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.
Genes Associated with Disturbed Cerebral Neurogenesis in the Embryonic Brain of Mouse Models of Down Syndrome
