Syngeneic Triple-negative Breast Cancer Research Articles

High-motility pro-tumorigenic monocytes drive macrophage enrichment in the tumor microenvironment.

Enrichment of tumor-associated macrophages (TAMΦs) in the tumor microenvironment correlates with worse clinical outcomes in triple-negative breast cancer (TNBC) patients, prompting the development of therapies to inhibit TAMΦ infiltration. However, the lackluster efficacy of CCL2-based chemotaxis blockade in clinical trials suggests that a new understanding of monocyte/macrophage infiltration may be necessary. Here we demonstrate that random migration, and not only chemotaxis, drives macrophage tumor infiltration. We identified tumor- associated monocytes (TAMos) that display a dramatically enhanced migration capability, induced rapidly by the tumor microenvironment, that drives effective tumor infiltration, in contrast to low-motility differentiated macrophages. TAMo, not TAMΦ, promotes cancer cell proliferation through activation of the MAPK pathway. IL-6 secreted both by cancer cells and TAMo themselves enhances TAMo migration by increasing dendritic protrusion dynamics and myosin- based contractility via the JAK2/STAT3 signaling pathway. Independent from CCL2 mediated chemotaxis, IL-6 driven enhanced migration and pro-proliferative effect of TAMo were validated in a syngeneic TNBC mouse model. Depletion of IL-6 in cancer cells significantly attenuated monocyte infiltration and reversed TAMo-induced cancer cell proliferation. This work reveals the critical role random migration plays in monocyte driven TAMΦ enrichment in a tumor and pinpoints IL-6 as a potential therapeutic target in combination with CCL2 to ameliorate current strategies against TAMΦ infiltration.

Targeting CD73 with flavonoids inhibits cancer stem cells and increases lymphocyte infiltration in a triple-negative breast cancer mouse model.

Chemotherapy remains the mainstay treatment for triple-negative breast cancer (TNBC) due to the lack of specific targets. Given a modest response of immune checkpoint inhibitors in TNBC patients, improving immunotherapy is an urgent and crucial task in this field. CD73 has emerged as a novel immunotherapeutic target, given its elevated expression on tumor, stromal, and specific immune cells, and its established role in inhibiting anti-cancer immunity. CD73-generated adenosine suppresses immunity by attenuating tumor-infiltrating T- and NK-cell activation, while amplifying regulatory T cell activation. Chemotherapy often leads to increased CD73 expression and activity, further suppressing anti-tumor immunity. While debulking the tumor mass, chemotherapy also enriches heterogenous cancer stem cells (CSC), potentially leading to tumor relapse. Therefore, drugs targeting both CD73, and CSCs hold promise for enhancing chemotherapy efficacy, overcoming treatment resistance, and improving clinical outcomes. However, safe and effective inhibitors of CD73 have not been developed as of now. We used in silico docking to screen compounds that may be repurposed for inhibiting CD73. The efficacy of these compounds was investigated through flow cytometry, RT-qPCR, CD73 activity, cell viability, tumorsphere formation, and other in vitro functional assays. For assessment of clinical translatability, TNBC patient-derived xenograft organotypic cultures were utilized. We also employed the ovalbumin-expressing AT3 TNBC mouse model to evaluate tumor-specific lymphocyte responses. We identified quercetin and luteolin, currently used as over-the-counter supplements, to have high in silico complementarity with CD73. When quercetin and luteolin were combined with the chemotherapeutic paclitaxel in a triple-drug regimen, we found an effective downregulation in paclitaxel-enhanced CD73 and CSC-promoting pathways YAP and Wnt. We found that CD73 expression was required for the maintenance of CD44highCD24low CSCs, and co-targeting CD73, YAP, and Wnt effectively suppressed the growth of human TNBC cell lines and patient-derived xenograft organotypic cultures. Furthermore, triple-drug combination inhibited paclitaxel-enriched CSCs and simultaneously improved lymphocyte infiltration in syngeneic TNBC mouse tumors. Conclusively, our findings elucidate the significance of CSCs in impairing anti-tumor immunity. The high efficacy of our triple-drug regimen in clinically relevant platforms not only underscores the importance for further mechanistic investigations but also paves the way for potential development of new, safe, and cost-effective therapeutic strategies for TNBC.

Abstract 708: Targeting aurora kinase B (AURKB) as a radiosensitizing strategy in syngeneic models of triple negative breast cancer (TNBC)

