Trisomy 21 enhances human fetal erythro-megakaryocytic development

Abstract Children with Down syndrome exhibit 2 related hematopoietic diseases: transient myeloproliferative disorder (TMD) and acute megakaryoblastic leukemia (AMKL). Both exhibit clonal expansion of blasts with biphenotypic erythroid and megakaryocytic features and contain somatic GATA1 mutations. While altered GATA1 inhibits erythro-megakaryocytic development, less is known about how trisomy 21 impacts blood formation, particularly in the human fetus where TMD and AMKL originate. We used in vitro and mouse transplantation assays to study hematopoiesis in trisomy 21 fetal livers with normal GATA1 alleles. Remarkably, trisomy 21 progenitors exhibited enhanced production of erythroid and megakaryocytic cells that proliferated excessively. Our findings indicate that trisomy 21 itself is associated with cell-autonomous expansion of erythro-megakaryocytic progenitors. This may predispose to TMD and AMKL by increasing the pool of cells susceptible to malignant transformation through acquired mutations in GATA1 and other cooperating genes.

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GATA1-Centered Genetic Network on Chromosome 21 Drives Down Syndrome Acute Megakaryoblastic Leukemia

Blood ◽

10.1182/blood.v124.21.4310.4310 ◽

2014 ◽

Vol 124 (21) ◽

pp. 4310-4310

Author(s):

Lena Stachorski ◽

Dirk Heckl ◽

Veera Raghavan Thangapandi ◽

Aliaksandra Maroz ◽

Dirk Reinhardt ◽

...

Keyword(s):

Down Syndrome ◽

Progenitor Cells ◽

Candidate Genes ◽

Cell Lines ◽

Trisomy 21 ◽

Fetal Liver ◽

Chromosome 21 ◽

Acute Megakaryoblastic Leukemia ◽

Megakaryoblastic Leukemia

Abstract Children with trisomy 21 (Down syndrome, DS) are predisposed to develop acute megakaryoblastic leukemia (DS-AMKL) as well as the antecedent transient leukemia (DS-TL). Mutations in the transcription factor GATA1 -leading to the exclusive expression of the shorter GATA1 isoform (GATA1s)- are present in nearly all children with DS-AMKL and DS-TL. GATA1s is both essential and sufficient to cause DS-TL in synergy with trisomy 21. To elucidate how the presence of an extra copy of chromosome 21 (hsa21) perturbs fetal hematopoiesis to provide a GATA1s-sensitive background during trisomy 21-associated leukemogenesis, we integrated an RNAi viability screening (512 shRNAmirs against 210 genes on hsa21) and a proteomics approach creating an hsa21 oncogenic network centered on GATA1s. shRNA-mediated knock-down of 42 genes conferred a profound selective growth disadvantage in DS-AMKL cell lines (CMK and CMY). A secondary functional validation screening confirmed 8 genes to specifically affect proliferation, cell viability, apoptosis or differentiation in GATA1s/trisomy-associated leukemia; whereas expression of 9 genes was also essential for proliferation and survival of erythroleukemia (K562) and non-DS-AMKL (M07) cell lines. Gain- and loss-of-function studies of 12 selected candidates (8 GATA1s/trisomy-specific oncogenes plus 4 global oncogenes) in CD34+ hematopoietic stem and progenitor cells (HSPCs) uncovered their regulatory function during megakaryopoiesis, erythropoiesis and myelopoiesis. Knockdown of four genes (USP25, BACH1, U2AF1 and C21orf33) inhibited megakaryocytic and erythroid in vitro differentiation, while enhancing myeloid differentiation. Inversely, ectopic expression of six genes (C21orf33, CHAF1B, IFNGR2, WDR4, RUNX1 or GABPA) resulted in a switch from erythroid to megakaryocytic differentiation. These 12 candidate genes acted synergistically to enhance the self-renewal efficiency of murine fetal liver cells in vitro. Pooled transduction of these genes increased the replating efficiency (more than 5 rounds) of fetal liver HSPCs whereas the colony-forming capacity was lost after second replating in the empty vector control. Further, 9 out of 12 candidate genes were overexpressed in DS-AMKL patient samples (n=23) compared to non-DS-AMKL (n=37; 1.3-fold to 2-fold) underscoring their relevance for the pathogenesis of DS-AMKL. Using an in vivo biotinylation approach to study the protein-protein interaction in DS-AMKL cells, we showed that bioGATA1 is associated with protein-complexes of 10 different hsa21-oncogenes, which are involved in splicing, deubiquitination and transcriptional regulation. Direct interactions with several of these factors are perturbed in N-terminal truncated GATA1s. Thus, we deciphered a complex interactive network on hsa21 around GATA1 positively regulating megakaryopoiesis. Deregulation of this network results in synergistic effects on hematopoietic differentiation, which can promote transformation of GATA1s-mutated fetal hematopoietic progenitor cells. Disclosures No relevant conflicts of interest to declare.

