Citations

Introduction: Clonal dendritic cell (DC) aggregates in chronic myelomonocytic leukemia (CMML) are seen in approximately 20-30% patients at diagnosis. Previously, these were thought to be comprised of CD123+ plasmacytoid DCs but our data has suggested that these are heterogenous and comprise of myeloid DCs, monocytes, and myeloid-derived suppressor cells. Furthermore, these aggregates uniformly express immune checkpoints such as indoleamine 2,3-dioxygenase-1 (IDO), associate with an IDO-specific systemic impact (increased trytophan catabolism), and expanded regulatory T cell population in CMML, suggesting a role in inducing immune tolerance. DC aggregates are also associated with increased risk of acute myeloid leukemia (AML) transformation and resistance to hypomethylating agents, highlighting that they are important for disease progression in CMML (Mangaonkar A et al. Blood Adv 2020). We performed this study to decipher the architecture of a CMML DC aggregate to understand the complex inter-cellular interactions, and assess their impact on the adaptive immune system. Methods: In this study, we used archival fresh-frozen paraffin-embedded (FFPE) bone marrow biopsy specimens obtained from CMML patients, and isolated monocytes (CD14+ magnetic bead sorting) and lymphocytes (CD14-ve fraction) from untreated CMML and age-matched normal control (NC) peripheral blood mononuclear cells (PBMCs). DCs were cultured from monocytes using a commercial protocol. Spatial architecture of the CMML BM microenvironment was interrogated through multiplex imaging of FFPE samples by co-detection by indexing (CODEX) using 30 markers (CCR6, CD107a, CD11c, CD20, CD21, CD31, CD3e, CD4, CD44, CD45, CD45RO CD56, CD68, CD8, E-Cadherin, FoxP3, HLA-DR, CD14, CD16, CD11b, CD33, CD15, CD66b, CD123, CD303, CD34, CD163 and IDO). Single-cell transcriptomic signatures were analyzed using the 10X single cell Fixed RNA profiling platform, and validated using bulk RNA seq (Illumina stranded mRNA prep) from isolated and cultured cells. Impact of DCs on lymphocytes was assessed via autologous mixed DC-T culture experiments. Results: BM FFPE samples from 5 CMML cases (3 with DC aggregates) were included for multiplex imaging via CODEX. Results confirm the significant interactions of CD8+ and CD4+ T cells with CD123+/HLA-DR+ DCs within a CMML DC aggregate (Fig. 1A). Upon quantitative assessment of cell-cell interactions, IDO+/CD8+T ( P=0.006), IDO+/MDSC ( P=0.005), and IDO+/macrophage ( P=0.001) interactions were significantly increased in CMML samples with versus without DC aggregates . Specific findings were validated using single-stain immunohistochemistry and multiplex immunoflourescence staining. Next, in order to assess transcriptomic signatures within BM at the single-cell level, we chose two cases; CMML (BM aspirate blast% 1, WBC 49.6 x 10 9/L, ASXL1/GATA2/SETBP1/SRSF2 mutations) and AML transformed from CMML (BM aspirate blast% 81, WBC 152 x 10 9/L, NPM1/DNMT3A/TET2 mutations). Approximately, 10,000 cells were obtained and analyzed with cell type annotation through human PBMC reference (Azimuth). Results show an expanded regulatory T cell population in AML compared to CMML (UMAP plot, Fig. 1B), suggestive of immune tolerance. In order to validate findings and perform functional experiments, we used sorted monocytes and cutured DCs from CMML and NC. DC phenotype was confirmed both through visual inspection and flow cytometry, and clonality was confirmed through Sanger sequencing (for previously identified mutations). Differential gene expression analysis through bulk RNAseq showed significant differences between CMML and NC lymphocytes with immunoinhibitory receptor, LILRB2 (log2fold change=7.05, adjusted P=0.0002) being one of the top five significantly upregulated genes, further confirming immune tolerance. Finally, mixed autologous DC-lymphocyte reaction (1:4 ratio) experiments demonstrated that DCs induce T cell tolerance in CMML (expansion of exhausted PD1/TIM3/LAG3+ CD8 T cells) when compared to NC. Conclusion: Clonal dendritic cell aggregates within the CMML bone marrow microenviroment interact with T cells and induce immune tolerance. This represents a novel mechanism of disease progression in CMML with potential therapeutic targets (IDO, LILRB2, and/or CD123).

