Syndrome Acute Megakaryoblastic Leukemia Research Articles

Neonatal and adult megakaryopoiesis

It has become increasingly clear that there are substantial biological differences between fetal/neonatal and adult megakaryopoiesis. Over the last 18 months, studies challenged the paradigm that neonatal megakaryocytes are immature and revealed a developmentally unique uncoupling of proliferation, polyploidization, and cytoplasmic maturation. Several studies also described substantial molecular differences between fetal/neonatal and adult megakaryocytes involving transcription factors, signaling pathways, cytokine receptors, and microRNAs. This review will summarize our current knowledge on the developmental differences between fetal/neonatal and adult megakaryocytes, and recent advances in the underlying molecular mechanisms, including differences in transcription factors, in the response to thrombopoietin (Tpo), and newly described developmentally regulated signaling pathways. We will also discuss the implications of these findings on the way megakaryocytes interact with the environment, the response of neonates to thrombocytopenia, and the pathogenesis of Down syndrome-transient myeloproliferative disorder (TMD) and Down syndrome-acute megakaryoblastic leukemia (DS-AMKL). A better characterization of the molecular differences between fetal/neonatal and adult megakaryocytes is critical to elucidating the pathogenesis of a group of disorders that selectively affect fetal/neonatal megakaryocyte progenitors, including the thrombocytopenia-absent radius (TAR) syndrome, Down syndrome-TMD or Down syndrome-AMKL, and the delayed platelet engraftment following cord blood transplantation.

Mosaic trisomy 21 and trisomy 14 as acquired cytogenetic abnormalities without GATA1 mutation in a pediatric non-down syndrome acute megakaryoblastic leukemia

One case of acute megakaryoblastic leukemia (AMKL) with trisomy 21, trisomy 14 and unmutated GATA1 gene in a phenotypically normal girl was reported. The patient experienced transient myelodysplasia before the onset of AMKL. The bone marrow blasts manifested typical morphology of megakaryoblast both by the May-Giemsa staining and under the electronic microscopy. Leukemic cells were positive for CD13, CD33, CD117, CD56, CD38, CD41 and CD61 in flow cytometry analysis. Cytogenetic study showed karyotype of 48, XX, +14, +21 in 40% metaphases. Known mutations of GATA1 gene in Down syndrome or acquired trisomy 21 were not detected in this case.

Ikaros Is a GATA Switch Target Gene That Restrains Megakaryopoiesis In Part by Blocking Notch Signaling.

AbstractAbstract 1602The hematopoietic transcription factor Ikaros regulates the development of lymphoid cells. In particular, Ikaros has been shown to suppress expression of Notch target genes in developing thymocytes. In addition, Ikaros-deficient animals fail to develop B cells and instead display a T cell lymphoproliferative disorder. Furthermore, alterations in Ikaros gene expression are associated with human lymphoid neoplasms and loss of Ikaros is associated with progression of myeloproliferative neoplasms to acute myeloid leukemia. However, the role of Ikaros in normal and malignant myelopoiesis is not well characterized. Therefore, we first analyzed myeloid development in Ikaros deficient mice and observed extramedullary hematopoiesis in the spleen and a striking thrombocytosis in the peripheral blood of 8–9 week-old Ikaros-null mice (2×106 platelets/μl, compared to 0.5×106 platelets/μl in wild-type littermates). Flow cytometry, histology and colony forming assays revealed that the bone marrow and spleen of young Ikaros-null mice harbored a substantial increase in the numbers of megakaryocytes and myeloid cells. We next investigated how Ikaros activity is reduced during terminal differentiation. Previous reports have shown that GATA1-deficient and GATA1s mutant megakaryocytes, which are associated with Down syndrome acute megakaryoblastic leukemia (DS-AMKL), express aberrantly high levels of Ikaros, suggesting that Ikaros is a target of GATA-1 repression during terminal megakaryocyte differentiation. By chromatin immunoprecipitation assays, we found multiple sites in the Ikaros locus that are bound by GATA-2 in proliferating progenitors and by GATA-1 in maturing megakaryocytes. Furthermore, we discovered that GATA-1s fails to occupy these conserved Ikaros loci. Together these results strongly suggest that GATA-1, but not GATA-1s, displaces GATA-2 from the Ikaros gene during differentiation and that this GATA switch leads to repression of Ikaros, in turn allowing for terminal maturation of megakaryocytes. Since Notch participates in specification of the megakaryocyte lineage, we then asked whether Ikaros inhibited megakaryopoiesis by interfering with Notch signaling. Retroviral transduction of full-length Ikaros in murine common myeloid progenitors, but not in LSK cells, reduced megakaryocyte development upon Notch stimulation in OP9-DL1 stromal co-cultures. Ectopic expression of Ikaros in the 6133 cell line, which models the t(1;22) subtype of AMKL and shows aberrant Notch target genes activation by the OTT-MAL fusion oncogene, inhibited proliferation and induced apoptosis. Genome wide expression profiling in transduced 6133 cells confirmed that Ikaros alters expression of several genes involved in the Notch and growth factor signaling pathways. These results indicate that Ikaros restricts megakaryocyte development and inhibits proliferation of OTT-MAL-transformed AMKL cells at least in part by suppressing Notch signaling. Together, our results suggest that a functional antagonism between the Notch pathway and Ikaros controls normal megakaryocyte development and that this axis is deregulated in AMKL, contributing to aberrant expansion of immature megakaryoblasts. Disclosures:No relevant conflicts of interest to declare.