Abstract Purpose: Triple negative breast cancer (TNBC) is an aggressive breast cancer (BC) subtype with few treatment options. Radiation therapy (RT) is a mainstay therapy for the treatment of BC, but local recurrence following RT therapy is common. Consequently, decreasing local recurrence in patients with TNBC is a critical clinical need. Prior work demonstrated that AURKB is overexpressed in TNBC, and over-expression correlates with poor prognosis. Here, we examined the effects of AURKB inhibition as a novel radiosensitizing strategy in syngeneic TNBC models. Methods: Cell viability assays were used to determine the half-maximal inhibitory concentrations (IC50) of the AURKB inhibitors Barasertib-HQPA and SP-96 72 hours post treatment. Clonogenic survival assays were used to assess the radiosensitivity of TNBC murine cell lines to AURKB inhibition. In these assays, AURKB inhibitors were delivered at sub-IC50 concentrations 24 hours prior to RT, and radiation enhancement ratios (rERs) were calculated. Immunofluorescent microscopy using DAPI stain assessed micronuclei. Propidium iodide staining to assess aneuploidy was completed via flow cytometry. To assess the radiosensitizing effects of AURKB inhibition in vivo, the 4T1 syngeneic TNBC cell line was injected bilaterally into Balb/c mice and treated with Barasertib and RT. Tumor volume was recorded twice weekly throughout the study. Results: Aurora kinase B inhibitors (500-750 nM Barasertib-HQPA, 100-200 nM SP-96) delivered 24 hours prior to radiation therapy induced radiosensitization in the murine TNBC cells 4T1 (rER: 1.24-1.56) and Py8119 (rER: 1.51-1.72). Mechanistically, combined AURKB inhibition and RT significantly increased micronuclei formation in 4T1 cells 24 hours after RT compared to vehicle control (p<0.0001). Furthermore, combined AURKB inhibition and RT induced aneuploidy in murine TNBC cell lines 24 hours after radiation therapy compared to vehicle control. Combined AURKB inhibition (Barasertib 25 mg/kg, IP) and RT (8 Gy RT in 1 fraction) significantly decreased tumor volume compared to mice that had received vehicle control (777 ± 61 mm3 vs 1623 ± 126 mm3; p <0.0001). Conclusion: AURKB inhibition induces radiosensitization in syngeneic models of TNBC and leads to increased micronuclei and aneuploidy, suggesting a mechanism of sensitization. These results suggest that AURKB is a potential radiosensitizing strategy for the treatment of triple negative disease. Ongoing studies are further refining the underlying mechanisms of AURKB inhibition and RT on the antitumoral immune response. Citation Format: Kassidy M. Jungles, Zhuwen Wang, Caroline Bishop, Cydnee Wilson, Meilan Liu, Jadyn James, Michael Green, James M. Rae, Corey W. Speers, Lori J. Pierce. Targeting aurora kinase B (AURKB) as a radiosensitizing strategy in syngeneic models of triple negative breast cancer (TNBC) [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 708.

Abstract 3015: HD-PTP mediates tumor suppression via modulation of anti-tumor immunity

Abstract The role of circulating extracellular vesicles in cancer biology is an area of intense research interest. Here, we reveal a tumor suppressor mechanism of HD-PTP (His Domain Protein Tyrosine Phosphatase, encoded by PTPN23), a protein associated with ESCRT (endosomal sorting complex required for transport), a complex that controls endosome sorting and exosome secretion. While loss of HD-PTP is known to be tumorigenic in mice and is frequently observed in human cancers including Triple Negative Breast Cancer (TNBC), the relevant pathways have remained obscure. We find that inhibition of HD-PTP in vivo promotes tumor progression selectively in a syngeneic, immune-competent TNBC model but not in a matched immune-deficient model. Through systematic profiling of the exosomal proteome in HD-PTP proficient and deficient cells, we identify PD-L1 as a key exosomal cargo selectively enriched due to loss of HD-PTP. Mechanistically, we show that inhibition of HD-PTP results in PD-L1 stabilization, its co-localization with the exosomal marker CD63, the enrichment of endosomal PD-L1 and subsequent increase in ExoPD-L1 release. Exosomes produced post HD-PTP loss inhibits antigen-dependent T cell activity in vitro, and HD-PTP inhibition in a syngeneic TNBC model promotes non-cell-autonomous, exosome- and PD-L1-dependent tumor progression, associated with reduced T cell infiltration and altered macrophage polarization. Consistent with these findings, low HD-PTP levels in human solid tumors are associated with decreased tumor infiltrating lymphocytes, poor response to immune checkpoint inhibition and inferior overall survival. Taken together, our studies unveil a novel tumor suppressor pathway controlling PD-L1 trafficking and ExoPD-L1 secretion, highlighting the critical roles of HD-PTP and ESCRT in anti-tumor immunity. Citation Format: Chenxu Guo, Sheng Sun, Nayana Thimmiah, Agustina Maccio, Siang Koh, Leif Ellisen. HD-PTP mediates tumor suppression via modulation of anti-tumor immunity [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 3015.

Abstract IA05: Leveraging preclinical models of triple negative breast cancer for translational research

Abstract We have developed multiple novel preclinical syngeneic triple negative breast cancer(TNBC) genetically engineered mouse (GEM) models, which have been characterized genomically and with respect to their immune microenvironments. By integrating the immunological characterization of murine syngeneic mammary tumor models with analyses of human breast cancer datasets, we have demonstrated a relationship between EMT and myeloid cells, specifically tumor-associated macrophages (TAMs). We also have leveraged our syngeneic GEM models to define the response to immune checkpoint blockade therapy (ICBT) with emphasis on the myeloid cell environment. We have used our GEM models to test single agent drugs along with standard-of-care therapies for appropriate durations. Finally, we have tested these treatment regimens on established metastases with sufficient tumor burdens. Specifically, we will present results illustrating how CSF-1R targeting therapy combined with metronomic chemotherapy enhances a B and T cell response for the treatment of metastatic TNBC. In additional we well discuss how targeting the RNA helicase eIF4A that unwinds the secondary structures in the 5’UTR of mRNAs to facilitate translation initiation triggers an interferon response to suppress TNBC. Citation Format: Jeffrey M Rosen, Diego Pedroza, Na Zhao, Xiang H-F Zhang, Charles M Perou. Leveraging preclinical models of triple negative breast cancer for translational research [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr IA05.

