Hematopoietic Transcription Factor Research Articles

Transcription factor regulation of ribosomal RNA in hematopoiesis.

Ribosomal RNAs (rRNAs) are transcribed within nucleoli from rDNA repeats by RNA Polymerase I (Pol I). There is variation in rRNA transcription rates across the hematopoietic tree, and leukemic blast cells have prominent nucleoli, indicating abundant ribosome biogenesis. The mechanisms underlying these variations are poorly understood. The purpose of this review is to summarize findings of rDNA binding and Pol I regulation by hematopoietic transcription factors. Our group recently used custom genome assemblies optimized for human and mouse rDNA mapping to map nearly 2200 ChIP-Seq datasets for nearly 250 factors to rDNA, allowing us to identify conserved occupancy patterns for multiple transcription factors. We confirmed known rDNA occupancy of MYC and RUNX factors, and identified new binding sites for CEBP factors, IRF factors, and SPI1 at canonical motif sequences. We also showed that CEBPA degradation rapidly leads to reduced Pol I occupancy and nascent rRNA in mouse myeloid cells. We propose that a number of hematopoietic transcription factors bind rDNA and potentially regulate rRNA transcription. Our model has implications for normal and malignant hematopoiesis. This review summarizes the literature, and outlines experimental considerations to bear in mind while dissecting transcription factor roles on rDNA.

Enhancement of PRMT6 binding to a novel germline GATA1 mutation associated with congenital anemia.

Mutations in the master hematopoietic transcription factor GATA1 are often associated with functional defects in erythropoiesis and megakaryopoiesis. In this study, we identified a novel GATA1 germline mutation (c.1162delGG, p.Leu387Leufs*62) in a patient with congenital anemia and occasional thrombocytopenia. The C-terminal GATA1, a rarely studied mutational region, undergoes frameshifting translation as a consequence of this double-base deletion mutation. To investigate the specific function and pathogenic mechanism of this mutant, in vitro mutant models of stable re-expression cells were generated. The mutation was subsequently validated to cause diminished transcriptional activity of GATA1 and defective differentiation of erythroid and megakaryocytes. Using proximity labeling and mass spectrometry, we identified selective alterations in the proximal protein networks of the mutant, revealing decreased binding to a set of normal GATA1-interaction proteins, including the essential co-factor FOG1. Notably, our findings further demonstrated enhanced recruitment of the protein arginine methyltransferase PRMT6, which mediates histone modification at H3R2me2a and represses transcription activity. We also found an enhanced binding of this mutant GATA1/PRMT6 complex to the transcriptional regulatory elements of GATA1's target genes. Moreover, treatment of the PRMT6 inhibitor MS023 could partially rescue the inhibited transcriptional and impaired erythroid differentiation caused by the GATA1 mutation. Taken together, our results provide molecular insights into erythropoiesis in which mutation leads to partial loss of GATA1 function and the broader role of PRMT6 and its inhibitor MS023 in congenital anemia, highlighting PRMT6 binding as a negative factor of GATA1 transcriptional activity in aberrant hematopoiesis.

MiR-1202 acts as anti-oncomiR in myeloid leukaemia by down-modulating GATA-1S expression.

Transient abnormal myelopoiesis (TAM) is a Down syndrome-related pre-leukaemic condition characterized by somatic mutations in the haematopoietic transcription factor GATA-1 that result in exclusive production of its shorter isoform (GATA-1S). Given the common hallmark of altered miRNA expression profiles in haematological malignancies and the pro-leukaemic role of GATA-1S, we aimed to search for miRNAs potentially able to modulate the expression of GATA-1 isoforms. Starting from an in silico prediction of miRNA binding sites in the GATA-1 transcript, miR-1202 came into our sight as potential regulator of GATA-1 expression. Expression studies in K562 cells revealed that miR-1202 directly targets GATA-1, negatively regulates its expression, impairs GATA-1S production, reduces cell proliferation, and increases apoptosis sensitivity. Furthermore, data from TAM and myeloid leukaemia patients provided substantial support to our study by showing that miR-1202 down-modulation is accompanied by increased GATA-1 levels, with more marked effects on GATA-1S. These findings indicate that miR-1202 acts as an anti-oncomiR in myeloid cells and may impact leukaemogenesis at least in part by down-modulating GATA-1S levels.

The ncBAF Complex Regulates Transcription in AML Through H3K27ac Sensing by BRD9.

The bromodomain-containing protein BRD9 is essential for AML cell viability, but it is unclear whether this requirement is due to the protein's role as an epigenetic reader. We inhibited this activity and identified altered gene-distal chromatin regulation and transcription consistent with a more mature myeloid cell state.

Cellular and Molecular Analysis of Hematopoietic Stem and Progenitor Cells in Shwachman-Diamond Syndrome

