Luminal Breast Tumors Research Articles

Loss of ASAP1 in the MMTV-PyMT model of luminal breast cancer activates AKT, accelerates tumorigenesis, and promotes metastasis

ASAP1 is a multi-domain adaptor protein that regulates cytoskeletal dynamics, receptor recycling and intracellular vesicle trafficking. Its expression is associated with poor prognosis in a variety of cancers, and can promote cell migration, invasion and metastasis. Although amplification and expression of ASAP1 has been associated with poor survival in breast cancer, we found that in the autochthonous MMTV-PyMT model of luminal breast cancer, ablation of ASAP1 resulted in an earlier onset of tumor initiation and increased metastasis. This was due to tumor cell-intrinsic effects of ASAP1 deletion, as ASAP1 deficiency in tumor, but not in stromal cells was sufficient to replicate the enhanced tumorigenicity and metastasis observed in the ASAP1-null MMTV-PyMT mice. Loss of ASAP1 in MMTV-PyMT mice had no effect on proliferation, apoptosis, angiogenesis or immune cell infiltration, but enhanced mammary gland hyperplasia and tumor cell invasion, indicating that ASAP1 can accelerate tumor initiation and promote dissemination. Mechanistically, these effects were associated with a potent activation of AKT. Importantly, lower ASAP1 levels correlated with poor prognosis and enhanced AKT activation in human ER+/luminal breast tumors, validating our findings in the MMTV-PyMT mouse model for this subtype of breast cancer. Taken together, our findings reveal that ASAP1 can have distinct functions in different tumor types and demonstrate a tumor suppressive activity for ASAP1 in luminal breast cancer.

Abstract P3-09-12: Breast cancer derived GATA3 mutations disrupt luminal transcriptional network

Abstract GATA3 has been identified as one of the most frequently mutated genes in breast cancer. In the METABRIC cohort, among 1,980 patient cases, 230 breast cancers harbored GATA3 mutations (~12%). 75% of the GATA mutations were observed in luminal breast tumors (47% in luminal A, 28% in luminal B). The recent genomic data in metastatic breast cancer also showed that the frequency of GATA3 somatic mutations was even higher in the metastatic breast cancer cohort. Lung, lymph nodes, and brain metastases were observed in the GATA3 mutant breast cancer patients. Based on these patient genomic data, GATA3 mutations have been considered as cancer drivers, yet the functional consequences of GATA3 mutations are underexplored. We and other groups previously identified that patients carrying GATA3 mutations have diverse clinical features. More than 70% of cases are small nucleotide deletions or insertions (indel), while less than 30% are missense mutations. By classifying the GATA3 indel mutations into 4 groups, we observed distinct clinical features. Somatic mutations found in the GATA3 second zinc-finger domain (ZnFn2) are significantly associated with poorer patient outcomes including worse patient survival. ZnFn2 mutations are predominantly found in luminal B breast tumors, while splice site mutations are frequently found in luminal A breast tumors and associated with better patient survival. These distinct clinical features clearly suggest the differential impacts of GATA3 mutations on breast cancer cells. To dissect the function of GATA3 mutants, we developed a GATA3 mutant cell line (R330fs/WT T47D cells) by CRISPR, which endogenously expresses a heterozygous R330 frame-shift (R330fs) mutant. R330fs mutations are found in multiple data cohorts. The majority (>90%) of GATA3 mutations are heterozygous. Therefore, this R330fs mutant T47D cell line mimics the type of alteration found in patients. Importantly, we found that the R330fs mutation induces transcriptional reprogramming in T47D luminal breast cancer cells leading to more aggressive phenotypes such as faster tumor growth. In the mutant cells, many epithelial marker genes (such as progesterone receptor and TFF1) were down-regulated while mesenchymal marker genes (such as TWIST1 and SNAI2) are up-regulated suggesting that R330fs induces a partial EMT in T47D cells. We also found that transcriptional changes in the R330fs mutant cells were strongly associated with redistribution of GATA3, Estrogen Receptor alpha, and FOXA1. Gene expression profiles in the other GATA3 mutant breast cancer cell lines also suggest similar genomic alterations. These results suggest the active roles of GATA3 mutations during breast cancer development. Citation Format: Motoki Takaku, Mika Saotome, Renju Nair. Breast cancer derived GATA3 mutations disrupt luminal transcriptional network [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P3-09-12.

Path to Clonal Theranostics in Luminal Breast Cancers.

Integrating tumor heterogeneity in the drug discovery process is a key challenge to tackle breast cancer resistance. Identifying protein targets for functionally distinct tumor clones is particularly important to tailor therapy to the heterogeneous tumor subpopulations and achieve clonal theranostics. For this purpose, we performed an unsupervised, label-free, spatially resolved shotgun proteomics guided by MALDI mass spectrometry imaging (MSI) on 124 selected tumor clonal areas from early luminal breast cancers, tumor stroma, and breast cancer metastases. 2868 proteins were identified. The main protein classes found in the clonal proteome dataset were enzymes, cytoskeletal proteins, membrane-traffic, translational or scaffold proteins, or transporters. As a comparison, gene-specific transcriptional regulators, chromatin related proteins or transmembrane signal receptor were more abundant in the TCGA dataset. Moreover, 26 mutated proteins have been identified. Similarly, expanding the search to alternative proteins databases retrieved 126 alternative proteins in the clonal proteome dataset. Most of these alternative proteins were coded mainly from non-coding RNA. To fully understand the molecular information brought by our approach and its relevance to drug target discovery, the clonal proteomic dataset was further compared to the TCGA breast cancer database and two transcriptomic panels, BC360 (nanoString®) and CDx (Foundation One®). We retrieved 139 pathways in the clonal proteome dataset. Only 55% of these pathways were also present in the TCGA dataset, 68% in BC360 and 50% in CDx. Seven of these pathways have been suggested as candidate for drug targeting, 22 have been associated with breast cancer in experimental or clinical reports, the remaining 19 pathways have been understudied in breast cancer. Among the anticancer drugs, 35 drugs matched uniquely with the clonal proteome dataset, with only 7 of them already approved in breast cancer. The number of target and drug interactions with non-anticancer drugs (such as agents targeting the cardiovascular system, metabolism, the musculoskeletal or the nervous systems) was higher in the clonal proteome dataset (540 interactions) compared to TCGA (83 interactions), BC360 (419 interactions), or CDx (172 interactions). Many of the protein targets identified and drugs screened were clinically relevant to breast cancer and are in clinical trials. Thus, we described the non-redundant knowledge brought by this clone-tailored approach compared to TCGA or transcriptomic panels, the targetable proteins identified in the clonal proteome dataset, and the potential of this approach for drug discovery and repurposing through drug interactions with antineoplastic agents and non-anticancer drugs.

