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Acute megakaryoblastic leukemia (AMKL) is a rare malignancy affecting megakaryocytes, platelet-producing cells that reside in the bone marrow. Children with Down syndrome (DS) are particularly prone to developing the disease and have a different age of onset, distinct genetic mutations, and better prognosis as compared with individuals without DS who develop the disease. Here, we discuss the contributions of chromosome 21 genes and other genetic mutations to AMKL, the clinical features of the disease, and the differing features of DS- and non-DS-AMKL. Further studies elucidating the role of chromosome 21 genes in this disease may aid our understanding of how they function in other types of leukemia, in which they are frequently mutated or differentially expressed. Although researchers have made many insights into understanding AMKL, much more remains to be learned about its underlying molecular mechanisms.

The Aurora kinases, which include Aurora A (AURKA), Aurora B (AURKB) and Aurora C (AURKC), are serine/threonine kinases required for the control of mitosis (AURKA and AURKB) and meiosis (AURKC). Since their discovery nearly twenty years ago, Aurora kinases have been studied extensively in cell and cancer biology 1. Several early studies found that Aurora kinases are amplified and overexpressed at the transcript and protein level in various malignancies, including several types of leukemia. These discoveries and others provided a rationale for the development of small molecule inhibitors of Aurora kinases as leukemia therapies. The first generation of Aurora kinase inhibitors did not fare well in clinical trials, owing to poor efficacy and high toxicity. However, the creation of second generation, highly selective Aurora kinase inhibitors has increased the enthusiasm for targeting these proteins in leukemia. This review will describe the functions of each Aurora kinase, summarize their involvement in leukemia and discuss inhibitor development and efficacy in leukemia clinical trials.

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.

We used high resolution mate-pair sequencing (HRMPS) in 15 patients with primary myelofibrosis (PMF): eight with normal karyotype and seven with PMF-characteristic cytogenetic abnormalities, including der(6)t(1;6)(q21–23;p21.3) (n = 4), der(7)t(1;7)(q10;p10) (n = 2), del(20)(q11.2q13.3) (n = 3), and complex karyotype (n = 1). We describe seven novel deletions/translocations in five patients (including two with normal karyotype) whose breakpoints were PCR-validated and involved MACROD2, CACNA2D4, TET2, SGMS2, LRBA, SH3D19, INTS3, FOP (CHTOP), SCLT1, and PHF17. Deletions with breakpoints involving MACROD2 (lysine deacetylase; 20p12.1) were recurrent and found in two of the 15 study patients. A novel fusion transcript was found in one of the study patients (INTS3-CHTOP), and also in an additional non-study patient with PMF. In two patients with der(6)t(1;6)(q21–23;p21.3), we were able to map the precise translocation breakpoints, which involved KCNN3 and GUSBP2 in one case and HYDIN2 in another. This study demonstrates the utility of HRMPS in uncovering submicroscopic deletions/translocations/fusions, and precise mapping of breakpoints in those with overt cytogenetic abnormalities. The overall results confirm the genetic heterogeneity of PMF, given the low frequency of recurrent specific abnormalities, identified by this screening strategy. Currently, we are pursuing the pathogenetic relevance of some of the aforementioned findings. Am. J. Hematol. 88:741–746, 2013. © 2013 Wiley Periodicals, Inc.

Drug resistance in acute lymphoblastic leukemia (ALL) remains a major problem warranting new treatment strategies. Wnt/catenin signaling is critical for the self-renewal of normal hematopoietic progenitor cells. Deregulated Wnt signaling is evident in chronic and acute myeloid leukemia; however, little is known about ALL. Differential interaction of catenin with either the Kat3 coactivator CREBBP (CREB-binding protein (CBP)) or the highly homologous EP300 (p300) is critical to determine divergent cellular responses and provides a rationale for the regulation of both proliferation and differentiation by the Wnt signaling pathway. Usage of the coactivator CBP by catenin leads to transcriptional activation of cassettes of genes that are involved in maintenance of progenitor cell self-renewal. However, the use of the coactivator p300 leads to activation of genes involved in the initiation of differentiation. ICG-001 is a novel small-molecule modulator of Wnt/catenin signaling, which specifically binds to the N-terminus of CBP and not p300, within amino acids 1–110, thereby disrupting the interaction between CBP and catenin. Here, we report that selective disruption of the CBP/β- and γ-catenin interactions using ICG-001 leads to differentiation of pre-B ALL cells and loss of self-renewal capacity. Survivin, an inhibitor-of-apoptosis protein, was also downregulated in primary ALL after treatment with ICG-001. Using chromatin immunoprecipitation assay, we demonstrate occupancy of the survivin promoter by CBP that is decreased by ICG-001 in primary ALL. CBP mutations have been recently identified in a significant percentage of ALL patients, however, almost all of the identified mutations reported occur C-terminal to the binding site for ICG-001. Importantly, ICG-001, regardless of CBP mutational status and chromosomal aberration, leads to eradication of drug-resistant primary leukemia in combination with conventional therapy in vitro and significantly prolongs the survival of NOD/SCID mice engrafted with primary ALL. Therefore, specifically inhibiting CBP/catenin transcription represents a novel approach to overcome relapse in ALL.

Primary myelofibrosis (PMF) is a clonal hematologic malignancy characterized by BM fibrosis, extramedullary hematopoiesis, circulating CD34+ cells, splenomegaly, and a propensity to evolve to acute myeloid leukemia. Moreover, the spleen and BM of patients harbor atypical, clustered megakaryocytes, which contribute to the disease by secreting profibrotic cytokines. Here, we have revealed that megakaryocytes in PMF show impaired maturation that is associated with reduced GATA1 protein. In investigating the cause of GATA1 downregulation, our gene-expression study revealed the presence of the RPS14-deficient gene signature, which is associated with defective ribosomal protein function and linked to the erythroid lineage in 5q deletion myelodysplastic syndrome. Surprisingly, reduced GATA1 expression and impaired differentiation were limited to megakaryocytes, consistent with a proproliferative effect of a GATA1 deficiency on this lineage. Importantly, expression of GATA1 effectively rescued maturation of PMF megakaryocytes. Together, these results suggest that ribosomal deficiency contributes to impaired megakaryopoiesis in myeloproliferative neoplasms.

The requirement that leukemic Gata1 mutations be present in cells harboring trisomy 21 led to the discovery that overexpression of ERG drives aberrant megakaryopoiesis. Given that constitutive PI3K/AKT signaling is a frequent component of hematologic malignancies and the relationship between AKT and Notch in this lineage, we studied the crosstalk between AKT signaling and ERG in megakaryopoiesis. We discovered that constitutive AKT signaling is associated with a dramatic increase in apoptosis of WT megakaryocytes (MKs), but that overexpression of ERG blocks AKT-induced death. We further found that Gata1 mutations protect MKs from activated AKT-induced apoptosis. As a consequence, however, the enhanced signaling inhibits differentiation of Gata1 mutant, but not WT, MKs. Gata1 mutant cells that overexpress ERG with hyperactive AKT are characterized by diminished FOXO1/3a expression and an increased dependency on the c-Jun pathway similar to that seen in acute megakaryoblastic leukemia (AMKL) cell lines, acute myeloid leukemia (AML) with knockdown of FOXO3a, or AML with expression of myristoylated Akt. Additionally, we found that the AKT allosteric inhibitor MK2206 caused reduced cell viability and proliferation of AMKL cell lines. The contribution of aberrant AKT signaling during the ontogeny of Down syndrome-transient myeloproliferative disorder/AMKL indicates that AKT is a therapeutic target in this form of AML.

