HEBP2-governed glutamine competition between tumor and macrophages dictates immunotherapy efficacy in triple-negative breast cancer.

Background: Anti-PD-1 antibody combined with antiangiogenic drugs have demonstrated antitumor activity in advanced triple negative breast cancer (TNBC). Anlotinib, an oral multi-targeted tyrosine kinase inhibitor (TKI), has synergistic effect with anti-PD-1 antibody. Preclinical studies showed that metronomic chemotherapy inhibited angiogenesis and enhanced immunotherapy efficacy in TNBC via modulation of tumor immune microenvironment. We hereby conducted a single-arm, multicenter, phase II trial to investigate the efficacy and safety of sintilimab (anti-PD-1 antibody) in combination with anlotinib plus metronomic chemotherapy as a potential novel therapeutic strategy in advanced TNBC and explore potential biomarkers. Methods: Forty-three cases were planning to be included in this trial. The eligible patients who had received no more than two lines of chemotherapy for metastatic disease were enrolled and received sintilimab (200 mg iv q3w) in combination with anlotinib (12 mg po d1-14 q3w) plus capecitabine (500 mg po, tid) or vinorelbine (40 mg po, tiw) until disease progression or intolerable toxicity. The primary endpoint is objective response rate (ORR) and secondary endpoints are disease control rate (DCR), progression free survival (PFS), and overall survival (OS). The safety profile has also been assessed. Blood samples collected at different time points of the baseline, first and second cycle post-treatment, and disease progression were used for next-generation sequencing of ctDNA containing 654 tumor-related genes. Results: As of July 2022, a total of 32 patients were enrolled, and 29 patients were evaluable for efficacy. 2 patients (6.9%) achieved complete response (CR). 6 patients (20.7%) achieved partial response (PR). The ORR is 27.6% (8/29) and DCR is 79.3% (23/29). The median PFS was not reached. The most common grade 1 or 2 adverse events (AEs) include elevated thyroid stimulating hormone (37.0%, 10/27), hand-foot syndrome (18.5%, 5/27), elevated aspartate aminotransferase (14.8%, 4/27), elevated bilirubin (11.1%, 3/27) and hypertension (11.1%, 3/27). Grade 3 AEs include elevated bilirubin (3.7%, 1/27) and hypertension (3.7%, 1/27). No grade 4 or 5 AEs occurred. By analyzing ctDNA mutations of blood samples in 10 patients at baseline, we found that genes with high mutation frequency were HLA-DRB5 (8/10, 80%), TP53 (7/10, 70%), HLA-DRB1 (5/10, 50%) and PIK3CA (4/10, 40%). Among these 10 patients, 2, 3 and 5 patients achieved PR, SD and PD, respectively. The number of gene mutations in patients with PD was higher than that in patients with PR or SD at baseline. This indicate that mutations in ctDNA may be associated with poor efficacy in advanced TNBC. But this still needs further verification. Dynamic analysis of gene mutations at different time points showed that the amplification of HLA-DRB5 or the elimination of KMT2D, RELN and TP53 occurred in patients with PR and SD, but not in patients with PD. Conclusions: Sintilimab in combination with anlotinib plus metronomic chemotherapy has shown favorable efficacy and acceptable safety profile in patients with advanced TNBC. The clinical significance of ctDNA dynamic monitoring needs further validation. Clinical trial information: ChiCTR2100044725 Citation Format: Huihui Li, Qiaorui Tan, Shujuan Sun, Dongdong Zhou, Bo Yu, Mu Su, Baojiang Li, Shu Fang, Ling Qiang, Guohua Ren, Bing Bu, Sha Yin, Xiaochu Man, Pengfei Qiu, Xinzhao Wang, Chao Li, Fangli Cao, Qian Shao, Dali Han, Lihua Song, Bingjie Fan, Baoxuan Zhang, Liang Xu, Xianguang Zhao, Yuqian Liao, Xuemei Xie, Lanping Liu. Efficacy and safety of sintilimab in combination with anlotinib plus metronomic chemotherapy in advanced triple negative breast cancer (SPACE): preliminary results of a single-arm, multicenter phase II trial [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 OT1-05-02.

