Triple-negative Breast Tumors Research Articles

Exploring the interplay between triple-negative breast cancer stem cells and tumor microenvironment for effective therapeutic strategies.

Triple-negative breast cancer (TNBC)is a highly aggressive and metastatic malignancy with poor treatment outcomes. The interaction between the tumor microenvironment (TME)and breast cancer stem cells (BCSCs)plays an important role in the development of TNBC. Owing to their ability of self-renewal and multidirectional differentiation, BCSCs maintain tumor growth, drive metastatic colonization, and facilitate the development of drug resistance. TME is the main factor regulating the phenotype and metastasis of BCSCs. Immune cells, cancer-related fibroblasts (CAFs),cytokines, mesenchymal cells, endothelial cells, and extracellular matrix within the TME form a complex communication network, exert highly selective pressure on the tumor, and provide a conducive environment for the formation of BCSC niches. Tumor growth and metastasis can be controlled by targeting the TME to eliminate BCSC niches or targeting BCSCs to modify the TME. These approaches may improve the treatment outcomes and possess great application potential in clinical settings. In this review, we summarized the relationship between BCSCs and the progression and drug resistance of TNBC, especially focusing on the interaction between BCSCs and TME. In addition, we discussed therapeutic strategies that target the TME to inhibit or eliminate BCSCs, providing valuable insights into the clinical treatment of TNBC.

CD163+ macrophages in the triple-negative breast tumor microenvironment are associated with improved survival in the Women’s Circle of Health Study and the Women’s Circle of Health Follow-Up Study

BackgroundTumor-associated macrophages (TAMs) are a prominent immune subpopulation in the tumor microenvironment that could potentially serve as therapeutic targets for breast cancer. Thus, it is important to characterize this cell population across different tumor subtypes including patterns of association with demographic and prognostic factors, and breast cancer outcomes.MethodsWe investigated CD163+ macrophages in relation to clinicopathologic variables and breast cancer outcomes in the Women’s Circle of Health Study and Women’s Circle of Health Follow-up Study populations of predominantly Black women with breast cancer. We evaluated 611 invasive breast tumor samples (507 from Black women, 104 from White women) with immunohistochemical staining of tissue microarray slides followed by digital image analysis. Multivariable Cox proportional hazards models were used to estimate hazard ratios for overall survival (OS) and breast cancer-specific survival (BCSS) for 546 cases with available survival data (median follow-up time 9.68 years (IQR: 7.43–12.33).ResultsWomen with triple-negative breast cancer showed significantly improved OS in relation to increased levels of tumor-infiltrating CD163+ macrophages in age-adjusted (Q3 vs. Q1: HR = 0.36; 95% CI 0.16–0.83) and fully adjusted models (Q3 vs. Q1: HR = 0.30; 95% CI 0.12–0.73). A similar, but non-statistically significant, association was observed for BCSS. Macrophage infiltration in luminal and HER2+ tumors was not associated with OS or BCSS. In a multivariate regression model that adjusted for age, subtype, grade, and tumor size, there was no significant difference in CD163+ macrophage density between Black and White women (RR = 0.88; 95% CI 0.71–1.10).ConclusionsIn contrast to previous studies, we observed that higher densities of CD163+ macrophages are independently associated with improved OS and BCSS in women with invasive triple-negative breast cancer.Trial registrationNot applicable.

Abstract PO4-06-11: Development of fully human anti-progranulin monoclonal antibodies as inhibitors of triple negative breast cancer cell growth and tumor formation

Abstract Triple negative breast cancer (TNBC) is characterized by invasiveness and poor survival. Identifying novel TNBC targeted therapies to potentiate standard of care (SOC) therapy, is an unmet need. In the past 4 years, several targeted therapies for TNBC have been approved by the Food and Drug Administration (FDA). However, the identification of targeted therapies against other biological drivers of TNBC remains important. Progranulin (PGRN/GP88) is a biological driver of tumorigenesis, survival, and drug resistance in breast cancer. PGRN/GP88 tissue expression is an independent prognostic factor of recurrence while elevated serum PGRN/GP88 level is associated with poor outcomes such as progression of disease and shortened survival. PGRN/GP88 expression is elevated in 30% TNBC. The importance of inhibiting PGRN/GP88 effect on the proliferation and tumor growth of triple negative breast cancer cells was previously established. In the current study, in association with Precision Antibody, we developed anti-PGRN/GP88 fully human monoclonal antibodies by immunizing TC transgenic humanized mice with recombinant human progranulin. Within 60 days since the start of immunization, several fully human monoclonal antibodies were successfully developed, characterized by several functional assays including neutralization, inhibition of PGRN/GP88 cell surface binding, proliferation, migration in MDA-MB-231 cells in vitro and in vivo. Results: The inhibition of PGRN/GP88 action by fully human monoclonal antibodies inhibited proliferation and migration in a dose-and time-dependent fashion in TNBC cells. Octet analysis determined Kd in the range of 10−11 M to 10−10 M. Transwell assay showed that anti-PGRN treatment inhibited migration In vivo xenograft studies with MDA-MB-231 cells injected in athymic nude mice showed that several fully human anti-PGRN antibodies inhibited tumor growth when compared to antibody control treated mice. In vivo dose response of AG01 in MDAMB-231 tumor bearing mice will be provided. Conclusion: PGRN/GP88 represents a therapeutic target for TNBC with two companion diagnostics (tissue test and ELISA to measure GP88 circulating levels). The use of the TC transgenic mice allows for the rapid development of fully human high affinity monoclonal antibodies bypassing the need for chimerization or humanization of mouse monoclonal antibodies. The development of fully human anti-progranulin antibodies provides novel targeted therapeutic option for TNBC. This work is supported by a an SBIR grant 5R44 CA 224718 from the National Cancer Institute to GS. Citation Format: Ginette Serrero, Jianping Dong, Jun Hayashi, Chun Dong. Development of fully human anti-progranulin monoclonal antibodies as inhibitors of triple negative breast cancer cell growth and tumor formation [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 PO4-06-11.