Imetelstat (GRN163L) is a specific telomerase inhibitor that has demonstrated clinical activity in patients with myeloproliferative neoplasms (MPN) and in patients with solid tumors. The antitumor effects were associated with the development of thrombocytopenia, one of the common side effects observed in patients treated with imetelstat. The events underlying these adverse effects are not apparent. In this report, we investigated the potential mechanisms that account for imetelstat's beneficial effects in MPN patients and the manner by which imetelstat treatment leads to a reduction in platelet numbers. Using a well-established system of ex vivo megakaryopoiesis, we demonstrated that imetelestat treatment affects normal megakaryocyte (MK) development by exclusively delaying maturation of MK precursor cells. By contrast, additional stages along MPN MK development were affected by imetelstat resulting in reduced numbers of assayable colony-forming unit MK and impaired MK maturation. In addition, treatment with imetelstat inhibited the secretion of fibrogenic growth factors by malignant but not by normal MK. Our results indicate that the delay observed in normal MK maturation may account for imetelstat-induced thrombocytopenia, while the more global effects of imetelstat on several stages along the hierarchy of MPN megakaryopoiesis may be responsible for the favorable clinical outcomes reported in MPN patients.

Although it is clear that trisomy 21 causes Down syndrome, the molecular events acting downstream of the trisomy remain ill defined. Using complementary genomics analyses, we identified the interferon pathway as the major signaling cascade consistently activated by trisomy 21 in human cells. Transcriptome analysis revealed that trisomy 21 activates the interferon transcriptional response in fibroblast and lymphoblastoid cell lines, as well as circulating monocytes and T cells. Trisomy 21 cells show increased induction of interferon-stimulated genes and decreased expression of ribosomal proteins and translation factors. An shRNA screen determined that the interferon-activated kinases JAK1 and TYK2 suppress proliferation of trisomy 21 fibroblasts, and this defect is rescued by pharmacological JAK inhibition. Therefore, we propose that interferon activation, likely via increased gene dosage of the four interferon receptors encoded on chromosome 21, contributes to many of the clinical impacts of trisomy 21, and that interferon antagonists could have therapeutic benefits. DOI: http://dx.doi.org/10.7554/eLife.16220.001, eLife digest Our genetic information is contained within structures called chromosomes. Down syndrome is caused by the genetic condition known as trisomy 21, in which a person is born with an extra copy of chromosome 21. This extra chromosome affects human development in many ways, including causing neurological problems and stunted growth. Trisomy 21 makes individuals more susceptible to certain diseases, such as Alzheimer’s disease and autoimmune disorders – where the immune system attacks healthy cells in the body – while protecting them from tumors and some other conditions. Since cells with trisomy 21 have an extra copy of every single gene on chromosome 21, it is expected that these genes should be more highly expressed – that is, the products of these genes should be present at higher levels inside cells. However, it was not clear which genes on other chromosomes are also affected by trisomy 21. Sullivan et al. aimed to identify which genes are affected by trisomy 21 by studying samples collected from a variety of individuals with, and without, this condition. Four genes in chromosome 21 encode proteins that recognize signal molecules called interferons, which are produced by cells in response to viral or bacterial infection. Interferons act on neighboring cells to regulate genes that prevent the spread of the infection, shut down the production of proteins and activate the immune system. Sullivan et al. show that cells with trisomy 21 produce high levels of genes that are activated by interferons and lower levels of genes required for protein production. In other words, the cells of people with Down syndrome are constantly fighting a viral infection that does not exist. Constant activation of interferon signaling could explain many aspects of Down syndrome, including neurological problems and protection against tumors. The next steps are to fully define the role of interferon signaling in the development of Down syndrome, and to find out whether drugs that block the action of interferons could have therapeutic benefits. DOI: http://dx.doi.org/10.7554/eLife.16220.002

PURPOSE OF REVIEW: Four years ago it was discovered that nearly all cases of transient myeloproliferative disorder and acute megakaryocytic leukemia in children with Down syndrome acquire mutations in the hematopoietic transcription factor gene GATA1. Studies within the past year, described within this review, have provided tremendous insights into the role of GATA1 mutations in these malignancies. RECENT FINDINGS: In the past year, our understanding of the molecular and cellular consequences of GATA1 mutations has been greatly enhanced. Most importantly, we have learned that these mutations, which result in the exclusive production of the short GATA1 isoform named GATA1s, have a distinct effect on fetal liver progenitors. In addition, multiple studies have shown that GATA1s can substitute for GATA1 in many aspects of megakaryocytic maturation. Finally, an important clinical study has revealed that GATA1 mutations alone are insufficient for leukemia. SUMMARY: Leukemia in children with Down syndrome requires at least three cooperating events--trisomy 21, a GATA1 mutation, and a third, as yet undefined, genetic alteration. Recent studies have provided tremendous insights into the GATA1 side of the story. Future experiments with human patient samples and mouse models will likely increase our awareness of the role of trisomy 21 in transient myeloproliferative disorder and acute megakaryocytic leukemia.

Every 2 years since 1978, an international group of scientists, physicians, and other researchers meet to discuss the latest developments in the underlying etiology, mechanisms of action, and developmental acquisition of cellular and systemic defects exhibited and elicited by the most common inherited human disorders, the hemoglobinopathies. The 19th Hemoglobin Switching Conference, held in September 2014 at St. John's College in Oxford, once again exceeded all expectations by describing cutting edge research in cellular, molecular, developmental, and genomic advances focused on these diseases. The conference comprised about 60 short talks over 3 days by leading investigators in the field. This meeting report describes the highlights of the conference.

Acute myelogenous leukemia (AML) is a high-risk hematopoietic malignancy caused by a variety of mutations, including genes encoding the cohesin complex. Recent studies have demonstrated that reduction in cohesin complex levels leads to enhanced self-renewal in hematopoietic stem and progenitors (HSPCs). We sought to delineate the molecular mechanisms by which cohesin mutations promote enhanced HSPC self-renewal as this represents a critical initial step during leukemic transformation. We verified that RNAi against the cohesin subunit Rad21 causes enhanced self-renewal of HSPCs in vitro through derepression of polycomb repressive complex 2 (PRC2) target genes, including Hoxa7 and Hoxa9. Importantly, knockdown of either Hoxa7 or Hoxa9 suppressed self-renewal, implying that both are critical downstream effectors of reduced cohesin levels. We further demonstrate that the cohesin and PRC2 complexes interact and are bound in close proximity to Hoxa7 and Hoxa9. Rad21 depletion resulted in decreased levels of H3K27me3 at the Hoxa7 and Hoxa9 promoters, consistent with Rad21 being critical to proper gene silencing by recruiting the PRC2 complex. Our data demonstrates that the cohesin complex regulates PRC2 targeting to silence Hoxa7 and Hoxa9 and negatively regulate self-renewal. Our studies identify a novel epigenetic mechanism underlying leukemogenesis in AML patients with cohesin mutations.

