Hematopoietic Stem Cell Pool Research Articles

HSPCs display within-family homogeneity in differentiation and proliferation despite population heterogeneity.

High-throughput single-cell methods have uncovered substantial heterogeneity in the pool of hematopoietic stem and progenitor cells (HSPCs), but how much instruction is inherited by offspring from their heterogeneous ancestors remains unanswered. Using a method that enables simultaneous determination of common ancestor, division number, and differentiation status of a large collection of single cells, our data revealed that murine cells that derived from a common ancestor had significant similarities in their division progression and differentiation outcomes. Although each family diversifies, the overall collection of cell types observed is composed of homogeneous families. Heterogeneity between families could be explained, in part, by differences in ancestral expression of cell surface markers. Our analyses demonstrate that fate decisions of cells are largely inherited from ancestor cells, indicating the importance of common ancestor effects. These results may have ramifications for bone marrow transplantation and leukemia, where substantial heterogeneity in HSPC behavior is observed.

A latent subset of human hematopoietic stem cells resists regenerative stress to preserve stemness.

Continuous supply of immune cells throughout life relies on the delicate balance in the hematopoietic stem cell (HSC) pool between long-term maintenance and meeting the demands of both normal blood production and unexpected stress conditions. Here we identified distinct subsets of human long-term (LT)-HSCs that responded differently to regeneration-mediated stress: an immune checkpoint ligand CD112lo subset that exhibited a transient engraftment restraint (termed latency) before contributing to hematopoietic reconstitution and a primed CD112hi subset that responded rapidly. This functional heterogeneity and CD112 expression are regulated by INKA1 through direct interaction with PAK4 and SIRT1, inducing epigenetic changes and defining an alternative state of LT-HSC quiescence that serves to preserve self-renewal and regenerative capacity upon regeneration-mediated stress. Collectively, our data uncovered the molecular intricacies underlying HSC heterogeneity and self-renewal regulation and point to latency as an orchestrated physiological response that balances blood cell demands with preserving a stem cell reservoir.

Pparγ1 Facilitates ErbB2-Mammary Adenocarcinoma in Mice.

Simple SummaryHER2, which is associated with clinically aggressive disease, is overexpressed in 15–20% of breast cancers (BC). Peroxisome proliferator-activated receptor γ (PPARγ), is expressed in a variety of malignancies. The aim of our study was to determine the function of endogenous Pparγ1 in the onset and progression of mammary tumors induced by ErbB2 in mice. Genetic deletion of Pparγ1 slowed the rate of tumor progression and death from ErbB2-induced mammary tumors. The deletion of Pparγ1 correlated with reduced pro-tumorigenic inflammation. We conclude ErbB2 collaborates with endogenous Pparγ1 in the onset and progression of mammary tumorigenesis.HER2, which is associated with clinically aggressive disease, is overexpressed in 15–20% of breast cancers (BC). The host immune system participates in the therapeutic response of HER2+ breast cancer. Identifying genetic programs that participate in ErbB2-induced tumors may provide the rational basis for co-extinction therapeutic approaches. Peroxisome proliferator-activated receptor γ (PPARγ), which is expressed in a variety of malignancies, governs biological functions through transcriptional programs. Herein, genetic deletion of endogenous Pparγ1 restrained mammary tumor progression, lipogenesis, and induced local mammary tumor macrophage infiltration, without affecting other tissue hematopoietic stem cell pools. Endogenous Pparγ1 induced expression of both an EphA2-Amphiregulin and an inflammatory INFγ and Cxcl5 signaling module, that was recapitulated in human breast cancer. Pparγ1 bound directly to growth promoting and proinflammatory target genes in the context of chromatin. We conclude Pparγ1 promotes ErbB2-induced tumor growth and inflammation and represents a relevant target for therapeutic coextinction. Herein, endogenous Pparγ1 promoted ErbB2-mediated mammary tumor onset and progression. PPARγ1 increased expression of an EGF-EphA2 receptor tyrosine kinase module and a cytokine/chemokine 1 transcriptional module. The induction of a pro-tumorigenic inflammatory state by Pparγ1 may provide the rationale for complementary coextinction programs in ErbB2 tumors.