Developmental stage-specific interplay of GATA1 and IGF signaling in fetal megakaryopoiesis and leukemogenesis

Oncogene-mediated transformation of hematopoietic cells has been studied extensively, but little is known about the molecular basis for restriction of oncogenes to certain target cells and differential cellular context-specific requirements for oncogenic transformation between infant and adult leukemias. Understanding cell type-specific interplay of signaling pathways and oncogenes is essential for developing targeted cancer therapies. Here, we address the vexing issue of how developmental restriction is achieved in Down syndrome acute megakaryoblastic leukemia (DS-AMKL), characterized by the triad of fetal origin, mutated GATA1 (GATA1s), and trisomy 21. We demonstrate overactivity of insulin-like growth factor (IGF) signaling in authentic human DS-AMKL and in a DS-AMKL mouse model generated through retroviral insertional mutagenesis. Fetal but not adult megakaryocytic progenitors are dependent on this pathway. GATA1 restricts IGF-mediated activation of the E2F transcription network to coordinate proliferation and differentiation. Failure of a direct GATA1-E2F interaction in mutated GATA1s converges with overactive IGF signaling to promote cellular transformation of DS fetal progenitors, revealing a complex, fetal stage-specific regulatory network. Our study underscores context-dependent requirements during oncogenesis, and explains resistance to transformation of ostensibly similar adult progenitors.

Developmental Stage-Specific Interplay Between GATA1 and IGF Signaling in Fetal Hematopoiesis and Leukemogenesis.

Abstract 386Oncogene-mediated transformation of hematopoietic cells has been extensively studied, but little is known about the molecular basis for restriction of oncogenes to certain target cells and differential cellular context-specific requirements for oncogenic transformation between infant and adult leukemias. Understanding cell type-specific interplay between signaling pathways and oncogenes is essential for developing targeted cancer therapies.To study how developmental restriction is achieved in Down syndrome acute megakaryoblastic leukemia (DS-AMKL), characterized by a triad of fetal origin, mutated GATA1 (GATA1s), and trisomy 21, we generated a DS-AMKL mouse model (G1s-mAMKL) through retroviral insertional mutagenesis. Using cross-species integrated genomic analysis, we demonstrate overactivity of insulin-like growth factor (IGF) signaling in murine and authentic human DS-AMKL. ShRNA mediated IGF1R knockdown in DS-AMKL cell lines (CMK, CMY) led to a dramatic reduction of transduced cells, whereas no substantial effect was observed in “control” cell lines, including K562, CHRF288-11, and M-07. Pharmacological activation and repression of IGF1R signaling followed by in vitro culture, CFU assays and intracellular phosphoprotein staining showed that in both murine and primary human AMKL cells expressing GATA1s, the IGF/IGF1R/mTOR pathway is highly activated and is the major mitogenic pathway for growth. We observed a significant delay of leukemia onset upon IGF1R knockdown after transplantation of G1s-mAMKL cells into RAG2-/- recipients. Gene set enrichment analysis revealed that the mitogenic activity of IGF1R is exerted at the transcriptional level by activation of E2F target genes. Using a genetic approach we demonstrate that fetal, but not adult, hematopoietic progenitor cells are dependent on IGF1R signaling, showing a developmental difference in the activity of this pathway. Gata1 restricts IGF-mediated activation of the E2F transcription network to coordinate proliferation and differentiation. In contrast, mutated Gata1s is insufficient to control the IGF1R induced proliferation in fetal megakaryocytic progenitors (MPs), which were stimulated or retrovirally transduced with IGFR ligands (IGF1 and IGF2). The hyperproliferative phenotype of Gata1s fetal MPs could be rescued by shRNA mediated repression of global E2F transcription activity.Thus, by focusing on DS-AMKL, which is restricted to neonates and infants, we have discovered fetal stage-specific interplay between insulin-like growth factor (IGF) signaling and lineage-determining transcription factor, GATA1. Acquired mutation in GATA1 perturbs the regulatory function of GATA1 on IGF pathway. Together with overactive IGF signaling, mutant GATA1 leads to malignant transformation of DS fetal progenitors, underscoring context-dependent requirements during oncogenesis and explaining resistance to transformation of ostensibly similar adult progenitors. Disclosures:No relevant conflicts of interest to declare.