The development of complex organisms requires the formation of diverse cell types from common stem and progenitor cells. GATA family transcriptional regulators and their dedicated co-factors, termed Friend of GATA (FOG) proteins, control cell fate and differentiation in multiple tissue types from Drosophila to man. FOGs can both facilitate and antagonize GATA factor transcriptional regulation depending on the factor, cell, and even the specific gene target. In this review, we highlight recent studies that have elucidated mechanisms by which FOGs regulate GATA factor function and discuss how these factors use these diverse modes of gene regulation to control cell lineage specification throughout metazoans.

GATA-1 and its cofactor FOG-1 are required for the differentiation of erythrocytes and megakaryocytes. In contrast, mast cell development requires GATA-1 and the absence of FOG-1. Through genome-wide comparison of the chromatin occupancy of GATA-1 and a naturally occurring mutant that cannot bind FOG-1 (GATA-1(V205G)), we reveal that FOG-1 intricately regulates the chromatin occupancy of GATA-1. We identified GATA1-selective and GATA-1(V205G)-selective binding sites and show that GATA-1, in the absence of FOG-1, occupies GATA-1(V205G)-selective sites, but not GATA1-selective sites. By integrating ChIP-seq and gene expression data, we discovered that GATA-1(V205G) binds and activates mast cell-specific genes via GATA-1(V205G)-selective sites. We further show that exogenous expression of FOG-1 in mast cells leads to displacement of GATA-1 from mast cell-specific genes and causes their downregulation. Together these findings establish a mechanism of gene regulation whereby a non-DNA binding cofactor directly modulates the occupancy of a transcription factor to control lineage specification.

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.

Purpose of review The discovery of the JAK2 V617F mutation in the classical myeloproliferative neoplasms (MPNs) essential thrombocytosis, polycythemia vera, and primary myelofibrosis has ushered in a new era of scientific discovery in these diseases, resulting in a molecular classification and an improved understanding of disease pathogenesis. Alongside this period of discovery has been the rapid development of targeted therapy and, here, we summarize results from clinical trials involving these small molecule Janus family of tyrosine kinase (JAK) inhibitors. Recent findings The JAK inhibitors consistently alleviate constitutional symptoms and reduce spleen size. Early phase testing indicates that some of these inhibitors have additional unique effects: INCB018424 results in a significant reduction in the level of pro-inflammatory cytokines; TG101348 may modify disease burden as assessed by JAK2 allele measurements; and CYT387 ameliorates anemia. Summary The initial enthusiasm for these agents has been tempered by recognition that JAK2 V617F may represent only one component of lesions driving the heterogeneity of the MPN. Clinical trial design cannot address important disease endpoints such as thrombosis or leukemia transformation, but it appears that JAK inhibitors will offer an important palliative option and because of the molecular complexity in these diseases, it might be rational to give these inhibitors along with other agents that target alternate mechanisms of the disease pathogenesis.

Relapse of drug-resistant acute lymphoblastic leukemia (ALL) has been associated with increased expression of survivin/BIRC5, an inhibitor of apoptosis protein, suggesting a survival advantage for ALL cells. In the present study, we report that inhibition of survivin in patient-derived ALL can eradicate leukemia. Targeting survivin with shRNA in combination with chemotherapy resulted in no detectable minimal residual disease in a xenograft model of primary ALL. Similarly, pharmacologic knock-down of survivin using EZN-3042, a novel locked nucleic acid antisense oligonucleotide, in combination with chemotherapy eliminated drug-resistant ALL cells. These findings show the importance of survivin expression in drug resistance and demonstrate that survivin inhibition may represent a powerful approach to overcoming drug resistance and preventing relapse in patients with ALL.

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.

Myeloid leukemia of Down syndrome (ML-DS) is a tractable human model of acute myeloid leukemia. A preleukemia phase, transient abnormal myelopoiesis (TAM) and silent TAM, occurs in 28% of neonates with Down Syndrome (Roberts et al. Blood 2013). TAM is caused by trisomy 21 and acquired mutations in GATA1 that result in a N-terminal truncated protein, GATA1s, in hematopoietic stem and progenitor cells (HSPCs) of fetal origin. ML-DS evolves from TAM by acquisition of additional genetic lesions. The nature of these lesions and the mechanism of transformation are incompletely understood. We performed exome sequencing and targeted resequencing of 141 ML-DS and 111 TAM patients to characterize the evolving mutational landscape from TAM to ML-DS. On average 1.6 acquired mutations were detected in ML-DS (in addition to GATA1 mutations), significantly more than in TAM (0.4 mutations per sample). Additional anticipated loss-of-function mutations acquired in ML-DS mainly affected cohesin components including CTCF (43% of patients), PRC2 components (13%), KANSL1 and other epigenetic regulators (14%). Conversely, anticipated gain-of-function mutations were most prevalent in signaling pathways, e.g. JAK kinases, MPL, KIT and RAS family members (40%). Importantly, we detected a novel recurrent hotspot mutation in 4% of patients (6/141 cases) in CSF2RB encoding the IL3-, IL5-, GM-CSF-receptor common beta chain. To test if the A455D/T variant in the CSF2RB transmembrane domain is a putative oncogenic driver, we ectopically expressed CSF2RBA455D in TF1 cells. Cells expressing CSF2RBA455D exhibited cytokine independent growth and STAT5 autonomous phosphorylation. In a CD34+-HSPC megakaryocytic differentiation assay, CSF2RBA455D blocked terminal megakaryocytic differentiation whilst increasing proliferation by 30-fold (P=0.046). Moreover, the median survival of NSG mice transplanted with CSF2RBA455DTF1 cells was shortened by 30 days compared to wild type TF1 cells (23 days compared to 53 days, P=0.0097). To experimentally test the potential of loss-of-function mutations to transform TAM to ML-DS, we performed an in vivo murine isogenic transplantation screen using Gata1s expressing fetal hematopoietic cells from Cas9-knockin mice. We tested variants in 22 genes, recurrently detected in ML-DS, with a pool of prevalidated gRNAs. This resulted in short latency (n=18 mice; median survival 36 days) and high penetrance (100%) leukemia. Leukemia was not detected in mice infected with control gRNAs. Leukemias had a typical ML-DS megakaryoblastic phenotype (CD117+ and CD41a+). Amplicon sequencing revealed on average 2.9 gRNAs per leukemia and high representation (61% of all leukemias) of gRNAs directed to the tumor suppressor Trp53, which was alone sufficient to induce leukemia with 100% penetrance. When excluding the Trp53 gRNA from pools, leukemic cells from moribund mice contained gRNAs against negative regulators of the RAS and JAK-STAT signaling cascade, such as Nf1, Cbl and Sh2b3 (70% of the mice), Ezh2, Asxl1, Kdm6a,Bcor and other epigenetic modifiers (85%) or Ctcf (15%), closely resembling the mutational landscape of ML-DS. In contrast to ML-DS, gRNAs targeting cohesion components, such as Rad21 and Stag2, were not present in any of the leukemias. In summary, we performed the largest genetic analysis of transforming events in ML-DS that cooperate with trisomy 21 and GATA1s and uncovered a previously undescribed activating mutation in CSR2B. We experimentally validated many of the loss-of-function mutations in a novel murine fetal leukemia assay for ML-DS. The field is now well-placed to study mechanisms of oncogenic cooperativity and identify novel therapeutic approaches for this leukemia. Disclosures Crispino: Scholar Rock: Research Funding; Forma Therapeutics: Research Funding.