Shwachman-Diamond syndrome (SDS) is an inherited multi-organ disorder with bone marrow failure (BMF), which may include neutropenia, anemia, and thrombocytopenia, as well as a high risk of myelodysplasia and leukemia. Hematological complications are common causes of morbidity and mortality in SDS. Around 90% of SDS patients have biallelic mutations in the Shwachman-Bodian-Diamond Syndrome ( SBDS) gene, which plays an important role in ribosome biogenesis and hematopoietic cell survival. In a collaborative study led by Dr. John Dick (Science 2016) novel hematopoietic progenitor subsets were identified (noted as F1, F2 and F3), of which the abundance, clonal capacity, and function in disease states remain unknown. We hypothesized that cellular and molecular analysis of hematopoietic stem and progenitor cells (HSPC) including these novel progenitors would provide valuable insights into the pathogenesis of BMF in SDS. Using multiparametric flow cytometry we quantified the following HSPC populations: hematopoietic stem cells (HSC), multipotent progenitors (MPP F1-F3), common myeloid progenitors (CMP F1-F3), megakaryocyte-erythroid progenitors (MEP F1-F3) and granulocyte-monocyte progenitor (GMP) in 13 SDS patients with SBDS mutations and 10 normal control donors. We found a significant reduction in CD34+/CD38- and CD34+/CD38+ cells in SDS patients. It is particularly noteworthy that the sub-populations of CMPs (F1 and F2), and MEPs (F2 and F3) were prominently decreased. These cells contribute to the production of myeloid, erythroid, and megakaryocytic cells with different proportions. Using single-cell colony-forming assays, we assessed the cloning efficiency of each HSPC, revealing that HSCs, MPPs and GMPs from SDS patients exhibited a significantly lower cloning efficiency compared to those from control subjects. Notably, the typical erythroid-committing progenitor cells (CMP F2, CMP F3, MEP F2, and MEP F3) in SDS collectively produced approximately 46% fewer erythroid cells per colony compared to control progenitors, while myeloid-committing progenitor cells (CMP F1, MEP F1) from these patients produced approximately 86% fewer myeloid cells, including neutrophils, per colony compared to the corresponding control cells. Further, HSCs and MPPs-F1 cells from SDS patients had a reduced cloning efficiency of approximately 22% and 27%, respectively, compared to control HSCs and MPP-F1 progenitors. These data suggest that multiple HSPC populations from SDS patients are characterized by reduced cell numbers, functional impairments, and dysregulation compared to normal subjects. Next, we performed transcriptome analysis of each HSPC by RNA sequencing. We revealed dysregulated pathways and genes with the potential to disrupt hematopoiesis and mediate BMF in SDS. Using KEGG pathway analysis, we found that downregulation of the ribosome pathway reached statistical significance in HSC, CMP F1 and MEP F3. Downregulation of the hematopoietic cell lineage pathway reached statistical significance in MEP F1-2 and MPP F1-2. Additionally, the TGF-beta signaling pathway was suppressed in CMP F1-3, MPP F1, and GMP, while the NF-kappa B signaling pathway, crucial for regulating the survival, activation and differentiation of innate immune cells and inflammatory T cells, was also found to be decreased in GMP. Interrogation of specific dysregulate genes, we found that the hematopoietic transcription factor, ZFP36 (ZFP36 ring finger protein) which involved in cellular response to cytokines and growth factors, was significantly downregulated in five subpopulations of SDS HSPCs, particularly noticeable in the four progenitors (CMP F1-3, MEP F2, and GMP) which produced substantially fewer colonies in SDS patients compared to controls. Additionally, we observed downregulation of TGFB2 and upregulation of NFKBIA and NFKBIB may inhibit the NF-kappa B signaling pathway in SDS patients. These alterations have the potential to influence the hematopoietic cell lineage pathway, leading to BMF. Functional assays are ongoing and aim to assess the impact of these dysregulated genes on formation and behavior of HSPCs. In summary, our data suggest that multiple SDS HSPC populations are characterized by reduced cell numbers and functional impairments. As well, we revealed dysregulated pathways and genes with the potential to disrupt hematopoiesis and mediate BMF in SDS.

Synergy of Stag2-Cohesin Loss Results in Expansion of Npm1c-Mutant Hematopoietic Stem and Progenitor Cells

STAG2 is a member of cohesin complex that is recurrently mutated in >10 cancers and is essential in maintaining the integrity of the 3D genome partitioning structure known as topologically structural domains (TADs). Our previous work has demonstrated that depletion of various cohesin factors, including Stag2, leads to increased hematopoietic stem and progenitor population (HSPC) self-renewal and myeloid-biased differentiation. Loss of Stag2 leads to impaired sub-TADs and affects key hematopoietic transcription factors, such as PU.1, to access and engage their target genes. Yet, the extent and cooperative transformational role of Stag2-cohesin with leukemia driver mutations, such as Npm1c, remains unexplored. To determine whether Stag2-cohesin regulates leukemic 3D chromatin organization, we generated dual Stag2 Δ Npm1 c/+ murine models with tamoxifen inducible UbcCreER T2. After 4 weeks, HSPCs (LSK cells; Lin-Sca1+Kit+) are increased in Stag2 Δ Npm1 c/+ double mutant mice ( Figure 1A). Within LSK, Stag2 Δ Npm1 c/+ but not Npm1 c/+ has marked expansion of the myeloid bias MPP2 (LSK+Flk2-Cd150+Cd48+) and MPP3 (LSK+Flk2-Cd150-Cd48+) compartment. Interestingly, Stag2 Δ single mutant exhibit LSK and MPP3 expansion at 16 weeks, suggesting the early expansion of HSPC compartment is the result of Stag2 ΔNpm1 c/+ synergistic acceleration. No difference in serial replating of LSK and GMP (LSK-Cd34+FcyRII+) cells were observed between Npm1 c/+ and Stag2 Δ Npm1 c/+. However, Stag2 Δ Npm1 c/+ LSK cells had impaired reconstitution capacity and myeloid biased output in primary and secondary transplantation. To determine if the blockage is specific to Stag2-cohesin loss, we generated the Smc3/Npm1c double mutant mice, where both Stag1-cohesin and Stag2-cohesin complexes were partially depleted upon heterozygous deletion of Smc3. Interestingly, there were no changes in LSK or MPP3 population in Smc3 +/- Npm1 c/+ mice, suggesting it is specific to Stag2-cohesin loss which obstructs differentiation in Npm1 c/+ HSPC. Mechanistically, we performed bulk ATAC-seq on sorted HSC LT (LSK+Flk2-Cd150+Cd48-) and MPP3 population, as poor long-term reconstitution suggests functional alteration in HSC LT. Comparing to WT, both Stag2 Δ and Stag2 Δ Npm1 c/+ HSC LT have increased chromatin accessibility, while only Stag2 Δ Npm1 c/+ MPP3 have persistent opening of chromatin. Motif analysis of increased accessible peaks identified RUNX2, GATA3 and MEIS1 are among the increased accessible region in Stag2 Δ Npm1 c/+ MPP3, which reflects the increased myeloid output in the transplantation. Recently, various group have suggested that Npm1c directly binds to chromatin and drives the overexpression of genes, especially at HOX clusters and MEIS1. This suggests a possible synergistic mechanism which Stag2-cohesin regulates chromatin accessibility of key TF loci, which influences normal and leukemic stem and progenitor differentiation. To comprehensively profile the LSK population, we performed scRNAseq at 4 weeks post mutation activation, results showed a marked immune pathway activation in Stag2 Δ Npm1 c/+ LSK cells. To inter cell fate transition, we employed CellDancer, an RNA velocity based algorithm and found that Stag2 Δ Npm1 c/+ MPP cells have altered differentiation trajectory and delayed kinetics at MPP3 stages, suggesting a differentiation block ( Figure 1B). In conjunction with our results, we postulate that Stag2-cohesin regulates TF interactions with regulatory elements during cell fate transitions. We are currently investigating the direct interaction of Stag2-cohesin with regulatory elements in Npm1 c/+ cells and compare to WT and Stag2 Δ Npm1 c/+ cells. We will investigate the 3D genome organization of Npm1 c/+ and Stag2 Δ Npm1 c/+ cells to determine whether Stag2 loss induces the phenotypic changes via Ctcf-dependent or independent manner.

