Stemness-associated cell states are linked to chemotherapy resistance in AML. We uncovered a direct mechanistic link between expression of the stem cell transcription factor GATA2 and drug resistance. The GATA-binding protein 2 (GATA2) plays a central role in blood stem cell generation and maintenance. We find substantial intra- and inter-patient variability in GATA2 expression across AML patient samples. GATA2 expression varies by molecular subtype and has been linked to outcome. In a murine model, KMT2A-MLL3 driven AML originating from a stem cell or immature progenitor cell population have higher Gata2 expression and are more resistant to the standard AML chemotherapy agent doxorubicin. Deletion of Gata2 resulted in more robust induction of p53 following exposure to doxorubicin. ChIP-Seq, RNA-Seq and functional studies revealed that GATA2 regulates the expression of RASSF4, a modulator of the p53 inhibitor MDM2. GATA2 and RASSF4 are anti-correlated in human cell lines and AML patient cell bulk and single cell expression datasets. Knockdown of Rassf4 in Gata2 low cells resulted in doxorubicin or nutlin-3 resistance. Conversely, overexpression of Rassf4 results in sensitization of cells expressing high levels of Gata2. Finally, doxorubicin and nutlin-3 are synergistic in Gata2-high murine AML, as well as AML patient samples. We discovered a previously unappreciated role for GATA2 in dampening p53-mediated apoptosis via transcriptional regulation of RASSF4, a modulator of MDM2. This role for GATA2 directly links the expression of a stemness associated transcription factor to chemotherapy resistance.

Stem cell transcriptional signatures are linked to poor outcomes in AML. Conventional chemotherapy (+/-HSCT) remains the only curative approach for AML, and survival largely reflects chemotherapy sensitivity. The mechanism by which a stem-cell-like transcriptional signature promotes chemotherapy resistance and relapse has not been determined. We identified a direct link between stemness and p53-mediated apoptosis involving a GATA2-RASSF4-MDM2-p53 axis. To interrogate transcriptional states that might mediate resistance in AML patients, we conducted single-cell RNA sequencing and single-cell ATAC sequencing on 10 pediatric AML samples. We found that expression of the stem/progenitor cell transcription factor GATA2 identified an immature cell population that partially overlaps with populations characterized by ERG and MECOM expression and chromatin accessibility. Using a Gata2 conditional retroviral KMT2A-MLLT3 mouse model, we established a model to study functional consequences of distinct GATA2 expression levels. This analysis revealed that AML originating from a murine stem cell or immature progenitor cell population showed higher Gata2 expression and were more resistant to standard AML chemotherapy agents. While GATA2 was not strictly required for leukemogenesis, Gata2high cells were more resistant to doxorubicin than Gata2low cells. In vitro treatment of bulk leukemias with doxorubicin resulted in selection for Gata2high cells. GATA2 expression was also increased in patient AML samples at relapse compared to initial diagnosis. Genetic inactivation of Gata2 profoundly enhanced chemosensitivity in Gata2high, but not Gata2low leukemias. Deletion of Gata2 in Gata2high cells increased activation of p53-mediated apoptosis in response to nutlin-3. Importantly, loss of Gata2 decreased MDM2 protein stability indicating that GATA2 may blunt the p53 response by enhancing MDM2 stabilization. ChIP-Seq and RNA-Seq in murine KMT2A-MLLT3 leukemias showed that neither p53 nor Mdm2 are transcriptionally regulated by GATA2. We next sought to identify a potential regulator of MDM2 stability. Integrated RNA-Seq and ChIP-Seq data nominated Rassf4, which was significantly upregulated upon Gata2 ablation, and GATA2 occupied its promoter. Similar to leukemia cells, decreasing Gata2 expression in normal hematopoietic stem cells increased Rassf4 expression, a pattern that is partially rescued by re-expressing Gata2. The RASSF family has been shown to promote apoptosis by modulating MDM2 protein stability. We functionally validated whether the relationship between high Gata2 expression and drug resistance is mediated by Rassf4 repression. Gata2-/- MA9 cells transduced with Rassf4 sgRNAs gained a competitive growth advantage under the pressure of nutlin-3a treatment versus cells transduced with non-targeting sgRNA indicating the emergence and expansion of drug-resistant cells. Conversely, overexpressing Rassf4 in Gata2high MA9 cells sensitized them to nutlin-3a and chemotherapy. Furthermore, GATA2 and RASSF4 expression inversely correlated in our AML patient sample scRNA-Seq dataset, as well as publicly available AML patient datasets, supporting the notion that a GATA2 mechanism to suppress RASSF4 applies to multiple AML subtypes. We evaluated whether MDM2 inhibitors, such as Idasanutlin, in combination with doxorubicin, could overcome the drug resistance seen in Gata2high leukemias. Combination therapy prolonged survival in vivo. In conclusion, we identified a novel role for GATA2 in blunting p53-mediated apoptosis via transcriptional repression of Rassf4, a regulator of MDM2 protein stability. Our study supports a model where the “volume control” of p53-mediated apoptosis by a stem cell transcription factor is an integral part of stemness, which is imparted on leukemic cells arising from a stem-cell-like cell-of-origin. Our findings provide a mechanistic explanation for the well-established, but thus far unexplained observation that the expression of HSC signatures are associated with poor outcomes in AML.