Abstract PO3-25-02: Circadian Rhythm Disruption Accelerates Aggressive Mammary Tumorigenesis by Altering Tumor Microenvironment

Abstract \Breast cancer is the most common cancer in women worldwide, leading to over 650,000 deaths worldwide. The circadian rhythm is a 24-hour biological rhythm that controls most cellular, hormonal, and behavioral processes in living organisms, which is why perturbation of these rhythms is linked to several devastating diseases such as cardiovascular diseases, diabetes, and cancer. In the mammary gland, circadian clocks regulate the rhythmic expression of numerous genes. Circadian rhythm disruption (CRD), which has been termed a "carcinogen" by the International Agency for Research in Cancer, is caused by different factors, one of them being shift work. Several epidemiological studies have revealed that women who work night shifts are predisposed to breast cancer. The aim of this study is to investigate the effect of circadian rhythm disruption on aggressive mammary tumorigenesis. To do this, we used genetically engineered mouse models of mammary tumors (HER2-tumor and triple-negative breast tumor, or TNBC). Chronic jetlag, which mimics shift work in mice and is known to cause CRD, was introduced in one group of mice by perturbing the normal light and dark cycles with an 8-hour advance every three days. Wheel-running, which is represented by an actogram, was used to confirm the circadian disruption in the CRD mice. CRD was found to accelerate tumor initiation with a significant increase in weight in the CRD group of mice. CRD enhanced tumor burden significantly compared to LD mice. Our results also showed high metastatic foci in the lungs of CRD mice compared to LD mice. A flow cytometry study showed a higher cancer stem cell population in CRD tumors. Mammosphere assays with tumor-derived cells showed that CRD increased the number and size of the mammospheres compared to the control tumors. The tumor cells were analyzed using single-cell RNA sequencing, which showed that CRD alters the tumor microenvironment by developing more immunosuppressive and "cold" tumors. The tumor-derived organoids (tumoroid) showed resistance to chemotherapeutics. Finally, bioinformatics analysis using single-cell RNA seq data from human breast tumors (TNBC, ER/PR, and HER2) showed that the malignant cells in TNBC tumors had a significantly higher circadian rhythm disrupted score (CRD score) compared to ER/PR tumors. Taken together, our data showed how circadian rhythm disruption affects TNBC tumorigenesis and drug response by altering the tumor microenvironment. Citation Format: Olajumoke Ogunlusi, Mrinmoy Sarkar, Tristan Nguyen, Devon Boland, James Cai, Arhit Chakrabarti, Bani Mallick, Alex Keene, Tapasree Roy Sarkar. Circadian Rhythm Disruption Accelerates Aggressive Mammary Tumorigenesis by Altering Tumor Microenvironment [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 PO3-25-02.

Abstract PO5-13-10: Determining tumor-driving cell signaling pathways in breast cancer: Support for targeted therapy selection

Abstract Introduction: Success of therapeutic interventions largely rely on the pathological and biological characteristics of the tumor and varies due to the heterogeneous nature of breast cancers. Signal transduction pathways play an important role and are often used as target for specific therapies like hormone treatment, PI3K inhibitors etc. Immunohistochemistry (IHC) is currently the first assessment to divide tumors for subtyping and treatment and, especially in more advanced disease, mutation analyses are often used. Both IHC and mutation analyses only analyze a specific part of a signal transduction pathway and do not correlate well with outcome. Therefore, there is a need for new approaches to look at signaling pathways in cancer. We have developed a methodology that measures and quantifies signal transduction pathways (STPs) activities to reveal potential tumor-driving STPs to identify new options for targeted therapy in breast cancer. Method: Using the mRNA-based OncoSIGNal pathway activity profiling qPCR test (InnoSIGN), STP activities of 7 pathways (AR, ER, PI3K, MAPK, HH, TGFβ and Notch) were measured and quantified on a scale from 0-100. By using a set of non-tumor epithelial breast tissue samples, a reference range for normal physiological pathway activity was defined. A threshold was set on the 95th percentile of this range. A pathway activity score above this threshold was considered to be high (activated). Using this methodology, 201 ER positive (based on IHC) and 64 triple negative FFPE breast tumor samples obtained from different study sites and commercial biobanks were analyzed, including samples from primary and metastatic lesions, to determine for each individual tumor its pathway activity profile and identify activated tumor-driving pathways. Results: In the 195 primary ER positive tumors, the ER pathway was found to be activated in 78% of the samples, even though all samples were strongly ER immunohistochemistry stain positive. In addition to ER pathway activation, occasionally other pathways were activated: PI3K in 37%, HH in 25%, MAPK in 19%, AR in 20%, TGFβ in 6%, and Notch in 1%, of these samples. Analysis of 27 metastatic samples of ER positive tumors revealed in only 63% an active ER pathway. Additionally, more frequently PI3K pathway was observed in 70% of the samples. Other frequencies of pathway activation were: AR in 7% and MAPK in 4%, No Notch and TGFβ activity was found in this cohort. In 76 of the 78 triple negative tumors (TNBC) the ER pathway was inactive as expected. Remarkably, 2 samples appeared to have an activated ER pathway. An activated MAPK pathway was found in 50%, PI3K in 49%, HH in 18%, AR in 14%, TGFβ in 5% and Notch in 3% of these TNBC tumors. Conclusion: Compared to ER positive primary tumors we observed in metastatic ER positive tumors less frequent activation of the ER and more frequent activation of the PI3K pathway, suggesting that the latter pathway is associated with a more aggressive tumor phenotype. The triple negative subtype is characterized predominantly by activation of the PI3K, MAPK, AR and/or HH pathways, which may create new options for personalized targeted therapy of this subtype. OncoSIGNal can be used to determine the tumor driving signaling pathways in breast cancer patients, guiding selection of personalized targeted therapies. Citation Format: Paul van de Wiel, Yvonne Wesseling-Rozendaal, Anke Pierik, Dianne van Strijp, Saskia Vermeer, Fatema Tashrifwala, Josette Hartnett, Robert Babkowski, Suzanne Rose. Determining tumor-driving cell signaling pathways in breast cancer: Support for targeted therapy selection [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 PO5-13-10.