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Activating JAK3 Mutations in Transient Myeloproliferative Disorder and Acute Megakaryoblastic Leukemia Accompanying Down Syndrome.

Blood ◽

10.1182/blood.v110.11.1620.1620 ◽

2007 ◽

Vol 110 (11) ◽

pp. 1620-1620

Author(s):

Tomohiko Sato ◽

Tsutomu Toki ◽

Rika Kanezaki ◽

Gang Xu ◽

Kiminori Terui ◽

...

Keyword(s):

Down Syndrome ◽

Cell Lines ◽

Sh2 Domain ◽

Myeloproliferative Disorder ◽

Early Event ◽

Therapeutic Effects ◽

Acute Megakaryoblastic Leukemia ◽

Megakaryoblastic Leukemia ◽

Transient Myeloproliferative Disorder ◽

Transforming Activity

Abstract Children with Down syndrome (DS) have an approximately 20-fold higher incidence of leukemia than the general population. The majority of leukemia cases associated with DS are acute megakaryoblastic leukemia (AMKL). Although GATA1 mutations have been found in almost all cases of transient myeloproliferative disorder (TMD) “a preleukemia” that may be present in as many as 10% of newborn infants with DS and AMKL accompanying DS (DS-AMKL), GATA1 mutation alone may not be sufficient for development of leukemia. Following identification of acquired activating JAK3 mutations in DS-AMKL, JAK3 mutations have been reported also in TMD patients. However, the frequency and functional consequence of JAK3 mutations in TMD remain unknown. To further understand how JAK3 mutations are involved in the development and/or progression of leukemia in DS, we screened TMD patients and two DS-AMKL cell lines for JAK3 mutation, and examined the functional consequences of these JAK3 mutations. In one out of the two DS-AMKL cell lines, MGS, we identified novel JAK3 mutations (JAK3Q501H mutation in the SH2 domain and JAK3R657Q mutation in the psuedokinase domain in the same allele). JAK3Q501H and JAK3R657Q each constitutively phosphorylates STAT5 and transformes Ba/F3 cells to factor-independent growth, whereas the double mutant (JAK3Q501H and JAK3R657Q) has more potent transforming activity than each mutant. Biochemichal analysis in Ba/F3 cells revealed that the degrees of phosphorylation of STAT5 in the cells transduced with each JAK3 mutant were correlated with its transforming activity. Although we previously identified a JAK3I87T mutation in one of two TMD patients, no JAK3 mutations were detected in another 9 TMD patients. Together with the previous results, we found JAK3 mutations in each of 11 TMD and 11 DS-AMKL patients. Although the number of the patients analyzed was small, these results indicate that there are no significant differences in the frequency of JAK3 mutations between TMD and DS-AMKL. In this study, we showed for the first time that the TMD patient-derived JAK3 mutation was also an activating one. JAK3I87T transformed Ba/F3 cells to factor-independent growth. Treatment with JAK3 inhibitors (WHI-P131 and WHI-P154) resulted in a significant decrease in the growth and viability of Ba/F3 cells expressing each activating JAK3 mutant. These results suggest that the JAK3 activating mutation is an early event during the development of AMKL in DS. Furthermore these results provide a proof-of-principle that JAK3 inhibitor should have therapeutic effects on the AMKL and TMD patients carrying the activating JAK3 mutations.

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A Myeloproliferative Disorder in the Tc1 Mouse Model of Down Syndrome

Blood ◽

10.1182/blood.v112.11.2790.2790 ◽

2008 ◽

Vol 112 (11) ◽

pp. 2790-2790

Author(s):

Kate A. Alford ◽

Lesley Vanes ◽

Zhe Li ◽

Stuart H. Orkin ◽

Elizabeth M. C. Fisher ◽

...

