P&F Projects

Hematopoietic stem cells (HSCs) are responsible for long-term maintenance and regeneration of the hematopoietic system. Loss of long-term (LT)-HSC function is a major contributor to decline in hematopoietic function with aging, leading to increased risk of infection, poor vaccination response, and increased susceptibility to hematologic malignancies. A number of LT-HSC-intrinsic alterations and LT-HSC-extrinsic changes in the bone marrow (BM) microenvironment have been associated with functional decline in aged LTHSCs, however, the initiating changes causing LT-HSC remain unclear. We took the novel approach of examining LT-HSC frequency and function in mice at a wide array of ages with the rationale that interventions to extend LT-HSC function will be most effective starting at or before the age of onset of functional hematopoietic decline. We found that canonical markers of LT-HSC aging significantly accumulate by middle age (9-12mo) in C57BL/6 mice, including increased phenotypic LT-HSC frequency, reduced regenerative capacity, myeloid lineage bias at both transcriptional and functional levels, increased gH2.AX staining, and loss of polarity of CDC42 and tubulin. Furthermore, we found by reciprocal transplantation studies of young LTHSCs into middle-aged recipient mice and middle-aged LT-HSCs into young recipient mice that LT-HSC extrinsic changes in the middle-aged BM microenvironment were necessary and sufficient to cause LT-HSC aging. By transcriptome analysis, we identified decreased IGF1 signaling in LT-HSCs as a candidate mechanism causing LT-HSC aging. We systematically identify mesenchymal stromal cells (MSCs) as the major local producer of IGF1 in the BM of young mice and determine that this production is diminished by middle age. To evaluate the specific effect of MSC-produced IGF1 on LT-HSCs, we co-cultured LT-HSCs with Igf1 conditional knockout MSCs, which was found to cause increased differentiation of LT-HSCs toward myeloid progenitor cells. In vivo, reduced IGF signaling in the BM microenvironment was modeled by transplantation of wild-type BM cells into Igf1 conditional knockout recipients, which phenocopied myeloid-biased hematopoiesis as observed in middle-aged mice. A similar myeloid-biased hematopoiesis phenotype was observed upon transplant of Igf1r conditional knockout LT-HSCs into wild-type recipients, supporting a model of direct communication between MSCs and LT-HSCs via IGF1 signaling. To determine whether restoration of IGF1 signaling had the capacity to rejuvenate middle-aged LT-HSCs, we applied short-term (18hr) in vitro treatment of recombinant IGF1. IGF1 treatment restored polarity of CDC42 and tubulin, decreased gH2.AX foci formation, and decreased myeloid-biased differentiation of middle-aged LT-HSCs both in vitro and in vivo. Transcriptional analysis identified an increase in mTOR signaling and decreased myeloid-biased LT-HSC and increased lymphoid-biased LT-HSC signatures. Other signatures upregulated upon IGF1 stimulation of middle-aged LT-HSCs included oxidative phosphorylation, cell cycle checkpoint and chromatin organization. We propose that restoration of local IGF1 signaling, or its downstream target pathways, represents an attractive prophylactic strategy for extending hematopoietic healthspan into older age. With the NIDDK-CCEH External Pilot Feasibility Grant, we propose to interrogate whether this same mechanism causes, and can be targeted to ameliorate, human HSC aging. This will provide critical preliminary data to enable translational and preclinical studies. We currently lack access to and direct funding for high-quality human samples to generate this preliminary data.

The long-term goal of my research is to elucidate the mechanisms that specify hematopoietic stem and progenitor cell fates. Such knowledge is critical for developing treatments for hematological diseases, as well as producing blood products for transfusion medicine. Currently there are many obstacles to overcome before we can manufacture a sufficient and safe supply of platelets and RBCs for the more than 10% of in-hospital patients receiving transfusions.

Both platelet-producing megakaryocytes (Mk) and RBCs can derive from the common bipotent progenitor called the Megakaryocytic-Erythroid Progenitor (MEP). Little is known regarding the mechanisms that control MEP fate specification, and a better understanding will inform in vitro derivation of platelets and RBCs.

Niche interactions, including communication between neighboring cells in the bone marrow, are key mediators of hematopoietic lineage commitment. Given the known role of bone marrow-derived macrophages (BMDM), bone marrow endothelial cells (BMEC) and mesenchymal stromal cells (MSC) in supporting hematopoietic stem cells, in conjunction with single cell RNAseq data from our lab showing MEP expression of cell surface proteins implicated in binding signals produced by these niche cells, I hypothesize that BMDM, BMEC and MSC regulate the fate of MEP. I propose to model the bone marrow niche in vitro and test the effects of BMDM, BMEC and MSC on MEP fate in collaboration with Dr. Beverly Torok-Storb. Her laboratory at the NIDDK-funded Fred Hutch Cooperative Center of Excellence in Hematology has successfully modeled a vascularized human bone marrow niche in vitro. With Dr. Torok-Storb’s support, I propose to establish an engineered, vascularized human bone marrow niche in vitro and grow MEP clonally to develop a model to elucidate the mechanism by which MEP self-renewal, or lineage commitment is regulated by BMDM, BMEC and MSC. Furthermore, I will explore the possibility of adapting this in vitro niche for live imaging to observe cell-cell interactions, as well as phenotypic/quantifiable features of MEP (cell division rate, motility, frequency of division types) that are influenced by these niche cells.