KPT-8602 Induced Hematopoietic Differentiation in U2AF1 S34F Mutant Cells and Myelodysplastic Syndromes Bone Marrow Stem and Progenitor Cells

Myelodysplastic syndromes (MDS) are a group of clonal bone marrow (BM) neoplasms characterized by ineffective hematopoiesis, cytopenia's, and a high risk of progression to acute myeloid leukemia. Current standard therapies include erythropoiesis stimulating agents for lower risk disease and DNMTi for higher risk MDS; however, less than half of patients respond and even the best responses are transient and non-curative. The majority of MDS patients harbor at least one of ~40 commonly found somatic gene mutations, most of which occur in splicing factor genes such as U2AF1, which also have non-canonical roles in nuclear export. KPT-8602 (Eltanexor) is a novel selective inhibitor of nuclear export (SINE) that acts by disrupting nuclear export of cargo relevant in MDS. We hypothesized that U2AF1 S34 F mutant cells would be selectively sensitive to treatment with KPT-8602. We created immortalized murine hematopoietic progenitor cells by retroviral transduction of stimulated bone marrow (BM) cells isolated from U2AF1 WT or U2AF1 S34F mice with a HOXB8_ER vector. Estrogen drives the hematopoietic transcription factor immortalizing the cells in a progenitor state, however, upon estrogen withdrawal the cells will differentiate and eventually die. This differentiation can be driven by treatment with specific growth factors such as erythropoietin (EPO) for erythroid differentiation or granulocyte macrophage colony stimulating factor (GM-CSF) for myeloid differentiation. KPT-8602 exposure induced differentiation and increased hematopoiesis in murine cell models and primary MDS samples. U2AF1 WT and U2AF1 S34F cells were plated in medium lacking estrogen and treated with GM-CSF (10nM) or EPO (1nM) with or without KPT-8602 (10nM) for 72h, then stained with Ly6C, CD11b, Ter119, and CD71 for flow cytometry analysis. In addition, we performed colony forming capacity assays in both the murine cells and primary MDS BM stem and progenitor cells and age-comparable healthy donors, with 14 day KPT-8602 exposure. Treatment with GM-CSF significantly increased the expression of Ly6C in both U2AF1 WT and U2AF1 S34F cells (WT fold change untreated vs GMCSF =1.9, p=0.03; S34F fold change untreated vs GMCSF =2.3, p<0.01). However, addition of KPT-8602 caused significantly more Ly6C expression than GM-CSF alone in the mutant cells (WT fold change GMCSF vs combination =3.1, p=0.06; S34F fold change GMCSF vs combination =4.0, p<0.01). There were no significant changes in CD11b expression with KPT-8602 treatment. We did not find a statistically significant increase in erythroid markers assessed by Ter-119 and CD71 after EPO treatment, however, KPT-8602 treatment alone showed a trend of increased expression of Ter-119 and CD71 in the mutant cells that fell just below statistical significance (Ter119 p=0.08 for WT, p=0.09 for S34F; CD71 p=0.21 for WT, p=0.06 for S34F). Additionally, we looked at selective toxicity of U2AF1 S34Fby WST-1 reagent and found no differences in toxicity between the WT or mutant cells with KPT-8602. However, U2AF1 WT and U2AF1 S34F progenitor cells showed an increase in colony forming capacity (n=1 with technical duplicates) when plated in the presence of KPT-8602 showing an average fold change in colony growth of 1.4 for U2AF1 WT and 1.3 for U2AF1 S34F underscoring the ability of KPT-8602 to induce differentiation. To confirm these results were not cell line artifacts, we next treated primary BM stem and progenitor cells from MDS patients (n=2) and healthy donors (n=4). MDS patient 1 (age 73y) was diagnosed with MDS-MLD-EB1 and harbored mutations in TP53, ASXL1, BCOR, TET2 and PPM1D. MDS patient 2 (age 79y) was diagnosed with MDS/MPN and harbored an SF3B1 mutation. Healthy donors (n=4) were all male and had a median age of 65y. Treatment with KPT-8602 (10nM) significantly increased burst and colony forming unit-erythroid (BFU-E/CFU-E) in MDS primary cells (p=0.02) with no significant increase in the healthy donors (p=0.57) (BFU-E/CFU-E fold increase was 1.1 for healthy donors and 3.5 for MDS). Collectively, these findings suggest that KPT-8602 promotes myeloid and erythroid differentiation and further validates the use of KPT-8602 as a therapeutic strategy for MDS. Future studies will fully elucidate the specific mechanisms by which KPT-8602 acts in the context of splicing factor mutations, and other commonly found MDS mutation types.

Elucidation of the Unique Function of GATA1s in the Development of Transient Abnormal Myeloproliferative Disorders

