3168–TARGETING JAK1 SIGNALING FOR MOLECULAR PREVENTION IN CLONAL HEMATOPOIESIS

Nature cardiovascular research

Clonal hematopoiesis (CH) is an independent risk factor for atherosclerotic cardiovascular disease. Murine models of CH suggest a central role of inflammasomes and IL-1β in accelerated atherosclerosis and plaque destabilization. Here we show using single-cell RNA sequencing in human carotid plaques that inflammasome components are enriched in macrophages, while the receptor for IL-1β is enriched in fibroblasts and smooth muscle cells (SMCs). To address the role of inflammatory crosstalk in features of plaque destabilization, we conducted SMC fate mapping in Ldlr−/− mice modeling Jak2VF or Tet2 CH treated with IL-1β antibodies. Unexpectedly, this treatment minimally affected SMC differentiation, leading instead to a prominent expansion of fibroblast-like cells. Depletion of fibroblasts from mice treated with IL-1β antibody resulted in thinner fibrous caps. Conversely, genetic inactivation of Jak2VF …

Cancer Research

Observational studies have implicated modifiable factors like smoking, alcohol intake, physical activity (PA), and sleep to cancer risk. Clonal hematopoiesis (CH), marked by clonal expansions of mutated hematopoietic progenitors with mosaic chromosomal alterations (mCAs) or clonal hematopoiesis of indeterminate potential (CHIP), has been associated with the risk of serval cancers and may shed light the interplay between these factors and early preneoplastic hematopoietic expansion.Leveraging genotyping array and whole-exome sequencing data of leukocyte-derived DNA from 485,028 participants without hematologic malignancies in the UK Biobank (UKBB), we performed genome-wide characterization of two common forms of CH—mCAs and CHIP. To assess associations between social deprivation, self-reported modifiable risk factors, and CH risk, we employed multivariable logistic regression models …

Blood advances

COI notes: STC has no paid advisory roles to declare. STC is volunteer member of ClinGen Expert Panels: Muscular Dystrophies and Myopathies GCEP and Limb Girdle Muscular Dystrophy VCEP. STC is named inventor of Intellectual Property (IP) relating to novel methods to identify splicing variants (PCT/AU2019/000141; PCT/AU2020/050234) owned jointly by The University of Sydney and Sydney Children's Hospitals Network. STC is Director of Frontier Genomics Pty (Australia) which has licenced this IP. STC performs this Director role outside of her UniSydney role and currently receives no consultancy fees or other remuneration for this role. Frontier Genomics has not traded and has no existing financial relationships that will benefit from publication of these data.

Cancer Research

Clonal hematopoiesis (CH) is the age-related clonal expansion of hematopoietic cells as a result of acquired mutations in driver genes, commonly referred to as clonal hematopoiesis of indeterminate potential (CHIP), or due to large-scale mosaic chromosomal alterations (mCAs). While the type and genomic location of CH exhibits varying associations with hematological parameters as well as lymphoid and myeloid malignancy risk, few studies have examined the co-occurrence of CH types. We characterized the frequency of co-occurring CH and examined the association of co-occurring CH with various cancer-related phenotypes to identify potential high-risk clones associated with hematologic cancer risk. We analyzed sequencing and genotyping array data from 453,807 participants in the UK Biobank to detect CHIP and mCAs. In total, 83,240 (18.3%) individuals had at least one detectable type of CH, with …

Pediatric Blood & Cancer

Phenotypic heterogeneity associated with a novel MECOM variant: Mild thrombocytopenia to hydrops fetalis. - Abstract - Europe PMC Sign in | Create an account https://orcid.org Europe PMC Menu About Tools Developers Help Contact us Helpdesk Feedback Twitter Blog Tech blog Developer Forum Europe PMC plus Search life-sciences literature (43,904,233 articles, preprints and more) Search Advanced search Feedback This website requires cookies, and the limited processing of your personal data in order to function. By using the site you are agreeing to this as outlined in our privacy notice and cookie policy. Abstract Full text Phenotypic heterogeneity associated with a novel MECOM variant: Mild thrombocytopenia to hydrops fetalis. Jaspersen SL 1 , Stacy AV 1 , McGlynn MC 1 , Gooch CF 2 , Wilson DB 1 , Bolton KL 3 Author information Affiliations 1. Department of Pediatrics, Division of Hematology and …