Abstract PO5-17-02: Triple-negative breast cancer as an untraceable tumor compared to other immunohistochemical subtypes: a cross-sectional study

Abstract Objective: To compare the form of presentation at diagnosis of triple negative breast tumors with other immunohistochemical subtypes, in relation to clinical examination or imaging examination. Methods: This is a cross sectional study that evaluated data related to the presentation of breast carcinomas at diagnosis (clinical examination or screening imaging examination findings), immunohistochemical subtype, histological grade and tumor size, in diagnosed and referred patients. for the Mastology service at Hospital São Paulo – Unifesp, in São Paulo – Brazil, in the period between 2012 and 2021, by analyzing data from the medical records of these patients. Results: Among the 699 invasive carcinomas diagnosed, 246 patients were diagnosed with luminal profile A (35.2%), 257 luminal B (36.8%), 58 luminal B HER-2+ (8.3%), 26 HER-2 enriched (3.7%) and 112 triple negative (16%). Triple negative carcinomas had a higher rate of diagnosis by clinical examination (86.6%), 6.65 times more likely to be diagnosed by clinical examination than luminal A, in addition to having a larger lesion. The triple negative and HER-2 enriched subtypes presented the worst scenarios of clinical stage at diagnosis. Among the tumors diagnosed by screening examination, those belonging to the luminal A were associated with nodules seen on imaging (in 45.1%) and the subtypes luminal B and luminal B HER-2+ with the appearance of microcalcifications. There were no cases of triple negatives involving microcalcifications in our sample. Asymmetries were more common in images of luminal B HER-2+ tumors. In the multivariate analysis, it was observed that only the histological grade and the Ki-67 value are related to the form of diagnosis. Tumors with histological grade 2 or 3 are more likely to be diagnosed by clinical examination and not be traceable in relation to histological grade 1. As for Ki-67, its increase of 1 percentage point leads to an increase of 1.3% in the chance finding on physical examination. There was no correlation of hormone receptors and HER-2 with the chance of the tumor not being screenable. Conclusions: triple negative subtype breast carcinomas present a higher risk of diagnosis by clinical examination compared to other immunohistochemical subtypes and, therefore, tend not to be traceable. Parameters such as high Ki-67 and histological grade correlate with this trend. Citation Format: Morgana Silva, Afonso Nazário, Vanessa Sanvido. Triple-negative breast cancer as an untraceable tumor compared to other immunohistochemical subtypes: a cross-sectional study [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 PO5-17-02.

Abstract PO4-13-11: Epigenetically defined sub-clonal heterogeneity drives therapy resistance in triple-negative breast cancer

Abstract Background Triple-negative breast cancer (TNBC) is a highly aggressive and heterogeneous disease with an average survival of less than 50%. Although immunotherapy is showing promising results in early TNBC, many patients do not respond, and chemotherapy remains the main treatment. High recurrence rates in TNBC are in part due to its inherent molecular heterogeneity and sub-clonal diversity, whereby cells present in minority sub-populations escape therapeutic pressure. Recurrent genomic alterations are not selected for upon therapy resistance in neoadjuvant chemotherapy (NAC) resistant TNBC, however sub-clonal transcriptionally defined cells are “primed” to drive resistance and are pre-existing, suggesting phenotypic heterogeneity is governed by inheritable activation of epigenetically defined regulatory regions. Here, we sought to characterise the sub-clonal and spatial diversity of chemotherapy resistant TNBC to identify the epigenetic hallmarks of lethal sub-populations of cells that may induce cell state transition, therapy resistance and ultimately metastatic progression. Methods Sixteen primary untreated triple-negative breast tumours (eight chemotherapy sensitive (ChemoS) and eight chemotherapy resistant (ChemoR, who died of their disease within three years)) were subjected to cell-matched high-depth multiome single nuclei RNA- and ATAC-sequencing. Driver DNA mutations and copy number alterations (CNA’s) were identified using a clinically validated targeted capture panel. Four tumours were subject to spatial RNA and ATAC profiling. Fourteen TNBC PDX models generated from patients with residual disease post NAC were subjected to single nuclei sequencing and used for validation. Meta-clustering of epithelial cells from the RNA profiles was used to identify sub-populations of cells harbouring distinct epigenetic and transcriptomic features enriched in ChemoR patients. Findings were validated in the BrighTNess TNBC NAC clinical trial and in chemotherapy treated TNBC patients from the SCAN-B study. Results Unsupervised meta-clustering (MC) of epithelial cells in G1 phase of the cell cycle identified nine meta-clusters across the dataset. A distinct immune-like MC was contributed by epithelial G1 cells, suggesting a cross-talk of epithelial cells with spatially proximal immune cells. Two MCs were dominated by cells from ChemoR patients and were defined by pathways related to hypoxia, EMT, and extra-cellular matrix signalling. Using scATAC profiles, we identified that the sub-populations of cells in the ChemoR dominant MCs were underpinned by both promoter and distal differential chromatin accessibility, that mapped to H3K27ac enhancer sites, suggesting a role of distal enhancer chromatin modification in regulating MC transcription. None of the meta-clusters were dominated by tumours with specific genetic mutations or CNA’s. Spatial ATAC and RNA profiling of a subset of tumours identified spatial epigenetically defined regions that showed distinct chromatin profiles, suggesting that the epigenetic regulation of genes that define the MCs are spatially distinct. ChemoR clusters were independently reproduced in a cohort of fourteen residual disease TNBC PDXs. Genes pertaining to the immune-like MC were associated with a good prognosis in independent cohorts of contemporary treated TNBC (SCAN-B) and associated with response to NAC in the BrighTNess trial. Marker genes from ChemoR dominant MCs were associated with poor prognosis and significant levels of residual disease following NAC. Conclusions Our multi-modal integrative analysis reveals unprecedented insight into the role of epigenetically defined spatially distinct, sub-clonal populations that pre-exist in treatment naïve TNBC and predict therapy response. This suggests pre-existing populations of cells have already acquired the epigenetic footprints and hence transcriptomic features to allow them to evade therapy. Citation Format: Fatemeh Ahmadimoghari, Yu Zhang, Ioanna Mavrommati, Shefali Thakur, Chris Starling, Ioannis Roxanis, Syed Haider, Rachael Natrajan. Epigenetically defined sub-clonal heterogeneity drives therapy resistance in triple-negative breast cancer [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 PO4-13-11.

