Citations

Introduction: ASXL1-mutant Chronic Myelomonocytic Leukemia (CMML) is a high-risk myeloid neoplasm characterized by epigenetic remodeling and overexpression of leukemogenic driver genes. DLK1 (delta-like homolog 1) is a non-canonical Notch ligand, crucial for the proliferation of early hematopoietic progenitor cells and its expression is controlled by maternal imprinting. DLK1 expression is restricted to select progenitor populations, but was found to be overexpressed in several malignancies including ASXL1-mutant CMML. Here we investigate the molecular mechanisms that drive the aberrant re-expression of DLK1 and postulate potential targeted therapeutic interventions. Methods: We performed bulk RNA- and ChIP-seq (H3K4me1/3, H3K27ac/me3, H2AK119ub, H2BK20ac) on bone marrow mononuclear cells from 20 patients with CMML (11 ASXL1-mutant, 9 -wildtype). All libraries were sequenced on an Illumina NovaSeq instrument. Differential gene expression and histone modification occupancy between ASXL1-mutant and -wildtype patients were assessed. Single-cell RNA-seq was performed for a subset of patients to investigate the cell populations of interest for DLK1 expression. To assess DLK1 allelic expression, we identified a single nucleotide polymorphism (SNP) in the coding region of DLK1 (rs1802710) and used cases which were confirmed heterozygous by Sanger sequencing. DLK1 transcript levels were quantified by RT-PCR, and the relative abundance of alleles was measured using digital droplet PCR. We designed primers to amplify the region around our SNP of interest and utilized high mismatch discrimination locked nucleic acid (LNA) probes labeled with FAM (reference SNP allele) and HEX (alternate SNP allele) to quantify specific alleles. The number of RNA molecules originating from each allele was quantified by counting the number of droplets that fluoresced with the corresponding probe colors. Illumina 850K EPIC microarrays were used to evaluate DNA methylation. Drug studies were carried out in progenitor colony forming assays derived from ASXL1-mutant patients (bone marrow mononuclear cells) with the dual BRD4 and p300 / CBP inhibitor EP31670 (62 nM, 24-hour exposure), quantifying DLK1 expression by real-time RT-PCR. Results: This study included 20 patients with CMML (median age at diagnosis 74 years). All ASXL1 mutations were typical hotspot mutations (most commonly Gly646Trpfs*12) and the variant allele frequencies were compatible with heterozygosity (median 0.41). The spectrum of co-mutations was typical and included TET2 (23%), SRSF2 (16%), CBL (7%), among others. DLK1 was strongly overexpressed in ASXL1-mutant patients (log2 fold-change +4.0, FDR < 0.001). Allele-specific gene expression studies (n = 3) revealed the re-expression of the imprinted maternal DLK1 allele in ASXL1-mutant patients. Single-cell gene expression studies (n = 5) demonstrated that the DLK1 overexpression is restricted to the hematopoietic progenitor cell population. Differential methylation analysis between patients with ASXL1-mutant clonal hematopoiesis (n = 4) and matched controls (n = 8) identified two hypermethylated CpG islands in the DLK1 promoter region (cg18122767, Δß = +8.6%, p = 0.021 and cg15831296, Δß = +5.8%, p = 0.008). Histone modification analysis identified DLK1 as a p300 target gene by mapping H2BK20ac onto established ASXL1-mutant-specific enhancer regions (H3K4me1 + H3K27ac co-occupancy). The distal enhancer EH38D2760182 was predicted to bind p300 and to govern the expression of DLK1. ChIP-seq experiment in K-562 and Kasumi-1 cells showed the binding of BRD4 and p300 to EH38D2760182, respectively. Targeting BRD4 and p300 using EP31670 led to the downregulation of DLK1 expression (mean fold-change = 0.569, p < 0.001, n = 3) in progenitor colony forming assays. Conclusions: We observed the aberrant re-expression of the maternally imprinted DLK1 allele in patients with ASXL1-mutant CMML. Both DLK1 promoter hypermethylation and the genotype-specific activation of enhancers by p300 were identified as putative regulatory mechanisms. The distal enhancer EH38D2760182 was found to bind BRD4 and p300 and the pharmacologic disruption of BRD4 / p300 activity in model systems led to the downregulation of DLK1. The clinical-grade dual inhibitor of BRD and CBP / p300 EP31670 is currently being investigated in patients with ASXL1-mutant chronic myeloid neoplasms (NCT05488548).