Transient abnormal myeloproliferative disorder (TAM) is a pre-leukemic condition that occurs at birth in children with Down syndrome. The responsible gene of TAM, GATA1, is a hematopoietic transcription factor integral to hematopoiesis. The GATA1 gene has two translation start sites, giving rise to two protein variants: GATA1-fl, a full-length protein, and GATA1s, an isoform lacking the N-terminal domain. Notably, TAM blasts express only the GATA1s due to a mutation in the GATA1 gene, as opposed to normal cells which express both proteins. This implies that the precancerous state is not triggered by the production of an aberrant protein butrather by the absence of the full-length protein and the exclusive expression of the minor isoform protein also present in normal cells. This is in stark contrast with the functions of many other oncogenes. Thus, the prevailing view is that TAM development involves a “loss of function” due to deficiency of GATA1-fl. However, some studies suggest that GATA1s may have unique functions that actively drive the TAM phenotype. Because of these conflicting findings reported, the precise pathomechanism of TAM development remains elusive, despite the identification of the responsible gene. To address this, we analyzed the function of GATA1s using an induced pluripotent stem cell (iPSC) model of TAM. We previously examined the effect of increasing GATA1s expression levels during early hematopoietic differentiation stage by stage using the TAM-iPSC model. We found that, in the absence of GATA1-fl protein, overexpression of GATA1s in early hematopoietic progenitor cells (HPC) shifted megakaryocytic differentiation toward myeloid lineage, intensifying TAM severity by expanding progenitor cells. These findings suggest that GATA1s has a unique function distinct from that of GATA1-fl. In this study, we investigated the function of GATA1s proteins as transcription factors using chromatin immunoprecipitation sequencing (ChIP-seq). Our results demonstrated that genes involved in hematopoiesis or cell proliferation belong to the gene cluster to which overexpressed GATA1s (O/E GATA1s) binds solely in the absence of GATA1-fl at the early HPC stage. Gene Ontology (GO) analysis further validated the significant enrichment of terms relevant to the TAM phenotype, including regulation of myeloid cell differentiation and negative regulation of cell differentiation. Intriguingly, the genes to which O/E GATA1s binds when GATA1-fl is present were unrelated to hematopoiesis or cell proliferation. These observations suggest that GATA1s uniquely binds to genes, instigating TAM onset under GATA1-fl deficient conditions, thus confirming TAM as a disorder hallmarked by the absence of GATA1-fl due to the GATA1 gene mutation. Our RNA-sequencing data indicate that, at the early HPC stage, O/E GATA1s only induces changes in binding to gene regions involved in TAM pathogenesis, without altering gene expression levels. In contrast, gene expression analyses of other later stage HPC fractions (megakaryocyte-erythroid progenitor-like cells: MEP-like cells), revealed increased STAT3 expression and enrichment of the JAK-STAT pathway and myeloid cell differentiation pathway. These results suggest that TAM development may be influenced by shifts in the gene-binding region of GATA1s in the early HPC, which, in turn, induces TAM in more differentiated MEP-like cells by skewing their differentiation towards the myeloid lineage. In conclusion, our study provides important insights into the pathological mechanisms of TAM pathogenesis, particularly with respect to the role of GATA1 in driving the TAM phenotype. the unique function of GATA1s and their specific binding to genes involved in TAM pathogenesis shed light on the complex interplay between GATA1s and TAM pathogenesis shed light on the complex interplay between GATA1s and TAM pathogenesis. Understanding these mechanisms may pave the way for targeted therapies aimed at modulating GATA1s activity and reducing the severity of TAM.

Genomic Correlates of Radiosensitivity in Diffuse Large B Cell Lymphoma

Introduction Radiotherapy (RT) is beneficial for select patients with localized diffuse large B cell lymphoma (DLBCL), in both the early stage and relapsed/refractory settings. Although molecular subtyping plays a key role in classification of DLBCL, genomic predictors of local response to RT have yet to be adequately explored. To our knowledge, there are no existing studies investigating the genomic landscape of DLBCL patients treated with RT. Therefore, we sought to identify genomic signatures of radiosensitivity in localized DLBCL patients. Methods We analyzed an institutional database of DLBCL patients treated with RT for localized measurable disease treated between 2007-2022. All patients underwent next-generation sequencing using an institutional 505-gene panel (MSK-Impact). Clinical and radiographic characteristics were obtained from chart and imaging review. All patients had measurable disease on CT scan at time of RT and included both Ann Arbor stage I/II patients referred for consolidative radiotherapy and those with isolated sites of relapsed/refractory disease. Patients did not receive any systemic therapy between RT start and time of response assessment. The public NCI LymphGen algorithm was used to further classify genomic subgroups. Using Fisher's exact test and logistic regression, we tested for univariable correlation between specific gene alterations and LymphGen subgroups with initial radiation response, defined by Lugano criteria and decrease in maximum SUV. Results We identified 46 patients including 15 (33%) tumors treated in the early-stage setting (13% definitively without prior chemotherapy, 87% as consolidation), while 31 (60%) were treated in the localized relapsed/refractory setting. The majority were GCB subtype (57%) while 28% were non-GCB and 15% had missing immunohistochemistry data. The median number of prior lines of systemic therapy was 1 (0-2) in early stage and 1 (1-7) in localized relapsed/refractory diseases. The median RT dose was 36 Gy (4-54), with 38 (83%) tumors receiving at least 30Gy. The median lesion SUV at the time of RT was 4.11 (1.1-46.0). Complete response (CR) was achieved in 41% of tumors at the time of first imaging assessment (median 3.1 months) with a median of 90.3% (42-100%) decrease in SUV. The most commonly altered individual genes were KMT2D (35%), MYD88 (28%), and TP53 (35%). LymphGen classified 22% of cases as EZB subtype, 13% as MCD, 2% as BN2, 2% as ST2, and the remaining 61% were unclassified. Mutations in ETV6 (13% of tumors) were associated with a higher CR rate in response to RT (83% mutated vs 35% wildtype, p=0.068). Lesions from ETV6 mutated cases had similar SUVs (p=0.74) and sizes (p=0.92) as wildtype cases. Alterations in genes from the LymphGen MCD subgroup were associated with greater reductions in SUV (ETV6 p<0.0001, PIM1 p<0.0001, MYD88 p<0.001, and TNFAIP3 p=0.02) The MCD subgroup trended towards increased odds of CR post-RT (log odds 1.9, p=0.08) and greater reduction of SUV (p=0.07) compared to the EZB subgroup. Conclusions To our knowledge this is the first study evaluating the role of LymphGen subgrouping on radiotherapy response, and the first study to examine genomic alterations in DLBCL patients treated with radiation. Genomic biomarkers of radiosensitivity are needed to identify radioresponsive tumors which may benefit from better integration of RT into existing salvage paradigms such as CAR T cells. Our data suggest that alterations in MCD LymphGen subgroup genes (ETV6, PIM1, MYD88, TNFAIP3) may be associated with better response to radiotherapy. ETV6 is a strong hematopoietic transcription factor known to be altered in many human leukemias and myelodysplastic syndromes. Its role in DLBCL is not well defined, with preclinical studies suggesting a distinct role compared to leukemias. Due to the limited sample size, these findings are hypothesis generating and warrant further study with larger, prospective cohorts to validate the ability of genomic signatures to predict RT response.