Lineage-specific silencing of PSAT1 induces serine auxotrophy and sensitivity to dietary serine starvation in luminal breast tumors.

SUMMARYA major challenge of targeting metabolism for cancer therapy is pathway redundancy, in which multiple sources of critical nutrients can limit the effectiveness of some metabolism-targeted therapies. Here, we analyze lineage-dependent gene expression in human breast tumors to identify differences in metabolic gene expression that may limit pathway redundancy and create therapeutic vulnerabilities. We find that the serine synthesis pathway gene PSAT1 is the most depleted metabolic gene in luminal breast tumors relative to basal tumors. Low PSAT1 prevents de novo serine biosynthesis and sensitizes luminal breast cancer cells to serine and glycine starvation in vitro and in vivo. This PSAT1 expression disparity preexists in the putative cells of origin of basal and luminal tumors and is due to luminal-specific hypermethylation of the PSAT1 gene. Our data demonstrate that luminal breast tumors are auxotrophic for serine and may be uniquely sensitive to therapies targeting serine availability.

High prevalence of APOA1/C3/A4/A5 alterations in luminal breast cancers among young women in East Asia

In East Asia, the breast cancer incidence rate among women aged <50 years has rapidly increased. Emerging tumors are distinctly characterized by a high prevalence of estrogen receptor (ER)–positive/human epidermal growth factor receptor (HER2)–negative cancer. In the present study, we identified unique genetic alterations in these emerging tumors. We analyzed gene copy number variations (CNVs) in breast tumors from 120 Taiwanese patients, and obtained public datasets of CNV and gene expression (GE). The data regarding CNV and GE were separately compared between East Asian and Western patients, and the overlapping genes identified in the comparisons were explored to identify the gene–gene interaction networks. In the age <50 years/ER + /HER2– subgroup, tumors of East Asian patients exhibited a higher frequency of copy number loss in APOA1/C3/A4/A5, a lipid-metabolizing gene cluster (33 vs. 10%, P < .001) and lower APOA1/C3/A4/A5 expressions than tumors of Western patients. These copy number loss related– and GE–related results were validated in another Taiwanese cohort and in two GE datasets, respectively. The copy number loss was significantly associated with poor survival among Western patients, but not among East Asian patients. Lower APOA1, APOC3, and APOA5 expressions were associated with higher ESTIMATE immune scores, indicating an abundance of tumor-infiltrating immune cells. In conclusion, APOA1/C3/A4/A5 copy number loss was more prevalent in luminal breast tumors among East Asian women aged <50 years, and its immunomodulatory effect on the tumor microenvironment possibly plays various roles in the tumor biology of East Asian patients.

Abstract 2838: Epigenetic regulator MLL3 mutations promote tumor metastasis by enhancing the epithelial-mesenchymal transition state

Abstract Epigenetic dysregulations have a recognized role in cancer pathogenesis. Histone methyltransferase MLL3 (mixed-lineage leukemia protein 3) (gene symbol - KMT2C) is one of the most frequently altered genes in human breast cancer. In addition to the loss-of-function mutations, the expression levels of MLL3 are downregulated significantly in the tumor, compared to normal breast samples. Consistent with these cancer genome findings, functional studies in mouse models and human cancer cells showed loss of MLL3 promotes tumor initiation and growth. However, the role of MLL3 mutation in metastasis has not been determined. Here we showed that the MLL3 levels were further reduced in distant metastases compared to primary tumors, suggesting a potential role of MLL3 loss in metastasis. We demonstrate that MLL3 loss markedly promoted distant metastasis through specifically enhancing the step of colonization during the metastatic cascade. MLL3 loss facilitated the acquisition of an epithelial/mesenchymal (E/M) transition state by cancer cells disseminated to the lung. These cells have a hybrid phenotype of epithelial and mesenchymal features. We further showed that the E/M transitional cells induced by MLL3 loss have greatly increased tumor-initiating ability and multi-organ metastatic ability. We further demonstrated that MLL3 lose facilitated luminal breast tumor cells to acquire the E/M hybrid features and promoted endocrine therapy resistance, suggesting that loss of MLL3 also promotes the epithelial-mesenchymal plasticity and therapeutic resistance. To identify vulnerability of MLL3-mutant cancer cells, we performed small molecule screening and discovered that MLL3-mutant cancer cells are markedly more sensitive to BET inhibitors than MLL3-WT cells. The in vivo study confirmed that the BET inhibitor repressed the MLL3-mutant tumor growth much more efficiently than MLL3-WT tumors. Furthermore, the BET inhibitor is effective in suppressing the growth of established MLL3-mutant lung metastasis. Taken together, our findings indicate that MLL3 mutations promotes metastasis and therapeutic resistance by enhancing epithelial-mesenchymal plasticity. However, these aggressive cancer cells are uniquely vulnerable to BET inhibition, suggesting a potential therapeutic strategy for MLL3-mutant cancers. Citation Format: Jihong Cui, Kai Ge, Wenjun Guo. Epigenetic regulator MLL3 mutations promote tumor metastasis by enhancing the epithelial-mesenchymal transition state [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2838.