Successful completion of this project will reveal potential role(s) of macrophages, endothelial cells and marrow stromal cells in the bone marrow niche on bipotent MEP fate, which has implications for advancing our basic understanding of hematopoietic progenitor lineage commitment and deriving blood products to improve outcomes for patients receiving transfusions. Results of these pilot studies will be used as preliminary data in future grant proposals to the NIDDK and other relevant funding agencies.

ATRX (α-thalassemia mental retardation, X-linked) is a member of the SWI/SNF family of chromatin remodelers. Mutations in ATRX lead to ATRX syndrome, a developmental disorder characterized by mental retardation and α-thalassemia. Exactly how these mutations lead to α-thalassemia, a loss of α-globin production due to repression of the HBA (hemoglobin α) gene, is yet unknown. We will investigate the sub-telomeric positioning of the HBA gene cluster (chr.16) and how telomeric cohesion impacts its expression in ATRX syndrome. We previously demonstrated that ATRX influences the cohesion at the telomeres through its interaction with a histone variant, macroH2A1.1. Interaction of ATRX with macroH2A1.1 sequesters the histone variant from localizing to the telomeres, allowing for cohesion resolution. Our preliminary data suggest that unlike WT ATRX, an ATRX patient mutation (ATRX L409S) does not interact with macroH2A1.1. We therefore hypothesize that ATRX L409S prevents α-globin gene expression by promoting persistent telomere cohesion. To test this hypothesis, we propose to create hiPSCs harboring the ATRX L409S mutations (Harvard NIDDK core) and differentiate them to erythroid lineage. Next, we will analyze the status of telomere cohesion and monitor the expression of the α-globin genes. These will be compared with WT hiPSCs and hiPSCs with ATRX knock downs (we have previously shown that reduction of ATRX leads to persistent cohesion). Finally, we will study the role of macroH2A1.1’s telomere localization on our phenotype. Successful remediation of the α-globin expression in the ATRX disease model by resolution of telomere cohesion will allow us to propose a treatment strategy to alleviate the α-globin deficiency. This will be through designing small molecules that that will mimic the ATRX domain, which can bind to and sequester macroH2A1.1 away from the telomeres, thereby potentially treat α-thalassemia in ATRX syndrome.

Macrophages play a critical role in mammalian iron metabolism as they are responsible for degrading senescent red blood cells and recycling iron back to plasma. They do this at a rate of 20-30 mg/day. Total body iron levels are approximately 3-4 g so that equates to about 1% of iron being recycled by macrophages/day, thus underscoring their important role in mammalian iron homeostasis. When iron is in excess macrophages store iron in cytosolic ferritin, which when iron is need can be broken down in the lysosome and iron released back to the cytosol for export into plasma. Iron in ferritin is stored in the Fe3+ state, but all iron transporters identified to date transport Fe2+, therefore, iron must be reduced to be exported from the lysosome. Reductases are found at the plasma membrane or in early endosomes (enterocyte Dcytb or Steap1-4) but the reductase involved in lysosomal iron reduction has not been identified. A candidate reductase for the lysosomal reductase,Cytb561a3 (Lcytb) was suggested years ago. While Lcytb is localized to the lysosome no evidence was provided that it is involved in lysosomal iron reduction. Using CrispR/Cas9 mutagenesis, we found that loss of Lcytb results in decreased iron export from lysosomes of RAW264.7 cells. We determined that RAW264.7 macrophages do not express Steap1, Steap2 or Steap4 mRNA. Again, using CrispR/Cas9, we determined that loss of the endosomal reductase Steap3 also decreases iron export from the lysosome and that loss of both is additive in limiting lysosomal iron export. This suggests that the mammalian lysosome can exist as an iron storage organelle similar to the vacuole in plants and yeast and that iron can be exported to the cytosol. We hypothesize that Lcytb and Steap3 are the reductases necessary for iron recycling in macrophages. Our preliminary results were done in an immortalized macrophage cell line RAW264.7 and we are currently confirming that Lcytb and Steap3 function in primary macrophages.

We utilized three cores sponsored by NIDDK (Utah – Metabolomics, Utah – Iron and Heme and Mutation Generation Detection Core) to determine the roles of these reductases in macrophage iron recycling and macrophage lysosome function.

