Children with Down syndrome have a 10-20-fold elevated risk of developing leukemia, particularly acute megakaryoblastic leukemia (AMKL). While a subset of pediatric AMKLs is associated with the 1;22 translocation and expression of a mutant fusion protein, 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) 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 hematopoiesis, one study has shown that it is required for normal development in vivo. Together, these findings indicate that loss of wildtype GATA1 constitutes one step in the pathogenesis of AMKL in Down syndrome.

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.

IntroductionOur understanding of the molecular pathogenesis of myeloid malignancies, most notably acute myeloid leukemia (AML) and chronic myeloid leukemia (CML), has largely resulted from the identification and characterization of recurrent chromosomal translocations. 1 However, in many patients with myeloproliferative neoplasms (MPNs) and chronic myelomonocytic leukemia (CMML), recurrent clonal cytogenetic abnormalities are not observed. More recently, DNA resequencing studies of candidate genes, 2 gene families, 3,4 and the cancer genome 5 in MPN, CMML, and AML have identified somatic mutations in FLT3, 6 JAK2, [7][8][9][10][11][12][13]14,15 and the RAS family of oncogenes. 16 These discoveries demonstrate activation of signal transduction pathways is a common pathogenic event in myeloid malignancies and have led to the development of molecularly targeted therapies. However, with the exception of CML, these therapies have yet to substantively improve outcomes for patients with myeloid malignancies. 17,18 This may reflect insufficient target inhibition, or, alternatively, this may indicate incomplete dependence on these activated pathways resulting from the presence of additional somatic mutations with prognostic, therapeutic, and biologic relevance.The role of TET (Ten-Eleven Translocation) family gene members in hematopoietic transformation was thought to be restricted to the involvement of TET1 as a translocation partner MLL-translocated AML, until the recent identification of inactivating mutations in TET2 in MPN and MDS patients. 19 We therefore sought to evaluate a large set of MPN, CMML, and AML samples for somatic TET2 alterations. We sequenced all coding exons of TET2 in 408 paired tumor/normal samples and then assessed the frequency of somatic TET2 mutations in 606 patients with MPN, CMML, and AML. We also investigated whether deletion or epigenetic inactivation of TET2 are observed in MPN and evaluated MPN patients for somatic mutations in TET1 and TET3. Methods Copy number analysis of TET1, TET2, and TET3A total of 207 MPN tumor samples were analyzed using Affymetrix 250K StyI Arrays. 20 The JAK2V617F-mutant AML cell lines HEL and SET2 were analyzed using Affymetrix 6.0 SNP Arrays. Methylation-specific polymerase chain reactionMethylation of 2 CpG islands in the promoter region of TET2 was assessed in 37 MPN patients and 4 JAK2V617F-positive leukemia cell lines (SET2, MBO2, HEL, UKE1). Methylation-specific polymerase chain reaction was performed as previously described (primers are listed in supplemental Table 1). 21 StatisticsStatistical analyses were performed using MedCalc (MedCalc). Results and discussionSequence analysis of all coding exons of TET2 in 408 paired tumor/normal samples identified 8 frameshift, 12 nonsense, and 37 nonsynonymous alterations not present in dbSNP. Analysis of germ line DNA distinguished between 31 somatic missense mutations and 6 unannotated SNPs (Table 1; supplemental Figure 1); all unannotated SNPs were observed in matched normal tissue in at least 2 samples. Al...

Tyrosine kinases are aberrantly activated in numerous malignancies, including acute myeloid leukemia (AML). To identify tyrosine kinases activated in AML, we developed a screening strategy that rapidly identifies tyrosine-phosphorylated proteins using mass spectrometry. This allowed the identification of an activating mutation (A572V) in the JAK3 pseudokinase domain in the acute megakaryoblastic leukemia (AMKL) cell line CMK. Subsequent analysis identified two additional JAK3 alleles, V722I and P132T, in AMKL patients. JAK3(A572V), JAK3(V722I), and JAK3(P132T) each transform Ba/F3 cells to factor-independent growth, and JAK3(A572V) confers features of megakaryoblastic leukemia in a murine model. These findings illustrate the biological importance of gain-of-function JAK3 mutations in leukemogenesis and demonstrate the utility of proteomic approaches to identifying clinically relevant mutations.