Mutations in GATA1 are seen in rare cases of dyserythropoietic anemia and in a subset of patients with Diamond Blackfan Anemia (DBA). Of note the truncation mutations in DBA, known as GATA1s, closely resemble those that are more commonly associated with acute megakaryoblastic leukemia in children with Down syndrome (DS). Studies with a mouse model of the Gata1s mutation revealed that replacement of the full-length protein by the shortened isoform led to a marked yet transient enhancement in megakaryopoiesis, similar in some respects to transient myeloproliferative disorder in DS. Furthermore, these mutant mice displayed impaired embryonic erythropoiesis but ostensibly no defects in adult hematopoiesis. In our efforts to better understand the connection between GATA1s and DBA, we comprehensively studied erythropoiesis in the Gata1s mouse strain. We observed a striking impairment in erythropoiesis in fetuses at E10.5 though E12.5, but saw improvement as the animals progressed through E14.5 and beyond. Defects included impaired terminal maturation and reduced numbers of erythroid progenitors, likely at the expense of expanded megakaryopoiesis. RNA-sequencing revealed that both erythroid genes and megakaryocytic genes were altered by the Gata1s mutation. Epiproteomic histone modification analysis further revealed there was an accumulation of H3K27 methylation in the R3 (CD71hiTer119hi) erythroid progenitor population, which suggests that GATA1 has a link to the epigenetic machinery that is altered in Gata1s mutant cells. Despite a global increase in H3K27me3, critical Gata2 regulatory elements in Gata1s mutant erythroid progenitors were marked by substantially less H3K27me3 than in wild-type littermates. Given that overexpression of GATA2 has been reported to impair erythropoiesis, we investigated whether reducing the GATA2 levels would ameliorate the phenotype. Indeed, we observed that haploinsufficiency for Gata2 rescued the erythroid defects of Gata1s fetuses. Next, to comprehensively study the effect of absence of the GATA1 N-terminus genome-wide, we performed Cleavage Under Targets and Release Using Nuclease (CUT&RUN) with H3K27me3, GATA1 or GATA1s antibodies on wild-type versus Gata1s expressing fetal erythroid cells. Our data indicated that there is a substantial reduction in H3K27me3 along regulatory elements of the Runx1 gene at the late stage (R3) of fetal erythropoiesis in Gata1s mice. Along with an increase in Runx1 expression we observed strong downregulation of Klf1, a repressive target of RUNX1. Thus, failure of GATA1s to facilitate trimethylation of Runx1 and Gata2 regulatory elements appears to cause the defects in erythroid cell and megakaryocyte development. In parallel, we performed an in-depth analysis of the phenotype of adult Gata1s mice and discovered that they have reduced red cell counts, lower hemoglobin and hematocrit, increased extramedullary hematopoiesis and impaired stress erythropoiesis compared to control littermates. Although there were significantly more megakaryocyte erythrocyte progenitors (MEPs, Lin-c-Kit+Sca-1-CD34-FcgR-) in Gata1s mouse bone marrow, there were fewer pre-colony-forming unit erythroid cells (preCFU-E, Lin-c-Kit+Sca-1-CD41-FcgR-CD150hiCD105hi), likely at the expense of expanded megakaryocyte progenitors (MkP, Lin-c-Kit+Sca-1-CD41+CD150hi).Gata1s mice also developed an MDS-like disease with age. Together, our integrated genomic analysis of transcriptome, GATA1/GATA1s chromatin binding profile and chromatin signature reveal that, although Gata1s mice do not precisely model DBA, they provide novel insights into the role of the N-terminus of GATA1 in gene transcriptional regulation, lineage determination and red blood cell maturation. Disclosures Crispino: Scholar Rock: Research Funding; Forma Therapeutics: Research Funding.

Signal transducer and activator of transcription 3 (STAT3) is a transcription factor that mediates signal transduction from the extracellular surface to the nucleus. Canonically, STAT3 is phosphorylated at Tyrosine 705 (Y705) by JAK family kinases, which promotes its dimerization and subsequent localization to the nucleus. However, the role of Serine 727 (S727) phosphorylation in regulating STAT3 activity varies across cell types and remains unclear in hematopoietic tissues particularly. Several studies indicate that phosphorylation at S727 is critical for optimal STAT3 function. For example, astrogliogenesis is regulated by enhancing STAT3 activity by phosphorylation of S727 by DYRK1A. Of note, DYRK1A is overexpressed in Down syndrome-acute lymphoblastic leukemia (DS-ALL), and has previously been found to phosphorylate substrates in order to prime them for downstream phosphorylation events. Given these findings, we hypothesized that the DYRK1A phosphorylation of STAT3 at S727 is critical for promoting DS-ALL. Furthermore, certain subtypes of ALL have high rates of JAK2 activation, namely DS-ALL and Philadelphia-like ALL (Ph-like ALL); we propose that STAT3 can effectively be targeted specifically in these subtypes. In order to elucidate the role of DYRK1A phosphorylation of STAT3, we treated cytokine-deprived murine pre-B cells with EHT1610, a selective DYRK1 inhibitor, or vehicle and then pulsed the cells with JAK-STAT activating cytokines. EHT1610-treated cells had diminished S727 phosphorylation compared to vehicle, regardless of cytokine pulse; however, only vehicle-treated cells regained Y705 phosphorylation after cytokine pulse. This suggests that S727 phosphorylation is cytokine-independent and is critical for maintenance of Y705 phosphorylation. We then generated flag-tagged STAT3 S727 phospho-mimetic (S727D/E) and phospho-deficient (S727A) alleles and transduced them into pre-B cells. We observed that the degree of Y705 phosphorylation is dependent on S727, as cells expressing S727A have reduced Y705 phosphorylation compared to wild-type STAT3. Additionally, overexpression of the phospho-deficient allele conferred a significant proliferative impairment compared to the phospho-mimetic alleles. As DS-ALL and Ph-like ALL often have JAK2-activating mutations, we next aimed to determine if loss of S727 phosphorylation would decrease ALL cell growth. Indeed, two human Ph-like ALL cell lines, MHH-CALL4 and MUTZ5, displayed decreased proliferation when overexpressing the S727A mutant. These cell lines were also sensitive to treatment with C188-9, a small molecule STAT3 inhibitor that is in clinical trials for various solid tumors. Additionally, we treated primary patient ALL samples with amplification of HSA21 segments ex vivo and found that DS-ALL samples were preferentially sensitive to STAT3 inhibition compared to HD-ALL or iAMP-ALL, suggesting that STAT3 is specifically a target in JAK2-activated ALL. Our study provides new and significant insights into the regulation of STAT3 by DYRK1A, and presents a new therapeutic target for ALL cells with JAK2 activating mutations. Disclosures Bourquin: Amgen: Other: Travel Support. Crispino:Scholar Rock: Research Funding; Forma Therapeutics: Research Funding.