Selective Ru(II)/benzoate complexes against triple-negative breast tumor cells and their interactions with DNA and BSA

New ruthenium(II) complexes bearing benzoate (AB) ligand with the general formula [Ru(AB)(bipy)(PP)]PF6, where 2,2′-bipyridine (bipy) and different diphosphine ligands, (PP) = 1,2′-bis(diphenylphosphine)ethane (dppe, 1), 1,3′-bis(diphenylphosphine)propane (dppp, 2) and 1,2′-bis(diphenylphosphine)ferrocene (dppf, 3), were synthesized. The compounds were characterized by molar conductivity, elemental analysis, cyclic voltammetry, infrared and UV–Vis spectroscopies, NMR, and by single-crystal X-ray diffraction for complexes 1 and 2. The complexes showed a weak interaction to CT-DNA through DNA minor groove, with Kb values at around 103-104 M−1. CT-DNA interaction assays by viscosity and circular dichroism (CD) suggested that the compounds do not significantly alter the secondary DNA structure. The complexes are cytotoxic against MDA-MB-231, MCF-7 (breast) and A549 (lung) tumor cell lines, with IC50 values in the range of 1 to 17 µM. The compounds 1-3 showed high selectivity against triple-negative breast tumor cells. Remarkably, complexes 1 and 3 show greater cytotoxic activity against cells than cisplatin, being promising agents for tumor treatment.

Abstract 1201: Pervasive HLA disruption fuels cancer evolution

Abstract Disruption of the human leukocyte antigen (HLA) molecules has important implications for immune evasion and tumor evolution. However, although genomic loss of HLA is frequent in non-small cell lung cancer (NSCLC) and in some types of breast cancer, the extent and importance of transcriptomic disruption to HLA presentation, including transcript repression and alternative splicing (AS), remains unclear. To address this, we developed MHC Hammer, a tool to call HLA allele-specific mutations, loss of heterozygosity (LOH), transcriptional repression, and alternative splicing. We first assessed HLA allelic expression and the prevalence of AS in normal tissue. Applying MHC Hammer to 510 normal lung samples and 407 normal breast samples from the Genotype-Tissue Expression cohort, we identified extensive heterogeneity in HLA allelic expression depending on both the allele and tissue type. In addition, HLA allelic AS, occurring in at least 20% of the allele transcripts, was observed in 19% of normal lung and 13% of normal breast samples. Next, we applied MHC Hammer to 413 TRACERx NSCLCs and 743 TCGA breast tumours. While mutations were uncommon, HLA LOH was a frequent event, occurring in 32% of lung adenocarcinomas (LUAD) and 57% of lung squamous cell carcinomas (LUSC). We observed a similar rate of HLA LOH in estrogen receptor positive (ER+) and negative (ER-) tumors (ER+: 14%, ER-: 15%), while in triple negative breast tumours we observed a higher rate of LOH (31%). Due to the heterogeneity in HLA expression observed in the normal samples, to measure tumor specific HLA repression and alternative splicing, we restricted our analysis to tumours with WES, tumor and tumor-adjacent normal RNAseq data. This resulted in a cohort of 88 NSCLCs from the TRACERx study as well as 44 ER+ breast tumours from the TCGA cohort. 61% of LUAD, 75% of LUSC and 35% of ER+ tumours harbored class I HLA transcriptional repression that could not be explained by underlying genomic loss. 27% of LUAD, 7% of LUSC and 55% of ER+ breast tumor patients exhibited no HLA disruption. Tumor specific AS affecting HLA exons 2, 3 or 4, potentially disrupting HLA antigen presentation, was identified in 8% of LUADs, 11% of LUSC and 7% of ER+ breast tumours. Tumor specific AS of exon 5, yielding soluble HLA molecules without a transmembrane domain was identified in 20% of LUAD, 14% of LUSCs and 18% of ER+ breast tumours. LUAD and LUSC tumor regions without HLA LOH or repression were enriched for somatic HLA AS events and alleles with somatic HLA AS had a higher neoantigen burden than those without. We found no relationship between survival and HLA expression or AS in the normal tissue. However, consistent with the importance of HLA dysfunction in tumor evolution, LUADs with low HLA expression in at least one tumor region were associated with shorter disease-free survival, HLA LOH was more common in LUAD’s that metastasized and HLA repression was enriched in LUSC primary regions seeding metastases. Citation Format: Clare Puttick, Thomas P. Jones, Michelle Leung, Oriol Pich, Felipe Galvez Cancino, Jiali Liu, Manuel Varas-Godoy, Andrew Rowan, Carlos Martinez-Ruiz, Robert Bentham, Krijn K. Dijkstra, Rachel Rosenthal, Nnennaya Kanu, Kevin Litchfield, Roberto Salgado, David A. Moore, Peter Van Loo, Mariam Jamal-Hanjani, Sergio A. Quezada, Nicholas McGranahan, Charles Swanton. Pervasive HLA disruption fuels cancer evolution [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 1201.

