Multiclonal competition reveals impaired cell fitness of GATA2 deficient cells [ATAC-seq]
GATA2 deficiency has been identified as a common hereditary cause of myelodysplastic syndromes (MDS) and acute myeloid leukemia in children. Penetrance and expressivity within affected families is often variable, suggesting that cooperating factors are required to trigger the disease. Somatic mutations in MDS driver genes (SETBP1, ASXL1) have been identified GATA2-MDS. The molecular mechanism that triggers the leukemic progression in GATA2 carriers remains unknown. Particularly, these questions remain unanswered: 1) whether GATA2 germline mutation itself is sufficient to trigger MDS/AML 2) whether SETBP1 and ASXL1 mutations induce the malignant transformation. Addressing these questions has been difficult due to the lack of faithful human disease models. Here I studied in vitro/in vivo engineered cord blood CD34+ cells carrying  GATA2 mutation alone or in combination with SETBP1/ASXL1 mutations in NSG mice to evaluate the engraftment capacity and the clonal evolution. Specifically, CRISPR/Cas9/rAAV6 was used to introduce the R398W mutation in CD34+ cells alone (GATA2) or in combination with SETBP1 and ASXL1 mutations (Multiplex). Primary and secondary transplantation showed similar multilineage constitution in all the conditions. Interestingly, genetic studies indicated that the predominant expanded clones in the multiplex condition carried SETBP1+ASXL1 mutations, while clones with only GATA2 mutation were lost. To study the impact of the mutations at transcriptomic level, scRNAseq is underway. In vitro data confirmed that cells carrying the GATA2 R398W mutation have an impaired clonogenic capacity and proliferation, recapitulating MDS patient phenotype. In summary, we developed a human model of clonal competition by CRISPR/Cas9 targeting CD34+ cells. Our findings strongly suggest that GATA2 R398W mutation is not sufficient to increase cell fitness, suggesting that co-operating of genetic, epigenetic, niche and stressor factors are necessary to trigger the disease. Overall design: CRISPR/Cas9 combined with rAAV6 was employed to knock in the GATA2-R398W-EGFP mutation in human CD34+ cells. Both GATA2-R398W-EGFP and control cells were cultured for ten days before being processed for ATAC-seq analysis