GATA1 Mutations Research Articles

Gata1s mutant mice display persistent defects in the erythroid lineage.

GATA1 mutations that result in loss of the N-terminal 83 amino acids are a feature of myeloid leukemia in children with Down syndrome, rare familial cases of dyserythropoietic anemia, and a subset of cases of Diamond-Blackfan anemia. The Gata1s mouse model, which expresses only the short GATA1 isoform that begins at methionine 84, has been shown to have a defect in hematopoiesis, especially impaired erythropoiesis with expanded megakaryopoiesis, during gestation. However, these mice reportedly did not show any postnatal phenotype. Here, we demonstrate that Gata1s mutant mice display macrocytic anemia and features of aberrant megakaryopoiesis throughout life, culminating in profound splenomegaly and bone marrow fibrosis. These data support the use of this animal model for studies of GATA1 deficiencies.

Myeloid Leukemia of Down Syndrome.

Myeloid leukemia of Down syndrome (ML-DS) is characterized by a distinct natural history and is classified by the World Health Organization (WHO) as an independent entity, occurring with unique clinical and molecular features. The presence of a long preleukemic, myelodysplastic phase, called transient abnormal myelopoiesis (TAM), precedes the initiation of ML-DS and is defined by unusual chromosomal findings. Individuals with constitutional trisomy 21 have a profound dosage imbalance in the hematopoiesis-governing genes located on chromosome 21 and thus are subject to impaired fetal as well as to neonatal erythro-megakaryopoiesis. Almost all neonates with DS develop quantitative and morphological hematological abnormalities, yet still only 5-10% of them present with one of the preleukemic or leukemic conditions of DS. The acquired mutations in the key hematopoietic transcription factor gene GATA1, found solely in cells trisomic for chromosome 21, are considered to be the essential step for the selective growth advantage of leukemic cells. While the majority of cases of TAM remain clinically 'silent' or undergo spontaneous remission, the remaining 20% to 30% of them progress into ML-DS until the age of 4 years. The hypersensitivity of ML-DS blasts to chemotherapeutic agents, including but not limited to cytarabine, and drugs' increased infectious and cardiac toxicity have necessitated the development of risk-adapted treatment protocols for children with ML-DS. Recent advances in cytogenetics and specific molecular mechanisms involved in the evolution of TAM and ML-DS are reviewed here, as well as their integration in the improvement of risk stratification and targeted management of ML-DS.

Advances in molecular characterization of pediatric acute megakaryoblastic leukemia not associated with Down syndrome; impact on therapy development.

Acute megakaryoblastic leukemia (AMKL) is a rare subtype of acute myeloid leukemia (AML) in which leukemic blasts have megakaryocytic features. AMKL makes up 4%-15% of newly diagnosed pediatric AML, typically affecting young children (less than 2years old). AMKL associated with Down syndrome (DS) shows GATA1 mutations and has a favorable prognosis. In contrast, AMKL in children without DS is often associated with recurrent and mutually exclusive chimeric fusion genes and has an unfavorable prognosis. This review mainly summarizes the unique features of pediatric non-DS AMKL and highlights the development of novel therapies for high-risk patients. Due to the rarity of pediatric AMKL, large-scale multi-center studies are needed to progress molecular characterization of this disease. Better disease models are also required to test leukemogenic mechanisms and emerging therapies.

Lineage skewing and genome instability underlie marrow failure in a zebrafish model of GATA2 deficiency

Inherited bone marrow failure associated with heterozygous mutations in GATA2 predisposes toward hematological malignancies, but the mechanisms remain poorly understood. Here, we investigate the mechanistic basis of marrow failure in a zebrafish model of GATA2 deficiency. Single-cell transcriptomics and chromatin accessibility assays reveal that loss of gata2a leads to skewing toward the erythroid lineage at the expense of myeloid cells, associated with loss of cebpa expression and decreased PU.1 and CEBPA transcription factor accessibility in hematopoietic stem and progenitor cells (HSPCs). Furthermore, gata2a mutants show impaired expression of npm1a, the zebrafish NPM1 ortholog. Progressive loss of npm1a in HSPCs is associated with elevated levels of DNA damage in gata2a mutants. Thus, Gata2a maintains myeloid lineage priming through cebpa and protects against genome instability and marrow failure by maintaining expression of npm1a. Our results establish a potential mechanism underlying bone marrow failure in GATA2 deficiency.

