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Cancer driver mutations often show distinct temporal acquisition patterns, but the biological basis for this, if any, remains unknown. RAS mutations occur invariably late in the course of acute myeloid leukaemia, upon progression or relapsed/refractory disease 1-6 . Here, by using human leukaemogenesis models, we first show that RAS mutations are obligatory late events that need to succeed earlier cooperating mutations. We provide the mechanistic explanation for this in a requirement for mutant RAS to specifically transform committed progenitors of the myelomonocytic lineage (granulocyte-monocyte progenitors) harbouring previously acquired driver mutations, showing that advanced leukaemic clones can originate from a different cell type in the haematopoietic hierarchy than ancestral clones. Furthermore, we demonstrate that RAS-mutant leukaemia stem cells (LSCs) give rise to monocytic disease, as observed frequently in patients with poor responses to treatment with the BCL2 inhibitor venetoclax. We show that this is because RAS-mutant LSCs, in contrast to RAS-wild-type LSCs, have altered BCL2 family gene expression and are resistant to venetoclax, driving clinical resistance and relapse with monocytic features. Our findings demonstrate that a specific genetic driver shapes the non-genetic cellular hierarchy of acute myeloid leukaemia by imposing a specific LSC target cell restriction and critically affects therapeutic outcomes in patients., (© 2024. The Author(s).)

Clonal hematopoiesis (CH) is a common premalignant state in the blood and confers an increased risk of blood cancers and all-cause mortality. Identification of therapeutic targets in CH has been hindered by the lack of an ex vivo platform amenable for studying primary hematopoietic stem and progenitor cells (HSPCs). Here, we utilize an ex vivo co-culture system of HSPCs with bone marrow endothelial cells to perform CRISPR/Cas9 screens in mutant HSPCs. Our data reveal that loss of the histone demethylase family members Kdm3b and Jmjd1c specifically reduces the fitness of Idh2- and Tet2-mutant HSPCs. Kdm3b loss in mutant cells leads to decreased expression of critical cytokine receptors including Mpl, rendering mutant HSPCs preferentially susceptible to inhibition of downstream JAK2 signaling. Our study nominates an epigenetic regulator and an epigenetically regulated receptor signaling pathway as genotype-specific therapeutic targets and provides a scalable platform to identify genetic dependencies in mutant HSPCs. Significance: Given the broad prevalence, comorbidities, and risk of malignant transformation associated with CH, there is an unmet need to identify therapeutic targets. We develop an ex vivo platform to perform CRISPR/Cas9 screens in primary HSPCs. We identify KDM3B and downstream signaling components as genotype-specific dependencies in CH and myeloid malignancies. See related commentary by Khabusheva and Goodell, p. 1768., (©2024 American Association for Cancer Research.)

Telomere biology disorders (TBD), caused by pathogenic germline variants in telomere-related genes, present with multi-organ disease and a predisposition to cancer. Clonal hematopoiesis (CH) as a marker of cancer development and survival in TBD is poorly understood. Here, we characterized the clonal landscape of a large cohort of 207 TBD patients with a broad range of age and phenotype. CH occurred predominantly in symptomatic patients and in signature genes typically associated with cancers: PPM1D, POT1, TERT promoter (TERTp), U2AF1S34, and/or TP53. Chromosome 1q gain (Chr1q+) was the commonest karyotypic abnormality. Clinically, multiorgan involvement and CH in TERTp, TP53, and splicing factor genes associated with poorer overall survival. Chr1q+, and splicing factor or TP53 mutations significantly increased the risk of hematologic malignancies, regardless of the clonal burden. Chr1q+ and U2AF1S34 mutated clones were pre-malignant events associated with the secondary acquisition of mutations in genes related to hematologic malignancies. Like known effects of Chr1q+ and TP53-CH, functional studies demonstrated that U2AF1S34 mutations primarily compensated for aberrant upregulation of TP53 and interferon pathways in telomere-dysfunctional hematopoietic stem cells, highlighting the TP53 pathway as a canonical route of malignancy in TBD. In contrast, somatic POT1/PPM1D/TERTp-CH had distinct trajectories unrelated to cancer development. With implications beyond TBD, our data show that telomere dysfunction is a strong selective pressure for CH. In TBD, CH is a poor prognostic marker associated with worse overall survival. The identification of key regulatory pathways that drive clonal transformation in TBD allows the identification of patients at a higher risk of cancer development., (Copyright © 2024 American Society of Hematology.)

Abstract: Increased eosinophil counts are associated with cardiovascular disease and may be an independent predictor of major cardiovascular events. However, the causality and underlying mechanisms are poorly understood. Genome-wide association studies have shown an association of a common LNK variant (R262W, T allele) with eosinophilia and atherothrombotic disorders. LNK(TT) reduces LNK function, and Lnk-deficient mice display accelerated atherosclerosis and thrombosis. This study was undertaken to assess the role of eosinophils in arterial thrombosis in mice with hematopoietic Lnk deficiency. Hematopoietic Lnk deficiency increased circulating and activated eosinophils, JAK/STAT signaling in eosinophils, and carotid arterial thrombosis with increased eosinophil abundance and extracellular trap formation (EETosis) in thrombi. Depletion of eosinophils by anti-Siglec-F antibody or by the ΔdbIGata1 mutation eliminated eosinophils in thrombi and markedly reduced thrombosis in mice with hematopoietic Lnk deficiency but not in control mice. Eosinophil depletion reduced neutrophil abundance and NETosis in thrombi without altering circulating neutrophil counts. To assess the role of Lnk specifically in eosinophils, we crossed Lnkf/f mice with eoCre mice. LnkΔeos mice displayed isolated eosinophilia, increased eosinophil activation, and accelerated arterial thrombosis associated with increased EETosis and NETosis in thrombi. DNase I infusion abolished EETs and neutrophil extracellular traps (NETs) in thrombi and reversed the accelerated thrombosis. Human induced pluripotent stem cell-derived LNK(TT) eosinophils showed increased activation and EETosis relative to isogenic LNK(CC) eosinophils, demonstrating human relevance. These studies show a direct link between eosinophilia, EETosis, and atherothrombosis in hematopoietic Lnk deficiency and an essential role of eosinophil LNK in suppression of arterial thrombosis., (© 2024 American Society of Hematology. Published by Elsevier Inc. All rights are reserved, including those for text and data mining, AI training, and similar technologies.)

