13652141, 2022, 4, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/bjh.18330 by Readcube (Labtiva Inc.), Wiley Online Library on [09/02/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License Received: 7 April 2022 | Accepted: 11 June 2022 DOI: 10.1111/bjh.18330 REVIEW GATA2 deficiency and MDS/AML: Experimental strategies for disease modelling and future therapeutic prospects Lili Kotmayer1 | Damia Romero-­Moya2 | Oskar Marin-­Bejar2 | Emilia Kozyra3,4 | Albert Català5,6 | Anna Bigas7,8 | Marcin W. Wlodarski3,9 | Csaba Bödör1 | 2,10,11 Alessandra Giorgetti 1 HCEMM-­SE Molecular Oncohematology Research Group, 1st Department of Pathology and Experimental Cancer Research, Semmelweis University, Budapest, Hungary 2 Regenerative Medicine Program, Institut d'Investigació Biomèdica de Bellvitge (IDIBELL), Barcelona, Spain 3 Division of Pediatric Hematology and Oncology, Department of Pediatrics and Adolescent Medicine, Medical Center, Faculty of Medicine, University of Freiburg, Freiburg, Germany 4 Faculty of Biology, University of Freiburg, Freiburg, Germany 5 Department of Hematology and Oncology, Institut de Recerca Sant Joan de Déu, Hospital Sant Joan de Deu, Barcelona, Spain 6 Biomedical Network Research Centre on Rare Diseases, Instituto de Salud Carlos III, Madrid, Spain 7 Cancer Research Program, Institut Hospital del Mar d'Investigacions Mèdiques, CIBERONC, Hospital del Mar, Barcelona, Spain 8 Josep Carreras Research Institute (IJC), Badalona, Barcelona, Spain 9 Department of Hematology, St. Jude Children's Research Hospital, Memphis, Tennessee, USA 10 Fondazione Pisana Per la Scienza ONLUS (FPS), San Giuliano Terme, Italy 11 Department of Pathology and Experimental Therapeutics, Faculty of Medicine and Health Sciences, Barcelona University, Barcelona, Spain Correspondence Alessandra Giorgetti, Bellvitge Institute Summary for Biomedical Research (IDIBELL), Hospital Duran i Reynals-­3rd floor Gran The importance of predisposition to leukaemia in clinical practice is being increasingly Via de l'Hospitalet, 199 08908 Hospitalet de recognized. This is emphasized by the establishment of a novel WHO disease category Llobregat, Barcelona, Spain. Email: agiorgetti@idibell.cat in 2016 called “myeloid neoplasms with germline predisposition”. A major syndrome Funding information within this group is GATA2 deficiency, a heterogeneous immunodeficiency syndrome Bundesministerium für Bildung und with a very high lifetime risk to develop myelodysplastic syndrome (MDS) and acute Forschung, Grant/Award Number: 2018-­123/01KU1904 and BMBF MyPred myeloid leukaemia (AML). GATA2 deficiency has been identified as the most common 01GM1911A; EU’s Horizon 2020 Research and Innovation Program, Grant/Award hereditary cause of MDS in adolescents with monosomy 7. Allogenic haematopoietic Number: 739593; Fondazione Pisana per stem cell transplantation is the only curative option; however, chances of survival de- la Scienza ONLUS, Grant/Award Number: FPS Grant 2018; Generalitat de Catalunya, crease with progression of immunodeficiency and MDS evolution. Penetrance and ex- Grant/Award Number: CERCA Programme and SLT011/18/00006; Hungarian National pressivity within families carrying GATA2 mutations is often variable, suggesting that Academy of Scientist Education; MSCA No co-­operating extrinsic events are required to trigger the disease. Predictive tools are lack- 101029927-­scGATA2track, Grant/Award Number: H2020-­MSCA-­IF-­2020; Ministerio ing, and intrafamilial heterogeneity is poorly understood; hence there is a clear unmet de Economía y Competitividad, Grant/Award Number: PID2020-­15591RB-­100; Nemzeti medical need. On behalf of the ERAPerMed GATA2 HuMo consortium, in this review Kutatási és Technológiai Hivatal, Grant/ we describe the genetic, clinical, and biological aspects of familial GATA2-­related MDS, Award Number: ED-­18-­1-­2019-­0 01, TKP2021-­ EGA-­24, TKP2021-­N VA-­15 and ÚNKP-­21-­2-­ highlighting the importance of developing robust disease preclinical models to improve I-­SE-­21; Vera and Joseph Dresner Foundation; Evans MDS Foundation; American Lebanese early detection and clinical decision-­making of GATA2 carriers. Syrian Associated Charities [Correction added on 06 July 2022, after first K EY WOR DS online publication: Affiliation 8 was updated acute myeloid leukaemia, blood cancer, GATA2 deficiency, myelodysplastic syndromes in this version.] This is an open access article under the terms of the Creative Commons Attribution-­NonCommercial-­NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-­commercial and no modifications or adaptations are made. © 2022 The Authors. British Journal of Haematology published by British Society for Haematology and John Wiley & Sons Ltd. 482 | wileyonlinelibrary.com/journal/bjh  Br J Haematol. 2022;199:482–495. 13652141, 2022, 4, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/bjh.18330 by Readcube (Labtiva Inc.), Wiley Online Library on [09/02/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License KOTMAYER et al.    | 483 I N T RODUC T ION approaches with in vitro and in vivo models. In this review, we provide an overview of the molecular background of Over a decade ago, the first germline pathogenic variants in GATA2 deficiency with an insight into clinical implications, the GATA2 gene with a predisposition to myelodysplastic and current and future challenges in the management of pa- syndrome (MDS) and acute myeloid leukaemia (AML) were tients with GATA2 predispositions. reported by Hahn and colleagues.1 Since then, a number of germline GATA2 variants across individual cases and fam- ilies with associated characteristic haematological and non- ROL E OF GATA 2 T R A NSCR I P T ION haematological phenotypes have been described. Together FAC TOR I N H A E M ATOPOI ET IC A N D they define GATA2 deficiency syndrome, a rare autosomal NON H A E M ATOPOI ET IC T IS SU E S dominant genetic disease with increased susceptibility to myeloid malignancies and other non-­malignant symptoms.2 GATA2 is a member of the GATA transcription factor fam- Clinical presentations of GATA2 deficiency syndrome may ily and is a master regulator of haematopoiesis and lym- range from severe bone marrow failure (BMF), myeloid ma- phatic angiogenesis by regulating target genes through lignancies, immunodeficiencies and congenital syndromic occupying WGATAR DNA structure motifs with two zinc features to a minority of asymptomatic cases due to partial finger domains (ZF1 and ZF2).4,25,26 ZF1 controls protein–­ or incomplete penetrance.3,4 protein interactions, while ZF2 is critical for regulating Despite the highly variable manifestations of GATA2 transcription through protein-­ DNA binding.22,27 GATA2 deficiency, associated phenotypes are now recognized as a has a major role in the early embryonic haematopoietic single monogenic syndrome, introduced in the revised 2016 specification through endothelial haematopoietic transi- WHO classification of haematopoietic tumours as the novel tion (EHT).28–­32 During postnatal haematopoiesis GATA2 is category of ”myeloid neoplasms with germline GATA2 mu- expressed at high levels in HSCs and early haematopoietic tations”. GATA2 deficiency syndrome encompasses cellular progenitors,31–­35 and promotes myeloid-­erythroid progeni- immunodeficiency disorders, namely MonoMac5,6 (mono- tor cell development11,21,27,36–­38 (Figure 1A). cytopenia and mycobacterial infections) and Emberger syn- Furthermore, GATA2 is expressed in the lymphatic endo- drome7,8 (myelodysplasia and lymphoedema), dendritic cell, thelium, instigating fetal lymphatic vessel valve development monocyte, B-­and NK-­lymphoid (DCML) deficiency 9,10 and and normal valve function in adults,24,39 and its mechano-­ myeloid malignancies, predominantly familial and primary sensitive expression in endothelial cell compartment also paediatric MDS and AML.1–­3,11,12 Germline GATA2 mu- implicates GATA2 in angiogenesis and vascular integri- tations have also been identified and linked to additional ty.25,40–­42 Several single nucleotide polymorphisms (SNPs) clinical manifestations such as chronic neutropenia,5 hypo- of GATA2 have been associated with coronary artery dis- cellular BMF,13,14 defects of megakaryopoiesis, B and NK cell ease implying its putative role in arterial development; how- deficiency,15,16 congenital auditory and neurological disor- ever, underlying molecular mechanisms involving GATA2 ders (predominantly sensorineural deafness and ADHD)17,18 are still poorly understood.24,43,44 Additionally, HSPCs and and recurrent miscarriages.5,17,19 endothelial components, high GATA2 protein levels were Despite the frequent and significant overlap among these observed during fetal neurodevelopment,45,46 as well as in clinical features and temporal changes in the clinical presen- selected types of differentiated cells, including neurons, tations over the disease course, no common pathognomonic androgen receptor-­expressing and endocrine cells,47–­49 and GATA2 phenotype has been defined to date. It suggests macrophages.50,51 that different unknown underlying co-­ operating factors (“stressor events”) in specific cellular contexts and processes are required for each GATA2-­related clinical manifesta- E X IST I NG A N D N E W tion, as recently proposed by Homan and colleagues.20,21 E X PE R I M E N TA L MODE L S TO ST U DY Amalgamation of an impaired GATA2 function and the GATA 2 F U NC T ION accumulation of stochastic biological or environmental stressors may (substantially) dysregulate a subset of GATA2-­ The role of GATA2 gene as master regulator of haemat- dependent processes, rattling normal cellular function. This opoietic system has been defined in 1994, when Orkin and mechanism has been proposed for haematopoietic stem cell colleagues showed that Gata2-­KO mice died at 10.5 days (HSC) exhaustion and BMF due to recurrent infections,13,22 post-­coitum (dpc), due to the collapse of primitive and de- myeloid malignancies due to acquired somatic alterations, finitive haematopoiesis.28,29 Notably, mouse Gata2-­null such as ASXL1 mutations or monosomy 723 and presumably endothelial cells failed to produce HSCs because of im- lymphoedema due to mechanical or inflammatory stresses paired EHT.28,31–­35 Formal demonstration of GATA2 func- of lymphatic vessels and valves during development.24 tion within HSC development, proliferation and survival The ERAPerMed GATA2 HuMo consortium is an inter- in adult haematopoiesis has been gleaned using Gata2 +/− national collaborative network aiming for the precise under- mice. Heterozygous Gata2 +/− mice survive and show a re- standing of GATA2 deficiency syndrome and subsequent duced number of HSCs both in the embryo and within adult myeloid transformation by combining clinical and genomic BM, and Gata2+/− HSCs are qualitatively defective.35,52 In GATA2 DEFICIENCY AND MDS/AML: EXPERIMENTAL STRATEGIES FOR DISEASE MODELLING 13652141, 2022, 4, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/bjh.18330 by Readcube (Labtiva Inc.), Wiley Online Library on [09/02/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License 484 |    AND FUTURE THERAPEUTIC PROSPECTS F I G U R E 1 (A) Role of the GATA2 transcription factor during embryonal haematopoietic development and postnatal haematopoiesis. GATA2 plays a major role in the early embryonic haematopoietic specification through endothelial haematopoietic transition, maintenance of long-­term haematopoietic stem cells (LT-­HSC) and erythroid differentiation. The GATA2 transcriptional factor also instigates fetal lymphatic vessel valve development and normal valve function in adults, and its high expression in endothelial compartments also implicates its tole in