Blood

Abstract JAK2 V617F (JAK2VF) clonal hematopoiesis (CH) has been associated with atherothrombotic cardiovascular disease (CVD). We assessed the impact of Jak2VF CH on arterial thrombosis and explored the underlying mechanisms. A meta-analysis of 3 large cohort studies confirmed the association of JAK2VF with CVD and with platelet counts and adjusted mean platelet volume (MPV). In mice, 20% or 1.5% Jak2VF CH accelerated arterial thrombosis and increased platelet activation. Megakaryocytes in Jak2VF CH showed elevated proplatelet formation and release, increasing prothrombogenic reticulated platelet counts. Gp1ba-Cre–mediated expression of Jak2VF in platelets (VFGp1ba) increased platelet counts to a similar level as in 20% Jak2VF CH mice while having no effect on leukocyte counts. Like Jak2VF CH mice, VFGp1ba mice showed enhanced platelet …

The I4 mission, the first all-civilian orbital flight mission, investigated the physiological effects of short duration spaceflight through a multi-omic approach. We analyzed telomere length, clonal hematopoiesis of indeterminate potential (CHIP), whole genome stability, cell-free DNA (cfDNA) cell lysis, and immune cell gene expression. Our results revealed telomere length dynamics similar to those observed in the NASA Twins Study and in astronauts spending 6 months on the ISS, with lengthening in space and shortening upon return to Earth. Our cell-type of origin analysis of cfDNA fragments revealed an increased presence of innate and adaptive immune cell signatures that persisted over a month after return to earth. No significant relationship between spaceflight and CHIP-related or whole genome abnormalities were observed. Longitudinal mitochondrial, ribosomal and immune function gene expression changes occurred across both adaptive and innate immune cells, suggesting adaptations to the space environment can extend months after return from spaceflight and alter immune function. Our findings provide valuable insights into the physiological consequences of short duration spaceflight and will serve as a reference point for future space tourism, low Earth-orbit (LEO) missions, and deep-space exploration.

The present disclosure provides for compositions and methods of use of CDK4/6 inhibitors to prevent therapy-related clonal hematopoiesis and associated myeloid neoplasms.

Journal of Lipid Research

The deposition of cholesterol-rich lipoproteins in the arterial wall triggers macrophage inflammatory responses, which promote atherosclerosis. The NLRP3 inflammasome aggravates atherosclerosis; however, cellular mechanisms connecting macrophage cholesterol accumulation to inflammasome activation are poorly understood. We investigated the mechanisms of NLRP3 inflammasome activation in cholesterol-loaded macrophages and in atherosclerosis-prone Ldlr−/− mice with defects in macrophage cholesterol efflux. We found that accumulation of cholesterol in macrophages treated with modified LDL or cholesterol crystals, or in macrophages defective in the cholesterol efflux promoting transporters ABCA1 and ABCG1, leads to activation of NLRP3 inflammasomes as a result of increased cholesterol trafficking from the plasma membrane to the ER, via Aster-B. In turn, the accumulation of cholesterol in the …

The Lancet Haematology

BackgroundSomatic mutations are frequently reported in individuals with cytopenia but without a confirmed haematological diagnosis (clonal cytopenia of undetermined significance; CCUS). These patients have an increased risk of progression to a myeloid malignancy and worse overall survival than those with no such mutations. To date, studies have been limited by retrospective analysis or small patient numbers. We aimed to establish the natural history of CCUS by prospectively investigating outcome in a large, well defined patient cohort.MethodsThis prospective cohort study was conducted at the Haematological Malignancy Diagnostic Service, a diagnostic laboratory in Leeds, UK. Patients aged at least 18 years who were referred for investigation of cytopenia were eligible for inclusion; those with a history of myeloid malignancy were not eligible. Targeted sequencing was conducted alongside routine clinical …