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.

Background: About 25% of patients with acute myocardial infarction complicated by cardiogenic shock (AMI-CS) have chronic total occlusion (CTO) at non-culprit artery. However, the prognostic impact of CTO in these patients is still unclear. Aims: We aimed to investigate the impact of CTO at non-culprit artery on 1-year mortality in patients with AMI-CS using the multicenter registry of cardiogenic shock. Methods: A total of 1,247 patients presented with cardiogenic shock (CS) were enrolled in the SMART-RESCUE registry between January 2014 and December 2018. After exclusion of non-ischemic cause shock, ischemic cardiomyopathy, failed or no revascularization, vasospasm, and patients undergoing coronary artery bypass surgery, 695 patients underwent percutaneous coronary intervention (PCI). Among these, we analyzed 536 patients with multivessel disease (MVD) according to the presence of CTO at non-culprit artery. The study endpoint was a 1-year all-cause mortality. We also examined the incidence of 1-year cardiac death, and in-hospital death or renal replacement therapy (RRT). Results: A 141 patients (26.3%) of study population had CTO lesion at non-culprit artery. The CTO group had more patients with diabetes mellitus, and lesser patients with ST-elevation myocardial infarction. Although initial left ventricular ejection fraction was lower in CTO group (28.5 vs. 34.1%, P=0.005), initial systolic blood pressure (73.8 vs. 73.2 mmHg, P=0.822) and the level of lactate (6.6 vs. 6.4 mmol/L, P=0.804) were similar in both groups. Venoarterial extracorporeal membrane oxygenation was applied in 206 patients (38.4%), and its use rate was comparable in 2 groups (40.4 vs. 37.7%, P=0.571). Pre- (32.9 vs. 22.9, P <0.001) and post-PCI SYNTAX score (16.7 vs. 5.3, P <0.001) was significantly higher in CTO group. In CTO group, 53 patients (37.6%) had CTO at left main or left anterior descending artery, and only 19 patients (13.5%) in CTO group received PCI for CTO. In multivariate analysis, all-cause mortality at 1-year was significantly higher in CTO group (51.8 vs. 40.5%, hazard ratio [HR] 1.44, 95% confidence interval [CI] 1.09-1.90, P=0.011). The incidences of 1-year cardiac death (45.4 vs. 32.4%, HR 1.55, 95% CI 1.15-2.10, P=0.004) and in-hospital death or RRT (46.8 vs. 36.5%, P=0.031) were also higher in CTO group. Conclusions: Non-culprit CTO lesion was associated with higher 1-year mortality in patients with AMI-CS and multivessel disease.

Abstract Background Malignant peripheral nerve sheath tumors (MPNSTs) can arise from atypical neurofibromas (ANF). Loss of the polycomb repressor complex 2 (PRC2) is a common event. Previous studies on PRC2-regulated genes in MPNST used genetic add-back experiments in highly aneuploid MPNST cell lines which may miss PRC2-regulated genes in NF1-mutant ANF-like precursor cells. A set of PRC2-regulated genes in human Schwann cells (SCs) has not been defined. We hypothesized that PRC2 loss has direct and indirect effects on gene expression resulting in MPNST, so we sought to identify PRC2-regulated genes in immortalized human Schwann cells (iHSCs). Methods We engineered NF1-deficient iHSCs with loss of function SUZ12 or EED mutations. RNA sequencing revealed 1327 differentially expressed genes to define PRC2-regulated genes. To investigate MPNST pathogenesis, we compared genes in iHSCs to consistent gene expression differences between ANF and MPNSTs. Chromatin immunoprecipitation sequencing was used to further define targets. Methylome and proteomic analyses were performed to further identify enriched pathways. Results We identified potential PRC2-regulated drivers of MPNST progression. Pathway analysis indicates many upregulated cancer-related pathways. We found transcriptional evidence for activated Notch and Sonic Hedgehog (SHH) signaling in PRC2-deficient iHSCs. Functional studies confirm that Notch signaling is active in MPNST cell lines, patient-derived xenografts, and transient cell models of PRC2 deficiency. A combination of MEK and γ-secretase inhibition shows synergy in MPNST cell lines. Conclusions We identified PRC2-regulated genes and potential drivers of MPNSTs. Our findings support the Notch pathway as a druggable target in MPNSTs. Our identification of PRC2-regulated genes and pathways could result in more novel therapeutic approaches.

Abstract Background Smoldering multiple myeloma (SMM), an asymptomatic precursor of multiple myeloma (MM), carries a variable risk of progression to MM. There is little consensus on the efficacy or optimal timing of treatment in SMM. We systematically reviewed the landscape of all clinical trials in SMM. We compared the efficacy of treatment regimens studied in SMM to results from these regimens when used in newly diagnosed multiple myeloma (NDMM), to determine whether the data suggest deeper responses in SMM versus NDMM. Methods All prospective interventional clinical trials for SMM, including published studies, meeting abstracts, and unpublished trials listed on ClinicalTrials.gov up to April 1, 2023, were identified. Trial-related variables were captured, including treatment strategy and efficacy results. Relevant clinical endpoints were defined as overall survival (OS) and quality of life. Results Among 45 SMM trials identified, 38 (84.4%) assessed active myeloma drugs, while 7 (15.6%) studied bone-modifying agents alone. Of 18 randomized trials in SMM, only one (5.6%) had a primary endpoint of OS; the most common primary endpoint was progression-free survival (n = 7, 38.9%). Among 32 SMM trials with available results, 9 (28.1%) met their prespecified primary endpoint, of which 5 were single-arm studies. Six treatment regimens were tested in both SMM and NDMM; 5 regimens yielded a lower rate of very good partial response rate or better (≥VGPR) in SMM compared to the corresponding NDMM trial (32% vs 63%, 43% vs 53%, 40% vs 63%, 86% vs 89%, 92% vs 95%, and 94% vs 87%, respectively). Conclusion In this systematic review of all prospective interventional clinical trials in SMM, we found significant variability in trial design, including randomization status, primary endpoints, and types of intervention used. Despite the statistical limitations, comparison of treatment regimens revealed no compelling evidence that the treatment is more effective when introduced early in SMM compared to NDMM.

Abstract Aims The Shock Academic Research Consortium (SHARC) recently proposed pragmatic consensus definitions to standardize classification of cardiogenic shock (CS) in registries and clinical trials. We aimed to describe contemporary CS epidemiology using the SHARC definitions in a cardiac intensive care unit (CICU) population. Methods and results The Critical Care Cardiology Trials Network (CCCTN) is a multinational research network of advanced CICUs coordinated by the TIMI Study Group (Boston, MA). Cardiogenic shock was defined as a cardiac disorder resulting in SBP &lt; 90 mmHg for ≥30 min [or the need for vasopressors, inotropes, or mechanical circulatory support (MCS) to maintain SBP ≥ 90 mmHg] with evidence of hypoperfusion. Primary aetiologic categories included acute myocardial infarction-related CS (AMI-CS), heart failure-related CS (HF-CS), and non-myocardial (secondary) CS. Post-cardiotomy CS was not included. Heart failure-related CS was further subcategorized as de novo vs. acute-on-chronic HF-CS. Patients with both cardiogenic and non-cardiogenic components of shock were classified separately as mixed CS. Of 8974 patients meeting shock criteria (2017–23), 65% had isolated CS and 17% had mixed shock. Among patients with CS (n = 5869), 27% had AMI-CS (65% STEMI), 59% HF-CS (72% acute-on-chronic, 28% de novo), and 14% secondary CS. Patients with AMI-CS and de novo HF-CS were most likely to have had concomitant cardiac arrest (P &lt; 0.001). Patients with AMI-CS and mixed CS were most likely to present in more severe shock stages (SCAI D or E; P &lt; 0.001). Temporary MCS use was highest in AMI-CS (59%). In-hospital mortality was highest in mixed CS (48%), followed by AMI-CS (41%), similar in de novo HF-CS (31%) and secondary CS (31%), and lowest in acute-on-chronic HF-CS (25%; P &lt; 0.001). Conclusion SHARC consensus definitions for CS classification can be pragmatically applied in contemporary registries and reveal discrete subpopulations of CS with distinct phenotypes and outcomes that may be relevant to clinical practice and future research.

