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There are few options for patients with relapse/refractory B-cell acute lymphoblastic leukemia (B-ALL), thus this is a major area of unmet medical need. Here, we reveal that inclusion of a poison exon in RBM39, which could be induced both by CDK9 or CDK9 independent CMGC (cyclin-dependent kinases, mitogen-activated protein kinases, glycogen synthase kinases, CDC-like kinases) kinase inhibition, is recognized by the nonsense-mediated mRNA decay (NMD) pathway for degradation. Targeting this poison exon in RBM39 with CMGC inhibitors lead to protein downregulation and inhibition of ALL growth, particularly in relapse/refractory B-ALL. Mechanistically, disruption of co-transcriptional splicing by inhibition of CMGC kinases including DYRK1A, or inhibition of CDK9, which phosphorylate the C-terminal domain of RNA polymerase II (Pol II), results in alteration of SF3B1 and Pol II association. Disruption of SF3B1 and transcriptional elongation complex alters Pol II pausing, which promotes the inclusion of a poison exon in RBM39. Moreover, RBM39 ablation suppresses the growth of human B-ALL, and targeting RBM39 with sulfonamides, which degrade RBM39 protein, showed strong anti-tumor activity in preclinical models. Our data reveal that relapse/refractory B-ALL is susceptible to pharmacologic and genetic inhibition of RBM39 and provide two potential strategies to target this axis., (Copyright © 2024 American Society of Hematology.)

Abstract: The spectrum of myeloid disorders ranges from aplastic bone marrow failure characterized by an empty bone marrow completely lacking in hematopoiesis to acute myeloid leukemia in which the marrow space is replaced by undifferentiated leukemic blasts. Recent advances in the capacity to sequence bulk tumor population as well as at a single-cell level has provided significant insight into the stepwise process of transformation to acute myeloid leukemia. Using models of progression in the context of germ line predisposition (trisomy 21, GATA2 deficiency, and SAMD9/9L syndrome), premalignant states (clonal hematopoiesis and clonal cytopenia of unknown significance), and myelodysplastic syndrome, we review the mechanisms of progression focusing on the hierarchy of clonal mutation and potential roles of transcription factor alterations, splicing factor mutations, and the bone marrow environment in progression to acute myeloid leukemia. Despite major advances in our understanding, preventing the progression of these disorders or treating them at the acute leukemia phase remains a major area of unmet medical need., (© 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.)

Myelofibrosis (MF) is a disease associated with high unmet medical needs because allogeneic stem cell transplantation is not an option for most patients, and JAK inhibitors are generally effective for only 2 to 3 years and do not delay disease progression. MF is characterized by dysplastic megakaryocytic hyperplasia and progression to fulminant disease, which is associated with progressively increasing marrow fibrosis. Despite evidence that the inflammatory milieu in MF contributes to disease progression, the specific factors that promote megakaryocyte growth are poorly understood. Here, we analyzed changes in the cytokine profiles of MF mouse models before and after the development of fibrosis, coupled with the analysis of bone marrow populations using single-cell RNA sequencing. We found high interleukin 13 (IL-13) levels in the bone marrow of MF mice. IL-13 promoted the growth of mutant megakaryocytes and induced surface expression of transforming growth factor β and collagen biosynthesis. Similarly, analysis of samples from patients with MF revealed elevated levels of IL-13 in the plasma and increased IL-13 receptor expression in marrow megakaryocytes. In vivo, IL-13 overexpression promoted disease progression, whereas reducing IL-13/IL-4 signaling reduced several features of the disease, including fibrosis. Finally, we observed an increase in the number of marrow T cells and mast cells, which are known sources of IL-13. Together, our data demonstrate that IL-13 is involved in disease progression in MF and that inhibition of the IL-13/IL-4 signaling pathway might serve as a novel therapeutic target to treat MF., (© 2022 by The American Society of Hematology.)