Stag2-Cohesin Loss Attenuates Flt3 ITD Myeloid Blast Expansion Yet Preserves Mutant HSC

FLT3 is mutated through an internal tandem duplication (ITD) in 20-25% of acute myeloid leukemia (AML), driving aberrant STAT5/AKT signaling and leukemogenesis. Murine modelling of ITD showed marked myeloid progenitor expansion and hematopoietic stem cell (HSC) exhaustion in various co-mutant settings. Here we present a Stag2 Δ Flt3 ITD model with stark reversal of these two aforementioned features. STAG2, a cohesin complex member, maintains the integrity of the 3D genome partitioning structure known as topologically structural domains. Loss of Stag2 impairs the access and engagement of key hematopoietic transcription factors such as PU.1 to their target genes. sAML is diagnosed when patients present leukemia with known history of hematological malignancies or chemotherapy/radiotherapy treatment. Comparing to de novo AML, sAML often arises in older patients and harbors a poor prognosis with a 5-year overall survival rate of <30%. Advances in genomic studies found various epigenetic mutations, such as STAG2, are often associated with sAML and AML-myelodysplastic related changes (MRC) subtype. STAG2 mutations are found in 14-20% of sAML cases and is suggested to reside within a dominant clone during the pre-leukemia phase, MDS to sAML transformation, such as with FLT3 ITD acquisition and persists during remission. To understand the mechanistic contribution of STAG2-cohesin loss with FLT3 ITD, we generated sequential mutagenesis murine models where the order of Stag2 and Flt3 ITD mutation is set as either ITD 1st Stag2 2nd ( de novo like) or Stag2 1stITD 2nd (sAML like) using tamoxifen-inducible Cre/Flpo recombinase or pIpC-inducible Mx1Cre. In the de novo like model, ITD is constitutively active then Stag2 is deleted when mice reach 6-8 weeks of age. Surprisingly, loss of Stag2 attenuates LSK to MP transition at 4 weeks post deletion, while MPP3 remains elevated, suggesting aberrant remodeling of myeloid differentiation. In the sAML like model, Stag2 is deleted via Mx1Cre and waited for 4 months to mimic the MDS phase, which is then followed by activation of ITD mutation via Flpo, which represents the MDS to sAML transformation. After activating both mutations, mice were followed for another 4 months before analyzing the hematopoietic stem and progenitor compartment. In contrast to the de novo like model, sequential Stag2 1stITD 2nd preserves the HSC population defined by either immunophenotyping or transcriptome via scRNAseq ( Figure 1). The mutant HSC is more quiescent but retains the capacity to reconstitute lethally irradiated recipients in the short term. Similar to the de novo like model, sequential Stag2 1stITD 2nd mice also exhibits a blocked myeloid differentiation. Comparing to ITD mutant, Stag2 1stITD 2nd LSK cells have decreased expression of Socs2 and Cish. While functionally determining the role of mutant HSC, we are performing RNA-seq during at early timepoints post ITD activation to determine how preceding Stag2 mutation could have altered the stem cell fate decision. Targeted therapy with inhibitors of FLT3 have had an overall survival benefit in FLT3-mutant AML, though the magnitude of effect has been modest. STAG2 mutations are more likely to be identified in poor responders to FLT3 inhibition as both reported by us with Pexidartinib treatment, as well as in the setting of Crenolanib treatment where expansion of the STAG2-mutant clone was observed during treatment. Thus, remodeling of the chromatin landscape though altered CTCF binding or cohesin function might impact leukemia identity in FLT3-mutant AML. Our data highlights an important regulatory role of Stag2-cohesin in Flt3 ITD mediated leukemogenesis, while generating a model that mimics the genetic evolution of sAML. This model will not only shed light on the sAML pathogenesis but also with creates a pre-clinical testing platform with potential therapeutic relevance.

A Novel Interaction between RUNX1 and the Splicing Factor PTBP1 Is Enriched in Leukemia Cells

RUNX1 and CBFβ form a critical hematopoietic transcription factor complex. RUNX1 is important not only for normal blood development, but also in leukemia. In the context of Inversion 16 acute myeloid leukemia (AML), RUNX1 is required for the activity of CBFβ-SMMHC (CM), the fusion oncoprotein generated by a chromosomal rearrangement and driver of this subtype of AML. The respective roles RUNX1 and CBFβ play in transcriptional regulation are well characterized but the role of other interacting partners is less well understood. To identify novel RUNX1 binding partners, we immunoprecipitated the transcription factor in mouse leukemia cells expressing CBFβ-SMMHC (CM+) and analyzed precipitated proteins by mass spectrometry. One of the proteins identified was the splicing factor Polypyrimidine Tract Binding Protein 1 (PTBP1). PTBP1 is an RNA binding protein that regulates alternative splicing of mRNAs. PTBP1 is required for normal hematopoiesis, including roles in stem cell maintenance, erythroid differentiation, and B-cell selection. However, a role for PTBP1 in leukemia has yet to be described. We confirmed the association of PTBP1 with RUNX1 via immunoprecipitation/western blot in mouse CM+ leukemia cells and the human Inv(16) cell line, ME-1. Next, we asked if this interaction is unique to inv(16) AML. We found that antibodies against RUNX1, but not isotype control antibodies, immunoprecipitated PTBP1 in a variety of myeloid (Molm-13, MV411, Kasumi-1) and lymphoid (Jurkat, MOLT-4, REH, SEM) human leukemia cell lines, indicating that this interaction occurs in multiple leukemia subtypes. To identify the region of RUNX1 required for the interaction with PTBP1, we used two deletion mutants found in patients with RUNX1-Familial Platelet Disorder ( RUNX1-FPD), RUNX1-R232 and RUNX1-Y287. We observed strong binding of PTBP1 with full length RUNX1, but not with either truncation mutant indicating that the RUNX1 C-terminus is required for interaction with PTBP1. To determine location of this novel interaction, we used proximity ligation assay (PLA). We found that the RUNX1:PTBP1 association occurred exclusively in the nucleus in both mouse and human leukemia cells. Next, we asked if this association is observed in healthy hematopoietic cells. By PLA, we found that the RUNX1:PTBP1 interaction is significantly increased in mouse leukemic cells compared to wild type lineage depleted (lin-) mouse bone marrow cells. To determine if the decrease of PLA signal in healthy bone marrow cells was due to spatial separation of PTBP1 and RUNX1, we performed immunofluorescence staining. We observed diffuse nuclear staining of both proteins in the majority of both healthy and leukemic cells. These results indicate that the interaction between RUNX1 and PTBP1 is significantly increased in leukemia cells, despite both proteins being expressed in similar patterns in both healthy and leukemic cells. During our experiments, we noticed large variations in PTBP1 expression levels in different mouse CM+ leukemia samples. To test whether PTBP1 levels correlate with a difference in leukemia stem cell activity, we performed colony assays using equal numbers of leukemia cells from samples with differing PTBP1 expression. We found that samples expressing high levels of PTBP1 resulted in a statistically significant increase in colony number. In addition, the colonies that formed from PTBP1 high samples were notably larger than those formed from PTBP1 low cells, implying a relationship between PTBP1 and leukemia stem cell self-renewal and proliferation. To determine if human AML samples show the same variability in PTBP1 levels, we performed western blot analysis using a panel of nine de novo and relapsed AML samples. Like our findings in mouse leukemia, human samples showed high variability in PTBP1 levels. We are currently addressing whether increased PTBP1 directly promotes leukemia progression. Collectively, our results demonstrate that PTBP1, a splicing regulator, is a novel RUNX1 interacting partner whose association with RUNX1 is significantly increased in leukemia cells. In addition, our data imply a potential role in leukemia stem cell proliferation, self-renewal and a role for PTBP1 in the splicing of RUNX1 target genes, perhaps generating leukemia specific splicing variants. This work has the potential to identify a new component of the RUNX1 transcriptional complex important for leukemogenesis.