angiogenesis. During postnatal haematopoiesis GATA2 is expressed at high levels in haematopoietic stem cells (HSCs) and early haematopoietic progenitors and induces myeloid-­erythroid progenitor cell development and differentiation through cell-­specific transcription factor complexes. (B) Impact of GATA2 mutations and phenotype-­modifying factors, and clinical phenotypes associated with GATA2 deficiency syndrome. Although different types of germline GATA2 variants have not been linked strongly to substantial phenotypes, some phenotypes seem to cluster within affected carrier families. AML, acute myeloid leukaemia; DCML, dendritic cell, monocyte, B-­and NK-­lymphoid deficiency; MDS, myelodysplastic syndrome. addition, in vivo studies at single cell level revealed a GATA2 Gata2b.62 Previous studies have indicated that Gata2a is im- dose-­dependent mechanism regulating HSC identity.53–­55 plicated in lymphovascular development with some expres- Furthermore, Gata2 haploinsufficiency also reduces sion in HSPCs in adult haematopoiesis,63–­65 whereas Gata2b granulocytes-­macrophage function but no other myeloid is mainly expressed in HE cells that give rise to HSCs in committed progenitors.56 Of note, most recently, others and adult haematopoiesis.66 In addition, Gata2a mutant for the us have demonstrated a primary role of GATA2 in promoting conserved enhancer i4, corresponding to the described +9.5 EHT also in a human background using engineered pluripo- enhancer in the mouse Gata2 locus, revealed that Gata2a is tent stem cells.28,30–­35,57 Importantly, GATA2 is highly ex- required upstream of Gata2b, regulating Runx1 and Gata2b pressed in HSCs and its expression is tightly regulated by expression in the haemogenic endothelium67 (Figure 2). conserved cis-­regulatory elements and other signalling path- Recent transcriptomic analyses have shown that Gata2b ways. Results of mouse studies based on targeted ablation of loss impaired progression of the myeloid transcriptional the far upstream Gata2–­77 enhancer showed that it abro- programme while increasing the lymphoid programme in gates the multilineage differentiation potential of fetal liver haematopoietic progenitors.68,69 Altogether these finding progenitor cells without affecting HSC emergence,38 which suggest that mammalian Gata2 functions are divided among is diminished by the intronic +9.5 enhancer deletion35,39,58,59 gata2a and gata2b in zebrafish. (Figure 2). Furthermore, Notch signals tightly regulate the Overall, these findings have contributed greatly to our precise level of expression of GATA2 required for generation understanding of the role of GATA2 in embryonic and adult of HSCs in the embryonic aorta.60,61 haematopoiesis. However, Gata2 +/− mice do not develop Zebrafish is another valuable model that have been exten- MDS and AML, probably due to the short lifespan of mice, sively used to study the function of GATA2 in haematopoi- the different external stressor factors or the absence of con- esis. A genome duplication event in early teleosts generated current somatic lesions as observed in GATA2-­related MDS two Gata2 paralogs in the zebrafish genome: Gata2a and patients. Furthermore, there is still lack of a knock-­in mouse 13652141, 2022, 4, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/bjh.18330 by Readcube (Labtiva Inc.), Wiley Online Library on [09/02/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License KOTMAYER et al.    | 485 F I G U R E 2 Schematics of different strategies of disease modelling GATA2 deficiency in vivo. (A) Zebrafish; (B) genetically engineered modified mouse (GEEM); (C) CD34+ genetic edited cells transplanted to immunodeficient mice; (D) in vitro induced pluripotent stem (iPS) cells derived from patients and (E) genome edited iPS cells. (D and E) are used for in vitro disease modelling (left). Experimental readout of the disease models (right). model, which allows the overexpression of GATA2 mutant biological reasoning behind the poor engraftment of MDS protein, mimicking the haematological manifestations ob- primary cells remains unclear and might be related to both served in human GATA2 carriers. Therefore, there is a clear the specific requirements for environmental factors and/ need for the development of better faithful mouse models or the intrinsic biological characteristic of individual sam- of GATA2 deficiency, including patient-­derived xenograft ples. This limitation might be overcome by the generation (PDX), to mimic better this complex disease. Nowadays, of a humanized BM-­ MSC-­derived ossicles (ectopic bone there are no PDX studies focusing on patients carrying organoid composed of human bone, stroma, and haemato- germline GATA2 mutations. Generally, the establishment poietic tissue) that provides a human microenvironment.74 of human preclinical models by xenotransplantation of A more consistent outcome was achieved by using MISTRG primary MDS cells in immunodeficient mice represent a mice, a humanized strains expressing human M-­CSF, IL-­3, bottleneck that hampers research of the mechanism under- GM-­CSF, SIRP alpha and thrombopoietin at physiological standing this disease. During the last decade, several labora- level.75 The selection of the most suitable mouse model can tories have tested different approaches including intratibial significantly affect the outcome of the research findings. The injection in NSG (NOD-­Scid-­IL-­2Rcnull) or NSG-­S (NSG/ pivotal question that remains unanswered is which mouse IL3/GM-­CSF/hSCF), cotransplantation with patient-­derived model should be used to study GATA2 deficiency. Another or normal mesenchymal stem cell (MSC).70 However, these limitation for the development of mouse disease models is methods showed low efficiency and poor engraftment the genetic heterogeneity and the low prevalence of GATA2 rate, with a skew towards