GATA2 Deficiency: Predisposition to Myeloid Malignancy and Hematopoietic Cell Transplantation.

GATA2 deficiency is a haploinsufficiency syndrome associated with a wide spectrum of disease, including severe monocytopenia and B and NK lymphopenia, predisposition to myeloid malignancies, human papillomavirus infections, and infections with opportunistic organisms, particularly nontuberculous mycobacteria, herpes virus, and certain fungi. GATA2 mutations have variable penetrance and expressivity with imperfect genotype-phenotype correlations. However, approximately 75% of patients will develop a myeloid neoplasm at some point. Allogeneic hematopoietic cell transplantation (HCT) is the only currently available curative therapy. Here, we review the clinical manifestations of GATA2 deficiency, characterization of the hematologic abnormalities and progression to myeloid malignancy, and current HCT practices and outcomes. Cytogenetic abnormalities are common with high rates of trisomy 8, monosomy 7, and unbalanced translocation der(1;7) and may suggest an underlying GATA2 deficiency in patients presenting with myelodysplastic syndrome (MDS). Mutations in ASXL1 and STAG2 are the most frequently encountered somatic mutations and are associated with lower survival probability. A recent report of 59 patients with GATA2 deficiency who underwent allogenic HCT with myeloablative, busulfan-based conditioning and post-transplant cyclophosphamide reported excellent overall and event-free survival of 85% and 82% with reversal of disease phenotype and low rates of graft versus host disease. Allogeneic HCT with myeloablative conditioning results in disease correction and should be considered for patients with a history of recurrent, disfiguring and/or severe infections, organ dysfunction, MDS with cytogenetic abnormalities, high-risk somatic mutations or transfusion dependence, or myeloid progression. Improved genotype/phenotype correlations are needed to allow for greater predictive capabilities.

ACase of GATA2 Deficiency with Pancytopenia and Recurrent Infections: To Transplant or Not to Transplant?

BackgroundGATA2 deficiency is an autosomal dominant disorder arising from various mutations in the GATA2 gene, which encodes a zinc finger transcription factor that is required for hematopoiesis and angiogenesis. Patients with GATA2 deficiency can present with a range of hematologic and immunologic manifestations. We present a case of GATA2 deficiency with recurrent transient pancytopenia who is also heterozygous for C9 deficiency. Case presentationOur 30-year-old female patient presented with febrile neutropenia (ANC 850) and rash. Interestingly, she reported a history of recurrent viral upper respiratory infections since childhood and had mononucleosis at the age of 5. In her mid-twenties, she started having recurrent sinusitis and pneumonias, requiring hospitalization and antibiotic therapy 1–2 times per year. She also had persistent pancytopenia for at least 5 years that transiently improved with transfusions. CBC ranged from Hgb 5.5–10.1 g/dL (mean 8), platelets 30–102 K/uL (mean 67), WBC 1.4–5.6 K/uL (mean 2.87), neutrophils 0.75–4.35 K/uL (mean 2), monocytes 0–0.07 K/uL (mean 0.02), lymphocytes 0.38–1.18 K/uL (mean 0.8). Bone marrow biopsy showed severely hypocellular bone marrow with trilineage hypoplasia without dysplasia or neoplasia. We initiated an immune workup that showed normal IgG, IgA and IgM levels; low B cell numbers; absent NK cells (CD56+); and preserved T cell numbers on flow cytometry. Strep pneumoniae IgG levels were nonprotective with less than 0.1 ug/mL for all serotypes. Genetic panel testing for primary immunodeficiency revealed a heterozygous pathogenic mutation in GATA2 (c.1081C>T (p.Arg361Cys). Upon multidisciplinary discussion with Hematology, decision was made to begin immunoglobulin replacement. Bone marrow transplant workup was initiated, but the team is hesitant to proceed with transplant at this time. DiscussionHematopoietic stem cell transplantation is the only treatment option with curative potential for this patient, but there are significant associated morbidity and mortality risks. Identifying the appropriate timing for transplantation remains a central focus of this patient's care. Close monitoring of the patient's overall clinical evolution including peripheral cell counts, bone marrow findings, and infection frequency and severity could help approximate the optimal transplantation timing after careful consideration of risks and benefits.

