Cancer can recur when a subset of tumor cells, termed persister cells, survive therapy and re-enter the cell cycle. Through single-nucleus multi-omic profiling (single-nucleus RNA sequencing [snRNA-seq] and single-nucleus assay for transposase-accessible chromatin by sequencing [snATAC-seq]) of (1) non-malignant fallopian tubes and (2) treatment-naive and (3) neoadjuvant-chemotherapy-treated samples from patients with high-grade serous ovarian carcinoma (HGSOC), we identify a persister cell signature defining the chemotherapy-tolerant state. The chromatin features of the signature are detectable in residual tumors after treatment and in treatment-naive tumors from patients who later develop resistance. Further, the signature independently predicts chemotherapy response in metastatic HGSOC and patient-derived xenograft models. Cells enriched in the persister state display a subset of genes primed for expression before treatment, an altered cell cycle, and stress-response programs associated with poor clinical outcomes. These findings suggest that an intrinsic regulatory program primes tumor cells toward chemotherapy tolerance and reveal new vulnerabilities that can be targeted with chromatin-modifying agents to prevent cancer recurrence.

Senescent cells promote tissue dysfunction in part through the senescence-associated secretory phenotype (SASP)1. Cytosolic mitochondrial nucleic acids activate innate immune signalling to initiate this inflammatory programme2,3. Here we show that mitochondrial metabolism provides a second layer of control that enables execution of the inflammatory programme. In senescent cells, the mitochondrial pyruvate-citrate-acetyl-CoA axis is upregulated, increasing the availability of acetyl-CoA to support histone acetylation at SASP genes. Whereas mitochondrial DNA-driven signalling activates inflammatory transcription factors, acetyl-CoA availability is required for robust transcription of SASP genes. Accordingly, enhancing acetyl-CoA levels promotes SASP gene expression, whereas inhibition of SLC25A1, the mitochondrial citrate exporter, reduces histone acetylation at SASP loci, limiting activity of this programme. In vivo, inhibition of SLC25A1 reduces chromatin accessibility at SASP loci, dampens inflammation and improves healthspan in aged mice. Together, these findings identify a mitochondrial metabolic checkpoint that enables the epigenetic execution of innate immune signalling, revealing a mechanism that selectively controls the inflammatory output of senescent cells.