A comprehensive transcriptome signature of murine hematopoietic stem cell aging

AbstractWe surveyed 16 published and unpublished data sets to determine whether a consistent pattern of transcriptional deregulation in aging murine hematopoietic stem cells (HSC) exists. Despite substantial heterogeneity between individual studies, we uncovered a core and robust HSC aging signature. We detected increased transcriptional activation in aged HSCs, further confirmed by chromatin accessibility analysis. Unexpectedly, using 2 independent computational approaches, we established that deregulated aging genes consist largely of membrane-associated transcripts, including many cell surface molecules previously not associated with HSC biology. We show that Selp (P-selectin), the most consistent deregulated gene, is not merely a marker for aged HSCs but is associated with HSC functional decline. Additionally, single-cell transcriptomics analysis revealed increased heterogeneity of the aged HSC pool. We identify the presence of transcriptionally “young-like” HSCs in aged bone marrow. We share our results as an online resource and demonstrate its utility by confirming that exposure to sympathomimetics or deletion of Dnmt3a/b molecularly resembles HSC rejuvenation or aging, respectively.

PU.1 enforces quiescence and limits hematopoietic stem cell expansion during inflammatory stress.

Hematopoietic stem cells (HSCs) are capable of entering the cell cycle to replenish the blood system in response to inflammatory cues; however, excessive proliferation in response to chronic inflammation can lead to either HSC attrition or expansion. The mechanism(s) that limit HSC proliferation and expansion triggered by inflammatory signals are poorly defined. Here, we show that long-term HSCs (HSCLT) rapidly repress protein synthesis and cell cycle genes following treatment with the proinflammatory cytokine interleukin (IL)-1. This gene program is associated with activation of the transcription factor PU.1 and direct PU.1 binding at repressed target genes. Notably, PU.1 is required to repress cell cycle and protein synthesis genes, and IL-1 exposure triggers aberrant protein synthesis and cell cycle activity in PU.1-deficient HSCs. These features are associated with expansion of phenotypic PU.1-deficient HSCs. Thus, we identify a PU.1-dependent mechanism triggered by innate immune stimulation that limits HSC proliferation and pool size. These findings provide insight into how HSCs maintain homeostasis during inflammatory stress.

Reconciling Flux Experiments for Quantitative Modeling of Normal and Malignant Hematopoietic Stem/Progenitor Dynamics

SummaryHematopoiesis serves as a paradigm for how homeostasis is maintained within hierarchically organized cell populations. However, important questions remain as to the contribution of hematopoietic stem cells (HSCs) toward maintaining steady state hematopoiesis. A number of in vivo lineage labeling and propagation studies have given rise to contradictory interpretations, leaving key properties of stem cell function unresolved. Using processed flow cytometry data coupled with a biology-driven modeling approach, we show that in vivo flux experiments that come from different laboratories can all be reconciled into a single unifying model, even though they had previously been interpreted as being contradictory. We infer from comparative analysis that different transgenic models display distinct labeling efficiencies across a heterogeneous HSC pool, which we validate by marker gene expression associated with HSC function. Finally, we show how the unified model of HSC differentiation can be used to simulate clonal expansion in the early stages of leukemogenesis.

Abstract PS17-58: Pparg1 induces an EGF-EphA2 receptor tyrosine kinase module to promote ErbB2- mammary adenocarcinoma in mice

Abstract ErbB2 is overexpressed in approximately 25% of human breast cancers, associated with clinically aggressive disease. No soluble ligand has been identified and the receptor is regulated by heterodimerization with other ErbB family receptors, including EGFR, and other receptor tyrosine kinases including EphA2. EGFR is activated by seven different growth factors including EGF and Amphiregulin. Downstream signaling modules required for ErbB2 induced tumorigenesis in genetically engineered mouse models (GEMM) include the phosphatidylinositol 3-kinase/Akt (PKB) pathway, the Ras/Raf/MEK/ERK1/2 pathway and the phospholipase C (PLCγ) pathways. ErbB2-mediated tumorigenesis involves activation of receptor tyrosine kinases, induction of cyclin D1/CDK activity, and functional restraint by tumor suppressors. The receptor tyrosine kinase EPH receptor A2 (EphA2), a member of the Eph RTK family, is overexpressed in aggressive breast cancer and EphA2 forms a complex with ErbB2 thereby enhancing ErbB2-induced tumor onset and progression. The host immune system participates in the therapeutic response of HER2+ breast cancer. The tumor microenvironment (TME) is regulated by chemokines and their G protein coupled receptors binds several ligands, including Cxcl5 which binds Cxcr2, to augment the pro-tumor immune response, tumor growth and metastasis. Identifying genetic programs that participate in ErbB2-induced tumors may provide the rational basis for co-extinction therapeutic approaches. Peroxisome proliferator-activated receptor γ (PPARγ), which is expressed in a variety of malignancies, governs biological functions through transcriptional programs. Herein, genetic deletion of endogenous Pparγ1 restrained mammary tumor progression, lipogenesis, and induced local mammary tumor F4/80+ tumor-associated macrophage infiltration, without affecting other tissue hematopoietic stem cell pools. Pparγ1 induced peroxisomal target genes in the mammary tumors as evidenced by increased expression of PEX-11, together with PPARGC1 and ESRR induced regulator, muscle 1, Perm1 (PGC-1 and ERR-induced Regulator in Muscle 1). Peroxisomes induced by Pparγ1, activated Type1 interferons (IFNs) and IFN-stimulated gene expression, including Cxcl5. Endogenous Pparγ1 induced expression of both an EphA2-Amphiregulin and an inflammatory INFγ -Cxcl5 signaling module. Pparγ1 bound directly to growth promoting and proinflammatory target genes in the context of chromatin. We conclude Pparγ1 promotes ErbB2-induced tumor growth and inflammation and represents a relevant target for therapeutic coextinction. Citation Format: Richard G Pestell, Xuanmao Jiao, Andrew V Kossenkov, Adam Ertel, Wei Tong, Zhao Zhang, Peter A McCue. Pparg1 induces an EGF-EphA2 receptor tyrosine kinase module to promote ErbB2- mammary adenocarcinoma in mice [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS17-58.