Triple-negative breast cancer (TNBC) is an aggressive subtype characterized by resistance to cytotoxic therapies and immunotherapies due ​to the overexpression of survival proteins such as A20 and HSP70, which sustain the epithelial-to-mesenchymal transition (EMT) and cancer stem cell (CSC) phenotypes. We previously demonstrated that TNF-α stimulation in TNBC cell lines induces A20 expression, which upregulates HSP70 and protects cells from apoptosis. Luminal cell lines (MCF7 and ZR75-1), by contrast, exhibit approximately 70% apoptosis upon TNF-α treatment, and overexpression of A20 confers resistance and promotes aggressive metastatic properties in xenograft models. In vitro studies revealed that HSP70 secretion into the conditioned media is TNF-α-dependent in the EMT6 TNBC cell line, but no secretion was observed in the 4T1 TNBC cell line. LIMBO chaperone-binding assays confirmed A20 as a potential client protein of HSP70, and inhibition of HSP70 with the specific inhibitor JG-231 suppressed A20 expression in TNF-α-treated TNBC cells, reducing EMT/CSC phenotypes. Moreover, the A20/HSP70 axis was found to attract tumor-infiltrating lymphocytes (TILs) while promoting immunosuppressive myeloid-derived suppressor cells (MDSCs), as shown by increased Ly6G-positive cells in syngeneic mouse models. In vivo experiments using the EMT6 syngeneic mouse model demonstrated that JG-231, a small molecule HSP70 inhibitor, alone did not reduce primary tumor weight or metastasis after mammary fat pad injection. However, when combined with anti-PD-L1 therapy, tumor weight was significantly reduced, accompanied by a decrease in Ly6G-positive MDSCs in the tumor microenvironment. These findings suggest that the combination of JG-231 and immune checkpoint inhibitors reprograms the immunosuppressive tumor microenvironment and enhances therapeutic efficacy. In the 4T1 syngeneic model, JG-231 in combination with anti-PD-L1 and cyclophosphamide (CTX) achieved a greater than 10-fold reduction in primary tumor growth and complete elimination of metastases, while single-agent treatments showed no significant effect. Our study highlights the role of the A20/HSP70 axis in driving resistance to immunotherapy and shaping an immunosuppressive TME. By targeting HSP70, we demonstrate dual benefits: direct suppression of tumor-intrinsic survival pathways and reprogramming of the TME to reduce MDSCs and enhance TIL activity. These findings establish a strong preclinical rationale for combining HSP70 inhibitors like JG-231 with immune checkpoint inhibitors and standard chemotherapies to achieve durable therapeutic responses in TNBC. Further investigations are warranted to evaluate the clinical potential of this combinatorial strategy in aggressive breast cancers. Citation Format: Ahmet B. Caglayan, Fulya Koksalar Alkan, Hilmi K. Alkan, Cordarryl Jones, Sukran Ozdatli Kurtulus, Elana Ishaya, Mahassen Elboraii, Hasan Korkaya. Targeting HSP70 enhances immunotherapy efficacy in triple-negative breast cancer by modulating tumor intrinsic and immunosuppressive pathways [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 3958.

Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer that is characterized by the absence of estrogen receptors (ERs), progesterone receptors (PRs), and human epidermal growth factor receptor 2 (HER2). Due to its immunosuppressive tumour microenvironment (TME) and low immune cell infiltration, TNBC typically exhibits poor responsiveness to immunotherapy. Recent relevant research has focused on using strategies to convert cold tumors into hot tumors to increase tumor immunogenicity and improve treatment efficacy. This review aims to summarize the biological characteristics of both cold and hot tumors and explore the mechanisms underlying the transformation from cold to hot tumors. Key strategies include modulation of the TME, enhancement of immune cell infiltration, and regulation of the inflammatory responses. Additionally, the roles of immune checkpoint inhibitors (ICIs), cytokine therapy, chimeric antigen receptor T-cell (CAR-T) therapy, and cancer vaccines in reprogramming the TME are discussed. Further, the emerging combination strategies, such as the integration of ICIs with chemotherapy, radiotherapy, and targeted therapies, have been evaluated for their potential to increase TNBC immunogenicity. Current preclinical and clinical evidence suggests that reprogramming the TME through targeted interventions significantly increases immune cell infiltration and antigen presentation, thereby improving the immunotherapy efficacy in TNBC. The combinations of ICIs with chemotherapy and radiotherapy have shown promise in shifting the TME toward an immunoresponsive state. Moreover, advances in the CAR-T-cell therapy, cytokine therapy, and cancer vaccines have offered novel approaches for overcoming immune resistance in TNBC. In conclusion, transforming cold tumors into hot tumors represents a promising therapeutic strategy for TNBC. Future research should focus on optimizing the treatment combinations, refining therapeutic timing and dosage, and integrating precision medicine approaches to achieve maximized clinical benefits. A deeper understanding of TME modulation and immune resistance mechanisms would facilitate the development of novel immunotherapeutic strategies to improve the survival outcomes and quality of life in TNBC patients.