Innate immune processes impact diverse hematopoietic stem and progenitor cell (HSPC) functions, yet how innate immune signaling networks are sensed by HSPC genomes and how HSPC genomic activity alters cell responsiveness to innate immune regulators are not fully understood. We demonstrated that E14.5 primary (-77 -/-) and immortalized (hi-77 -/-) GATA2-deficient fetal progenitors from Gata2 -77 enhancer deletion mice upregulate Interferon-gamma (IFNγ) and Toll-like receptor (TLR) signaling components (Johnson et al. JEM 2020) and are hypersensitive to IFNγ and TLR signaling (Tran et al. iScience 2023). Combinatorial signaling involving both pathways regulates genes that are not regulated by individual pathways and amplifies responses beyond that induced by single pathways. Innate immune-activated genes harbor motifs for signal-dependent transcription factors, including the ETS factor PU.1, and GATA2 deficiency elevates PU.1 activity to upregulate B-lineage and myeloid genes (Jung et al. JCI 2023). We hypothesize that PU.1 links innate immune signaling networks and HSPC genome control. In new multi-omic studies, we asked if PU.1 is required to sense exclusively IFNγ or TLR signaling, both pathways independently or combinatorially, or is not essential. We used hi-77 -/- progenitors lacking a PU.1 enhancer ( Spi1URE-/-) with ~2-fold lower PU.1 vs. control hi-77 -/- progenitors. RNA-seq was conducted with hi-77 -/- and hi-77 -/-; Spi1URE-/- progenitors, with or without IFNγ, TLR1/2 agonist Pam 3CSK 4, or both agents for 4 h. hi-77 -/-; Spi1URE-/- progenitors lost the responsiveness of 27 TLR-activated genes and retained the responsiveness of 6 TLR-activated genes. 80% of IFNγ-activated genes (115 of 144) retained IFNγ-responsiveness when PU.1 was reduced. In hi-77 -/-; Spi1URE-/- progenitors, 68.2% (148 of 217 genes) retained their responsiveness to combinatorial signaling. These studies revealed the disproportionate importance of PU.1 for TLR- vs. IFNγ-mediated transcriptional control and a PU.1 requirement for combinatorial signaling at the majority of loci. Elevated IFNγ and TLR1/2 signaling in GATA2-deficient fetal progenitors is associated with increased monocytic and reduced granulocytic progenitors (Johnson et al. JEM 2020; Blood Adv 2022). Ablation of Irf8, encoding an IFNγ pathway component, partially normalizes the granulocytic progenitor deficit (Johnson et al. Blood Adv. 2022). Since GATA2 deficiency creates crosstalk between IFNγ and TLR1/2 pathways to elevate cytokine/chemokine production (Tran et al . iScience 2023), we asked if attenuating TLR signaling reverses the differentiation defect. As MYD88 Innate Immune Signal Transduction Adaptor (MYD88) is crucial for TLR1/2 signaling, we generated -77 +/-; Myd88-/- mice. Timed matings were conducted to obtain -77 +/+, -77 -/-, Myd88-/-, and -77 -/-; Myd88-/- E14.5 embryos. Myd88 ablation in -77 -/- embryos did not reverse the skewed granulocytic vs. monocytic progenitor ratio. To determine if MYD88 loss impacts -77 -/- progenitor function, we analyzed CMP and GMP populations from E14.5 embryos with CFU assays. In comparison with -77 +/+, -77 -/- CMP and Myd88-/- CMP produced fewer CFU-G colonies (1.9-fold decrease, P = 0.0097 and 2.0-fold decrease, P = 0.007, respectively). Surprisingly, -77 -/-; Myd88-/- CMP exhibited a further reduction in CFU-G colonies (5.2-fold decrease, P < 0.0001). The CFU-G colonies produced by -77 +/-; Myd88-/- CMP resembled that of -77 +/+ CMP. Thus, GATA2 and MYD88 synergistically promote CFU-G production from CMP, and a single -77 allele maintains CMP activity to produce CFU-G without Myd88. Myd88 deletion did not affect the reduced CFU-G nor the increased CFU-M with -77 -/- GMP. We are testing if TLR1/2 signaling is not required to maintain balanced fetal liver progenitor populations or if another signaling adapter nullifies the impact of MYD88 loss. Our studies revealed a mechanism underlying the hypersensitivity of GATA2-deficient fetal progenitors to innate immune signaling, and MYD88 loss further decreases the reduced CFU-G generated from -77 -/- CMP. We are testing the hypothesis that GATA2 restricts assembly of PU.1-containing chromatin complexes and analyzing models to unveil functional ramifications of elevated innate immune signaling in single HSPCs.