Molecular characterization of luminal breast tumors in African American women.

550 Background: Racial disparities in breast cancer (BC) mortality are attributed to later stage diagnoses and a higher incidence of triple-negative BC among African American (AA) women. In previous work, we showed that AA women with ER+ BC are more likely to develop biologically aggressive disease and are more likely to die from early stage, ER+ BC than non-Hispanic White women (Hoskins et al, JAMA Oncol, 2021). The underlying molecular drivers of this disparity are unknown. Here we report the molecular characterization of a series of luminal BC from AA women. Methods: Consecutive breast tumor specimens received in the Pathology Department underwent next generation sequencing (NGS). Unstained FFPE tissue sections were macro-dissected to isolate tumor cells, and nucleic acids were extracted using commercially available kits. DNA and RNA sequencing libraries were prepared with the Oncomine Comprehensive Assay v3 (OCAv3) (Thermo Fisher), which includes 161 driver genes and detects SNVs, CNVs, INDELs and gene fusions. Sequencing was performed on the Ion S5XL sequencer. Sequencing reads were mapped to the UCSC human genome build GRCh37/hg19 using Torrent Suite™ software (version 5.10; Thermo Fisher). Data analysis and variant calling was performed using the Ion Reporter analysis tool. Results: We identified 60 somatic driver gene alterations in luminal tumors from 35 AA patients (primary tumors, n = 26; metastatic tumors, n = 9). Recurrently altered genes identified in &gt; 5% of tumors are listed in the Table. The most frequently altered gene was PIK3CA (42% of tumors). ESR1 gene fusions were seen in 25% of tumors. Interestingly, an equal frequency of ESR1 fusions were detected in primary (27%) and metastatic (22%) tumors, in contrast to activating mutations which are found in recurrent tumors following treatment with aromatase inhibitors. ARID1A alterations were identified in 17% of primary tumors. ARID1A encodes a subunit of the SWI/SNF chromatin remodeling complex. Alterations in ARID1A confer endocrine resistance, and are enriched in recurrent tumors in the literature. We also found a high number of CNVs in members of the FGF gene family (36% of tumors), which are also associated with resistance to endocrine therapy. An in silico analysis comparing our findings with publicly available datasets will be presented. Conclusions: This study of somatic driver gene alterations in a consecutive series of luminal breast tumors from AA patients found a higher than expected frequency of alterations in genes associated with endocrine resistance in untreated primary tumors, suggesting a partial explanation for racial disparities in survival.[Table: see text]

Comprehensive omic characterization of breast cancer in Mexican-Hispanic women

Breast cancer is a heterogeneous pathology, but the genomic basis of its variability remains poorly understood in populations other than Caucasians. Here, through DNA and RNA portraits we explored the molecular features of breast cancers in a set of Hispanic-Mexican (HM) women and compared them to public multi-ancestry datasets. HM patients present an earlier onset of the disease, particularly in aggressive clinical subtypes, compared to non-Hispanic women. The age-related COSMIC signature 1 was more frequent in HM women than in those from other ancestries. We found the AKT1E17K hotspot mutation in 8% of the HM women and identify the AKT1/PIK3CA axis as a potentially druggable target. Also, HM luminal breast tumors present an enhanced immunogenic phenotype compared to Asiatic and Caucasian tumors. This study is an initial effort to include patients from Hispanic populations in the research of breast cancer etiology and biology to further understand breast cancer disparities.

Hormonal Receptor Immunochemistry Heterogeneity and 18F-FDG Metabolic Heterogeneity: Preliminary Results of Their Relationship and Prognostic Value in Luminal Non-Metastatic Breast Cancers.

IntroductionWe aimed to investigate whether 18F-FDG PET metabolic heterogeneity reflects the heterogeneity of estrogen receptor (ER) and progesterone receptor (PR) expressions within luminal non-metastatic breast tumors and if it could help in identifying patients with worst event-free survival (EFS).Materials and methodsOn 38 PET high-resolution breast bed positions, a single physician drew volumes of interest encompassing the breast tumors to extract SUVmax, histogram parameters and textural features. High-resolution immunochemistry (IHC) scans were analyzed to extract Haralick parameters and descriptors of the distribution shape. Correlation between IHC and PET parameters were explored using Spearman tests. Variables of interest to predict the EFS status at 8 years (EFS-8y) were sought by means of a random forest classification. EFS-8y analyses were then performed using univariable Kaplan-Meier analyses and Cox regression analysis. When appropriate, Mann-Whitney tests and Spearman correlations were used to explore the relationship between clinical data and tumoral PET heterogeneity variables.ResultsFor ER expression, correlations were mainly observed with 18F-FDG histogram parameters, whereas for PR expression correlations were mainly observed with gray-level co-occurrence matrix (GLCM) parameters. The strongest correlations were observed between skewness_ER and uniformity_HISTO (ρ = −0.386, p = 0.017) and correlation_PR and entropy_GLCM (ρ = 0.540, p = 0.001), respectively. The median follow-up was 6.5 years and the 8y-EFS was 71.0%. Random forest classification found age, clinical stage, SUVmax, skewness_ER, kurtosis_ER, entropy_HISTO, and uniformity_HISTO to be variables of importance to predict the 8y-EFS. Univariable Kaplan-Meier survival analyses showed that skewness_ER was a predictor of 8y-EFS (66.7 ± 27.2 versus 19.1 ± 15.2, p = 0.018 with a cut-off value set to 0.163) whereas other IHC and PET parameters were not. On multivariable analysis including age, clinical stage and skewness_ER, none of the parameters were independent predictors. Indeed, skewness_ER was significantly higher in youngest patients (ρ = −0.351, p = 0.031) and in clinical stage III tumors (p = 0.023).ConclusionA heterogeneous distribution of ER within the tumor in IHC appeared as an EFS-8y prognosticator in luminal non-metastatic breast cancers. Interestingly, it appeared to be correlated with PET histogram parameters which could therefore become potential non-invasive prognosticator tools, provided these results are confirmed by further larger and prospective studies.