Striking efficacy of a vaccine targeting TOP2A for triple-negative breast cancer immunoprevention

Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer that has a poor prognosis. TOP2A is a key enzyme in DNA replication and is a therapeutic target for breast and other cancers. TOP2A-specific Th1-promoting epitopes with optimal binding affinity to MHC II were identified using a combined scoring system. The multi-peptide TOP2A vaccine elicited a robust immunologic response in immunized mice, as demonstrated by the significant production of Th1 cytokines from immunized animals’ splenocytes stimulated in vitro with TOP2A peptides. Anti-tumor efficacy of the TOP2A vaccine was demonstrated in a syngeneic TNBC mouse model, in which pre-graft preventive vaccination was associated with significantly decreased tumor growth as compared to adjuvant control. In a genetically engineered mouse (GEM) model of TNBC, vaccinated animals demonstrated a significant reduction in tumor incidence and average tumor volume compared to adjuvant control. Finally, we examined TCR sequences in CD4 tumor Infiltrating lymphocytes (TIL) from vaccinated mice and found that the TIL contained TCR sequences specific to the three vaccine peptides. These data indicate that our newly developed multi-peptide TOP2A vaccine is highly immunogenic, elicits TILs with vaccine specific TCRs, and is highly effective in preventing and intercepting TNBC development and progression in vivo.

CD25high Effector Regulatory T Cells Hamper Responses to PD-1 Blockade in Triple-Negative Breast Cancer.

An imbalance between effector CD8+ T cells and CD25high effector Tregs marks immunosuppressive microenvironments in αPD-1-resistant TNBC and can be reversed through effector Treg depletion to increase αPD-1 efficacy.

Abstract 3608: MLL1 regulates cytokine-driven cell migration and metastasis

Abstract Cell migration, particularly in three-dimensional (3D) environments, is a critical requirement for metastasis - the spread of cancer cells from a primary tumor to distant sites. Despite causing the vast majority of cancer-related deaths, targeting metastasis remains challenging clinically. Cancer cell migration can be triggered and maintained by elevated levels of cytokines, which are presumed to be predominantly secreted by immune cells in the tumor microenvironment. However, recent work has highlighted the importance and impact of cytokine production by cancer cells themselves. While the signaling cascades that regulate cell migration have been studied extensively, the epigenetic regulation of cell migration is still poorly understood. MLL1 is a histone methyltransferase, which along with its scaffold protein Menin, is integral to methylation at the histone 3 lysine 4 (H3K4) site. Here, we show that the MLL1-Menin interaction is essential for the migration of cancer cells and that the disruption of this interaction impairs cell migration and metastasis. MLL1 depletion reduces 3D cell migration in vitro and led to lesser metastatic burden and prolonged survival in vivo. Reduced lung metastatic burden was observed even after accounting for the difference in the growth rate of primary tumors. Mechanistically, MLL1-Menin interaction controls actin filament assembly via the IL-6/pSTAT3/Arp3 axis and acto-myosin contractility via the TGF-β1/Gli2/ROCK1/2/pMLC2 axis, which together regulate dynamic protrusion generation and 3D cell migration. MLL1-Menin inhibition also decreases cell proliferation by inhibiting mitosis and cell cycle-related pathways. MLL1 depletion decreases primary tumor growth, metastatic spread, and metastatic outgrowth in mouse models of triple-negative breast cancer (TNBC), thus improving survival. When comparing metastatic burden at a threshold tumor size, rather than at a timepoint, MLL1 depletion still lowered metastatic burden. Combining an MLL1-Menin inhibitor with Paclitaxel, a standard TNBC chemotherapeutic, abrogated tumor growth and metastasis in a syngeneic TNBC mouse model. Overall, our studies show that MLL1 is an epigenetic regulator of 3D cancer cell migration, which exerts its effects by controlling the production of the key cytokines IL-6 and TGF-β1. Our in vivo studies demonstrate the potential of targeting the MLL1-Menin interaction in metastasis prevention and its potential to be combined with currently administered chemotherapeutics. Citation Format: Praful R. Nair, Ludmila Danilova, Estibaliz Gómez-de-Mariscal, Dongjoo Kim, Rong Fan, Arrate Muñoz-Barrutia, Elana Fertig, Denis Wirtz. MLL1 regulates cytokine-driven cell migration and metastasis. [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 3608.