Mutations in GATA1, which lead to expression of the GATA1s isoform that lacks the GATA1 N terminus, are seen in patients with Diamond-Blackfan anemia (DBA). In our efforts to better understand the connection between GATA1s and DBA, we comprehensively studied erythropoiesis in Gata1s mice. Defects in yolks sac and fetal liver hematopoiesis included impaired terminal maturation and reduced numbers of erythroid progenitors. RNA-sequencing revealed that both erythroid and megakaryocytic gene expression patterns were altered by the loss of the N terminus, including aberrant upregulation of Gata2 and Runx1. Dysregulation of global H3K27 methylation was found in the erythroid progenitors upon loss of N terminus of GATA1. Chromatin-binding assays revealed that, despite similar occupancy of GATA1 and GATA1s, there was a striking reduction of H3K27me3 at regulatory elements of the Gata2 and Runx1 genes. Consistent with the observation that overexpression of GATA2 has been reported to impair erythropoiesis, we found that haploinsufficiency of Gata2 rescued the erythroid defects of Gata1s fetuses. Together, our integrated genomic analysis of transcriptomic and epigenetic signatures reveals that, Gata1 mice provide novel insights into the role of the N terminus of GATA1 in transcriptional regulation and red blood cell maturation which may potentially be useful for DBA patients., (© 2019 by The American Society of Hematology.)

Ras mutations are commonly observed in juvenile myelomonocytic leukemia (JMML) and chronic myelomonocytic leukemia (CMML). JMML and CMML transform into acute myeloid leukemia (AML) in about 10% and 50% of patients, respectively. However, how additional events cooperate with Ras to promote this transformation are largely unknown. We show that absence of the ubiquitin-specific peptidase 22 (USP22), a component of the Spt-Ada-GCN5-acetyltransferase chromatin-remodeling complex that is linked to cancer progression, unexpectedly promotes AML transformation in mice expressing oncogenic Kras G12D/+ USP22 deficiency in Kras G12D/+ mice resulted in shorter survival compared with control mice. This was due to a block in myeloid cell differentiation leading to the generation of AML. This effect was cell autonomous because mice transplanted with USP22-deficient Kras G12D/+ cells developed an aggressive disease and died rapidly. The transcriptome profile of USP22-deficient Kras G12D/+ progenitors resembled leukemic stem cells and was highly correlated with genes associated with poor prognosis in AML. We show that USP22 functions as a PU.1 deubiquitylase by positively regulating its protein stability and promoting the expression of PU.1 target genes. Reconstitution of PU.1 overexpression in USP22-deficient Kras G12D/+ progenitors rescued their differentiation. Our findings uncovered an unexpected role for USP22 in Ras-induced leukemogenesis and provide further insights into the function of USP22 in carcinogenesis., (© 2018 by The American Society of Hematology.)

Competing Interests: Conflict-of-interest disclosure: The author declares no competing financial interests.

GATA family proteins play essential roles in development of many cell types, including hematopoietic, cardiac, and endodermal lineages. The first three factors, GATAs 1, 2, and 3, are essential for normal hematopoiesis, and their mutations are responsible for a variety of blood disorders. Acquired and inherited GATA1 mutations contribute to Diamond-Blackfan anemia, acute megakaryoblastic leukemia, transient myeloproliferative disorder, and a group of related congenital dyserythropoietic anemias with thrombocytopenia. Conversely, germ line mutations in GATA2 are associated with GATA2 deficiency syndrome, whereas acquired mutations are seen in myelodysplastic syndrome, acute myeloid leukemia, and in blast crisis transformation of chronic myeloid leukemia. The fact that mutations in these genes are commonly seen in blood disorders underscores their critical roles and highlights the need to develop targeted therapies for transcription factors. This review focuses on hematopoietic disorders that are associated with mutations in two prominent GATA family members, GATA1 and GATA2 ., (© 2017 by The American Society of Hematology.)