GATA2 at 10: Emergence of Genotype-Phenotye correlations

GATA2 mutations cause adult-onset bone-marrow failure characterized by cytopenias, infections and increased risk of leukemia. We reviewed hospital records and referrals from 232 GATA2 mutation positive individuals from 122 families to gather hematopoietic and syndromic features.Mutations were categorized by effect on GATA2 protein: missense after the 2nd Zinc-finger (C-term, n = 10); missense in the 2nd Zinc-Finger (2nd Zf, n = 104); mutations producing stable truncated protein (truncation, n = 26); null alleles (mRNA instability, large deletions, n = 42); or enhancer (n = 50). Median age at first symptom presentation for the cohort was 20 years.Kaplan-Meier analysis for symptom onset showed differences in age at presentation across truncation and null mutations (median 15 and 18 years, respectively), 2nd Zf (22 years) and enhancer mutations (45 years). A similar analysis of four recurrently mutated 2nd ZF amino acids revealed later symptom onset for R361 and R398 mutations (median 22 and 34 years, respectively) compared to R396 or T354 (16 and 19 years, respectively). Mixed-effect Cox regression analysis corrected for relatedness across patients and confirmed these findings.There was decreased penetrance in enhancer (27/50, 54% symptomatic) and 2nd Zf (85/104, 81.7%) compared to truncation (24/26, 92.3%) and null (39/40, 97.5%) mutation patients. Grouping mutations by median onset before (null, truncation, R396, T354) or after (R361, R398, enhancer) age 20 revealed significantly increased incidence of cytopenias, HPV, myelodysplastic syndrome and hearing loss among the early onset mutation group. Additionally, age of cytopenia or myelodysplasia syndrome diagnosis was significantly younger in the early onset group (P = 0.0019, P = 0.0012 respectively).Males had more monosomy 7 (11/92 males, 4/142 females, P < 0.0001). Nontuberculous mycobacterial infection was the presenting symptom in 14/92 males vs 7/142 females (P = 0.0188). Warts/anogenital HPV were common (93/234, 39.7%). Non-hematologic manifestations included hearing loss (46/232; 19.8%), lymphedema (30/232; 12.8%), and non-hematologic malignancies (46/232, 19.8%), the majority being HPV-related ano-genital cancers. Hematologic malignancy occurred in 26/234 patients (11%).This comprehensive analysis demonstrates that GATA2 mutation type, and specific mutations, affect penetrance and expression. Gender is an independent risk for specific infectious complications and cytogenetic abnormalities.

Megakaryoblasts in pleural fluid in neonate with transient leukaemia of Down's syndrome.