Abstract 6077: Chimeric RNAs are abundantly expressed in precancerous breast tissue of sporadic breast cancer patients

Abstract Background: Immuno-prevention efforts in breast cancer (BC) have been hampered by the lack of known neoantigenic targets that can be used for a cancer prevention vaccine. RNA fusions represent attractive candidates as these are more immunogenic than insertion/deletion mutations and are substantial source of neoantigens. The objective of this study was to characterize chimeric RNAs in precancerous breast tissue. Methodology: Novel RNA fusions were identified by mining the sequence reads from RNA sequencing of 25 Her2 positive, 25 hormone receptor positive (HR) and 25 triple negative (TN) breast tumors and paired histologically normal tissues by Qiagen’s CLC pipeline. Breast samples from women undergoing breast reduction surgery were used as controls to exclude normal tissue associated chimeric mRNAs from analysis. Neoantigen prediction was performed by MHCnuggets. Chimeric mRNAs were validated by PCR. Ribosomal profiling was performed to assess if fusion transcripts are engaged with ribosomes. Results: A median of 36 novel RNA fusions (range 7-877) were identified in the tumor samples. In line with high genomic instability of TNBC subtype of BC, about 1/3 of the TN tumors expressed greater than 300 RNA fusions. In order to identify RNA fusions that are relevant for BC prevention, we investigated the presence of RNA fusions in the paired histologically normal samples as compared to the index tumor. We restricted these analyses to top 20 fusion transcripts that were present across the set of 75 tumor samples and also detected in 1 or more of the TCGA samples. Interestingly, we found that more than 1/3 of the histologically normal samples express at least 1 of the top 20 RNA fusions. When compared to the corresponding tumor, we found up to 50% of the paired normal tissue expressed RNA fusions identified in the tumor, supporting a role for these chimeric mRNA as an early molecular change during breast tumorigenesis. To validate the robustness of the prediction pipeline, PCR validation was performed for the chimeric mRNA with highest prevalence, NSFP1- LRRC37A2 which confirmed the presence of the transcript in 72% (13/18) of the predicted positive patient samples. MHCnuggets pipeline predicted 15 neopeptides from intergenically spliced chimeric NSFP1 [Exon 1-13] - LRRC37A2 [Exon 2-14], ¼ of which we validated to be immunogenic by ELISA. In order to confirm that the translation of the chimeric NSFP1- LRRC37A2 transcript, we investigated their enrichment in polysome enriched fraction obtained from a BC cell line confirmed to express the fusion transcript by PCR. Enrichment of transcripts in the polysomal fraction confirmed that these transcripts are likely to be translated. Conclusions: RNA fusions are frequently present in at risk breast tissue and are the source of substantial number of neoantigens. These RNA fusion- derived neoantigens may provide novel opportunities for developing vaccines for BC prevention. Citation Format: Anjana Bhardwaj, Shiyanth Thevasagayampillai, Sakuni Rankothgedera, Prisha Verma, Micah B. Castillo, Alexander C. Koh, Constance Albarracin, Preethi H. Gunaratne, Isabelle Bedrosian. Chimeric RNAs are abundantly expressed in precancerous breast tissue of sporadic breast cancer patients [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 6077.

Abstract 309: AHNAK nucleoprotein 2 tune oxidative stress and resistance to chemotherapy in triple negative breast cancer

Abstract Background: Resistance to chemotherapy is a major reason for the poorerprognosis in patients with triple negative breast cancer (TNBC). Growing evidence suggests that oxidative stress induced by reactive oxygen species (ROS) is an important factor in both tumourprogression and responses to chemotherapies, however, many signalling pathways and key molecular underlying the regulation of cancer cells to the ROS metabolism remains unclear. Aim: (i) To investigate the mechanistic role of AHNAK nucleoprotein 2 (AHNAK2) in TNBC and its links with breast cancer mortality, particularly caused by chemotherapeutic resistance; and (ii) to test a hypothesis that AHNAK2 facilitates the abnormal activation of ROSscavenging system, result in the resistance to chemotherapy in TNBC. Methods: The expression level of AHNAK2 was determined by immunohistochemical staining and analysed the mRNA expression profiles from The Cancer Genome Atlas of triple negative breast tumors. Roles of AHNAK2 in cancer cell growth, metastasis, chemotherapeutic resistanceand activation of oxygen scavenging systemwere determined by molecular and cell biology methods. A jetPEI nanocarrier was used as the vehicle for anti-AHNAK2 siRNAs in mouse models of cancer therapeutics. Results: AHNAK2 is a novel triple negative breast cancer target, which is overexpressed in TNBC tissues and significantly associated with clinical pathology of chemotherapeutic resistance and poor prognosis of tumour patients. Mechanistically, AHNAK2 antagonizes the ROS accumulation induced by epirubicinvia activating JAK-STAT signaling pathway and transportingphosphorylated STAT1 & STAT4 to the nucleus to promote the expression of SOD2, a key gene in ROS metabolism.And epirubicin enhanced AHNAK2 expression by induced the accumulation of ROS,which hence the Sp1-driven transcription of AHNAK2,result inde novo resistance. Furthermore, AHNAK2-specific siRNAs encapsulated by jetPEI nanocarriers prominently inhibit tumor growth and epirubicin-induced resistance in vivo. Conclusions: AHNAK2 plays a key role in resistance to chemotherapyby regulating the oxidative stressand ROS metabolism,representing a future biomarker and therapeutic target of TNBC. Citation Format: Na Hao. AHNAK nucleoprotein 2 tune oxidative stress and resistance to chemotherapy in triple negative breast cancer [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 309.