Aurora kinase A (AURKA) is a therapeutic target in acute megakaryocytic leukemia. However, its requirement in normal hematopoiesis and megakaryocyte development has not been extensively characterized. Based on its role as a cell cycle regulator, we predicted that an Aurka deficiency would lead to severe abnormalities in all hematopoietic lineages. Here we reveal that loss of Aurka in hematopoietic cells causes profound cell autonomous defects in the peripheral blood and bone marrow. Surprisingly, in contrast to the survival defects of nearly all hematopoietic lineages, deletion of Aurka was associated with increased differentiation and polyploidization of megakaryocytes both in vivo and in vitro. Furthermore, in contrast to other cell types examined, megakaryocytes continued DNA synthesis after loss of Aurka. Thus, like other cell cycle regulators such as Aurkb and survivin, Aurka is required for hematopoiesis, but is dispensable for megakaryocyte endomitosis. Our work supports a growing body of evidence that the megakaryocyte endomitotic cell cycle differs significantly from the proliferative cell cycle., (© 2015 by The American Society of Hematology.)

Children with Down syndrome (DS) are at increased risk for acute myeloid leukemias (ML-DS) characterized by mixed megakaryocytic and erythroid phenotype and by acquired mutations in the GATA1 gene resulting in a short GATA1s isoform. The chromosome 21 microRNA (miR)-125b cluster has been previously shown to cooperate with GATA1s in transformation of fetal hematopoietic progenitors. In this study, we report that the expression of miR-486-5p is increased in ML-DS compared with non-DS acute megakaryocytic leukemias (AMKLs). miR-486-5p is regulated by GATA1 and GATA1s that bind to the promoter of its host gene ANK1. miR-486-5p is highly expressed in mouse erythroid precursors and knockdown (KD) in ML-DS cells reduced their erythroid phenotype. Ectopic expression and KD of miR-486-5p in primary fetal liver hematopoietic progenitors demonstrated that miR-486-5p cooperates with Gata1s to enhance their self renewal. Consistent with its activation of AKT, overexpression and KD experiments showed its importance for growth and survival of human leukemic cells. Thus, miR-486-5p cooperates with GATA1s in supporting the growth and survival, and the aberrant erythroid phenotype of the megakaryocytic leukemias of DS., (© 2015 by The American Society of Hematology.)

Most heritable anemias are caused by mutations in genes encoding globins, red blood cell (RBC) membrane proteins, or enzymes in the glycolytic and hexose monophosphate shunt pathways. A less common class of genetic anemia is caused by mutations that alter the functions of erythroid transcription factors (TFs). Many TF mutations associated with heritable anemia cause truncations or amino acid substitutions, resulting in the production of functionally altered proteins. Characterization of these mutant proteins has provided insights into mechanisms of gene expression, hematopoietic development, and human disease. Mutations within promoter or enhancer regions that disrupt TF binding to essential erythroid genes also cause anemia and heritable variations in RBC traits, such as fetal hemoglobin content. Defining the latter may have important clinical implications for de-repressing fetal hemoglobin synthesis to treat sickle cell anemia and β thalassemia. Functionally important alterations in genes encoding TFs or their cognate cis elements are likely to occur more frequently than currently appreciated, a hypothesis that will soon be tested through ongoing genome-wide association studies and the rapidly expanding use of global genome sequencing for human diagnostics. Findings obtained through such studies of RBCs and associated diseases are likely generalizable to many human diseases and quantitative traits., (© 2014 by The American Society of Hematology.)

Acute megakaryoblastic leukemia (AMKL) is more frequently observed in Down syndrome (DS) patients, in whom it is often preceded by a transient myeloproliferative disorder (TMD). The development of DS-TMD and DS-AMKL requires not only the presence of the trisomy 21 but also that of GATA1 mutations. Despite extensive studies into the genetics of DS-AMKL, the importance of epigenetic deregulation in this disease has been unexplored. We performed DNA methylation profiling at different stages of development of DS-AMKL and analyzed the dynamics of the epigenetic program. Early genome-wide DNA methylation changes can be detected in trisomy 21 fetal liver mononuclear cells, prior to the acquisition of GATA1 mutations. These early changes are characterized by marked loss of DNA methylation at genes associated with developmental disorders, including those affecting the cardiovascular, neurological, and endocrine systems. This is followed by a second wave of changes detected in DS-TMD and DS-AMKL, characterized by gains of methylation. This new wave of hypermethylation targets a distinct set of genes involved in hematopoiesis and regulation of cell growth and proliferation. These findings indicate that the final epigenetic landscape of DS-AMKL is the result of sequential and opposing changes in DNA methylation occurring at specific times in the disease development.

