Transient leukemia (TL) is evident in 5–10% of all neonates with Down syndrome (DS) and associated with N-terminal truncating GATA1-mutations (GATA1s). Here we report that TL cell clones generate abundant eosinophils in a substantial fraction of patients. Sorted eosinophils from patients with TL and eosinophilia carried the same GATA1s-mutation as sorted TL-blasts, consistent with their clonal origin. TL-blasts exhibited a genetic program characteristic of eosinophils and differentiated along the eosinophil lineage in vitro. Similarly, ectopic expression of Gata1s, but not Gata1, in wild-type CD34+-hematopoietic stem and progenitor cells induced hyperproliferation of eosinophil promyelocytes in vitro. While GATA1s retained the function of GATA1 to induce eosinophil genes by occupying their promoter regions, GATA1s was impaired in its ability to repress oncogenic MYC and the pro-proliferative E2F transcription network. ChIP-seq indicated reduced GATA1s occupancy at the MYC promoter. Knockdown of MYC, or the obligate E2F-cooperation partner DP1, rescued the GATA1s-induced hyperproliferative phenotype. In agreement, terminal eosinophil maturation was blocked in Gata1Δe2 knockin mice, exclusively expressing Gata1s, leading to accumulation of eosinophil precursors in blood and bone marrow. These data suggest a direct relationship between the N-terminal truncating mutations of GATA1 and clonal eosinophilia in DS patients.
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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