Abstract 2627: PanCancer epigenetic regulators of lymphocyte activation states

Abstract Several immune cell types have been successfully targeted for immunomodulatory therapy; however, the efficacies of such therapies are hindered by incompatible immune cell activation states. CD8+ T-cell exhaustion is one of the most studied T-cell states in the context of cancer and was shown to limit checkpoint blockade efficacy. Although CD8+ T-cell exhaustion studies in mice showed this state is tightly regulated by chromatin accessibility of cis-regulatory elements (CREs) and transcription factor activation, the exploration of these mechanisms, their prevalence, and their specificity in human cancers remains limited. In addition, chromatin accessibility signatures of other lymphocyte (CD4+ and NK) states frequently found in solid tumors have not been carefully characterized in a pan-cancer setting. Here we introduce the first single-nuclei atlas of pan-cancer and disease-specific cis- and trans-regulatory elements in tumor infiltrating lymphocytes. We profiled ~140K T-cell and NK cell nuclei with snATAC-seq and snRNA-seq from 227 tumor and normal samples from eleven cancer types. We distinguished two major subpopulations among exhausted CD8+ T-cells: GZMK expressing (CD8+ GZMK+ Tex) and ITGAE+ tissue-resident (CD8+ Trm ex). CD8+ GZMK+ Tex was enriched in clear cell renal cell carcinoma, while CD8+ Trm ex was found in all other cancers. Both exhausted groups featured high motif accessibility and expression of NR4A1, NFATC2, and NFKB2 transcription factors (previously reported to regulate exhaustion), but differed in EOMES motif accessibility and gene expression. We have identified other lymphocyte subpopulations showing exhaustion signatures, including CD4+ follicular helpers (CD4+ Tfh), CD4+ regulatory cells (CD4+ Tregs), and weakly cytotoxic tissue-resident NK cells (trNK weak). All these subpopulations showed increased expression and motif accessibility of the NR4A1 transcription factor, highlighting its role in regulating dysfunctional states not only in CD8+ T-cells, but also in CD4+ and NK cells. Additionally, CD4+ Tfh and Tregs showed enhanced activity of NFKB1, NFKB2, and POU2F2, and trNK weak cells had increased activity of NR4A2 and NR2F2. To put our results into clinical perspective, we have collected cohorts of renal cancer, melanoma and triple-negative breast tumors treated with immunotherapy in clinical trial settings, and showed elevated GZMK expression in exhausted T-cells in responders compared to non-responders. Citation Format: Alla Karpova, Nadezhda V. Terekhanova, Siqi Chen, Reyka G. Jayasinghe, Andrew Houston, Wagma Caravan, Ryan C. Fields, Li Ding. PanCancer epigenetic regulators of lymphocyte activation states [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 2627.

Zirconium-containing nanoscale coordination polymers for positron emission tomography and fluorescence-guided cargo delivery to triple-negative breast tumors

Nanoscale coordination polymer (NCP) is a class of hybrid materials formed by self-assembly of metal ions and organic ligands through coordination. The applications of NCP in biomedicine are quite extensive due to the diversity choice of metal ions and organic ligands. Here we designed Zr-P1 NCP based on Zr4+ selected as metal ion nodes and tetrakis(4-carboxyphenyl) ethylene as bridging ligands. Zr-P1 NCP was modified with functionalized pyrene derived polyethylene glycol (Py-PAA-PEG-Mal) on the surface and further conjugated with cRGD for active targeting of integrin αvβ3 overexpressed in triple-negative breast cancer. Doxorubicin was loaded on Zr-P1 NCP with encapsulation efficiency up to 22 % for the treatment of triple negative breast cancer. 89Zr-P1 NCP can be used for in vivo tumor imaging due to the fluorescence properties resulting from the enhanced aggregation-induced Emission (AIE) behavior of P1 ligands and its positron emission tomography (PET) capability. Cellular evaluation indicated that the functionalized Zr-P1@PEG-RGD presented a good function for tumor cell targeting imaging and doxorubicin could be targeted to triple negative breast cancer when it was loaded onto Zr-P1@PEG-RGD, which corroborated with the in vivo results. In summary, 89Zr-P1@PEG-RGD can serve as a biocompatible nanoplatform for fluorescence and PET image-guided cargo delivery. Statement of SignificanceNanoscale coordination polymer (NCP) is a class of hybrid materials formed by self-assembly of metal ions and organic ligands through coordination. The diversity of available metals and ligand structures upon NCP synthesis plays an advantage in establishing multimodal imaging platforms. Here we designed 89Zr-P1@PEG-RGD NCP based on Zr4+ selected as metal ion nodes and tetrakis(4-carboxyphenyl) ethylene as bridging ligands. 89Zr-P1@PEG-RGD nanomaterials have positron emission tomography (PET) capability due to the incorporation of zirconium-89, which can be used for in vivo tumor imaging with high sensitivity. The chemotherapeutic drug DOX was loaded on Zr-P1 NCP for the treatment of triple-negative breast cancer, and dual modality imaging can provide visual guidance for drug delivery.

Teadenol B as a Component of Microorganism-Fermented Tea Extract Inhibited Breast Cancers by Promoting Autophagy.

Breast cancer is a significant threat to life and health, which needs more safe and effective drugs to be explored. Teadenol B is a characteristic chemical component of microbial fermented tea. This study discovered that teadenol B could exhibit obvious inhibitory effects on all four different clinical subtype characteristics of breast cancer cells. Proteomic studies show that deoxycytidine triphosphate deaminase (DCTD), which could block DNA synthesis and repair DNA damage, had the most significant and consistent reduction in all four types of breast cancer cells with the treatment of teadenol B. Considering MDA-MB-231 cells exhibit poor clinical prognosis and displayed substantial statistical differences in KEGG pathway enrichment analysis results, we investigated its impact on the size and growth of MDA-MB-231 triple-negative breast tumors transplanted into nude mice and demonstrated that teadenol B significantly suppressed tumor growth without affecting body weight significantly. Finally, we found that the conversion of LC3-I to LC3-II in MDA-MB-231 increased significantly with teadenol B treatment. This proved that teadenol B could be a strong autophagy promotor, which explained the down-regulation of DCTD to some extent and may be the potential mechanism underlying teadenol B's anti-breast cancer effects. This finding provides new evidence for drinking fermented tea to prevent breast cancer and highlights the potential of teadenol B as a novel therapeutic option for breast cancer prevention and treatment, necessitating further investigations to clarify its exact target and the details involved.