Nbs1-mediated DNA damage repair pathway regulates haematopoietic stem cell development and embryonic haematopoiesis.

ObjectivesDNA damages pose threats to haematopoietic stem cells (HSC) maintenance and haematopoietic system homeostasis. Quiescent HSCs in adult mouse bone marrow are resistant to DNA damage, while human umbilical cord blood‐derived proliferative HSCs are prone to cell death upon ionizing radiation. Murine embryonic HSCs proliferate in foetal livers and divide symmetrically to generate HSC pool. How murine embryonic HSCs respond to DNA damages is not well‐defined.Materials and methodsMice models with DNA repair molecule Nbs1 or Nbs1/p53 specifically deleted in embryonic HSCs were generated. FACS analysis, in vitro and in vivo HSC differentiation assays, qPCR, immunofluorescence and Western blotting were used to delineate roles of Nbs1‐p53 signaling in HSCs and haematopoietic progenitors.ResultsNbs1 deficiency results in persistent DNA breaks in embryonic HSCs, compromises embryonic HSC development and finally results in mouse perinatal lethality. The persistent DNA breaks in Nbs1 deficient embryonic HSCs render cell cycle arrest, while driving a higher rate of cell death in haematopoietic progenitors. Although Nbs1 deficiency promotes Atm‐Chk2‐p53 axis activation in HSCs and their progenies, ablation of p53 in Nbs1 deficient HSCs accelerates embryonic lethality.ConclusionsOur study discloses that DNA double‐strand repair molecule Nbs1 is essential in embryonic HSC development and haematopoiesis. Persistent DNA damages result in distinct cell fate in HSCs and haematopoietic progenitors. Nbs1 null HSCs tend to be maintained through cell cycle arrest, while Nbs1 null haematopoietic progenitors commit cell death. The discrepancies are mediated possibly by different magnitude of p53 signaling.

JMJD6 promotes self-renewal and regenerative capacity of hematopoietic stem cells

AbstractLifelong multilineage hematopoiesis critically depends on rare hematopoietic stem cells (HSCs) that reside in the hypoxic bone marrow microenvironment. Although the role of the canonical oxygen sensor hypoxia-inducible factor prolyl hydroxylase has been investigated extensively in hematopoiesis, the functional significance of other members of the 2-oxoglutarate (2-OG)-dependent protein hydroxylase family of enzymes remains poorly defined in HSC biology and multilineage hematopoiesis. Here, by using hematopoietic-specific conditional gene deletion, we reveal that the 2-OG–dependent protein hydroxylase JMJD6 is essential for short- and long-term maintenance of the HSC pool and multilineage hematopoiesis. Additionally, upon hematopoietic injury, Jmjd6-deficient HSCs display a striking failure to expand and regenerate the hematopoietic system. Moreover, HSCs lacking Jmjd6 lose multilineage reconstitution potential and self-renewal capacity upon serial transplantation. At the molecular level, we found that JMJD6 functions to repress multiple processes whose downregulation is essential for HSC integrity, including mitochondrial oxidative phosphorylation (OXPHOS), protein synthesis, p53 stabilization, cell cycle checkpoint progression, and mTORC1 signaling. Indeed, Jmjd6-deficient primitive hematopoietic cells display elevated basal and maximal mitochondrial respiration rates and increased reactive oxygen species (ROS), prerequisites for HSC failure. Notably, an antioxidant, N-acetyl-l-cysteine, rescued HSC and lymphoid progenitor cell depletion, indicating a causal impact of OXPHOS-mediated ROS generation upon Jmjd6 deletion. Thus, JMJD6 promotes HSC maintenance and multilineage differentiation potential by suppressing fundamental pathways whose activation is detrimental for HSC function.