Bioinformatics

Motivation The acquisition of somatic mutations in hematopoietic stem and progenitor stem cells with resultant clonal expansion, termed clonal hematopoiesis (CH), is associated with increased risk of hematologic malignancies and other adverse outcomes. CH is generally present at low allelic fractions, but clonal expansion and acquisition of additional mutations leads to hematologic cancers in a small proportion of individuals. With high depth and high sensitivity sequencing, CH can be detected in most adults and its clonal trajectory mapped over time. However, accurate CH variant calling is challenging due to the difficulty in distinguishing low frequency CH mutations from sequencing artifacts. The lack of well-validated bioinformatic pipelines for CH calling may contribute to lack of reproducibility in studies of CH. Results Here, we developed ArCH, an Artifact filtering Clonal H …

Nature Cardiovascular Research

Older men with loss of the Y chromosome are more susceptible to heart failure but the responsible genes have not been identified. A study now shows that loss of a single Y chromosome gene in bone marrow cells induces heart failure by switching cardiac macrophages from an inflammatory to a fibrogenic pattern of gene activity.

Clinical Cancer Research

Purpose Clonal hematopoiesis (CH) is thought to be the origin of myeloid neoplasms (MN). Yet, our understanding of the mechanisms driving CH progression to MN and clinical risk prediction of MN remains limited. The human proteome reflects complex interactions between genetic and epigenetic regulation of biological systems. We hypothesized that the plasma proteome might predict MN risk and inform our understanding of the mechanisms promoting MN development. Experimental Design We jointly characterized CH and plasma proteomic profiles of 46,237 individuals in the UK Biobank at baseline study entry. During 500,036 person-years of follow-up, 115 individuals developed MN. Cox proportional hazard regression was used to test for an association between plasma protein levels and MN risk. Results We identified 115 proteins associated with …

Cancer Research

Background: Breast radiotherapy (RT) is the standard of care for patients with early-stage breast cancer (BC) who undergo breast-conserving surgery (BCS). However, the magnitude of benefit of RT is less clear in BCS patients with low-risk disease who receive effective systemic therapy. Among patients with early-stage HER2-positive (HER2+) BC, 10-year locoregional recurrence has been reported as low as 1.5% following BCS, adjuvant chemotherapy and HER2-targeted therapy, and RT. Given these exceedingly favorable outcomes, with the addition of HER2-directed therapy, we seek to evaluate the feasibility of omitting RT among patients with early-stage HER2+ BC following BCS and appropriate systemic therapy. Methods: This is a phase III randomized trial for patients ≥40 years with early-stage, node-negative HER2+ (IHC/FISH) BC treated with BCS with negative margins and sentinel lymph node biopsy …

JCO Precision Oncology

PURPOSEClonal hematopoiesis (CH), the expansion of clones in the hematopoietic system, has been linked to different internal and external features such as aging, genetic ancestry, smoking, and oncologic treatment. However, the interplay between mutations in known cancer predisposition genes and CH has not been thoroughly examined in patients with solid tumors.METHODSWe used prospective tumor-blood paired sequencing data from 46,906 patients who underwent Memorial Sloan Kettering-Integrated Mutation Profiling of Actionable Cancer Targets (MSK-IMPACT) testing to interrogate the associations between CH and rare pathogenic or likely pathogenic (P/LP) germline variants.RESULTSWe observed an enrichment of CH-positive patients among those carrying P/LP germline mutations and identified a significant association between P/LP germline variants in ATM and CH. Germline and CH …

Blood, The Journal of the American Society of Hematology

In this issue of Blood, Arends et al find an association between mutations causing clonal hematopoiesis (CH) and large artery atherosclerosis and white matter lesions in patients with ischemic stroke. 1 In a prospective study, patients with CH showed an increase in recurrent vascular events, stroke, and death compared with those without CH. The event rate is related to clone size, specific CH mutations, and the presence of multiple mutations. The study supports a causal role of CH in large artery atherosclerotic cardiovascular disease (CVD).CH has recently emerged as a major genetic risk factor for CVD. 2 CH arises when somatic mutations in leukemogenic genes endow a fitness advantage to hematopoietic stem and progenitor cells, leading to the clonal expansion of blood cells. CH is common among older people, occurring in> 10% of people aged> 70 years old. 2, 3 CH commonly involves mutations in genes …