Children with Down syndrome develop a unique congenital clonal megakaryocytic proliferation disorder (transient myeloproliferative disorder [TMD]). It is caused by an expansion of fetal megakaryocyte-erythroid progenitors (MEPs) triggered by trisomy of chromosome 21 and is further enhanced by the somatic acquisition of a mutation in GATA1. These mutations result in the expression of a short-isoform GATA1s lacking the N-terminal domain. To examine the hypothesis that the Hsa21 ETS transcription factor ERG cooperates with GATA1s in this process, we generated double-transgenic mice expressing hERG and Gata1s. We show that increased expression of ERG by itself is sufficient to induce expansion of MEPs in fetal livers. Gata1s expression synergizes with ERG in enhancing the expansion of fetal MEPs and megakaryocytic precursors, resulting in hepatic fibrosis, transient postnatal thrombocytosis, anemia, a gene expression profile that is similar to that of human TMD and progression to progenitor myeloid leukemia by 3 months of age. This ERG/Gata1s transgenic mouse model also uncovers an essential role for the N terminus of Gata1 in erythropoiesis and the antagonistic role of ERG in fetal erythroid differentiation and survival. The human relevance of this finding is underscored by the recent discovery of similar mutations in GATA1 in patients with Diamond-Blackfan anemia.

The transcription factor Ikaros regulates the development of hematopoietic cells. Ikaros-deficient animals fail to develop B cells and display a T-cell malignancy, which is correlated with altered Notch signaling. Recently, loss of Ikaros was associated with progression of myeloproliferative neoplasms to acute myeloid leukemia and increasing evidence shows that Ikaros is also critical for the regulation of myeloid development. Previous studies showed that Ikaros-deficient mice have increased megakaryopoiesis, but the molecular mechanism of this phenomenon remains unknown. Here, we show that Ikaros overexpression decreases NOTCH-induced megakaryocytic specification, and represses expression of several megakaryocytic genes including GATA-1 to block differentiation and terminal maturation. We also demonstrate that Ikaros expression is differentially regulated by GATA-2 and GATA-1 during megakaryocytic differentiation and reveal that the combined loss of Ikzf1 and Gata1 leads to synthetic lethality in vivo associated with prominent defects in erythroid cells and an expansion of megakaryocyte progenitors. Taken together, our observations demonstrate an important functional interplay between Ikaros, GATA factors, and the NOTCH signaling pathway in specification and homeostasis of the megakaryocyte lineage.

There are many examples of transcription factor families whose members control gene expression profiles of diverse cell types. However, the mechanism by which closely related factors occupy distinct regulatory elements and impart lineage specificity is largely undefined. Here we demonstrate on a genome wide scale that the hematopoietic GATA factors GATA-1 and GATA-2 bind overlapping sets of genes, often at distinct sites, as a means to differentially regulate target gene expression and to regulate the balance between proliferation and differentiation. We also reveal that the GATA switch, which entails a chromatin occupancy exchange between GATA2 and GATA1 in the course of differentiation, operates on more than one-third of GATA1 bound genes. The switch is equally likely to lead to transcriptional activation or repression; and in general, GATA1 and GATA2 act oppositely on switch target genes. In addition, we show that genomic regions co-occupied by GATA2 and the ETS factor ETS1 are strongly enriched for regions marked by H3K4me3 and occupied by Pol II. Finally, by comparing GATA1 occupancy in erythroid cells and megakaryocytes, we find that the presence of ETS factor motifs is a major discriminator of megakaryocyte versus red cell specification.

Erythroid cells and megakaryocytes are derived from a common precursor, the megakaryocyte-erythroid progenitor. Although these 2 closely related hematopoietic cell types share many transcription factors, there are several key differences in their regulatory networks that lead to differential gene expression downstream of the megakaryocyte-erythroid progenitor. With the advent of next-generation sequencing and our ability to precisely define transcription factor chromatin occupancy in vivo on a global scale, we are much closer to understanding how these 2 lineages are specified and in general how transcription factor complexes govern hematopoiesis.