Background: DNMT3A and TET2 are epigenetic regulator genes commonly mutated in age-related clonal hematopoiesis (CH). Despite having opposed epigenetic functions, these mutations are associated with increased all-cause mortality and a low risk for hematological neoplasms. In this study, we make use of a natural inflammatory response occurring during COVID-19 to define the impact of these mutations on inflammation and outcomes. Methods: A cohort of 243 community-based patients (Olmsted County, MN) with COVID-19 were identified. CH status was determined on PBMC using error corrected sequencing (lower detection limit 0.5% VAF). In a subset of patients, we use multi-omics interrogation of the genome, transcriptome, and epigenome at single cell (sc) resolution along with DNA methylation, in PBMC, from patients with COVID-19 and CH. Results: We detected 97 CH mutations in 72 (29.6%) of 243 patients with COVID-19. The most frequent being DNMT3A (n=30, 30%) and TET2 (n=26, 28%). The presence of CH negatively impacted overall survival when restricted to TET2 and DNMT3A mutations. However, only DNMT3Amt CH retained age and comorbidity independent prognostication (HR 2.84, p = 0.022). We then assessed for changes in DNA methylation using Illumina MethylationEPIC array on patients with COVID-19 and DNMT3A (n=3) or TET2mt (n=4) CH. Site specific DNA methylation changes revealed significant loss of methylation in DNMT3Amt CH in comparison to TET2mt patients, with 10,944 hypomethylated and 1,160 hypermethylated sites. We found that actively transcribed states (Tx, TxWk) were more commonly hypomethylated in DNMT3Amt CH while enhancers (Enh) and promoters (TssA, TssAFlnk) were more commonly hypomethylated in TET2mt CH. We then conducted scDNA-seq in DNMT3Amt (n=3) and TET2mt (n=1) CH in COVID-19 and observed that while in TET2mt CH, mutations were largely restricted to classical and intermediate monocytes, in DNMT3Amt CH, mutations were seen in myeloid and lymphoid lineage cells. We performed scRNA-seq on patients with COVID-19 and DNMT3A (n=3) or TET2mt (n=6) CH. Differential gene expression analysis identified 1,569 upregulated genes in DNMT3Amt patients and 205 downregulated. Pathway analysis demonstrated an upregulation of genes involved in T cell survival, function, and inflammation in DNMT3Amt patients compared to TET2mt patients with a significant overexpression of GIMAP1, GIMAP4, IRF2, IL32 in multiple cell types. We correlated transcriptomic changes with chromatin accessibility using the 10X Genomics Multiome platform to profile both gene expression and open chromatin from the same samples. Analysis of global distribution of cut sites and differentially accessible peaks showed increased chromatin accessibility in DNMT3Amt CH, especially CD4+ T lymphocytes and NK cells. Conclusion: We demonstrate an age, sex, and comorbidity-independent adverse impact on mortality associated with DNMT3Amt CH in the context of COVID-19 and highlight transcriptomic and epigenetic differences between DNMT3A and TET2mt CH, with single cell resolution. Citation Format: Jenna A Fernandez, Wazim Mohammed Ismail, Moritz Binder, Terra Lasho, Susan M. Geyer, Amelia Mazzone, Christy M. Finke, Abhishek Mangaonkar, Jeong-Heon Lee, Liguo Wang, Vernadette A. Simon, Fariborz Rakhshan Rohakthar, Amik Munankarmy, Jonathan J. Harrington, Melissa R. Snyder, Keith D. Robertson, Alexandre Gaspar-Maia, Mrinal Patnaik. Divergent impacts of DNMT3A and TET2 mutant clonal hematopoiesis on COVID-19 related outcomes [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: DNA Methylation, Clonal Hematopoiesis, and Cancer; 2025 Feb 1-4; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2025;85(3 Suppl):Abstract nr A012.