Background: The selective AURKA inhibitor alisertib (MLN8237) exhibits disease modifying activity in murine models of myelofibrosis by eradicating atypical megakaryocytes resulting in reduction of marrow fibrosis (Nat Med 2015). Here, we present long term follow-up results from the investigator initiated pilot study of alisertib in patients with myelofibrosis (clinical trials.gov Identifier NCT 02530619). Methods: 24 patients with DIPSS intermediate 1, intermediate-2, or high risk myelofibrosis who were in need of therapy, refractory/intolerant or unlikely to respond to JAK inhibitors with neutrophil count ≥ 1 x109/L, and platelet count ≥ 50 x109/L, received alisertib (provided by Millennium Pharmaceuticals Inc) at a dose of 50 mg twice daily for one week every 21 days. Toxicity assessment was performed by the standard common terminology criteria (Version 4.0). Response was assessed by the international working group for myelofibrosis research and treatment (IWGMRT) criteria. Correlative studies included assessments of JAK2V617F, CALR, and MPL mutant allele burden, degree of fibrosis and GATA1 expression in bone marrow samples obtained pre and post therapy. Results: We enrolled 17 patients with primary myelofibrosis, 4 with post essential thrombocythemia myelofibrosis and 3 with post polycythemia vera myelofibrosis. Median age was 72 years with 66% males. 79% of patients were DIPSS intermediate risk, and the remainder were high risk with 15 patients (62.5%) having received prior JAK inhibitor therapy. Driver mutational status was as follows; 58% JAK2V617F, 29% CALR, and 13% MPL mutated. At study entry, 54% of patients demonstrated palpable splenomegaly ≥ 5 cm below the left costal margin, 54% were transfusion dependent with all patients experiencing constitutional symptoms. At the time of data cut-off, patients received a median of 7.5 cycles (range; 1-29 cycles) of therapy. The 7 patients presently on study have received a median of 23 cycles (range; 8-29 cycles). Reasons for treatment discontinuation included progressive disease/lack of response in 11 (65%) patients, toxicity in 4 (24%) patients and refusal of further therapy in 2 (11%) patients.Safety and Efficacy assessments The most common treatment-emergent grade 3/4 adverse events included neutropenia (42%), thrombocytopenia (29%) and anemia (21%), with 4% each experiencing neutropenic fever, diarrhea, vertigo, elevated creatinine and elevated alanine aminotransferase. 22 patients were considered for response evaluation with 4 of 14 patients (29%) with palpable splenomegaly ≥ 5 cm achieving a spleen response, 1 of 13 patients (8%) becoming transfusion independent, and 5 of 22 patients (23%) experiencing symptom response with ≥ 50% reduction in the MPN-SAF total symptom score. However, when response assessment was restricted to 13 patients who had received a minimum of 5 cycles of therapy, spleen responses were observed in 4 of 7 (57%) patients, 1 of 5 (20%) achieved transfusion independence and 5 of 13 (38%) achieved symptom response. All patients presenting with leukocytosis (n=4) and thrombocytosis (n=2) had resolution with therapy. Of the 7 patients presently on study, four patients continue to demonstrate symptom response, two patients with both spleen and symptom response, and another patient with sustained anemia response. Correlative assessments We compared the intensity of staining of GATA1, a factor that is required for maturation, in sequential bone marrow biopsies from six patients at baseline and after a minimum of five cycles and observed a striking increase in the numbers of GATA1-positive megakaryocytes in five of six cases (Figure 1a). In addition, we observed a one grade reduction in marrow fibrosis in 4 of 6 paired samples (Figure 1b). This reduction in fibrosis was accompanied by sustained responses to the drug. Finally, we compared JAK2, MPL or CALR mutant allele burden in eight paired baseline and cycle 5 or 6 samples and observed decreases in 4 of 8 patients (Figure 1c). Conclusions: Alisertib is safe and well tolerated in patients with myelofibrosis with prolonged administration up to 1.7 years. In addition to providing clinical benefit, alisertib restored normal morphology and GATA1 expression in atypical megakaryocytes and reduced marrow fibrosis and mutant allele burdens. These findings demonstrate that AURKA inhibition should be further explored as a therapeutic option in myelofibrosis. Figure 1. Figure 1. Disclosures Swords: AbbVie: Employment. Watts:Jazz Pharma: Consultancy, Speakers Bureau; Takeda: Research Funding. Frankfurt:Celgene, Jazz, Agios: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; AbbVie: Membership on an entity's Board of Directors or advisory committees. Altman:Cyclacel: Other: payment to the institution to conduct clinical trial work; Epizyme: Other: payment to the institution to conduct clinical trial work; Ariad: Other: payment to the institution to conduct clinical trial work; Bayer: Other: payment to the institution to conduct clinical trial work; Celator: Other: payment to the institution to conduct clinical trial work; FujiFilm: Other: payment to the institution to conduct clinical trial work; Celgene: Membership on an entity's Board of Directors or advisory committees, Other: payment to the institution to conduct clinical trial work; Agios: Other: Payment to the institution to conduct the trial ; Astellas Pharma: Other; Genetech: Other: Payment to the institution to conduct clinical trial work; Syros: Membership on an entity's Board of Directors or advisory committees; Incyte: Other: payment to the institution to conduct clinical trial work; GSK: Other: payment to the institution to conduct clinical trial work; Immune Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees; Janssen Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Boeringer Ingelheim: Other: payment to the institution to conduct clinical trial work; Novartis: Membership on an entity's Board of Directors or advisory committees; Pfizer: Other: payment to the institution to conduct clinical trial work. Rampal:Celgene: Honoraria; Stemline: Research Funding; Incyte: Honoraria, Research Funding; Constellation: Research Funding; Jazz: Consultancy, Honoraria. Giles:Actuate Therapeutics Inc: Employment, Equity Ownership. Crispino:Forma Therapeutics: Research Funding; Scholar Rock: Research Funding.