Lymphocytes play a critical role in adaptive immunity and defense mechanisms, but the molecular mechanisms by which hematopoietic stem and progenitor cells differentiate into T and B lymphocytes are not fully established. Pioneer studies identify several transcription factors essential for lymphoid lineage determination. Yet, many questions remain unanswered about how these transcription factors interact with each other and with chromatin at different developmental stages. This interaction regulates a network of genes and proteins, promoting lymphoid lineage differentiation while suppressing other lineages. Throughout this intricate biological process, any genetic or epigenetic interruptions can derail normal differentiation trajectories, potentially leading to various human pathologic conditions. Here, we summarize recent advances in understanding lymphoid cell development, which was the focus of the Winter 2024 International Society for Experimental Hematology webinar.

Human GATA2 genetic variation causes GATA2 deficiency syndrome, which can lead to myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). While pathogenic GATA2 variants alter the important zinc fingers and inter-zinc finger spacer, cellular aberrations underlying the disease predisposition are not established (Bresnick et al., 2020). Furthermore, as many questions exist regarding how GATA2 controls genome function, ascribing the functional significance of variants can be challenging. Our analysis of GATA2 variants in the United States population through the All of Us Research Program database (All of Us Research Program Genomics Investigators, 2024) revealed rare zinc finger variants, e.g., C295S, R308W, T356N (1/490,000+ alleles), that had not been described. Common variants included A164T (91,211/490,000+) and P250A (962/490,000+) in the N-terminus. To advance genetic curation, we established mouse and human genetic rescue systems to generate functional signatures that discriminate GATA2 from pathogenic variants. GATA2 regulates its expression through two enhancers (-77 kb and +9.5 kb) required for hematopoietic development and function (Johnson et al., 2012; 2015). We are identifying essential GATA2-regulated enhancers genome-wide and described the importance of the GATA2-activated Cebpe +6 kb enhancer (Katsumura et al., 2024). Deletion of this enhancer in progenitors downregulated C/EBPε, expression of a GATA2-regulated gene cohort, and, surprisingly, genes regulated by the pathogenic variant T354M, which exhibits loss-of-function and gain-of-function activities. This analysis identified C/EBPε as a cooperating transcription factor with GATA2 and T354M, and Cebpe regulation did not discriminate GATA2 from T354M activity. Using multiomics with the genetic rescue system in murine Gata2 -77-enhancer-deleted myeloid progenitors (hi-77-/-) with ~75% lower GATA2, we identified metrics that discriminate GATA2 from a pathogenic variant. We compared how GATA2 and a human germline GATA2 pathogenic variant (9 amino acid insertion between the zinc fingers, 9aa-Ins) function genome-wide (Jung et al., 2023). GATA2 deficiency in hi-77-/- cells upregulated expression of innate immune genes, including Il6st and Il6ra encoding cytokine receptor subunits IL6ST and IL6RA. GATA2, but not 9aa-Ins, reversed elevated Il6st and Il6ra expression and IL-6/STAT3 signaling. ATAC-seq revealed accessible chromatin 24 kb and 30 kb upstream of Il6st in hi-77-/- cells or hi-77-/- with 9aa-ins, but not wild type cells or hi-77-/- rescued with GATA2. To ask whether these sites are enhancers mediating IL-6 signaling, CRISPR-Cas9 gene editing was used to delete the sequences in hi-77-/- cells. Il6st expression decreased 13-fold (P<0.0001) and IL-6/STAT3 signaling decreased 85% (P<0.0001). Thus, GATA2 deficiency commissions enhancers that elevate Il6st expression and IL-6 signaling. To determine whether GATA2 deficiency commissions enhancers essential for other GATA2-repressed cytokine/chemokine receptor genes, we analyzed accessible chromatin in hi-77-/- cells at all cytokine/chemokine receptor genes and identified prospective enhancers that are GATA2-, but not 9aa-Ins-, regulated. These included the Csf1r +2.6 kb enhancer (Rojo et al., 2019). As enhancer decommissioning has utility to discriminate GATA2 from a pathogenic variant, additional clinical variants are being analyzed. We are also analyzing whether variants are defective in utilizing transcription factors and coregulators (C/EBPε and SMARCD2 for activation and RUNX1 and PU.1 for repression) that functionally interact with GATA2 at enhancers, and are extending murine analyses to human with a GATA2+/- HUDEP2 erythroid progenitor rescue system. In aggregate, diverse metrics related to genome regulation, with and without inflammation, as well as subcellular localization and protein stability, are being used to innovate a machine learning-based classifier strategy to ascribe whether any variant most closely resembles GATA2 or pathogenic variants. Using a minimal metric cohort that rigorously segregates GATA2 from clinical variants, we will streamline assays to ensure rapid, yet definitive, variant curation, compatible with clinical decision making. This curation strategy is being extended to decipher genetic variation in other genes that create an MDS and AML predisposition.