Abstract A070: Examining patient-specific responses to PARP inhibitors in a novel, human induced pluripotent stem cell-based model of breast cancer

Abstract Patient-derived human induced pluripotent stem cells (hiPSCs) are a powerful tool for disease modeling because hiPSCs provide a scalable source of cells that retain patient-specific genomic variants. Recently, somatic cell reprogramming has been applied to modeling cancer: cancer cells are reprogrammed into hiPSCs, then re-differentiated into the relevant cancer cell lineage. hiPSCs derived from cancer cells retain tumor- and subclone-specific genomic variants and drug sensitivities, providing a unique tool to deconstruct the contributions of tumor heterogeneity to drug response. While hiPSCs have been shown to model various hematological malignancies, no study to date has examined the potential of hiPSCs to model breast cancer. We demonstrate for the first time that primary human breast cancer cells can be reprogrammed into hiPSCs by transiently overexpressing canonical reprogramming factors. Using an optimized reprogramming methodology, we generated a biobank of >200 hiPSC lines from 10 breast tumor samples, including hormone receptor positive, HER2 positive, and triple negative breast tumors. Breast tumor-derived hiPSCs retain patient-specific oncogenic variants. Additionally, we present a protocol to differentiate hiPSCs into mammary epithelial cells (hiPSC-MECs) and mammary-like organoids for in vitro disease modeling. hiPSC-MECs are proliferative and exhibit a gene expression profile that closely resembles primary MECs. Finally, we demonstrate that this breast tumor model can be used to assess patient-specific responses to PARP inhibitors. We show that hiPSC-MECs derived from BRCA1 and BRCA2 mutant tumors are deficient in homology-directed repair and are more sensitive to PARP inhibitor treatment. This work establishes the foundation of hiPSCs as a novel disease modeling platform for breast cancer and highlights the potential of tumor-derived hiPSCs to predict patient and subclone-specific drug responses. Citation Format: Carly Weddle, Paul Burridge. Examining patient-specific responses to PARP inhibitors in a novel, human induced pluripotent stem cell-based model of breast cancer [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 A070.

Abstract B059: Epigenetically defined sub-clonal heterogeneity drives therapy resistance in triple-negative breast cancer

Abstract Background: Triple-negative breast cancer (TNBC) is a highly aggressive and heterogeneous disease with an average survival of less than 50%. Although immunotherapy is showing promising results, many patients do not respond, and chemotherapy remains the main treatment. High TNBC recurrence rates are in part due to its molecular heterogeneity. Here, we sought to characterise the sub-clonal and spatial diversity of chemotherapy resistant TNBC to identify the epigenetic hallmarks of lethal sub-populations of cells that may induce cell state transition, therapy resistance and ultimately metastatic progression. Methods: Sixteen primary untreated triple-negative breast tumours (eight chemotherapy sensitive (ChemoS) and eight chemotherapy resistant (ChemoR, who died of their disease within three years)) were subjected to cell-matched high-depth multiome single nuclei RNA- and ATAC-sequencing. Four tumours were subject to spatial RNA and ATAC profiling. Fourteen TNBC PDX models generated from patients with residual disease post NAC were subjected to single nuclei sequencing and used for validation. Meta-clustering of epithelial cells from the RNA profiles was used to identify sub-populations of cells harbouring distinct epigenetic and transcriptomic features enriched in ChemoR patients. Findings were validated in the BrighTNess TNBC NAC clinical trial and in chemotherapy treated TNBC patients from the SCAN-B study. Results: Unsupervised meta-clustering (MC) of epithelial cells in G1 phase of the cell cycle identified nine MC’s across the dataset, including a distinct immune-like MC. Two MCs were dominated by cells from ChemoR patients and were defined by hypoxia, EMT, and extra-cellular matrix signalling. Sub-populations of cells in the ChemoR dominant MCs were underpinned by both promoter and distal differential chromatin accessibility, that mapped to H3K27ac enhancer sites, suggesting a role of enhancer regulation of MC transcription. Spatial ATAC and RNA profiling of a subset of tumours identified spatially epigenetically defined regions that showed distinct chromatin profiles, suggesting that the epigenetic regulation of genes that define the MCs are spatially distinct. ChemoR clusters were independently reproduced in a cohort of fourteen residual disease TNBC PDXs. Genes pertaining to the immune-like MC were associated with a good prognosis in independent cohorts of contemporary treated TNBC (SCAN-B) and with response to NAC in the BrighTNess trial. Marker genes from ChemoR dominant MCs were associated with poor prognosis and significant levels of residual disease following NAC. Conclusions: Our multi-modal integrative analysis reveals unprecedented insight into the role of epigenetically defined spatially distinct, sub-clonal populations that pre-exist in treatment naïve TNBC and predict therapy response. This suggests pre-existing populations of cells have already acquired the epigenetic footprints and hence transcriptomic features to allow them to evade therapy. Citation Format: Fatemeh Ahmadimoghari, Yu Zhang, Ioanna Mavrommatis, Shafali Thakur, Chris Starling, Ioannis Roxanis, Syed Haider, Rachael Natrajan. Epigenetically defined sub-clonal heterogeneity drives therapy resistance in triple-negative breast cancer [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 B059.

Photo-enhanced catalytic activity enabled by core–shell nanozymes for combination breast cancer therapy

Enhanced catalytic therapy of triple-negative 4T1 breast tumors through the application of photo-enhanced peroxidase (POD)-like and catalase (CAT)-like activities combined with chemotherapy could be enabled by core–shell nanozymes. Therefore, the dual POD and CAT-like activities of the core–shell iron oxide (Fe3O4)-@gold (IO@Au) nanozymes loaded with epirubicin (EPI) were evaluated in this study for photo-enhanced combination therapy. The core–shell nanozymes induced oxidative stress and apoptosis, and these effects were enhanced upon application of PDT, as demonstrated by ROS and caspase-3 activity assays. Additionally, it was shown that by reducing the expression of HIF-1α gene and upregulation of caspase-3 gene, core–shell nanozymes with and without PDT were able to induce a significant reduction in tumor weight, when compared to free EPI. The bio-distribution of the drug-loaded into core–shell nanozymes showed a higher accumulation of the EPI in the 4T1 breast tumor when compared to samples treated with free EPI. Overall core–shell nanozymes with catalytic-like activity can be used as potential platforms for photo-enhanced combination therapy.

