scATAnno: Automated Cell Type Annotation for single-cell ATAC Sequencing Data.

Recent advances in single-cell epigenomic techniques have created a growing demand for scATAC-seq analysis. One key analysis task is to determine cell type identity based on the epigenetic data. We introduce scATAnno, a python package designed to automatically annotate scATAC-seq data using large-scale scATAC-seq reference atlases. This workflow generates the reference atlases from publicly available datasets enabling accurate cell type annotation by integrating query data with reference atlases, without the use of scRNA-seq data. To enhance annotation accuracy, we have incorporated KNN-based and weighted distance-based uncertainty scores to effectively detect cell populations within the query data that are distinct from all cell types in the reference data. We compare and benchmark scATAnno against 7 other published approaches for cell annotation and show superior performance in multiple data sets and metrics. We showcase the utility of scATAnno across multiple datasets, including peripheral blood mononuclear cell (PBMC), Triple Negative Breast Cancer (TNBC), and basal cell carcinoma (BCC), and demonstrate that scATAnno accurately annotates cell types across conditions. Overall, scATAnno is a useful tool for scATAC-seq reference building and cell type annotation in scATAC-seq data and can aid in the interpretation of new scATAC-seq datasets in complex biological systems.

Combination Therapies to Improve the Efficacy of Immunotherapy in Triple-negative Breast Cancer.

Immune checkpoint inhibition combined with chemotherapy is currently approved as first-line treatment for patients with advanced PD-L1-positive triple-negative breast cancer (TNBC). However, a significant proportion of metastatic TNBC is PD-L1-negative and, in this population, chemotherapy alone largely remains the standard-of-care and novel therapeutic strategies are needed to improve clinical outcomes. Here, we describe a triple combination of anti-PD-L1 immune checkpoint blockade, epigenetic modulation thorough bromodomain and extra-terminal (BET) bromodomain inhibition (BBDI), and chemotherapy with paclitaxel that effectively inhibits both primary and metastatic tumor growth in two different syngeneic murine models of TNBC. Detailed cellular and molecular profiling of tumors from single and combination treatment arms revealed increased T- and B-cell infiltration and macrophage reprogramming from MHCIIlow to a MHCIIhigh phenotype in mice treated with triple combination. Triple combination also had a major impact on gene expression and chromatin profiles shifting cells to a more immunogenic and senescent state. Our results provide strong preclinical evidence to justify clinical testing of BBDI, paclitaxel, and immune checkpoint blockade combination.

JAK-STAT Signaling in Inflammatory Breast Cancer Enables Chemotherapy-Resistant Cell States.

Inflammatory breast cancer (IBC) is a difficult-to-treat disease with poor clinical outcomes due to high risk of metastasis and resistance to treatment. In breast cancer, CD44+CD24- cells possess stem cell-like features and contribute to disease progression, and we previously described a CD44+CD24-pSTAT3+ breast cancer cell subpopulation that is dependent on JAK2/STAT3 signaling. Here we report that CD44+CD24- cells are the most frequent cell type in IBC and are commonly pSTAT3+. Combination of JAK2/STAT3 inhibition with paclitaxel decreased IBC xenograft growth more than either agent alone. IBC cell lines resistant to paclitaxel and doxorubicin were developed and characterized to mimic therapeutic resistance in patients. Multi-omic profiling of parental and resistant cells revealed enrichment of genes associated with lineage identity and inflammation in chemotherapy-resistant derivatives. Integrated pSTAT3 chromatin immunoprecipitation sequencing and RNA sequencing (RNA-seq) analyses showed pSTAT3 regulates genes related to inflammation and epithelial-to-mesenchymal transition (EMT) in resistant cells, as well as PDE4A, a cAMP-specific phosphodiesterase. Metabolomic characterization identified elevated cAMP signaling and CREB as a candidate therapeutic target in IBC. Investigation of cellular dynamics and heterogeneity at the single cell level during chemotherapy and acquired resistance by CyTOF and single cell RNA-seq identified mechanisms of resistance including a shift from luminal to basal/mesenchymal cell states through selection for rare preexisting subpopulations or an acquired change. Finally, combination treatment with paclitaxel and JAK2/STAT3 inhibition prevented the emergence of the mesenchymal chemo-resistant subpopulation. These results provide mechanistic rational for combination of chemotherapy with inhibition of JAK2/STAT3 signaling as a more effective therapeutic strategy in IBC. SIGNIFICANCE: Chemotherapy resistance in inflammatory breast cancer is driven by the JAK2/STAT3 pathway, in part via cAMP/PKA signaling and a cell state switch, which can be overcome using paclitaxel combined with JAK2 inhibitors.

FOXR2 Is an Epigenetically Regulated Pan-Cancer Oncogene That Activates ETS Transcriptional Circuits.

Forkhead box R2 (FOXR2) is a forkhead transcription factor located on the X chromosome whose expression is normally restricted to the testis. In this study, we performed a pan-cancer analysis of FOXR2 activation across more than 10,000 adult and pediatric cancer samples and found FOXR2 to be aberrantly upregulated in 70% of all cancer types and 8% of all individual tumors. The majority of tumors (78%) aberrantly expressed FOXR2 through a previously undescribed epigenetic mechanism that involves hypomethylation of a novel promoter, which was functionally validated as necessary for FOXR2 expression and proliferation in FOXR2-expressing cancer cells. FOXR2 promoted tumor growth across multiple cancer lineages and co-opted ETS family transcription circuits across cancers. Taken together, this study identifies FOXR2 as a potent and ubiquitous oncogene that is epigenetically activated across the majority of human cancers. The identification of hijacking of ETS transcription circuits by FOXR2 extends the mechanisms known to active ETS transcription factors and highlights how transcription factor families cooperate to enhance tumorigenesis. SIGNIFICANCE: This work identifies a novel promoter that drives aberrant FOXR2 expression and delineates FOXR2 as a pan-cancer oncogene that specifically activates ETS transcriptional circuits across human cancers. See related commentary by Liu and Northcott, p. 2977.

Repurposing Erythrocytes as a "Photoactivatable Bomb": A General Strategy for Site-Specific Drug Release in Blood Vessels.

Tumor vasculature has long been considered as an extremely valuable therapeutic target for cancer therapy, but how to realize controlled and site-specific drug release in tumor blood vessels remains a huge challenge. Despite the widespread use of nanomaterials in constructing drug delivery systems, they are suboptimal in principle for meeting this demand due to their easy blood cell adsorption/internalization and short lifetime in the systemic circulation. Here, natural red blood cells (RBCs) are repurposed as a remote-controllable drug vehicle, which retains RBC's morphology and vessel-specific biodistribution pattern, by installing photoactivatable molecular triggers on the RBC membrane via covalent conjugation with a finely tuned modification density. The molecular triggers can burst the RBC vehicle under short and mild laser irradiation, leading to a complete and site-specific release of its payloads. This cell-based vehicle is generalized by loading different therapeutic agents including macromolecular thrombin, a blood clotting-inducing enzyme, and a small-molecule hypoxia-activatable chemodrug, tirapazamine. In vivo results demonstrate that the repurposed "anticancer RBCs" exhibit long-term stability in systemic circulation but, when tumors receive laser irradiation, precisely releases their cargoes in tumor vessels for thrombosis-induced starvation therapy and local deoxygenation-enhanced chemotherapy. This study proposes a general strategy for blood vessel-specific drug delivery.

CoBRA: Containerized Bioinformatics Workflow for Reproducible ChIP/ATAC-seq Analysis.

Chromatin immunoprecipitation sequencing (ChIP-seq) and the Assay for Transposase-Accessible Chromatin with high-throughput sequencing (ATAC-seq) have become essential technologies to effectively measure protein-DNA interactions and chromatin accessibility. However, there is a need for a scalable and reproducible pipeline that incorporates proper normalization between samples, correction of copy number variations, and integration of new downstream analysis tools. Here we present Containerized Bioinformatics workflow for Reproducible ChIP/ATAC-seq Analysis (CoBRA), a modularized computational workflow which quantifies ChIP-seq and ATAC-seq peak regions and performs unsupervised and supervised analyses. CoBRA provides a comprehensive state-of-the-art ChIP-seq and ATAC-seq analysis pipeline that can be used by scientists with limited computational experience. This enables researchers to gain rapid insight into protein-DNA interactions and chromatin accessibility through sample clustering, differential peak calling, motif enrichment, comparison of sites to a reference database, and pathway analysis. CoBRA is publicly available online at https://bitbucket.org/cfce/cobra.

Mitochondria-acting nanomicelles for destruction of cancer cells via excessive mitophagy/autophagy-driven lethal energy depletion and phototherapy.

Mitophagy is a specific self-protective autophagic process that degrades damaged or dysfunctional mitochondria, and is generally considered to reduce the effectiveness of mitochondria-targeted therapies. Here, we report an energy depletion-based anticancer strategy by selectively activating excessive mitophagy in cancer cells. We fabricate a type of mitochondria-targeting nanomicelles via the self-assembly of D-α-tocopheryl polyethylene glycol 1000 succinate (TPGS) and dc-IR825 (a near-infrared cyanine dye and a photothermal agent). The TPGS/dc-IR825 nanomicelles enable mitochondrial damage in cancer cells, which, for self-protection, activate two autophagic pathways, (1) mitophagy and (2) adenosine triphosphate (ATP) shortage-triggered autophagy. However, the excessive mitophagy/autophagy activities far surpass the degradative capacity of autolysosomes, leading to the formation of micrometer-sized vacuoles and degradation blockage. Immunofluorescence staining and Western blot analysis reveal that the nanomicelle-treated cancer cells are under severe ATP shortage, which eventually causes substantial cell death. Moreover, the nanomicelles intravenously injected into tumor-bearing mice show high tumor accumulation, long tumor retention, and inhibit the tumor growth by inducing excessive mitophagy/autophagy and energy depletion in tumor cells. Additional near-infrared laser irradiation treatment further enhances the in vitro and in vivo anticancer efficiencies of the nanomicelles, due to the excellent photothermal and photodynamic effects of dc-IR825. We believe that this work highlights the important role of mitophagy/autophagy in treating cancers.