FLIP is a well-established suppressor of death receptor-mediated apoptosis. To define its essential in vivo role in myeloid cells, we generated and characterized mice with Flip conditionally deleted in the myeloid lineage. Myeloid specific Flip-deficient mice exhibited growth retardation, premature death, and splenomegaly with altered architecture and extramedullary hematopoiesis. They also displayed a dramatic increase of circulating neutrophils and multiorgan neutrophil infiltration. In contrast, although circulating inflammatory monocytes were also significantly increased, macrophages in the spleen, lymph nodes, and the peritoneal cavity were reduced. In ex vivo cultures, bone marrow progenitor cells failed to differentiate into macrophages when Flip was deleted. Mixed bone marrow chimera experiments using cells from Flip-deficient and wild-type mice did not demonstrate an inflammatory phenotype. These observations demonstrate that FLIP is necessary for macrophage differentiation and the homeostatic regulation of granulopoiesis.

Enucleation of mammalian erythroblasts is a process whose mechanism is largely undefined. The prevailing model suggests that nuclear extrusion occurs via asymmetric cytokinesis. To test this hypothesis, we treated primary erythroblasts with inhibitors of cytokinesis, including blebbistatin, hesperadin, and nocodazole, and then assayed for enucleation. Although these agents inhibited cell-cycle progression and subsequent enucleation when added early in culture, they failed to block enucleation proper when added to postmitotic cells. These results suggest that contraction of the actomyosin ring is not essential for nuclear expulsion. Next, by ultrastructural examination of primary erythroblasts, we observed an accumulation of vacuoles in the cytoplasm proximal to the extruding nucleus. This finding led us to hypothesize that vesicle trafficking contributes to erythroblast enucleation. Here, we show that chemical inhibitors of vesicle trafficking block enucleation of primary erythroblasts without affecting differentiation, cell division, or apoptosis. Moreover, knock-down of clathrin inhibited the enucleation of late erythroblasts. In contrast, vacuolin-1, a small molecule that induces vacuole formation, increased the percentage of enucleated cells. Together, these results illustrate that vesicle trafficking, specifically the formation, movement, and subsequent coalescence of vacuoles at the junction of the nucleus and the cytoplasm, is a critical component of mammalian erythroblast enucleation.

GATA-1 and PU.1 are essential hematopoietic transcription factors that control erythromegakaryocytic and myelolymphoid differentiation, respectively. These proteins antagonize each other through direct physical interaction to repress alternate lineage programs. We used immortalized Gata1(-) erythromegakaryocytic progenitor cells to study how PU.1/Sfpi1 expression is regulated by GATA-1 and GATA-2, a related factor that is normally expressed at earlier stages of hematopoiesis. Both GATA factors bind the PU.1/Sfpi1 gene at 2 highly conserved regions. In the absence of GATA-1, GATA-2 binding is associated with an undifferentiated state, intermediate level PU.1/Sfpi1 expression, and low-level expression of its downstream myeloid target genes. Restoration of GATA-1 function induces erythromegakaryocytic differentiation. Concomitantly, GATA-1 replaces GATA-2 at the PU.1/Sfpi1 locus and PU.1/Sfpi1 expression is extinguished. In contrast, when GATA-1 is not present, shRNA knockdown of GATA-2 increases PU.1/Sfpi1 expression by 3-fold and reprograms the cells to become macrophages. Our findings indicate that GATA factors act sequentially to regulate lineage determination during hematopoiesis, in part by exerting variable repressive effects at the PU.1/Sfpi1 locus.