Abstract: Megakaryocytes (MKs) undergo directional migration from the proliferative osteoblastic niche within the bone marrow (BM) environment to the capillary-rich vascular niche for platelet production and release into the pulmonary circulation. This process is regulated, in part by dynamins, large GTPase proteins that regulate cellular functions such as endocytosis, vesicle transport and cell migration. Additional functions of dynamins include the formation of actin-rich structures, such as lamellipodia and dorsal membrane ruffles, invadopodia and podosomes. Previous studies have shown that mutations in Dynamin 2 (DNM2) cause thrombocytopenia in humans. To explore the function of dynamins in megakaryocyte migration and platelet production in more depth, we monitored the response of cells to chemotaxis SDF1α gradient signal by a microfluidic device-based approach. We observed an impaired directional migration by both human megakaryocytic cell lines and primary cells treated either with dynasore, a small molecule inhibitor of dynamins, or shRNA knockdown of Dynamin 2 and 3 (DNM2, DNM3). Since directional cell migration is tightly regulated by actin cytoskeletal rearrangements, we next measured actin polymerization and RhoA activity. We observed a profound decrease in the F-actin and Rho GTPase activity upon loss of DNM2 and DNM3 function. Next, since the response to chemoattractant signal is navigated by SDF1 through its receptor CXCR4, we explored the CXCR4 receptor response to ligand in dynamin defective megakaryocytes. Interestingly we observed an increase in CXCR4 expression in the dynasore treated primary human cells. Additionally, combined inhibition of DNM2 and DNM3 or over expression of dominant negative Dnm2-K44A or GTPase-defective DNM3 decreased the active β1- integrin (ITGB1) activity, which indicates a decrease in the integrin mediated endo/exocytic cycling during cell migration. Finally, to understand the role of dynamin in endosome recycling, we assayed the distribution of Rab11, a marker of recycling endosomes. We noticed an abnormal clustered staining pattern of Rab11 in dynasore-treated MKs which is indicative of a disruption in recycling pathways. This observation suggests decreased recruitment of the recycling pathway in dynasore-treated cells. Altogether, in this study we demonstrate that dynamins regulate MKs directional migration towards the SDF1α chemotaxis signal in the bone marrow and governs endocytosis and cell receptor trafficking. Disclosures Crispino: Scholar Rock: Research Funding; Forma Therapeutics: Research Funding.

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.

CHAF1B is the p60 subunit of the chromatin assembly factor (CAF1) complex, which is responsible for assembly of histones H3.1/H4 heterodimers at the replication fork during S phase. Here we report that CHAF1B is required for normal hematopoiesis while its overexpression promotes leukemia. CHAF1B has a pro-leukemia effect by binding chromatin at discrete sites and interfering with occupancy of transcription factors that promote myeloid differentiation, such as CEBPA. Reducing Chaf1b activity by either heterozygous deletion or overexpression of a CAF1 dominant negative allele is sufficient to suppress leukemogenesis in vivo without impairing normal hematopoiesis.

The transcriptional co‐factor Friend of GATA1 (FOG‐1) has been shown to interact with subunits of the nucleosome remodelling and histone deacetylase (NuRD) complex through a specific motif located at its N‐terminus. To test the importance of FOG‐1/NuRD interaction for haematopoiesis in vivo , we generated mice with a mutation that specifically disrupts FOG‐1/NuRD interaction (FOG‐1 R3K5A ). Homozygous FOG‐1 R3K5A mice were found to have splenomegaly, extramedullary erythropoiesis, granulocytosis and thrombocytopaenia secondary to a block in megakaryocyte maturation. FOG‐1 R3K5A/R3K5A megakaryocytes and erythroid progenitors expressed increased levels of GATA2, showing that FOG‐1/NuRD interaction is required for the earlier described ‘GATA Switch’. In addition, ablation of FOG‐1/NuRD interaction led to inappropriate expression of mast cell and eosinophil‐specific genes in the megakaryocyte and erythroid lineages. Chromatin immunoprecipitation experiments revealed that the NuRD complex was not properly recruited to a mast cell gene promoter in FOG‐1 R3K5A/R3K5A megakaryocytes, suggesting that FOG‐1/NuRD interaction is required for the direct suppression of mast cell gene expression. Taken together, these results underscore the importance of the FOG‐1/NuRD interaction for the re‐enforcement of lineage commitment during erythropoiesis and megakaryopoiesis in vivo .

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.

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.

Primary myelofibrosis (PMF) is characterized by bone marrow fibrosis, myeloproliferation, extramedullary hematopoiesis, splenomegaly and leukemic progression. Moreover, the bone marrow and spleens of individuals with PMF contain large numbers of atypical megakaryocytes that are postulated to contribute to fibrosis through the release of cytokines, including transforming growth factor (TGF)-β. Although the Janus kinase inhibitor ruxolitinib provides symptomatic relief, it does not reduce the mutant allele burden or substantially reverse fibrosis. Here we show through pharmacologic and genetic studies that aurora kinase A (AURKA) represents a new therapeutic target in PMF. Treatment with MLN8237, a selective AURKA inhibitor, promoted polyploidization and differentiation of megakaryocytes with PMF-associated mutations and had potent antifibrotic and antitumor activity in vivo in mouse models of PMF. Moreover, heterozygous deletion of Aurka was sufficient to ameliorate fibrosis and other PMF features in vivo. Our data suggest that megakaryocytes drive fibrosis in PMF and that targeting them with AURKA inhibitors has the potential to provide therapeutic benefit.

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.

The development of the complete repertoire of blood cells from a common progenitor, the hematopoietic stem cell, is a tightly controlled process that is regulated, in part, by the activity of lineage specific transcription factors. Despite our knowledge of these factors, the mechanisms that regulate the formation and growth of distinct, but closely related lineages, such as erythroid cells and megakaryocytes, remain largely uncharacterized. Here we show that Survivin, a member of the inhibitor of apoptosis (IAP) family that also plays an essential role in cytokinesis, is differentially expressed during erythroid versus megakaryocyte development. Erythroid cells express Survivin throughout their maturation, up to the terminal stage of differentiation (orthochromatic), even after the cells exit the cell cycle. This is surprising because Survivin is generally expressed in a cell cycle dependent manner and not thought to be expressed in terminally differentiated cells. In contrast, purified murine megakaryocytes express nearly 5-fold lower levels of Survivin mRNA compared to erythroid cells. To investigate whether Survivin is involved in the differentiation and/or survival of hematopoietic progenitors, we infected primary mouse bone marrow cells with retroviruses harboring either the human Survivin cDNA or a mouse Survivin shRNA, and then induced erythroid and megakaryocyte differentiation in both liquid culture and colony-forming assays. These studies revealed that overexpression of Survivin promoted the terminal differentiation of red blood cells, while its reduction, by RNA interference, inhibited their differentiation. In contrast, downregulation of Survivin facilitated the expansion of megakaryocytes, and its overexpression antagonized megakaryocyte formation. In addition, consistent with a role for survivin in erythropoiesis, downregulation of Survivin expression in MEL cells led to a block in terminal differentiation. Finally, since caspase activity is known to be required for erythroid maturation, we investigated whether survivin associated with cleaved caspase-3 in erythroid cells. Immunofluorescence revealed that Survivin and cleaved caspase-3 co-localized to discrete foci within the cytoplasm of erythroid cells at the orthochromatic stage of development. Based on these findings, we hypothesize that Survivin cooperates with cleaved caspase-3 in terminal maturation of red blood cells. Together, our findings demonstrate that Survivin plays multiple, distinct roles in hematopoiesis.