RUNX1 Haplodeficiency Reduces Platelet Endocytosis of Albumin and Fibrinogen and Impairs Megakaryocyte Intracellular Trafficking

Introduction:RUNX1 is a major hematopoietic transcription factor that regulates megakaryopoiesis and platelet formation and function. Germline RUNX1 haplodeficiency (RHD) is characterized by thrombocytopenia, impaired platelet function and granule deficiencies. Our previous studies in a patient with RUNX1 mutation showed that platelet albumin and fibrinogen were decreased (Sun et al, Blood 103: 948-54, 2004) .Human platelet α-granules contain numerous proteins, some synthesized (e.g., PF4 and VWF) and others (e.g., fibrinogen, albumin and IgG) are incorporated by endocytosis, a process poorly understood in platelets/megakaryocytes (MK). Methods: To understand the mechanisms leading to the decreased platelet albumin and fibrinogen, we performed studies on endocytosis in platelets from RHD patients, megakaryocytic HEL cells and MK differentiated from CD34 + hematopoietic stem and progenitor cells (HSPCs). We studied endocytosis of fluorescent-labeled albumin, fibrinogen and IgG in platelet suspensions (0-90 min) and in PMA-treated megakaryocytic HEL cells (up to 24 hrs) using flow cytometry and immunofluorescence microscopy, and assessed proteins linked to endocytosis. Results:Platelet uptake of albumin, fibrinogen and IgG was time- and concentration dependent. Uptake of all 3 was decreased in two patients (father and daughter) with RHD (c.352-1 G>T) (mean fluorescent intensity <50% of normal). In HEL cells, uptake of albumin and fibrinogen was time- and concentration-dependent. On siRNA RUNX1 knockdown (KD), uptake/retention of albumin and fibrinogen over 24 hrs was increased compared to control siRNA cells. Caveolin-1 and flotillin-1, two caveolae-associated membrane proteins linked to endocytosis, and LAMP2 (linked to lysosomal trafficking) and RAB11 (linked to recycling endosomes) were increased on RUNX1 KD on immunoblotting. Caveolin -1 downregulation (siRNA) in RUNX1-deficient HEL cells abrogated the increase in uptake of albumin, but not fibrinogen. At 30 min, in control cells albumin was strongly colocalized with caveolin-1 on immunofluorescence studies. This colocalization was abolished on CAV-1 KD. With RUNX1 KD there was a decrease in colocalization with caveolin-1, and an increase with flotillin-1 both at 30 (Figure 1a) and 120 min. Thus, RUNX1 KD alters endocytosis and trafficking of albumin from caveolin-1 to flotillin-1 bearing vesicles. In experiments where HEL cells were incubated in medium without albumin for 24 hours, cell albumin decreased over time in both control cells and RUNX1-deficient cells with lower levels in the latter. LAMP2 levels were increased on RUNX1 KD, which may provide a mechanism (lysosomal degradation) for this. With respect to fibrinogen, at 30 or 120 min, there was no colocalization of fibrinogen with caveolin-1 or flotillin-1, indicating fibrinogen uptake is not caveolin-dependent. In control cells, there was low/moderate fibrinogen colocalization with RAB11 (marker for recycling endosomes); this was markedly increased with RUNX1 KD (Figure 1b), along with increased RAB11 level on immunoblotting, suggesting that RUNX1 KD is associated with altered fibrinogen trafficking towards recycling endosomes. Recently, IFITM3 has been implicated in platelet fibrinogen uptake in nonviral sepsis. We found HEL cell IFITM3 was increased on RUNX1 KD; however, IFITM3 siRNA KD did not decrease fibrinogen uptake. We studied endocytosis in in vitro differentiated MKs for 12 days from human CD34 + HSPCs; upon lentiviral shRNA KD of RUNX1 the uptake of albumin and fibrinogen was increased compared to control cells . Conclusions: We provide the first evidence that 1) platelet endocytosis of albumin and fibrinogen is impaired in RHD and 2) RUNX1-deficient megakaryocytic cells have enhanced endocytosis but defective trafficking of albumin and fibrinogen, mediated by distinct mechanisms, and associated with increases in caveolin 1, flotillin-1, RAB11, LAMP2 and IFITM3. These studies provide new insights into mechanisms governing endocytosis by platelets and MKs relevant to the α-granule deficiencies in RHD.

Sustained MYB Activity Is Necessary for Oncogenic Transcription in KMT2A-Rearranged Acute Lymphoblastic Leukemia through Enhancer-Promoter Interactions and Epigenetic Modifications at Enhancers