Disease alleles that activate signal transduction are common in myeloid malignancies; however, there are additional unidentified mutations that contribute to myeloid transformation. Based on the recent identification of TET2 mutations, we evaluated the mutational status of TET1, TET2, and TET3 in myeloproliferative neoplasms (MPNs), chronic myelomonocytic leukemia (CMML), and acute myeloid leukemia (AML). Sequencing of TET2 in 408 paired tumor/normal samples distinguished between 68 somatic mutations and 6 novel single nucleotide polymorphisms and identified TET2 mutations in MPN (27 of 354, 7.6%), CMML (29 of 69, 42%), AML (11 of 91, 12%), and M7 AML (1 of 28, 3.6%) samples. We did not identify somatic TET1 or TET3 mutations or TET2 promoter hypermethylation in MPNs. TET2 mutations did not cluster in genetically defined MPN, CMML, or AML subsets but were associated with decreased overall survival in AML (P = .029). These data indicate that TET2 mutations are observed in different myeloid malignancies and may be important in AML prognosis.

Survivin is a member of the chromosome passenger complex, which plays an important role in chromosome alignment, separation, and cytokinesis. Although survivin is required for the proliferation and survival of hematopoietic stem and progenitor cells, the extent to which it is necessary for endomitosis of megakaryocytes remains controversial. To determine whether survivin is required for polyploidization, we analyzed mice with a megakaryocyte-specific deletion. PF4-Cre/survivin(fl/fl) mice harbored normal platelet counts with megakaryocytes that reached ploidy states comparable with those of control littermates. The CD41(+) cells within these animals showed little excision but increased annexin V staining, implying that survivin is required for survival of megakaryocyte progenitors in vivo. In contrast, megakaryocytes in which survivin was excised ex vivo showed robust excision and an increased degree of polyploidization. These results demonstrate that survivin is necessary for survival of megakaryocyte progenitors, but is not required for polyploidization of committed megakaryocytes.

ETS2 and ERG are transcription factors, encoded on human chromosome 21 (Hsa21), that have been implicated in human cancer. People with Down syndrome (DS), who are trisomic for Hsa21, are predisposed to acute megakaryoblastic leukemia (AMKL). DS-AMKL blasts harbor a mutation in GATA1, which leads to loss of full-length protein but expression of the GATA-1s isoform. To assess the consequences of ETS protein misexpression on megakaryopoiesis, we expressed ETS2, ERG, and the related protein FLI-1 in wild-type and Gata1 mutant murine fetal liver progenitors. These studies revealed that ETS2, ERG, and FLI-1 facilitated the expansion of megakaryocytes from wild-type, Gata1-knockdown, and Gata1s knockin progenitors, but none of the genes could overcome the differentiation block characteristic of the Gata1-knockdown megakaryocytes. Although overexpression of ETS proteins increased the proportion of CD41(+) cells generated from Gata1s-knockin progenitors, their expression led to a significant reduction in the more mature CD42 fraction. Serial replating assays revealed that overexpression of ERG or FLI-1 immortalized Gata1-knockdown and Gata1s knockin, but not wild-type, fetal liver progenitors. Immortalization was accompanied by activation of the JAK/STAT pathway, commonly seen in megakaryocytic malignancies. These findings provide evidence for synergy between alterations in GATA-1 and overexpression of ETS proteins in aberrant megakaryopoiesis.

Children with Down syndrome (DS) show a spectrum of clinical anomalies, including cognitive impairment, cardiac malformations, and craniofacial dysmorphy. Moreover, hematologists have also noted that these children commonly show macrocytosis, abnormal platelet counts, and an increased incidence of transient myeloproliferative disease (TMD), acute megakaryocytic leukemia (AMKL), and acute lymphoid leukemia (ALL). In this review, we summarize the clinical manifestations and characteristics of these leukemias, provide an update on therapeutic strategies and patient outcomes, and discuss the most recent advances in DS-leukemia research. With the increased knowledge of the way in which trisomy 21 affects hematopoiesis and the specific genetic mutations that are found in DS-associated leukemias, we are well on our way toward designing improved strategies for treating both myeloid and lymphoid malignancies in this high-risk population.

