CRISPR-engineered human GATA2 deficiency model uncovers mitotic dysfunction and premature aging in HSPCs, impairing hematopoietic fitness

Abstract

GATA2 deficiency is a monogenic transcriptopathy disorder characterized by bone marrow failure (BMF), immunodeficiency, and a high risk of developing myelodysplastic neoplasms (MDS) and acute myeloid leukemia (AML). Although informative mouse models have been developed, the mechanisms by which GATA2 haploinsufficiency drives disease initiation in humans remain incompletely understood. To address this, we developed a novel humanized model using CRISPR/Cas9 technology to knock-in GATA2-R398W variant in primary cord blood CD34+ cells. Additionally, we introduced specific mutations in SETBP1 and ASXL1 to model distinct premalignant stages of GATA2 deficiency. Through clonal competition and serial transplantation assays, we demonstrated that human CD34 cells harboring the GATA2 mutation exhibit significantly reduced fitness in vivo when competing with wild-type cells. Notably, this fitness disadvantage persists even when GATA2 mutations are combined with oncogenic SETBP1 and ASXL1 drivers, underscoring the dominant, deleterious effect of GATA2 deficiency on hematopoietic stem cell function. Functional in vitro analyses revealed that GATA2-R398W mutation impairs cell proliferation, disrupts cell cycle progression, and induces mitotic defects, which may contribute to hematopoietic stem/progenitor cell loss and impaired self-renewal. Transcriptomic profiles of GATA2-mutant cells revealed that these functional defects are associated with reduced HSC self-renewal capacity and upregulation of the pre-aging phenotype. Our work highlights the feasibility of generating a human GATA2 deficiency model suitable for studying the biological consequences of various GATA2 variants and the generation of a platform to test potential phenotype-rescuing therapeutics.

This work was supported by ERA PerMed GATA2-HuMo Funding Mechanism (Spain: Acció Instrumental de SLT011/18/00006 of the Department of Health of the Government of Catalonia), Ministerio de Ciencia e Innovación, which is part of Agencia Estatal de Investigación (AEI), through the Retos Investigación grant, number PID2020-115591RB-I00/ https://doi.org/10.13039/501100011033, and PID2023-146290OB-I00, Fundació La Marató TV3 228/C/2020, Award no. AC23_2/00040 by ISCIII through AES 2023 and within the European Joint Programme Rare Diseases framework, Instituto de Salud Carlos III (ISCIII), "Programa FORTALECE del Ministerio de Ciencia e Innovación", through the project number FORT23/00032 and Finançat per el Departament de Recerca i Universitats de la Generalitat de Catalunya i l'AGAUR (expedient 2021 SGR 00888) to AG. Funding for this project was provided in part by an EHA Research Grant award granted by the European Hematology Association (KOG-202109-01162) and the European Union's Horizon 2020 research and innovation programme under the Marie Sklodowska-Curie grant agreement No 101029927 to OM-B. DR-M was supported by Deutsche José Carreras Leukämie-Stiftung, DJCLS 13 R/2022. JP and MD were supported by Fundació La Marató TV3 228/C/2020. MM-M was supported by the TC030-AND/2022 Predoctoral grant from the Government of Andorra. OM-B is currently supported by the Ramón y Cajal contract (RYC2021-032129-I) funded by AEI/European Social Fund UE. The work in the LP laboratory was supported by "la Caixa" Foundation, LCF-PR-HR24-00150 and by PID2023-151556OB-I00 and CNS2024-154742 funded by MICIU/AEI/10.13039/501100011033 and, as appropriate, by "ESF Investing in your future", by "ESF+" or by "European Union NextGenerationEU/PRTR". The work in AB laboratory was funded by ERA PerMed-Departament de Salut, Generalitat de Catalunya (SLT011/18/00007) and the EJP RD-Instituto de Salud Carlos III (AC23_2/00014). The work of CC and OM was supported by the Spanish Ministry of Economy and Competitiveness/European Union NextGenerationEU (PID2022-142966OB-I00) and the Deutsche José Carreras Leukämie-Stiftung (DJCLS 15 R/2023) to OM. CC is supported by a predoctoral fellowship from the Spanish Ministry of Economy and Competitiveness (PID2022-142966OB-I00). The work of MP was supported by a Ramón y Cajal contract of the Spanish Ministry of Science, Innovation and Universities (Grant: RYC2018-024564-I funded by MICIU/AEI/10.13039/501100011033 and by "El FSE invierte en tu futuro"). The work in the MP laboratory was supported by the project PID2022-139580OB-I00 funded by MICIU/AEI/10.13039/501100011033 and FEDER and by "ERDF A way of making Europe", by the European Union. MWW was supported by Evans MDS DRG grant and American Society of Hematology bridge award. Authors thank the members of the Advanced Optical Microscopy facility from UB (Scientific and Technological Centers, Universitat de Barcelona [CCiTUB]) for their technical support. We thank the CERCA Programme/Generalitat de Catalunya for institutional support.