drives growth. We also discovered that TNBC expresses both wild-type ErbB-2 (WTErbB-2) and alternative ErbB-2 isoform c (ErbB-2c). Here, we revealed that the inhibitors of the retrograde transport Retro-2 and its cyclic derivative Retro-2.1 evict both WTErbB-2 and ErbB-2c from the nucleus of BC cells and tumors. Using BC cells from several molecular subtypes, as well as normal breast cells, we demonstrated that Retro-2 specifically blocks proliferation of BC cells expressing NErbB-2. Importantly, Retro-2 eviction of both ErbB-2 isoforms from the nucleus resulted in a striking growth abrogation in multiple TNBC preclinical models, including tumor explants and xenografts. Our mechanistic studies in TNBC cells revealed that Retro-2 induces a differential accumulation of WTErbB-2 at the early endosomes and the plasma membrane, and of ErbB-2c at the Golgi, shedding new light both on Retro-2 action on endogenous protein cargoes undergoing retrograde transport, and on the biology of ErbB-2 splicing variants. In addition, we revealed that the presence of a functional signal peptide and a nuclear export signal (NES), both located at the N-terminus of WTErbB-2, and absent in ErbB-2c, accounts for the differential subcellular distribution of ErbB-2 isoforms upon Retro-2 treatment. Our present discoveries provide evidence for the rational repurposing of Retro-2 as a novel therapeutic agent for TNBC.

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.

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.

Abstract 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

Background Trastuzumab resistance is an important clinical issue. Although a plethora of resistance mechanisms have been characterized, few have been shown to be actionable. We have demonstrated that soluble TNFα isoform (sTNFα) upregulates mucin 4 (MUC4) expression, which shields the trastuzumab epitope on HER2, hindering its therapeutic effect in vitro and in vivo.1,2 Since the success of trastuzumab treatment relies on immune response, we addressed the role of MUC4 on modulating the tumor immune infiltrate to foster immune evasion in sTNFα-induced trastuzumab-resistant HER2-positive (HER2+) breast cancer. Methods De novo trastuzumab-resistant JIMT-1 and KPL-4 cell lines were engineered to express a doxycycline-inducible MUC4 shRNA (JIMT-1-shMUC4 and KPL-4-shMUC4, respectively). Female nude mice bearing these s.c. tumors (~100 mm3), were treated i.p with IgG or trastuzumab (5mg/kg), a dominant negative (DN) sTNFα inhibitor (10 mg/kg) or trastuzumab+DN (n=4-6 per group). After 3 weeks of treatment, tumor-infiltrating immune cells were studied by immunofluorescence and flow cytometry. For macrophage and NK cell depletion, clodronate or anti-asialo GM1 was used, respectively. ADCP was studied using parental JIMT-1 cells pre-cultured for 48h with DN (10 μg/ml) or vehicle and then co-cultured with human macrophages for 1.5 h. A cohort of 91 HER2+ breast cancer patients treated with trastuzumab was used to correlate tumor MUC4 expression with tumor-infiltrating lymphocytes (TILs). Results Upon MUC4 silencing through doxycycline induction, trastuzumab antitumor effect was reinstated (80% or 85% tumor growth inhibition, JIMT-1-shMUC4 or KPL-4-shMUC4, respectively; p<0.0001). The addition of DN did not further decrease tumor burden. In the absence of doxycycline, trastuzumab+DN inhibited tumor growth and modified the immunosuppressive tumor milieu, increasing M1-like macrophage polarization (p<0.01) and NK cell degranulation (p<0.01). In MUC4-silenced tumors, trastuzumab treatment alone mimics this tumor infiltrate. Depletion experiments revealed a cross-talk between macrophages and NK cells necessary for trastuzumab+DN antitumor effect. When MUC4 was silenced, trastuzumab antitumor effect was lost upon macrophage depletion, but it was preserved when NK cells were absent. Furthermore, JIMT-1 cells pre-treated with DN were more susceptible to trastuzumab-dependent cellular phagocytosis (p<0.05). Finally, MUC4 expression in HER2+ breast cancer negatively correlated with TILs (p=0.004), reflecting "immune desert" tumors. Conclusions In all, we conclude that sTNFα isoform blockade is able to tackle MUC4 expression and, together with trastuzumab, triggers an effective antitumor immune response that relies on M1-macrophage-NK cell collaboration. These 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. References Mercogliano MF, De Martino M, Venturutti L, et al. TNFalpha-Induced Mucin 4 Expression Elicits Trastuzumab Resistance in HER2-Positive Breast Cancer. Clin Cancer Res 2017 23:636–48. Bruni S, De Martino M, Mauro FL, et al. Soluble TNFα induced mucin 4 is a mediator of trastuzumab resistance and of an immunosuppressive tumor microenvironment in HER2+ breast cancer. J. Immunotherapy Cancer 7, 2019;283:O39. doi:10.1186/s40425-019-0764-0 Ethics Approval Patient samples were collected with the patient9s informed consent and with Helsinki approval from Hospital Fernández (CEI # 201629) and Henry Moore Institute of Oncology, (Buenos Aires, Argentina) and from Hospital Oncológico Provincial de Córdoba (Cordoba, Argentina). All animal studies were conducted in accordance with the highest standards of animal care as outlined by the NIH Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee (IACUC) of IBYME.

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.

Triple negative breast cancer (TNBC) is clinically defined by the absence of estrogen and progesterone receptors and the lack of membrane overexpression or gene amplification of the receptor tyrosine kinase ErbB-2/HER2. Due to its heterogeneity, clinical biomarkers and targeted therapies for this disease remain elusive, and chemotherapy has been the standard of care for early and metastatic TNBC. ErbB-2 is classically located at the membrane of BC cells, where it triggers signalling cascades and promotes oncogenesis. However, we have demonstrated that ErbB-2 is also localized in the nucleus (NErbB-2) of TNBC cells and primary tumors, from where it drives growth. We also discovered that TNBC expresses both wild-type ErbB-2 (WTErbB-2) and alternative ErbB-2 isoform c (ErbB-2c). ErbB-2 migrates to the nucleus via retrograde transport. The small molecule Retro-2 is a non-toxic inhibitor of the retrograde transport route that protects cells from the deleterious effects of toxins and viruses. Here, we revealed that Retro-2 evicts both WTErbB-2 and ErbB-2c from the nuclei. Using BC models from several molecular subtypes, we demonstrated that Retro-2 specifically halts the proliferation of cells expressing NErbB-2 in a dose-dependent manner, whilst did not inhibit cell proliferation in the ErbB-2-negative MCF10A normal breast cell line. Additionally, Retro-2 decreased the expression of genes induced by NErbB-2 (cyclin D1 and Erk5) and promoted cell cycle arrest at G0/G1 phase and apoptosis. Even more, in preclinical models (including xenografts and tumor explants), Retro-2 treatment resulted in the eviction of NErbB-2 and abrogation of tumor growth. Our mechanistic studies demonstrated that Retro-2 induces a differential accumulation of WTErbB-2 at the early endosomes and plasma membrane, and of ErbB-2c at the Golgi, further preventing its sorting to the endoplasmic reticulum. These findings shed light both on Retro-2 action on endogenous protein cargoes undergoing retrograde transport and on the biology of ErbB-2 splicing variants. Together, our present discoveries provide evidence for the rational repurposing of Retro-2 as a novel therapeutic agent for TNBC. Citation Format: Santiago Madera, Franco Izzo, Maria F. Chervo, Agustina Dupont, Violeta A. Chiauzzi, Sofia Bruni, Ezequiel Petrillo, Diego Montero, Sharon Merin, Maria F. Mercogliano, Cecilia J. Proietti, Roxana Schillaci, Rosalia I. Cordo Russo, Patricia V. Elizalde. Blockade of retrograde transport in triple negative breast cancer excludes ErbB-2 isoforms from the nucleus and abrogates tumor growth [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 344.