Nature cardiovascular research

Clonal hematopoiesis (CH) increases the risk of atherosclerotic cardiovascular disease possibly due to increased plaque inflammation. Human studies suggest that limitation of interleukin-6 (IL-6) signaling could be beneficial in people with large CH clones, particularly in TET2 CH. Here we show that IL-6 receptor antibody treatment reverses the atherosclerosis promoted by Tet2 CH, with reduction of monocytosis, lesional macrophage burden and macrophage colony-stimulating factor 1 receptor (CSF1R) expression. IL-6 induces expression of Csf1r in Tet2-deficient macrophages through enhanced STAT3 binding to its promoter. In mouse and human Tet2-deficient macrophages, IL-6 increases CSF1R expression and enhances macrophage survival. Treatment with the CSF1R inhibitor PLX3397 reversed accelerated atherosclerosis in Tet2 CH mice. Our study demonstrates the causality of IL-6 signaling in Tet2 CH …

Blood

BackgroundMyeloid neoplasms (MN), including acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS), are hematologic malignancies that are highly resistant to therapy and confer a poor prognosis. Recent studies have revealed that hematopoietic stem cells and progenitor cells carrying preleukemic mutations, defined as clonal hematopoiesis (CH), are the cells of origin in MN. CH can occur many years before the diagnosis of MN. Multiple mutations, mutations with a high variant allele frequency (VAF), and mutations in TP53, spliceosome genes, and isocitrate dehydrogenase 1/2 ( IDH1/2) have been identified as high-risk features for CH progression to MN. Individuals with cytopenias who harbor acquired mutations in their blood yet do not meet criteria for hematologic malignancy, an entity called clonal cytopenia of undetermined significance (CCUS), have an even higher risk of developing MN …

Leukemia Research

Background And Aims: Clonal hematopoiesis (CH) while common with aging, confers a high relative risk of hematologic malignancies (HM). Large genome-wide association studies have identified several common germline predisposition loci for CH. Here we sought to characterize the contribution of rare germline genetic mutations to CH and HM.Methods: We profiled blood sequencing data from 479,117 individuals drawn from the UK Biobank (UKBB), the Cancer Genome Atlas (TCGA) and the Memorial Sloan Kettering (MSK) IMPACT cohort for CH and for pathogenic/likely pathogenic (P/LP) germline mutations in 241 known/hypothesized cancer predisposition genes.Results: Overall, 8.5% of individuals in the UKBB harbored a germline P/LP mutation in autosomal dominant genes (5.2% if restricted to HM-related genes). Rare variants in 16 genes (8 of which are novel) were associated with increased risk of CH …

Cancer Prevention Research

Background: Clonal hematopoiesis (CH), the presence of acquired mutations in leukemia driver genes, promotes systemic inflammation and is common among aging men. Prostate cancer, a subset of which is lethal, also develops in aging men. We hypothesized that CH contributes to development of lethal prostate cancer. Methods: We conducted nested case-cohort studies for metastatic prostate cancer and prostate cancer-specific death (lethal prostate cancer) within the prospective Health Professionals Follow-up Study. First, we followed 1155 men free of prostate cancer and cardiovascular disease at blood draw (1993-1995) for development of lethal prostate cancer over up to 26 years. Second, we followed 532 men with incident non-metastatic prostate cancer for development of lethal prostate cancer. We sequenced blood DNA from 1488 participants for putative CH driver mutations in the 9 most common CH …

Experimental Hematology

Myeloproliferative neoplasms (MPN) are driven by hyperactivation of JAK-STAT signaling but can demonstrate skewed hematopoiesis upon acquisition of additional somatic mutations. Here, using primary MPN samples and engineered embryonic stem cells, we demonstrate that mutations in JAK2 induce a significant increase in erythroid colony formation, whereas mutations in additional sex combs-like 1 (ASXL1) led to erythroid colony defect. RNA-sequencing revealed upregulation of protein arginine methyltransferase 6 (PRMT6) induced by mutant ASXL1. Furthermore, genetic perturbation of PRMT6 exacerbated MPN disease burden including leukemic engraftment and splenomegaly in patient-derived xenograft models, highlighting a novel tumor-suppressive function of PRMT6. However, augmented erythroid potential and bone marrow human CD71+ cells following PRMT6 knockdown were reserved only to …