the lymphoid lineage.71–­73 The deficiency that limits the access to patient primary cells. A GATA2 DEFICIENCY AND MDS/AML: EXPERIMENTAL STRATEGIES FOR DISEASE MODELLING 13652141, 2022, 4, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/bjh.18330 by Readcube (Labtiva Inc.), Wiley Online Library on [09/02/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License 486 |    AND FUTURE THERAPEUTIC PROSPECTS possible solution could be the generation of GATA2 defi- protein–­protein interactions21,92 and DNA binding.1,10,90,92 ciency preclinical mouse models using engineered human Monoallelic missense mutations of the ZFs have been re- haematopoietic CD34+ cells (both from cord blood and ported mostly, albeit not exclusively, within the extended mobilized peripheral blood). The ongoing improvement of ZF2 domain with only a few confirmed cases of germline CRISPR/Cas9 genomic engineering technologies enable the variants affecting ZF1. Intriguingly, unlike germline LOF manipulation of genes in primary CD34+ cells, facilitating GATA2 variants, somatic GATA2 mutations of adult AML the development of preclinical models to study the molecu- affecting the ZFs occur with a preference for ZF1 and can lar mechanisms and the clonal evolution of haematological exhibit gain-­of–­function (GOF) effects. 22,93,94 Germline diseases76–­78 (Figure 2). Furthermore, those models would variants in ZF1, such as p.Ala318Thr,65 p.His313Tyr and be fundamental tools to identify genetic subtype that are p.Leu315Pro17 are recognized as LOF alterations and in sensitive or resistant to therapeutic agents. In parallel, the most cases associated with paediatric MDS and accompa- induced pluripotent stem cells (hiPSCs)-­ based approach nying symptoms of underlying GATA2 deficiency. Notably, represents an alternative cellular platform for mechanistic a novel germline missense mutation in ZF1 (p.Asn317Ser) studies and drug screening.79–­82 Recently, we reported the was recently reported by Rütsche and colleagues in a pa- generation of human iPSC carrying two of the most recur- tient with JAK2 positive primary myelofibrosis (PM) and rent GATA2 mutations in paediatric MDS patients.83 The pancytopenia, although the role of GATA2 in the patho- availability of differentiated haematopoietic progenitor cells genesis of PM remains unclear.95 from iPSCs carrying GATA2 mutations will allow a com- Recurrent missense mutations in the extended ZF2 re- prehensive investigation of effects of these mutations as well gion diminishing DNA binding, including hotspot vari- as of secondary oncogenic somatic alterations (Figure 2). ants p.Thr354Met,1,6,10,96–­98 p.Arg396Trp/Gln3–­5,17,98–­101 and Indeed, hiPSC model offers the possibility to study the step- p.Arg398Trp,6,9,10,98,101 have been reported across a number wise progression of GATA2-­related MDS in vitro through of individuals and families and seem to be almost exclusively the sequential introduction of secondary oncogenic genetic familial. Interestingly, similar to somatic GATA2 mutations lesions by CRISPR/Cas9 gene editing, as already shown for p.Leu359Val92,93 and p.Arg307Trp,102 the germline muta- adult AML.84 Importantly, the iPSC-­based approach allows tions p.Thr354Met and p.Cys373Arg were both reported to the characterization of transcriptional programs driving exhibit gain of binding affinity to a GATA2 partner protein specific stage malignant transition and the identification of (PU.1 encoded by SPI1 gene). This might suggest an addi- possible prognostic markers for early therapeutic targeting tional mechanism to LOF driving leukemogenesis, namely (Figure 2). context-­dependent gain of function.20,92 Apart from missense variants, there are a few reports of synonymous GATA2 mutations in a handful of GATA2-­ T Y PE S OF DISE A SE - ­C AUSI NG deficient cases to date. Despite the unaltered amino acid GE R M L I N E GATA 2 M U TAT IONS composition, the variants result in RNA degradation and can be categorized as null alleles. The variant p.Thr117=, Hereditary genetic alterations disrupting GATA2 expression first described by Wehr and colleagues,103–­105 along with and protein function are pivotal in leukemogenesis by sabo- four additional mutations, namely p.Leu217=, p.Gly327=, taging normal haematopoiesis leading to GATA2 deficiency p.Ala341 = and p.Phe472=, reported by Kozyra and col- and subsequent myeloid malignancies. Approximately one-­ leagues,104 was found to strongly alter splicing and induce third of all germline GATA2 variants are passed on through selective loss of messenger RNA.20,104,105 Although sub- autosomal dominant inheritance (familial), while at least stantial phenotypic changes may support the putative role two-­t hirds of the mutations occur de novo.4,85,86 Mutational of synonymous GATA2 mutations, sequencing of GATA2 landscape of GATA2 deficiency includes a steadily increasing mRNA to confirm monoallelic loss and in silico analyses number of variants with approximately 500 published cases are essential to assess the actual impact of these seemingly harbouring roughly 180 different familial or de novo ger- innocuous, albeit potentially damaging variants on GATA2 mline mutations and partial or whole gene deletions.11,20,87 expression for each individual case.20,104 The majority of reported pathogenic or probably pathogenic Regulatory variants (in approximately 10% of patients) germline variants are inactivating (null) or loss-­of-­f unction constitute an important group of germline mutations affect- (LOF) resulting in haploinsufficiency (~60% of all GATA2 ing the GATA2 intronic +9.5 enhancer site and thus leading deficient cases) either through truncating (nonsense), to haploinsufficiency via decreased GATA2 expression in frameshift or splicing mutations and deletions.1,19,22,27,88–­91 HSPCs. Consequently, damaging mutations and deletions of These