Innate immune signaling protects against pathogens, controls hematopoietic development, and functions in oncogenesis, yet the relationship between these mechanisms is undefined. Downregulating the GATA2 transcription factor in fetal hematopoietic progenitor cells upregulates genes encoding innate immune regulators, increases Interferon-γ (IFNγ) signaling, and disrupts differentiation. We demonstrate that deletion of an enhancer that confers GATA2 expression in fetal progenitors elevated Toll-like receptor (TLR) TLR1/2 and TLR2/6 expression and signaling. Rescue by expressing GATA2 downregulated elevated TLR signaling. IFNγ amplified TLR1/2 and TLR2/6 signaling in GATA2-deficient progenitors, synergistically activating cytokine/chemokine genes and elevating cytokine/chemokine production in myeloid cell progeny. Genomic analysis of how innate immune signaling remodels the GATA2-deficient progenitor transcriptome revealed hypersensitive responses at innate immune genes harboring motifs for signal-dependent transcription factors and factors not linked to these mechanisms. As GATA2 establishes a transcriptome that constrains innate immune signaling, insufficient GATA2 renders fetal progenitor cells hypersensitive to innate immune signaling.

Purpose of review Recent discoveries have provided evidence for mechanistic links between the master regulator of hematopoiesis GATA2 and the key component of interferon and innate immunity signaling pathways, interferon-regulatory factor-8 (IRF8). These links have important implications for the control of myeloid differentiation in physiological and pathological states. Recent findings GATA2 deficiency resulting from loss of the Gata2 −77 enhancer in progenitors triggers an alarm that instigates the transcriptional induction of innate immune signaling and distorts a myeloid differentiation program. This pathological alteration renders progenitors hyperresponsive to interferon γ, toll-like receptor and interleukin-6 signaling and impaired in granulocyte-macrophage colony-stimulating factor signaling. IRF8 upregulation in −77 −/− progenitors promotes monocyte and dendritic cell differentiation while suppressing granulocytic differentiation. As PU.1 promotes transcription of Irf8 and other myeloid and B-lineage genes, GATA2-mediated repression of these genes opposes the PU.1-dependent activating mechanism. Summary As GATA2 deficiency syndrome is an immunodeficiency disorder often involving myelodysplastic syndromes and acute myeloid leukemia, elucidating how GATA2 commissions and decommissions genome activity and developmental regulatory programs will unveil mechanisms that go awry when GATA2 levels and/or activities are disrupted.

We previously demonstrated that a subset of acute myeloid leukemia (AML) patients with concurrent RAS pathway and TP53 mutations have an extremely poor prognosis and that most of these TP53 mutations are missense mutations. Here, we report that, in contrast to the mixed AML and T cell malignancy that developed in NrasG12D/+ p53–/– (NP–/–) mice, NrasG12D/+ p53R172H/+ (NPmut) mice rapidly developed inflammation-associated AML. Under the inflammatory conditions, NPmut hematopoietic stem and progenitor cells (HSPCs) displayed imbalanced myelopoiesis and lymphopoiesis and mostly normal cell proliferation despite MEK/ERK hyperactivation. RNA-Seq analysis revealed that oncogenic NRAS signaling and mutant p53 synergized to establish an NPmut-AML transcriptome distinct from that of NP–/– cells. The NPmut-AML transcriptome showed GATA2 downregulation and elevated the expression of inflammatory genes, including those linked to NF-κB signaling. NF-κB was also upregulated in human NRAS TP53 AML. Exogenous expression of GATA2 in human NPmut KY821 AML cells downregulated inflammatory gene expression. Mouse and human NPmut AML cells were sensitive to MEK and NF-κB inhibition in vitro. The proteasome inhibitor bortezomib stabilized the NF-κB–inhibitory protein IκBα, reduced inflammatory gene expression, and potentiated the survival benefit of a MEK inhibitor in NPmut mice. Our study demonstrates that a p53 structural mutant synergized with oncogenic NRAS to promote AML through mechanisms distinct from p53 loss.