Experimental Hematology

Sickle cell disease (SCD) is a genetic disorder characterized by abnormal hemoglobin and deformation of red blood cells (RBCs), leading to complications and reduced life expectancy. This study developed an in vitro assessment, the Sickle Erythrocyte Health Index, using quantitative phase imaging (QPI) and machine learning to model the health of RBCs in people with SCD. The health index combines assessment of cell deformation, sickle-shaped classification, and membrane flexibility to evaluate erythrocyte health. Using QPI and image processing, the percentage of sickle-shaped cells and membrane flexibility were quantified. Statistically significant differences were observed between individuals with and without SCD, indicating the impact of underlying pathophysiology on erythrocyte health. Additionally, sodium metabisulfite led to an increase in sickle-shaped cells and a decrease in flexibility in the sickle …

Experimental Hematology

During the recent coronavirus disease 2019 (COVID-19) pandemic several patients with β-thalassemia have been infected by severe acute respiratory syndrome coronavirus (SARS-CoV-2), and most patients were vaccinated against SARS-CoV-2. Recent studies demonstrate an impact of SARS-CoV-2 infection on the hematopoietic system. The main objective of this study was to verify the effects of exposure of erythroid precursor cells (ErPCs) from patients with β-thalassemia to SARS-CoV-2 spike protein (S-protein) and the BNT162b2 vaccine. Erythropoietin (EPO)-cultured ErPCs have been either untreated or treated with S-protein or BNT162b2 vaccine. The employed ErPCs were from a β-thalassemia cellular Biobank developed before the COVID-19 pandemic. The genotypes were β+-IVSI-110/β+-IVSI-110 (one patient),  β039/β+-IVSI-110 (3 patients), and β039/ β039 (2 patients). After treatment with S-protein …

Experimental Hematology

Erythroid terminal differentiation and maturation depend on an enormous energy supply. During periods of fasting, ketone bodies from the liver are transported into circulation and utilized as crucial fuel for peripheral tissues. However, the effects of fasting or ketogenesis on erythroid behavior remain unknown. Here, we generated a mouse model with insufficient ketogenesis by conditionally knocking out the gene encoding the hepatocyte-specific ketogenic enzyme hydroxymethylglutary-CoA synthase 2 (Hmgcs2 KO). Intriguingly, erythroid maturation was enhanced with boosted fatty acid synthesis in the bone marrow of a hepatic Hmgcs2 KO mouse under fasting conditions, suggesting that systemic ketogenesis has a profound effect on erythropoiesis. Moreover, we observed significantly activated fatty acid synthesis and mevalonate pathways along with reduced histone acetylation in immature erythrocytes under a …

Experimental Hematology

When hematopoietic cells are overwhelmed with ionizing radiation (IR) DNA damage, the alternative non-homologous end-joining (aNHEJ) repair pathway is activated to repair stressed replication forks. While aNHEJ can rescue cells overwhelmed with DNA damage, it can also mediate chromosomal deletions and fusions, which can cause mis-segregation in mitosis and resultant aneuploidy. We previously reported that a hematopoietic microRNA, miR223-3p normally represses aNHEJ. We found that miR223−/− mice have increased survival of hematopoietic stem and progenitor cells (HSPC) after sub-lethal IR. However, this came at the cost of significantly more genomic aberrancies, with miR223−/− hematopoietic progenitors having increased metaphase aberrancies, including chromothripsis, and increased sequence abnormalities, especially deletions, which is consistent with aNHEJ. These data imply that …

Experimental Hematology

During the recent coronavirus disease 2019 (COVID-19) pandemic several patients with β-thalassemia have been infected by severe acute respiratory syndrome coronavirus (SARS-CoV-2), and most patients were vaccinated against SARS-CoV-2. Recent studies demonstrate an impact of SARS-CoV-2 infection on the hematopoietic system. The main objective of this study was to verify the effects of exposure of erythroid precursor cells (ErPCs) from patients with β-thalassemia to SARS-CoV-2 spike protein (S-protein) and the BNT162b2 vaccine. Erythropoietin (EPO)-cultured ErPCs have been either untreated or treated with S-protein or BNT162b2 vaccine. The employed ErPCs were from a β-thalassemia cellular Biobank developed before the COVID-19 pandemic. The genotypes were β+-IVSI-110/β+-IVSI-110 (one patient),  β039/β+-IVSI-110 (3 patients), and β039/ β039 (2 patients). After treatment with S-protein …