confirmed germline variants along with less common intron 4 (NM_032638 isoform) perturb the dose-­dependent in-­frame deletions and insertions are scattered throughout transactivation activity of the Gata2 intronic enhancer dis- the coding region of GATA2.3,21 rupting haematopoietic cell development and cell differen- In addition to variants leading to premature trans- tiation fates.35,38,58,59 Interestingly, no germline variants of lation termination prior or within ZF2, missense vari- the distant upstream Gata2–­77 enhancer site have been de- ants within ZF2 represent a large proportion of germline scribed to date, although current clinical platforms (panel GATA2 variants (~30%) instigating LOF via disrupting and exome sequencing) do not account for such regulatory 13652141, 2022, 4, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/bjh.18330 by Readcube (Labtiva Inc.), Wiley Online Library on [09/02/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License KOTMAYER et al.    | 487 regions. Future studies using whole genome sequencing have Apart from general symptoms including fever, fatigue the potential to uncover undefined causes of GATA2 defi- and weight loss, further extra-­haematological phenotypes ciency, that is, in hitherto unknown regulatory regions. of GATA2 deficiency encompass premature birth, develop- Rare cases of whole gene deletions,97,106,107 in-­frame de- mental delays and congenital malformations,5,17,56,114,115 and letions1,6,9,98 and missense mutations located outside of high incidence of primary lymphedema and lymphadenop- relevant protein domains6,108,109 have also been reported, al- athy in the GATA2 deficient patient population underlying though data and functional studies on their putative impact the crucial role of GATA2 in lymphatic angiogenesis and (presumably LOF) are still absent.89 Recently, a novel type lymphatic valve development.97 of germline in-­frame GATA2 variants has been reported by Cavalcante de Andrade Silva and colleagues in a patient with characteristic features of GATA2 deficiency, including GE NOT Y PE –­P H E NOT Y PE bilateral lower limb lymphoedema, haematological manifes- C OR R E L AT IONS I N tations, and recurrent infections. The familial in-­frame in- GATA 2 DE F ICI E NC Y sertion of nine amino acids between ZF1 and ZF2 increased the spacing between the two ZFs abrogating target gene reg- Despite the rising number of reported cases with GATA2 ulation and cell differentiation.110 deficiency, different types of germline GATA2 variants have not been linked strongly to substantial phenotypes, clinical outcomes or age of onset, although some phenotypes seem C L I N IC A L PH E NOT Y PE S to cluster within affected carrier families (Figure 1B). Some A S SO CI AT E D W I T H analyses have shown missense GATA2 mutations to be associ- GATA 2 DE F ICI E NC Y ated with increased rates of myeloid malignancies compared to nonsense and frameshift variants12,17,86 with a predomi- Detailed description of the clinical presentations of GATA2 nance of leukaemia in patients harbouring p.Thr354Met.20 deficiency syndrome is beyond the scope of this review, However, this association was not confirmed in a cohort of therefore we refer to comprehensive reviews discussing 173 European MDS patients with germline GATA2 vari- GATA2-­associated phenotypes20,89 and previously pub- ants, examined on behalf of the GATA2 HuMo consortium; lished large cohort studies.4,17 cases with null alleles (encompassing nonsense, frameshift Myeloid malignancy is the most common phenotype truncating, splice site, whole gene deletion and synonymous associated with germline GATA2 mutations, manifest- -­RNA deleterious variants) had a higher prevalence of high-­ ing in roughly 80% of reported carriers at a median age of risk MDS (including leukaemia) in comparison to missense approximately 20 years,1,4,5,20,89 (Figure 1B). GATA2 defi- mutation carriers (unpublished observations of the EWOG-­ ciency is considered the most common germline predispo- MDS study group). Recurrent or severe infections and un- sition in paediatric MDS, accounting for 15% of cases with derlying cellular immunodeficiencies have been linked to MDS with excess blasts, and 7% of overall paediatric MDS variants leading to premature translation termination and within the co-­operative European Working Group of MDS deletions,12,86 although interestingly, missense mutations in Childhood (EWOG-­MDS) cohort of 426 patients pub- p.Arg398Trp and p.Arg396Gln, have also recently been as- lished by Wlodarski and colleagues.4 These numbers were sociated with immunodeficiency by Homan and colleagues validated in a larger cohort of 669 patients from the EWOG-­ based on analysis of reported cases.20 MDS registry, where GATA2 deficiency was detected in 7% Emberger syndrome has been exclusively reported in of MDS cases and was mutually exclusive with germline cases with regulatory, premature termination or LOF mis- SAMD9/SAMD9L mutations (another driver of paediatric sense variants diminishing DNA binding and transactiva- marrow failure and MDS) which accounted for 8%.111 tion activity (p.Arg361Leu, p.Cys373Arg, p.Arg396Gln), Immunological phenotypes of GATA2 predisposition implying the crucial role of haploinsufficiency in lymph- most commonly present as cellular immunodeficiencies edema development.17,20,24,86,92 Underlying molecular mech- with subsequent recurrent or atypical mycobacterial, viral, anism for normal lymphatic vessel valve development and and fungal infections and autoimmunity. Immune dysfunc- defects of the lymphatic vasculature in GATA2 deficiency tion is often the initial presentation in this patient popu- was first proposed by Kazenwadel and colleagues.24 They lation; however, pre-­existing immunodeficiency may also demonstrated that GATA2 levels in lymphatic endothelial be observed