Project description: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.

Project description:Clinical GATA2 deficiency syndromes arise from germline haploinsufficiency inducing mutations in GATA2, resulting in immunodeficiency that evolves to myelodysplastic syndrome (MDS)/acute myeloid leukemia (AML). How GATA2 haploinsufficiency disrupts the function and transcriptional network of hematopoietic stem/progenitors (HSCs/HSPCs) to facilitate the shift from immunodeficiency sequalae to pre-leukemia is poorly characterised. Using a conditional mouse model harboring a single allele deletion of Gata2 when HSCs emerge in utero, we identify pervasive defects in HSPC differentiation from young adult Gata2 haploinsufficient mice during B-cell maturation, early erythroid specification, megakaryocyte maturation to platelets and inflammatory cell generation. Gata2 haploinsufficiency abolishes HSC self-renewal and multi-lineage differentiation capacity following transplantation. These alterations closely associate with deregulated DNA damage responses and inflammatory signalling conveyed from Gata2 haploinsufficient HSCs. We also identify functional interplay between Gata2 and Asxl1, a driver of DNA damage and inflammation and, notably, a recurrent secondary mutation found in GATA2 haploinsufficiency disease progression to MDS/AML. shRNA mediated knockdown of Asxl1 in Gata2 haploinsufficient HSPCs led to a differentiation block in committed progenitor formation beyond that in Gata2 haploinsufficient HSPCs. By analysis of HSCs from young adult compound Gata2/Asxl1 haploinsufficient mice, we discover hyperproliferation of double haploinsufficent HSCs, which are also functionally compromised in transplantation compared to their single haploinsufficient counterparts. Through both Gata2/Asxl1 dependent and unique transcriptional programs, HSCs from young adult compound Gata2/Asxl1 haploinsufficient mice fortify deregulated DNA damage responses and inflammatory signalling in HSCs initiated in Gata2 haploinsufficient mice and establish a broad pre-leukemic program. Our data reveal how Gata2 haploinsufficiency initially drives deregulation of HSC genome integrity and suggest the mechanisms of how secondary mutations like ASXL1 take advantage of HSC genomic instability to nurture a pre-leukemic state in GATA2 haploinsufficiency syndromes.

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Multiclonal competition reveals impaired cell fitness of GATA2 deficient cells [ATAC-seq]

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