Defining the Regulatory Logic of Breast Cancer Using Single-Cell Epigenetic and Transcriptome Profiling.

Annotation of the cis-regulatory elements that drive transcriptional dysregulation in cancer cells is critical to improving our understanding of tumor biology. Herein, we present a compendium of matched chromatin accessibility (scATAC-seq) and transcriptome (scRNA-seq) profiles at single-cell resolution from human breast tumors and healthy mammary tissues processed immediately following surgical resection. We identify the most likely cell-of-origin for luminal breast tumors and basal breast tumors and then introduce a novel methodology that implements linear mixed-effects models to systematically quantify associations between regions of chromatin accessibility (i.e. regulatory elements) and gene expression in malignant cells versus normal mammary epithelial cells. These data unveil regulatory elements with that switch from silencers of gene expression in normal cells to enhancers of gene expression in cancer cells, leading to the upregulation of clinically relevant oncogenes. To translate the utility of this dataset into tractable models, we generated matched scATAC-seq and scRNA-seq profiles for breast cancer cell lines, revealing, for each subtype, a conserved oncogenic gene expression program between in vitro and in vivo cells. Together, this work highlights the importance of non-coding regulatory mechanisms that underlie oncogenic processes and the ability of single-cell multi-omics to define the regulatory logic of BC cells at single-cell resolution.

Single-Cell Transcriptional and Epigenetic Profiles of Male Breast Cancer Nominate Salient Cancer-Specific Enhancers.

Male breast cancer represents about 1% of all breast cancer diagnoses and, although there are some similarities between male and female breast cancer, the paucity of data available on male breast cancer makes it difficult to establish targeted therapies. To date, most male breast cancers (MBCs) are treated according to protocols established for female breast cancer (FBC). Thus, defining the transcriptional and epigenetic landscape of MBC with improved resolution is critical for developing better avenues for therapeutic intervention. In this study, we present matched transcriptional (scRNA-seq) and epigenetic (scATAC-seq) profiles at single-cell resolution of two treatment naïve MBC tumors processed immediately after surgical resection. These data enable the detection of differentially expressed genes between male and female breast tumors across immune, stromal, and malignant cell types, to highlight several genes that may have therapeutic implications. Notably, MYC target genes and mTORC1 signaling genes were significantly upregulated in the malignant cells of MBC compared to the female counterparts. To understand how the regulatory landscape of MBC gives rise to these male-specific gene expression patterns, we leveraged the scATAC-seq data to systematically link changes in chromatin accessibility to changes in gene expression within each cell type. We observed cancer-specific rewiring of several salient enhancers and posit that these enhancers have a higher regulatory load than lineage-specific enhancers. We highlight two examples of previously unannotated cancer-cell-specific enhancers of ANXA2 and PRDX4 gene expression and show evidence for super-enhancer regulation of LAMB3 and CD47 in male breast cancer cells. Overall, this dataset annotates clinically relevant regulatory networks in male breast tumors, providing a useful resource that expands our current understanding of the gene expression programs that underlie the biology of MBC.

A multi-omic dissection of super-enhancer driven oncogenic gene expression programs in ovarian cancer.

The human genome contains regulatory elements, such as enhancers, that are often rewired by cancer cells for the activation of genes that promote tumorigenesis and resistance to therapy. This is especially true for cancers that have little or no known driver mutations within protein coding genes, such as ovarian cancer. Herein, we utilize an integrated set of genomic and epigenomic datasets to identify clinically relevant super-enhancers that are preferentially amplified in ovarian cancer patients. We systematically probe the top 86 super-enhancers, using CRISPR-interference and CRISPR-deletion assays coupled to RNA-sequencing, to nominate two salient super-enhancers that drive proliferation and migration of cancer cells. Utilizing Hi-C, we construct chromatin interaction maps that enable the annotation of direct target genes for these super-enhancers and confirm their activity specifically within the cancer cell compartment of human tumors using single-cell genomics data. Together, our multi-omic approach examines a number of fundamental questions about how regulatory information encoded into super-enhancers drives gene expression networks that underlie the biology of ovarian cancer.

Enhancer RNA Transcription Is Essential for a Novel CSF1 Enhancer in Triple-Negative Breast Cancer.

Enhancers are critical regulatory elements in the genome that help orchestrate spatiotemporal patterns of gene expression during development and normal physiology. In cancer, enhancers are often rewired by various genetic and epigenetic mechanisms for the activation of oncogenes that lead to initiation and progression. A key feature of active enhancers is the production of non-coding RNA molecules called enhancer RNAs, whose functions remain unknown but can be used to specify active enhancers de novo. Using a combination of eRNA transcription and chromatin modifications, we have identified a novel enhancer located 30 kb upstream of Colony Stimulating Factor 1 (CSF1). Notably, CSF1 is implicated in the progression of breast cancer, is overexpressed in triple-negative breast cancer (TNBC) cell lines, and its enhancer is primarily active in TNBC patient tumors. Genomic deletion of the enhancer (via CRISPR/Cas9) enabled us to validate this regulatory element as a bona fide enhancer of CSF1 and subsequent cell-based assays revealed profound effects on cancer cell proliferation, colony formation, and migration. Epigenetic silencing of the enhancer via CRISPR-interference assays (dCas9-KRAB) coupled to RNA-sequencing, enabled unbiased identification of additional target genes, such as RSAD2, that are predictive of clinical outcome. Additionally, we repurposed the RNA-guided RNA-targeting CRISPR-Cas13 machinery to specifically degrade the eRNAs transcripts produced at this enhancer to determine the consequences on CSF1 mRNA expression, suggesting a post-transcriptional role for these non-coding transcripts. Finally, we test our eRNA-dependent model of CSF1 enhancer function and demonstrate that our results are extensible to other forms of cancer. Collectively, this work describes a novel enhancer that is active in the TNBC subtype, which is associated with cellular growth, and requires eRNA transcripts for proper enhancer function. These results demonstrate the significant impact of enhancers in cancer biology and highlight their potential as tractable targets for therapeutic intervention.

A multi-omic single-cell landscape of human gynecologic malignancies.

Deconvolution of regulatory mechanisms that drive transcriptional programs in cancer cells is key to understanding tumor biology. Herein, we present matched transcriptome (scRNA-seq) and chromatin accessibility (scATAC-seq) profiles at single-cell resolution from human ovarian and endometrial tumors processed immediately following surgical resection. This dataset reveals the complex cellular heterogeneity of these tumors and enabled us to quantitatively link variation in chromatin accessibility to gene expression. We show that malignant cells acquire previously unannotated regulatory elements to drive hallmark cancer pathways. Moreover, malignant cells from within the same patients show substantial variation in chromatin accessibility linked to transcriptional output, highlighting the importance of intratumoral heterogeneity. Finally, we infer the malignant cell type-specific activity of transcription factors. By defining the regulatory logic of cancer cells, this work reveals an important reliance on oncogenic regulatory elements and highlights the ability of matched scRNA-seq/scATAC-seq to uncover clinically relevant mechanisms of tumorigenesis in gynecologic cancers.