The level of survivin was reported to be scarce in mouse megakaryocytes (MKs) compared with erythroid cells. Considering this finding and previously reported in vitro data showing decreased MK ploidy upon retroviral-mediated overexpression of survivin, we sought to examine whether ectopic survivin expression in the MK lineage might alter ploidy level in vivo. Here we report the generation of 2 tissue specific hematopoietic transgenic mouse models, one expressing survivin in both the erythroid and MK lineages and the other expressing survivin solely in the MK lineage. Survivin protein overexpression was confirmed in MKs and erythrocytes. Surprisingly, analysis of both transgenic mouse lines showed no detectable changes in MK number, ploidy level, and platelet and erythrocyte counts, as compared with control mice. We conclude that elevated survivin expression does not alter MK/erythroid lineage development and that elevated survivin, alone, does not interfere with MK ploidy in vivo.

Children with Down syndrome (DS) display macrocytosis, thrombocytosis, and a 500-fold increased risk of developing megakaryocytic leukemia; however, the specific effects of trisomy 21 on hematopoiesis remain poorly defined. To study this question, we analyzed blood cell development in the Ts65Dn mouse model of DS. Ts65Dn mice are trisomic for 104 orthologs of Hsa21 genes and are the most widely used mouse model for DS. We discovered that Ts65Dn mice display persistent macrocytosis and develop a myeloproliferative disease (MPD) characterized by profound thrombocytosis, megakaryocyte hyperplasia, dysplastic megakaryocyte morphology, and myelofibrosis. In addition, these animals bear distorted hematopoietic stem and myeloid progenitor cell compartments compared with euploid control littermates. Of the 104 trisomic genes in Ts65Dn mice, Aml1/Runx1 attracts considerable attention as a candidate oncogene in DS-acute megakaryoblastic leukemia (DS-AMKL). To determine whether trisomy for Aml1/Runx1 is essential for MPD, we restored disomy at the Aml1/Runx1 locus in the Ts65Dn strain. Surprisingly, trisomy for Aml1/Runx1 is not required for megakaryocyte hyperplasia and myelofibrosis, suggesting that trisomy for one or more of the remaining genes can promote this disease. Our studies demonstrate the potential of DS mouse models to improve our understanding of chromosome 21 gene dosage effects in human hematologic malignancies.

Loss of a whole chromosome 5 or a deletion of the long arm, del(5q), is a recurring abnormality in myelodysplastic syndromes (MDSs) and acute myeloid leukemia (AML). To identify a leukemia-related gene on chromosome 5, we previously delineated a 970-kb segment of 5q31 that is deleted in all patients examined, and prepared a transcript map of this region. EGR1 is a candidate tumor suppressor gene within the commonly deleted segment of 5q, and encodes a zinc finger transcription factor. To test the hypothesis that loss of function of Egr1 is an initiating event in the pathogenesis of AML/MDS, Egr1-deficient mice were treated with a potent DNA alkylating agent, N-ethyl-nitrosourea (ENU), to induce secondary cooperating mutations. Egr1(+/-) and Egr1(-/-) mice treated with ENU developed immature T-cell lymphomas (CD4(+), CD8(+)) or a myeloproliferative disorder (MPD) at increased rates and with shorter latencies than that of wild-type littermates. The MPD was characterized by an elevated white blood cell count, anemia, and thrombocytopenia with ineffective erythropoiesis. Biallelic mutations of Egr1 were not observed in MPDs in Egr1(+/-) mice. Our data suggest that haploinsufficiency for Egr1 plays a role in murine leukemogenesis, and in the development of AML/MDS characterized by abnormalities of chromosome 5.