retrospectively in more than half of the cases cells are regulated via mechanic stimuli, including oscil- with myeloid malignancy and thus is strongly suggestive for latory fluid flow116 and extracellular matrix-­induced ten- GATA2-­related MDS/AML.22,89 Prominent clinical mani- sion42 driving valve morphogenesis and vessel sprouting, festations of GATA2 deficiency also include repeatedly ob- respectively. Stimuli-­dependent expression and cell-­specific served pulmonary alveolar proteinosis (PAP), and interstitial transcription factor complexes of GATA2 in lymphatic en- pulmonary diseases112 and high incidence of solid tumours dothelial cells seem to be profoundly impacted by haploin- and precancerous lesions with the majority of the cancers sufficiency compared to HSPCs. arising from underlying viral infections, particularly HPV Based on a handful of cases, large, whole gene deletions and EBV.3,6,15,100,113 and losses of chromosome 3q21.3 resulting in GATA2 GATA2 DEFICIENCY AND MDS/AML: EXPERIMENTAL STRATEGIES FOR DISEASE MODELLING 13652141, 2022, 4, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/bjh.18330 by Readcube (Labtiva Inc.), Wiley Online Library on [09/02/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License 488 |    AND FUTURE THERAPEUTIC PROSPECTS haploinsufficiency have been linked to dysmorphisms, de- SETBP1 and ASXL1 mutations.89,132 Unlike in cases with velopmental and neurological defects, monocytopenia and germline CEBPA, DDX41 and RUNX1 predispositions,103–­105 subsequent infections.12,91,97 biallelic mutations are uncommon as somatic GATA2 muta- Regulation and transcription factors interacting with tions mainly within ZF1 seem to be rare secondary events in GATA2 vary greatly in selected biological systems and in the GATA2-­driven myeloid neoplasia.20,127,133 steady state versus stress, providing a possible explanation Similar to biological and environmental stresses, com- to phenotype–­ genotype clustering in GATA2 deficiency. monly seen somatically mutated genes and recurrent cyto- Although it has been noted that substantial clinical presen- genetic alterations are putative “stressor events” that may tations and defects of certain organs are related to distinct constitute pathogenic triggers for clonal evolution and sub- types of GATA2 variants, in vitro studies on GATA2 func- sequent disease progression/evolution to AML.21 Therefore, tion in different cellular contexts and processes are required there is a strong, and today still unmet need for comprehen- to provide novel insight to underlying molecular mecha- sive screening of concurrent somatic aberrations in GATA2 nisms of phenotypic complexity.20,21,86 deficiency, especially in individuals and families with unex- pected clinical presentations and incomplete penetrance. SOM AT IC A BE R R AT IONS I N M Y E L OI D M A L IGNA NCI E S W I T H T H E R A PEU T IC STA N DA R D OF C A R E GATA 2 PR E DISPOSI T ION Despite the increasing number of cases with GATA2 defi- There are a number of recurrent cytogenetic aberrations and ciency syndrome, currently there are no comprehensive somatic mutations in a host of genes that occur commonly guidelines and recommendations on the diagnosis and with GATA2 deficiency and subsequent myeloid malignan- clinical management of individuals and families carry- cies. Monosomy 7 is the most frequently described clonal ing germline GATA2 mutations. Similar to other germline cytogenetic aberration. It has been reported in nearly half of cancer predisposition syndromes, suspicion of GATA2 de- the GATA2 deficient cases with associated myeloid malignan- ficiency arises in cases with unusually early age of onset of cies20 and together with the deletion of 7q17 and unbalanced myeloid malignancies, particularly MDS/AML, but positive translocation der(1;7)(q10;p10)3,14,117 uniformly result in the family history may also be evocative.134 Although in some monoallelic loss of chromosome 7q conferring poor progno- patients MDS/AML develops without pre-­ existing extra- sis.89,118 Among paediatric MDS with monosomy 7, 37% of haematopoietic phenotypes, others may show progressive cases were found to carry germline GATA2 mutations; this as- symptoms indicative for GATA2 deficiency, such as cytope- sociation is even more striking in adolescents with monosomy nias, generalized warts and monocytopenia, mycobacterial 7 where GATA2 deficiency is found in 72% of patients.4,118,119 infections, recurrent viral (predominantly HPV) infections, Similarly, the association between der(1;7) and GATA2 and lymphedema.4,86,135 Cytogenetic testing of BM biopsy deficiency was reiterated in a recent study by Kozyra and samples in cases with MDS/AML or unexplained cytopenias colleagues, demonstrating the majority (73%) of primary may be informative, as concurrent monosomy 7 and trisomy MDS with der(1;7) have an underlying GATA2 deficiency.120 8 can also point towards GATA2 deficiency.89 Isolated trisomy 8 is the second most common aneuploidy in Early diagnosis of GATA2 deficiency based on clinical GATA2 mutation carriers, occurring in approximately 20% presentation and/or genetic testing for germline GATA2 of the cases.1,3–­6,17,20,98 Other cytogenetic events, such as tri- mutations (with conventional bidirectional Sanger sequenc- somy 21,1,97,121 have also been encountered alone (isolated) ing or next generation sequencing methods) is pivotal in or in combination with other aberrations; however, due avoiding non-­curative therapies and especially immunosup- to the lack of routine screening their prevalence is largely pression due to underlying HSPC defects and immunode- unknown to date. Contrary to MDS of adulthood, loss of ficiency.89 Patients with high risk of evolution to advanced 5q and complex karyotypes are generally not reported in MDS or AML and/or high-­risk karyotypes, such as mono- GATA2 