Twin baby boy born at 35+2 weeks gestation with a birth weight of 2070 grams via emergency cesarean section secondary to abnormal CTG and foetal distress. Full blood count obtained at birth showed haemoglobin 159 g/L, total white cell count 99.54 × 109/L platelet count 779 × 109/L. Manual differential showed an absolute neutrophil count of 2.01 × 109/L absolute lymphocyte count of 45.27 × 109/L and blast count of 50.3 × 109/L (50%). Initial peripheral blood smear morphology review revealed a thrombocytosis, platelet anisocytosis and micromegakaryocytes. It also demonstrated neutropenia, myeloid left shift, large blasts with a high N/C ratio and nucleoli. Additionally, numerous nucleated red blood cells were seen with marked red cell dysplasia including binucleate forms and abnormal haemoglobinisation. There was no prenatal diagnosis of Down's syndrome (DS). The above blood results along with the baby's clinical signs raised suspicion of DS and subsequently transient leukaemia of DS. This baby was then transferred to the regional neonatal intensive care unit on day 2 of life. On admission, he was noted to have bilateral pleural effusion, synthetic liver dysfunction with elevated creatinine and ammonia levels as well as ascites. He was intubated, and ventilated and required inotropic support. Due to adverse features of elevated white cell count, hepatosplenomegaly, pleural effusion as well as ascites [1] and following the multidisciplinary team (MDT) discussion and parental consent, the patient was commenced on low dose cytarabine for 7 days. He tolerated that well, however, did experience mild cytopenias and pyrexic episodes, followed by recovery during the following two weeks with a reduction in pleural effusions, hepatosplenomegaly and reduction in oxygen requirement which enabled successful extubation and commencement of continuous positive airway pressure. Unfortunately, on day 19 of life he was noted to have slowly increasing oxygen requirements with re-accumulation of pleural effusions and increasing hepatosplenomegaly as well as increasing blast count in peripheral blood. He proceeded to require intubation and ventilation again as well as placement of a chest drain to remove pleural fluid to improve respiratory compliance. A review of pleural fluid morphology had shown frequent ‘blast-like’ cells; these were medium-sized with a high N/C ratio with finely condensed chromatin nucleoli and budded basophilic cytoplasm thought to most likely represent megakaryoblasts [2]. As a result of the deterioration, a decision by MDT was made to give another cycle of low-dose cytarabine on days 1 through 5. Following the second cycle of cytarabine, the patient again experienced mild cytopenias and pyrexic episodes, was treated with antibiotics and supportive management and transfusions of packed red cells. He had complete count recovery and no blast population identified morphologically on peripheral blood film on day 26 of the second cycle of cytarabine. His GATA1 mutation was subsequently found to be positive. He continued to improve and was discharged home on day 67 of life Figures 1-3. Karolina Chmielowiec—wrote the manuscript, Gary Beattie—manuscript review, obtained image, obtained and analysed flow cytometry data, Kathryn Clarke—manuscript review, obtained and analysed flow cytometry, Ellen McCloy—manuscript review, morphology review, Bethany Mitchell—manuscript review, morphology review, Christine Macartney—manuscript review, supervising consultant. All authors discussed the results and commented on the manuscript. None. The authors declare no conflict of interest. No funding was required for this submission. Not applicable. Not Applicable. Not Applicable All authors have contributed to the acquisition, analysis and interpretation of data as well as either drafting the paper or revising it critically. All authors approved the submitted and final versions. For original data, please contact [email protected]

Successful allogeneic hematopoietic stem cell transplantation for myelodysplastic neoplasms complicated with secondary pulmonary alveolar proteinosis and Behçet's disease harboring GATA2 mutation.

Myelodysplastic neoplasms (MDS) are defined by cytopenia and morphologic dysplasia originating from clonal hematopoiesis. They are also frequently complicated with diseases caused by immune dysfunction, such as Behçet's disease (BD) and secondary pulmonary alveolar proteinosis (sPAP). MDS with both BD and sPAP is extremely rare, and their prognosis is poor. In addition, haploinsufficiency of the hematopoietic transcription factor gene GATA2 is recognized as a cause of familial MDS and is frequently complicated by sPAP. Herein, we report a case of MDS combined with both BD and sPAP in association with GATA2 deficiency in a Japanese woman. Because she developed progressive leukopenia and macrocytic anemia during BD treatment at the age of 61, she underwent a bone-marrow examination and was diagnosed with MDS. She subsequently developed sPAP. At the age of 63, she underwent allogeneic hematopoietic stem cell transplantation (allo-HSCT). Since allo-HSCT, she has maintained complete remission of MDS as well as the symptoms of BD and sPAP. Furthermore, we performed whole exome sequencing and identified the GATA2 Ala164Thr germline mutation. These findings suggest that patients with MDS, BD and sPAP should be considered for early allo-HSCT.

Generation of 2 isogenic clones from a patient with Trisomy 21 and a GATA1 mutation

Trisomy 21 (T21), or Down Syndrome (DS), is a common chromosomal disorder resulting from a third copy of chromosome 21 (HSA21). Transient myeloproliferative disorder (TMD) is a pre-leukemic condition that occurs only in neonates with DS and is characterized by a mutation in the transcription factor GATA1 that results in a truncated protein (GATA1s). We generated a pair of isogenic T21 lines derived from a patient with TMD that differ only in GATA1 status. The iPSC lines were characterized for pluripotency, differentiation potential, and genomic stability. These lines are a valuable resource for studying T21 hematopoietic diseases.

Validation of the prognostic significance of the 2022 European LeukemiaNet risk stratification system in intensive chemotherapy treated aged 18 to 65 years patients with de novo acute myeloid leukemia.