Experimental Hematology

Epigenetic regulators, such as the polycomb repressive complex 2 (PRC2), play a critical role in both normal development and carcinogenesis. Mutations and functional dysregulation of PRC2 complex components, such as EZH2, are implicated in various forms of cancer and associated with poor prognosis. This study investigated the epigenetic vulnerabilities of acute myeloid leukemia (AML) and myelodysplastic/myeloproliferative disorders (MDS/MPN) by performing a chemical probe screen in patient cells. Paradoxically, we observed increased sensitivity to EZH2 and embryonic ectoderm development (EED) inhibitors in AML and MDS/MPN patient cells harboring EZH2 mutations. Expression analysis indicated that EZH2 inhibition elicited upregulation of pathways responsible for cell death and growth arrest, specifically in patient cells with mutant EZH2. The identified EZH2 mutations had drastically reduced …

Experimental Hematology

During the recent coronavirus disease 2019 (COVID-19) pandemic several patients with β-thalassemia have been infected by severe acute respiratory syndrome coronavirus (SARS-CoV-2), and most patients were vaccinated against SARS-CoV-2. Recent studies demonstrate an impact of SARS-CoV-2 infection on the hematopoietic system. The main objective of this study was to verify the effects of exposure of erythroid precursor cells (ErPCs) from patients with β-thalassemia to SARS-CoV-2 spike protein (S-protein) and the BNT162b2 vaccine. Erythropoietin (EPO)-cultured ErPCs have been either untreated or treated with S-protein or BNT162b2 vaccine. The employed ErPCs were from a β-thalassemia cellular Biobank developed before the COVID-19 pandemic. The genotypes were β+-IVSI-110/β+-IVSI-110 (one patient),  β039/β+-IVSI-110 (3 patients), and β039/ β039 (2 patients). After treatment with S-protein …

Experimental Hematology

Myeloproliferative neoplasms (MPN) are driven by hyperactivation of JAK-STAT signaling but can demonstrate skewed hematopoiesis upon acquisition of additional somatic mutations. Here, using primary MPN samples and engineered embryonic stem cells, we demonstrate that mutations in JAK2 induce a significant increase in erythroid colony formation, whereas mutations in additional sex combs-like 1 (ASXL1) led to erythroid colony defect. RNA-sequencing revealed upregulation of protein arginine methyltransferase 6 (PRMT6) induced by mutant ASXL1. Furthermore, genetic perturbation of PRMT6 exacerbated MPN disease burden including leukemic engraftment and splenomegaly in patient-derived xenograft models, highlighting a novel tumor-suppressive function of PRMT6. However, augmented erythroid potential and bone marrow human CD71+ cells following PRMT6 knockdown were reserved only to …

Experimental Hematology

HighlightsMassive endocytosis by endothelial cells was found in intestinal AL amyloidosis.Endothelial cells likely pump out the amyloid fibrils to form tissue amyloidosis.Amyloidosis is established using the physiologic function of endothelial cells.No mechanistic lead is known for establishing AL amyloid deposits in organs. We here report an electron microscopic (EM) analysis in a case of intestinal AL amyloidosis before initiating treatment for amyloidosis. The dense deposits of amyloid fibrils are concentrated around the small blood vessels in the submucosal area of intestinal tissue. Surprisingly, we observed endothelial cells (ECs) of blood vessels containing plenty of endocytotic (pinocytotic) and transcytotic vesicles at the luminal side and above the basement membrane, indicating the one-way active trafficking of either the immunoglobulin (Ig) light chain or preassembled amyloid fibrils from the luminal side of …