Endomitosis is a unique form of cell cycle used by megakaryocytes, in which the latter stages of mitosis are bypassed so that the cell can increase its DNA content and size. Although several transcription factors, including GATA-1 and RUNX-1, have been implicated in this process, the link between transcription factors and polyploidization remains undefined. Here we show that GATA-1-deficient megakaryocytes, which display reduced size and polyploidization, express nearly 10-fold less cyclin D1 and 10-fold increased levels of p16 compared with their wild-type counterparts. We further demonstrate that cyclin D1 is a direct GATA-1 target in megakaryocytes, but not erythroid cells. Restoration of cyclin D1 expression, when accompanied by ectopic overexpression of its partner Cdk4, resulted in a dramatic increase in megakaryocyte size and DNA content. However, terminal differentiation was not rescued. Of note, polyploidization was only modestly reduced in cyclin D1-deficient mice, likely due to compensation by elevated cyclin D3 expression. Finally, consistent with an additional defect conferred by increased levels of p16, inhibition of cyclin D-Cdk4 complexes with a TAT-p16 fusion peptide significantly blocked polyploidization of wild-type megakaryocytes. Together, these data show that GATA-1 controls growth and polyploidization by regulating cyclin D-Cdk4 kinase activity.

GATA1 is mutated in patients with 2 different disorders. First, individuals with a GATA1 mutation that blocks the interaction between GATA-1 and its cofactor Friend of GATA-1 (FOG-1) suffer from dyserythropoietic anemia and thrombocytopenia. Second, children with Down syndrome who develop acute megakaryoblastic leukemia harbor mutations in GATA1 that lead to the exclusive expression of a shorter isoform named GATA-1s. To determine the effect of these patient-specific mutations on GATA-1 function, we first compared the gene expression profile between wild-type and GATA-1-deficient megakaryocytes. Next, we introduced either GATA-1s or a FOG-binding mutant (V205G) into GATA-1-deficient megakaryocytes and assessed the effect on differentiation and gene expression. Whereas GATA-1-deficient megakaryocytes failed to undergo terminal differentiation and proliferated excessively in vitro, GATA-1s-expressing cells displayed proplatelet formation and other features of terminal maturation, but continued to proliferate aberrantly. In contrast, megakaryocytes that expressed V205G GATA-1 exhibited reduced proliferation, but failed to undergo maturation. Examination of the expression of megakaryocyte-specific genes in the various rescued cells correlated with the observed phenotypic differences. These studies show that GATA-1 is required for both normal regulation of proliferation and terminal maturation of megakaryocytes, and further, that these functions can be uncoupled by mutations in GATA1.

GATA-1 is the founding member of a transcription factor family that regulates growth and maturation of a diverse set of tissues. GATA-1 is expressed primarily in hematopoietic cells and is essential for proper development of erythroid cells, megakaryocytes, eosinophils, and mast cells. Although loss of GATA-1 leads to differentiation arrest and apoptosis of erythroid progenitors, absence of GATA-1 promotes accumulation of immature megakaryocytes. Recently, we and others have reported that mutagenesis of GATA1 is an early event in Down syndrome (DS) leukemogenesis. Acquired mutations in GATA1 were detected in the vast majority of patients with acute megakaryoblastic leukemia (DS-AMKL) and in nearly every patient with transient myeloproliferative disorder (TMD), a "preleukemia" that may be present in as many as 10% of infants with DS. Although the precise pathway by which mutagenesis of GATA1 contributes to leukemia is unknown, these findings confirm that GATA1 plays an important role in both normal and malignant hematopoiesis. Future studies to define the mechanism that results in the high frequency of GATA1 mutations in DS and the role of altered GATA1 in TMD and DS-AMKL will shed light on the multistep pathway in human leukemia and may lead to an increased understanding of why children with DS are markedly predisposed to leukemia.

As many as 10% of infants with Down syndrome (DS) present with transient myeloproliferative disorder (TMD) at or shortly after birth. TMD is characterized by an abundance of blasts within the peripheral blood and liver, and notably undergoes spontaneous remission in the majority of cases. TMD may be a precursor to acute megakaryoblastic leukemia (AMKL), with an estimated 30% of TMD patients developing AMKL within 3 years. We recently reported that mutations in the transcription factor GATA1 are associated with DS-AMKL. To determine whether the acquisition of GATA1 mutations is a late event restricted to acute leukemia, we analyzed GATA1 in DNA from TMD patients. Here we report that GATA1 is mutated in the TMD blasts from every infant examined. These results demonstrate that GATA1 is likely to play a critical role in the etiology of TMD, and mutagenesis of GATA1 represents a very early event in DS myeloid leukemogenesis.