Introduction: ASXL1-mutant (mt) Chronic Myelomonocytic Leukemia (CMML) is an aggressive myeloid neoplasm characterized by increased proliferation, resistance to epigenetic therapies, and poor survival. Epigenetic remodeling and up-regulation of leukemogenic driver genes are hallmarks of ASXL1mt CMML. Truncating mutations in ASXL1 activate enhancers that drive the expression of key leukemogenic driver genes such as HOXA9 and its co-factor MEIS1. Here, we investigate the chromatin accessibility of these leukemogenic enhancers by ASXL1 genotype at single-cell resolution (GoT-ChA-seq). Methods: After IRB approval, we performed bulk RNA-seq and ChIP-seq (H3K4me1, H3K4me3, H3K27ac, H3K27me3, H2AK119ub, H2BK20ac) on bone marrow (BM) mononuclear cells from 40 patients with CMML (19 ASXL1mt, 21 -wildtype/wt). After quality control, differential analyses of gene expression and histone modification occupancy between ASXL1mt and wt patients were conducted. A consensus peak calling framework was used to define genomic regions with ASXL1mt-specific histone modifications. Canonical enhancers were defined as genomic regions with a co-occupancy of H3K4me1 and H3K27ac. We then added regions marked by H2BK20ac to capture regulatory regions associated with p300 (Narita et al. Nat Genet 2023). We used an in silico prediction strategy to associate regulatory regions marked by strong histone modification occupancy with highly up-regulated leukemogenic driver genes (HOXA6-10, MEIS1). The ReMap database (Hammal et al. Nucleic Acids Res 2022) was queried for transcription factor binding in the genomic regions of interest. We then performed GoT-ChA-seq (Izzo et al. Nature 2024) to investigate chromatin accessibility for ASXL1mt (n=3) and wildtype BM single cells from CMML patients. In vitro colony forming assays were conducted on primary CMML BM cells (11- ASXL1mt and 9 wt CMML), assessing the efficacy of EP31670, a novel, oral, dual inhibitor of p300/BRD4. Results: This study included 40 patients with CMML, 19 ASXL1mt and 21 wt. The variant allele frequencies for ASXL1 were compatible with heterozygosity (median 0.41) and TET2 mutations were balanced between the two groups. We identified 149 candidate regulatory regions correlating with the expression of up-regulated target genes of interest (HOXA6-10, MEIS1) by bulk ChIP- and RNA-seq. Publicly available ChIP-seq data from myeloid cell lines demonstrated enrichment of p300 (in THP-1 cells, enrichment effect size +2.58, p=0.002) and BRD4 (in K-562 cells, enrichment effect size +1.58, p=0.045) in these candidate regulatory regions. Next, we interrogated the chromatin accessibility of ASXL1mt (n=13620) and ASXL1wt (n=24269) single cells from 3 CMML patients using GoT-ChA-seq. Differential accessibility analysis revealed 144 genomic regions, all of which were preferentially accessible in ASXL1mt cells compared to ASXL1wt cells (p<0.05 for all regions). Four of these preferentially accessible genomic regions (p<0.001 for all regions) overlapped with the previously identified candidate regulatory regions for MEIS1. The H3K27ac and H2BK20ac occupancy levels in these regions was strongly associated with the expression of MEIS1 (median Pearson r=0.73, p=1.16x10-7). These candidate regulatory regions were annotated in ENCODE as distal and proximal enhancers. Targeting this biology with the dual p300/BRD4 inhibitor EP31670 in progenitor colony forming assays led to a preferential therapeutic effect in ASXL1mt BM samples (median IC50 = 25nM) compared to in ASXL1wt samples (median IC50 = 74nM, p=0.03). Treatment with EP31670 was also associated with increased differentiation (increased blast forming units-erythroid; BFU-E) and organization of colonies in ASXL1mt vs wt CMML. Conclusions: ASXL1mt CMML is characterized by the up-regulation of several leukemogenic driver genes including HOXA9 and its co-factor MEIS1, secondary to the activation of genotype-specific enhancers. The preferential accessibility of these enhancers can be defined between ASXL1mt and ASXL1wt cells within patients. The enhancers of interest interact with p300 and BRD4 in myeloid cell lines and targeting them with the novel dual p300/BRD4 inhibitor, EP31670, induces therapeutic responses at low nanomolar drug concentrations in vitro, preferentially in ASXL1-mutant cells. These data solidify the biological rationale for targeting p300/BRD4 in ASXL1mt chronic myeloid neoplasms for therapeutic benefit (NCT05488548).

Cancer can recur when a subset of tumor cells, termed persister cells, survive therapy and re-enter the cell cycle. The cellular lineages that give rise to persister cells and the mechanisms that confer the persister state remain poorly understood. Through single-cell multiomic profiling (snRNA-seq and snATAC-seq) on a cohort of (1) non-malignant fallopian tube, (2) treatment-naïve, and (3) neoadjuvant chemotherapy (NACT)-treated high-grade serous ovarian cancer (HGSOC) patient samples, we identified an epigenetic signature that defines the chemotherapy-tolerant persister state. The changes in chromatin accessibility characterizing the signature were identified in residual NACT tumors but are also present in treatment-naive samples from patients who later developed resistance. Furthermore, this epigenetic signature independently predicted chemotherapy response in patient-derived xenograft models of HGSOC and in a separate patient cohort. Cells enriched in the persister state arose from multiple lineages and displayed activation of oncogenic pathways, including altered stress responses, epithelial to mesenchymal transition, and changes to the cell cycle promoting quiescence. Finally, we identified a subset of genes that are epigenetically primed for expression before treatment and are upregulated after treatment. These findings suggest that an intrinsic epigenetic program primes tumor cells towards chemotherapy tolerance and reveal new vulnerabilities that could be exploited to delay or prevent cancer recurrence. Mihai G. Dumbrava, Wazim M. Ismail, Leticia Sandoval, Amelia Mazzone, Syed Mohammed Musheer Aalam, Megan L. Ritting, Xiaonan Hou, Yiwen Xie, Shariska Harrington, Scott H. Kaufmann, Nagarajan Kannan, S. John Weroha, Alexandre Gaspar-Maia. Single cell multiomic analysis of high-grade serous ovarian carcinoma reveals an intrinsic epigenetic program that primes chemotherapy tolerance in persister cells [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Ovarian Cancer Research; 2025 Sep 19-21; Denver, CO. Philadelphia (PA): AACR; Cancer Res 2025;85(18_Suppl):Abstract nr PR008.