Cell type-specific transcription factors governing hematopoietic stem and progenitor cell transitions establish networks containing hundreds of genes and proteins. Network complexity renders it challenging to discover essential versus modulatory or redundant components. This scenario is exemplified by GATA2 mechanisms that control hematopoiesis during embryogenesis. Loss of a far upstream Gata2 enhancer (-77) disrupts the GATA2-dependent genetic network governing hematopoietic progenitor cell differentiation (Johnson KD. et al., Sci. Adv., 2015). The aberrant network includes the transcription factor Interferon Regulatory Factor-8 and a host of innate immune regulators, including Toll-like receptors (TLRs) (Johnson KD. et al., J. Exp. Med., 2020). Mutant embryonic progenitors lose the capacity to balance production of diverse hematopoietic progeny and generate excessive monocytic progeny. As IRF8 is vitally important for monocytic and dendritic cell differentiation (Yanez A. and Goodridge H., Curr. Opin. Hematol., 2016), we asked whether IRF8 is essential, contributory, or inconsequential. Using a double-mutant genetic rescue in vivo system, we demonstrated that reducing Irf8, in the context of the -77 mutant allele, reversed granulocytic deficiencies and the excessive accumulation of dendritic cell-committed progenitors. In -77 -/- E14.5 fetal livers, monocyte progenitors (MPs) increased 2.3-fold (P = 0.006), granulocyte progenitors (GPs) decreased 2.2-fold (P = 0.003) and common dendritic cell progenitors (CDPs) increased 10.2-fold (P = 0.021) relative to wildtype littermates. Ablating Irf8 in -77 mutants (-77 -/-; Irf8-/-) restored MPs and CDPs to wildtype levels and reversed the GP deficiency; further increasing GPs 4.2-fold relative to wildtype (P = 0.0009). Despite many dysregulated components that control vital transcriptional, signaling and immune processes, the aberrant elevation of a single transcription factor deconstructed the embryonic hematopoiesis program. We analyzed the mechanistic and biological implications of IRF8 dysregulation concomitant with ectopic upregulation of other innate immune genes (including Toll-like receptors (TLRs) in GATA2-deficient embryonic progenitors. In principle, such genes might function upstream, downstream, or in parallel with IRF8. Based on TLR upregulation and TLR roles in progenitor mechanisms (Nagai Y. et al., Immunity, 2006; Schuettpelz L. et al., Leukemia, 2014; Caiado F. et al., J. Exp. Med., 2021), we tested whether GATA2 deficiency in embryonic progenitors impacts cellular responsiveness to TLR ligands. Wild type and -77 enhancer-mutant progenitors were treated with increasing concentrations of the TLR1/2 ligand Pam 3CSK 4. The mutant progenitors were hypersensitive to Pam 3CSK 4, which resulted in supra-physiological induction of Tnf expression (2.8-fold at 34 nM, P = 0.004; 3.2-fold at 68 nM, P = 0.0003). Quantitative analyses indicated that hypersensitivity reflected increased Pam 3CSK 4 efficacy, but not potency. GATA2 re-expression in the mutant progenitors attenuated the elevated IRF8 expression and TLR signaling, normalizing Tnf and Ccl3 expression to a level comparable to that of wild type progenitors. In GATA2-rescued mutant progenitors, Tnf and Ccl3 expression decreased 3.9-fold (P = 0.005) and 2.5-fold (P = 0.047), respectively. Thus, GATA2 suppresses TLR signaling in embryonic progenitors. Ongoing studies are elucidating the mechanistic interconnections between IRF8- and TLR-dependent inflammatory networks in GATA2 deficiency during embryonic and adult hematopoiesis in cell populations and single cells, relationships between murine and human mechanisms, and the impact of targeted interventions that modulate these mechanisms.