Heme is an essential cofactor for many cellular processes in mammalian cells including oxygen binding and delivery, redox reactions, detoxification, and regulation of transcription and translation. While all cells in mammals have the ability to synthesize heme de novo, the levels to which heme is required by different cell types vary greatly with developing erythrocytes synthesizing a large quantity (~109 molecules per cell) for hemoglobin production. Thus in the developing erythrocytes heme and globin synthesis must be coordinated in order to avoid pathologic conditions including thalassemias, porphyrias, and anemias. The regulation of heme synthesis is not well understood, with most studies focused at the transcriptional level of heme synthesis enzymes. Recent data has demonstrated that the mitochondrial enzymes of the pathway exist in situ as a complex, or metabolon, and that this metabolon is important in regulating porphyrin and heme synthesis. In addition to heme synthesis enzymes, other proteins involved in intermediary metabolism, mitochondrial structure and dynamics, and mitochondrial metabolite transport were also found to interact with the heme metabolon. While some of these proteins have known cellular functions, how they interact with the metabolon to regulate porphyrin and heme synthesis and homeostasis is unclear. We hypothesize that several of these interacting proteins serve crucial roles in the regulation of substrate synthesis and/or delivery, heme synthesis enzyme activity, and trafficking of completed heme. Herein we propose the creation of human erythroid cell lines in which metabolon components have been disrupted by CRISPR-Cas genome editing. These cells will be analyzed for differences in levels of various heme synthesis metabolites including heme, porphyrins IX, and other porphyrin synthesis intermediates. Cells will also be analyzed for alterations in peripheral metabolite pools such as amino acids, TCA cycle intermediates, and redox metabolites. Data resulting from these experiments will serve as preliminary data for further study of the metabolon in erythroid and non-erythroid cells through R01 funding from the NIDDK. Outcomes of these studies will further illuminate the processes by which heme synthesis and the numerous pathways linked thereto are regulated and coordinated. Importantly, this work could lend insight to potential treatments for conditions including anemias, porphyrias, and thalassemias.

Macrophages are key players in heme and iron metabolism as well as immune homeostasis. They exhibit remarkable phenotypic and functional plasticity, reflected by their capacity to integrate diverse signals from the microenvironment and acquire distinct phenotypes. According to a simple dichotomous nomenclature, macrophages are defined as pro-inflammatory ‘classically activated’ M1 or anti-inflammatory ‘alternatively activated’ M2. M1 macrophages produce pro-inflammatory cytokines, reactive oxygen species (ROS) and nitric oxide (NO), express markers such as MHCII, CD86 and iNOS, and show bactericidal activity. Conversely, M2 macrophages are characterized by high expression of the mannose receptor CD206, produce anti-inflammatory cytokines, have immune-regulatory functions and are involved in cell growth control, matrix remodeling and tissue repair. Recently, we described the ability of heme and iron to induce an M1-like phenotypic switching of macrophages, which is prevented by heme and iron scavengers (e.g., hemopexin, transferrin, chelators). Heme-induced M1-like pro-inflammatory macrophages are of patho-physiologic relevance for hemolytic disorders and have been implicated in hepatic fibrosis in SCD (sickle cell disease). SCD is hallmarked by high circulating free heme and depletion of the heme scavenger Hemopexin as a result of intravascular hemolysis. Moreover, SCD is characterized by a chronic inflammatory state, which likely contributes to a number of complications associated with the disease. We suggest that M1 macrophage skewing triggered by free iron, heme and hemolysis is responsible for the chronic sterile inflammation in SCD and we believe that targeting the cellular and molecular mechanisms leading to macrophage phenotypic shift might be of therapeutic value in this disease. Although the underlying mechanisms have not been fully elucidated, our preliminary data indicate a role for TLR4 activation, ROS and NO production, and Arginase-1 suppression in heme/iron-driven M1 M polarization, suggesting a cell metabolic switching towards glycolysis. Emerging evidence on immunometabolism highlight the implication of metabolic intermediates in modulating and reprogramming macrophage immune functions. In this Pilot and Feasibility Program, we aim, in collaboration with the Metabolomics Core of the Center for Iron and Heme Disorders (CIHD) of the University of Utah, at exploring the link between metabolic skewing and immune reprogramming of macrophages by iron sources, with the hypothesis that cell inflammatory phenotypic switching is mediated by a specific metabolic response and adaptation to free heme and iron. We will test this concept by performing metabolic profiling of in vitro and ex vivo macrophages exposed to heme and iron. Finally, we will assess the metabolic profile of macrophages isolated from SCD mice, with the hypothesis that heme-triggered metabolic skewing determines macrophage phenotypic switch and hence contributes to chronic inflammation in this hemolytic condition.