Aberrant enhancer activity is a hallmark of many cancers including KMT2A-rearranged acute lymphoblastic leukemia (ALL), an aggressive leukemia subtype with an event-free survival of 19-45% in infant-ALL (<1 year of age) and 50-59% in childhood-ALL cases. Targeting enhancer activity may provide a novel therapeutic strategy for this hard-to-treat disease. Transcription factors are key effectors of enhancer activity. They recognise and bind to specific DNA sequences at enhancers, driving the expression of target genes from these distal loci. MYB is an important hematopoietic transcription factor that is commonly overexpressed in KMT2A-rearranged leukemias and is required for leukemia survival, where loss of MYB has been shown to abrogate the leukemia phenotype. However, the precise mechanism by which MYB contributes to enhancer function, and the stages of enhancer activation at which it acts are unknown. We employed a well-characterized chromatin anchoring system to interrogate MYB function and determine whether it can recruit specific enhancer activities de novo. By fusing the MYB transactivation domain (MYB TA) to the Tet repressor (TetR) DNA binding domain, MYB TA is directed to an array of Tet operator ( TetO) sequences incorporated into a neutral genomic region devoid of native gene regulatory elements. We found that MYB TA alone is sufficient to recruit key enhancer-associated proteins, including P300, Brd4 and Mediator to the TetO locus. We observed MYB TA-dependent deposition of H3K27ac, increased chromatin accessibility and transcription not just at the TetO locus, but remarkably, also at regions more than 50kb away. Using the chromosome conformation capture (3C) technique NG Capture-C, we observed MYB TA-induced DNA interactions between TetO and the distal sites of transcription, as well as more long-range interactions up to 400kb distal to where MYB TA is bound. Together, these observations suggest that MYB TA binding alone is sufficient to establish an enhancer-like regulatory element, which activates distant cryptic promoters. This activity is dependent on the continued presence of MYB TA, as disruption of MYB TA binding results in a rapid loss of transcription and acquired chromatin features, implicating MYB in enhancer maintenance as well as initiation. To confirm our observations in a disease context, we tested the requirement for MYB at endogenous enhancers in SEM cells, a model of KMT2A-rearranged ALL. By stably introducing a degradation tag (FKBP12 F36V) to the endogenous MYB gene, we were able to rapidly induce protein degradation and observe the immediate effects on chromatin features. Using transient transcriptome sequencing, we noted significant transcriptional downregulation of known MYB target genes, including BCL2, MYC, LMO4 and CDK6. We also observed decreases in enhancer H3K27ac levels and eRNA transcription, most marked at MYB-bound loci. To investigate the requirement for MYB in maintaining physical proximity between enhancer and promoter, we performed the base pair-resolution 3C technique Micro-Capture-C (MCC). MCC revealed highly punctate interactions between enhancer elements and their target promoters. At BCL2 and MYC, MYB degradation results in a reduction in interactions with the promoter at discrete regions within the enhancers. This argues for highly localised MYB-dependent activity at enhancers, driving interaction with and activation of distal target promoters. Overall, our data argue for a role for MYB in establishing enhancers de novo, indicating that MYB overexpression may directly result in onco-enhancer activity at key genes in vivo. At a subset of MYB-bound enhancers, the continued presence of MYB is absolutely required to maintain enhancer activity and oncogene upregulation. Further exploration of associated co-factors that mediate MYB-dependent enhancer activity may identify novel targets for therapeutic exploitation.

Dynamic Expression of Erg Controls Fetal-to-Adult Maturation of the Hematopoietic System

Many blood diseases are biased in their age of onset toward infancy, childhood, or mature adulthood. This occurs in concert with underlying changes in the properties of hematopoietic stem and progenitor cells (HSPCs) over the course of development, maturation, and aging that tailor the output of effector blood cells to age-specific physiology. Understanding how blood formation changes over the course of a lifetime would form a foundation upon which to investigate age-related determinants of manifestations of blood diseases. A growing body of work has shown that the heterochronic Lin28b/ let-7 switch defines fetal versus adult definitive HSPC states. In previous work, we found that the Polycomb repressive complex 1 component Cbx2 is a downstream target of let-7 that functions to specify the temporal maturation state of the hematopoietic system. In our current study, we find that Cbx2 represses the master hematopoietic stem cell (HSC) transcription factor (TF) Erg in fetal HSPCs, providing a mechanism by which the Lin28b/ let-7 axis integrates with the core HSC TF circuitry. During developmental maturation, Erg is upregulated during the fetal-to-adult HSC transition in humans (5.5-fold), mice (3.8-fold), and zebrafish (4.9-fold). Moreover, by analyzing existing ATAC-seq datasets, we find that Erg motifs are highly enriched in chromatin regions specifically active in adult relative to fetal HSPCs (P < 0.0001). Together, these findings suggest age-biased role(s) for Erg in hematopoiesis. We therefore hypothesized that since Erg is a key regulator of self-renewal and lineage output in HSCs, Erg likely serves stage-specific roles in HSCs. To test this, we either suppressed Erg expression in adult HSPCs or ectopically activated Erg in fetal HSPCs to determine consequences on HSPC function. We found that ectopic expression of Erg in HSPCs reprogrammed to the fetal state by expression of LIN28B resulted in blunting of fetal-like lineage biases. In adult mice, loss of a gene dose of Erg resulted in persistence of hallmarks of the fetal HSC state among blood effector cells and fetal-like HSPC distributions into maturity. During the normal process of fetal-to-adult maturation, HSCs transition from a highly proliferative to a quiescent state. We find that early postnatal Erg +/- HSCs do not effectively transition to quiescence and maintain active fetal-like self-renewal, which is leads to impairment in HSC function and susceptibility to replicative exhaustion later in adulthood. Mechanistically, we find that adult Erg +/- HSPCs ectopically maintain fetal-like gene expression profiles and patterns of promoter-enhancer looping. This is associated with failure to repress self-renewal promoting TFs that are normally expressed only in fetal HSPCs. Together, these findings demonstrate that master hematopoietic TFs and their associated networks are tightly titrated to implement age-appropriate rates self-renewal and mature cell output in HSPCs. Our results confirm heterochronic roles for Erg in defining age-tailored hematopoiesis on schedule with normal development and maturation and form a basis for applying this understanding of temporal hematopoietic maturation to disease models.

Abnormalities in red blood cell production and pathogenesis of anemia in the progression of rock bream iridovirus (RBIV)

Rock bream iridovirus (RBIV), belonging to Megalocytivirus, causes severe mortality in rock bream. Almost all deaths associated with RBIV are accompanied by splenic enlargement and anemia. Although red blood cells (RBCs) are involved in the immune response against viral infections, their involvement in rock bream has not yet been studied in terms of the immune response against RBIV. In this study, the viral replication patterns, blood characteristics and anemia-related factors were evaluated in rock bream post RBIV infection. The virus-infected RBCs of rock bream demonstrated similarities in the expression levels of hemoglobins (HGB) (α and β), cytokine-dependent hematopoietic cell linker (CLNK) and hematopoietic transcription factor GATA (GATA), with significantly decreasing levels from 4 days post infection (dpi) to 17 (dpi), when the viral replication was at its peak. This suggests that the expression of blood-related genes is inadequate for HGB synthesis and RBC production, thereby causing anemia leading to death. Moreover, the levels of complete blood cell count (CBC) indicators, such as RBCs, HGB and hematocrit (HCT), significantly decreased from 10 to 17 dpi. This phenomenon suggests that blood-related gene expression and/or RBC-, HGB- and HCT-related levels are critical factors in RBIV-induced anemia and disease progression. These results highlight the significance of blood-mediated immune responses against RBIV infection in rock bream. Understanding blood-related gene levels to identify blood-related immune response interactions in rock bream will be useful for development of future strategies in controlling RBIV diseases in rock bream.

