Characterization Of Oncogenes On Chromosome 21 Identified By shRNA-Based Viability Screening

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 hematopoietic transcription factor GATA1 have been found in nearly all children with DS-AMKL and DS-TL, but not in other malignancies. Recent whole genome sequencing efforts strongly supported the hypothesis that the triad of fetal origin, trisomy 21 and GATA1s-mutation are sufficient to cause DS-TL. Thus, the presence of an extra copy of hsa21 perturbs fetal hematopoiesis to provide a GATA1s sensitive background during leukemogenesis. To decipher the deregulated oncogenic gene network on hsa21, we conducted a shRNA-based viability screening.

GATA1s-mutated DS-AMKL cell line CMK as well as non-DS-AML cell lines (K562, M-07) as control were lentivirally transduced and the effect of the knock-down was evaluated by cell viability and proliferation assays. Upon knock-down we found 42 genes conferring a profound selective growth disadvantage in DS-AMKL cell lines. Interestingly, 31 out of those candidate genes are located in one particular chromosomal region (21q22.1-21q22.3) and in addition 11 (out of 14 tested) are overexpressed in DS-AMKL compared to non-DS-AMKL.

In a secondary functional validation screening the effects of the knock-down on the cell lines were analyzed by competition assays, apoptosis assays and cell viability assays as well as colony forming assays. Furthermore, differentiation and morphology were characterized using immunophenotyping and cytospins, respectively. We could demonstrate that the potential oncogenes participate in different cellular processes affecting proliferation, cell viability, apoptosis or differentiation.

To further delineate the impact of 11 selected candidates on normal hematopoiesis, we characterized their effects in gain- and loss-of-function studies (confirmed by qRT-PCR) using CD34⁺ hematopoietic stem and progenitor cells (HSPCs). Four of those genes (USP25 [ubiquitin specific peptidase 25], BACH1 [BTB and CNC homology 1, basic leucine zipper transcription factor 1], U2AF1 [U2 small nuclear RNA auxiliary factor 1] and C21orf33) inhibited megakaryocytic and erythroid in vitro differentiation upon knockdown. The fraction of cells expressing early and late megakaryocytic markers CD41 and CD42b or early erythroid marker CD36 was reduced by 2-20-fold (P<0.001). Inversely, the knock-down of those four genes and two other genes (ATP5O [ATP synthase, H+ transporting, mitochondrial F1 complex, O subunit] and C21orf45) enhanced the myeloid differentiation propensity of CD34⁺-HSPCs (2.1-13.4-fold increase of CD14⁺-monocytic cells, P<0.001). The opposite effect was observed in gain-of-function studies. Ectopic expression of six genes (hU2AF1, mC21orf33, hIFNGR2 [interferon gamma receptor 2], hWDR4 [WD repeat domain 4] or mGABPA [GA binding protein transcription factor, alpha subunit 60kDa]) resulted in a radical switch in lineage commitment with a drastic change from erythroid to megakaryocytic differentiation (CD41⁺ 1.7-2.4-fold increase, P<0.001, CD235a⁺ [late erythroid marker] 3-300-fold reduction, P<0.001).

Thus, we found a remarkable number of genes regulating erythroid and megakaryocytic differentiation as well as proliferation in normal hematopoiesis. Given the genetic background during trisomy 21-mediated leukemogenesis, we propose a complex interactive network located in one particular region on hsa21. Deregulation of this network might result in synergistic effects on hematopoietic differentiation, which promotes transformation of GATA1s-mutated fetal hematopoietic progenitor cells.