Abstract Rationale Although many studies have examined gene expression in lung tissue, the gene regulatory processes underlying emphysema are still not well understood. Finding efficient nonimaging screening methods and disease-modifying therapies has been challenging, but knowledge of the transcriptomic features of emphysema may help in this effort. Objectives Our goals were to identify emphysema-associated biological pathways through transcriptomic analysis of bulk lung tissue, to determine the lung cell types in which these emphysema-associated pathways are altered, and to detect unique and overlapping transcriptomic signatures in blood and lung samples. Methods Using RNA-sequencing data from 446 samples in the Lung Tissue Research Consortium and 3,606 blood samples from the COPDGene study, we examined the transcriptomic features of chest computed tomography–quantified emphysema. We also leveraged publicly available lung single-cell RNA-sequencing data to identify cell types showing chronic obstructive pulmonary disease–associated differential expression of the emphysema pathways found in the bulk analyses. Measurements and Main Results In the bulk lung RNA-sequencing analysis, 1,087 differentially expressed genes and 34 dysregulated pathways were significantly associated with emphysema. We observed alternative splicing of several genes and increased activity in pluripotency and cell barrier function pathways. Lung tissue and blood samples shared differentially expressed genes and biological pathways. Multiple lung cell types displayed dysregulation of epithelial barrier function pathways, and distinct pathway activities were observed among various macrophage subpopulations. Conclusions This study identified emphysema-related changes in gene expression and alternative splicing, cell type–specific dysregulated pathways, and instances of shared pathway dysregulation between blood and lung.

ObjectivesThere have been significant advances in the management of large (≥20 mm) laterally spreading tumors (LSTs) or nonpedunculated colorectal polyps; however, there is a lack of clear consensus on the management of these lesions with significant geographic variability especially between Eastern and Western paradigms. We aimed to provide an international consensus to better guide management and attempt to homogenize practices.MethodsTwo experts in interventional endoscopy spearheaded an evidence‐based Delphi study on behalf of the World Endoscopy Organization Colorectal Cancer Screening Committee. A steering committee comprising six members devised 51 statements, and 43 experts from 18 countries on six continents participated in a three‐round voting process. The Grading of Recommendations, Assessment, Development and Evaluations tool was used to assess evidence quality and recommendation strength. Consensus was defined as ≥80% agreement (strongly agree or agree) on a 5‐point Likert scale.ResultsForty‐two statements reached consensus after three rounds of voting. Recommendations included: three statements on training and competency; 10 statements on preresection evaluation, including optical diagnosis, classification, and staging of LSTs; 14 statements on endoscopic resection indications and technique, including statements on en bloc and piecemeal resection decision‐making; seven statements on postresection evaluation; and eight statements on postresection care.ConclusionsAn international expert consensus based on the current available evidence has been developed to guide the evaluation, resection, and follow‐up of LSTs. This may provide guiding principles for the global management of these lesions and standardize current practices.

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.