The European LeukemiaNet (ELN) recently proposed a revised recommendation for the diagnosis and management of acute myeloid leukemia (AML) in adults, recognized as ELN-2022. However, validation in a large real-world cohort remains lacking. In this study, we aimed to validate the prognostic relevance of the ELN-2022 in a cohort of 809 de novo, non-M3, younger (ages 18-65 years) AML patients receiving standard chemotherapy. The risk categories of 106 (13.1%) patients were reclassified from that determined using ELN-2017 to that determined using ELN-2022. The ELN-2022 effectively helped distinguish patients as favorable, intermediate, and adverse risk groups in terms of remission rates and survival. Among patients who achieved first complete remission (CR1), allogeneic transplantation was beneficial for those in the intermediate risk group, but not for those in the favorable or adverse risk groups. We further refined the ELN-2022 system by re-categorizing AML patients with t(8;21)(q22;q22.1)/RUNX1::RUNX1T1 with KIThigh , JAK2 or FLT3-ITDhigh mutations into the intermediate risk subset, AML patients with t(7;11)(p15;p15)/NUP98::HOXA9 and AML patients with co-mutated DNMT3A and FLT3-ITD into the adverse risk subsets, and AML patients with complex or monosomal karyotypes, inv (3)(q21.3q26.2) or t(3;3)(q21.3;q26.2)/GATA2,MECOM(EVI1) or TP53 mutation into the very adverse risk subset. The refined ELN-2022 system performed effectively to distinguish patients as favorable, intermediate, adverse, and very adverse risk groups. In conclusion, the ELN-2022 helped distinguish younger, intensively treated patients into three groups with distinct outcomes; the proposed refinement of ELN-2022 may further improve risk stratification among AML patients. Prospective validation of the new predictive model is necessary.

Heterogeneity analysis of the CEBPAdm AML based on bZIP region mutations.

Patients with double-mutated CEBPA (CEBPAdm) AML were stratified into favorable risk group, however, few studies have investigated the heterogeneity of different CEBPAdm types in detail. In this study, we analyzed 2211 newly diagnosed AML and identified CEBPAdm in 10.8% of the patients. Within the CEBPAdm cohort, 225 of 239 patients (94.14%) presented with bZIP region mutations (CEBPAdmbZIP) while 14 of 239 patients (5.86%) without bZIP region mutation (CEBPAdmnonbZIP). Analysis of the accompanied molecular mutations showed statistically different incidences of GATA2 mutations between the CEBPAdmbZIP group and the CEBPAdmnonbZIP group (30.29% vs 0%). In the analysis of outcomes, patients with CEBPAdmnonbZIP were associated with shorter overall survival (OS) censored at hematopoietic stem cell transplantation (HSCT) during CR1 compared to those with CEBPAdmbZIP (hazard ratio (HR) = 3.132, 95% confidence interval (CI) = 1.229-7.979, P = .017). Refractory or relapsed AML (R/RAML) patients with CEBPAdmnonbZIP were associated with shorter OS compared to those with CEBPAdmbZIP (HR = 2.881, 95% CI = 1.021-8.131, P = .046). Taken together, AML with CEBPAdmbZIP and CEBPAdmnonbZIP showed different outcomes and might be regarded as distinctive AML entities.

De novo intronic GATA1 mutation leads to diamond-blackfan anemia like disease.

GATA1 is required for normal erythropoiesis. Exonic/intronic GATA1 mutations causes Diamond-Blackfan Anemia (DBA)-like disease. Herein, we present a case of a 5-year-old boy with anemia of unknown etiology. Whole-exome sequencing revealed a de novo GATA1 c.220 + 1G>C mutation. The reporter gene assay revealed that such mutations did not affect on GATA1 transcriptional activity. The normal transcription of GATA1 was disturbed, as evidenced by increased expression of the shorter GATA1 isoform. RDDS prediction analysis revealed that abnormal GATA1 splicing might be the underlying mechanism disrupting GATA1 transcription, thereby impairing erythropoiesis. Prednisone treatment significantly improved erythropoiesis, evidenced by increased hemoglobin and reticulocyte counts.

Somatic genetic alterations predict hematological progression in GATA2 deficiency.

