Imaging Flow Cytometry and Convolutional Neural Network-Based Classification Enable Discrimination of Hematopoietic and Leukemic Stem Cells in Acute Myeloid Leukemia.

Acute myeloid leukemia (AML) is a heterogenous blood cancer with a dismal prognosis. It emanates from leukemic stem cells (LSCs) arising from the genetic transformation of hematopoietic stem cells (HSCs). LSCs hold prognostic value, but their molecular and immunophenotypic heterogeneity poses challenges: there is no single marker for identifying all LSCs across AML samples. We hypothesized that imaging flow cytometry (IFC) paired with artificial intelligence-driven image analysis could visually distinguish LSCs from HSCs based solely on morphology. Initially, a seven-color IFC panel was employed to immunophenotypically identify LSCs and HSCs in bone marrow samples from five AML patients and ten healthy donors, respectively. Next, we developed convolutional neural network (CNN) models for HSC-LSC discrimination using brightfield (BF), side scatter (SSC), and DNA images. Classification using only BF images achieved 86.96% accuracy, indicating significant morphological differences. Accuracy increased to 93.42% when combining BF with DNA images, highlighting differences in nuclear morphology, although DNA images alone were inadequate for accurate HSC-LSC discrimination. Model development using SSC images revealed minor granularity differences. Performance metrics varied substantially between AML patients, indicating considerable morphologic variations among LSCs. Overall, we demonstrate proof-of-concept results for accurate CNN-based HSC-LSC differentiation, instigating the development of a novel technique within AML monitoring.

The hematopoietic microenvironment of the fetal liver and transient abnormal myelopoiesis associated with Down syndrome: A review.

Transient abnormal myelopoiesis (TAM) in neonates with Down syndrome is a distinct form of leukemia or preleukemia that mirrors the hematological features of acute megakaryoblastic leukemia. However, it typically resolves spontaneously in the early stages. TAM originates from fetal liver (FL) hematopoietic precursor cells and emerges due to somatic mutations in GATA1 in utero. In TAM, progenitor cells proliferate and differentiate into mature megakaryocytes and granulocytes. This process occurs both in vitro, aided by hematopoietic growth factors (HGFs) produced in the FL, and in vivo, particularly in specific anatomical sites like the FL and blood vessels. The FL's hematopoietic microenvironment plays a crucial role in TAM's pathogenesis and may contribute to its spontaneous regression. This review presents an overview of current knowledge regarding the unique features of TAM in relation to the FL hematopoietic microenvironment, focusing on the functions of HGFs and the pathological features of TAM.

Mapping genotypes to chromatin accessibility profiles in single cells.

In somatic tissue differentiation, chromatin accessibility changes govern priming and precursor commitment towards cellular fates1-3. Therefore, somatic mutations are likely to alter chromatin accessibility patterns, as they disrupt differentiation topologies leading to abnormal clonal outgrowth. However, defining the impact of somatic mutations on the epigenome in human samples is challenging due to admixed mutated and wild-type cells. Here, to chart how somatic mutations disrupt epigenetic landscapes in human clonal outgrowths, we developed genotyping of targeted loci with single-cell chromatin accessibility (GoT-ChA). This high-throughput platform links genotypes to chromatin accessibility at single-cell resolution across thousands of cells within a single assay. We applied GoT-ChA to CD34+ cells from patients with myeloproliferative neoplasms with JAK2V617F-mutated haematopoiesis. Differential accessibility analysis between wild-type and JAK2V617F-mutant progenitors revealed both cell-intrinsic and cell-state-specific shifts within mutant haematopoietic precursors, including cell-intrinsic pro-inflammatory signatures in haematopoietic stem cells, and a distinct profibrotic inflammatory chromatin landscape in megakaryocytic progenitors. Integration of mitochondrial genome profiling and cell-surface protein expression measurement allowed expansion of genotyping onto DOGMA-seq through imputation, enabling single-cell capture of genotypes, chromatin accessibility, RNA expression and cell-surface protein expression. Collectively, we show that the JAK2V617F mutation leads to epigenetic rewiring in a cell-intrinsic and cell type-specific manner, influencing inflammation states and differentiation trajectories. We envision that GoT-ChA will empower broad future investigations of the critical link between somatic mutations and epigenetic alterations across clonal populations in malignant and non-malignant contexts.

Age-specific induction of mutant p53 drives clonal hematopoiesis and acute myeloid leukemia in adult mice.

The investigation of the mechanisms behind p53 mutations in acute myeloid leukemia (AML) has been limited by the lack of suitable mouse models, which historically have resulted in lymphoma rather than leukemia. This study introduces two new AML mouse models. One model induces mutant p53 and Mdm2 haploinsufficiency in early development, showing the role of Mdm2 in myeloid-biased hematopoiesis and AML predisposition, independent of p53. The second model mimics clonal hematopoiesis by inducing mutant p53 in adult hematopoietic stem cells, demonstrating that the timing of p53 mutation determines AML vs. lymphoma development. In this context, age-related changes in hematopoietic stem cells (HSCs) collaborate with mutant p53 to predispose toward myeloid transformation rather than lymphoma development. Our study unveils new insights into the cooperative impact of HSC age, Trp53 mutations, and Mdm2 haploinsufficiency on clonal hematopoiesis and the development of myeloid malignancies.

Hemangiosarcoma Cells Promote Conserved Host-derived Hematopoietic Expansion.

Hemangiosarcoma and angiosarcoma are soft-tissue sarcomas of blood vessel-forming cells in dogs and humans, respectively. These vasoformative sarcomas are aggressive and highly metastatic, with disorganized, irregular blood-filled vascular spaces. Our objective was to define molecular programs which support the niche that enables progression of canine hemangiosarcoma and human angiosarcoma. Dog-in-mouse hemangiosarcoma xenografts recapitulated the vasoformative and highly angiogenic morphology and molecular characteristics of primary tumors. Blood vessels in the tumors were complex and disorganized, and they were lined by both donor and host cells. In a series of xenografts, we observed that the transplanted hemangiosarcoma cells created exuberant myeloid hyperplasia and gave rise to lymphoproliferative tumors of mouse origin. Our functional analyses indicate that hemangiosarcoma cells generate a microenvironment that supports expansion and differentiation of hematopoietic progenitor populations. Furthermore, gene expression profiling data revealed hemangiosarcoma cells expressed a repertoire of hematopoietic cytokines capable of regulating the surrounding stromal cells. We conclude that canine hemangiosarcomas, and possibly human angiosarcomas, maintain molecular properties that provide hematopoietic support and facilitate stromal reactions, suggesting their potential involvement in promoting the growth of hematopoietic tumors. SIGNIFICANCE: We demonstrate that hemangiosarcomas regulate molecular programs supporting hematopoietic expansion and differentiation, providing insights into their potential roles in creating a permissive stromal-immune environment for tumor progression.

Ultraviolet Light-Emitting Diode Radiation Kills Leukemia Cells Without Affecting Hematopoiesis of Hematopoietic Stem Cells in Mice.

OBJECTIVES: The eradication of leukemia cells while sparing hematopoietic stem cells in the graft before autologous hematopoietic stem cell transplant is critical to prevention of leukemia relapse. Proliferating cells have been shown to be more prone to apoptosis than differentiated cells in response to ultraviolet radiation; however, whether leukemia cells are more sensitive to ultraviolet LED radiation than hematopoietic stem cells remains unclear. MATERIALS AND METHODS: We compared the in vitro responses between murine leukemia L1210 cells and murine hematopoietic stem cells to 280-nm ultraviolet LED radiation. We also investigated the effects of ultraviolet LED radiation on the tumorigenic and metastatic capacity of L1210 cells and hematopoietic stem cell hematopoiesis in a mouse model of hematopoietic stem cell transplant. RESULTS: L1210 cells were more sensitive to ultraviolet LED radiation than hematopoietic stem cells in vitro, as evidenced by significantly reduced colony formation rates and cell proliferation rates, along with remarkably increased apoptosis rates in L1210 cells. Compared with corresponding unirradiated cells, ultraviolet LED-irradiated L1210 cells failed to generate palpable tumors in mice, whereas ultraviolet LED-irradiated bone marrow cells restored hematopoiesis in vivo. Furthermore, transplant with an irradiated mixture of L1210 cells and bone marrow cells showed later onset of leukemia, milder leukemic infiltration, and prolonged survival in mice, compared with unirradiated cell transplant. CONCLUSIONS: Our results suggest that ultraviolet LED radiation can suppress the proliferative and tumorigenic abilities of leukemia cells without reducing the hematopoietic reconstitution capacity of hematopoietic stem cells, serving as a promising approach to kill leukemia cells in autograft before autologous hematopoietic stem cell transplant.

Forged in the fire: Lasting impacts of inflammation on hematopoietic progenitors.

Quiescence and differentiation of hematopoietic stem and progenitor cells (HSPC) can be modified by systemic inflammatory cues. Such cues can not only yield short-term changes in HSPCs such as in supporting emergency granulopoiesis but can also promote lasting influences on the HSPC compartment. First, inflammation can be a driver for clonal expansion, promoting clonal hematopoiesis for certain mutant clones, reducing overall clonal diversity, and reshaping the composition of the HSPC pool with significant health consequences. Second, inflammation can generate lasting cell-autonomous changes in HSPCs themselves, leading to changes in the epigenetic state, metabolism, and function of downstream innate immune cells. This concept, termed "trained immunity," suggests that inflammatory stimuli can alter subsequent immune responses leading to improved innate immunity or, conversely, autoimmunity. Both of these concepts have major implications in human health. Here we reviewed current literature about the lasting effects of inflammation on the HSPC compartment and opportunities for future advancement in this fast-developing field.

Chromatin organization in myelodysplastic syndrome.

Disordered chromatin organization has emerged as a new aspect of the pathogenesis of myelodysplastic syndrome (MDS). Characterized by lineage dysplasia and a high transformation rate to acute myeloid leukemia (AML), the genetic determinant of MDS is thought to be the main driver of the disease's progression. Among the recurrently mutated pathways, alterations in chromatin organization, such as the cohesin complex, have a profound impact on hematopoietic stem cell (HSC) function and lineage commitment. The cohesin complex is a ring-like structure comprised of structural maintenance of chromosomes (SMC), RAD21, and STAG proteins that involve three-dimensional (3D) genome organization via loop extrusion in mammalian cells. The partial loss of the functional cohesin ring leads to altered chromatin accessibility specific to key hematopoietic transcription factors, which is thought to be the molecular mechanism of cohesin dysfunction. Currently, there are no specific targeting agents for cohesin mutant MDS/AML. Potential therapeutic strategies have been proposed based on the current understanding of cohesin mutant leukemogenesis. Here, we will review the recent advances in investigation and targeting approaches against cohesin mutant MDS/AML.

Lethal and sublethal effects of programmed cell death pathways on hematopoietic stem cells.

Programmed cell death is an evolutionally conserved cellular process in multicellular organisms that eliminates unnecessary or rogue cells during development, infection, and carcinogenesis. Hematopoietic stem cells (HSCs) are a rare, self-renewing, and multipotent cell population necessary for the establishment and regeneration of the hematopoietic system. Counterintuitively, key components necessary for programmed cell death induction are abundantly expressed in long-lived HSCs, which often survive myeloablative stress by engaging a prosurvival response that counteracts cell death-inducing stimuli. Although HSCs are well known for their apoptosis resistance, recent studies have revealed their unique vulnerability to certain types of programmed necrosis, such as necroptosis and ferroptosis. Moreover, emerging evidence has shown that programmed cell death pathways can be sublethally activated to cause nonlethal consequences such as innate immune response, organelle dysfunction, and mutagenesis. In this review, we summarized recent findings on how divergent cell death programs are molecularly regulated in HSCs. We then discussed potential side effects caused by sublethal activation of programmed cell death pathways on the functionality of surviving HSCs.

Loss of hematopoietic progenitors heterogeneity is an adverse prognostic factor in lower-risk myelodysplastic neoplasms.

Myelodysplastic neoplasms (MDS) are characterized by clonal evolution starting from the compartment of hematopoietic stem and progenitors cells (HSPCs), leading in some cases to leukemic transformation. We hypothesized that deciphering the diversity of the HSPCs compartment may allow for the early detection of an emergent sub-clone that drives disease progression. Deep analysis of HSPCs repartition by multiparametric flow cytometry revealed a strong disorder of the hematopoietic branching system in most patients at diagnosis with different phenotypic signatures closely related to specific MDS features. In two independent cohorts of 131 and 584 MDS, the HSPCs heterogeneity quantified through entropy calculation was decreased in 47% and 46% of cases, reflecting a more advanced state of the disease with deeper cytopenias, higher IPSS-R risk and accumulation of somatic mutations. We demonstrated that patients with lower-risk MDS and low CD34 + CD38+HSPCs entropy had an adverse outcome and that this parameter is as an independent predictive biomarker for progression free survival, leukemia free survival and overall survival. Analysis of HSPCs repartition at diagnosis represents therefore a very powerful tool to identify lower-risk MDS patients with a worse outcome and valuable for clinical decision-making, which could be fully integrated in the MDS diagnostic workflow.

Epigenetics of hematopoietic stem cell aging.

PURPOSE OF REVIEW: The development of new antiaging medicines is of great interest to the current elderly and aging population. Aging of the hematopoietic system is attributed to the aging of hematopoietic stem cells (HSCs), and epigenetic alterations are the key effectors driving HSC aging. Understanding the epigenetics of HSC aging holds promise of providing new insights for combating HSC aging and age-related hematological malignancies. RECENT FINDINGS: Aging is characterized by the progressive loss of physiological integrity, leading to impaired function and increased vulnerability to death. During aging, the HSCs undergo both quantitative and qualitative changes. These functional changes in HSCs cause dysregulated hematopoiesis, resulting in anemia, immune dysfunction, and an increased risk of hematological malignancies. Various cell-intrinsic and cell-extrinsic effectors influencing HSC aging have also been identified. Epigenetic alterations are one such mechanism. SUMMARY: Cumulative epigenetic alterations in aged HSCs affect their fate, leading to aberrant self-renewal, differentiation, and function of aged HSCs. In turn, these factors provide an opportunity for aged HSCs to expand by modulating their self-renewal and differentiation balance, thereby contributing to the development of hematological malignancies.

Myelodysplastic neoplasms dissected into indolent, leukaemic and unfavourable subtypes by computational clustering of haematopoietic stem and progenitor cells.

Myelodysplastic neoplasms (MDS) encompass haematological malignancies, which are characterised by dysplasia, ineffective haematopoiesis and the risk of progression towards acute myeloid leukaemia (AML). Myelodysplastic neoplasms are notorious for their heterogeneity: clinical outcomes range from a near-normal life expectancy to leukaemic transformation or premature death due to cytopenia. The Molecular International Prognostic Scoring System made progress in the dissection of MDS by clinical outcomes. To contribute to the risk stratification of MDS by immunophenotypic profiles, this study performed computational clustering of flow cytometry data of CD34+ cells in 67 MDS, 67 AML patients and 49 controls. Our data revealed heterogeneity also within the MDS-derived CD34+ compartment. In MDS, maintenance of lymphoid progenitors and megakaryocytic-erythroid progenitors predicted favourable outcomes, whereas expansion of granulocyte-monocyte progenitors increased the risk of leukaemic transformation. The proliferation of haematopoietic stem cells and common myeloid progenitors with downregulated CD44 expression, suggestive of impaired haematopoietic differentiation, characterised a distinct MDS subtype with a poor overall survival. This exploratory study demonstrates the prognostic value of known and previously unexplored CD34+ populations and suggests the feasibility of dissecting MDS into a more indolent, a leukaemic and another unfavourable subtype.

Loss of Dnmt3a increases self-renewal and resistance to pegIFN-α in JAK2-V617F-positive myeloproliferative neoplasms.

ABSTRACT: Pegylated interferon alfa (pegIFN-α) can induce molecular remissions in patients with JAK2-V617F-positive myeloproliferative neoplasms (MPNs) by targeting long-term hematopoietic stem cells (LT-HSCs). Additional somatic mutations in genes regulating LT-HSC self-renewal, such as DNMT3A, have been reported to have poorer responses to pegIFN-α. We investigated whether DNMT3A loss leads to alterations in JAK2-V617F LT-HSC functions conferring resistance to pegIFN-α treatment in a mouse model of MPN and in hematopoietic progenitors from patients with MPN. Long-term treatment with pegIFN-α normalized blood parameters and reduced splenomegaly and JAK2-V617F chimerism in single-mutant JAK2-V617F (VF) mice. However, pegIFN-α in VF;Dnmt3aΔ/Δ (VF;DmΔ/Δ) mice worsened splenomegaly and failed to reduce JAK2-V617F chimerism. Furthermore, LT-HSCs from VF;DmΔ/Δ mice compared with VF were less prone to accumulate DNA damage and exit dormancy upon pegIFN-α treatment. RNA sequencing showed that IFN-α induced stronger upregulation of inflammatory pathways in LT-HSCs from VF;DmΔ/Δ than from VF mice, indicating that the resistance of VF;DmΔ/Δ LT-HSC was not due to failure in IFN-α signaling. Transplantations of bone marrow from pegIFN-α-treated VF;DmΔ/Δ mice gave rise to more aggressive disease in secondary and tertiary recipients. Liquid cultures of hematopoietic progenitors from patients with MPN with JAK2-V617F and DNMT3A mutation showed increased percentages of JAK2-V617F-positive colonies upon IFN-α exposure, whereas in patients with JAK2-V617F alone, the percentages of JAK2-V617F-positive colonies decreased or remained unchanged. PegIFN-α combined with 5-azacytidine only partially overcame resistance in VF;DmΔ/Δ mice. However, this combination strongly decreased the JAK2-mutant allele burden in mice carrying VF mutation only, showing potential to inflict substantial damage preferentially to the JAK2-mutant clone.

HSPA9/mortalin inhibition disrupts erythroid maturation through a TP53-dependent mechanism in human CD34+ hematopoietic progenitor cells.

Myelodysplastic syndromes (MDS) are a heterogeneous group of clonal hematopoietic stem cell malignancies characterized by abnormal hematopoietic cell maturation, increased apoptosis of bone marrow cells, and anemia. They are the most common myeloid blood cancers in American adults. The full complement of gene mutations that contribute to the phenotypes or clinical symptoms in MDS is not fully understood. Around 10%-25% of MDS patients harbor an interstitial heterozygous deletion on the long arm of chromosome 5 [del(5q)], creating haploinsufficiency for a large set of genes, including HSPA9. The HSPA9 gene encodes for the protein mortalin, a highly conserved heat shock protein predominantly localized in mitochondria. Our prior study showed that knockdown of HSPA9 induces TP53-dependent apoptosis in human CD34+ hematopoietic progenitor cells. In this study, we explored the role of HSPA9 in regulating erythroid maturation using human CD34+ cells. We inhibited the expression of HSPA9 using gene knockdown and pharmacological inhibition and found that inhibition of HSPA9 disrupted erythroid maturation as well as increased expression of p53 in CD34+ cells. To test whether the molecular mechanism of HSPA9 regulating erythroid maturation is TP53-dependent, we knocked down HSPA9 and TP53 individually or in combination in human CD34+ cells. We found that the knockdown of TP53 partially rescued the erythroid maturation defect induced by HSPA9 knockdown, suggesting that the defect in cells with reduced HSPA9 expression is TP53-dependent. Collectively, these findings indicate that reduced levels of HSPA9 may contribute to the anemia observed in del(5q)-associated MDS patients due to the activation of TP53.

Phenotypic Analysis of Hematopoietic Stem and Progenitor Cell Populations in Acute Myeloid Leukemia Based on Spectral Flow Cytometry, a 20-Color Panel, and Unsupervised Learning Algorithms.

The analysis of hematopoietic stem and progenitor cell populations (HSPCs) is fundamental in the understanding of normal hematopoiesis as well as in the management of malignant diseases, such as leukemias, and in their diagnosis and follow-up, particularly the measurement of treatment efficiency with the detection of measurable residual disease (MRD). In this study, I designed a 20-color flow cytometry panel tailored for the comprehensive analysis of HSPCs using a spectral cytometer. My investigation encompassed the examination of forty-six samples derived from both normal human bone marrows (BMs) and patients with acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS) along with those subjected to chemotherapy and BM transplantation. By comparing my findings to those obtained through conventional flow cytometric analyses utilizing multiple tubes, I demonstrate that my innovative 20-color approach enables a more in-depth exploration of HSPC subpopulations and the detection of MRD with at least comparable sensitivity. Furthermore, leveraging advanced analytical tools such as t-SNE and FlowSOM learning algorithms, I conduct extensive cross-sample comparisons with two-dimensional gating approaches. My results underscore the efficacy of these two methods as powerful unsupervised alternatives for manual HSPC subpopulation analysis. I expect that in the future, complex multi-dimensional flow cytometric data analyses, such as those employed in this study, will be increasingly used in hematologic diagnostics.

Ferritin-mediated mitochondrial iron homeostasis is essential for the survival of hematopoietic stem cells and leukemic stem cells.

Iron metabolism plays a crucial role in cell viability, but its relationship with adult stem cells and cancer stem cells is not fully understood. The ferritin complex, responsible for intracellular iron storage, is important in this process. We report that conditional deletion of ferritin heavy chain 1 (Fth1) in the hematopoietic system reduced the number and repopulation capacity of hematopoietic stem cells (HSCs). These effects were associated with a decrease in cellular iron level, leading to impaired mitochondrial function and the initiation of apoptosis. Iron supplementation, antioxidant, and apoptosis inhibitors reversed the reduced cell viability of Fth1-deleted hematopoietic stem and progenitor cells (HSPCs). Importantly, leukemic stem cells (LSCs) derived from MLL-AF9-induced acute myeloid leukemia (AML) mice exhibited reduced Fth1 expression, rendering them more susceptible to apoptosis induced by the iron chelation compared to normal HSPCs. Modulating FTH1 expression using mono-methyl fumarate increased LSCs resistance to iron chelator-induced apoptosis. Additionally, iron supplementation, antioxidant, and apoptosis inhibitors protected LSCs from iron chelator-induced cell death. Fth1 deletion also extended the survival of AML mice. These findings unveil a novel mechanism by which ferritin-mediated iron homeostasis regulates the survival of both HSCs and LSCs, suggesting potential therapeutic strategies for blood cancer with iron dysregulation.

Spatial Mapping of Hematopoietic Clones in Human Bone Marrow.

Clonal hematopoiesis (CH) is the expansion of somatically mutated cells in the hematopoietic compartment of individuals without hematopoietic dysfunction. Large CH clones (i.e., >2% variant allele fraction) predispose to hematologic malignancy, but CH is detected at lower levels in nearly all middle-aged individuals. Prior work has extensively characterized CH in peripheral blood, but the spatial distribution of hematopoietic clones in human bone marrow is largely undescribed. To understand CH at this level, we developed a method for spatially aware somatic mutation profiling and characterized the bone marrow of a patient with polycythemia vera. We identified the complex clonal distribution of somatic mutations in the hematopoietic compartment, the restriction of somatic mutations to specific subpopulations of hematopoietic cells, and spatial constraints of these clones in the bone marrow. This proof of principle paves the way to answering fundamental questions regarding CH spatial organization and factors driving CH expansion and malignant transformation in the bone marrow. SIGNIFICANCE: CH occurs commonly in humans and can predispose to hematologic malignancy. Although well characterized in blood, it is poorly understood how clones are spatially distributed in the bone marrow. To answer this, we developed methods for spatially aware somatic mutation profiling to describe clonal heterogeneity in human bone marrow. See related commentary by Austin and Aifantis, p. 139.

Zeb1 maintains long-term adult hematopoietic stem cell function and extramedullary hematopoiesis.

Emerging evidence implicates the epithelial-mesenchymal transition transcription factor Zeb1 as a critical regulator of hematopoietic stem cell (HSC) differentiation. Whether Zeb1 regulates long-term maintenance of HSC function remains an open question. Using an inducible Mx-1-Cre mouse model that deletes conditional Zeb1 alleles in the adult hematopoietic system, we found that mice engineered to be deficient in Zeb1 for 32 weeks displayed expanded immunophenotypically defined adult HSCs and multipotent progenitors associated with increased abundance of lineage-biased/balanced HSC subsets and augmented cell survival characteristics. During hematopoietic differentiation, persistent Zeb1 loss increased B cells in the bone marrow and spleen and decreased monocyte generation in the peripheral blood. In competitive transplantation experiments, we found that HSCs from adult mice with long-term Zeb1 deletion displayed a cell autonomous defect in multilineage differentiation capacity. Long-term Zeb1 loss perturbed extramedullary hematopoiesis characterized by increased splenic weight and a paradoxical reduction in splenic cellularity that was accompanied by HSC exhaustion, lineage-specific defects, and an accumulation of aberrant, preleukemic like c-kit+CD16/32+ progenitors. Loss of Zeb1 for up to 42 weeks can lead to progressive splenomegaly and an accumulation of Gr-1+Mac-1+ cells, further supporting the notion that long-term expression of Zeb1 suppresses preleukemic activity. Thus, sustained Zeb1 deletion disrupts HSC functionality in vivo and impairs regulation of extramedullary hematopoiesis with potential implications for tumor suppressor functions of Zeb1 in myeloid neoplasms.