Spliceosome machinery mutations are common early mutations in myeloid malignancies; however, effective targeted therapies against them are still lacking. In the current study, we used an in vitro high-throughput drug screen among four different isogenic cell lines and identified RKI-1447, a Rho-associated protein kinase inhibitor, as selective cytotoxic effector of SRSF2 mutant cells. RKI-1447 targeted SRSF2 mutated primary human samples in xenografts models. RKI-1447 induced mitotic catastrophe and induced major reorganization of the microtubule system and severe nuclear deformation. Transmission electron microscopy and 3D light microscopy revealed that SRSF2 mutations induce deep nuclear indentation and segmentation that are apparently driven by microtubule-rich cytoplasmic intrusions, which are exacerbated by RKI-1447. The severe nuclear deformation in RKI-1447-treated SRSF2 mutant cells prevents cells from completing mitosis. These findings shed new light on the interplay between microtubules and the nucleus and offers new ways for targeting pre-leukemic SRSF2 mutant cells., Competing Interests: The authors declare no competing interests., (© 2024 The Author(s).)

RNA splicing factor (SF) gene mutations are commonly observed in patients with myeloid malignancies. Here we showed that SRSF2- and U2AF1-mutant leukemias are preferentially sensitive to PARP inhibitors (PARPi), despite being proficient in homologous recombination repair. Instead, SF-mutant leukemias exhibited R-loop accumulation that elicited an R-loop-associated PARP1 response, rendering cells dependent on PARP1 activity for survival. Consequently, PARPi induced DNA damage and cell death in SF-mutant leukemias in an R-loop-dependent manner. PARPi further increased aberrant R-loop levels, causing higher transcription-replication collisions and triggering ATR activation in SF-mutant leukemias. Ultimately, PARPi-induced DNA damage and cell death in SF-mutant leukemias could be enhanced by ATR inhibition. Finally, the level of PARP1 activity at R-loops correlated with PARPi sensitivity, suggesting that R-loop-associated PARP1 activity could be predictive of PARPi sensitivity in patients harboring SF gene mutations. This study highlights the potential of targeting different R-loop response pathways caused by spliceosome gene mutations as a therapeutic strategy for treating cancer., Significance: Spliceosome-mutant leukemias accumulate R-loops and require PARP1 to resolve transcription-replication conflicts and genomic instability, providing rationale to repurpose FDA-approved PARP inhibitors for patients carrying spliceosome gene mutations., (©2023 American Association for Cancer Research.)

Background: Clonal hematopoiesis (CH) has emerged as an independent risk factor for atherosclerotic cardiovascular disease, with activation of macrophage inflammasomes as a potential underlying mechanism. The NLRP3 (NLR family pyrin domain containing 3) inflammasome has a key role in promoting atherosclerosis in mouse models of Tet2 CH, whereas inhibition of the inflammasome product interleukin-1β appeared to particularly benefit patients with TET2 CH in CANTOS (Cardiovascular Risk Reduction Study [Reduction in Recurrent Major CV Disease Events]). TET2 is an epigenetic modifier that decreases promoter methylation. However, the mechanisms underlying macrophage NLRP3 inflammasome activation in TET2 (Tet methylcytosine dioxygenase 2) deficiency and potential links with epigenetic modifications are poorly understood., Methods: We used cholesterol-loaded TET2-deficient murine and embryonic stem cell-derived isogenic human macrophages to evaluate mechanisms of NLRP3 inflammasome activation in vitro and hypercholesterolemic Ldlr - / - mice modeling TET2 CH to assess the role of NLRP3 inflammasome activation in atherosclerosis., Results: Tet2 deficiency in murine macrophages acted synergistically with cholesterol loading in cell culture and with hypercholesterolemia in vivo to increase JNK1 (c-Jun N-terminal kinase 1) phosphorylation and NLRP3 inflammasome activation. The mechanism of JNK (c-Jun N-terminal kinase) activation in TET2 deficiency was increased promoter methylation and decreased expression of the JNK-inactivating dual-specificity phosphatase Dusp10 . Active Tet1-deadCas9-targeted editing of Dusp10 promoter methylation abolished cholesterol-induced inflammasome activation in Tet2 -deficient macrophages. Increased JNK1 signaling led to NLRP3 deubiquitylation and activation by the deubiquitinase BRCC3 ( BRCA1/BRCA2 -containing complex subunit 3). Accelerated atherosclerosis and neutrophil extracellular trap formation (NETosis) in Tet2 CH mice were reversed by holomycin, a BRCC3 deubiquitinase inhibitor, and also by hematopoietic deficiency of Abro1, an essential scaffolding protein in the BRCC3-containing cytosolic complex. Human TET2 - / - macrophages displayed increased JNK1 and NLRP3 inflammasome activation, especially after cholesterol loading, with reversal by holomycin treatment, indicating human relevance., Conclusions: Hypercholesterolemia and TET2 deficiency converge on a common pathway of NLRP3 inflammasome activation mediated by JNK1 activation and BRCC3-mediated NLRP3 deubiquitylation, with potential therapeutic implications for the prevention of cardiovascular disease in TET2 CH., Competing Interests: Disclosures Dr Tall is a consultant for Amgen, CSL Behring, Astra Zeneca, and Foresite Laboratories, and is on the Scientific Advisory Board of Staten Biotech, Fortico Biotech, and Beren Therapeutics.

In a recent study, Martin-Rufino and colleagues combined massively parallel base editing in primary human hematopoietic stem and progenitor cells (HSPCs) with functional and single-cell transcriptomic readouts. A series of proof-of-principle experiments highlight the breadth of applications made possible with this approach, which range from gene therapy and immunotherapy, to characterizing single nucleotide variants., Competing Interests: Declaration of interests The author declares no competing interests., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

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 accelerated atherosclerosis, identifies IL-6-induced CSF1R expression as a critical mechanism and supports blockade of IL-6 signaling as a potential therapy for CH-driven cardiovascular disease., Competing Interests: Competing interests A.R.T. is a consultant for Amgen, CSL Behring, AstraZeneca and Foresite Laboratories, and is on the scientific advisory board of Staten Biotechnology, Fortico Biotech and Beren Therapeutics. P.L. is an unpaid consultant to, or involved in clinical trials for Amgen, AstraZeneca, Baim Institute, Beren Therapeutics, Esperion Therapeutics, Genentech, Kancera, Kowa Pharmaceuticals, MedImmune, Merck, Moderna, Novo Nordisk, Novartis, Pfizer and Sanofi Regeneron. P.L. is a member of the scientific advisory board for Amgen, Caristo Diagnostics, Cartesian Therapeutics, CSL Behring, DalCor Pharmaceuticals, Dewpoint Therapeutics, Elucid Bioimaging, Kancera, Kowa Pharmaceuticals, Olatec Therapeutics, MedImmune, Novartis, PlaqueTec, TenSixteen Bio, Soley Thereapeutics and XBiotech. His laboratory has received research funding in the last 2 years from Novartis, Novo Nordisk and Genentech. P.L. is on the board of directors of XBiotech. He has a financial interest in XBiotech, a company developing therapeutic human antibodies, in TenSixteen Bio, a company targeting somatic mosaicism and CHIP to discover and develop new therapeutics to treat age-related diseases, and in Soley Therapeutics, a biotechnology company that is combining artificial intelligence with molecular and cellular response detection to discover and develop new drugs, currently focusing on cancer therapeutics. His interests were reviewed and are managed by Brigham and Women’s Hospital and Mass General Brigham in accordance with their conflict-of-interest policies. The other authors declare no competing interests.

SF3B1K700E is the most frequent mutation in myelodysplastic syndrome (MDS), but the mechanisms by which it drives MDS pathogenesis remain unclear. We derived a panel of 18 genetically matched SF3B1K700E- and SF3B1WT-induced pluripotent stem cell (iPSC) lines from patients with MDS with ring sideroblasts (MDS-RS) harboring isolated SF3B1K700E mutations and performed RNA and ATAC sequencing in purified CD34+/CD45+ hematopoietic stem/progenitor cells (HSPCs) derived from them. We developed a novel computational framework integrating splicing with transcript usage and gene expression analyses and derived a SF3B1K700E splicing signature consisting of 59 splicing events linked to 34 genes, which associates with the SF3B1 mutational status of primary MDS patient cells. The chromatin landscape of SF3B1K700E HSPCs showed increased priming toward the megakaryocyte- erythroid lineage. Transcription factor motifs enriched in chromatin regions more accessible in SF3B1K700E cells included, unexpectedly, motifs of the TEA domain (TEAD) transcription factor family. TEAD expression and transcriptional activity were upregulated in SF3B1-mutant iPSC-HSPCs, in support of a Hippo pathway-independent role of TEAD as a potential novel transcriptional regulator of SF3B1K700E cells. This study provides a comprehensive characterization of the transcriptional and chromatin landscape of SF3B1K700E HSPCs and nominates novel mis-spliced genes and transcriptional programs with putative roles in MDS-RS disease biology., (© 2022 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

In this issue of Cell Stem Cell, Reilly et al. propose loss of LMNB1, the gene encoding lamin B1, often deleted in MDS/AML, as a novel genetic basis for the abnormal nuclear shape of neutrophils (known as acquired Pelger-Huët anomaly) and a cause of HSPC fate alterations promoting malignancy., Competing Interests: Declaration of interests The author declares no competing interests., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Mutations in splicing factors (SF) are the predominant class of mutations in myelodysplastic syndrome (MDS), but convergent downstream disease drivers remain elusive. To identify common direct targets of missplicing by mutant U2AF1 and SRSF2, we performed RNA sequencing and enhanced version of the cross-linking and immunoprecipitation assay in human hematopoietic stem/progenitor cells derived from isogenic induced pluripotent stem cell (iPSC) models. Integrative analyses of alternative splicing and differential binding converged on a long isoform of GNAS (GNAS-L), promoted by both mutant factors. MDS population genetics, functional and biochemical analyses support that GNAS-L is a driver of MDS and encodes a hyperactive long form of the stimulatory G protein alpha subunit, Gαs-L, that activates ERK/MAPK signaling. SF-mutant MDS cells have activated ERK signaling and consequently are sensitive to MEK inhibitors. Our findings highlight an unexpected and unifying mechanism by which SRSF2 and U2AF1 mutations drive oncogenesis with potential therapeutic implications for MDS and other SF-mutant neoplasms., Significance: SF mutations are disease-defining in MDS, but their critical effectors remain unknown. We discover the first direct target of convergent missplicing by mutant U2AF1 and SRSF2, a long GNAS isoform, which activates G protein and ERK/MAPK signaling, thereby driving MDS and rendering mutant cells sensitive to MEK inhibition. This article is highlighted in the In This Issue feature, p. 587., (©2021 American Association for Cancer Research.)

Acute myeloid leukemia (AML) is a clonal hematopoietic stem and progenitor cell malignancy characterized by poor clinical outcomes. Major histocompatibility complex class I polypeptide-related sequence A and B (MICA/B) are stress proteins expressed by cancer cells, and antibody-mediated inhibition of MICA/B shedding represents a novel approach to stimulate immunity against cancers. We found that the MICA/B antibody 7C6 potently inhibits the outgrowth of AML in 2 models in immunocompetent mice. Macrophages were essential for therapeutic efficacy, and 7C6 triggered antibody-dependent phagocytosis of AML cells. Furthermore, we found that romidepsin, a selective histone deacetylase inhibitor, increased MICB messenger RNA in AML cells and enabled subsequent stabilization of the translated protein by 7C6. This drug combination substantially increased surface MICA/B expression in a human AML line, pluripotent stem cell-derived AML blasts and leukemia stem cells, as well as primary cells from 3 untreated patients with AML. Human macrophages phagocytosed AML cells following treatment with 7C6 and romidepsin, and the combination therapy lowered leukemia burden in a humanized model of AML. Therefore, inhibition of MICA/B shedding promotes macrophage-driven immunity against AML via Fc receptor signaling and synergizes with an epigenetic regulator. These results provide the rationale for the clinical testing of this innovative immunotherapeutic approach for the treatment of AML., (© 2022 by The American Society of Hematology.)

Despite the initial success of some drugs and vaccines targeting COVID-19, understanding the mechanism underlying SARS-CoV-2 disease pathogenesis remains crucial for the development of further approaches to treatment. Some patients with severe Covid-19 experience a cytokine storm and display evidence of inflammasome activation leading to increased levels of IL-1β and IL-18; however, other reports have suggested reduced inflammatory responses to Sars-Cov-2. In this study we have examined the effects of the Sars-Cov-2 envelope (E) protein, a virulence factor in coronaviruses, on inflammasome activation and pulmonary inflammation. In cultured macrophages the E protein suppressed inflammasome priming and NLRP3 inflammasome activation. Similarly, in mice transfected with E protein and treated with poly(I:C) to simulate the effects of viral RNA, the E protein, in an NLRP3-dependent fashion, reduced expression of pro-IL-1β, levels of IL-1β and IL-18 in broncho-alveolar lavage fluid, and macrophage infiltration in the lung. To simulate the effects of more advanced infection, macrophages were treated with both LPS and poly(I:C). In this setting the E protein increased NLRP3 inflammasome activation in both murine and human macrophages. Thus, the Sars-Cov-2 E protein may initially suppress the host NLRP3 inflammasome response to viral RNA while potentially increasing NLRP3 inflammasome responses in the later stages of infection. Targeting the Sars-Cov-2 E protein especially in the early stages of infection may represent a novel approach to Covid-19 therapy., (© 2021. The Author(s).)

Background: LNK/SH2B3 inhibits Janus kinase/signal transducer and activator of transcription (JAK/STAT) signaling by hematopoietic cytokine receptors. Genome-wide association studies have shown association of a common single nucleotide polymorphism in LNK (R262W, T allele) with neutrophilia, thrombocytosis, and coronary artery disease. We have shown that LNK(TT ) reduces LNK function and that LNK-deficient mice display prominent platelet-neutrophil aggregates, accelerated atherosclerosis, and thrombosis. Platelet-neutrophil interactions can promote neutrophil extracellular trap (NET) formation. The goals of this study were to assess the role of NETs in atherosclerosis and thrombosis in mice with hematopoietic Lnk deficiency., Methods: We bred mice with combined deficiency of Lnk and the NETosis-essential enzyme PAD4 (peptidyl arginine deiminase 4) and transplanted their bone marrow into Ldlr -/- mice. We evaluated the role of LNK in atherothrombosis in humans and mice bearing a gain of function variant in JAK2 (JAK2 V617F )., Results: Lnk -deficient mice displayed accelerated carotid artery thrombosis with prominent NETosis that was completely reversed by PAD4 deficiency. Thrombin-activated Lnk -/- platelets promoted increased NETosis when incubated with Lnk -/- neutrophils compared with wild-type platelets or wild-type neutrophils. This involved increased surface exposure and release of oxidized phospholipids (OxPL) from Lnk -/- platelets, as well as increased priming and response of Lnk -/- neutrophils to OxPL. To counteract the effects of OxPL, we introduced a transgene expressing the single-chain variable fragment of E06 (E06-scFv). E06-scFv reversed accelerated NETosis, atherosclerosis, and thrombosis in Lnk -/- mice. We also showed increased NETosis when human induced pluripotent stem cell-derived LNK(TT ) neutrophils were incubated with LNK(TT ) platelet/megakaryocytes, but not in isogenic LNK(CC ) controls, confirming human relevance. Using data from the UK Biobank, we found that individuals with the JAK2 VF mutation only showed increased risk of coronary artery disease when also carrying the LNK R262W allele. Mice with hematopoietic Lnk +/- and Jak2 VF clonal hematopoiesis showed accelerated arterial thrombosis but not atherosclerosis compared with Jak2 VF Lnk +/+ controls., Conclusions: Hematopoietic Lnk deficiency promotes NETosis and arterial thrombosis in an OxPL-dependent fashion. LNK(R262W) reduces LNK function in human platelets and neutrophils, promoting NETosis, and increases coronary artery disease risk in humans carrying Jak2 VF mutations. Therapies targeting OxPL may be beneficial for coronary artery disease in genetically defined human populations.

Human cancers arise through the sequential acquisition of somatic mutations that create successive clonal populations. Human cancer evolution models could help illuminate this process and inform therapeutic intervention at an early disease stage, but their creation has faced significant challenges. Here, we combined induced pluripotent stem cell (iPSC) and CRISPR-Cas9 technologies to develop a model of the clonal evolution of acute myeloid leukemia (AML). Through the stepwise introduction of three driver mutations, we generated iPSC lines that, upon hematopoietic differentiation, capture distinct premalignant stages, including clonal hematopoiesis (CH) and myelodysplastic syndrome (MDS), culminating in a transplantable leukemia, and recapitulate transcriptional and chromatin accessibility signatures of primary human MDS and AML. By mapping dynamic changes in transcriptomes and chromatin landscapes, we characterize transcriptional programs driving specific transitions between disease stages. We identify cell-autonomous dysregulation of inflammatory signaling as an early and persistent event in leukemogenesis and a promising early therapeutic target., Competing Interests: Declaration of interests D.T.S. serves on the scientific advisory board of Kurome Therapeutics. E.P.P. has received honoraria from Celgene and Merck and research support from Incyte for research not related to this study., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Clonal haematopoiesis, which is highly prevalent in older individuals, arises from somatic mutations that endow a proliferative advantage to haematopoietic cells. Clonal haematopoiesis increases the risk of myocardial infarction and stroke independently of traditional risk factors 1 . Among the common genetic variants that give rise to clonal haematopoiesis, the JAK2 V617F (JAK2 VF ) mutation, which increases JAK-STAT signalling, occurs at a younger age and imparts the strongest risk of premature coronary heart disease 1,2 . Here we show increased proliferation of macrophages and prominent formation of necrotic cores in atherosclerotic lesions in mice that express Jak2 VF selectively in macrophages, and in chimeric mice that model clonal haematopoiesis. Deletion of the essential inflammasome components caspase 1 and 11, or of the pyroptosis executioner gasdermin D, reversed these adverse changes. Jak2 VF lesions showed increased expression of AIM2, oxidative DNA damage and DNA replication stress, and Aim2 deficiency reduced atherosclerosis. Single-cell RNA sequencing analysis of Jak2 VF lesions revealed a landscape that was enriched for inflammatory myeloid cells, which were suppressed by deletion of Gsdmd. Inhibition of the inflammasome product interleukin-1β reduced macrophage proliferation and necrotic formation while increasing the thickness of fibrous caps, indicating that it stabilized plaques. Our findings suggest that increased proliferation and glycolytic metabolism in Jak2 VF macrophages lead to DNA replication stress and activation of the AIM2 inflammasome, thereby aggravating atherosclerosis. Precise application of therapies that target interleukin-1β or specific inflammasomes according to clonal haematopoiesis status could substantially reduce cardiovascular risk.

RUNX1 familial platelet disorder (RUNX1-FPD) is an autosomal dominant disorder caused by a monoallelic mutation of RUNX1, initially resulting in approximately half-normal RUNX1 activity. Clinical features include thrombocytopenia, platelet functional defects, and a predisposition to leukemia. RUNX1 is rapidly degraded through the ubiquitin-proteasome pathway. Moreover, it may autoregulate its expression. A predicted kinetic property of autoregulatory circuits is that transient perturbations of steady-state levels result in continued maintenance of expression at adjusted levels, even after inhibitors of degradation or inducers of transcription are withdrawn, suggesting that transient inhibition of RUNX1 degradation may have prolonged effects. We hypothesized that pharmacological inhibition of RUNX1 protein degradation could normalize RUNX1 protein levels, restore the number of platelets and their function, and potentially delay or prevent malignant transformation. In this study, we evaluated cell lines, induced pluripotent stem cells derived from patients with RUNX1-FPD, RUNX1-FPD primary bone marrow cells, and acute myeloid leukemia blood cells from patients with RUNX1 mutations. The results showed that, in some circumstances, transient expression of exogenous RUNX1 or inhibition of steps leading to RUNX1 ubiquitylation and proteasomal degradation restored RUNX1 levels, thereby advancing megakaryocytic differentiation in vitro. Thus, drugs retarding RUNX1 proteolytic degradation may represent a therapeutic avenue for treating bleeding complications and preventing leukemia in RUNX1-FPD., (© 2021 by The American Society of Hematology.)

Patient-derived induced pluripotent stem cells (iPSCs) have recently provided a new way to model acute myeloid leukemia (AML) and other myeloid malignancies. Here, we describe methods for the generation of patient-derived iPSCs from leukemia cells and for their subsequent directed in vitro differentiation into hematopoietic cells that recapitulate features of leukemia stem cells (LSCs) and leukemic blasts.

Purpose of Review: Myeloid malignancies comprise a spectrum of genetically heterogeneous disorders marked by the stepwise acquisition of somatic mutations and clonal evolution. The blood and bone marrow of patients typically consists of a mix of different clones and subclones along the path of clonal evolution that cannot be deconvoluted with most current approaches. Here, we review the application of induced pluripotent stem cell (iPSC) technology to the study of the clonal architecture and clonal evolution of these diseases, focusing on myelodysplastic syndromes and acute myeloid leukemia., Recent Findings: Reprogramming to pluripotency allows capture of the genomes of single somatic cells into stable iPSC lines. In addition, precise genome editing can introduce specific driver mutations, isolated, and in combinations, into normal iPSCs. Studies utilizing these approaches have elucidated the clonal composition and mutational order in patients with myeloid neoplasms. Importantly, they have also enabled functional interrogation of the cellular and molecular consequences of individual mutations and their combinations and allowed testing of the effects of drugs on distinct disease clones., Summary: Human iPSCs are important tools to elucidate the mechanisms of progression from normal to malignant haematopoiesis and empower drug testing and drug discovery.

The reprogramming of cancer cells into induced pluripotent stem cells (iPSCs) can capture entire cancer genomes, and thus create genetically faithful models of human cancers. By providing stringent genetically clonal conditions, iPSC modeling can also unveil non-genetic sources of cancer heterogeneity and provide a unique opportunity to study them separately from genetic sources, as we recently showed in an iPSC-based model of acute myeloid leukemia (AML). Genetically clonal iPSCs, derived from a patient with AML, reproduce, upon hematopoietic differentiation, phenotypic and functional heterogeneity with all the hallmarks of a leukemia stem cell (LSC) hierarchy. Here we discuss the lessons that can be learned about the LSC state, its plasticity, stability and genetic and epigenetic determinants from iPSC modeling. We also discuss the practical and translational implications of exploiting AML-iPSCs to prospectively isolate large numbers of iLSCs for large-scale experiments, such as screens, and for discovery of new therapeutic targets specific to AML LSCs., (Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.)

Recurrent chromosomal deletions spanning several megabases are often found in hematological malignancies. The ability to engineer deletions in model systems to functionally study their effects on the phenotype would enable, first, determination of whether a given deletion is pathogenic or neutral and, second, identification of the critical genes. Incomplete synteny makes modeling of deletions of megabase scale challenging or impossible in the mouse or other model organisms. Furthermore, despite the breakthroughs in targeted nuclease technologies in recent years, engineering of megabase-scale deletions remains challenging and has not been achieved in normal diploid human cells. Large deletions of the long arm of chromosome 7 (chr7q) occur frequently in myelodysplastic syndrome (MDS) and are associated with poor prognosis. We previously found that we can model chr7q deletions in human induced pluripotent stem cells (iPSCs) using a modified Cre-loxP strategy. However, this strategy did not afford control over the length and boundaries of the engineered deletions, which were initiated through random chromosome breaks. Here we developed strategies enabling the generation of defined and precise chromosomal deletions of up to 22 Mb, using two different strategies: "classic" Cre-loxP recombination and CRISPR/Cas9-mediated DNA cleavage. As proof of principle, we illustrate that phenotypic characterization of the hematopoiesis derived from these iPSCs upon in vitro differentiation allows further definition of the critical region of chr7q whose hemizygosity impairs hematopoietic differentiation potential. The strategies we present here can be broadly applicable to engineering of diverse chromosomal deletions in human cells., (Copyright © 2020 ISEH -- Society for Hematology and Stem Cells. Published by Elsevier Inc. All rights reserved.)

Leukemia stem cells (LSCs) are believed to have more distinct vulnerabilities than the bulk acute myeloid leukemia (AML) cells, but their rarity and the lack of universal markers for their prospective isolation hamper their study. We report that genetically clonal induced pluripotent stem cells (iPSCs) derived from an AML patient and characterized by exceptionally high engraftment potential give rise, upon hematopoietic differentiation, to a phenotypic hierarchy. Through fate-tracking experiments, xenotransplantation, and single-cell transcriptomics, we identify a cell fraction (iLSC) that can be isolated prospectively by means of adherent in vitro growth that resides on the apex of this hierarchy and fulfills the hallmark features of LSCs. Through integrative genomic studies of the iLSC transcriptome and chromatin landscape, we derive an LSC gene signature that predicts patient survival and uncovers a dependency of LSCs, across AML genotypes, on the RUNX1 transcription factor. These findings can empower efforts to therapeutically target AML LSCs., Competing Interests: Declaration of Interests E.P.P. has received honoraria from Celgene and Merck and research support from Incyte. M.G.K. has received consultant fees from Acent Therapeutics and research support from 28-7. These disclosures are not directly related to the present study., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

The reprogramming of human somatic cells into induced pluripotent stem cells (iPSCs) a little over a decade ago raised exciting prospects to transform the study and potentially also the therapy of human diseases. iPSC models have now been created for a multitude of hematologic diseases, including malignancies. Here we discuss practical aspects of iPSC modeling of malignant diseases, review recent studies, and discuss the new opportunities that iPSC models offer, as well as their current limitations and prospects for future development., (Copyright © 2019 Cold Spring Harbor Laboratory Press; all rights reserved.)

Cancer-associated mutations in genes encoding RNA splicing factors (SFs) commonly occur in leukemias, as well as in a variety of solid tumors, and confer dependence on wild-type splicing. These observations have led to clinical efforts to directly inhibit the spliceosome in patients with refractory leukemias. Here, we identify that inhibiting symmetric or asymmetric dimethylation of arginine, mediated by PRMT5 and type I protein arginine methyltransferases (PRMTs), respectively, reduces splicing fidelity and results in preferential killing of SF-mutant leukemias over wild-type counterparts. These data identify genetic subsets of cancer most likely to respond to PRMT inhibition, synergistic effects of combined PRMT5 and type I PRMT inhibition, and a mechanistic basis for the therapeutic efficacy of PRMT inhibition in cancer., (Copyright © 2019. Published by Elsevier Inc.)

Induced pluripotent stem cells (iPSCs) are derived from somatic cells through a reprogramming process, which converts them to a pluripotent state, akin to that of embryonic stem cells. Over the past decade, iPSC models have found increasing applications in the study of human diseases, with blood disorders featuring prominently. Here, we discuss methodological aspects pertaining to iPSC generation, hematopoietic differentiation and gene editing, and provide an overview of uses of iPSCs in modeling the cell and gene therapy of inherited genetic blood disorders, as well as their more recent use as models of myeloid malignancies. We also discuss the strengths and limitations of iPSCs compared to model organisms and other cellular systems commonly used in hematology research., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2019. Published by The Company of Biologists Ltd.)

Monosomy 7 or deletion of 7q (del(7q)) are common clonal cytogenetic abnormalities associated with high grade myelodysplastic syndrome (MDS) arising in inherited and acquired bone marrow failure. Current non-transplant approaches to treat marrow failure may be complicated by stimulation of clonal outgrowth. To study the biological consequences of del(7q) within the context of a failing marrow, we generated induced pluripotent stem cells (iPSCs) derived from patients with Shwachman Diamond Syndrome (SDS), a bone marrow failure disorder with MDS predisposition, and genomically engineered a 7q deletion. The TGFβ pathway was the top differentially regulated pathway in transcriptomic analysis of SDS versus SDSdel(7q) iPSCs. SMAD2 phosphorylation was increased in SDS relative to wild type cells consistent with hyperactivation of the TGFbeta pathway in SDS. Phospho-SMAD2 levels were reduced following 7q deletion in SDS cells and increased upon restoration of 7q diploidy. Inhibition of the TGFbeta pathway rescued hematopoiesis in SDS-iPSCs and in bone marrow hematopoietic cells from SDS patients while it had no impact on the SDSdel(7q) cells. These results identified a potential targetable vulnerability to improve hematopoiesis in an MDS-predisposition syndrome, and highlight the importance of the germline context of somatic alterations to inform precision medicine approaches to therapy.

Modeling human diseases with patient-derived induced pluripotent stem cells (iPSCs) offers unique research opportunities and is particularly attractive for hematology research. Whereas monogenic inherited blood diseases featured prominently among the first proof-of-principle studies of iPSC modeling, malignant hematologic disorders have been off to a slower start. This has been due to challenges in the derivation of iPSCs from cancer cells and the need to establish robust differentiation protocols and to standardize phenotypic assays of iPSC-derived hematopoiesis. Recent studies of iPSC modeling of myeloid malignancies exploited the clonal heterogeneity of patient samples to derive genetically matched normal controls and recapitulate the clonal evolution of the disease. Comparisons of the malignant phenotypes and molecular signatures of primary leukemic cells, derived iPSCs, and their hematopoietic progeny stress the importance of the cell-of-origin in oncogenesis and enable investigation of the interplay between cell identity and the cancer genome. Larger collections of genetically diverse iPSC lines and more readily scalable hematopoietic differentiation protocols, ideally mimicking adult bone marrow-derived hematopoiesis, would further empower applications of iPSC modeling in myeloid malignancy in the future. Nevertheless, with recent progress in this field, the stage is set for the wider adoption of this model system by the hematology community., (Copyright © 2018 ISEH -- Society for Hematology and Stem Cells. Published by Elsevier Inc. All rights reserved.)

Connecting specific cancer genotypes with phenotypes and drug responses constitutes the central premise of precision oncology but is hindered by the genetic complexity and heterogeneity of primary cancer cells. Here, we use patient-derived induced pluripotent stem cells (iPSCs) and CRISPR/Cas9 genome editing to dissect the individual contributions of two recurrent genetic lesions, the splicing factor SRSF2 P95L mutation and the chromosome 7q deletion, to the development of myeloid malignancy. Using a comprehensive panel of isogenic iPSCs-with none, one, or both genetic lesions-we characterize their relative phenotypic contributions and identify drug sensitivities specific to each one through a candidate drug approach and an unbiased large-scale small-molecule screen. To facilitate drug testing and discovery, we also derive SRSF2-mutant and isogenic normal expandable hematopoietic progenitor cells. We thus describe here an approach to dissect the individual effects of two cooperating mutations to clinically relevant features of malignant diseases., (Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.)

In the version of this article initially published, in the Methods, the Gene Expression Omnibus accession code for H3K36me3 ChIP-seq data was incorrectly given as GSM1003585 instead of GSM733725. The error has been corrected in the HTML, PDF and print versions of the article.

The version of the Supplementary Text and Figures file initially posted was missing Supplementary Tables 1-6 and the Supplementary Note and used incorrect versions of the supplementary figures.

TET enzymes oxidize 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), which can lead to DNA demethylation. However, direct connections between TET-mediated DNA demethylation and transcriptional output are difficult to establish owing to challenges in distinguishing global versus locus-specific effects. Here we show that TET1, TET2 and TET3 triple-knockout (TKO) human embryonic stem cells (hESCs) exhibit prominent bivalent promoter hypermethylation without an overall corresponding decrease in gene expression in the undifferentiated state. Focusing on the bivalent PAX6 locus, we find that increased DNMT3B binding is associated with promoter hypermethylation, which precipitates a neural differentiation defect and failure of PAX6 induction during differentiation. dCas9-mediated locus-specific demethylation and global inactivation of DNMT3B in TKO hESCs partially reverses the hypermethylation at the PAX6 promoter and improves differentiation to neuroectoderm. Taking these findings together with further genome-wide methylation and TET1 and DNMT3B ChIP-seq analyses, we conclude that TET proteins safeguard bivalent promoters from de novo methylation to ensure robust lineage-specific transcription upon differentiation.

Myeloid malignancy is increasingly viewed as a disease spectrum, comprising hematopoietic disorders that extend across a phenotypic continuum ranging from clonal hematopoiesis to myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). In this study, we derived a collection of induced pluripotent stem cell (iPSC) lines capturing a range of disease stages encompassing preleukemia, low-risk MDS, high-risk MDS, and secondary AML. Upon their differentiation, we found hematopoietic phenotypes of graded severity and/or stage specificity that together delineate a phenotypic roadmap of disease progression culminating in serially transplantable leukemia. We also show that disease stage transitions, both reversal and progression, can be modeled in this system using genetic correction or introduction of mutations via CRISPR/Cas9 and that this iPSC-based approach can be used to uncover disease-stage-specific responses to drugs. Our study therefore provides insight into the cellular events demarcating the initiation and progression of myeloid transformation and a new platform for testing genetic and pharmacological interventions., (Copyright © 2017 Elsevier Inc. All rights reserved.)

In recent years, breakthroughs in human pluripotent stem cell (hPSC) research, namely cellular reprogramming and the emergence of sophisticated genetic engineering technologies, have opened new frontiers for cell and gene therapy. The prospect of using hPSCs, either autologous or histocompatible, as targets of genetic modification and their differentiated progeny as cell products for transplantation, presents a new paradigm of regenerative medicine of potential tremendous value for the treatment of blood disorders, including beta-thalassemia (BT) and sickle cell disease (SCD). Despite advances at a remarkable pace and great promise, many roadblocks remain before clinical translation can be realistically considered. Here we discuss the theoretical advantages of cell therapies utilizing hPSC derivatives, recent proof-of-principle studies and the main challenges towards realizing the potential of hPSC therapies in the clinic.

Together with recent advances in the processing and culture of human tissue, bioengineering, xenotransplantation and genome editing, Induced pluripotent stem cells (iPSCs) present a range of new opportunities for the study of human cancer. Here we discuss the main advantages and limitations of iPSC modeling, and how the method intersects with other patient-derived models of cancer, such as organoids, organs-on-chips and patient-derived xenografts (PDXs). We highlight the opportunities that iPSC models can provide beyond those offered by existing systems and animal models and present current challenges and crucial areas for future improvements toward wider adoption of this technology.

Transgenesis of human pluripotent stem cells (hPSCs) can enable and empower a variety of studies in stem cell research, including lineage tracing and functional genetics studies. While in recent years much progress has been made in the development of tools for gene targeting, little attention has been given to the identification of sites in the human genome where transgenes can be inserted and reliably expressed. In order to find human genomic sites capable of supporting long-term and high-level transgene expression in hPSCs, we performed a lentiviral screen in human induced pluripotent stem cells (iPSCs). We isolated 40 iPSC clones each harboring a single vector copy and characterized the level of transgene expression afforded by each unique integration site. We selected one clone, LiPS-A3 with an integration site in chromosome 15 maintaining robust expression without silencing and demonstrate that different transgenes can be inserted therein rapidly and efficiently through recombinase-mediated cassette exchange (RMCE). The LiPS-A3 line can greatly facilitate the insertion of reporter and other genes in hPSCs. Targeting transgenes in the LiPS-A3S genomic locus can find broad applications in stem cell research and possibly cell and gene therapy.