Although physicians have known for many decades that children with Down syndrome are predisposed to developing transient myeloproliferative disorder (TMD) and acute megakaryoblastic leukemia (AMKL), many questions regarding these disorders remain unresolved. First, what is the relationship between TMD and AMKL? Second, what specific genetic alterations contribute to the leukemic process? Finally, what factors lead to the increased predisposition to these myeloid disorders? In this review I will summarize important new insights into the biology of TMD and AMKL gained from the recent discovery that GATA1, a gene that encodes an essential hematopoietic transcription factor, is mutated in the leukemic blasts from nearly all patients with these malignancies. In addition, I will discuss whether assaying for the presence of a GATA1 mutation can aid in the diagnosis of these and related megakaryoblastic leukemias. Future research aimed at defining the activity of mutant GATA-1 protein and identifying interacting factors encoded by chromosome 21 will likely lead to an even greater understanding of this intriguing leukemia.

GATA1 mutations and trisomy 21 are inextricably linked in the neonatal leukemia of children with Down syndrome (DS). A recent report by Banno et al. (2016) sheds new light on the mechanism of the synergy between these genetic alterations by modeling hematopoietic abnormalities with patient-derived induced pluripotent stem cells.

To analyse individual factors that may contribute to leukemic transformation in vivo, we have developed a murine model of leukemogenesis based on the early hematopoietic precursor cell FL5.12. FL5.12 cells are interleukin-3 (IL-3) dependent for growth, proliferation, and survival. Relative resistance to cell death following IL-3 withdrawal can be conferred by either overexpression of the Bcl-x(L) apoptotic inhibitor, or constitutive activation of the serine/threonine kinase Akt. The ability of Bcl-x(L) or a constitutively active myristylated Akt to promote leukemic transformation of FL5.12 cells was compared in athymic nu(+)/nu(+) mice. Bcl-x(L) alone could not promote leukemic transformation, but mice injected with FL5.12 cells overexpressing Bcl-x(L) and a dominant-negative p53 construct developed leukocytosis and blastic infiltration of lymph nodes, spleen, and liver with features of a high-grade lymphoid malignancy. In contrast to the cells injected into these animals, cell lines derived from the mice were able to proliferate in the absence of IL-3, and were found to have constitutively activated Akt. This constitutive activation was associated with a variety of alterations of the signaling pathway regulating Akt activity, including alterations of PTEN mRNA and protein expression. In addition, some of these leukemic clones demonstrated concurrent constitutive upregulation of ERK activity. A constitutively active Akt construct introduced into FL5.12 cells promoted similar clonal expansion in vivo, with emergence of clonal IL-3-independent proliferation. Bcl-x(L) and Akt appeared to function cooperatively in this model, enhancing rapid clonal outgrowth in vivo relative to Akt alone. These results implicate activated Akt and growth-factor independence in leukemogenic transformation, and demonstrate the potential for in vivo analysis of genetic determinants of leukemogenesis.

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.

Thompson et al. identify the dual specificity tyrosine-regulated kinase 1A (DYRK1A) as a new player in the control of lymphopoiesis. Loss of DYRK1A in mice results in Cyclin D3 stabilization and failure to repress E2F target genes, thus impairing cell cycle exit and proper pre–B and pre–T cell differentiation., Pre–B and pre–T lymphocytes must orchestrate a transition from a highly proliferative state to a quiescent one during development. Cyclin D3 is essential for these cells’ proliferation, but little is known about its posttranslational regulation at this stage. Here, we show that the dual specificity tyrosine-regulated kinase 1A (DYRK1A) restrains Cyclin D3 protein levels by phosphorylating T283 to induce its degradation. Loss of DYRK1A activity, via genetic inactivation or pharmacologic inhibition in mice, caused accumulation of Cyclin D3 protein, incomplete repression of E2F-mediated gene transcription, and failure to properly couple cell cycle exit with differentiation. Expression of a nonphosphorylatable Cyclin D3 T283A mutant recapitulated these defects, whereas inhibition of Cyclin D:CDK4/6 mitigated the effects of DYRK1A inhibition or loss. These data uncover a previously unknown role for DYRK1A in lymphopoiesis, and demonstrate how Cyclin D3 protein stability is negatively regulated during exit from the proliferative phases of B and T cell development.

The transcription factor GATA-1 and its cofactor FOG-1 are essential for the normal development of erythroid cells and megakaryocytes. FOG-1 can stimulate or inhibit GATA-1 activity depending on cell and promoter context. How the GATA-1-FOG-1 complex controls the expression of distinct sets of gene in megakaryocytes and erythroid cells is not understood. Here, we examine the molecular basis for the megakaryocyte-restricted activation of the alphaIIb gene. FOG-1 stimulates GATA-1-dependent alphaIIb gene expression in a manner that requires their direct physical interaction. Transcriptional output by the GATA-1-FOG-1 complex is determined by the hematopoietic Ets protein Fli-1 that binds to an adjacent Ets element. Chromatin immunoprecipitation experiments show that GATA-1, FOG-1 and Fli-1 co-occupy the alphaIIb promoter in vivo. Expression of several additional megakaryocyte-specific genes that bear tandem GATA and Ets elements in their promoters also depends on the physical interaction between GATA-1 and FOG-1. Our studies define a molecular context for transcriptional activation by GATA-1 and FOG-1, and may explain the occurrence of tandem GATA and Ets elements in the promoters of numerous megakaryocyte-expressed genes.

Children with Down syndrome have a 10–20-fold elevated risk of developing leukemia, particularly acute megakaryoblastic leukemia (AMKL)1. While a subset of pediatric AMKLs is associated with the 1;22 translocation and expression of a mutant fusion protein2,3, the genetic alterations that promote Down syndrome–related AMKL (DS-AMKL) have remained elusive. Here we show that leukemic cells from every individual with DS-AMKL that we examined contain mutations in GATA1, encoding the essential hematopoietic transcription factor GATA1 (GATA binding protein 1 or globin transcription factor 1). Each mutation results in the introduction of a premature stop codon in the gene sequence that encodes the amino-terminal activation domain. These mutations prevent synthesis of full-length GATA1, but not synthesis of a shorter variant that is initiated downstream. We show that the shorter GATA1 protein, which lacks the N-terminal activation domain, binds DNA and interacts with its essential cofactor Friend of GATA1 (FOG1; encoded by ZFPM1)4 to the same extent as does full-length GATA1, but has a reduced transactivation potential. Although some reports suggest that the activation domain is dispensable in cell-culture models of hematopoiesis5,6, one study has shown that it is required for normal development in vivo7. Together, these findings indicate that loss of wildtype GATA1 constitutes one step in the pathogenesis of AMKL in Down syndrome.

With the increasing popularity of the yeast two-hybrid screen, a large number of protein–protein interactions have been identified. In many cases, single proteins have been found to associate with a large number of cofactors. For example, the hematopoietic transcription factor GATA-1 interacts with a multitude of other nuclear proteins, including Friend of GATA-1 (FOG-1), EKLF, CBP/p300, and Lmo2. Furthermore, p300, besides associating with GATA-1, interacts with at least seven other hematopoietic transcription factors. Despite the numerous pairwise and higher-order interactions identified, assessment of their contribution to transcriptional control has been lacking. Here we describe a strategy that can be applied to assess the functional relevance of any protein–protein association. This approach involves the creation of altered specificity mutants though the use of a combination of two yeast two-hybrid screens. Once altered specificity factors are obtained, a researcher can then proceed to functional assays that address the role of a specific protein–protein interaction.