Keyword(s):

Down Syndrome ◽

Mouse Model ◽

Extramedullary Hematopoiesis ◽

Myeloproliferative Disorder ◽

Chromosome 21 ◽

Gene Encoding ◽

Megakaryoblastic Leukemia ◽

Cell Counts ◽

Hematopoietic Disorders

Abstract Down syndrome (DS) children have a one in ten chance of being diagnosed with leukemia within the first ten years of life. Acute megakaryoblastic leukemia (AMKL) is a subtype of acute myeloid leukemia (AML) that accounts for nearly 50% of these leukemias. AMKL is associated with a self-regressing neoplasia found almost exclusively in DS newborns called Transient Myeloproliferative Disorder (TMD). In all cases of TMD and DS-AMKL, leukemic blast cells show mutations in the gene encoding the hematopoietic transcription factor GATA1, resulting in production of a truncated form of the protein called GATA1s. Mutations in GATA1 are not seen in non-DS-AMKL or other DS leukemias and it is clear both trisomy of human chromosome 21 (HSA21) and a mutation in GATA1 are required for the development of both TMD and AMKL. However, it is unknown which genes on HSA21 need to be trisomic in order to predispose an individual with DS to AMKL. Our group has generated mice (termed the Tc1 mice) that contain an almost complete, freely segregating copy of HSA21. These mice display phenotypic features of DS. We have examined adult hematopoiesis in these mice. Blood samples taken from a cohort of Tc1 mice were examined from 4 weeks until 60 weeks of age. Complete blood cell counts show that whilst the mice do not develop leukemia they displayed persistent macrocytosis and had reduced erythrocyte numbers. Crossing the Tc1 mice with mice that express GATA1s protein did not perturb or exacerbate this phenotype. Over the age of 15 months more than 50% of Tc1 mice examined were found to have developed splenomegaly. These mice displayed megakaryocyte hyperplasia and had increased numbers of cells of the erythroid lineage. In vitro colony forming assays demonstrated an increase in the frequency of megakaryocytic and granulocyte-macrophage progenitors in the spleen, consistent with extramedullary hematopoiesis. In the bone marrow, no abnormalities were seen in the lineage-, c-Kit+, Sca1+ (LSK) compartment, however there was a significant increase in the percentage of common myeloid progenitors (CMP) and a corresponding decrease in megakaryocyte-erythrocyte progenitors (MEP). This suggests a possible block in development from CMP to MEP. These data demonstrate defects in hematopoietic development in a proportion of adult Tc1 mice. However, preliminary data suggest that these mice do not develop a neonatal myeloproliferative disorder that is comparable with human TMD. It may be that the phenotype seen in the adult Tc1 mice is due to defects in hematopoietic progenitors that are different to those responsible for development of TMD and DS-AMKL. This mouse model may therefore provide a useful tool to examine the role of HSA21 genes in adult hematopoietic disorders.

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High Frequency of GATA1 Mutations in Childhood Non-Down Syndrome Acute Megakaryoblastic Leukemia

Blood ◽

10.1182/blood.v120.21.888.888 ◽

2012 ◽

Vol 120 (21) ◽

pp. 888-888 ◽

Cited By ~ 1

Author(s):

Katarina Reinhardt ◽

C. Michel Zwaan ◽

Michael Dworzak ◽

Jasmijn D.E. de Rooij ◽

Gertjan Kaspers ◽

...

Keyword(s):