Hematopoietic Transcription Factor RUNX1 is Essential for Promoting Macrophage-Myofibroblast Transition in Non-Small-Cell Lung Carcinoma.

Macrophage-myofibroblast transition (MMT) is a newly discovered pathway for mass production of pro-tumoral cancer-associated fibroblasts (CAFs) in non-small cell lung carcinoma (NSCLC) in a TGF-β1/Smad3 dependent manner. Better understanding its regulatory signaling in tumor microenvironment (TME) may identify druggable target for the development of precision medicine. Here, by dissecting the transcriptome dynamics of tumor-associated macrophage at single-cell resolution, a crucial role of a hematopoietic transcription factor Runx1 in MMT formation is revealed. Surprisingly, integrative bioinformatic analysis uncovers Runx1 as a key regulator in the downstream of MMT-specific TGF-β1/Smad3 signaling. Stromal Runx1 level positively correlates with the MMT-derived CAF abundance and mortality in NSCLC patients. Mechanistically, macrophage-specific Runx1 promotes the transcription of genes related to CAF signatures in MMT cells at genomic level. Importantly, macrophage-specific genetic deletion and systemic pharmacological inhibition of TGF-β1/Smad3/Runx1 signaling effectively prevent MMT-driven CAF and tumor formation in vitro and in vivo, representing a potential therapeutic target for clinical NSCLC.

A transcriptional network governing ceramide homeostasis establishes a cytokine-dependent developmental process

Transcriptional mechanisms controlling developmental processes establish and maintain proteomic networks, which can govern the levels of intracellular small molecules. Although dynamic changes in bioactive small molecules can link transcription factor and genome activity with cell state transitions, many mechanistic questions are unresolved. Using quantitative lipidomics and multiomics, we discover that the hematopoietic transcription factor GATA1 establishes ceramide homeostasis during erythroid differentiation by regulating genes encoding sphingolipid metabolic enzymes. Inhibiting a GATA1-induced sphingolipid biosynthetic enzyme, delta(4)-desaturase, or disrupting ceramide homeostasis with cell-permeable dihydroceramide or ceramide is detrimental to erythroid, but not myeloid, progenitor activity. Coupled with genetic editing-based rewiring of the regulatory circuitry, we demonstrate that ceramide homeostasis commissions vital stem cell factor and erythropoietin signaling by opposing an inhibitory protein phosphatase 2A-dependent, dual-component mechanism. Integrating bioactive lipids as essential components of GATA factor mechanisms to control cell state transitions has implications for diverse cell and tissue types.

BloodChIP Xtra: an expanded database of comparative genome-wide transcription factor binding and gene-expression profiles in healthy human stem/progenitor subsets and leukemic cells.

The BloodChIP Xtra database (http://bloodchipXtra.vafaeelab.com/) facilitates genome-wide exploration and visualization of transcription factor (TF) occupancy and chromatin configuration in rare primary human hematopoietic stem (HSC-MPP) and progenitor (CMP, GMP, MEP) cells and acute myeloid leukemia (AML) cell lines (KG-1, ME-1, Kasumi1, TSU-1621-MT), along with chromatin accessibility and gene expression data from these and primary patient AMLs. BloodChIP Xtra features significantly more datasets than our earlier database BloodChIP (two primary cell types and two cell lines). Improved methodologies for determining TF occupancy and chromatin accessibility have led to increased availability of data for rare primary cell types across the spectrum of healthy and AML hematopoiesis. However, there is a continuing need for these data to be integrated in an easily accessible manner for gene-based queries and use in downstream applications. Here, we provide a user-friendly database based around genome-wide binding profiles of key hematopoietic TFs and histone marks in healthy stem/progenitor cell types. These are compared with binding profiles and chromatin accessibility derived from primary and cell line AML and integrated with expression data from corresponding cell types. All queries can be exported to construct TF-gene and protein-protein networks and evaluate the association of genes with specific cellular processes.

Differential transcriptional control of hematopoiesis in congenital and cyclic neutropenia patients harboring ELANE mutations.

Mutations in the ELANE gene, encoding the neutrophil elastase (NE) protein, are responsible for most cyclic neutropenia (CyN) cases and approximately 25% of congenital neutropenia (CN) cases. In CN and in CyN, a median of 2.8% of CD34+ cells were early CD49f+ hematopoietic stem cells (eHSC) that did not express ELANE and thus escape from the unfolded protein response (UPR) caused by mutated NE. In CyN, the CD49f+ cells respond to granulocyte colony-stimulating factor (G-CSF) with a significant upregulation of the hematopoietic stem cell-specific transcription factors, C/EBPα, MLL1, HOXA9, MEIS1, and HLF during the ascending arm of the cycle, resulting in the differentiation of myeloid cells to mature neutrophils at the cycle peak. However, NE protein released by neutrophils at the cycle's peak caused a negative feedback loop on granulopoiesis through the proteolytic digestion of G-CSF. In contrast, in CN patients, CD49f+ cells failed to express mRNA levels of HSC-specific transcription factors mentioned above. Rescue of C/EBPα expression in CN restored granulopoiesis.

Single-cell RNA sequencing of a new transgenic t(8;21) preleukemia mouse model reveals regulatory networks promoting leukemic transformation.

T(8;21)(q22;q22), which generates the AML1-ETO fusion oncoprotein, is a common chromosomal abnormality in acute myeloid leukemia (AML) patients. Despite having favorable prognosis, 40% of patients will relapse, highlighting the need for innovative models and application of the newest technologies to study t(8;21) leukemogenesis. Currently, available AML1-ETO mouse models have limited utility for studying the pre-leukemic stage because AML1-ETO produces mild hematopoietic phenotypes and no leukemic transformation. Conversely, overexpression of a truncated variant, AML1-ETO9a (AE9a), promotes fully penetrant leukemia and is too potent for studying pre-leukemic changes. To overcome these limitations, we devised a germline-transmitted Rosa26 locus AE9a knock-in mouse model that moderately overexpressed AE9a and developed leukemia with long latency and low penetrance. We observed pre-leukemic alterations in AE9a mice, including skewing of progenitors towards granulocyte/monocyte lineages and replating of stem and progenitor cells. Next, we performed single-cell RNA sequencing to identify specific cell populations that contribute to these pre-leukemic phenotypes. We discovered a subset of common myeloid progenitors that have heightened granulocyte/monocyte bias in AE9a mice. We also observed dysregulation of key hematopoietic transcription factor target gene networks, blocking cellular differentiation. Finally, we identified Sox4 activation as a potential contributor to stem cell self-renewal during the pre-leukemic stage.