VPS4A is a highly evolutionarily conserved ATPase that assists the Endosomal Sorting Complex Required for Transport (ESCRT)-III to perform membrane scission in a variety of cellular processes including formation of endosomal multivesicular bodies (MVBs) and the abscission step of cytokinesis. We recently identified mutations in VPS4A as a cause of congenital dyserythropoietic anemia (CDA) in several unrelated patients with a syndrome of dyserythropoiesis, hemolytic anemia, and neurodevelopmental delay. Dominant negative (DN) VPS4A mutations in the ATPase domain were associated with BM morphology resembling CDA-I with binucleated erythroblasts and cytoplasmic bridges, likely due to cytokinesis failure as a result of VPS4A loss of function. In peripheral blood from patients with VPS4A mutations, mature red blood cells retained the transferrin receptor (TfR/CD71) suggesting loss of VPS4A impacted TfR processing and reticulocyte maturation, likely through its role in endosomal vesicle trafficking. Knockdown or dominant negative expression of VPS4A in mammalian cell lines has been shown to inhibit receptor internalization, recycling, and degradation in a cell type-specific and pathway-dependent manner with the potential to impact transferrin uptake or downstream signaling from cytokine receptors. Given the profound defects of erythropoiesis in these patients, we hypothesize that loss of VPS4A activity in the erythroid lineage causes endolysosomal dysfunction that perturbs critical erythroid-specific signaling pathways and membrane receptor degradation causing ineffective erythropoiesis and production of unstable red blood cells (RBCs). The goal of this project will be to determine the mechanisms by which VPS4A loss of function disrupts normal erythropoiesis and reticulocyte maturation. In aim 1, we will evaluate erythroblast endosomal and lysosomal compartments by immunofluoresence and identify targets of signaling dysregulation by gene expression analysis of erythroblasts cultured from iPSCs expressing DN VPS4A mutants. In aim 2, we will evaluate TfR downregulation in patient and normal reticulocytes and its secretion in exosomes, establishing for the first time in humans the contribution of VPS4A and ESCRT-III to this process. This work will demonstrate a novel role for VPS4A and ESCRT-III in erythropoiesis signaling and reticulocyte maturation and the results of these experiments will provide critical preliminary data to produce a competitive K01 or R01 application aimed at further understanding these novel mechanisms essential for erythropoiesis.

Heme is the prosthetic group of hemoglobin (HGB). Eighty percent of body heme is produced by red blood cell (RBC) precursors to support HGB production. Heme is synthesized in eight enzymatic steps. The first enzyme, 5-aminolevulinic acid synthase 2, ALAS2, is rate limiting in RBC precursors. As such, ALAS2 is called the “gatekeeper” of heme biosynthesis. In mitochondria, ALAS2 condenses glycine and succinyl co-enzyme A to form 5-aminolevulinic acid (ALA), the monomeric precursor of heme and all other porphyrins.

Partial loss-of-function mutations in ALAS2 cause X-linked Sideroblastic Anemia (XLSA) which is associated with pathologic erythroid mitochondrial iron accumulation. C-terminal deletions of ALAS2, found in patients with X-linked Protoporphyria (XLPP), increase ALAS2 enzymatic activity and ALA production causing free protoporphyrin IX (PPIX) accumulation (erythroid protoporphyria) leading to photosensitivity and liver disease. Excess PPIX may also result from a deficiency in ferrochelatase (FECH), the last enzyme in heme biosynthesis that incorporates iron into PPIX

ALAS2 expression is massively upregulated as erythroid precursors mature. A key regulator of ALAS2 expression is encoded in the 5’UTR of its mRNA—a stem-loop iron responsive element (IRE) that couples ALAS2 expression to erythroid iron levels. In the absence of Iron Regulatory Proteins, are thought to bind the ALAS2-IRE and inhibit ALAS2 translation, limiting ALA and thus heme production. In several Congenital Sideroblastic Anemias (CSAs) other than XLSA, IRP1 IRE-binding activity is inappropriately increased, which is thought to repress ALAS2 expression and contribute to the pathogenesis of the diseases.

Here, we have constructed several mouse lines with mutations in Alas2, including mice with XLSA and IRE deletions. We will use these models to better understand the control of ALAS2 expression by IRPs during normal and pathological erythropoiesis and how ALAS2 misregulation or regulation through the IRE can contribute to the pathogenesis of CSA and erythroid porphyrias.

Imaging of cells is critical to our understanding of cellular architecture and function. Until recently, limitations due to the wavelength of light made it impossible to image cells with standard light microscopy at less than 200 nm resolution, far larger than that needed to resolve the molecular makeup of subcellular structures in blood cells. Over the past 10-15 years, super-resolution microscopy techniques have improved upon resolution, but at the expense of requiring special microscopes and complex analysis algorithms, low throughputs and having limited abilities to image hematology specimens, including blood cells and bone marrow tissues. To overcome the current limitations of super-resolution microscopy in hematology, we propose to establish expansion microscopy protocol for the visualization of ultrastructure in all blood cell types and explore its potential toward highly multiplexed nanoscopy of hematology specimens. Specific examples of application will focus on macrophages, megakaryocytes and platelets but are applicable to all blood cell types. We will distribute the tools we develop freely and to instruct the community on their usage.