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-4 knock-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. Transcriptional activity of the GATA-factors is modulated through interaction with nuclear proteins, including zinc finger proteins of the Kruppel and FOG/Ushaped families, general coactivators (p300 and CBP), the myocardial-expressed protein Nkx2.5, and NF-AT3 (Durocher and Nemer 1998;Mackay and Crossley 1998;Blobel 2000;Molkentin 2000). Whereas the specificity and in vivo functional relevance of many of these interactions are incompletely defined, the association of GATA-1 with FOG-1 has been examined in detail. FOG-1 interacts with GATA-1 in hematopoietic cells and regulates the ability of GATA-1 to promote terminal differentiation of erythroid cells and megakaryocytes (Tsang et al. 1997). Mutation of specific residues within the conserved N-terminal zinc finger of GATA-1, such as V205G, disrupts binding to FOG-1, preserves DNA-binding properties of GATA-1, and renders GATA-1 unable to promote terminal differentiation of red blood cells (Crispino et al. 1999). Furthermore, mutation of Val 205 in humans leads to congenital dyserythropoietic anemia and thrombocytopenia (Nichols et al. 2000). Taken together, these findings demonstrate that direct physical association of GATA-1 and FOG-1 is essential for GATA-1's roles in transcription and, critical for the experiments reported herein, identifies a specific residue of the N finger that mediates cofactor interaction.GATA-4, GATA-5, and GATA-6, nonhematopoietic expressed factors, are implicated in development of heart, endoderm, and intestinal epithelia, where they are expressed in an overlapping and dynamic fashion Bossard and Zaret 1998;Gao et al. 1998;Koutsourakis et al. 1999;Parmacek and Leiden 1999;Molkentin 2000). GATA-4 has been extensively studied in the context of heart development, as it is present in precardiac splanchnic mesoderm and binds to and activates promoters and enhancers of numerous myocardial-expressed genes . In its absence, mouse embryos die by E7.0-9.5, with failure of ventral morphogenesis leading to cardiac bifida (Kuo et al. 1997;Molkentin et al. 1997). The deat...

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.

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 DSleukemia research. With the increased knowledge of the way in which trisomy 21 affects hematopoiesis and the specific genetic mutations that are found in DSassociated leukemias, we are well on our way toward designing improved strategies for treating both myeloid and lymphoid malignancies in this high-risk population. (Blood. 2009;113:2619-2628) IntroductionDown syndrome (DS), or constitutional trisomy 21, is the most common human aneuploidy, with an incidence of 1 in 700 births. Nearly 80 different clinical phenotypes have been identified in people with DS, including cognitive impairment, craniofacial dysmorphy, gastrointestinal tract abnormalities, congenital heart defects, endocrine abnormalities, neuropathology leading to dementia, and immunologic defects. With respect to the hematopoietic system, children with DS frequently show macrocytosis, abnormalities in platelet counts, and an increased prevalence of leukemia. 1,2 The incidence of acute lymphoblastic leukemia (ALL; the most common leukemia in childhood) in children with DS is approximately 20-fold higher than in the general population, while the incidence of acute megakaryoblastic leukemia (AMKL) is 500-fold higher. 2 Furthermore, it has been estimated that between 4% and 10% of infants with DS are born with transient myeloproliferative disease (TMD), a clonal disease that is characterized by immature megakaryoblasts in the fetal liver and peripheral blood. 3,4 Although TMD spontaneously disappears in most cases, it is regarded as a preleukemic syndrome; approximately 20% of children diagnosed with TMD develop DS-AMKL within 4 years. The natural history of leukemia in children with DS suggests that trisomy 21 directly and functionally contributes to the malignant transformation of hematopoietic cells. It is important to note, however, that DS is not a classic genomic instability syndrome, as the overall risk of developing cancer, in particular solid tumors, is lower in these people. 5 In line with these data, experiments with a mouse model of DS showed that trisomy for orthologs of about half of the genes on chromosome 21 led to a significant reduction in the number of adenomatous polyposis coli (multiple intestinal neoplasia [APC(min)]-mediated intestinal tumors. 6 To better understand the impact of trisomy 21 on hematopoiesis, studies have been undertaken with human fetal liver cells as well as animal and cell-line models to determine the c...