Background: Recently, immune check-point inhibitors have shown efficacy in triple-negative breast cancer (TNBC). Since Hedgehog (Hh) signalling mediates crosstalk between breast cancer cells and tumor-infiltrating immune cells, we investigated the mechanisms by which Hh can modulate PDL1 expression and the tumor-immune microenvironment. Methods: TNBC tumors from untreated patients were subjects to PDL1 and Gli1 expression analysis by IHC. The correlation of Hh pathway activation and PDL1 expression was assessed in TNBC cells treated with the SMO-inhibitor NVP-LDE225. The main aim was to study the PDL1/Gli1 cross-talk. Results: The expression of PDL1 and Gli1, the major indicator for the canonical Hh signaling activation, were assessed by IHC in a tissue microarray (TMA) of TNBC samples from 237 untreated patients. In 203/237 cases we could analyze both PDL1 and Gli1 protein expression. A significant correlation between PDL1 and Gli1 expression was found. Indeed, of 77/203 (38%) PDL1 positive tumors 42/77 (54%) expressed Gli1. In order to explore correlations between other Hh pathway members and PDL1 in TNBCs, we interrogated the open-access database TCGA; PDL1 positive TNBCs showed high levels of SMO and PTCH1, Hh pathway receptors (Q-value 0.018 and 0.010). To better understand the link between Hh pathway and PDL1, we selected a panel of TNBC cell lines; the pharmacological Hh pathway inhibition led to a reduction of PDL1 protein and mRNA. On the other hand, engineered cells harbouring Gli1 overexpression showed higher levels of PDL1. Therefore, we hypothesized that Hh pathway could be involved in the PDL1 transcriptional modulation. To address this issue, we performed a luciferase reporter assay and a ChIP analysis following by PCR amplification of the PDL1 gene promoter. We found that Gli1 binds the PDL1 promoter, indicating that Gli1 transcriptionally enhances PDL1 expression. Gli1 expression was evaluated also in 4T1 cells, a TNBC murine model; furthermore, in 4T1 cells PDL1 protein expression was inhibited by NVP-LDE225. In vivo experiment will be performed in Balb/C mice orthotopically xenografted with 4T1 cells to confirm the role of Hh pathway to modulate the PDL1 expression and the tumor microenviroment in vivo. Conclusions: Our results suggest that Hh pathway has a crucial role in cancer immune evasion trough PDL-1 modulation. Due to their ability to target both tumor cells and the tumor microenvironment, Hh inhibitors could represent promising therapeutics to be clinically investigated in Gli1 overexpressing TNBC patients. Citation Format: Pietro De Placido, Concetta Di Mauro, Daniela Esposito, Ada Pesapane, Stefania Belli, Fabiana Napolitano, Antonio Santaniello, Priscilla Cascetta, Annachiara Carratù, Eleonora Mozzillo, Roberta Marciano, Alberto Servetto, Roberto Bianco, Luigi Formisano. Hedgehog pathway is involved in cancer immune surveillance through PDL1 modulation [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P5-04-17.

e13142 Background: Triple-negative breast cancer (TNBC) patients often face a grim prognosis, primarily due to their resistance to chemotherapy. Carboplatin is a common treatment for TNBC, particularly effective in BRCA1/2 mutations, but its efficacy varies in BRCA1/2 wild-type patients. A previous study identified GPRC5A expression as a biomarker for carboplatin chemotherapy in ovarian cancer, yet its association with carboplatin efficacy in TNBC remains unknown. This study aims to investigate whether GPRC5A can serve as a biomarker for predicting carboplatin efficacy in TNBC and to elucidate the underlying mechanisms. Methods: Analyzing clinical data from the past 5 years in Division of Breast & Thyroid Surgery in Jiangsu Province Hospital, we identified 199 TNBC patients treated with carboplatin. GPRC5A expression in TNBC tumors was assessed via immunohistochemistry (IHC) and evaluated by three individual pathologists. Expression levels were categorized as low (low), low (mild), high (moderate), and high (high). The Chi-square test was employed to assess the correlation between GPRC5A expression and carboplatin efficacy. TNBC-derived organoids were used to validate the relationship between GPRC5A expression and carboplatin sensitivity. Results: Among the 199 TNBC patients, 31 (17.2%) had BRCA1/2 mutations. The pathologic complete response (pCR) rate in BRCA1/2 mutation patients receiving carboplatin was 83.9%, compared to 61.3% in BRCA1/2 wild-type patients (p=0.016). Analyzing the relationship between GPRC5A expression and BRCA1/2 gene mutations in 145 patient samples revealed no significant association (p=0.37). Irrespective of BRCA1/2 mutation status, patients with low/mild GPRC5A expression had a higher pCR rate (74.1%) compared to those with moderate/high GPRC5A expression (45%, p<0.001). TNBC-derived organoids treated with carboplatin showed a cell survival rate of 31.25% (±8.5%) in low GPRC5A expression organoids and 56.25% (±12.6%) in high GPRC5A expression organoids (p=0.017). Conclusions: Our study demonstrates a correlation between GPRC5A expression and BRCA1/2 mutation status in TNBC. Furthermore, TNBC patients with low GPRC5A expression exhibited a more favorable response to carboplatin treatment, suggesting GPRC5A could be a potential biomarker for predicting carboplatin efficacy in TNBC patients.