Deficiency of Rbpj Leads to Defective Stress-Induced Hematopoietic Stem Cell Functions and Hif Mediated Activation of Non-canonical Notch Signaling Pathways.

Deregulated notch signaling has been associated with human pathobiology. However, functions of notch pathways in hematopoiesis remain incompletely understood. Here, we ablated canonical notch pathways, through genetic deletion of Rbpj, in hematopoietic stem cells (HSCs). Our data identified that loss of canonical notch results in normal adult HSC pool, at steady state conditions. However, HSC maintenance and functions in response to radiation-, chemotherapy-, and cytokine- induced stress were compromised in the absence of canonical notch. Rbpj deficient HSCs exhibit decreased proliferation rates and elevated expression of p57Kip2. Surprisingly, loss of Rbpj resulted in upregulation of key notch target genes and augmented binding of Hes1 to p57 and Gata2 promoters. Further molecular analyses identified an increase in notch activity, elevated expression and nuclear translocation of Hif proteins, and augmented binding of Hif1α to Hes1 promoter in the absence of Rbpj. These studies, for the first time, identify a previously unknown role for non-canonical notch signaling and establish a functional link between Hif and Notch pathways in hematopoiesis.

Stable colony-stimulating factor 1 fusion protein treatment increases hematopoietic stem cell pool and enhances their mobilisation in mice

BackgroundPrior chemotherapy and/or underlying morbidity commonly leads to poor mobilisation of hematopoietic stem cells (HSC) for transplantation in cancer patients. Increasing the number of available HSC prior to mobilisation is a potential strategy to overcome this deficiency. Resident bone marrow (BM) macrophages are essential for maintenance of niches that support HSC and enable engraftment in transplant recipients. Here we examined potential of donor treatment with modified recombinant colony-stimulating factor 1 (CSF1) to influence the HSC niche and expand the HSC pool for autologous transplantation.MethodsWe administered an acute treatment regimen of CSF1 Fc fusion protein (CSF1-Fc, daily injection for 4 consecutive days) to naive C57Bl/6 mice. Treatment impacts on macrophage and HSC number, HSC function and overall hematopoiesis were assessed at both the predicted peak drug action and during post-treatment recovery. A serial treatment strategy using CSF1-Fc followed by granulocyte colony-stimulating factor (G-CSF) was used to interrogate HSC mobilisation impacts. Outcomes were assessed by in situ imaging and ex vivo standard and imaging flow cytometry with functional validation by colony formation and competitive transplantation assay.ResultsCSF1-Fc treatment caused a transient expansion of monocyte-macrophage cells within BM and spleen at the expense of BM B lymphopoiesis and hematopoietic stem and progenitor cell (HSPC) homeostasis. During the recovery phase after cessation of CSF1-Fc treatment, normalisation of hematopoiesis was accompanied by an increase in the total available HSPC pool. Multiple approaches confirmed that CD48−CD150+ HSC do not express the CSF1 receptor, ruling out direct action of CSF1-Fc on these cells. In the spleen, increased HSC was associated with expression of the BM HSC niche macrophage marker CD169 in red pulp macrophages, suggesting elevated spleen engraftment with CD48−CD150+ HSC was secondary to CSF1-Fc macrophage impacts. Competitive transplant assays demonstrated that pre-treatment of donors with CSF1-Fc increased the number and reconstitution potential of HSPC in blood following a HSC mobilising regimen of G-CSF treatment.ConclusionThese results indicate that CSF1-Fc conditioning could represent a therapeutic strategy to overcome poor HSC mobilisation and subsequently improve HSC transplantation outcomes.

Thrombopoietin regulates mitochondria homeostasis for hematopoietic stem cell maintenance

Cell cycle quiescence is a fundamental property of hematopoietic stem cells (HSCs). Quiescent HSCs form a healthy pool of cells that serve as a reserve for massive HSC expansion under various conditions of stress. We previously reported that thrombopoietin (THPO) maintains quiescent HSCs and stimulates mitochondrial metabolism, megakaryocyte-lineage differentiation, and proliferation of HSCs. The underlying mechanism by which THPO balances its contradictory effect of promoting proliferation or quiescence on HSCs remains unknown. This review explores the role of THPO signaling in HSC differentiation and quiescence regulation. We present our data, which suggests that a THPO-independent HSC subpopulation sustaining a low mitochondrial metabolic profile reverts to quiescence and regains stem cell potential with external stimuli. There is a possibility that THPO-independent HSCs form a non-quiescent reserve HSC pool from which quiescent HSCs originate in the adult bone marrow.

Chronic lymphocytic leukemia increases the pool of peripheral blood hematopoietic stem cells and skews differentiation.