Abstract P4-07-12: Development of Triple-negative breast cancer (TNBC) syngeneic models and TROP2-directed antibody-drug conjugate (ADC) surrogate to model therapeutic combinations

Abstract Background: Sacituzumab govitecan (SG, Trodelvy®) is a human trophoblast cell surface antigen 2 (TROP-2) directed antibody drug conjugate (ADC) coupled to an active form of irinotecan (SN-38) via our novel hydrolyzable linker (CL2A). SG is the only FDA-approved ADC treatment for TNBC patients in the second-line setting. TROP-2 is a transmembrane protein encoded by the tumor-associated calcium signal transducer 2 (TACSTD2) gene and highly expressed in TNBC, an aggressive type of cancer accounting for approximately 15% of all breast cancers. TROP-2 overexpression is associated with poor survival and relapse, but its biological function in TNBC remains poorly understood. Hypothesis/rationale: To better understand TROP-2 and TROP-2-directed ADC biology, we developed and characterized TROP2high vs TROP2low TNBC syngeneic tumors and an SG surrogate directed to murine TROP-2. Experimental design: We established 2 syngeneic TNBC models with differential TROP-2 expression: 4T1 cells were flow sorted into high (>95%) vs low (< 7%) TROP-2 expressors and EMT6 cells were transduced with a murine TACSTD2-encoding lentivirus. Balb/c mice were subcutaneously implanted with 0.5 × 10 E6 TROP-2high, TROP-2low, or parental tumor cells (4T1 or EMT6). Tumor immunophenotyping and transcriptomic analyses were performed 15 and 24 days after implantation. An SG mouse surrogate was engineered to mimic SG, using an anti-TROP-2 antibody (Rab64) that cross-reacts with human and murine TROP-2 covalently attached to SN-38 by the CL2A linker. SG surrogate activity was characterized in vitro and in 4T1 syngeneic models. Results: SG surrogate demonstrated high affinity for human and mouse TROP-2 (KD=1.1 and 1.4 nM, respectively) with SN-38 release rates and PK similar to that of SG. Flow cytometry analysis after bulk cell sorting of 4T1 or lentivirus transduction of EMT6 confirmed high TROP2 expression after at least 3 in vitro passages. Fifteen days after subcutaneous implantation, flow cytometry analysis of tumor single-cell suspensions revealed significant differences in immune infiltrates between 4T1-derived tumor groups (n=5/group; mean percentages in TROP2high vs TROP2low 4T1-derived tumors of cells expressing CD45: 65% vs 10%, P < 0.0001; CD8: 5.5% vs 1%, P = 0.0033; CD4: 22% vs 4%, P = 0.0055; macrophages: 12.5% vs 2.5%, P = 0.0002; myeloid cells: 52% vs 75%, P = 0.0066). In addition, TROP2high 4T1-derived tumors were smaller and had significantly less necrosis than TROP2low and unsorted 4T1-derived tumors 25 days after implantation. Finally, transcriptomics analyses of TROP2high vs TROP2low 4T1-derived tumors demonstrated the association of TACSTD2 expression levels with regulation of distinct molecular pathways. Conclusion: Syngeneic tumors derived from 4T1 cells with differential TROP2 expression levels are associated with differential cellular states and tumor microenvironment composition. In contrast, no significant phenotypic changes were observed in tumors derived from TACSTD2-transduced compared with mock-transduced EMT6 cells. Taken together, these results suggest that expression of the TACSTD2 gene is associated with, but not causative of, different tumor phenotypic states. Additional studies to investigate TROP-2 expression as a correlative marker of patient prognosis and the antitumor immune response are warranted. The effects of in vivo treatment with an SG surrogate on 4T1 tumor growth and immune phenotype will be discussed at the time of the presentation. Citation Format: Chih-Chien Chou, Jordan Kardos, Becky Yang, Jessica Orf, Rutwij Dave, Yurong Lai, Chingwei V. Lee, Giuseppe A. Papalia, Kelli Boyd, Lauri Diehl, Nathalie Scholler. Development of Triple-negative breast cancer (TNBC) syngeneic models and TROP2-directed antibody-drug conjugate (ADC) surrogate to model therapeutic combinations [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 P4-07-12.