Splicing factor (SF) mutations are of significant interest in myelodysplastic syndromes (MDS) as they are frequent, early occurring and associated with clinical outcome. However, the molecular mechanisms underlying the proliferative advantage of SF-mutant cells and the consequences on RNA biology are still not fully understood. Moreover, despite recent efforts, small-molecule splicing modulators currently in clinical trials are progressing slowly towards the clinic. Dissecting the pathways activated by SF mutations will deepen our understanding of MDS pathophysiology and accelerate the development of more efficient targeted therapies. We have identified that mutations in the splicing factor U2AF1 are directly linked to increased formation of stress granules (SGs), biomolecular condensates that improve cellular adaptation to stress and whose upregulation has been reported in disease. Analysis of splicing alterations related to SRSF2 mutations also revealed a significant enrichment in SG-related terms. These collective data lay the foundation for a new paradigm by which SF mutations promote the ability of the cell to withstand stress. SG perturbations may therefore represent a novel therapeutic vulnerability in SF-mutant MDS. The proposed project aims at further understanding the effect of U2AF1 and SRSF2 mutations on stress granules and additional biomolecular condensates in MDS cells. We will characterize SG perturbations at the functional level by immunofluorescence and super-resolution microscopy, and at the RNA/protein level by SG isolation and long-read RNA-seq or mass spectrometry (AIM 1). We will perform targeted single-cell RNA-seq to establish a direct link between SF-mutant genotype and SG perturbation, investigating cell specific changes in SG RNAs (AIM 2). Finally, we will evaluate by confocal microscopy the effect of SFmutations on Cajal bodies, nuclear condensates implicated in RNA metabolic processes (AIM 3). The results of this project will set the stage to understand clonal advantage of SF-mutant cells. In subsequent studies we will functionally modulate SG formation to probe SGs as novel therapeutic targets in MDS.

Myelodysplastic syndromes (MDS) are a heterogeneous group of age-related blood disorders which can broadly be characterized by the accumulation of immature hematopoietic progenitors in the bone marrow. Patients normally present with cytopenia, anemia and are at high risk for leukemic transformation. Many mutations have been linked to hematopoietic failure in MDS, with components of the spliceosome being most prevalent. The most common of these mutations occurs in the U2 snRNP associated protein SF3B1, where a series of “hot-spot” mutations are clustered within the HEAT domain, a region known to directly associate with the pre-mRNA. These mutations alter in global splicing patterns, the most frequent event being use of cryptic 3´ splice sites (3´ss) which largely result in inclusion of premature termination codons in the final mRNA transcript, thus initiating nonsense mediated decay. It is believed that SF3B1 mutation typically occurs in the founding clone and drives disease etiology, therefore much work has been done to parse the downstream consequences of SF3B1 mutation. Thus far, no one target has yet to fully recapitulate disease phenotypes or oncogenic transformation. We hypothesize that SF3B1 mutation causes differentiation defects and premature blast retention through transcriptomic deregulation as a result aberrant splicing of the CDK8 pre-mRNA. As a member of the mediator complex, CDK8 has been shown to function as a regulatory protein in transcriptional activation and elongation of pathway specific genes. We hypothesize that CDK8 deregulation leads to an inability to activate the gene networks critical for initiating lineage commitment and differentiation in hematopoietic stem cells. Herein, we propose to interrogate CDK8’s function as a regulator of hematopoiesis and its role in MDS pathogenesis.

All cells in a developing embryo have essentially the same DNA sequences in their genomes. In order for cells to become differentiated from one another, cell type-specific gene expression programs must become activated, while programs for alternate lineage must be suppressed. This “lineage specification” process is poorly understood. Our findings show that Oct1 deficiency 1) impairs mouse development, in particular the development of mesoderm and blood progenitors, and 2) cause improper mesodermal differentiation of mouse embryonic stem cells in vitro. This proposal tests the role of human OCT1 in embryonic blood cell development by developing, for the first time, an in vitro model.

Hepcidin regulates systemic iron homeostasis by controlling dietary iron absorption, the release of iron from iron recycling macrophages, and the release of iron from hepatic stores. During pregnancy in humans and rodents, maternal hepcidin is profoundly suppressed, which is thought to maximize dietary iron absorption and mobilization of iron from stores for transfer to the developing fetus. Augmenting maternal hepcidin in mouse pregnancy by administration hepcidin analogs led to severe embryo anemia or even death. Thus, maternal hepcidin suppression is essential for maternal and embryo iron homeostasis and health. Despite its importance, the mechanism(s) responsible for hepcidin suppression remain elusive. We identified the placenta as a source of a pregnancy-related hepcidin suppressor; conditioned media from primary human trophoblasts robustly suppressed hepcidin in the hepatic cell line Hep3B. Using standard orthogonal multi-step protein purification of trophoblast supernatant followed by LC-MS analysis we identified 228 candidate proteins. To date, we tested five candidate proteins, chosen based on their expression profiles during pregnancy and known associations with the BMP-SMAD signaling pathway, a key regulator of hepcidin expression in the liver. These studies identified Netrin-1 as a novel regulator of hepcidin. Although a role for netrin-1 in iron homeostasis has not been described previously, its receptor neogenin, is a known regulator of hepcidin. This project aims to 1) determine if netrin-1 is required for hepcidin suppression in pregnancy in vivo; 2) define the mechanism of netrin-1-mediated hepcidin suppression in vitro; and 3) test additional proteins identified in our screen for ability to regulate hepcidin. When completed, these studies will provide fundamental insight into the regulation of iron homeostasis during and even outside of pregnancy, with broad translational potential for treatment of iron disorders.

5-aminolevulinic acid synthase 2 (ALAS2) is the first and rate-limiting enzyme of erythroid heme biosynthesis. It combines glycine and succinyl-CoA to form 5-aminolevulinic acid (ALA), the precursor of porphyrins and heme. ALAS2 is essential in mammals, its expression being precisely regulated during erythropoiesis. Inherited ALAS2 defects cause two rare diseases: X-linked Sideroblastic Anemia (XLSA) and X-linked Protoporphyria (XLPP), due to loss-of-function and gain-of-function mutations, respectively. Male XLSA patients have a microcytic hypochromic anemia of variable severity, characterized by abnormal mitochondrial iron deposits in nucleated and enucleated erythroid cells, termed ring sideroblasts and siderocytes, respectively. In two-thirds of patients, the anemia responds to pyridoxine (vitamin B6) supplementation. Both male and female XLPP patients develop acute photosensitivity, due to accumulation of free protoporphyrin (PP) in erythrocytes. No curative treatment, other than hematopoietic stem cell transplantation, is available for XLSA or XLPP. To better understand the physiopathology of heme disorders and to develop potential treatments for XLSA and XLPP, we employed CRISPR/Cas9 gene targeting technology, to established 3 common XLSA (ALAS2 p.R170H, p.R411H and p.R452H) and 1 XLPP (ALAS2 p.Q548X) mutations in mice. The natural history of the disease was determined for each strain over the course of one year. In addition, the consequences of dietary vitamin B6 deficiency and supplementation was analyzed. Knock-in ALAS2 mouse strains with viable loss and gain of function mutations express the key features of the corresponding human diseases. By flow cytometry (BD Celesta, BV605), we have been able to demonstrate free protoporphyrin accumulation in red blood cells and erythroblasts from the XLPP model. We have, however, been limited in our ability to quantify zinc protoporphyrin (which is characteristically increased in XLPP, and in case of iron deficiency) due to an overlap between zinc and free protoporphyrin with the Helena Laboratories Protofluor-Z instrument. To better phenotype our new XLSA and XLPP model, we desire to employ the gold standard assays provided by the CCEH Iron and Heme Core at the University of Utah to quantify erythrocyte, serum, and hepatic protoporphyrins (free and zinc) and heme in our mouse models, with or without vitamin B6 restriction. In addition, we would like to measure erythroid Alas activity in both the disease models.

Erythropoiesis is a complex process that occurs in erythroblastic islands within the bone marrow niche. Erythroblastic island are composed a central macrophage surrounded by developing erythroblasts and are essential for in vivo erythroid differentiation. Until recently this complex interaction has not been modeled in vitro thus limiting our knowledge of erythropoiesis to isolated cells in a liquid medium with a group of growth factors. The Torok-Storb laboratory has recently developed a system for the in vitro formation of erythroblastic island from engineered bone marrow-derived CD34+ cells. Herein we propose to use this system to understand transcriptomics and proteomics at the single cell level for the later stages of erythropoiesis in normal and inflammatory states. For our first aim, we will establish a time line for the induction of heme synthesis during erythropoiesis. For our second aim, we will characterize the impact that a lipopolysaccharide (LPS)-induced inflammatory response has on heme synthesis and differentiation of the erythroblasts within the erythroblastic island. Outcomes from this pilot and feasibility studies will be a better understanding of the temporal induction of heme synthesis enzymes in erythroblasts within erythroblastic islands. Our data will establish the utility of this system to address questions in a more physiologically relevant environment and will also provide the first experimental examination/quantification of the impact of inflammation on erythropoiesis in erythroblastic islands.

The role of the micro-environment or niche in regulating stem cell activity has been a major area of research and is of critical importance for uncovering therapeutic strategies that inhibit its ability to direct malignant transformation. Gaining a deeper understanding of this interaction is paramount to inhibiting myeloproliferation and reversing immune senescence, which are observed in a large number of individuals within our ageing population, impairing their innate immune system’s ability to fight disease. If the niche can indeed affect lineage commitment of hematopoietic stem cells, this has profound implications in pursuing therapeutic avenues that target the micro-environment. The current proposal aims to gather evidence for the role of the sympathetic nervous system, an important niche contributor, in affecting hematopoietic stem cell lineage choice. To gain fundamentally new insight into this dynamic process, standard optogenetic tools used to manipulate neuronal activity in the brain will be adapted for use in the bone marrow and validated extensively. Precise in vivo spatio-temporal control of sympathetic nerve activity will be employed and the response of surrounding chemokine CXCl12 positive stromal cells tracked in real time. Extended optogenetic stimulation will be performed to assess the effect of increased sympathetic nerve activity on hematopoietic stem cell fate. Along with the planned K01 grant entitled “Probing hematopoietic-stromal crosstalk by spatial transcriptomics and optogenetic manipulation of the niche: potential new therapeutic strategies for the reversal of immune senescence will be uncovered.

Hematopoietic stem cells (HSCs) originate during a transient window of embryonic development from specialized endothelial cells, termed hemogenic endothelium (HE), in a process referred to as the endothelial to hematopoietic transition (EHT). Essential properties that define HSCs, such as the ability to self-renew, home to the bone marrow, and provide multilineage hematopoiesis, must be acquired during EHT to generate HSCs that are capable of long-term, multilineage hematopoietic reconstitution following transplantation. Previuosly, our lab engineered a novel vascular niche model of the embryonic AGM (aorta-gonad-mesonephros region), where the first HSCs arise from HE, to recapitulate the process of EHT and HSC formation in vitro. To understand the molecular programs that regulate acquisition of HSC-defining properties during EHT, we performed single cell RNA-sequencing (scRNA-seq) on embryonic hemogenic precursors during their transition from HE to HSC in vivo and in vitro in the AGM vascular niche. Ordering of cells in pseudotime based on their transcriptional profiles recapitulated gene expression dynamics during EHT and enabled us to identify novel genes whose temporal expression suggests a role in HSC specification and self-renewal. To begin to explore the roles of these genes in HSC development, we propose to use a CRISPR/Cas9-based approach to test the consequence of gene knockout during HSC formation from HE in the AGM vascular niche in vitro. The studies proposed here will not only provide critical proof-of-concept data for this approach, but also allow us to begin to screen for novel regulators of HSC development, advancing the longer-term goal of engineering HSCs for therapeutic applications. Ultimately, the data and reagents generated from this proposal will be a cornerstone for future fellowship/training grant applications.

The essentiality of iron for life sustaining processes, coupled with its capacity to promote damaging free radical production, has made it a desirable target for health promotion and disease prevention, respectively. One such approach may be through the modulation of ferroptosis, a form of iron-mediated programmed cell death. However, we must first understand how cells manipulate the homeostatic regulators of iron metabolism to promote disease before we can fully harness iron’s therapeutic potential. The iron regulatory proteins 1 and 2 (IRP1 and IRP2) are the master regulators of intracellular iron homeostasis because they coordinate the expression of proteins involved in iron storage, uptake, and utilization. Yet, the roles and regulation of IRPs during cellular ferroptosis remain unknown. The long-term goal of the Montgomery lab is to understand how acquisition of exaggerated amounts of iron promote metabolic perturbations that lead to iron-mediated disease progression. The primary objective of this work is to establish how IRP mRNA binding activity influences cellular sensitivity to ferroptotic cell death. The central hypothesis is that activation of IRP mRNA binding promotes cellular iron accumulation and facilitates ferroptotic cell death. In this proposal we aim to utilize CRISPR technology to individually knockout each IRP to establish the extent to which IRP1 and IRP2 mRNA binding activity are required for ferroptosis. Our preliminary data indicates that increased IRP mRNA binding enhances sensitivity to ferroptotic cell death, and that mutations in the ferroptosis suppressor, TP53, may promote ferroptosis sensitivity in an IRP-dependent manner. Thus, we also propose to use ICP-MS to investigate IRP- and mutant TP53-dependent differences in cellular iron accumulation following induction of ferroptosis.

In human aging there is a reduction in the diversity of individual hematopoietic stem and progenitor cell (HSPC) clones due to an accumulation of recurrent genetic mutations, including in ASXL1, DNMT3A, and TET2. This reduction in clonal diversity, termed clonal hematopoiesis (CH), increases with age and is associated with an increased risk of hematological malignancies and cardiovascular disease. Currently, there are no treatments for CH. A therapeutic goal is to re-establish a balanced clonal output by outcompeting the mutant HSPCs. A major barrier to identifying therapeutic options for CH is a lack of an experimental model to study its progression in an endogenous niche in vivo. To address this gap, we developed a system whereby mosaic mutagenesis allows prospective establishment of CH in vivo in a zebrafish model. This system is combined with fluorescent labeling of HSPC clones which allows identification of the dominant clone by its relative expansion compared to non-dominant clones and isolation of this clone via flow cytometry for further analyses. The overarching hypothesis guiding this proposal is that there are metabolic vulnerabilities in mutant HSPCs in CH that can be targeted to result in decreased competitive potential. Preclinical data suggest intensive metabolic interventions targeting reductive/oxidative (redox) metabolism may improve age-related HSPC dysfunction. The proposal aims to 1) identify key metabolic alterations in clonally dominant HSPCs using untargeted metabolomics and RNA-sequencing in the zebrafish, and 2) determine how altering redox metabolism affects CH development and HSPC function using color barcoding, metabolomics, and lineage output measures. Combining our novel system of modeling CH, in vivo, in a manner in which different HSPC populations can be isolated for downstream metabolic analysis will offer a new layer of biological understanding of mechanism of clonal competitiveness and elucidate therapeutic opportunities in CH.