Key Points CLL increases the pool of ST-HSCs in the peripheral blood. Differentiation of CLL-derived peripheral blood HSPCs is skewed toward the myeloid lineage.

Loss of Nupr1 promotes engraftment by tuning the quiescence threshold of hematopoietic stem cell repository via regulating p53-checkpoint pathway.

Hematopoietic stem cells (HSC) are dominantly quiescent under homeostasis, which is a key mechanism of maintaining the HSC pool for life-long hematopoiesis. Dormant HSC are poised to be immediately activated in certain conditions and can return to quiescence after homeostasis has been regained. At present, the molecular networks of regulating the threshold of HSC dormancy, if existing, remain largely unknown. Here, we show that deletion of Nupr1, a gene preferentially expressed in HSC, activated quiescent HSC under homeostasis, which conferred a competitive engraftment advantage for these HSC without compromising their stemness or multi-lineage differentiation capacity in serial transplantation settings. Following an expansion protocol, the Nupr1-/- HSC proliferated more robustly than their wild-type counterparts in vitro. Nupr1 inhibits the expression of p53 and rescue of this inhibition offsets the engraftment advantage. Our data reveal a new role for Nupr1 as a regulator of HSC quiescence, which provides insights for accelerating the engraftment efficacy of HSC transplantation by targeting the HSC quiescence-controlling network.

Hematopoietic Stem Cells Are Endowed with Erythroid Signature in Beta-Thalassemia

Background Beta-thalassemia (Bthal) is a genetic disorder due to mutations in the ß-globin gene, leading to a reduced or absent production of HbA, which interferes with erythroid cell maturation and limits normal red cell production. Patients are affected by severe anemia, hepatosplenomegaly, and skeletal abnormalities due to rapid expansion of the erythroid compartment in bone marrow (BM) caused by ineffective erythropoiesis. In a classical view of hematopoiesis, the blood cell lineages arise via a hierarchical scheme starting with multipotent stem cells that become increasingly restricted in their differentiation potential through oligopotent and then unipotent progenitors. In human, novel purification strategies based on differential expression of CD49f and CD90 enrich for long-term (49f+) and short-term (49f−) repopulating hematopoietic stem cells (HSCs), with distinct cell cycle properties, but similar myeloid (My) and lymphoid (Ly) potential. In this view, it has been proposed that erythroid (Ery) and megakaryocytic (Mk) fates branch off directly from CD90-/49f− multipotent progenitors (MPPs). Recently, a new study suggested that separation between multipotent (Ery/My/Ly) long-term repopulating cells (Subset1, defined as CLEC9AhighCD34low) and cells with only My/Ly and no Ery potential (Subset2, defined as CLEC9AlowCD34high)occurs within the phenotypic HSC/MPP and CD49f+ HSCs compartment. Aims A general perturbed and stress condition is present in the thalassemic BM microenvironment. Since its impact on the hematopoietic cell subpopulations is mostly unknown, we will investigate which model of hematopoiesis/erythropoiesis occurs in Bthal. Moreover, since Beta-Thalassemia is an erythropoietic disorder, it could be considered as a disease model to study the 'erythroid branching' in the hematopoietic hierarchy. Methods We defined by immunophenotype and functional analysis the lineage commitment of most primitive HSC/MPP cells in patients affected by this pathology compared to healthy donors (HDs). Furthermore, in order to delineate the transcriptional networks governing hematopoiesis in Beta-thalassemia, RNAseq analysis was performed on sorted hematopoietic subpopulations from BM of Bthal patients and HDs. By droplet digital PCR on RNA purified from mesenchymal stromal cells of Bthal patients, we evaluated the expression levels of some niche factors involved in the regulation of hematopoiesis and erythropoiesis. Moreover, the protein levels in the BM plasma were analyzed by performing ELISA. Results Differences in the primitive compartment were observed with an increased proportion of multipotent progenitors in Bthal patients compared to HDs. The Subset1 compartment is actually endowed with an enhanced Ery potential. Focusing on progenitors (CD34+ CD38+) and using a new sorting scheme that efficiently resolved My, Ery, and Mk lineage fates, we quantified the new My (CD71-BAH1-/+) and Ery (CD71+ BAH1-/+) subsets and found a reduction of Ery subset in Bthal samples. We can hypothesize that the erythroid-enriched subsets are more prone to differentiate quickly due to the higher sensitivity to Epo stimuli or other bone marrow niche signals. Gene set enrichment analysis, perfomed on RNAseq data, showed that Bthal HSC/MPP presented negative enrichment of several pathways related to stemness and quiescence. Cellular processes involved in erythropoiesis were found altered in Bthal HSC. Moreover, some master erythroid transcription factors involved were overrepresented in Bthal across the hematopoietic cascade. We identified the niche factors which affect molecular pathways and the lineage commitment of Bthal HSCs. Summary/Conclusions Overall, these data indicate that Bthal HSCs are more cycling cells which egress from the quiescent state probably towards an erythroid differentiation, probably in response to a chronic BM stimulation. On the other hand,some evidences support our hypothesis of an 'erythroid branching' already present in the HSC pool, exacerbated by the pathophysiology of the disease. Disclosures No relevant conflicts of interest to declare.

Loss of Dnmt3a Confers Resistance to Pegifnα in JAK2-V617F Mouse Model

Myeloproliferative neoplasms (MPNs) are clonal hematological malignancies, which are initiated and maintained by a pool of malignant long-term hematopoietic stem cells (LT-HSCs). A recurrent gain-of-function mutation in JAK2-V617F (VF) is present in a majority of patients with MPN. Treatment with pegylated interferon alpha (pegIFNα) is currently the only therapy known to reduce JAK2-V617F+ LT-HSCs and to induce a complete molecular remission (CMR) in a subset of patients. Long-term follow-up studies suggested, that patients with additional mutations in epigenetic modifiers, such as DNMT3A, have poorer responses to pegIFNα and are less likely to achieve CMR. We therefore hypothesized that DNMT3A loss leads to alterations in JAK2-V617F HSCs functions conferring resistance to pegIFNα treatment. To test the hypothesis we performed competitive transplantations of bone marrow cells from VF or VF;Dnmt3a-/- mice, which also co-express the GFP reporter mixed with wildtype competitor (WT) cells in 1:20 ratio (Figure 1A). After 6 weeks, recipients mice were randomized and treated for 16 weeks with mouse pegIFNα (25µg/kg; s.c.; once per week) or saline (Figure 1A). Complete blood counts and GFP chimerism monitored every 4 weeks showed, that pegIFNα was able to achieve normalization of peripheral blood parameters and reduce GFP chimerism in both, VF and VF;Dnmt3a-/- chimeric mice. In VF chimeric mice treated with pegIFNα we observed a significant reduction of splenomegaly (Figure 1B) and normalization of bone marrow MPN histopathology compared to saline, however pegIFNα treatment in double-mutant VF;Dnmt3a-/- did not improve histopathological features and had tendency to worsen splenomegaly. GFP chimerism in LT-HSCs and committed hematopoietic progenitors was reduced only in VF chimeric mice, but not in double mutant mice. Chronic activation and ensuing replication-induced DNA damage is a critical factor for the functional decline and depletion of mutant LT-HSCs upon treatment with pegIFNα. Our functional analysis shows that LT-HSCs and early progenitors from double-mutant VF;Dnmt3a-/- mice were less prone to accumulate reactive oxygen species, DNA damage and exit dormancy upon treatment with pegIFNα, compared to VF (Figure 1B). In series of secondary transplantations at increasingly limiting dilutions, bone marrow cells from VF mice treated with pegIFNα showed reduced repopulation capacity resulting in a milder MPN phenotype and lower mutant LT-HSC chimerism in the secondary recipients. In contrast, bone marrow cells from VF;Dnmt3a-/- double mutant mice treated with pegIFNα gave rise to a more aggressive disease phenotype compared to saline in secondary (Figure 1C) and also tertiary transplantations (not shown). Recipients transplanted with BM cells from VF;Dnmt3a-/- mice treated with pegIFNα also showed increased frequencies of mutant LT-HSCs compared to saline. Our results suggest that the accelerating effects of pegIFNα therapy inflicted on the LT-HSC of VF;Dnmt3a-/- double mutant mice are stably inherited upon secondary and tertiary transplantations. Overall, our results demonstrate that loss of Dnmt3a mitigate functional attrition of JAK2-V617F+ LT-HSCs upon chronic pegIFNα treatment and create vastly expanded pool of resistant LT-HSCs and point towards possible detrimental effects of pegIFNα therapy in patients with additional loss-of-function mutation in DNMT3A. Disclosures No relevant conflicts of interest to declare.

YY1 Promotes SCF/c-Kit Signaling and HSC Self-Renewal in Fetal Hematopoiesis

Fetal hematopoietic stem cells (HSCs) exhibit markedly different properties as compared to adult HSCs including cell surface marker expression, proliferation state and repopulation capacity. Changes of HSC activity in postnatal mice is defined by a process of decreasing in cell cycle and entering into quiescence. Yin Yang 1 (YY1) is a ubiquitous transcription factor and mammalian Polycomb Group (PcG) Protein with important functions to regulate embryonic development, lineage differentiation and cell proliferation. While homozygote deletion of YY1 in mice results in lethality at the peri-implantation stage, heterozygote deletion of YY1 causes severe developmental defects. By conditionally deleting YY1 in adult hematopoietic system, our previous study showed that YY1 is an essential regulator for adult hematopoiesis by promoting HSC long-term self-renewal and maintaining adult HSC quiescence. In contrast to adult HSCs, in which quiescence is a fundamental characteristic, over 95% of fetal HSCs are in an active cell cycle to rapidly generate homeostatic levels of blood cells for oxygen transport and immune system development in the growing organism. Herein, we assessed whether YY1 was also required for maintaining fetal HSC pool and regulating fetal HSC functions, and what was the underlying mechanism by which YY1 regulated fetal HSCs. To test how loss-of-function of YY1 impacted fetal hematopoiesis, Yy1f/f mice in which the Yy1 promoter region and exon 1 are flanked by loxP sites, were crossed to Vav-Cre mice to generate heterozygous Yy1f/+ Vav-cre mice. The Vav promoter drives Cre recombinase expression specifically in fetal liver hematopoietic cell starting at day E11.5. Yy1f/+ Vav-cre mice were then subsequently bred with Yy1f/f mice to generate homozygous Yy1f/f Vav-cre mice. Among 141 pups resulting from breeding Yy1f/fto Yy1f/+ Vav-Cre, only 7 were Yy1f/f Vav-Cre (n=7) and was significantly lower than the estimated number (n=35) according to the Mendelian ratio (P<0.05). All Yy1f/f Vav-Cre pups died within 72 hours after birth, which supported essential role of YY1 in fetal hematopoiesis and survival. At E14.5 of fetal development, Yy1f/f Vav-Cre fetuses had reduced numbers of hematopoietic stem and progenitor cells in the liver. In addition, YY1 deficient fetal HSCs failed to self-renew in primary and secondary bone marrow transplantation assays. Colony formation assay showed that fetal liver cells from Yy1f/f Vav-Cre mice failed to form CFU-GEMM, CFU-GU and BFU-E compared to Vav-Cre control. While YY1 promotes SCF/c-Kit signaling in adult HSCs, it does not impact c-Kit cell surface expression in early T cell progenitors (unpublished data). To assess YY1 impact on SCF/c-Kit axle in fetal HSCs, c-Kit transcript level, c-Kit median fluorescence intensity and phosphorylated AKT were measured. Similar as its function in adult HSCs, YY1 deficient fetal HSCs had decreased Kit transcript expression, decreased c-Kit cell surface expression and decreased SCF/c-Kit signaling. Our results supported that YY1 is required for maintaining a continuous pool of HSCs in fetal liver and is critical for fetal HSC long-term self-renewal and differentiation. Similar as its function in adult HSCs, YY1 promotes SCF/c-Kit signaling in fetal HSCs. Disclosures No relevant conflicts of interest to declare.

Fzr1 Preserves Hematopoietic Stem Cell Quiescence through Inhibiting Runx1

Regulated blood production is achieved through the hierarchical organization of dormant hematopoietic stem cell (HSC) subsets that differ in self-renewal potential and division frequency, with long-term (LT)-HSCs dividing the least. FZR1, as a regulatory subunit of anaphase promoting complex/cyclosome (APC), is a master regulator of cell cycle, but little is known with regard to its role in HSC quiescence. Thus, we examined the function of Fzr1 in HSCs during steady-state hematopoiesis and under stress. We demonstrate that Fzr1 deletion led to perturbed hematopoiesis with an approximately 2-fold decrease in HSC pool size, as consequence of Fzr1 loss driving HSC from quiescence into rapid cycling (~70.1% to ~28.4%), elevating proliferation (~4.58% to ~18.5%) and apoptosis (~0.23% to ~2.86%) in HSC. As shown by serial bone marrow transplantation and competitive repopulation assays, self-renewal capacity and regenerative capacity were impaired, but differentiation of HSCs was not affected post Fzr1 deletion. Mechanistically, Fzr1 loss led to upregulation of Runx1 expression, in line with the protein expression, the ubiquitin sites of the gene was also obviouly decreased, knockdown Runx1 can rescue the Fzr1-deficient phenotype in HSC. Together, our data therefore support that Fzr1 acts a positive regulator of HSC quiescence and self-renewal capacity through inhibiting Runx1 expression. Disclosures No relevant conflicts of interest to declare.

Nuclear Protein DEK Governs Quiescence and Metabolic Homeostasis of Hematopoietic Stem Cells By Shaping Chromatin Accessibility

Hematopoietic stem cells (HSCs) must achieve a balance between quiescence and activation that fulfils the demands for hematopoiesis without compromising long-term maintenance of HSCs. DEK, a chromatin architectural factor, is involved in chromatin remodeling, transcriptional regulation and DNA replication, and is implicated in genetic and epigenetic regulation of gene expression. Here, we identified that DEK is a critical regulator of HSCs quiescence. Deletion of DEK in mice resulted in abnormal hematopoiesis with an obvious decreased HSC pool size (~3700 to ~1700 cells/mouse), associated with apparent reduction in the proportion of HSCs in G0 phase as compared to control HSCs (~72% to ~57%). As shown by serial bone marrow transplantation and competitive repopulation assays, deletion of DEK impaired the self-renewal capacity of HSCs. Mechanistically, deficiency of DEK in HSC altered chromatin accessibility landscape, resulting in increased transcription of activation-specific genes (including Akt1/2,Ccnb2, and Rps6) and decreased transcription of quiescence-specific genes (including p21 and Gata2), leading to excessively activated Akt-mTOR signaling and elevated metabolism of HSC. Targeting the Akt-mTOR pathway efficiently abrogated the impaired quiescence and the increased metabolism of HSC in DEK-deficient mice, and partially rescued the long-term functions of HSC. Further, DEK regulated chromatin accessibility of HSC by recruiting the co-repressor NCoR1 to repress acetylation of histone 3 at lysine 27. Collectively, our findings revealed crucial functions of DEK in HSC quiescence maintenance and disclosed a new link between chromatin remodelers, epigenetic modification, gene transcription, and HSC homeostasis. Disclosures No relevant conflicts of interest to declare.

Reversing Clonal Hematopoiesis and Associated Atherosclerotic Disease By Targeted Antibody-Drug-Conjugate (ADC) Conditioning and Transplant

Cardiovascular disease (CVD) is the leading cause of death worldwide. Recently, age-related clonal hematopoiesis (CH) has been recognized as a risk factor for CVD of comparable magnitude to smoking, hypertension and hypercholesteremia. While these other risk factors can be mitigated by pharmacological intervention or lifestyle changes, there are no such strategies in place for CH. As CH is initiated by mutations in hematopoietic stem cells (HSCs), a hematopoietic stem cell transplantat (HSCT) could serve as a curative therapy. However, stem cell transplantation is associated with significant toxicity due in part from current conditioning regimens. There is also no evidence that depletion of the disease-driving clones impacts established atherosclerosis. We developed an antibody drug conjugate (ADC) targeting murine CD45. In the context of stem cell transplantation, the CD45-ADC efficiently depletes endogenous HSCs as well as mature leukocytes while enabling rapid engraftment of an infused stem cell graft. In addition, the CD45-ADCs are not based on broad-acting genotoxic agents that lead to long-lasting health risks. We decided to test if CD45-ADC and HSCT could halt atherosclerosis progression through elimination Tet2 knockout HSCs and their disease propagating myeloid progeny. To model CH associated atherosclerosis, LDLR knockout mice were transplanted with 20% CFP labeled wild-type (WT) or Tet2 knockout bone marrow. A single dose of isotype- or CD45-ADC was delivered after 6 weeks of atherosclerosis development and was followed by an infusion of WT CD45.1 bone marrow. As has been reported before, we observed in the isotype-ADC treated animals that Tet2 deficiency leads to a competitive advantage over WT cells. Tet2 knockout cells contributed to peripheral blood chimerism at successively increasing levels and mice harboring the knockout graft showed a significant expansion of their HSC population. Despite their obvious advantage, Tet2 deficient HSC were as efficiently depleted as their WT counterparts upon CD45-ADC and HSCT. Peripheral blood and bone marrow chimerism were similar in WT and Tet2 knockout hosts and the expanded HSC pool was successfully curbed 6 weeks following the intervention. More importantly, CD45-ADC also depleted cells in the atherosclerotic plaques as efficiently as in blood in both WT and Tet2 mutant recipients. This resulted in a significant reduction of myeloid cell infiltration in CD45-ADC conditioned and transplanted knockout hosts and ultimately lead to drastically reduced plaque size in these animals. In conclusion, these data demonstrate that CD45-ADC and HSCT efficiently replaces the disease driving myeloid cells in the atherosclerosis plaques leading to an overall reduction in disease burden. CD45-ADC and transplantation may thus offer a novel therapy for CH and its associated morbidities. Disclosures Palchaudhuri: Magenta Therapeutics: Current Employment. Hyzy:Magenta Therapeutics: Current Employment, Current equity holder in publicly-traded company. Proctor:Magenta Therapeutics: Current Employment. Gillard:Magenta Therapeutics: Current Employment. Boitano:Magenta Therapeutics: Ended employment in the past 24 months, Patents & Royalties. Cooke:Magenta Therapeutics: Ended employment in the past 24 months. Scadden:Magenta Therapeutics: Consultancy, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees.