Targeting SRSF2 mutations in leukemia with RKI-1447: A strategy to impair cellular division and nuclear structure.

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.

R-Loop Accumulation in Spliceosome Mutant Leukemias Confers Sensitivity to PARP1 Inhibition by Triggering Transcription-Replication Conflicts.

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.

Hematopoietic and eosinophil-specific LNK(SH2B3) deficiency promote eosinophilia and arterial thrombosis.

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. GWAS 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 ∆dbIGata1 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. Lnk∆eos 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 NETs in thrombi and reversed the accelerated thrombosis. Human iPSC-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.

BRCC3-Mediated NLRP3 Deubiquitylation Promotes Inflammasome Activation and Atherosclerosis in Tet2 Clonal Hematopoiesis.

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-1ß 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.

Blockade of IL-6 signaling alleviates atherosclerosis in Tet2-deficient clonal hematopoiesis.

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.

Base editors dissect genetic variants in human hematopoietic cells on a large scale.

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.

Patient-Derived iPSCs Faithfully Represent the Genetic Diversity and Cellular Architecture of Human Acute Myeloid Leukemia.

The reprogramming of human acute myeloid leukemia (AML) cells into induced pluripotent stem cell (iPSC) lines could provide new faithful genetic models of AML, but is currently hindered by low success rates and uncertainty about whether iPSC-derived cells resemble their primary counterparts. Here we developed a reprogramming method tailored to cancer cells, with which we generated iPSCs from 15 patients representing all major genetic groups of AML. These AML-iPSCs retain genetic fidelity and produce transplantable hematopoietic cells with hallmark phenotypic leukemic features. Critically, single-cell transcriptomics reveal that, upon xenotransplantation, iPSC-derived leukemias faithfully mimic the primary patient-matched xenografts. Transplantation of iPSC-derived leukemias capturing a clone and subclone from the same patient allowed us to isolate the contribution of a FLT3-ITD mutation to the AML phenotype. The results and resources reported here can transform basic and preclinical cancer research of AML and other human cancers. SIGNIFICANCE: We report the generation of patient-derived iPSC models of all major genetic groups of human AML. These exhibit phenotypic hallmarks of AML in vitro and in vivo, inform the clonal hierarchy and clonal dynamics of human AML, and exhibit striking similarity to patient-matched primary leukemias upon xenotransplantation. See related commentary by Doulatov, p. 252. This article is highlighted in the In This Issue feature, p. 247.

MDS/AML with del5q: An acquired "laminopathy"?

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.

Patient-specific MDS-RS iPSCs define the mis-spliced transcript repertoire and chromatin landscape of SF3B1-mutant HSPCs.

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.

Integrative RNA-omics Discovers GNAS Alternative Splicing as a Phenotypic Driver of Splicing Factor-Mutant Neoplasms.

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.

MICA/B antibody induces macrophage-mediated immunity against acute myeloid leukemia.

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.

Modulation of the NLRP3 inflammasome by Sars-CoV-2 Envelope protein.

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-1ß 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-1ß, levels of IL-1ß 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.

Oxidized Phospholipids Promote NETosis and Arterial Thrombosis in LNK(SH2B3) Deficiency.

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 (JAK2V617F). 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 JAK2VF mutation only showed increased risk of coronary artery disease when also carrying the LNK R262W allele. Mice with hematopoietic Lnk+/- and Jak2VF clonal hematopoiesis showed accelerated arterial thrombosis but not atherosclerosis compared with Jak2VFLnk+/+ 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 Jak2VF mutations. Therapies targeting OxPL may be beneficial for coronary artery disease in genetically defined human populations.

The AIM2 inflammasome exacerbates atherosclerosis in clonal haematopoiesis.

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 factors1. Among the common genetic variants that give rise to clonal haematopoiesis, the JAK2V617F (JAK2VF) mutation, which increases JAK-STAT signalling, occurs at a younger age and imparts the strongest risk of premature coronary heart disease1,2. Here we show increased proliferation of macrophages and prominent formation of necrotic cores in atherosclerotic lesions in mice that express Jak2VF 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. Jak2VF lesions showed increased expression of AIM2, oxidative DNA damage and DNA replication stress, and Aim2 deficiency reduced atherosclerosis. Single-cell RNA sequencing analysis of Jak2VF lesions revealed a landscape that was enriched for inflammatory myeloid cells, which were suppressed by deletion of Gsdmd. Inhibition of the inflammasome product interleukin-1ß 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 Jak2VF macrophages lead to DNA replication stress and activation of the AIM2 inflammasome, thereby aggravating atherosclerosis. Precise application of therapies that target interleukin-1ß or specific inflammasomes according to clonal haematopoiesis status could substantially reduce cardiovascular risk.

Restoring RUNX1 deficiency in RUNX1 familial platelet disorder by inhibiting its degradation.

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.

Sequential CRISPR gene editing in human iPSCs charts the clonal evolution of myeloid leukemia and identifies early disease targets.

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.

Studying clonal evolution of myeloid malignancies using induced pluripotent stem cells.

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.

Modeling Leukemia Stem Cells with Patient-Derived Induced Pluripotent Stem Cells.

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.

Engineering of targeted megabase-scale deletions in human induced pluripotent stem cells.

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.