ERG Is a Megakaryocytic Oncogene

Ets-related gene (ERG) is a member of the ETS transcription factor gene family located on Hsa21. ERG is known to have a crucial role in establishing definitive hematopoiesis and is required for normal megakaryopoiesis. Truncated forms of ERG are associated with multiple cancers such as Ewing's sarcoma, prostate cancer, and leukemia as part of oncogenic fusion translocations. Increased expression of ERG is highly indicative of poor prognosis in acute myeloid leukemia and ERG is expressed in acute megakaryoblastic leukemia (AMKL); however, it is unclear if expression of ERG per se has a leukemogenic activity. We show that ectopic expression of ERG in fetal hematopoietic progenitors promotes megakaryopoiesis and that ERG alone acts as a potent oncogene in vivo leading to rapid onset of leukemia in mice. We observe that the endogenous ERG is required for the proliferation and maintenance of AMKL cell lines. ERG also strongly cooperates with the GATA1s mutated protein, found in Down syndrome AMKL, to immortalize megakaryocyte progenitors, suggesting that the additional copy of ERG in trisomy 21 may have a role in Down syndrome AMKL. These data suggest that ERG is a hematopoietic oncogene that may play a direct role in myeloid leukemia pathogenesis.

Developmental stage-specific interplay between GATA1 and IGF signaling during fetal hematopoiesis and leukemogenesis

Fetal and adult hematopoiesis are distinct. Signaling pathways and their coordination with lineage-specific transcription during fetal hematopoiesis and their subversion during leukemogenesis are poorly understood. Down syndrome acute megakaryoblastic leukemia (AMKL) is characterized by a triad of fetal origin, mutated GATA1 (GATA1s), and trisomy 21, providing a unique model system to address these issues. We used a cross-species integrated genomic approach to understand how developmental specificity is achieved in DS-AMKL and demonstrate overactivity of IGF signaling in both a DS-AMKL mouse-model -generated through retroviral insertional mutagenesis- and human DS-AMKL. Fetal, but not adult, hematopoietic progenitor cells are dependent on IGF signaling, which exerts its mitogenic potential by activation of the E2F transcription network. The lineage determining transcription factor GATA1 restricts this activation to coordinate proliferation and differentiation. Perturbation of this feature in mutated GATA1s, in convergence with overactivated IGF signaling, commonly deregulates cell-cycle control and contributes to malignant transformation, revealing fetal context-dependent requirements during DS leukemogenesis.

GATA-2 Controls Proliferation of Megakaryocyte Progenitors and Its Expression Contributes to Human DS-AMKL.

GATA2 is an essential transcription factor that regulates multiple aspects of hematopoiesis. Mutations in GATA2 are associated with blast crisis phase of chronic myelogenous leukemia (CML) while overexpression of GATA2 is a hallmark of Down syndrome acute megakaryoblastic leukemia (DS-AMKL), a malignancy that is defined by the combination of trisomic chromosome 21 and a GATA1 mutation. Here, we show thatGATA2 is essential for megakaryocyte development from both wild-type and GATA-1mutant hematopoietic progenitors. Furthermore, we reveal that GATA2 is indispensable for cell cycle progression and that its expression cooperates with activated JAK3 to promote TPO-independent expansion of megakaryocytes. Genome-wide microarray analysis revealed that GATA2 regulates a wide set of genes including cell cycle regulators and lineage-specific genes that are essential for terminal differentiation of megakaryocytes. Of note, a subset of genes within the GATA2 signature is contained within human DS-AMKL patient specimens. These observations implicate overexpression of GATA2 in the aberrant gene expression and proliferation of AMKL. Taken together, our data demonstrate thatGATA2 is a critical regulator of normal and malignant megakaryopoiesis.

ETS2 and ERG1 Promote Megakaryopoiesis and Immortalize GATA1 Knockdown (ΔneoΔHS) Fetal Liver Progenitors: Implications for Leukemogenesis in Down Syndrome.