Myelofibrosis (MF) is characterized by hyperactivation of thrombopoietin (TPO) signaling, which induces a RPS14 deficiency that de-regulates GATA1 in megakaryocytes by hampering its mRNA translation. As mice carrying the hypomorphic Gata1low mutation, which reduces the levels of Gata1 mRNA in megakaryocytes, develop MF, we investigated whether the TPO axis is hyperactive in this model. Gata1low mice contained two times more Tpo mRNA in liver and TPO in plasma than wild-type littermates. Furthermore, Gata1low LSKs expressed levels of Mpl mRNA (five times greater than normal) and protein (two times lower than normal) similar to those expressed by LSKs from TPO-treated wild-type mice. Gata1low marrow and spleen contained more JAK2/STAT5 than wild-type tissues, an indication that these organs were reach of TPO-responsive cells. Moreover, treatment of Gata1low mice with the JAK inhibitor ruxolitinib reduced their splenomegaly. Also in Gata1low mice activation of the TPO/MPL axis was associated with a RSP14 deficiency and a discordant microarray ribosome signature (reduced RPS24, RPS26 and SBDS expression). Finally, electron microscopy revealed that Gata1low megakaryocytes contained poorly developed endoplasmic reticulum with rare polysomes. In summary, Gata1low mice are a bona fide model of MF, which recapitulates the hyperactivation of the TPO/MPL/JAK2 axis observed in megakaryocytes from myelofibrotic patients.

Acute myeloid leukemia (AML) is a hematologic malignancy that is the most common type of acute leukemia diagnosed in adults and the second most common type in children. The overall survival is poor and treatment is associated with significant complications and even death. In addition, a significant number of patients will not respond to therapy or relapse. In this review, several new signaling proteins aberrantly regulated in AML are described, including CREB, Triad1, Bcl-2 family members, Stat3, and mTOR/MEK. Identifying more effective and less toxic agents will provide novel approaches to treat AML.

GATA-family transcription factors are critical to the development of diverse tissues. In particular, GATA-4 has been implicated in formation of the vertebrate heart. As the mouse Gata-4knock-out is early embryonic lethal because of a defect in ventral morphogenesis, the in vivo function of this factor in heart development remains unresolved. To search for a requirement for Gata4 in heart development, we created mice harboring a single amino acid replacement in GATA-4 that impairs its physical interaction with its presumptive cardiac cofactor FOG-2. Gata4ki/ki mice die just after embryonic day (E) 12.5 exhibiting features in common with Fog2−/− embryos as well as additional semilunar cardiac valve defects and a double-outlet right ventricle. These findings establish an intrinsic requirement for GATA-4 in heart development. We also infer that GATA-4 function is dependent on interaction with FOG-2 and, very likely, an additional FOG protein for distinct aspects of heart formation.

The commitment of multipotent cells to particular developmental pathways requires specific changes in their transcription factor complement to generate the patterns of gene expression characteristic of specialized cell types. We have studied the role of the GATA cofactor Friend of GATA (FOG) in the differentiation of avian multipotent hematopoietic progenitors. We found that multipotent cells express high levels of FOG mRNA, which were rapidly down-regulated upon their C/EBPβ-mediated commitment to the eosinophil lineage. Expression of FOG in eosinophils led to a loss of eosinophil markers and the acquisition of a multipotent phenotype, and constitutive expression of FOG in multipotent progenitors blocked activation of eosinophil-specific gene expression by C/EBPβ. Our results show that FOG is a repressor of the eosinophil lineage, and that C/EBP-mediated down-regulation of FOG is a critical step in eosinophil lineage commitment. Furthermore, our results indicate that maintenance of a multipotent state in hematopoiesis is achieved through cooperation between FOG and GATA-1. We present a model in which C/EBPβ induces eosinophil differentiation by the coordinate direct activation of eosinophil-specific promoters and the removal of FOG, a promoter of multipotency as well as a repressor of eosinophil gene expression.

Haematopoietic development is regulated by nuclear protein complexes that coordinate lineage-specific patterns of gene expression. Targeted mutagenesis in embryonic stem cells and mice has revealed roles for the X-linked gene Gata1 in erythrocyte and megakaryocyte differentiation. GATA-1 is the founding member of a family of DNA-binding proteins that recognize the motif WGATAR through a conserved multifunctional domain consisting of two C4-type zinc fingers. Here we describe a family with X-linked dyserythropoietic anaemia and thrombocytopenia due to a substitution of methionine for valine at amino acid 205 of GATA-1. This highly conserved valine is necessary for interaction of the amino-terminal zinc finger of GATA-1 with its essential cofactor, FOG-1 (for friend of GATA-1; refs 9-12). We show that the V205M mutation abrogates the interaction between Gata-1 and Fog-1, inhibiting the ability of Gata-1 to rescue erythroid differentiation in an erythroid cell line deficient for Gata-1 (G1E). Our findings underscore the importance of FOG-1:Gata-1 associations in both megakaryocyte and erythroid development, and suggest that other X-linked anaemias or thrombocytopenias may be caused by defects in GATA1.

GATA-1 and FOG (Friend of GATA-1) are each essential for erythroid and megakaryocyte development. FOG, a zinc finger protein, interacts with the amino (N) finger of GATA-1 and cooperates with GATA-1 to promote differentiation. To determine whether this interaction is critical for GATA-1 action, we selected GATA-1 mutants in yeast that fail to interact with FOG but retain normal DNA binding, as well a compensatory FOG mutant that restores interaction. These novel GATA-1 mutants do not promote erythroid differentiation of GATA-1- erythroid cells. Differentiation is rescued by the second-site FOG mutant. Thus, interaction of FOG with GATA-1 is essential for the function of GATA-1 in erythroid differentiation. These findings provide a paradigm for dissecting protein-protein associations involved in mammalian development.

Down syndrome confers a 20-fold increased risk of B cell acute lymphoblastic leukemia (B-ALL), and polysomy 21 is the most frequent somatic aneuploidy among all B-ALLs. Yet the mechanistic links between chromosome 21 triplication and B-ALL remain undefined. Here we show that germline triplication of only 31 genes orthologous to human chromosome 21q22 confers mouse progenitor B cell self renewal in vitro, maturation defects in vivo and B-ALL with either the BCR-ABL fusion protein or CRLF2 with activated JAK2. Chromosome 21q22 triplication suppresses histone H3 Lys27 trimethylation (H3K27me3) in progenitor B cells and B-ALLs, and 'bivalent' genes with both H3K27me3 and H3K4me3 at their promoters in wild-type progenitor B cells are preferentially overexpressed in triplicated cells. Human B-ALLs with polysomy 21 are distinguished by their overexpression of genes marked with H3K27me3 in multiple cell types. Overexpression of HMGN1, a nucleosome remodeling protein encoded on chromosome 21q22 (refs. 3,4,5), suppresses H3K27me3 and promotes both B cell proliferation in vitro and B-ALL in vivo.