Germline GATA2 Mutations (R986Q and R398W) Inhibit Endothelial Development and Augment Myeloid Differentiation during Early Human Hematopoiesis

Germline heterozygous GATA2 mutations underlie an autosomal dominant predisposition to myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML), often with an aggressive disease course and poor outcome. Penetrance and expressivity within GATA2 families is often variable, suggesting that additional aberrations are required to trigger the development of the disease. The only curative option is the hematopoietic stem cell transplantation; therefore there is a clear unmet medical need. A mechanistic understanding of how GATA2 haploinsufficiency affects hematopoietic development and promotes myeloid malignant transformation is hindered by the limited number of cases reported and by the lack of appropriate human disease model system. Here we show the somatic and germline changes found in 12 Spanish GATA2-deficient carriers using a deep whole exome sequencing analysis (mean coverage 300X). Interestingly, our data show that mutations in relevant genes, involved in the RTK-RAS pathways, are recurrent in our cohort. In addition, we observed somatic mutations in SETBP1, RUNX1, EZH2 and STAG2 consistent with previous reports. Next, we studied the impact of two of the most recurrent germline GATA2 mutations associated with MDS (R396Q and R389W) using a human iPSC-based disease model. Applying a CRISPR/Cas9-mediated genome editing strategy we generated two hiPSC-GATA2 mutant lines and differentiated them toward blood progenitors. Hematopoietic differentiation was assessed using a stroma-free approach based on the generation of embryoid bodies (EBs), where hiPSCs are specified first into Vecad+CD43-CD73- hemogenic endothelial progenitors (HEPs) and then into CD34+CD43+CD45+ hematopoietic and myeloid CD33+CD14+ progenitors in a stage-specific manner. Data collected from FACS analyses at day 10 of EB development revealed a pronounced (3-fold; P<0.05) decrease in Vecad+CD43- cells development in hiPSC-GATA2Mut, when compared with isogenic control. Interestingly, the decrease of Vecad+ cells was due to a marked reduction of endothelial progenitors (Vecad+CD43-CD73-) in hiPSC-GATA2Mut, while the fraction of HEPs (Vecad+CD43-CD73-) did not show significant differences between hiPSCs-GATA2^Mut and their isogenic control. This finding supports the statement that 11-30% of people carrying GATA2 mutations develop lymphedema in the first decade of life. Moreover, FACS analyses at day 15 of EB development showed that GATA2 mutations lead to a block of early hematopoietic progenitors maturation (CD34+CD43+CD45+) and to an augment of myeloid progenitors’ compartment (CD33+/CD14+), which is in line with a low-risk MDS stage in childhood. The increased hematopoietic output of hiPSC-GATA2Mut lines could be related to the higher proliferation/survival of the emerging HPCs or CD33+cells. However, cell cycle analysis of CD34+ and CD33+ cells revealed no significant differences among hiPSC-GATA2Mut and control. These data suggest a specific effect of GATA2 mutation on blood differentiation rather than on proliferation. Finally, to assess the in vivo impact of GATA2 mutations, first we introduced the GATA2 R398W mutation in Cord Blood CD34+ cells by CRISPR/cas9 system. Then, 300.000 CD34+ nucleofected cells were intra-bone marrow transplanted (IBMT) into irradiated (2.5 Gy) 8-10-week-old NSG mice. No disease symptoms were observed up to 13 weeks. However, the presence of GATA2 R398W mutation enhanced hematopoietic engraftment 2-fold as compared with control cells (36% vs 16% in PB and 85% vs 67% in BM), suggesting that GATA2 R398W mutation sustains robust engraftment.

In summary, our preliminary data suggest that RTK-RAS pathway mutations might represent a class of driver events in GATA2-deficient carriers. Moreover we described for the first time a functional impact of GATA2 R398W and R396Q mutations during early human hematopoietic development revealing how hiPSC-based hematopoietic differentiation represents a promising system to study the molecular and cellular mechanisms underlying the myeloid transformation in GATA2 carriers.