Abstract: DNMT3A and TET2 are epigenetic regulator genes commonly mutated in age-related clonal hematopoiesis (CH). Despite having opposed epigenetic functions, these mutations are associated with increased all-cause mortality and a low risk for progression to hematologic neoplasms. Although individual impacts on the epigenome have been described using different model systems, the phenotypic complexity in humans remains to be elucidated. Here, we make use of a natural inflammatory response occurring during coronavirus disease 2019 (COVID-19), to understand the association of these mutations with inflammatory morbidity (acute respiratory distress syndrome [ARDS]) and mortality. We demonstrate the age-independent, negative impact of DNMT3A mutant (DNMT3Amt) CH on COVID-19–related ARDS and mortality. Using single-cell proteogenomics we show that DNMT3A mutations involve myeloid and lymphoid lineage cells. Using single-cell multiomics sequencing, we identify cell-specific gene expression changes associated with DNMT3A mutations, along with significant epigenomic deregulation affecting enhancer accessibility, resulting in overexpression of interleukin-32 (IL-32), a proinflammatory cytokine that can result in inflammasome activation in monocytes and macrophages. Finally, we show with single-cell resolution that the loss of function of DNMT3A is directly associated with increased chromatin accessibility in mutant cells. Hence, we demonstrate the negative prognostic impact of DNMT3Amt CH on COVID-19–related ARDS and mortality. DNMT3Amt CH in the context of COVID-19, was associated with inflammatory transcriptional priming, resulting in overexpression of IL32. This overexpression was secondary to increased chromatic accessibility, specific to DNMT3Amt CH cells. DNMT3Amt CH can thus serve as a potential biomarker for adverse outcomes in COVID-19.

Cancer can recur when a subset of tumor cells, denoted here as persister cells, are able to survive therapy and re-enter the cell cycle. The precise mechanisms that confer the persister state and whether it is characteristic of a subgroup of cells or arises from multiple cellular lineages remain poorly understood. We hypothesize that an epigenetic signature underlies the drug-tolerant persister state, characterized by transcriptional and chromatin accessibility changes that promote survival of residual cancer following chemotherapy. To identify clinically relevant features of persister cells in untreated tumors and residual disease, we performed single-cell multiomic profiling (snRNA+snATAC) on a cohort of non-malignant fallopian tube, treatment-naïve, and neoadjuvant chemotherapy (NACT)-treated high-grade serous ovarian cancer (HGSOC) samples. We identified differences in gene expression and open chromatin between naïve and residual patient tumors following chemotherapy. Although only a small proportion of the differentially expressed genes enriched in residual HGSOC overlapped with established gene sets for chemo-response and patient prognosis, the epigenomic analysis revealed activity of several DNA-binding factors that are both enriched upon chemotherapy and also high in resistant tumors prior to treatment. From this analysis, we identified an epigenetic signature that precedes expression and defines the persister state. This epigenetic signature also correlated with chemotherapy sensitivity and resistance using patient-derived xenograft models of HGSOC. Gene regulatory networks driven by the persister signature are involved in the activation of oncogenic pathways, including changes to the cell cycle promoting quiescence and stress response. Further study of the persister cells identified by this epigenetic signature may increase understanding of the mechanisms underlying persister cell survival and reveal new vulnerabilities that could be exploited to delay or prevent cancer recurrence. ### Competing Interest Statement The authors have declared no competing interest. Mayo Clinic Ovarian Cancer SPORE grant, P50 CA136393 DOD Ovarian Cancer Research Program, Ovarian Cancer Academy, W81XWH2110475 Canadian Institutes of Health Research Doctoral Foreign Study Award, Foundation for Women’s Wellness, https://ror.org/009j61e89 Mayo Medical Scientist Training Program, T32 GM145408 Paul Calabresi Program in Clinical/Translational Research, K12CA090628

e17535 Background: High-grade serous ovarian carcinoma (HGSOC) is the most frequent and deadliest type of ovarian cancer (OC). With the recent advances in transcriptomic and epigenomic profiling of cancer at single cell resolution, it has become clear that intra-tumor heterogeneity driven by genetic and epigenetic factors may be the basis of drug resistance. Current treatments are not homogeneously effective against all the cancer cell subpopulations, thus enabling resistance. With single cell sequencing technology, characterization of cell signatures and regulatory mechanisms may translate to prognostic markers and novel drug targets. Methods: The transcriptomic and epigenomic landscape of HGSOC was mapped for five tumor samples from debulking surgeries and one tissue patient-derived xenograft (PDX) using the 10X genomics sequencing platform. This strategy permits the combination of single cell ATAC-seq and single cell RNA-seq within the same nucleus (Multiome). Isolation of nuclei, library preparation and sequencing were performed following 10X genomics protocols. Sample quality and library quality were assessed by nuclear morphology and fragment size analysis, respectively. Sequenced reads were mapped to the human genome and downstream analysis including copy number variant prediction, pathway enrichment, motif enrichment and gene regulatory network analysis were performed. The sampled patients continue to be followed for clinical outcomes, such as response to therapy. Clinical data including patient age, grade, and stage of cancer, debulking status, CA-125 levels, and neoadjuvant status, were used in conjunction with the genomics data to characterize patient specific molecular regulatory signatures. Results: Cells from the six samples (total N= 26,421) were projected via UMAP. Immune and stromal cells were shown to cluster by cell-type while cancer cells clustered by patient. Cancer cells were identified as cycling cells, ciliated cells, cells with enrichment of the JAK-STAT signaling pathway, and cells exhibiting cancer stemness signatures. Transcription factor motifs and binding-site enrichment in open chromatin regions reveal transcription regulation-based subpopulations. Using the combined gene expression and open chromatin information from these cells, there is potential to uncover the genetic regulatory network(s) that drives treatment resistance. Conclusions: With single cell technology, specific clusters of cancer and tumor microenvironment cells can be classified. Beyond characterizing patient specific signatures, multiome enables the discovery of genomic, transcriptomic and epigenomic signatures that provide insight in tumor progression and treatment resistance.

Voltage-gated ion channels (VGICs) orchestrate electrical activities that drive mechanical functions in contractile tissues such as the heart and gut. In turn, contractions change membrane tension and impact ion channels. VGICs are mechanosensitive, but the mechanisms of mechanosensitivity remain poorly understood. Here, we leverage the relative simplicity of NaChBac, a prokaryotic voltage-gated sodium channel from Bacillus halodurans, to investigate mechanosensitivity. In whole-cell experiments on heterologously transfected HEK293 cells, shear stress reversibly altered the kinetic properties of NaChBac and increased its maximum current, comparably to the mechanosensitive eukaryotic sodium channel NaV1.5. In single-channel experiments, patch suction reversibly increased the open probability of a NaChBac mutant with inactivation removed. A simple kinetic mechanism featuring a mechanosensitive pore opening transition explained the overall response to force, whereas an alternative model with mechanosensitive voltage sensor activation diverged from the data. Structural analysis of NaChBac identified a large displacement of the hinged intracellular gate, and mutagenesis near the hinge diminished NaChBac mechanosensitivity, further supporting the proposed mechanism. Our results suggest that NaChBac is overall mechanosensitive due to the mechanosensitivity of a voltage-insensitive gating step associated with the pore opening. This mechanism may apply to eukaryotic VGICs, including NaV1.5.

Ovarian cancer is one of the most common gynecologic malignancies, with high-grade serous carcinoma (HGSC) as the most frequent and deadliest type of ovarian cancer (OC). With the recent advances in cancer epigenomic profiling, it has become clear that intra-tumor heterogeneity driven by genetic and epigenetic factors may be the basis of resistance, where current treatments could efficiently target sub-populations of cells while a sub-group may be resistant, or prone to develop resistance. With epigenomic profiling and single cell sequencing technology, we aim to characterize cell signatures that translate as prognostic markers and novel drug targets. We have mapped the epigenomic landscape of HGSC in 12 patient-derived xenografts (PDX), and their metastatic counterparts (circulating tumor cells and ascites present in the PDX models) using an assay for transposase-accessible chromatin (or ATAC-seq) and compared it with normal fallopian tube organoids to identify tumor and metastatic-specific regulatory elements. We also performed RNA-seq to validate potential transcriptional dependencies associated with resistance to chemotherapy. In order to identify potentially chemotherapy-resistant subpopulations and further validate the activity of some of the transcription factors that can be driving resistance, we performed single cell multiomic profiling using the 10x Multiome sequencing platform (scRNA+scATAC from the same nuclei) in HGSC patient samples and PDX. Finally, we performed immunohistochemistry of patient tissue slides for target markers and correlated it with overall survival in OC patients. As previously suggested, a subset of cancer clonal cells can drive treatment resistance and metastasis and result in poor patient outcomes. Data derived from PDX ATAC-seq showed a cancer stem cell signature driven by SOX2 in metastatic samples compared to the paired PDX primary tumor, suggesting a role for SOX2 in disease progression. Interestingly, SOX2 expression in epithelial cells in the Multiome analysis is more patient-specific than other stem cell markers, like CD133. We also observed that SOX2 expression negatively correlated with prognosis. These data, taken together, point out the promising role of SOX2 as a prognostic and targetable marker for HGSC. Citation Format: Leticia Assad Maia Sandoval, Wazim Mohammed Ismail, Amelia Mazzone, Amik Munankarmy, Jagneet Kaur, Tsering Tashi, Xiaonan Hou, Nagarajan Kannan, Ann Oberg, Scott H. Kaufmann, Saravut John Weroha, Alexandre Gaspar-Maia. Single cell multiomic profiling of high-grade serous carcinoma. [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 4724.

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.

Considerable efforts have been made to characterize active enhancer elements, which can be annotated by accessible chromatin and H3 lysine 27 acetylation (H3K27ac). However, apart from poised enhancers that are observed in early stages of development and putative silencers, the functional significance of cis -regulatory elements lacking H3K27ac is poorly understood. Here we show that macroH2A histone variants mark a subset of enhancers in normal and cancer cells, which we coined ‘macro-Bound Enhancers’, that modulate enhancer activity. We find macroH2A variants localized at enhancer elements that are devoid of H3K27ac in a cell type-specific manner, indicating a role for macroH2A at inactive enhancers to maintain cell identity. In following, reactivation of macro-bound enhancers is associated with oncogenic programs in breast cancer and their repressive role is correlated with the activity of macroH2A2 as a negative regulator of BRD4 chromatin occupancy. Finally, through single cell epigenomic profiling of normal mammary stem cells derived from mice, we show that macroH2A deficiency facilitates increased activity of transcription factors associated with stem cell activity.

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.

Myeloid neoplasms are clonal hematopoietic stem cell disorders driven by the sequential acquisition of recurrent genetic lesions. Truncating mutations in the chromatin remodeler ASXL1 (ASXL1 MT ) are associated with a high-risk disease phenotype with increased proliferation, epigenetic therapeutic resistance, and poor survival outcomes. We performed a multi-omics interrogation to define gene expression and chromatin remodeling associated with ASXL1 MT in chronic myelomonocytic leukemia (CMML). ASXL1 MT are associated with a loss of repressive histone methylation and increase in permissive histone methylation and acetylation in promoter regions. ASXL1 MT are further associated with de novo accessibility of distal enhancers binding ETS transcription factors, targeting important leukemogenic driver genes. Chromatin remodeling of promoters and enhancers is strongly associated with gene expression and heterogenous among overexpressed genes. These results provide a comprehensive map of the transcriptome and chromatin landscape of ASXL1 MT CMML, forming an important framework for the development of novel therapeutic strategies targeting oncogenic cis interactions.

Background: With the advent of triplet and quadruplet induction therapies, response rates and survival outcomes have improved significantly in multiple myeloma. However, a minority of patients does not achieve deep responses to these anti-myeloma therapies and survival outcomes for these patients remain dismal. The molecular mechanisms of primary therapeutic resistance remain poorly understood and novel therapeutic approaches are needed for these high-risk patients. Methods: We generated single-cell gene expression and chromatin accessibility profiles at the time of diagnosis from 16 patients with multiple myeloma using the 10X Genomics Multiome platform. Viably frozen CD138+ bone marrow plasma cells were thawed and washed before removing dead cells and isolating nuclei according to the manufacturer's protocols. RNA- and ATAC-seq libraries were constructed per the manufacturer's user guides. GEX and ATAC libraries were sequenced separately on an Illumina HiSeq 4000 instrument before demultiplexing, alignment to the reference genome, and post-alignment quality control. The Cell Ranger pipelines with default parameters were used to trim reads, align, count unique molecular identifiers, and call cells, transcripts, and peaks. Downstream processing was carried out using Seurat and Signac. Doublets were identified by DoubletFinder and batch effects were corrected using Harmony. After quality control for feature counts and mitochondrial gene expression, there were a total of 16188 single cells: 6139 cells from 6 patients resistant to quadruplet induction (Dara-Ixa-Rd), 4841 cells from 4 patients resistant to triplet induction (VRd), and 5208 cells from 6 patients responding to quadruplet / triplet induction. Primary resistance was defined as not achieving at least a very good partial response (VGPR+ after a minimum of 4 cycles of therapy). Expression of the up-regulated resistance genes in single cells was measured by calculating a module score, with higher numbers indicating higher expression (Tirosh et al. Science 2016;352(6282):189-196). We used a score cut-off of 0.200 to define increased expression of resistance genes. Results: Half of the patients were female, the median age at diagnosis was 61 years (42-80), and half of them had high-risk cytogenetics. There was differential gene expression between cells from quadruplet-resistant and -responsive patients (1272 genes, FDR<0.05). Likewise, there was differential gene expression triplet-resistant and -responsive patients (1354 genes, FDR<0.05). There were 212 genes up- and 222 down-regulated in both comparisons (shared resistance genes), an overlap unlikely to have arisen by chance (p<2.22x10-16). Functional annotation of these shared differentially expressed genes revealed up-regulation of GTPase (FDR=4.01x10-4), kinase (FDR=4.39x10-3), and transferase (FDR=1.65x10-3) activity in cells from resistant patients. Conversely, there was down-regulation of genes involved in ubiquitin (FDR=6.63x10-13) and ubiquitin-like protein ligase binding (FDR=2.50x10-12) as well as unfolded protein binding (FDR=1.21x10-10). Expectedly, the mean expression of the shared up-regulated resistance genes was higher among quadruplet- (score=0.261) and triplet-resistant patients (score=0.212) compared to responding patients (score=0.096, Figure 1A). This difference in gene expression was driven by larger subpopulations of cells with increased expression of resistance genes: Quadruplet-resistant (39.2% of cells) and triplet-resistant (37.5% of cells), compared to patients responding to induction therapy (17.2% of cells, Figure 1B). The subpopulations of cells with increased expression of resistance genes were characterized by increased accessibility in Spi-B (FDR=2.55x10-2) and RAR-γ (FDR=1.86x10-3) binding sites. The shared up-regulated genes included known drug resistance genes such as IKZF1 and viable therapeutic targets including CCND3, RASA1, AKT3, CDK8, and ATM. Conclusions: Single-cell interrogation of the transcriptome and epigenome of patients with primary resistant multiple myeloma suggests an overexpression of actionable drug targets and increased cell proliferation in cell subpopulations. There is biological rationale for the investigation of novel individualized therapeutic approaches like IKZF1 degradation and targeted kinase inhibition in this patient population. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Interstitial cells of Cajal (ICCs) generate electrical slow waves, which are required for normal gastrointestinal motility. The mechanisms for generation of normal pacemaking are not fully understood. Normal gastrointestinal contractility- and electrical slow-wave activity depend on the presence of extracellular HCO3-. Previous transcriptional analysis identified enrichment of mRNA encoding the electrogenic Na+/HCO3- cotransporter (NBCe1) gene (Slc4a4) in pacemaker myenteric ICCs in mouse small intestine. We aimed to determine the distribution of NBCe1 protein in ICCs of the mouse gastrointestinal tract and to identify the transcripts of the Slc4a4 gene in mouse and human small intestinal tunica muscularis. We determined the distribution of NBCe1 immunoreactivity (NBCe1-IR) by immunofluorescent labeling in mouse and human tissues. In mice, NBCe1-IR was restricted to Kit-positive myenteric ICCs of the stomach and small intestine and submuscular ICCs of the large intestine, that is, the slow wave generating subset of ICCs. Other subtypes of ICCs were NBCe1-negative. Quantitative real-time PCR identified >500-fold enrichment of Slc4a4-207 and Slc4a4-208 transcripts ["IP3-receptor-binding protein released by IP3" (IRBIT)-regulated isoforms] in Kit-expressing cells isolated from KitcreERT2/+, Rpl22tm1.1Psam/Sj mice and from single GFP-positive ICCs from Kittm1Rosay mice. Human jejunal tunica muscularis ICCs were also NBCe1-positive, and SLC4A4-201 and SLC4A4-204 RNAs were >300-fold enriched relative to SLC4A4-202. In summary, NBCe1 protein expressed in ICCs with electrical pacemaker function is encoded by Slc4a4 gene transcripts that generate IRBIT-regulated isoforms of NBCe1. In conclusion, Na+/HCO3- cotransport through NBCe1 contributes to the generation of pacemaker activity in subsets of ICCs.NEW & NOTEWORTHY In this study, we show that the electrogenic Na+/HCO3- cotransporter, NBCe1/Slc4a4, is expressed in subtypes of interstitial cells of Cajal (ICCs) responsible for electrical slow wave generation throughout the mouse gastrointestinal tract and is absent in other types of ICCs. The transcripts of Slc4a4 expressed in mouse ICCs and human gastrointestinal smooth muscle are the regulated isoforms. This indicates a key role for HCO3- transport in generation of gastrointestinal motility patterns.

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

The Medical Genome Facility Genome Analysis Core has successfully performed single-cell transcriptome experiments for more than 6 years. Using the Fluidigm C1 and 10x Genomics Chromium platforms, we have captured and labeled single cells from over 470 samples. We continue to expand the number of procedures we perform for Mayo Clinic investigators, and this year we are making available the 10x Genomics ATAC-Seq (Assay for Transposase-Accessible Chromatin using sequencing) protocol. In collaboration with the Gaspar Maia lab in the Mayo Clinic College of Medicine, we have run pilot experiments charting regulatory elements in an ovarian cancer PDX model. Here we discuss our methods and present some promising preliminary results.

Objective This study was designed to evaluate the roles of microRNAs (miRNAs) in slow transit constipation (STC). Design All human tissue samples were from the muscularis externa of the colon. Expression of 372 miRNAs was examined in a discovery cohort of four patients with STC versus three age/sex-matched controls by a quantitative PCR array. Upregulated miRNAs were examined by quantitative reverse transcription qPCR (RT-qPCR) in a validation cohort of seven patients with STC and age/sex-matched controls. The effect of a highly differentially expressed miRNA on a custom human smooth muscle cell line was examined in vitro by RT-qPCR, electrophysiology, traction force microscopy, and ex vivo by lentiviral transduction in rat muscularis externa organotypic cultures. Results The expression of 13 miRNAs was increased in STC samples. Of those miRNAs, four were predicted to target SCN5A, the gene that encodes the Na+ channel NaV1.5. The expression of SCN5A mRNA was decreased in STC samples. Let-7f significantly decreased Na+ current density in vitro in human smooth muscle cells. In rat muscularis externa organotypic cultures, overexpression of let-7f resulted in reduced frequency and amplitude of contraction. Conclusions A small group of miRNAs is upregulated in STC, and many of these miRNAs target the SCN5A-encoded Na+ channel NaV1.5. Within this set, a novel NaV1.5 regulator, let-7f, resulted in decreased NaV1.5 expression, current density and reduced motility of GI smooth muscle. These results suggest NaV1.5 and miRNAs as novel diagnostic and potential therapeutic targets in STC.