Germline GATA2 mutations predispose to myeloid malignancies resulting from the progressive acquisition of additional somatic mutations. Here we describe clinical and biological features of 78 GATA2-deficient patients. Hematopoietic stem and progenitor cell phenotypic characterization revealed an exhaustion of myeloid progenitors. Somatic mutations in STAG2, ASXL1 and SETBP1 genes along with cytogenetic abnormalities (monosomy 7, trisomy 8, der(1;7)) occurred frequently in patients with GATA2 germline mutations. Patients were classified into three hematopoietic spectra based on bone marrow cytomorphology. No somatic additional mutations were detected in patients with normal bone marrow (spectrum 0), whereas clonal hematopoiesis mediated by STAG2 mutations was frequent in those with a hypocellular and/or myelodysplastic bone marrow without excess blasts (spectrum 1). Finally, SETBP1, RAS pathway and RUNX1 mutations were predominantly associated with leukemic transformation stage (spectrum 2), highlighting their implications in the transformation process. Specific somatic alterations, potentially providing distinct selective advantages to affected cells, are therefore associated with the clinical/hematological evolution of GATA2 syndrome. Our study not only suggests that somatic genetic profiling will help clinicians in their management of patients, but will also clarify the mechanism of leukemogenesis in the context of germline GATA2 mutations.

Predicting Modifiers of Genotype-Phenotype Correlations in Craniofacial Development.

Most human birth defects are phenotypically variable even when they share a common genetic basis. Our understanding of the mechanisms of this variation is limited, but they are thought to be due to complex gene-environment interactions. Loss of the transcription factor Gata3 associates with the highly variable human birth defects HDR syndrome and microsomia, and can lead to disruption of the neural crest-derived facial skeleton. We have demonstrated that zebrafish gata3 mutants model the variability seen in humans, with genetic background and candidate pathways modifying the resulting phenotype. In this study, we sought to use an unbiased bioinformatic approach to identify environmental modifiers of gata3 mutant craniofacial phenotypes. The LINCs L1000 dataset identifies chemicals that generate differential gene expression that either positively or negatively correlates with an input gene list. These chemicals are predicted to worsen or lessen the mutant phenotype, respectively. We performed RNA-seq on neural crest cells isolated from zebrafish across control, Gata3 loss-of-function, and Gata3 rescue groups. Differential expression analyses revealed 551 potential targets of gata3. We queried the LINCs database with the 100 most upregulated and 100 most downregulated genes. We tested the top eight available chemicals predicted to worsen the mutant phenotype and the top eight predicted to lessen the phenotype. Of these, we found that vinblastine, a microtubule inhibitor, and clofibric acid, a PPAR-alpha agonist, did indeed worsen the gata3 phenotype. The Topoisomerase II and RNA-pol II inhibitors daunorubicin and triptolide, respectively, lessened the phenotype. GO analysis identified Wnt signaling and RNA polymerase function as being enriched in our RNA-seq data, consistent with the mechanism of action of some of the chemicals. Our study illustrates multiple potential pathways for Gata3 function, and demonstrates a systematic, unbiased process to identify modifiers of genotype-phenotype correlations.

RUNX1 isoform disequilibrium promotes the development of trisomy 21–associated myeloid leukemia

AbstractGain of chromosome 21 (Hsa21) is among the most frequent aneuploidies in leukemia. However, it remains unclear how partial or complete amplifications of Hsa21 promote leukemogenesis and why children with Down syndrome (DS) (ie, trisomy 21) are particularly at risk of leukemia development. Here, we propose that RUNX1 isoform disequilibrium with RUNX1A bias is key to DS-associated myeloid leukemia (ML-DS). Starting with Hsa21-focused CRISPR–CRISPR-associated protein 9 screens, we uncovered a strong and specific RUNX1 dependency in ML-DS cells. Expression of the RUNX1A isoform is elevated in patients with ML-DS, and mechanistic studies using murine ML-DS models and patient-derived xenografts revealed that excess RUNX1A synergizes with the pathognomonic Gata1s mutation during leukemogenesis by displacing RUNX1C from its endogenous binding sites and inducing oncogenic programs in complex with the MYC cofactor MAX. These effects were reversed by restoring the RUNX1A:RUNX1C equilibrium in patient-derived xenografts in vitro and in vivo. Moreover, pharmacological interference with MYC:MAX dimerization using MYCi361 exerted strong antileukemic effects. Thus, our study highlights the importance of alternative splicing in leukemogenesis, even on a background of aneuploidy, and paves the way for the development of specific and targeted therapies for ML-DS, as well as for other leukemias with Hsa21 aneuploidy or RUNX1 isoform disequilibrium.

Leukemogenesis in infants and young children with trisomy 21.

Children with Down syndrome (DS) have a greater than 100-fold increased risk of developing acute myeloid leukemia (ML) and an approximately 30-fold increased risk of acute lymphoblastic leukemia (ALL) before their fifth birthday. ML-DS originates in utero and typically presents with a self-limiting, neonatal leukemic syndrome known as transient abnormal myelopoiesis (TAM) that is caused by cooperation between trisomy 21-associated abnormalities of fetal hematopoiesis and somatic N-terminal mutations in the transcription factor GATA1. Around 10% of neonates with DS have clinical signs of TAM, although the frequency of hematologically silent GATA1 mutations in DS neonates is much higher (~25%). While most cases of TAM/silent TAM resolve without treatment within 3 to 4 months, in 10% to 20% of cases transformation to full-blown leukemia occurs within the first 4 years of life when cells harboring GATA1 mutations persist and acquire secondary mutations, most often in cohesin genes. By contrast, DS-ALL, which is almost always B-lineage, presents after the first few months of life and is characterized by a high frequency of rearrangement of the CRLF2 gene (60%), often co-occurring with activating mutations in JAK2 or RAS genes. While treatment of ML-DS achieves long-term survival in approximately 90% of children, the outcome of DS-ALL is inferior to ALL in children without DS. Ongoing studies in primary cells and model systems indicate that the role of trisomy 21 in DS leukemogenesis is complex and cell context dependent but show promise in improving management and the treatment of relapse, in which the outcome of both ML-DS and DS-ALL remains poor.

Cellular and metabolic characteristics of pre-leukemic hematopoietic progenitors with GATA2 haploinsufficiency.

Mono-allelic germline disruptions of the transcription factor GATA2 result in a propensity for developing myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML), affecting more than 85% of carriers. How a partial loss of GATA2 functionality enables leukemic transformation years later is unclear. This question has remained unsolved mainly due to the lack of informative models, as Gata2 heterozygote mice do not develop hematologic malignancies. Here we show that two different germline Gata2 mutations (TgErg/Gata2het and TgErg/Gata2L359V) accelerate AML in mice expressing the human hematopoietic stem cell regulator ERG. Analysis of Erg/Gata2het fetal liver and bone marrow-derived hematopoietic cells revealed a distinct pre-leukemic phenotype. This was characterized by enhanced transition from stem to progenitor state, increased proliferation, and a striking mitochondrial phenotype, consisting of highly expressed oxidative-phosphorylation-related gene sets, elevated oxygen consumption rates, and notably, markedly distorted mitochondrial morphology. Importantly, the same mitochondrial gene-expression signature was observed in human AML harboring GATA2 aberrations. Similar to the observations in mice, non-leukemic bone marrows from children with germline GATA2 mutation demonstrated marked mitochondrial abnormalities. Thus, we observed the tumor suppressive effects of GATA2 in two germline Gata2 genetic mouse models. As oncogenic mutations often accumulate with age, GATA2 deficiency-mediated priming of hematopoietic cells for oncogenic transformation may explain the earlier occurrence of MDS/AML in patients with GATA2 germline mutation. The mitochondrial phenotype is a potential therapeutic opportunity for the prevention of leukemic transformation in these patients.

Mechanism of KIT gene regulation by GATA1 lacking the N-terminal domain in Down syndrome–related myeloid disorders

