Kim, Minsuk

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.

Abstract Background DNMT3A and TET2 are epigenetic regulator genes commonly mutated in age-related clonal hematopoiesis (CH). Despite having opposing epigenetic functions, these mutations are associated with increased all-cause mortality and a low risk for progression to hematological neoplasms. While individual impacts on the epigenome have been described using different model systems, the phenotypic complexity in humans remains to be elucidated. Results 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 and mortality. We demonstrate the age-independent, negative impact of DNMT3A mutant CH on COVID-19-related cytokine release severity and mortality. Using single cell proteogenomics we show that DNMT3A mutations involve myeloid and lymphoid 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 IL32, 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. Conclusions We demonstrate the negative prognostic impact of DNMT3Amt CH on COVID-19 related inflammatory morbidity and mortality. DNMT3Amt CH involves myeloid and lymphoid cells and 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 serve as a potential biomarker for adverse inflammatory outcomes.

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 Background: SARS-Cov-2 infections are associated with increased mortality and morbidity, largely due to inflammatory cascades and cytokine release syndrome (CRS). Clonal hematopoiesis of indeterminate potential (CHIP) is defined by the presence and subsequent expansion of somatic, leukemia-associated driver mutations in apparently healthy individuals with normal blood counts. CHIP has been associated with increased inflammation, with cytokines such as IL1-b, IL6 and TNF-a being elevated at baseline in affected individuals. We hypothesized that the presence of CHIP in patients with COVID-19 would result in excessive inflammation-related mortality and morbidity. Methods: We used the Mayo Clinic COVID-19 database to identify patients with COVID-19 on whom peripheral blood mononuclear cells (PBMC) were available for research use (IRB approved). We carried out target-capture next generation sequencing for 220 CH related genes, by previously described methods (1000 x coverage, variant allele fraction/VAF detection limit >0.5%; Kusne Y et al AJH 2021). CHIP was defined by the presence of a CH mutation with a VAF>1% in an individual with normal baseline blood counts. Demographics, blood counts, and inflammatory markers (CRP and cytokine levels- ELISA assay) at COVID-19 diagnosis and during follow-up (as clinically indicated) were collected. COVID-19 disease severity was classified based on the presence and severity of CRS, graded using the Penn Grading Scale (Porter et. al., 2018), and the WHO ordinal scale (WHO Blueprint, 2020). We used Fisher's exact test and the Wilcoxon rank sum test to compare categorical and continuous variables. Survival analysis was performed using the Kaplan-Meier method. We accounted for differences in age and sex using multivariable-adjusted proportional hazards regression models. Results: Seventy-two CHIP mutations were detected in 56 (25%) of the 227 patients with COVID-19 that had PBMC available; median age 69 years (range; 42-99 years), 61% male. Fifteen (26%) patients had 2 CHIP mutations, while 1 patient had 3 CHIP mutations. Common mutations encountered included DNMT3A (32%) , TET2 (19%) , SF3B1 (8%) , ASXL1 (6%) , MPL (5%) , and TP53 (5%; Figure 1A). COVID-19 patients with CHIP were older in age (median 69 vs 57 years; p<0.0001) and had higher baseline MCP-1 (p=0.04) levels. However, there were no differences in sex, comorbidities, blood counts, IL1-b, IL6 and TNF-a levels between the two groups. The median follow-up for the entire cohort was 9 months. The relative change from baseline in blood counts and inflammatory markers (CRP and cytokines) during follow-up was similar in CHIP and non-CHIP patients, with the exception that COVID-19-onset neutropenia was more common in CHIP patients (8% vs 1%; p=0.017) compared to those without CHIP. At last follow up neutropenia had resolved in all patients. Both groups had comparable number of patients with CRS (61% CHIP vs 53% non-CHIP patients, p=0.354, Figure 1B), however, CHIP patients had more severe CRS (median Penn Grade 3 versus 2 in non-CHIP, p=0.018, Figure 1C). Based on the WHO ordinal scale, CHIP patients were more likely to experience hospitalization with severe disease and death (61% versus 45% in non-CHIP, p = 0.049). Moreover, COVID-19 CHIP patients experienced worse overall survival in comparison to patients without CHIP (median 13.1 months vs not reached, p<0.001, Figure 1D). This association remained consistent after adjusting for age and sex at the time of COVID-19 diagnosis (HR 3.15, 95% 1.32 - 7.55, p = 0.010). At last follow-up, 22 deaths were documented: 13 (23%) in patients with CHIP and 9 (5%) in the non-CHIP group (p=0.02), with the primary cause for mortality being hypoxic respiratory failure (62% in CHIP vs 44% non-CHIP, p=0.04). Conclusions: In this study, we observe an age-independent impact of CHIP on COVID-19 associated inflammatory morbidity (CRS) and mortality (hypoxemic respiratory failure). We are currently carrying out detailed single cell (ssDNA, RNA and ATAC-seq) and proteomic studies (O-link PEA assays) to better elucidate this pathophysiology. Figure 1 Figure 1. Disclosures Patnaik: Kura Oncology: Research Funding; StemLine: Research Funding.

Colorectal adenomas are precursors of CRC. Recently, the gut microbiota, i.e., the collection of microbes residing in our gut, has been recognized as a key player in CRC development. There have been a number of gut microbiota profiling studies for colorectal adenoma and CRC; however, fewer studies have considered the gut metabolome, which serves as the chemical interface between the host and gut microbiota. Here, we conducted a gut metabolome profiling study of colorectal adenoma and CRC and analyzed the metabolomic profiles together with paired microbiota composition profiles. We found several chemical signatures of colorectal adenoma that were associated with some gut microbes and potentially indicative of future CRC. This study highlights potential early-driver metabolites in CRC pathogenesis and guides further targeted experiments and thus provides an important stepping stone toward developing better CRC prevention strategies.

Primary sclerosing cholangitis (PSC) is a pathogenically complex, chronic, fibroinflammatory disorder of the bile ducts without known etiology or effective pharmacotherapy. Emerging in vitro and in vivo evidence support fundamental pathophysiologic mechanisms in PSC centered on enterohepatic circulation. To date, no studies have specifically interrogated the chemical footprint of enterohepatic circulation in PSC. Herein, we evaluated the metabolome and lipidome of portal venous blood and bile obtained at the time of liver transplantation in patients with PSC (n = 7) as compared to individuals with noncholestatic, end-stage liver disease (viral, metabolic, etc. (disease control, DC, n = 19)) and to nondisease controls (NC, living donors, n = 12). Global metabolomic and lipidomic profiling was performed on serum derived from portal venous blood (portal serum) and bile using ultraperformance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) and differential mobility spectroscopy-mass spectroscopy (DMS-MS; complex lipid platform). The Mann–Whitney U test was used to identify metabolites that significantly differed between groups. Principal-component analysis (PCA) showed significant separation of both PSC and DC from NC for both portal serum and bile. Metabolite set enrichment analysis of portal serum and bile demonstrated that the liver-disease cohorts (PSC and DC) exhibited similar enrichment in several metabolite categories compared to NC. Interestingly, the bile in PSC was uniquely enriched for dipeptide and polyamine metabolites. Finally, analysis of patient-matched portal serum and biliary metabolome revealed that these biological fluids were more homogeneous in PSC than in DC or NC, suggesting aberrant bile formation and enterohepatic circulation. In summary, PSC and DC patients exhibited alterations in several metabolites in portal serum and bile, while PSC patients exhibited a unique bile metabolome. These specific alterations in PSC are amenable to hypothesis testing and, potentially, therapeutic pharmacologic manipulation.