Transcription Factor ◽

Down Syndrome ◽

Trisomy 21 ◽

Mutation Screening ◽

Study Group ◽

Acute Megakaryoblastic Leukemia ◽

Monosomy 7 ◽

Megakaryoblastic Leukemia ◽

Exon 1 ◽

Gata1 Mutation

Abstract Abstract 888 Introduction: Pediatric acute megakaryoblastic leukemia (AMKL) occurred in 6.6% (84/1271) of the children enrolled to the AML-BFM98 and 2004 studies. Despite a similar phenotype in morphology and immunophenotype, AMKL shows a heterogenous cytogenetic distribution (normal karyotype 23%, complex karyotype 21%, t(1;22) 9%; MLL-rearrangement 8%; monosomy 7 5%, trisomy 8 5%; other aberrations 29%). Mutations of the hematopoietic transcription factor GATA1 have been identified in almost all children suffering myeloid leukemia of Down syndrome (ML-DS). In addition, GATA1 mutations (GATA1mut) could be identified in children with trisomy 21 mosaic. Here, AMKL without evidence of Down syndrome or Down syndrome mosaic were analyzed for mutations in exon 1, 2 or 3 of the transcription factor GATA1. Patients: Seventy-one children from the AML-BFM Study group (n=51; 2000–2011), the Netherlands (n=10), France (n=3) and Scandinavia (n=7) were included. Within the AML-BFM Group the 51 analyzed patients showed similar characteristics compared to the total cohort of 84 children with AMKL of the AML-BFM 98 and 2004 studies. AMKL was confirmed according to the WHO classification by genetics (t(1;22)); morphology and immunophenotyping. Table 1a) summarizes the patientxs characteristics and b) the cytogenetic results. Methods: For GATA1 mutation screening genomic DNA was amplified by PCR reaction for exon 1, 2, and 3. PCR amplicons were analyzed by direct sequencing or following denaturing high-performance liquid chromatography (WAVE). Results: Seven different GATA1 mutations were detected in 8 children (11.1%; table 2). In all GATA1mut leukemia, a trisomy 21 within the leukemic blasts could be detected. Seven out of these 8 children and all other 64 AMKL patients have been treated with intensive chemotherapy regimens according the study group protocols. The results are given in table 2. All achieved continuous complete remission (CCR; 0.4 to 4.2 years). In one newborn with typical morphology and immunophenotype a GATA1mut associated transient leukemia was supposed. The child achieved CCR (follow-up 6 years). In total, allogeneic stem cell transplantation in 1st CR was performed in 6 children with AMKL (GATA1mut leukemia n=1). Conclusions: GATA1 mutations occurred in 11% of children with AMKL without any symptoms or evidence of trisomy 21 or trisomy 21 mosaic. GATA1 mutations are associated with a trisomy 21 within the leukemic blasts. Although non-response occurred, prognosis was significant better compared to other AMKL. Therefore, analysis of GATA1 mutation in infant AMKL is strongly recommended. Whether treatment reduction similar to ML-DS Down syndrome is feasible needs to be confirmed. Disclosures: No relevant conflicts of interest to declare.

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Bone marrow microenvironment in acute megakaryoblastic leukemia of children with down syndrome

Experimental Hematology ◽

10.1016/j.exphem.2017.06.125 ◽

2017 ◽

Vol 53 ◽

pp. S66

Author(s):

Theresa Hack ◽

Stephanie Sendker ◽

Nils von Neuhoff ◽

Dirk Reinhardt ◽

Mareike Rasche

Keyword(s):

Bone Marrow ◽

Down Syndrome ◽

Bone Marrow Microenvironment ◽

Acute Megakaryoblastic Leukemia ◽

Megakaryoblastic Leukemia ◽

Children With Down Syndrome

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The biology of pediatric acute megakaryoblastic leukemia

Blood ◽

10.1182/blood-2015-05-567859 ◽

2015 ◽

Vol 126 (8) ◽

pp. 943-949 ◽

Cited By ~ 59

Author(s):

Tanja A. Gruber ◽

James R. Downing

Keyword(s):

Trisomy 21 ◽

Myeloid Leukemia ◽

Poor Prognosis ◽

Somatic Mutations ◽

Newly Diagnosed ◽

Acute Megakaryoblastic Leukemia ◽

Pediatric Acute Myeloid Leukemia ◽

Megakaryoblastic Leukemia ◽

Children With Down Syndrome ◽

Gata1 Mutation

Abstract Acute megakaryoblastic leukemia (AMKL) comprises between 4% and 15% of newly diagnosed pediatric acute myeloid leukemia patients. AMKL in children with Down syndrome (DS) is characterized by a founding GATA1 mutation that cooperates with trisomy 21, followed by the acquisition of additional somatic mutations. In contrast, non–DS-AMKL is characterized by chimeric oncogenes consisting of genes known to play a role in normal hematopoiesis. CBFA2T3-GLIS2 is the most frequent chimeric oncogene identified to date in this subset of patients and confers a poor prognosis.

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