Abstract B046: MLL1 regulates cytokine-driven cell migration and metastasis

Abstract Cell migration, particularly in three-dimensional (3D) environments, is a critical requirement for metastasis - the spread of cancer cells from a primary tumor to distant sites. Despite causing the vast majority of cancer-related deaths, targeting metastasis remains challenging clinically. Cancer cell migration can be triggered and maintained by elevated levels of cytokines, which are presumed to be predominantly secreted by immune cells in the tumor microenvironment. However, recent work has highlighted the importance and impact of cytokine production by cancer cells themselves. While the signaling cascades that regulate cell migration have been studied extensively, the epigenetic regulation of cell migration is still poorly understood. MLL1 is a histone methyltransferase, which along with its scaffold protein Menin, is integral to methylation at the histone 3 lysine 4 (H3K4) site. Here, we show that the MLL1-Menin interaction is essential for the migration of cancer cells and that the disruption of this interaction impairs cell migration and metastasis. MLL1 depletion reduces 3D cell migration in vitro and led to lesser metastatic burden and prolonged survival in vivo. Reduced lung metastatic burden was observed even after accounting for the difference in the growth rate of primary tumors. Mechanistically, MLL1-Menin interaction controls actin filament assembly via the IL-6/pSTAT3/Arp3 axis and acto-myosin contractility via the TGF-β1/Gli2/ROCK1/2/pMLC2 axis, which together regulate dynamic protrusion generation and 3D cell migration. MLL1-Menin inhibition also decreases cell proliferation by inhibiting mitosis and cell cycle-related pathways. MLL1 depletion decreases primary tumor growth, metastatic spread, and metastatic outgrowth in mouse models of triple-negative breast cancer (TNBC), thus improving survival. Combining an MLL1-Menin inhibitor with Paclitaxel, a standard TNBC chemotherapeutic, abrogated tumor growth and metastasis in a syngeneic TNBC mouse model. Overall, our studies show that MLL1 is an epigenetic regulator of 3D cancer cell migration, which exerts its effects by controlling the production of the key cytokines IL-6 and TGF-β1. Our in vivo studies demonstrate the potential of targeting the MLL1-Menin interaction in metastasis prevention and its potential to be combined with currently administered chemotherapeutics. Citation Format: Praful R. Nair, Ludmila Danilova, Estibaliz Gómez-de-Mariscal, Dongjoo Kim, Rong Fan, Arrate Muñoz-Barrutia, Elana Fertig, Denis Wirtz. MLL1 regulates cytokine-driven cell migration and metastasis [abstract]. In: Proceedings of the AACR Special Conference: Cancer Metastasis; 2022 Nov 14-17; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_2):Abstract nr B046.

Separate and combined effects of advanced age and obesity on mammary adipose inflammation, immunosuppression and tumor progression in mouse models of triple negative breast cancer.

Advanced age and obesity are independent risk and progression factors for triple negative breast cancer (TNBC), which presents significant public health concerns for the aging population and its increasing burden of obesity. Due to parallels between advanced age- and obesityrelated biology, particularly adipose inflammation, we hypothesized that advanced age and obesity each accelerate mammary tumor growth through convergent, and likely interactive, mechanisms. To test this hypothesis, we orthotopically transplanted murine syngeneic TNBC cells into the mammary glands of young normoweight control (7 months), young diet-induced obese (DIO), aged normoweight control (17 months), and aged DIO female C57BL/6J mice. Here we report accelerated tumor growth in aged control and young DIO mice, compared with young controls. Transcriptional analyses revealed, with a few exceptions, overlapping patterns of mammary tumor inflammation and tumor immunosuppression in aged control mice and young DIO mice, relative to young controls. Moreover, aged control and young DIO tumors, compared with young controls, had reduced abundance ofcytotoxic CD8 T cells. Finally, DIO in advanced age exacerbated mammary tumor growth, inflammation and tumor immunosuppression. These findings demonstrate commonalities in the mechanisms driving TNBC in aged and obese mice, relative to young normoweight controls. Moreover, we found that advanced age and DIO interact to accelerate mammary tumor progression. Given the US population is getting older and more obese, age- and obesity-related biological differences will need to be considered when developing mechanism-based strategies for preventing or controlling breast cancer.

Abstract IA015: Immunoprevention of triple negative breast cancer by TOP2A derived peptide vaccination

Abstract Top2A is a key enzyme involved in DNA replication and is a therapeutic target for several cancer types including breast cancer. Overexpression of Top2A has been observed in both human and mouse triple-negative breast cancer (TNBC). The present study evaluated both immunogenicity and antitumor efficacy of a newly formulated multi-peptide vaccine targeting multiple epitopes of the Top2A protein. Top2A-specific MHC II epitopes with optimal binding affinity were identified using a combined scoring system, which predicted their potential to elicit a Th1 immune response. The formulated vaccine contained top three Top2A peptides, which elicited the strongest immunologic response and showed 100% sequence homology between human and mouse. Anti-tumor efficacy of the Top2A vaccine was initially evaluated in a syngeneic TNBC mouse model, in which pre-graft preventive vaccination was associated with significantly decreased tumor growth as compared to the adjuvant controls. The Top2A peptide vaccine exhibited striking efficacy in a genetically engineered TNBC mouse model (C3(1)/Tag), reducing tumor burden by >90% when compared with adjuvant alone. Splenocytes collected from vaccinated animals showed a robust immunologic response to the immunizing peptides. There were no overt toxicities observed with the Top2A vaccination. To explore potential mechanisms underlying the anti-tumor response induced by Top2A vaccine treatment, scTCR-seq of tumors in both control and Top2A vaccine groups revealed new T cell clones as a consequence of Top2A vaccination. Furthermore, in vitro stimulation of these splenocytes by the vaccinated Top2A peptides resulted in the secretion of cytokines indicative of Th1 responses but with minimal secretion of Th2-related cytokines. Our data indicate that the newly developed multi-peptide Top2A vaccine is immunogenic and efficacious in the prevention of TNBC development and progression in vivo. Citation Format: Sang Beom Lee, Jing Pan, Donghai Xiong, Katie Palen, Bryon Johnson, Jeffrey E. Green, Shizuko Sei, Robert H. Shoemaker, Ronald A. Lubet, Yian Wang, Ming You. Immunoprevention of triple negative breast cancer by TOP2A derived peptide vaccination [abstract]. In: Proceedings of the Second Biennial NCI Meeting: Translational Advances in Cancer Prevention Agent Development (TACPAD); 2022 Sep 7-9. Philadelphia (PA): AACR; Can Prev Res 2022;15(12 Suppl_2): Abstract nr IA015.