Targeting Receptor Tyrosine Kinases as a Novel Strategy for the Treatment of Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) comprises a group of aggressive and heterogeneous breast carcinoma. Chemotherapy is the mainstay for the treatment of triple-negative tumors. Nevertheless, the success of chemotherapeutic treatments is limited by their toxicity and development of acquired resistance leading to therapeutic failure and tumor relapse. Hence, there is an urgent need to explore novel targeted therapies for TNBC. Receptor tyrosine kinases (RTKs) are a family of transmembrane receptors that are key regulators of intracellular signaling pathways controlling cell proliferation, differentiation, survival, and motility. Aberrant activity and/or expression of several types of RTKs have been strongly connected to tumorigenesis. RTKs are frequently overexpressed and/or deregulated in triple-negative breast tumors and are further associated with tumor progression and reduced survival in patients. Therefore, targeting RTKs could be an appealing therapeutic strategy for the treatment of TNBC. This review summarizes the current evidence regarding the antitumor activity of RTK inhibitors in preclinical models of TNBC. The review also provides insights into the clinical trials evaluating the use of RTK inhibitors for the treatment of patients with TNBC.

Detection of KRAS Mutations in Triple-negative Breast Cancers by Polymerase Chain Reaction

Abstract Background: Recently, it has been proven that KRAS gene mutation analysis is an additional in vitro diagnostic method for determining colorectal cancer patients who will not respond to anti-epidermal growth factor receptor (EGFR) therapy. KRAS mutation status assessment may be important in other EGFR-overexpressed cancers, such as breast cancer tumors. Only 5% of breast tumors are KRAS-mutated, yet 60% of triple-negative breast tumors express EGFR, making them potential targets for EGFR inhibitors. To provide a biological foundation for assessing anti-EGFR therapy, we aimed to examine the frequency of KRAS mutations in triple-negative breast cancer (TNBC). Materials and Methods: Forty breast cancer cases were evaluated. These 40 cases have undergone immunohistochemistry for estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2 markers which exhibited a negative reaction and were ascertained as triple negative. The mutation status of KRAS was determined by real-time polymerase chain reaction (RT-PCR) to detect wild or mutant types of KRAS. Results: In our study, we observed that, out of the total 40 cases of TNBCs, 14 (35%) cases were of wild type and 26 (65%) cases were mutant type detected by RT-PCR. Conclusion: According to our study, KRAS mutations were detected in TNBCs, and EGFR inhibitors may be effective in the treatment of these tumors, which overexpress EGFR in around 65% of cases.

Epigenomic Profiling Advises Therapeutic Potential of Leukotriene Receptor Inhibitors for a Subset of Triple-Negative Breast Tumors.

Triple-negative breast cancer (TNBC) is the most aggressive molecular subtype, with a poor survival rate compared to others subtypes. For a long time, chemotherapy was the only systemic treatment for TNBC, and the identification of actionable molecular targets might ultimately improve the prognosis for TNBC patients. We performed a genome-wide analysis of DNA methylation at CpG islands on a collection of one hundred ten breast carcinoma samples and six normal breast tissue samples using reduced representation bisulfite sequencing with the XmaI restriction enzyme (XmaI-RRBS) and identified a subset of TNBC samples with significant hypomethylation at the LTB4R/LTB4R2 genes' CpG islands, including CpG dinucleotides covered with cg12853742 and cg21886367 HumanMethylation 450K microarray probes. Abnormal DNA hypomethylation of this region in TNBC compared to normal samples was confirmed by bisulfite Sanger sequencing. Gene expression generally anticorrelates with promoter methylation, and thus, the promoter hypomethylation detected and confirmed in our study might be revealed as an indirect marker of high LTB4R/LTB4R2 expression using a simple methylation-sensitive PCR test. Analysis of RNA-seq expression and DNA methylation data from the TCGA dataset demonstrates that the expression of the LTB4R and LTB4R2 genes significantly negatively correlates with DNA methylation at both CpG sites cg12853742 (R = -0.4, p = 2.6 × 10-6; R = -0.21, p = 0.015) and cg21886367 (R = -0.45, p = 7.3 × 10-8; R = -0.24, p = 0.005), suggesting the upregulation of these genes in tumors with abnormal hypomethylation of their CpG island. Kaplan-Meier analysis using the TCGA-BRCA gene expression and clinical data revealed poorer overall survival for TNBC patients with an upregulated LTB4R. To this day, only the leukotriene inhibitor LY255283 has been tested on an MCF-7/DOX cell line, which is a luminal A breast cancer molecular subtype. Other studies compare the effects of Montelukast and Zafirlukast (inhibitors of the cysteinyl leukotriene receptor, which is different from LTB4R/LTB4R2) on the MDA-MB-231 (TNBC) cell line, with high methylation and low expression levels of LTB4R. In our study, we assess the therapeutic effects of various drugs (including leukotriene receptor inhibitors) with the DepMap gene effect and drug sensitivity data for TNBC cell lines with hypomethylated and upregulated LTB4R/LTB4R2 genes. LY255283, Minocycline, Silibinin, Piceatannol, Mitiglinide, 1-Azakenpaullone, Carbetocin, and Pim-1-inhibitor-2 can be considered as candidates for the additional treatment of TNBC patients with tumors demonstrating LTB4R/LTB4R2 hypomethylation/upregulation. Finally, our results suggest that the epigenetic status of leukotriene B4 receptors is a novel, potential, predictive, and prognostic biomarker for TNBC. These findings might improve individualized therapy for TNBC patients by introducing new therapeutic adjuncts as anticancer agents.