Children with Down syndrome (DS) are at high risk of transient abnormal myelopoiesis (TAM) and myeloid leukemia of DS (ML-DS). GATA1 mutations are detected in almost all TAM and ML-DS samples, with exclusive expression of short GATA1 protein (GATA1s) lacking the N-terminal domain (NTD). However, it remains to be clarified how GATA1s is involved with both disorders. Here, we established the K562 GATA1s (K562-G1s) clones expressing only GATA1s by CRISPR/Cas9 genome editing. The K562-G1s clones expressed KIT at significantly higher levels compared to the wild type of K562 (K562-WT). Chromatin immunoprecipitation studies identified the GATA1-bound regulatory sites upstream of KIT in K562-WT, K562-G1s clones and two ML-DS cell lines; KPAM1 and CMK11-5. Sonication-based chromosome conformation capture (3C) assay demonstrated that in K562-WT, the − 87 kb enhancer region of KIT was proximal to the − 115 kb, − 109 kb and + 1 kb region, while in a K562-G1s clone, CMK11-5 and primary TAM cells, the − 87 kb region was more proximal to the KIT transcriptional start site. These results suggest that the NTD of GATA1 is essential for proper genomic conformation and regulation of KIT gene expression, and that perturbation of this function might be involved in the pathogenesis of TAM and ML-DS.

Multiplex CRISPR/Cas9 Gene Editing in Human Stem Cells to Model Clonal Competition in GATA2 Deficiency

GATA2 deficiency has been identified as a common hereditary cause of myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) in children. Penetrance and expressivity within affected families is often variable, suggesting that cooperating factors are required to trigger the disease. Somatic mutations in MDS driver genes (i.e. SETBP1, ASXL1) have been identified by us and others in GATA2-MDS. Despite recent studies identifying germline genetics and acquired mutations in GATA2 deficiency, the understanding of the molecular mechanism that triggers the leukemic progression remains still unknown. Particularly, it is still unanswered the following biological questions: 1) whether GATA2 germline mutation itself is sufficient to trigger MDS/AML 2) whether SETBP1 and ASXL1 mutations induce the malignant transformation. Addressing these questions has been challenging due to the absence of trustworthy human disease models. Here, we used CRISPR/Cas9 genome engineering of primary Cord Blood (CB) CD34+ cells followed by serial transplantation into immunodeficient mice to study the genetic malignant effect of GATA2 (loss-of-function R398W mutation), SETBP1 (gain-of-function D868N) and ASXL1 (loss-of-function G646X) mutations. Specifically, CRISPR/Cas9 in combination with recombinant adeno-associated virus serotype 6 (rAAV6) were used to knock-in a modified GATA2 exon 6 (exon6-T2A-GFP) carrying the R398W mutation in CB-CD34+ cells alone (referred to as GATA2) or in combination with mutations in SETBP1 and ASXL1 (TRIPLE). Mock-treated CD34+ cells were nucleofected without RNP, rAAV6 or ssODN (Control). Four days after the nucleofection, ~11% of cells were GFP positive in GATA2 condition (n=3) and ~7% in TRIPLE condition (n=3), which directly correlates with the percentage of GATA2-R398W mutant cells. Next, manipulated CD34+ cells (Control, GATA2 and TRIPLE) were transplanted into sublethally irradiated NSG mice (n=24). Sixteen weeks after transplantation, 18/24 mice showed more than 10% of human cell engraftment (hCD45) in the bone marrow (BM). Moreover, a multilineage reconstitution (hCD34 for HPCS, hCD33 for myeloid cells and hCD19 for lymphoid cells) was observed in all the conditions. In order to dissect the clonal architecture of concurrent mutations, we performed single-cell DNA-sequencing for GATA2, SETBP1 and ASXL1 in hundreds of human CD45+ engrafted cells derived from colony forming units (CFU). Fifty of a total of 118 colonies analyzed (43%) had targeted editing of at least one gene (GATA2, SETBP1 or ASXL1). Interestingly, in TRIPLE condition 13/51 (24%) colonies, more than one gene was edited. To study the long-term clonal evolution, we performed secondary transplants.Nine out of 15 mice reached the end point of 24 weeks. We noted that the predominant expanded clones were carrying either SETBP1 or SETBP1+ASXL1 mutations, while clones with only GATA2 mutation were lost in both GATA2 and TRIPLE conditions. To study the impact of the mutations at transcriptomic level, single cell RNA-seq from primary and secondary hCD45+/CD19- engrafted cells was performed (the analysis is underway). In summary, we developed a new in vivo human model of GATA2 deficiency and GATA2-MDS-specific mutations by CRISPR/Cas9 targeting of CD34+ cells. Our findings strongly suggest that GATA2 R398W mutation is not sufficient to increase the fitness of the mutant cells, suggesting that a co-operation of genetic, epigenetic and stressor factors is necessary to trigger the clonal expansion. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal