Acute megakaryoblastic leukemia of Down syndrome (DS-AMKL) is a model of clonal evolution from a preleukemic transient myeloproliferative disorder requiring both a trisomy 21 (T21) and a GATA1 s mutation to a leukemia driven by additional driver mutations. We modeled the megakaryocyte differentiation defect through stepwise gene editing of GATA1s , SMC3 +/– , and MPL W515K , providing 20 different T21 or disomy 21 (D21) induced pluripotent stem cell (iPSC) clones. GATA1s profoundly reshaped iPSC-derived hematopoietic architecture with gradual myeloid-to-megakaryocyte shift and megakaryocyte differentiation alteration upon addition of SMC3 and MPL mutations. Transcriptional, chromatin accessibility, and GATA1-binding data showed alteration of essential megakaryocyte differentiation genes, including NFE2 downregulation that was associated with loss of GATA1s binding and functionally involved in megakaryocyte differentiation blockage. T21 enhanced the proliferative phenotype, reproducing the cellular and molecular abnormalities of DS-AMKL. Our study provides an array of human cell–based models revealing individual contributions of different mutations to DS-AMKL differentiation blockage, a major determinant of leukemic progression.

EZH2, the enzymatic component of PRC2, has been identified as a key factor in hematopoiesis. EZH2 loss of function mutations have been found in myeloproliferative neoplasms, more particularly in myelofibrosis, but the precise function of EZH2 in megakaryopoiesis is not fully delineated. Here, we show that EZH2 inhibition by small molecules and shRNA induces MK commitment by accelerating lineage marker acquisition without change in proliferation. Later in differentiation, EZH2 inhibition blocks proliferation, polyploidization and decreases proplatelet formation. EZH2 inhibitors similarly reduce MK polyploidization and proplatelet formation in vitro and platelet level in vivo in a JAK2V617F background. In transcriptome profiling, the defect in proplatelet formation was associated with an aberrant actin cytoskeleton regulation pathway, whereas polyploidization was associated with an inhibition of expression of genes involved in DNA replication and repair, and an upregulation of CDK inhibitors, more particularly CDKN1A and CDKN2D. The knockdown of CDKN1A and at a lesser extend of CDKN2D could partially rescue the percentage of polyploid MKs. Moreover, H3K27me3 and EZH2 ChIP assays revealed that only CDKN1A is a direct EZH2 target while CDKN2D expression is not directly regulated by EZH2 suggesting that EZH2 controls MK polyploidization directly through CDKN1A and indirectly through CDKN2D.