ETS2 and ERG are two ETS family transcription factors encoded on chromosome 21 that have been implicated in leukemia and other types of cancer. In the setting of trisomy 21 (e.g. in individuals with Down syndrome) there is a significant increase in the propensity for leukemia, including AMKL (acute megakaryoblastic leukemia). It has been theorized that genes located on chromosome 21 may contribute to the predisposition of children with Down syndrome for pediatric AMKL, possibly by collaborating with mutations in the transcriptional regulator GATA1. To test the oncogenic potential of these genes in the hematopoietic system, ETS2 and the ERG isoform, ERG1, were over-expressed in both wild-type and GATA1 knockdown (ΔneoΔHS) fetal liver hematopoietic progenitors and the effects on growth and differentiation were studied. We discovered that over-expression of either ETS2 or ERG1 in wild-type progenitors increased the formation of CD41 positive megakaryocytes by 2–3 fold in liquid culture. However, whereas ETS2 appeared to primarily promote the early stages of megakaryopoiesis, ERG1 over-expression led to a dramatic rise in the percentage of cells expressing CD42, a marker of more mature megakaryocytes. Ectopic ERG1 expression also affected the ploidy profile, with nearly all cells containing ≥4N DNA content. Quantitative real time RT-PCR analysis of lineage specific genes confirmed that expression of megakaryocyte genes, including GPIIb (CD41), platelet factor 4 (PF4), and ß1-tubulin, was significantly increased in undifferentiated cells over-expressing either ETS2 or ERG1. These results show that over-expression of ETS2 or ERG1 rapidly initiates the megakaryocyte program. Over-expression of either ETS2 or ERG1 in GATA1 knockdown fetal liver cells similarly led to a 2.5–5 fold increase in the formation of CD41 positive megakaryocytes in liquid culture; however, neither ETS2 or ERG1 could circumvent the differentiation block that is characteristic of the GATA1 knockdown strain, including the inability to form proplatelet extensions. We next assessed the capacity of ETS2 or ERG1 over-expression to alter the colony forming ability of wild-type and GATA1 knockdown fetal liver progenitors. Both ETS2 and ERG1 expression led to a significant increase in CFU-MK derived from wild-type progenitors, as evidenced by an increase in the number of acetylcholinesterase positive colonies. In contrast, neither gene altered the formation of CFU-MK when over-expressed in the GATA1 knockdown progenitors, which already show a prominent enhancement in megakaryocyte colony formation over wild-type fetal liver cells. Finally, serial re-plating assays performed with GATA1 knockdown fetal liver cells showed that ectopic expression of either ETS2 or ERG1 could immortalize hematopoietic progenitors, whereas only ERG1 could enhance colony re-plating of wild-type cells. These findings provide evidence for a specific synergy between GATA-1 deficiency and ETS2 over-expression and suggest a distinct oncogenic potential for each factor. Overall, these results in primary murine fetal liver cells demonstrate that ETS2 and ERG1 promote the megakaryocyte lineage and facilitate immortalization of GATA1 knockdown cells, consistent with a potential oncogenic role in Down syndrome AMKL for these two ETS family genes located on chromosome 21.

Trisomy 21 Expands the Megakaryocyte-Erythroid Progenitor Compartment in Human Fetal Liver-Implications for Down Syndrome AMKL.