Histone‐lysine N‐methyltransferase SETD2 (SETD2), the sole histone methyltransferase that catalyzes trimethylation of lysine 36 on histone H3 (H3K36me3), is often mutated in clear cell renal cell carcinoma (ccRCC). SETD2 mutation and/or loss of H3K36me3 is linked to metastasis and poor outcome in ccRCC patients. Epithelial‐to‐mesenchymal transition (EMT) is a major pathway that drives invasion and metastasis in various cancer types. Here, using novel kidney epithelial cell lines isogenic for SETD2, we discovered that SETD2 inactivation drives EMT and promotes migration, invasion, and stemness in a transforming growth factor‐beta‐independent manner. This newly identified EMT program is triggered in part through secreted factors, including cytokines and growth factors, and through transcriptional reprogramming. RNA‐seq and assay for transposase‐accessible chromatin sequencing uncovered key transcription factors upregulated upon SETD2 loss, including SOX2, POU2F2 (OCT2), and PRRX1, that could individually drive EMT and stemness phenotypes in SETD2 wild‐type (WT) cells. Public expression data from SETD2 WT/mutant ccRCC support the EMT transcriptional signatures derived from cell line models. In summary, our studies reveal that SETD2 is a key regulator of EMT phenotypes through cell‐intrinsic and cell‐extrinsic mechanisms that help explain the association between SETD2 loss and ccRCC metastasis.

The snATAC + snRNA platform allows epigenomic profiling of open chromatin and gene expression with single-cell resolution. The most critical assay step is to isolate high-quality nuclei to proceed with droplet-base single nuclei isolation and barcoding. With the increasing popularity of multiomic profiling in various fields, there is a need for optimized and reliable nuclei isolation methods, mainly for human tissue samples. Herein we compared different nuclei isolation methods for cell suspensions, such as peripheral blood mononuclear cells (PBMC, n = 18) and a solid tumor type, ovarian cancer (OC, n = 18), derived from debulking surgery. Nuclei morphology and sequencing output parameters were used to evaluate the quality of preparation. Our results show that NP-40 detergent-based nuclei isolation yields better sequencing results than collagenase tissue dissociation for OC, significantly impacting cell type identification and analysis. Given the utility of applying such techniques to frozen samples, we also tested frozen preparation and digestion (n = 6). A paired comparison between frozen and fresh samples validated the quality of both specimens. Finally, we demonstrate the reproducibility of scRNA and snATAC + snRNA platform, by comparing the gene expression profiling of PBMC. Our results highlight how the choice of nuclei isolation methods is critical for obtaining quality data in multiomic assays. It also shows that the measurement of expression between scRNA and snRNA is comparable and effective for cell type identification.

Abstract Background: COVID-19 causes significant morbidity and mortality, albeit with considerable heterogeneity among affected individuals. It remains unclear which host factors determine disease severity and survival. Given the propensity of clonal hematopoiesis (CH) to promote inflammation in healthy individuals, we investigated its effect on COVID-19 outcomes. Methods: We performed a multi-omics interrogation of the genome, epigenome, transcriptome, and proteome of peripheral blood mononuclear cells from COVID-19 patients (n=227). We obtained clinical data, laboratory studies, and survival outcomes. We determined CH status and TET2-related DNA methylation. We performed single-cell proteogenomics to understand clonal composition in relation to cell phenotype. We interrogated single-cell gene expression in isolation and in conjunction with DNA accessibility. We integrated these multi-omics data to understand the effect of CH on clonal composition, gene expression, methylation of cis-regulatory elements, and lineage commitment in COVID-19 patients. We performed shRNA knockdowns to validate the effect of one candidate transcription factor in myeloid cell lines. Results: The presence of CH was strongly associated with COVID-19 severity and all-cause mortality, independent of age (HR 3.48, 95% CI 1.45-8.36, p=0.005). Differential methylation of promoters and enhancers was prevalent in TET2-mutant, but not DNMT3A-mutant CH. TET2-mutant CH was associated with enhanced classical/intermediate monocytosis and single-cell proteogenomics confirmed an enrichment of TET2 mutations in these cell types. We identified cell-type specific gene expression changes associated with TET2 mutations in 102,072 single cells (n=34). Single-cell RNA-seq confirmed the skewing of hematopoiesis towards classical and intermediate monocytes and demonstrated the downregulation of EGR1 (a transcription factor important for monocyte differentiation) along with up-regulation of the lncRNA MALAT1 in monocytes. Combined scRNA-/scATAC-seq in 43,160 single cells (n=18) confirmed the skewing of hematopoiesis and up-regulation of MALAT1 in monocytes along with decreased accessibility of EGR1 motifs in known cis-regulatory elements. Using myeloid cell lines for functional validation, shRNA knockdowns of EGR1 confirmed the up-regulation of MALAT1 (in comparison to wildtype controls). Conclusions: CH is an independent prognostic factor in COVID-19 and skews hematopoiesis towards monocytosis. TET2-mutant CH is characterized by differential methylation and accessibility of enhancers binding myeloid transcriptions factors including EGR1. The ensuing loss of EGR1 expression in monocytes causes MALAT1 overexpression, a factor known to promote monocyte differentiation and inflammation. These data provide a mechanistic insight to the adverse prognostic impact of CH in COVID-19. Citation Format: Moritz Binder, Terra L. Lasho, Wazim Mohammed Ismail, Nana A. Ben-Crentsil, Jenna A. Fernandez, Minsuk Kim, Susan M. Geyer, Amelia Mazzone, Christy M. Finke, Abhishek A. Mangaonkar, Jeong-Heon Lee, Kwan Hyun Kim, Vernadette A. Simon, Fariborz Rakhshan Rohakthar, Amik Munankarmy, Susan M. Schwager, Jonathan J. Harrington, Melissa R. Snyder, Nathalie M. Droin, Eric Solary, Keith D. Robertson, Eric D. Wieben, Eric Padron, Nicholas Chia, Alexandre Gaspar-Maia, Mrinal M. Patnaik. Enhancer deregulation inTET2-mutant clonal hematopoiesis is associated with increased COVID-19 severity and mortality [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 806.

Abstract Understanding the function of non-coding genomic sequence variants represents a challenge for biomedicine. Many diseases are products of gene-by-environment interactions with complex mechanisms. This study addresses these themes by mechanistic characterization of non-coding variants that influence gene expression only after drug or hormone exposure. Using glucocorticoid signaling as a model system, we integrated genomic, transcriptomic, and epigenomic approaches to unravel mechanisms by which variant function could be revealed by hormones or drugs. Specifically, we identified cis-regulatory elements and 3D interactions underlying ligand-dependent associations between variants and gene expression. One-quarter of the glucocorticoid-modulated variants that we identified had already been associated with clinical phenotypes. However, their affected genes were ‘unmasked’ only after glucocorticoid exposure and often with function relevant to the disease phenotypes. These diseases involved glucocorticoids as risk factors or therapeutic agents and included autoimmunity, metabolic and mood disorders, osteoporosis and cancer. For example, we identified a novel breast cancer risk gene, MAST4, with expression that was repressed by glucocorticoids in cells carrying the risk genotype, repression that correlated with MAST4 expression in breast cancer and treatment outcomes. These observations provide a mechanistic framework for understanding non-coding genetic variant-chemical environment interactions and their role in disease risk and drug response.

AbstractAlternative polyadenylation (APA) is a common genetic regulatory mechanism that generates distinct 3′ ends for RNA transcripts. Changes in APA have been associated with multiple biological processes and disease phenotypes. However, the role of hormones and their drug analogs in APA remains largely unknown. In this study, we investigated transcriptome‐wide the impact of glucocorticoids on APA in 30 human B‐lymphoblastoid cell lines. We found that glucocorticoids could regulate APA for a subset of genes, possibly by changing the expression of 142 RNA‐binding proteins, some with known APA‐regulating properties. Interestingly, genes with glucocorticoid‐mediated APA were enriched in viral translation‐related pathways, while genes with glucocorticoid‐mediated expression were enriched in interferon and interleukin pathways, suggesting that glucocorticoid‐mediated APA might result in functional consequences distinct from gene expression. For example, glucocorticoids, a pharmacotherapy for severe COVID‐19, were found to change the APA but not the expression of LY6E, an important antiviral inhibitor in coronavirus diseases. Glucocorticoid‐mediated APA was also cell‐type‐specific, suggesting an action of glucocorticoids that may be unique to immune regulation. We also observed evidence for genotype‐dependent glucocorticoid‐mediated APA (referred to as pharmacogenomic‐alterative polyadenylation quantitative trait loci), providing potential functional mechanisms for a series of common genetic variants that had previously been associated with immune disorders, but without a clear mechanism. In summary, this study reports a series of observations regarding the impact of glucocorticoids on APA, raising the possibility that this mechanism might have implications for both disease pathophysiology and drug therapy.

Abstract The human genome encodes large numbers of non-coding RNAs, including divergent antisense transcripts at transcription start sites (TSSs). However, molecular mechanisms by which divergent antisense transcription is regulated have not been detailed. Here, we report a novel ZWC complex composed of ZC3H4, WDR82 and CK2 that suppresses divergent antisense transcription. The ZWC complex preferentially localizes at TSSs of active genes through direct interactions of ZC3H4 and WDR82 subunits with the S5p RNAPII C-terminal domain. ZC3H4 depletion leads to increased divergent antisense transcription, especially at genes that naturally produce divergent antisense transcripts. We further demonstrate that the ZWC complex phosphorylates the previously uncharacterized N-terminal acidic domain of SPT5, a subunit of the transcription-elongation factor DSIF, and that this phosphorylation is responsible for suppressing divergent antisense transcription. Our study provides evidence that the newly identified ZWC-DSIF axis regulates the direction of transcription during the transition from early to productive elongation.

Time-restricted feeding improves glucose homeostasis through epigenetic control of pancreatic β cell function.