Fn14-Directed DART Nanoparticles Selectively Target Neoplastic Cells in Preclinical Models of Triple-Negative Breast Cancer Brain Metastasis.

Triple-negative breast cancer (TNBC) patients with brain metastasis (BM) face dismal prognosis due to the limited therapeutic efficacy of the currently available treatment options. We previously demonstrated that paclitaxel-loaded PLGA-PEG nanoparticles (NPs) directed to the Fn14 receptor, termed "DARTs", are more efficacious than Abraxane─an FDA-approved paclitaxel nanoformulation─following intravenous delivery in a mouse model of TNBC BM. However, the precise basis for this difference was not investigated. Here, we further examine the utility of the DART drug delivery platform in complementary xenograft and syngeneic TNBC BM models. First, we demonstrated that, in comparison to nontargeted NPs, DART NPs exhibit preferential association with Fn14-positive human and murine TNBC cell lines cultured in vitro. We next identified tumor cells as the predominant source of Fn14 expression in the TNBC BM-immune microenvironment with minimal expression by microglia, infiltrating macrophages, monocytes, or lymphocytes. We then show that despite similar accumulation in brains harboring TNBC tumors, Fn14-targeted DARTs exhibit significant and specific association with Fn14-positive TNBC cells compared to nontargeted NPs or Abraxane. Together, these results indicate that Fn14 expression primarily by tumor cells in TNBC BMs enables selective DART NP delivery to these cells, likely driving the significantly improved therapeutic efficacy observed in our prior work.

Intestinal Microbiota Influence Doxorubicin Responsiveness in Triple-Negative Breast Cancer.

Simple SummaryTriple-negative breast cancer (TNBC) is a highly aggressive tumor with a poor prognosis and limited treatment options. Finding new approaches to improve TNBC outcomes and response to treatment is critical. Using the 4T1 murine TNBC model, we show the effect of neoadjuvant chemotherapy in modulating gut microbiota contents. In addition, gut microbiota could be used in the future as a predictive biomarker for doxorubicin responsiveness. Modulating gut microbiota through antibiotics, diet-derived fecal microbiota transplantation, or by exogenous LPS administration impact tumor growth, response to treatment, and metastasis formation. Therefore, harnessing gut microbiota contents could be considered a promising approach in affecting triple-negative breast cancer responsiveness to chemotherapy treatment.Triple-negative breast cancer (TNBC) is highly aggressive with a poor 5-year survival rate. Targeted therapy options are limited and most TNBC patients are treated with chemotherapy. This study aimed to determine whether doxorubicin (Dox) shifts the gut microbiome and whether gut microbiome populations influence chemotherapeutic responsiveness. Female BALB/c mice (n = 115) were injected with 4T1-luciferase cells (a murine syngeneic TNBC model) and treated with Dox and/or antibiotics, high-fat diet-derived fecal microbiota transplant (HFD-FMT), or exogenous lipopolysaccharide (LPS). Metagenomic sequencing was performed on fecal DNA samples. Mice that received Dox were stratified into Dox responders or Dox nonresponders. Mice from the Dox responders and antibiotics + Dox groups displayed reduced tumor weight and metastatic burden. Metagenomic analysis showed that Dox was associated with increased Akkermansia muciniphila proportional abundance. Moreover, Dox responders showed an elevated proportional abundance of Akkermansia muciniphila prior to Dox treatment. HFD-FMT potentiated tumor growth and decreased Dox responsiveness. Indeed, lipopolysaccharide, a structural component of Gram-negative bacteria, was increased in the plasma of Dox nonresponders and FMT + Dox mice. Treatment with exogenous LPS increases intestinal inflammation, reduces Dox responsiveness, and increases lung metastasis. Taken together, we show that modulating the gut microbiota through antibiotics, HFD-FMT, or by administering LPS influenced TNBC chemotherapy responsiveness, lung metastasis, and intestinal inflammation.

Abstract 2401: Investigating the role of type II IL4 receptor in breast cancer brain metastasis