Path to Clonal Theranostics in Luminal Breast Cancers

Integrating tumor heterogeneity in the drug discovery process is a key challenge to tackle breast cancer resistance. Identifying protein targets for functionally distinct tumor clones is particularly important to tailor therapy to the heterogeneous tumor subpopulations and achieve clonal theranostics. For this purpose, we performed an unsupervised, label-free, spatially resolved shotgun proteogenomic guided by MALDI mass spectrometry imaging (MSI) on 124 selected tumor clonal areas from early luminal breast cancers, tumor stroma, and breast cancer metastases. 2868 proteins were identified. The main protein classes found in the clonal proteome dataset were enzymes, cytoskeletal proteins, membrane-traffic, translational or scaffold proteins, or transporters. As a comparison, gene-specific transcriptional regulators, chromatin related proteins or transmembrane signal receptor were more abundant in the TCGA dataset. Moreover, 26 mutated proteins have been identified. Similarly, expanding the search to alternative proteins databases retrieved 126 alternative proteins in the clonal proteome dataset . Most of these alternative proteins were coded mainly from non-coding RNA. To fully understand the molecular information brought by our approach and its relevance to drug target discovery, the clonal proteomic dataset was further compared to the TCGA breast cancer database and two transcriptomic panels, BC360 (nanoString®) and CDx (Foundation One®). We retrieved 139 pathways in the clonal proteome dataset. Only 55% of these pathways were also present in the TCGA dataset, 68% in BC360 and 50% in CDx. Seven of these pathways have been suggested as candidate for drug targeting, 22 have been associated with breast cancer in experimental or clinical reports, the remaining 19 pathways have been understudied in breast cancer. Among the anticancer drugs, 35 drugs matched uniquely with the clonal proteome dataset, with only 7 of them already approved in breast cancer. The number of target and drug interactions with non-anticancer drugs (such as agents targeting the cardiovascular system, metabolism, the musculoskeletal or the nervous systems) was higher in the clonal proteome dataset (540 interactions) compared to TCGA (83 interactions), BC360 (419 interactions), or CDx (172 interactions). Many of the protein targets identified and drugs screened were clinically relevant to breast cancer and are in clinical trials. Thus, we described the non-redundant knowledge brought by this clone-tailored approach compared to TCGA or transcriptomic panels, the targetable proteins identified in the clonal proteome dataset, and the potential of this approach for drug discovery and repurposing through drug interactions with antineoplastic agents and non-anticancer drugs.

Abstract PO-220: Comprehensive "omic" portraits of breast cancer in Mexican-Hispanic women

Abstract Breast cancer is a relevant public health problem that affects millions of women worldwide. Breast tumors are recognized as a clinically and molecular heterogeneous pathology, but the genomic basis of its variability remains poorly understood, mainly regarding the comprehensive of ethnicity/racial disparities and their consequences in genomics studies. Moreover, most of the comprehensive molecular characterizations are predominantly centered on Caucasian population. Thus, we deeply characterized the molecular architecture of breast cancer in a set of Mexican patients through genomic and transcriptomic portraits. We explored the molecular features of breast cancers in Mexican-Hispanic (HM) women and compared them with public multi-ethnic datasets. HM patients presented an earlier onset of the disease, in particular in the aggressive Basal-like/triple negative and Luminal B subtypes. The significant E17K hotspot mutation in AKT1 gene occurred in 8% of the HM tumors, an enriched prevalence exclusively seen in this dataset. An integrative analysis also revealed an important role of the AKT1/PIK3CA axis as a potentially druggable target and indicate an aggressive biology. Computational analysis allowed us to identify age-related COSMIC (v2) signature 1 to be present in a much broader spectrum of HM women than in those from other ethnicities. On the basis of transcriptome immunogenomic analysis across human population we identified that luminal breast tumors with HM ancestry presented an enhanced immunogenic phenotype than their Asiatic and Caucasian counterparts. To the best of our knowledge, this dataset represents the largest breast cancer genomics characterization of breast tumors in Mexican women, and, provides novel insights into molecular phenotypes and mechanisms underlying carcinogenesis in a group of Mexican Hispanic women. In conclusion, this study provides a conceptual framework to better understand ethnic disparities of breast cancer. Citation Format: Sandra Lorena Romero-Cordoba, Ivan Salido-Guadarrama, Rosa Rebollar-Vega, Alberto Tenorio-Flores, Verónica Bautista-Piña, Felipe Villegas-Carlos, Carlos Dominguez-Reyes, Alfredo Hidalgo-Miranda. Comprehensive "omic" portraits of breast cancer in Mexican-Hispanic women [abstract]. In: Proceedings of the AACR Virtual Conference: Thirteenth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2020 Oct 2-4. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(12 Suppl):Abstract nr PO-220.

ER\u03b1-related chromothripsis enhances concordant gene transcription on chromosome 17q11.1-q24.1 in luminal breast cancer

BackgroundChromothripsis is an event of genomic instability leading to complex chromosomal alterations in cancer. Frequent long-range chromatin interactions between transcription factors (TFs) and targets may promote extensive translocations and copy-number alterations in proximal contact regions through inappropriate DNA stitching. Although studies have proposed models to explain the initiation of chromothripsis, few discussed how TFs influence this process for tumor progression.MethodsThis study focused on genomic alterations in amplification associated regions within chromosome 17. Inter−/intra-chromosomal rearrangements were analyzed using whole genome sequencing data of breast tumors in the Cancer Genome Atlas (TCGA) cohort. Common ERα binding sites were defined based on MCF-7, T47D, and MDA-MB-134 breast cancer cell lines using univariate K-means clustering methods. Nanopore sequencing technology was applied to validate frequent rearrangements detected between ATC loci on 17q23 and an ERα hub on 20q13. The efficacy of pharmacological inhibition of a potentially druggable target gene on 17q23 was evaluated using breast cancer cell lines and patient-derived circulating breast tumor cells.ResultsThere are five adjoining regions from 17q11.1 to 17q24.1 being hotspots of chromothripsis. Inter−/intra-chromosomal rearrangements of these regions occurred more frequently in ERα-positive tumors than in ERα-negative tumors. In addition, the locations of the rearrangements were often mapped within or close to dense ERα binding sites localized on these five 17q regions or other chromosomes. This chromothriptic event was linked to concordant upregulation of 96 loci that predominantly regulate cell-cycle machineries in advanced luminal tumors. Genome-editing analysis confirmed that an ERα hub localized on 20q13 coordinately regulates a subset of these loci localized on 17q23 through long-range chromosome interactions. One of these loci, Tousled Like Kinase 2 (TLK2) known to participate in DNA damage checkpoint control, is an actionable target using phenothiazine antipsychotics (PTZs). The antiproliferative effect of PTZs was prominent in high TLK2-expressing cells, compared to low expressing cells.ConclusionThis study demonstrates a new approach for identifying tumorigenic drivers from genomic regions highly susceptible to ERα-related chromothripsis. We found a group of luminal breast tumors displaying 17q-related chromothripsis for which antipsychotics can be repurposed as treatment adjuncts.

Abstract A88: RUNX1-deficiency in luminal mammary epithelial cells leads to development of a unique type of immune “hot” ER+ mammary tumor

Abstract Genetic mutations in cancer cells play key roles in shaping the tumor immune microenvironment. RUNX1 is a transcription factor (TF) essential for estrogen receptor (ER)+ luminal mammary epithelial cells (MECs) and is mutated in a significant portion of human ER+ luminal breast cancers. It is also an important regulator of immune cells, raising an intriguing question as to whether its loss-of-function mutations found in ER+ breast cancers affect both MECs and immune cells. To address this, we established an in vivo mouse model by inducing loss of both RUNX1 and p53 in luminal MECs, upon intraductal injection of a Cre-expressing adenovirus under the control of the Keratin 8 promoter (Ad-K8-Cre) to mammary glands of female mice carrying their conditional knockout alleles. Compared to similarly injected wild-type (WT) control mice or mice with induced loss of p53 alone, induced loss of both led to hyperproliferation of Runx1/Trp53-null MECs and eventually development of mammary tumors after a long latency (a tumor-free period of ~9-13 months after Ad-K8-Cre injection). Characterization of these tumors revealed that they are ER+ mammary tumors with extensive leukocyte infiltration. CD45+ leukocytes in them constituted ~32-84% of total live tumor cells, which is unusual for a mouse cancer model. Among CD45+ cells, ~45% were macrophages and ~15% were CD3+ T cells. Among tumor-infiltrating myeloid cells (CD45+CD11b+), many were myeloid-derived suppressor cells (MDSCs). In addition, significant differential expression of PD-1 on CD3+ T cells (38.5% vs. 7.3%) and PD-L1 on CD45- cells (2.7% vs. 0.3%) was observed in these Runx1/Trp53-null tumors, when compared to WT controls. This immune profile suggests an immunosuppressive microenvironment in these tumors. The tumor immune phenotype induced by RUNX1-loss in our model is supported by genomic data from human breast cancers analyzed by the TIMER online tool (Li et al., Cancer Res 77(21):e108-e110). The TIMER analysis showed that in human breast cancers, levels of most infiltrating immune cell subtypes (e.g., macrophages, T cells) in RUNX1-mutated cases are higher than those of RUNX1-WT cases; in contrast, a similar analysis for GATA3-mutated cases exhibited the opposite. As both RUNX1 and GATA3 are key TFs for luminal MECs and their mutations are both found in luminal breast cancers, the data from both human cases and our mouse model suggest that RUNX1-mutated breast cancers may represent a unique, immune “hot” subtype of ER+ luminal breast tumors. ER+ breast cancers are typically immune “cold” and do not respond to immune checkpoint blockade (ICB) well. Our data suggest that RUNX1-mutated breast cancers may represent a subset of ER+ breast cancers that may respond to ICB-based therapy. Collectively, our data suggest that loss of RUNX1 promotes development of ER+ breast cancer and modulates the immune microenvironment; a better understanding of how these two processes are interconnected may provide novel strategies to treat breast cancer with RUNX1-mutation. Citation Format: Dongxi Xiang, Zhe Li. RUNX1-deficiency in luminal mammary epithelial cells leads to development of a unique type of immune “hot” ER+ mammary tumor [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr A88.

Abstract P3-03-02: Integrative proteogenomic analyses identifies CPT1A and fatty acid oxidation as a potential therapeutic strategy in hormone receptor positive breast cancer

Abstract Background: Clinically, approximately two-thirds of the nearly 250,000 breast cancer cases diagnosed each year in the United States are hormone receptor positive (HR+), luminal breast tumors. Slower growing luminal breast tumors are often successfully treated using endocrine based therapies resulting in a relatively good prognosis for these patients. However more highly proliferative luminal tumors are often resistant, or become resistant, to current therapies leading to a worse outcome for these patients. As such, there is a critical need to identify genomic alterations and dysregulated signaling pathway in this subset of tumors that may represent novel therapeutic opportunities. Methods: In order to identify genetic events responsible for tumorigenesis and to identify potential drug-able genetic alterations and/or pathways associated with high levels of proliferation in HR+ tumors, we used an integrative genomics-based strategy to interrogate orthogonal genome-wide data from more than 2,500 patients from the TCGA, METABRIC, CPTAC studies. Data from a genome-wide RNAi screen was used to delineate essential from non-essential genes in HR+ breast cancer cell lines. Genetic and pharmacological-based in vitro and in vivo studies were used to demonstrate the impact identified candidate genes on cell viability, proliferation, colony formation, spheroid formation and tumor growth rate. Results: Integrative genomic analyses of human breast tumors identified DNA amplification as well as mRNA and protein over-expression of carnitine palmitoyltransferase 1A (CPT1A) in highly proliferative luminal tumors. Further in silico analyses demonstrated that high CPT1A expression corresponded with protein markers of proliferation in human tumors and is prognostic in HR+ tumors in both the TCGA and METABRIC cohorts. Since CPT1A is the rate limiting enzyme responsible for fatty acid import into the mitochondria during Fatty Acid β Oxidation (FAO), these data indicate that highly proliferative luminal tumors may utilize FAO as a prominent energy source. As such, we hypothesized that inhibiting CPT1A and/or FAO in this subset of tumors or cell lines may represent a potential therapeutic opportunity. Consistent with our hypothesis, analyses of data from a genome-wide RNAi screen in 27 breast cancer cell lines suggested that HR+ cells lines are dependent on CPT1A expression. Importantly, we experimentally demonstrated that in vitro genetic silencing of CPT1A or pharmacological inhibition of FAO in ER+ breast cancer cell lines results in reduced cell viability and increased cell death as well as decreased spheroid formation. Importantly, our data indicated that in vivo inhibition of CPT1A resulted in decreased tumor volume and growth rate in orthotopic xenograft tumor models. Conclusions: In this study, integrative proteogenomic analyses identified amplification and over-expression of CPT1A in highly proliferative ER+ breast tumors. In vitro and in vivo studies demonstrated that CPT1A is an essential oncogenic driver in HR+/luminal breast cancer and establish CTP1A/FAO inhibition as a putative therapeutic strategy for treatment of these tumors. Citation Format: Michael L Gatza, Vrushank Bhatt, Kimberly Parker, Guarav Mehta, Christen Khella, Yanxiang Guo, Nidhi Jariwala. Integrative proteogenomic analyses identifies CPT1A and fatty acid oxidation as a potential therapeutic strategy in hormone receptor positive breast cancer [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 P3-03-02.

Abstract P1-10-23: Prediction of pathological complete response (pCR) upon neoadjuvant chemotherapy by MammaTyper® pCR score

Abstract Background: Neoadjuvant chemotherapy (NACT) (+/- anti HER2) is the preferred therapy for triple negative and HER2 positive subtype breast tumors. In Luminal subtype breast tumors, NACT is used to downstage large inoperable tumors and enable breast conserving therapy. However, this subgroup of patients may be overtreated as many Luminal subtype tumors do not respond to NACT due to the low rate of proliferating tumor cells. Herein, we tested the MammaTyper® pCR score as a predictor of therapy success measured as pCR in a retrospective analysis of archived samples from a single center. Methods: 85 FFPE biopsy samples from the years 2012-2018 were sourced from the archive. Samples with &amp;gt;20% tumor cell content were subjected to RNA extraction. Relative mRNA expression levels of ERBB2, ESR1, PGR and MKI67 were determined by RT-qPCR using the CE-IVD MammaTyper® kit. The continuous expression values were integrated into a prediction score (pCR-score) using a pre-defined algorithm and cutoff. The association of continuous and binary pCR-score results with pCR (defined as ypT0/Tis) and partial response was analyzed. Results: Marker positivity rates of the 75 samples included in the final analysis were 62.7% for ER, 53.3% for PR, 40.0% for HER2 and 94.7% for Ki67 (≥20% positive cells). 42.7% of patients were pre-menopausal and all samples except one were grade 3. pCR rates over all samples and in hormone receptor (HR) positive/HER2 negative patients only were 48.0% and 20.0%, respectively. Binary pCR-score result was significantly associated with pCR over all patients (Sensitivity: 88.9%, Specificity: 51.3%, PPV: 62.8%, NPV: 83.3%) and also in HR positive/HER2 negative patients only (Sensitivity: 83.3%, Specificity: 70.8%, PPV: 41.7%, NPV: 94.4%). ROC analysis revealed a good association of the continuous pCR-score with achievement pCR over all patients (AUC=0.756) and in the subgroup of HR+/HER2- patients (AUC=0.774). pCR rates according to St Gallen surrogate subtype definition were similar for IHC and RT-qPCR defined subtypes in Triple Negative (80.0% and 78.6% respectively) and in the HER2+ non luminal subtype (75.0%, and 70.0% respectively). In tumors with incomplete response the continuous pCR-score was significantly associated with residual tumor size (Spearman rs: -0.477 p-value: 0.0021) and %-decrease of tumor size (Spearman rs: 0.388, p-value: 0.0147). Conclusion: The MammaTyper® pCR score may serve as a standardized tool to predict response to NACT based on a pre-treatment biopsy. For patients with inoperable Luminal tumors and low predicted probability of pCR, neo-adjuvant aromatase inhibitor alone or combined with the new generation of TKIs such as CDK4/6 inhibitors or Pi3KCa/mTOR inhibitors may be an alternative for downstaging of tumors. Citation Format: Silvana Saracchini, Anna Bassini, Wally Marus, Serena Corsetti, Ilaria Specogna, Manuela Bertola, Elvia Micheli, Ralph M Wirtz, Mark Laible, Ugur Sahin, Daniele Generali, Sandro Sulfaro. Prediction of pathological complete response (pCR) upon neoadjuvant chemotherapy by MammaTyper® pCR score [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 P1-10-23.

Abstract P4-04-03: High-throughput epigenetic screening in hormone receptor positive (HR+) breast cancer cells

Abstract Background &amp; Objective: Substantial progress has been made in treating HR+ metastatic breast cancer patients with CDK4/6, mTOR and PIK3CA inhibitors (all FDA approved), but there are no current available therapies to directly restore sensitivity to endocrine therapy once patients’ cells develop epigenetic suppression of ER pathways. CCCTC-binding factor (CTCF), which regulates higher-order chromatin organization and estrogen reception (ER) transcription, is altered by hemizygous deletion or mutation in more than 50% of HR+ luminal type breast tumors. The goal of this project is to characterize an epigenetic signature in CTCF-altered breast cancers to identify novel epigenetic targets for therapeutics development. Targeting CTCF influenced pathways with or without its chaperones may restore sensitivity to endocrine therapy in patients with HR+ breast cancer. Methods: We identified patient-derived cell lines from the Cancer Cell Line Encyclopedia (CCLE) with known CTCF gene copy number. Selected cell lines were tested for proliferation with a custom siRNA library targeting 1,300 genes involved in epigenetic regulation while in the presence of different concentrations of estradiol, tamoxifen and fulvestrant. We confirmed presence of CTCF protein by western blot and mRNA expression by real-time PCR. Proliferation of the lines in vitro was quantified by the MTT assay. Further functional annotation and pathway enrichment analysis of the identified genes were performed by using the g:ProfileR package. Results: We have screened more than 60 breast cancer cell lines and focused on six HR+ luminal (3 each “a” and “b” type) lines with different levels of CTCF copy number; a normal breast epithelial line was used as control. We observed that cell growth responded to anti-estrogen therapy differently based on the copy number of CTCF. Cells with high copy number of CTCF are resistant to endocrine therapy, the sensitivity was restored by silencing CTCF using siRNA. We then selected two cell lines with low and high copy number of CTCF for further high-throughput screening with the epigenetic siRNA library. A total of 67 and 35 candidate sensitizer target genes, and 56 and 44 candidate enhancer target genes were prioritized in cells with low and high copy number of CTCF, respectively. Functional annotation and pathway enrichment analysis of the identified genes showed that cells with low copy number of CTCF had apoptosis related hits, such as BCL family and caspase genes; however, cells with high copy number of CTCF were found to relate to other epigenetic regulators such as DNMTs and CHD. This suggests that CTCF might form complexes with other epigenetic proteins to form abnormal chromatin configuration with abnormal regulation of ER pathway as consequence. Conclusions: To our knowledge, this is the first epigenetic library screen of HR+ luminal breast cancer cells with responses categorized by differential CTCF copy number status. We have confirmed CTCF as an important protein in predicting response to endocrine therapy and identified possible interactions between CTCF, DNMT and CHD. We conclude that development of therapeutics against targets identified in our screen to reverse endocrine resistance may be achievable for treatment of luminal breast cancers that undertake epigenetic deregulation. Keywords: Breast cancer; Hormone receptor positive; epigenetic screening Citation Format: Fengting Yan, Olga Nikolova, VK Gadi. High-throughput epigenetic screening in hormone receptor positive (HR+) breast cancer cells [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 P4-04-03.

Abstract P4-05-17: ESR1 related chromothripsis enhances concordant gene transcription on chromosome 17q11.1-q24.1 in luminal breast cancer

Abstract Chromothripsis is an event of genomic instability leading to complex chromosomal alterations in cancer. Although studies have proposed models to explain its initiation, few discussed how transcription factors (TFs) influence chromothripsis for tumor progression. Frequent long-range chromatin interactions between TFs and targets may promote extensive translocations and copy-number alterations in proximal contact regions through inappropriate DNA stitching. Herein we surveyed genomic alterations of chromosome 17 in 1,014 breast tumors in the Cancer Genome Atlas (TCGA) cohort and found five adjoining regions from 17q11.1 to 17q24.1 being hotspots of chromothripsis. Inter-/intra-chromosomal rearrangements of these regions occurred more frequently in ERα-positive tumors than in ERα-negative tumors. In addition, the rearrangement sites were often mapped within or close to dense estrogen receptor 1 (ESR1) sites localized on the regions or other chromosomes. This chromothriptic event was linked to concordant upregulation of 96 loci that predominantly regulate cell-cycle machineries in advanced luminal tumors. Genome-editing analysis confirmed that an ESR1 transcription hub localized on 20q13 coordinately regulates a subset of these loci localized on 17q23 through long-range chromosome interactions. One of these loci, Tousled Like Kinase 2 (TLK2) known to participate in DNA damage checkpoint control, is an actionable target using phenothiazine antipsychotics (PTZs). The antiproliferative effect of PTZs was prominent in high TLK2-expressing cells, compared to low expressing cells. Collectively, we identified a group of luminal breast tumors displaying 17q-related chromothripsis for which antipsychotics can be repurposed as treatment adjuncts. Citation Format: Chun-Lin Lin. ESR1 related chromothripsis enhances concordant gene transcription on chromosome 17q11.1-q24.1 in luminal breast cancer [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 P4-05-17.

Immune gene expression profiling reveals heterogeneity in luminal breast tumors

BackgroundHeterogeneity of immune gene expression patterns of luminal breast cancer (BC), which is clinically heterogeneous and overall considered as low immunogenic, has not been well studied especially in non-European populations. Here, we aimed at characterizing the immune gene expression profile of luminal BC in an Asian population and associating it with patient characteristics and tumor genomic features.MethodsWe performed immune gene expression profiling of tumor and adjacent normal tissue in 92 luminal BC patients from Hong Kong using RNA-sequencing data and used unsupervised consensus clustering to stratify tumors. We then used luminal patients from The Cancer Genome Atlas (TCGA, N = 564) and a Korean breast cancer study (KBC, N = 112) as replication datasets.ResultsBased on the expression of 130 immune-related genes, luminal tumors were stratified into three distinct immune subtypes. Tumors in one subtype showed higher level of tumor-infiltrating lymphocytes (TILs), characterized by T cell gene activation, higher expression of immune checkpoint genes, higher nonsynonymous mutation burden, and higher APOBEC-signature mutations, compared with other luminal tumors. The high-TIL subtype was also associated with lower ESR1/ESR2 expression ratio and increasing body mass index. The comparison of the immune profile in tumor and matched normal tissue suggested a tumor-derived activation of specific immune responses, which was only seen in high-TIL patients. Tumors in a second subtype were characterized by increased expression of interferon-stimulated genes and enrichment for TP53 somatic mutations. The presence of three immune subtypes within luminal BC was replicated in TCGA and KBC, although the pattern was more similar in Asian populations. The germline APOBEC3B deletion polymorphism, which is prevalent in East Asian populations and was previously linked to immune activation, was not associated with immune subtypes in our study. This result does not support the hypothesis that the germline APOBEC3B deletion polymorphism is the driving force for immune activation in breast tumors in Asian populations.ConclusionOur findings suggest that immune gene expression and associated genomic features could be useful to further stratify luminal BC beyond the current luminal A/B classification and a subset of luminal BC patients may benefit from checkpoint immunotherapy, at least in Asian populations.

Abstract 5214: Targeting the estrogen receptor pathway in luminal breast cancer through inhibition of p300/CBP

Abstract Luminal breast cancer represents approximately two thirds of all breast cancer cases and is characterized by the expression of hormone receptors, such as the estrogen receptor alpha (ER). ERα is a member of the steroid nuclear receptor family and is involved in a hormonal signaling pathway that drives tumor growth through upregulation of genes involved in cell cycle progression, such as MYC and CCND1. Antagonists of ER function are the standard of care treatment for ER+ luminal breast cancer. However, resistance to ER antagonists is common in advanced breast cancer cases. Therefore, there is an urgent need to investigate new therapeutics that can be utilized to treat tumors that are resistant to traditional therapies. p300/CBP are two paralogous acetyltransferases that catalyze histone 3, lysine 18 and 27 acetylation (H3K18ac and H3K27ac) at promoters and enhancers to promote gene expression. ER mediated transcription is critically reliant upon the recruitment of co-activators, including p300/CBP. However, the mechanistic role of p300/CBP catalytic activity in globally regulating ER mediated transcription remains poorly understood. Inhibition of p300/CBP, as a critical co-activator of ER, potentially represents an applicable strategy for inhibiting ER function in luminal breast tumors. Importantly, the catalytic histone acetyltransferase (HAT) domain of p300/CBP is under intense investigation as a target of small molecule therapeutics in cancer. We analyzed publicly available data and report that p300/CBP are upregulated at the mRNA level in breast cancer, including the luminal subtypes, and that their expression negatively correlates with patient survival. We also found that pharmacologic inhibition of p300/CBP HAT activity in ER+ cell lines potently suppresses ER mediated transcription, downregulates ER, c-Myc and Cyclin D1 protein levels and prevents estrogen induced growth in vitro. Studies are underway to understand how p300/CBP regulates ER signaling using genetic, pharmacologic, and biochemical methods to assess the therapeutic effects of pharmacologic p300/CBP inhibition on ER+ breast cancer. (Supported by Bankhead-Coley Research Program, and James and Esther King Biomedical Research Program, Florida Department of Health) Citation Format: Aaron Waddell, Iqbal Mahmud, Guimei Tian, Daiqing Liao. Targeting the estrogen receptor pathway in luminal breast cancer through inhibition of p300/CBP [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5214.

The prognosis comparison of different molecular subtypes of breast tumors after radiotherapy and the intrinsic reasons for their distinct radiosensitivity.

Radiotherapy can increase the cell cycle arrest that promotes apoptosis, reduces the risk of tumor recurrence and has become an irreplaceable component of systematic treatment for patients with breast cancer. Substantial advances in precise radiotherapy unequivocally indicate that the benefits of radiotherapy vary depending on intrinsic subtypes of the disease; luminal A breast cancer has the highest benefit whereas human epidermal growth factor receptor 2 (HER2)-positive and triple negative breast cancer (TNBC) are affected to a lesser extent irrespective of the selection of radiotherapy strategies, such as conventional whole-breast irradiation (CWBI), accelerated partial-breast irradiation (APBI), and hypofractionated whole-breast irradiation (HWBI). The benefit disparity correlates with the differential invasiveness, malignance, and radiosensitivity of the subtypes. A combination of a number of molecular mechanisms leads to the strong radioresistant profile of HER2-positive breast cancer, and sensitization to irradiation can be induced by multiple drugs or compounds in luminal disease and TNBC. In this review, we aimed to summarize the prognostic differences between various subtypes of breast tumors after CWBI, APBI, and HWBI, the potential reasons for drug-enhanced radiosensitivity in luminal breast tumors and TNBC, and the robust radioresistance of HER2-positive cancer.