Children with Down syndrome (DS) have a uniquely high frequency of acute megakaryoblastic leukemia (AMKL)- ~500-fold increased compared to children without trisomy 21 (T21). At least two genetic events are required but are not sufficient for DS-AMKL: T21 and N-terminal truncating mutations in the key megakaryocytic transcription factor GATA1. This tight association of T21 with GATA1 mutations and the development of AMKL in a narrow temporal window (fetal life-5yrs) makes DS-AMKL a highly informative model of multi-hit leukemogenesis in which the first steps occur in utero. However, the individual contributions of T21 and mutant GATA1 in the leukemogenesis are unclear. To specifically investigate the role of T21 in DS-AMKL and why leukemia-initiation is confined to fetal (or early post-natal) life we have studied fetal hemopoiesis in DS during the second and third trimester in 16 fetuses (gestational age 15–37 weeks) where an antenatal diagnosis of DS with T21 was made by amniotic fluid fetal cell karyotyping. Samples of fetal blood (n=13), fetal liver (n=9) and fetal bone marrow (n=8) were screened for mutations in the GATA1 gene genomic DNA by DHPLC or direct sequencing (sensitivity of detecting a GATA1 mutation is 1–5% by DHPLC). No GATA1 mutations were detected. This allowed us to study the impact of T21 independent of GATA1 mutation on fetal hemopoiesis. DS fetuses showed marked qualitative and quantitative abnormalities in hemopoiesis. While the total number of CD34+ cells in DS and normal fetal liver were comparable, DS fetuses had a striking increase in bi-potential megakaryocyte-erythroid progenitors (MEP; CD34+CD38+FcgloCD45RA+− 74.4% vs 27.0% of fetal liver CD34+/CD38+ cells. Peripheral blood from all DS fetuses studied compared to normal fetal blood samples showed dysmegakaryopoiesis (abnormally shaped and/or giant platelets and MK fragments), dyserythropoiesis (macrocytes, poikilocytes, basophilic stippling), increased numbers of blast cells and also had an increased percentage of MEPs − 40.3% vs 26.9%. By contrast, there was no difference in the number of MEP nor erythroid or MK lineage morphology in DS fetal bone marrow compared to normal fetal bone marrow. CD34+ cells from DS fetal liver and fetal blood expressed both fl GATA1 and GATA1s mRNA indicating that dysmegakaryopoiesis and erythropoiesis were not due to lack of expression of fl GATA1. These data indicate, for the first time, that T21 by itself profoundly disturbs megakaryopoiesis and erythropoiesis and leads to an increased of frequency of MEP. This has important implications since it provides a testable hypothesis for the role of T21 in the initiating step of AMKL, namely that T21 expands a fetal liver-derived progenitor compartment which forms a substrate upon which GATA1 mutations then confer a further selective advantage.

GATA Transcription Factors in Hematologic Disease

GATA family transcription factors play essential roles in broad developmental settings. GATA-1, one of the hematopoietically expressed members, is required for normal erythroid and megakaryocytic differentiation. Over the past few years, mutations in the gene encoding GATA-1 have been linked to several human hematologic disorders, including X-linked dyserythropoietic anemia and thrombocytopenia, X-linked thrombocytopenia and beta-thalassemia, and Down syndrome acute megakaryoblastic leukemia. This review summarizes the role of GATA-1 during normal hematopoiesis and discusses how disease-associated mutations may affect its function.

Mutations in GATA1 in both transient myeloproliferative disorder and acute megakaryoblastic leukemia of Down syndrome

Mutations in transcription factors often contribute to human leukemias by providing a block to normal differentiation. To determine whether mutations in the hematopoietic transcription factor GATA1 are associated with leukemia, we assayed for alterations in the GATA1 gene in bone marrow samples from patients with various subtypes of acute leukemia. Here we summarize our findings that GATA1 is mutated in the leukemic blasts of patients with Down syndrome acute megakaryoblastic leukemia (DS-AMKL). We did not find mutations in GATA1 in leukemic cells of DS patients with other types of acute leukemia, or in other patients with AMKL who did not have DS. Furthermore, we did not detect GATA1 mutations in DNAs from over 75 other patients with acute leukemia or from 21 healthy individuals. Since the GATA1 mutations were restricted to DS-AMKL, we also investigated whether GATA1 was altered in the “preleukemia” of DS, transient myeloproliferative disorder (TMD). TMD is a common myeloid disorder that affects 10% of DS newborns and evolves to AMKL in nearly 30% patients. We detected GATA1 mutations in TMD blasts from every infant examined. Together, these results demonstrate that GATA1 is likely to play a critical role in the etiology of TMD and DS-AMKL, and that mutagenesis of GATA1 represents a very early event in DS myeloid leukemogenesis. We hypothesize that disruption of normal GATA-1 function is an essential step in the initiation of megakaryoblastic leukemia in DS.

GATA1 mutations in transient leukemia and acute megakaryoblastic leukemia of Down syndrome

AbstractChildren with constitutional trisomy 21 (Down syndrome) have an approximately 500-fold increased risk of developing acute megakaryoblastic leukemia (AMKL), a form of acute myeloid leukemia. Unique to newborn infants with Down syndrome is a transient leukemia (TL), also referred to as transient myeloproliferative syndrome, that undergoes spontaneous remission in the majority of cases but in approximately 20% is followed by AMKL later in life. Recently mutations of the gene encoding the hematopoietic transcription factor GATA1 were shown to be specific for AMKL of Down syndrome. Here, we demonstrate that GATA1 mutations are present in blasts of TL and show the identical GATA1 mutation in sequential samples collected from a patient during TL and subsequent AMKL. These findings suggest a model of malignant transformation in Down syndrome AMKL in which GATA1 mutations are an early event and AMKL arises from latent TL clones following initial apparent remission.