Experimental Hematology

Germline mutations of THPO were reported as causes of hereditary thrombocythemia. Six previously reported distinct sites of the mutation were clustered at the 5`-untranslated region or the exon 3 splicing donor site of the THPO gene. Each mutation was identified in an independent pedigree, and the differences between the mutations were not compared. We cloned six distinct THPO mutations (THPO c.-47delG, THPO c.-31G>T, THPO c.13G>A, THPO c.13+1G>A, THPO c.13+2T>C, and THPO c.13+5G>A) and compared the molecular mechanisms that underlie the increased production of THPO protein. At the transcript level, all of the mutations except THPO c.-47delG showed an exon 3 skipping transcript, including two mutations (THPO c.-31G>T and THPO c.13+5G>A) that were distant from the splicing donor site. THPO c.-47delG showed the same full-length transcript as that of the wild-type transcript. At the …

Experimental Hematology

Emerging evidence implicates the epithelial-mesenchymal transition (EMT) transcription factor Zeb1 as a critical regulator of hematopoietic stem cell (HSC) differentiation. Whether Zeb1 regulates long-term maintenance of HSC function remains an open question. Through use of an inducible Mx-1 Cre mouse model that deletes conditional Zeb1 alleles in the adult hematopoietic system, we found that mice engineered to be deficient in Zeb1 for 32 weeks displayed expanded immunophenotypically defined adult HSCs and multi-potent progenitors associated with increased abundance of lineage-biased/balanced HSC subsets and augmented cell survival characteristics. During hematopoietic differentiation, persistent Zeb1 loss increased B-cells in the bone marrow and spleen and decreased monocyte generation in the peripheral blood. Using competitive transplantation experiments, we found that HSCs from adult …

Experimental Hematology

Myelodysplastic/myeloproliferative diseases of childhood cause a relevant disease burden, and many of these diseases may have a fatal course. The use of next-generation sequencing (NGS) has led to the identification of novel genetic variants in patients with these diseases, advancing our understanding of the underlying pathophysiology. However, novel mutations can often only be interpreted as variants of unknown significance (VUS), hindering adequate diagnosis and the use of a targeted therapy. To improve variant interpretation and test targeted therapies in a preclinical setting, we are using a rapid zebrafish embryo model that allows functional evaluation of the novel variant and possible therapeutic approaches within days. Thereby, we accelerate the translation from genetic findings to treatment options. Here, we establish this workflow on a novel in-frame tandem duplication in NRAS (c.192_227dup; p …

Experimental Hematology

Myeloproliferative neoplasms (MPN) are driven by hyperactivation of JAK-STAT signaling but can demonstrate skewed hematopoiesis upon acquisition of additional somatic mutations. Here, using primary MPN samples and engineered embryonic stem cells, we demonstrate that mutations in JAK2 induce a significant increase in erythroid colony formation, whereas mutations in additional sex combs-like 1 (ASXL1) led to erythroid colony defect. RNA-sequencing revealed upregulation of protein arginine methyltransferase 6 (PRMT6) induced by mutant ASXL1. Furthermore, genetic perturbation of PRMT6 exacerbated MPN disease burden including leukemic engraftment and splenomegaly in patient-derived xenograft models, highlighting a novel tumor-suppressive function of PRMT6. However, augmented erythroid potential and bone marrow human CD71+ cells following PRMT6 knockdown were reserved only to …

Experimental Hematology

The revised International Prognostic Index (R-IPI) is an important prognostic tool in diffuse large B cell lymphoma (DLBCL); however, outcomes can vary markedly within R-IPI groups, and additional prognostic markers are needed. We conducted a prospective observational study to evaluate the circulating immature myeloid (IM) cell subsets and cytokine profiles of 31 patients with newly diagnosed DLBCL before and after chemoimmunotherapy. Among circulating IM cells, myeloid-derived suppressor cells (MDSCs) were the predominant cell type (73.8% ± 26%). At baseline, circulating monocytic MDSCs (M-MDSCs) and polymorphonuclear MDSCs (PMN-MDSCs) were predominantly mutually exclusive. Patients with DLBCL clustered into three distinct immunotypes according to MDSC levels and subtype predominance: M-MDSChigh, PMN-MDSChigh, and MDSClow. The M-MDSChigh immunotype was associated …

Experimental Hematology

Sickle cell disease (SCD) is a genetic disorder characterized by abnormal hemoglobin and deformation of red blood cells (RBCs), leading to complications and reduced life expectancy. This study developed an in vitro assessment, the Sickle Erythrocyte Health Index, using quantitative phase imaging (QPI) and machine learning to model the health of RBCs in people with SCD. The health index combines assessment of cell deformation, sickle-shaped classification, and membrane flexibility to evaluate erythrocyte health. Using QPI and image processing, the percentage of sickle-shaped cells and membrane flexibility were quantified. Statistically significant differences were observed between individuals with and without SCD, indicating the impact of underlying pathophysiology on erythrocyte health. Additionally, sodium metabisulfite led to an increase in sickle-shaped cells and a decrease in flexibility in the sickle …

Experimental Hematology

GATA2 coding and noncoding germline mutations cause GATA2 deficiency syndrome, often leading to myelodysplastic syndrome and acute myeloid leukemia. We demonstrated that GATA2 deficiency in murine fetal liver progenitors resulting from loss of Gata2 -77 enhancer upregulates components of Interferon-gamma (IFNg) and Toll-like receptor (TLR) signaling pathways. Using a genetic rescue system, we demonstrated that GATA2-deficient fetal progenitors are hypersensitive to IFNg and TLR1/2 agonist through signaling crosstalk to establish an aberrant transcriptional state. This elevated innate immune signaling is associated with disrupted hematopoiesis and retention of monocytic differentiation ex vivo. GATA2 deficiency elevated expression of select B-lineage and myeloid genes via a PU.1-dependent mechanism. To test the hypothesis that PU.1 is required to establish an ectopic signaling state in GATA2 …

Experimental Hematology

Bone marrow transplantation (BMT) is a widely used treatment for a variety of hematological diseases. However, prior to BMT, recipients are conditioned with total body irradiation (TBI) and/or chemotherapy to eliminate their hematopoietic system, which can also damage the niche, including mesenchymal stem and progenitor cells (MSPCs). The niche is an important microenvironment for the maintenance and regulation of stem cell function, including hematopoietic stem cells.The aim of our study was to investigate the effects of BMT on MSPCs and their subsequent recovery. We found that 10 months after BMT, MSPCs exhibited long-term damage, including decreased frequency (CFU-F), accumulation of DNA damage and an increase in senescent cells. In addition, we observed over-activation of CDC42 and damage to the actin cytoskeleton in BMT MSPCs, resulting in abnormal mitochondrial control and …

Experimental Hematology

Diamond Blackfan anemia (DBA) is an inherited bone marrow failure syndrome characterized by failure of erythropoiesis at the progenitor stages, congenital anomalies, and predisposition to cancer. DBA is most frequently due to mutations in genes encoding a ribosomal protein, the most common one being mutations in RPS19. There is a paucity of in vivo murine models of DBA.Using CRISPR/Cas9 technologies, we generated a conditional knockout of Rps19 by flanking exon 2 with 2 loxP sites and studied hematopoiesis in this model. Vav-iCre;Rps19fl/+ mice are born at expected Mendelian ratios with severe macrocytic anemia. Surprisingly, and unlike any other model generated to date, these mice present with reticulocytopenia, a critical characteristic of patients with DBA and die by post-natal day 10 (P10), due to bone marrow failure and decreased splenic stress erythropoiesis. To understand the role of …

Experimental Hematology

Efficient treatment of Acute Myeloid Leukemia (AML) patients remains a challenge despite recent advances. Here using a CRISPRi screen targeting chromatin factors, we identified BPTF as an essential regulator of AML cell survival. We demonstrate that BPTF forms an alternative NURF chromatin remodeling complex with SMARCA5 and BAP18, which regulates the accessibility of a large set of insulator regions in leukemic cells. This ensures efficient CTCF binding and boundary formation between topologically associated domains that is essential for maintaining the leukemic transcriptional programs. We also demonstrate that the well-studied PHD2-BROMO chromatin reader modules of BPTF are dispensable for leukemic cell growth. Taken together, our results uncover how the alternative NURF complex contributes to leukemia and provide a rationale for its targeting in AML.