Abstract Breast-to-brain metastasis is a devastating disease complication for which there are currently few treatment options. Among breast cancer subtypes, patients with triple-negative breast cancer (TNBC) and HER2+ breast cancer experience elevated rates of brain metastasis. The cytokines IL4 and IL13 are overexpressed by many human solid tumors. Both are associated with invasive and metastatic phenotypes; IL13 is additionally consequential in primary brain tumors. These cytokines signal through type I (IL4 only) and type II (IL4/IL13) IL4 receptors (IL4R). Importantly, expression of type II IL4R correlates with poor patient prognosis in basal-like (i.e., triple-negative) breast cancer, and HER2+ tumors display elevated type II IL4R expression. Our laboratory has shown that genetic loss of IL4R signaling in tumor cells attenuates lung and liver metastasis in mice. Additionally, we recently identified a role for IL4R signaling in modulating glycosylation in tumor cells. We hypothesize that IL4R signaling influences TNBC brain metastasis at least in part via modulating glycosylation. In a pilot mouse study, pharmacological blockade of IL4R after intracardiac injection of syngeneic TNBC cells resulted in attenuation of brain metastatic growth and circulating tumor cell burden. We have also observed significant changes in the expression of a sialyltransferase (ST8SIA1) that has been implicated in primary brain tumors, as well as in the abundance of several sialic acid-containing protein species in breast cancer cells following treatment with IL4 or IL13. Further, IL4 stimulation increases the overall glycosylation of several proteins of interest, including a glucose transporter and a glutamine transporter, without altering gene expression. Overall, our data suggest that type II IL4R signaling promotes metastasis, potentially through a mechanism involving protein glycosylation. Blockade of this receptor may be a useful therapeutic approach for patients with breast cancer brain metastases. Citation Format: Wendy Bindeman, Demond Williams, Ebony Hargrove-Wiley, Barbara Fingleton. Investigating the role of type II IL4 receptor in breast cancer brain metastasis [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 2401.

The PxIxIT motif of the RCAN3 inhibits angiogenesis and tumor progression in Triple Negative breast cancer in immunocompetent mice.

RCAN proteins are endogenous regulators of the calcineurin-cytosolic nuclear factor of activated T cells (CN-NFATc) pathway that bind CN through similar conserved motifs PxIxIT and LxVP of the NFATc family. RCAN1 and RCAN3 protein levels were reported to correlate with overall survival of breast cancer patients. We additionally provided supporting results about RCAN3 role on cancer showing that overexpression of the native PxIxIT sequence of RCAN3-derived R3 peptide (PSVVVH, EGFP-R3178-210) dramatically inhibits tumor growth and tumor angiogenesis in an orthotopic mouse model of Triple Negative Breast Cancer (TNBC) in nude mice. On the other hand, RCAN3 protein and its derived peptide EGFP-R3178-210 bind to CN and inhibit NFAT-mediated cytokine gene expression without affecting CN phosphatase activity suggesting that RCAN3 and EGFP-R3178-210 peptide have tumor suppressor and immunosuppressant activity. Due to the known relationship between tumor development and immune system, as well as the relevance of CN-NFATc in the regulation of the immune system, in the present study we decided to assess the effect of EGFP-R3178-210 peptide in an orthotopic syngeneic TNBC mouse model, in order to ensure that the role of RCAN3 as immunosuppressant do not override its tumor suppressor activity. Our results evidence that EGFP-R3178-210 peptide displays an inhibitory potential on tumor growth and tumor angiogenesis similar to those obtained in the previous orthotopic TNBC model. These results highlight the importance of the RCAN3 peptide as a tumor suppressor protein and totally complement our previous results, indicating that this antitumor activity role is maintained in the presence of a complete functional immune system.

Ephrin receptor A10 monoclonal antibodies and the derived chimeric antigen receptor T cells exert an antitumor response in mouse models of triple-negative breast cancer

Expression of the receptor tyrosine kinase ephrin receptor A10 (EphA10), which is undetectable in most normal tissues except for the male testis, has been shown to correlate with tumor progression and poor prognosis in several malignancies, including triple-negative breast cancer (TNBC). Therefore, EphA10 could be a potential therapeutic target, likely with minimal adverse effects. However, no effective clinical drugs against EphA10 are currently available. Here, we report high expression levels of EphA10 in tumor regions of breast, lung, and ovarian cancers as well as in immunosuppressive myeloid cells in the tumor microenvironment. Furthermore, we developed anti-EphA10 monoclonal antibodies (mAbs) that specifically recognize cell surface EphA10, but not other EphA family isoforms, and target tumor regions precisely in vivo with no apparent accumulation in other organs. In syngeneic TNBC mouse models, we found that anti-EphA10 mAb clone #4 enhanced tumor regression, therapeutic response rate, and T cell–mediated antitumor immunity. Notably, the chimeric antigen receptor T cells derived from clone #4 significantly inhibited TNBC cell viability in vitro and tumor growth in vivo. Together, our findings suggest that targeting EphA10 via EphA10 mAbs and EphA10-specific chimeric antigen receptor–T cell therapy may represent a promising strategy for patients with EphA10-positive tumors.