deficiency.11,22 somy 7, should undergo timely HSCT with selected con- Recurrent somatic mutations in SETBP1, ASXL1, CBL, ditioning regimens. Patients with stable disease (e.g. MDS EZH2, KRAS/NRAS, JAK3, STAG2, RUNX1, and PTPN11 with refractory cytopenia of childhood), without progress- and STAG2 have been described in cases with GATA2-­ ing immunodeficiencies underlying recurrent infections, driven myeloid malignancies.88,96,101,106,122–­131 LOF ASXL1 transfusion-­dependency and MDS with karyotypic aber- mutations are reported in approximately one-­third of all rations or dysplasia may qualify for the watch and wait ap- patients with AML/MDS and similarly to SETBP1 variants, proach, although progressive disease can be expected over are strongly linked to monosomy 7 and thus associated with time in the majority of cases.3,11,89,136–­138 General recom- a more advanced disease state.96,98 Interestingly, single-­cell mendations for surveillance include frequent evaluation level analyses reported by Pastor Loyola and colleagues re- of complete blood counts, annual BM biopsies with cyto- vealed a non-­random acquisition of these secondary events, genetic testing, analysis of lymphocyte subsets and immu- implying monosomy 7 as an early somatic event in the MDS noglobulins, and screening for subclinical presentations of founding clone, followed by the acquisition of concomitant pulmonary disorders (predominantly PAP) and (HPV-­or 13652141, 2022, 4, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/bjh.18330 by Readcube (Labtiva Inc.), Wiley Online Library on [09/02/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License KOTMAYER et al.    | 489 F I G U R E 3 Isolation of haematopoietic stem cells (HSCs) of a GATA2mut patient and gene correction using the CRISPR/Cas9 system. The induced pluripotent stem cell (iPSC) function can be further evaluated after transplantation into mouse model. EBV-­associated) cancers as part of yearly skin and gynaeco- of healthy individuals carrying GATA2 variants is recom- logical examinations.86 mended by the EWOG-­MDS group to assess GATA2-­related Currently, allogenic HSCT is the only curative approach cytogenetic aberrations.86,88 Similar to patients with stable for patients with GATA2 deficiency. There is consensus that disease, further management of asymptomatic carriers may in high-­risk cases, the ideal point in time for pre-­emptive include evaluation of complete blood counts, pulmonary HSCT is during the hypocellular phase of MDS prior to se- function tests and surveillance for malignancy and immu- vere complications, such as invasive infections or evolution nodeficiencies.88,141 Notably, critical role of screening for to MDS-­EB and AML.4,11,22,88,89,113,136 Myeloablative con- inherited variants in relatives is also becoming generally ditioning regimens for HSCT are the preferred standard recognized during HSCT donor selection in order to avoid approach in paediatric MDS with GATA2 predisposition, donor-­derived myeloid malignancies conferring dismal but reduced intensity conditioning regimens have also been outcome.20,142 successfully used in some cases.4,11,86,89,121,139 Based on the results of EWOG-­MDS cohort study, overall outcomes were not influenced by the presence of germline GATA2 variants POT E N T I A L NOV E L T H E R A PI E S with a comparably favourable 5-­year overall survival of 66% in patients with MDS who had already acquired monosomy GATA2-­related MDS/AML constitute a complex disorder 7. Rates of infectious complications were also comparable to with a profound impact on the quality of life of patients the control GATA2 wild-­t ype group (66% vs. 61%),4 but de- and their life expectancy. Due to the phenotypic diversity, spite the non-­inferior results, HSCT in GATA2 deficiency clinical overlap, and the evolving phenotype of this disease, remains challenging due to underlying immunodeficiencies achieving a timely diagnosis is often difficult. This holdup and comorbidities threatening the outcome of HSCT.86,137 has a direct impact on patient's management. The only cu- Interestingly, Hoffman and colleagues recently reported rative option for these patients is allogenic HSCT, usually a higher incidence of thrombotic events and neurological performed in the setting of advanced MDS or severe immu- complications in a cohort of patients with GATA2-­driven nodeficiency. The overall outcome after HSCT is favourable paediatric MDS undergoing HSCT with myeloablative con- for the early stages of haematological disease but declines ditioning, although graft versus host disease (GVHD), graft considerably in patients with advanced MDS. In addition, failure and treatment related mortality rates were compara- the lack of suitable donor and associated mortality and mor- ble to patients with non-­GATA2-­driven MDS/AML.140 bidity can hinder this procedure in a number of patients. Considering highly variable phenotypes and incomplete Correction of patients' HSCs by gene therapy could represent penetrance of GATA2 deficiency, genetic testing of inher- a promising alternative to allogenic HSCT, due to the pro- ited GATA2 variants should be performed in family mem- liferative advantage of corrected cells (Figure 3). This phe- bers to identify asymptomatic carriers.20 Although data and nomenon has been recently described in one unique case, in recommendations on early HSCT in phenotypically “silent” whom spontaneous somatic genetic rescue in GATA2 gene cases are still absent to date, baseline BM aspirate evaluation conferred selective advantage of the corrected HSCs and GATA2 DEFICIENCY AND MDS/AML: EXPERIMENTAL STRATEGIES FOR DISEASE MODELLING 13652141, 2022, 4, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/bjh.18330 by Readcube (Labtiva Inc.), Wiley Online Library on [09/02/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License 490 |    AND FUTURE THERAPEUTIC PROSPECTS annulled the effect of pathogenic GATA2 mutation.143 These therapy may be beneficial for haematopoiesis and to prevent findings strongly indicate that having few corrected HSCs other clinical complications.143 might be sufficient to restore haematopoiesis in GATA2 In this context, with the breakthroughs in CRISPR/Cas9 carriers. It is important to highlight that the expression of technology, it is an imaginable scenario where GATA2 carri- GATA2 gene is tightly regulated in HSCs and during leu- ers at early stage of the disease can be actively assisted by an kaemia progression. Therefore, classical lentiviral trans- innovative tailored and precise gene-­based treatment. The genic approaches do not represent the best option as gene reinfusion of corrected autologous HSCs would represent a therapy method to treat GATA2 deficiency. Advanced gene promising and less toxic alternative treatment for GATA2-­ editing technology via homologous recombination (HR) has related MDS. However, a multidisciplinary effort is still paved the way to the development of precision medicine and needed to establish the clonal origin of leukemogenesis in outcome-­driven therapy for individual patients.78,144,145 This GATA2 carriers in order to select the appropriate time frame allows the specific correction of the mutated gene, maintain- to treat these patients by gene editing. ing the physiological expression of the gene and eliminat- ing the integration of exogenous DNA material elsewhere. AU T HOR C ON T R I BU T ION S Moreover, gene editing based on CRISPR/Cas9 and large HR LK, DRM, CB and AG wrote the manuscript, OM-­B pre- donor deliver by adeno-­associated viral vector of serotype 6 pared figures and reviewed the manuscript, EK, AC, AB, (rAVV6) has already revealed promising clinical results, as MWW reviewed and edited the manuscript. recently shown in haematological diseases such as sickle cell disease and pyruvate kinase deficiency.76,144,146,147 AC K N OW L E D G E M E N T S Based on these advances we can speculate that the op- This work was supported by ERA PerMed GATA2-­ timization of a repair mechanism by large HR donor tem- HuMo Funding Mechanism (Spain: Acció instrumental plates covering different exons could provide treatment for de SLT011/18/00006 of the Department of Health of the a substantial number of GATA2 patients allowing effective Government of Catalonia, to AG and AB; Hungary: ED-­18-­ and sustainable translation in the clinical arena (Figure 3). 1-­2019-­001 grant from the National Research, Development Importantly, recurrent somatic mutations in MDS driver and Innovation Office to CB; and Germany: German genes (i.e. SETBP1, ASXL1, STAG2 and RAS pathways) Federal Ministry of Education and Research (BMBF) has been identified by us and others in GATA2-­MDS pa- 2018-­123/01KU1904 to MWW), ÚNKP-­21-­2-­I-­SE-­21 and tients .88,96,101,106,122–­130 Therefore, a preliminary genomic Hungarian National Academy of Scientist Education grant to screening for additional somatic mutations should be imple- KL, TKP2021-­N VA-­15, TKP2021-­EGA-­24 and EU's Horizon ment as standard procedure for each GATA2 deficient pa- 2020 research and innovation programme under grant tient. This may serve as prognostic markers predicting the agreement no. 739593 and Elixir Hungary to CB, MSCA risk of relapse, and thus be crucial in guiding treatment strat- No 101029927-­scGATA2track (H2020-­MSCA-­IF-­2020) to egy. Finally, a possible technical obstacle for clinical applica- OM-­B, the Spanish Ministry of Economy, Industry, and tion of gene editing correction of GATA2 mutations is the Competitiveness (MINECO PID2020-­ 15591RB-­ 100), La off-­target effects (OTEs) that might occur in undesired parts Marató de TV3 (202001-­32), FPS Grant 2018 by Fondazione of the genome. Therefore, detection of OTEs with screening Pisana per la Scienza ONLUS and CERCA Programme/ strategies, such as GUIDE-­seq or CIRCLE-­seq,148,149 should Generalitat de Catalunya for institutional support to AG, Vera be used before any clinical translation of this gene therapy and Joseph Dresner Foundation, Evans MDS Foundation, approach. American Lebanese Syrian Associated Charities, and BMBF MyPred 01GM1911A to MWW. Open access funding ena- bled and organized by ProjektDEAL. C ONC LUSION C OM PE T I N G I N T E R E S T GATA2 deficiency is a monogenic disorder with complex The authors declare that they have no known competing fi- clinical manifestations and a very high propensity for MDS/ nancial interests or personal relationships that could have AML development. Carriers of GATA2 mutations show appeared to influence the work reported in this study. nearly complete life-­time penetrance towards the develop- ment of myeloid neoplasia; however, it is not clear what factors DATA AVA I L A BI L I T Y S TAT E M E N T (genetic, epigenetic, or inflammatory) modify the pheno- All data used in this manuscript was collected using the type leading to variable penetrance observed for identical PubMed database (accessed: 20 April 2022). mutations. Due to the phenotypic diversity, clinical overlap and evolving phenotype of this disease, a timely diagnosis is ORC I D often difficult to achieve. Therefore, a crucial unmet need Lili Kotmayer https://orcid.org/0000-0002-5089-7659 for novel, genotype-­specific, more efficacious therapies for Damia Romero-­Moya https://orcid. GATA2 deficient patients remain. Recently, Catto and col- org/0000-0001-9947-3577 leagues suggested that early recovery of haematopoiesis in Oskar Marin-­Bejar https://orcid. GATA2 deficiency either by transplant or potentially by gene org/0000-0002-6889-6566 13652141, 2022, 4, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/bjh.18330 by Readcube (Labtiva Inc.), Wiley Online Library on [09/02/2023]. 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