Genomic safe harbors (GSHs) are sites in the genome able to accommodate the integration of new genetic material in a manner that ensures that the newly inserted genetic elements: (i) function predictably and (ii) do not cause alterations of the host genome posing a risk to the host cell or organism. GSHs are thus ideal sites for transgene insertion whose use can empower functional genetics studies in basic research and therapeutic applications in human gene therapy. Currently, no fully validated GSHs exist in the human genome. Here, we review our formerly proposed GSH criteria and discuss additional considerations on extending these criteria, on strategies for the identification and validation of GSHs, as well as future prospects on GSH targeting for therapeutic applications. In view of recent advances in genome biology, gene targeting technologies, and regenerative medicine, gene insertion into GSHs can potentially catalyze nearly all applications in human gene therapy.

Human pluripotent stem cells (hPSCs) hold great promise for cell therapy. However, a major concern is the risk of tumor formation by residual undifferentiated cells contaminating the hPSC-derived cell product. Suicide genes could safeguard against such adverse events by enabling elimination of cells gone astray, but the efficacy of this approach has not yet been thoroughly tested. Here, we engineered a lentivirally encoded herpes simplex virus thymidine kinase (HSVtk) with expression restricted to undifferentiated hPSCs through regulation by the let7 family of miRNAs. We show that induced pluripotent stem cells (iPSCs) expressing a let7-regulated HSVtk transgene are selectively killed by ganciclovir (GCV), whereas differentiated cells are fully protected. However, in contrast to previous studies, we find that in vivo GCV administration results in longer latency but does not prevent teratoma formation by iPSCs expressing either a constitutive or a let7-regulated HSVtk, without evidence of silencing of the HSVtk. Clonal analyses of iPSCs expressing HSVtk revealed frequent emergence of GCV resistance which, at least in some cases, could be attributed to preexisting inactivating mutations in the HSVtk coding sequence, selected for upon GCV treatment. Our findings have important consequences for the future use of suicide genes in hPSC-based cell therapies.

Chromosomal deletions associated with human diseases, such as cancer, are common, but synteny issues complicate modeling of these deletions in mice. We use cellular reprogramming and genome engineering to functionally dissect the loss of chromosome 7q (del(7q)), a somatic cytogenetic abnormality present in myelodysplastic syndromes (MDS). We derive del(7q)- and isogenic karyotypically normal induced pluripotent stem cells (iPSCs) from hematopoietic cells of MDS patients and show that the del(7q) iPSCs recapitulate disease-associated phenotypes, including impaired hematopoietic differentiation. These disease phenotypes are rescued by spontaneous dosage correction and can be reproduced in karyotypically normal cells by engineering hemizygosity of defined chr7q segments in a 20-Mb region. We use a phenotype-rescue screen to identify candidate haploinsufficient genes that might mediate the del(7q)- hematopoietic defect. Our approach highlights the utility of human iPSCs both for functional mapping of disease-associated large-scale chromosomal deletions and for discovery of haploinsufficient genes.

Epigenetic regulation of key transcriptional programs is a critical mechanism that controls hematopoietic development, and, thus, aberrant expression patterns or mutations in epigenetic regulators occur frequently in hematologic malignancies. We demonstrate that the Polycomb protein L3MBTL1, which is monoallelically deleted in 20q- myeloid malignancies, represses the ability of stem cells to drive hematopoietic-specific transcriptional programs by regulating the expression of SMAD5 and impairing its recruitment to target regulatory regions. Indeed, knockdown of L3MBTL1 promotes the development of hematopoiesis and impairs neural cell fate in human pluripotent stem cells. We also found a role for L3MBTL1 in regulating SMAD5 target gene expression in mature hematopoietic cell populations, thereby affecting erythroid differentiation. Taken together, we have identified epigenetic priming of hematopoietic-specific transcriptional networks, which may assist in the development of therapeutic approaches for patients with anemia., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)

The long-term risk of malignancy associated with stem cell therapies is a significant concern in the clinical application of this exciting technology. We report a cancer-selective strategy to enhance the safety of stem cell therapies. Briefly, using a cell engineering approach, we show that aggressive cancers derived from human or murine induced pluripotent stem cells (iPSCs) and embryonic stem cells (ESCs) are strikingly sensitive to temporary MYC blockade. On the other hand, differentiated tissues derived from human or mouse iPSCs can readily tolerate temporary MYC inactivation. In cancer cells, endogenous MYC is required to maintain the metabolic and epigenetic functions of the embryonic and cancer-specific pyruvate kinase M2 isoform (PKM2). In summary, our results implicate PKM2 in cancer's increased MYC dependence and indicate dominant MYC inhibition as a cancer-selective fail-safe for stem cell therapies., (Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.)

Oct4 is an essential regulator of pluripotency in vivo and in vitro in embryonic stem cells, as well as a key mediator of the reprogramming of somatic cells into induced pluripotent stem cells. It is not known whether activation and/or repression of specific genes by Oct4 is relevant to these functions. Here, we show that fusion proteins containing the coding sequence of Oct4 or Xlpou91 (the Xenopus homolog of Oct4) fused to activating regions, but not those fused to repressing regions, behave as Oct4, suppressing differentiation and promoting maintenance of undifferentiated phenotypes in vivo and in vitro. An Oct4 activation domain fusion supported embryonic stem cell self-renewal in vitro at lower concentrations than that required for Oct4 while alleviating the ordinary requirement for the cytokine LIF. At still lower levels of the fusion, LIF dependence was restored. We conclude that the necessary and sufficient function of Oct4 in promoting pluripotency is to activate specific target genes., (Copyright © 2012 The Authors. Published by Elsevier Inc. All rights reserved.)

Many applications in human pluripotent stem cell (PSC) research require the genetic modification of PSCs to express a transgene in a stable and dependable manner. Random transgene integration commonly results in unpredictable and heterogeneous expression. We describe a protocol for the derivation of clonal populations of human embryonic stem cells or induced pluripotent stem cells (iPSCs) expressing a transgene from a single copy of an integrated lentiviral vector that is mapped to the genome. Using optimized transduction conditions, followed by single-cell subcloning and a round of antibiotic selection, we find that approximately half of the colonies retrieved contain a single vector copy. After expansion, the majority of these are confirmed to be clonal. The vector/genomic DNA junction is sequenced and the unique integration site is mapped to the genome. This protocol enables the efficient derivation of genetically modified PSCs containing an integrated transgene at a known genomic site in ∼7 weeks.