Abstract P145: The RNA helicase EIF4A is a therapeutic vulnerability in triple-negative breast cancer

Abstract Background: Translational control of oncoprotein expression has been implicated in the pathogenesis of multiple solid tumors. The eukaryotic translation initiation factor 4A (eIF4A) is an RNA helicase that catalyzes the unwinding of secondary structure in the 5′-untranslated region (5′-UTR) of mRNAs to facilitate translation initiation. eIF4A is an essential gene in many cancer cell lines and multiple oncogenes or anti-apoptotic proteins that contain structured 5’-UTRs have been shown to require enhanced eIF4A activity for translation. Zotatifin (eFT226) is a selective eIF4A inhibitor that has been shown previously to inhibit tumor growth in several immunocompromised mouse models and is currently in Phase I clinical trials as a single agent (NCT04092673). In this study we investigated the effects of Zotatifin both as a single agent and in combination with standard-of-care chemotherapy on tumor progression in several syngeneic triple-negative breast cancer (TNBC) mouse models. Methods: The activity of Zotatifin was assessed as single agent in eight different syngeneic p53-null mouse TNBC models across several intrinsic molecular subtypes. Synergism of Zotatifin with chemotherapy was evaluated in three responsive models. In addition, a Zotatifin-resistant model was developed following prolonged drug treatment. Drug response mechanisms and the effect on the tumor immune microenvironment were determined by tandem mass tag mass spectrometry, reverse phase protein array (RPPA) and mass cytometry. Results: Zotatifin treatment slowed tumor growth in six out of eight syngeneic TNBC models without obvious toxicity. Mass spectrometry of acutely treated tumors revealed that Zotatifin inhibited proliferative and stem cell signaling pathways including E2F targets, G2/M checkpoints, and NOTCH signaling, and induced proteins involved in Interferon-α and Interferon-γ responses. Mass cytometry analyses showed that Zotatifin suppressed the infiltration of M2 macrophages and neutrophils and promoted the infiltration of conventional dendritic cells to the tumor microenvironment. Interestingly, combination therapy of Zotatifin with chemotherapy significantly slowed tumor growth and improved survival. However, drug resistance emerged after prolonged treatment. The resistant tumors strikingly displayed a mesenchymal histology and an increased activity of the PI3K-AKT-mTOR pathway as determined by RPPA analysis. Combination treatment of Zotatifin with the mTOR inhibitor Everolimus prolonged survival. Conclusions: eIF4A is a therapeutic vulnerability in TNBC. Zotatifin inhibits tumor progression both through tumor-intrinsic mechanisms and by targeting the tumor immune microenvironment. Synergism between Zotatifin and chemotherapy supports the clinical potential of combination therapies. Citation Format: Na Zhao, Jeffrey Mark Rosen. The RNA helicase EIF4A is a therapeutic vulnerability in triple-negative breast cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P145.

Abstract P108: In vivo CRISPR screens identify E3 ligase COP1 as a modulator of macrophage infiltration and cancer immunotherapy target

Abstract Despite remarkable clinical efficacies of immune checkpoint blockade (ICB) in cancer treatment, ICB benefits in triple-negative breast cancer (TNBC) remain limited. Through pooled in vivo CRISPR knockout (KO) screens in syngeneic TNBC mouse models, we found that inhibition of the E3 ubiquitin ligase COP1 in cancer cells decreases the secretion of macrophage-associated chemokines, reduces tumor macrophage infiltration, enhances tumor immunity and ICB response. Transcriptomics, epigenomics, and proteomics analyses revealed COP1 functions through proteasomal degradation of the C/ebpδ protein. COP1 substrate TRIB2 functions as a scaffold linking COP1 and C/ebpδ, which leads to polyubiquitination of C/ebpδ. COP1 inhibition stabilizes C/ebpδ to suppress the expression of macrophage chemoattractant genes. Our integrated approach implicates COP1 as a target for improving cancer immunotherapy efficacy by regulating chemokine secretion and macrophage infiltration in the TNBC tumor microenvironment. Citation Format: Xiaoqing Wang, Collin Tokheim, Shengqing S. Gu, Binbin Wang, Qin Tang, Yihao Li, Nicole Traugh, Zexian Zeng, Yi Zhang, Boning Zhang, Jingxin Fu, Tengfei Xiao, Wei Li, Clifford Meyer, Peng Jiang, Paloma Cejas, Klothilda Lim, Henry Long, Myles Brown, X. Shirley Liu. In vivo CRISPR screens identify E3 ligase COP1 as a modulator of macrophage infiltration and cancer immunotherapy target [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P108.

In vivo CRISPR screens identify the E3 ligase Cop1 as a modulator of macrophage infiltration and cancer immunotherapy target

Despite remarkable clinical efficacy of immune checkpoint blockade (ICB) in cancer treatment, ICB benefits for triple-negative breast cancer (TNBC) remain limited. Through pooled invivo CRISPR knockout (KO) screens in syngeneic TNBC mouse models, we found that deletion of the E3 ubiquitin ligase Cop1 in cancer cells decreases secretion of macrophage-associated chemokines, reduces tumor macrophage infiltration, enhances anti-tumor immunity, and strengthens ICB response. Transcriptomics, epigenomics, and proteomics analyses revealed that Cop1 functions through proteasomal degradation of the C/ebpδ protein. The Cop1 substrate Trib2 functions as a scaffold linking Cop1 and C/ebpδ, which leads to polyubiquitination of C/ebpδ. In addition, deletion of the E3 ubiquitin ligase Cop1 in cancer cells stabilizes C/ebpδ to suppress expression of macrophage chemoattractant genes. Our integrated approach implicates Cop1 as a target for improving cancer immunotherapy efficacy in TNBC by regulating chemokine secretion and macrophage infiltration in the tumor microenvironment.