Background: Neratinib is a potent, irreversible pan-HER inhibitor that inhibits the ErbB family members EGFR, HER2, and HER4 and downstream signal transduction of these receptors. Triple-negative breast cancer (TNBC) is a heterogeneous disease that lacks druggable levels of receptors for estrogen, progesterone and HER2 and therefore challenging to treat. There is evidence that some cases of TNBC have activated signaling pathways mediated by ErbB family members that may contribute to aggressive behavior. The purpose of this preclinical study was to identify and validate kinases whose targeting may enhance the antitumor activity of neratinib in TNBC cell lines. Methods: In vitro proliferation assays were used to evaluate the efficacy of neratinib in TNBC cell lines. Baseline and post-neratinib-treatment expression of EGFR and phosphorylated EGFR (phospho-EGFR) were assessed via Western blot analysis in 18 TNBC cell lines. Reverse-phase protein array (RPPA) was used to profile and validate the signaling networks induced by neratinib. To identify potential targets or pathways that may synergize with neratinib treatment, we performed high-throughput RNA interference (HT RNAi) screening using a 709-kinome library. CellTiter-Blue, sulforhodamine B, and soft-agar assays were performed to evaluate the antiproliferative effect of neratinib alone and with target inhibitor. Mammary fat pad xenograft models were used to evaluate the efficacy of neratinib alone or with inhibitor in vivo. Results: In vitro proliferation assays showed that the half-maximal inhibitory concentration (IC50) of neratinib in tested TNBC cell lines ranged from 0.16 µM to 1.25 µM. RPPA and Western blot analyses revealed that the efficacy of neratinib correlated with phospho-EGFR expression levels across the TNBC cell lines tested (R2 = 0.3245). Among the tested TNBC cell lines, SUM149 cells (PIK3CA wild-type) were selected for high throughput RNAi screening because this cell line has high EGFR expression and is moderately sensitive to neratinib (IC50 = 0.35 µM). We identified the 40 most relevant kinase targets by the sensitivity index analysis, and further pathway analysis identified PI3K/AKT/mTOR (drug: everolimus) and MAPK (drug: trametinib) as major canonical pathways whose targeting enhanced the cytotoxic effect of neratinib. Everolimus (mTOR inhibitor) produced a strong antiproliferative effect when combined with neratinib in most tested TNBC cell lines (12 of 15 cell lines; combination index [CI] values, 0.1-0.5) and was more effective in PIK3CA-mutated compared to wildtype cell lines. Trametinib (MEK inhibitor) showed a moderate antiproliferative effect (effective in 10 of 15 cell lines; CI values, 0.2-0.9). Synergistic antitumor effects of neratinib combined with everolimus or with trametinib were also observed in anchorage-independent growth conditions (P < 0.05). In vivo experiments demonstrated that neratinib plus everolimus and neratinib plus trametinib combinations inhibited tumor growth in the SUM149 xenograft model for than single drug (neratinib, 42.3% growth inhibition; everolimus, 29.7%; trametinib, 47.1%; neratinib plus everolimus, 69.7%; neratinib plus trametinib, 77.7%; P < 0.0001). Conclusion: Combining neratinib with everolimus or with trametinib enhanced the antitumor effects of these drugs in TNBC regardless of PIK3CA mutation status, and clinical investigations evaluating these combination regimens for the treatment of TNBC are warranted. Citation Format: Jangsoon Lee, Troy Pearson, Huey Liu, Jon A. Fuson, Toshiaki Iwase, Irmina Diala, Alshad S. Lalani, Lisa D. Eli, Debu Tripathy, Bora Lim, Naoto T. Ueno. Identification of novel molecules that enhance neratinib efficacy in triple-negative breast cancer by high-throughput RNA interference [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS4-07.

Background: Immune checkpoint inhibitors (ICIs) targeting the PD-1/PD-L1 axis have emerged as constituting a new pillar for breast cancer; however, they benefit only a subset of patients, and predictive biomarkers are urgently needed. While dendritic cells (DCs) are essential for the orchestration of antitumor immune response, the clinical value of DCs in breast cancer immunotherapy remains unclear. An improved understanding of the functional diversity of DC subsets, as well as their relationship with ICI response, would eventually improve the efficacy of ICIs in breast cancer patients. Objective: We sought to comprehensively characterize DC heterogeneity within the tumor microenvironment, identify distinct DC subsets associated with the response to ICIs, and define clinically applicable predictive biomarkers for ICI therapy. Methods: We performed a comprehensive single-cell RNA sequencing (scRNA-seq) analysis of 53 breast tumors receiving ICIs or chemotherapy in two retrospective studies (NCT03197389 and GSE169246) to identify specific DC populations associated with ICI responsiveness. Next, we harnessed multiplex immunohistochemistry (n = 186), multiparametric flow cytometry of fresh tissues (n = 87), large consecutive transcriptomic datasets (The Cancer Genome Atlas [TCGA] and Fudan University Shanghai Cancer Center [FUSCC]), and in vivo experiments to illustrate the molecular portraits and clinical relevance of the identified DC subsets. Additionally, except for the two scRNA-seq datasets, we leveraged another four independent cohorts (NCT03805399, NCT04613674, NCT04129996, GSE124821) and assessed the clinical utility of the established clinically applicable biomarkers derived from prior analyses to predict response following treatment with ICIs or chemotherapy. Results: By obtaining single-cell transcriptome data of 1,955 high-quality DCs, we identified five distinct DC subpopulations (CLEC9A+, CD1A+, CLEC10A+, CCL19+, and LILRA4+). We then illustrated that a specific CCL19+ DC population, but not whole DCs or other subsets, was associated with ICI responsiveness, particularly in triple-negative breast cancer (TNBC). Mechanistically, a potent CD8+ T-cell response was unleashed, rendering tumors susceptible to PD-1 blockade in vivo and confirming their putative immunomodulatory capacity. This phenomenon was only observed in CCL19+ DCs but not in CCL19- DCs, indicating CCL19 as a specific marker and could reflect the infiltration and functional phenotype of CCL19+ DCs in breast tumors. Finally, by integrating six independent ICI therapy cohorts, we demonstrated that baseline CCL19 in both tumor and blood could predict better response and survival in TNBC patients receiving ICIs, but not so for chemotherapy. Conclusions: Our results provide important insights into the relationship between the clinical outcome of ICIs and DC heterogeneity at single-cell resolution, support the development of therapies modulating CCL19+ DCs to trigger antitumor CD8+ T-cell immunity, and suggest baseline CCL19 levels as a noninvasive predictive biomarker for patients receiving ICIs, that are potentially applicable in the clinic. In light of these results, we suggest CCL19+ DC modulation as a possible precision immunotherapy approach for cancer patients. Citation Format: Song-Yang Wu, Si-Wei Zhang, Ding Ma, Xi Jin, Yi-Zhou Jiang, Zhi-Ming Shao. PD4-05 Integrated multi-cohort profiling identifies CCL19+ dendritic cells to potentiate immunotherapy efficacy in triple-negative breast cancer [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 PD4-05.

Background: Invasive lobular carcinoma (ILC) is the second most common type of invasive breast cancer and accounts for 10-15% of all cases. Though ILC has distinct clinical, prognostic and molecular features, studies are limited and include few patients. ILCs show a decreased response to neoadjuvant chemotherapy and an increased resistance to endocrine therapy. Thus, there is a great need to identify alternative therapies, such as immunotherapy, that could improve overall survival. Success of immunotherapy largely depends on tumor immunogenicity which varies with histologic type. Determination of predictive and prognostic biomarkers for ILC will help determine who can benefit the most. Our study investigates canonical markers of immunogenicity - PD-L1 expression and Tumor Mutational Burden (TMB) - in patients with ILC compared to invasive ductal carcinoma (IDC). We further correlate these markers in different subtypes i.e. hormone receptor positive (HR+), HER2 positive (HER2+), and in triple negative breast cancers (TNBC). We also analyze differences in immune cell profiles constituting the tumor microenvironment (TME) and differences in genomic alterations between ILC and IDC. Methods: A retrospective data analysis was performed to identify breast cancer tumors with ILC or IDC histology profiled at Caris Life Sciences (Phoenix, AZ) that were tested for PD-L1 by SP142 assay and had whole transcriptome sequencing data available. ILC and IDC cases were further subtyped as HR+ and HER2+ and TNBC. PD-L1 expression in immune cell was assessed using Ventana PD-L1 (SP142) histochemical assay and PD-L1 expression in tumor cell was assessed by laboratory developed test using SP142 clone with staining higher than 2+ considered positive. TMB was measured by counting somatic non-synonymous missense mutations on the 592 gene panel (Nextseq) next generation sequencing (NGS) assay, and ≥ 10 mutations/megabase (mut/Mb) was considered high. Using the whole transcriptome RNA sequencing (NovaSeq) data we analyzed the difference in immune cell profiles constituting the TME using a computational RNA deconvolution approach. NGS was used to identify significant differences in genomic alterations between ILC and IDC. Results: We identified 1,359 breast cancer patients (ILC: 194 vs IDC: 1,165). Among ILC, 79% were HR +, 11% TNBC, <2% HER2+ and the rest were of unclear subtype, compared to 55% HR +, 31% TNBC, 10% HER2+ in the IDC group. PD-L1 expression in immune cells was lower in ILC (PD-L1≥ 1% ILC: 13% vs. IDC: 35% p <0.0001; PD-L1>10% ILC: 1% vs. IDC: 5% p=0.017). PD-L1 expression in tumor cells was also lower in ILC (ILC 1% vs IDC 5%; p =0.0136). All subtypes of ILC had lower PD-L1 expression in immune and tumor cells when compared to IDC. TMB was similar and comparable in IDC and ILC (median TMB 7 mut/Mb). Assessment of TME showed a significantly increased abundance of cytotoxic lymphocytes and monocytic cells in IDC, and stromal endothelial cells in ILC. Androgen receptor (AR) expression, and mutations in CDH1 (ILC 76% vs IDC 2%) and PIK3CA (ILC 44% vs IDC 30%) were more common in ILC. CDH1 mutation was significantly higher in both TN ILC (ILC 60% vs IDC 1%; p<0.05) and HR+ ILC (77% vs IDC 2%; p<0.05) subtypes of ILC. TP53 mutation was lower in ILC (ILC 26% vs IDC 65%) irrespective of subtype. Conclusion: In summary, PD-L1 expression in immune cells and tumor cells was lower in ILC, however TMB was comparable between ILC and IDC. Immune cell profiling supports a cold or less immunogenic TME for ILC. A composite immune biomarker may be able to better characterize immunogenicity of ILC. Citation Format: Upama Giri, Joanne Xiu, Tatiana Karadimitriou, Sofi Castanon, Foluso Ademuyiwa, Paula Pohlmann, Elias Obeid, Jasgit Sachdev, Elisa Krill-Jackson, Michael Simon, Antoinette Tan, Neelima Denduluri, Lee Schwartzberg, Michael Korn, Evanthia T Roussos Torres. Molecular evaluation of immunogenicity and genomic alterations in invasive lobular breast cancer [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS17-27.

A prodrug nanodevice co-delivering docetaxel and ROR1 siRNA for enhanced triple negative breast cancer therapy.

Background: Triple-negative breast cancer (TNBC) accounts for approximately 15% of breast cancers and is associated with a poorer prognosis and higher rate of distant recurrence compared to receptor positive breast cancer. Due to an absence of effective targeted therapies, cytotoxic chemotherapy remains the standard of care for TNBC patients. However, the aggressive nature of these tumors makes preventing progression to metastatic disease a priority in therapeutic strategy. Inhibition of lymphatic activation represents an attractive approach to reduce peritumoral lymphatic vessel recruitment and attenuate cancer cell invasion to lymph nodes and subsequent metastasis to distant sites. However, little is currently known regarding the role of lymphatics in TNBC and how common therapeutic strategies can influence lymphatic reactivity. We hypothesized that inhibiting lymphatic activation in combination with standard of care chemotherapy will enhance therapeutic response in TNBC. Methods: Our lab has developed a novel 3D tissue-engineered tri-culture system that recapitulates the tumor-lymphatic interface within the breast tumor microenvironment. It consists of a porous tissue culture insert on which we seed human lymphatic endothelial cells (LECs) to form a monolayer on the underside. Atop the insert, we place Collagen I gel with human mammary fibroblasts and human breast cancer cells labeled with fluorescent Cell Tracker dye. After gelation, flow is applied via a pressure head and the system equilibrates overnight (16-18h). Drugs are applied via flow and then flushed from the system with basal media. After 48h from first application, gels are removed, dissociated, and analyzed via flow cytometry for death [Live/Dead and apoptosis (Caspase 3/7)], while the inserts are analyzed via microscopy for invaded cancer cells. Animal experiments were performed according to guidelines set forth by the UVA Animal Care and Use Committee. Twenty female Balb/c mice were implanted with 250,000 4T1 cells and treated with either anti-VEGFR3 antibody or IgG control and docetaxel (n=5 per group) or saline control once tumors were palpable. Tumors were dissected and dissociated once they reached 150 mm3, and analyzed via flow cytometry for stromal cell populations. Lungs were analyzed for metastases by cryosectioning and H&E staining. Results: We used our in vitro system to screen the common chemotherapeutics docetaxel, doxorubicin, 5-FU, and carboplatin across three common TNBC cell lines MDAMB231, HCC1806, and HCC38. We found that the presence of LECs significantly increases TNBC cell survival and invasiveness, shifting EC50s for cell death 10-1000 fold (EC50 shift nonlinear regression, p<0.05 for +/- LECs) and doubling to tripling invasion of cancer cells (analysis by two-way ANOVA, p<0.05). This poor response to therapy can be attenuated by treatment with the VEGFR3 inhibitor MAZ51. In vivo, we have seen that adjuvant anti- VEGFR3 therapy can reduce lymphatic endothelial cell populations in the tumor stroma with and without docetaxel chemotherapy when analyzed by flow cytometry (n=4 per group, p<0.05). Additionally, combination-treated mice showed reduced metastasis to the lung. Conclusion: Together, these data could indicate the effectiveness of anti-VEGFR3 therapy as an addition to chemotherapy to reduce metastatic progression of TNBC by limiting tumor-induced and therapy-induced lymphangiogenesis. Studies are ongoing to optimize and understand the ability of this adjuvant therapeutic strategy to increase therapeutic efficacy in TNBC. Citation Format: Alexandra H. Harris, Jennifer M. Munson. Lymphatics in triple-negative breast cancer: Contribution to chemotherapeutic efficacy in vitro and in vivo. [abstract]. In: Proceedings of the AACR Special Conference: Function of Tumor Microenvironment in Cancer Progression; 2016 Jan 7–10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2016;76(15 Suppl):Abstract nr B16.

Background: Apoptosis resistance is a hallmark of cancer, and apoptosis regulators have been targeted for cancer treatment. A class of such regulators is inhibitor of apoptosis proteins (IAPs), which suppress caspase activity by inducing degradation of active caspases or blocking interaction of caspases with their substrates. Dysregulated IAP expression has been reported in many cancers, including breast cancer, and is involved in chemoresistance. IAPs have therefore become attractive targets for cancer treatment. Birinapant (TL32711), a biindole-based bivalent mimetic of second mitochondria-derived activator of caspase (SMAC, an endogenous antagonist of IAPs), has been shown to effectively activate apoptotic signaling by targeting IAPs and has potential application for treatment of multiple cancers. In addition, birinapant showed synergistic anti-tumor effects with several widely used chemotherapeutic agents in various cancers. Here, we assessed whether birinapant synergizes with commonly used anti-cancer drugs, including entinostat (class I histone deacetylase inhibitor), cisplatin, paclitaxel, voxtalisib (PI3K inhibitor), dasatinib (Src inhibitor), everolimus (mTOR inhibitor), and gemcitabine, in triple-negative breast cancer (TNBC). Among these drugs, gemcitabine strongly synergized with birinapant (combination index [CI] <1). We hypothesize that birinapant enhances anti-tumor efficacy of gemcitabine in TNBC by inducing intrinsic pathway-dependent apoptosis. Methods: The in vitro anti-tumor efficacy of birinapant, gemcitabine, and their combination was determined using CellTiter-Blue viability and soft agar colony formation assays. The CI (CalcuSyn software) was used to quantify the synergy. The effects of drug treatment on cell cycle distribution were analyzed by flow cytometry; the effects on apoptosis were assessed by annexin V-PE and 7-AAD staining, and induction of apoptosis was further confirmed by pan-caspase inhibitor treatment; and the effects on IAP degradation were examined by Western blotting. The in vivo antitumor efficacy was determined using TNBC xenograft mouse models. Results: Birinapant as a single agent had varying anti-proliferation effects in TNBC cells (IC50 = 0.21 to >20 µM). It had no synergistic effects with entinostat, cisplatin, paclitaxel, voxtalisib, dasatinib, or everolimus but strongly synergized with gemcitabine (CI <1 µM) in tested TNBC cells. Birinapant and gemcitabine synergistically inhibited the growth of TNBC cells by activating the intrinsic apoptosis pathway, accomplished by inducing degradation of cIAP2 and XIAP, which led to apoptotic cell death. Furthermore, birinapant significantly enhanced anti-tumor effectiveness of gemcitabine in TNBC xenograft mouse models (n = 10; P < 0.05) by inducing apoptosis. Conclusion: Our findings demonstrate the therapeutic potential of birinapant for enhancing the anti-tumor efficacy of gemcitabine in TNBC by targeting the IAP family of proteins. Citation Format: Xuemei Xie, Jangsoon Lee, Troy Pearson, Alexander Y. Lu, Debu Tripathy, Gayathri R. Devi, Chandra Bartholomeusz, Naoto T. Ueno. Birinapant enhances gemcitabine's anti-tumor efficacy in triple-negative breast cancer by inducing intrinsic pathway-dependent apoptosis [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 548.

BackgroundThe CD155-TIGIT axis, a breast cancer progression biomarker, underscored neoadjuvant chemotherapy (NAC) response variability in triple-negative breast cancer (TNBC), urging biomarker-based patient stratification for timely therapy.MethodsThirty-nine TNBC patients who received NAC were recruited. The expression of TIGIT, CD155, CD226, and CD96 on tumoral and stromal cells in the tumor microenvironment was detected by immunohistochemistry, and their relationships with NAC response were explored.Results10.3% patients exhibited grade 1 (G1) response to NAC, and 20.5% achieved a complete pathological response. Notably, CD155 and CD96 were predominantly detected on tumor cells, whereas CD226 and TIGIT were predominantly detected on stromal cells. The expression of these markers did not significantly correlate with response to NAC (p > 0.05), and each individual marker lacked predictive power for determining NAC therapeutic efficacy (p > 0.05). However, a specific combination of tumoral cells expression of CD226(≥4%), CD155(≥40%), and CD96(≥35%), coupled with TIGIT expression on tumoral (<35%) and stromal cells (<12.5%), was able to identify patients with G1 response to NAC.ConclusionExpression levels of TIGIT/CD155/CD226/CD96 on tumoral and stromal cells might collectively serve as predictive biomarkers for NAC response in TNBC. This implied that CD155-TIGIT axis could be prospectively applied clinically to identify NAC-resistant TNBC patients.

Abstract Background: Triple-negative breast cancer (TNBC) remains a formidable challenge in oncology due to its aggressive nature and limited therapeutic options. Our previous investigations have elucidated the role of miR-20a-5p in promoting TNBC cell proliferation, invasion, and migration while inhibiting apoptosis through the RUNX3/Bim/p21 pathway. We have also demonstrated a positive correlation between tissue-expressed miR-20a-5p and circulating miR-20a-5p (cirmiR-20a-5p) in TNBC patients, with elevated cirmiR-20a-5p levels associated with poorer prognosis, suggesting its potential as a prognostic biomarker. While immune checkpoint inhibitors show promise for TNBC treatment, resistance mechanisms remain poorly understood. This study builds upon these findings to investigate the role of cancer cell-derived exosomal microRNA-20a-5p (miR-20a-5p) in modulating CD8+ T-cell function and conferring resistance to anti-programmed cell death 1 (PD-1) therapy in TNBC.Methods: We employed a comprehensive approach combining in vitro cellular assays, in vivo mouse models, and clinical sample analyses. Circulating miR-20a-5p levels were quantified using qRT-PCR in plasma samples from TNBC patients, patients with relapsed TNBC, and healthy donors. Exosomes were isolated from TNBC cell lines and characterized using nanoparticle tracking analysis and transmission electron microscopy. The functional impact of exosomal miR-20a-5p on CD8+ T cells was assessed through cytokine production assays and cytotoxicity tests. Mechanistic studies utilized luciferase reporter assays and Western blotting to confirm direct targeting of NPAT and RUNX3 by miR-20a-5p. A humanized mouse model was employed to evaluate the impact of miR-20a-5p overexpression on anti-PD-1 therapy resistance. Immunohistochemistry was performed on TNBC tissue samples to assess the correlation between cirmiR-20a-5p levels and CD8+ T-cell infiltration. Results: Circulating miR-20a-5p levels were significantly elevated in TNBC patients compared to healthy donors (3.2-fold increase, p&amp;lt;0.001) and further increased in patients with relapsed TNBC (1.8-fold higher than primary TNBC, p&amp;lt;0.01). High cirmiR-20a-5p levels correlated with poor prognosis (HR=2.3, 95% CI: 1.8-2.9, p&amp;lt;0.001). TNBC cell-derived exosomes contained high levels of miR-20a-5p, suppressing CD8+ T-cell function by reducing cytokine production (IFNγ, TNF-α, granzyme B, and perforin) and tumor cell killing capacity. Mechanistically, miR-20a-5p directly targeted the 3'-UTR of nuclear protein ataxia-telangiectasia (NPAT) and RUNX3, decreasing their expression in CD8+ T cells and TNBC cells, respectively. In the humanized mouse model, tumors overexpressing miR-20a-5p exhibited resistance to anti-PD-1 therapy, with reduced CD8+ T-cell infiltration and MHC class I expression. Plasma exosomal miR-20a-5p levels were 2.5-fold higher in TNBC patients resistant to anti-PD-1 therapy compared to responders (p&amp;lt;0.001). Immunohistochemistry revealed a significant negative correlation between cirmiR-20a-5p levels and CD8+ T-cell infiltration in TNBC tissues (r = -0.68, p&amp;lt;0.001). Conclusions: This study identifies exosomal miR-20a-5p as a novel mediator of CD8+ T-cell dysfunction and anti-PD-1 therapy resistance in TNBC. The miR-20a-5p/NPAT and miR-20a-5p/RUNX3 axes represent previously unrecognized mechanisms by which TNBC cells suppress anti-tumor immunity and promote tumor progression. Our findings suggest that circulating miR-20a-5p may serve as a potential biomarker for predicting immunotherapy response and prognosis in TNBC patients. Furthermore, targeting these miR-20a-5p-mediated pathways could be a promising strategy to enhance immunotherapy efficacy in TNBC, potentially improving outcomes for this aggressive breast cancer subtype. These results provide a strong rationale for further investigation of miR-20a-5p as a therapeutic target and prognostic marker in TNBC, opening new avenues for personalized medicine approaches in this challenging malignancy. Citation Format: Xiangdong BaiI, Guohui Han, Weina Li, Feng Li, Yating Hao, Li Huang, Peng Bu. Cancer cell-derived exosomal miR-20a-5p inhibits CD8+ T-cell function and confers anti-programmed cell death 1 therapy resistance in triple-negative breast cancer [abstract]. In: Proceedings of the San Antonio Breast Cancer Symposium 2024; 2024 Dec 10-13; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2025;31(12 Suppl):Abstract nr P1-09-24.

Sciatic nerve stimulation enhances NK cell cytotoxicity through dopamine signaling and synergizes immunotherapy in triple-negative breast cancer.

In the present study, a novel series of 3-pyrimidinylazaindoles were designed and synthesized using a bioinformatics strategy as cyclin-dependent kinases CDK2 and CDK9 inhibitors, which play critical roles in the cell cycle control and regulation of cell transcription. The present approach gives new dimensions to the existing SAR and opens a new opportunity for the lead optimizations from comparatively inexpensive starting materials. The study led to the identification of the alternative lead candidate 4ab with a nanomolar potency against CDK2 and CDK9 and potent antiproliferative activities against a panel of tested tumor cell lines along with a better safety ratio of ∼33 in comparison to reported leads. In addition, the identified lead 4ab demonstrated a good solubility and an acceptable in vivo PK profile. The identified lead 4ab showed an in vivo efficacy in mouse triple-negative breast cancer (TNBC) syngeneic models with a TGI (tumor growth inhibition) of 90% without any mortality growth inhibition in comparison to reported leads.