Over the last five years, single-cell analysis has become a powerful approach for resolving complex mixtures of cells found in normal and diseased tissues. For hematopoiesis, application of single-cell RNA sequencing, in particular, has begun to reshape our notions of the hematopoietic hierarchy and the heterogeneity of blood progenitors. However, these studies have largely been limited to assaying polyA containing RNA and biased toward detection of abundant mRNAs. There has also been limited application of these approaches to characterize differences in cell-based products used for hematopoietic stem cell transplantations. In this pilot, we will use a new single-cell genomic technology developed at the Fred Hutch, dubbed scCUT&Tag, to resolve chromatin landscapes in donor-derived hematopoietic stem and progenitor cell (HSPC) populations. This technology will allow us to assess active (H3K4me2) and repressive (H3K27me3) histone marks in human CD34+ HSPC populations derived from BM, G-CSF mobilization, and after in vitro expansion. We propose that this analysis will provide a more accurate view of HSC and early/late progenitor commitment and also better address whether specific epigenetic landscapes are associated with HSPC source or manipulation (BM, peripheral blood, in vitro, donor, sex). If successful, this pilot will create a key data resource for chromatin marks to better resolve single cell states and developmental trajectories in HSPCs and address key questions about the fidelity of epigenetic states in differently derived donor CD34+ populations.

This proposal seeks to characterize the role of alternative protein isoforms of Mitoferrin-1 in regulating delivery of iron to mitochondria for synthesis of hemoglobin. Erythroid maturation is characterized by a rapid ramp-up of heme and globin synthesis. Dynamic changes to the transcriptome and processing of RNA (including intron retention or IR) accompanies this process. One of the erythroid-specific genes that demonstrates high levels of intron retention is SLC25A37A (encoding Mitoferrin 1). Mitoferrin 1 is critical to transporting ferrous iron from the intermembrane mitochondrial space to mitochondrial matrix for conjugation to protoporphyrin to form heme. Given the redox potential of excess free iron, this process needs to be tightly regulated. We hypothesize that intron retention of SLC25A37 transcript critically regulates the amount of physiologically active Mitoferrin 1 delivered to mitochondria, thereby limiting iron-mediated oxidative damage. Our preliminary results show that the SLC25A37 transcript variant with intron retention (SLC25A37-IR) is translated to previously uncharacterized C- and N- terminally truncated isoforms. Based on the structural domains of SLC proteins, we posit that these shorter isoforms are functionally distinct from the canonical full-length protein. In this application, we seek to definitively test these hypotheses through novel in vitro models of human erythroid maturation. HUDEP-2 is a nontransformed human erythroid cell line responsive to erythropoietin and capable of terminal erythroid differentiation, which can be genome-edited for C- and N-terminal epitope tagging. We will determine how relative abundance of Mitoferrin 1 isoforms change during erythroid maturation, and their subcellular localization (mitochondrial vs. cytoplasmic). Using gain of function and loss of function approaches, we will then determine how perturbation of intron retention affects erythroid maturation and mitochondrial iron delivery. The project will utilize the Iron and Heme Core at University of Utah for iron quantification experiments. The proposal is designed to generate critical preliminary data to support a competitive K08 application from the PI Dr. Boddu, a fellow in Hematology-Oncology and aspiring physician-scientist.

The current dogma is that mammalian hepatic heme levels are controlled by the fine balance between heme biosynthesis, regulated by the rate-controlling heme biosynthetic enzyme 5-aminolevulinic acid synthase1 (ALAS1), and heme catabolism via heme oxygenase. Recent studies have identified several eukaryotic heme/heme precursor transporters and heme chaperones, giving rise to the emerging notion that inter- and intra-cellular heme trafficking pathways also contribute to the maintenance of heme homeostasis. Yet, the pathways and mechanisms that mediate hepatic heme homeostasis outside of the context of heme biosynthesis and catabolism are currently poorly understood. Our recent preliminary studies have revealed an unexpected and novel finding that adult mice with essentially no hepatic ALAS activity (designated Alas1/2KO mice) maintain near-normal hepatocyte heme and hemoprotein activity levels, strongly indicating that hepatic heme homeostasis involves previously unappreciated mechanism(s).Thus, the overall goal of the proposed studies is to use the Alas1/2KO mouse model to elucidate the mechanism(s) that are involved in maintaining hepatocyte heme homeostasis, particularly in the absence of Alas1-driven heme synthesis. To this end, Aim1 will characterize the Alas1/2KO mice in detail using biochemical and immunohistochemical approaches. Aim 2 will investigate the mechanism(s) by which the ALAS-deficient hepatocytes maintain heme, focusing on the two most probable mechanisms, that they acquire: 1) a heme precursor, and/or 2) heme itself, specifically via macrophage-mediated heme recycling of senescent erythrocytes. When completed, these studies should improve our understanding of how hepatic heme homeostasis is maintained in health and disease. In particular, they may provide insights into the pathogenesis of the acute neurovisceral attacks that occur in the four acute hepatic porphyrias, which are currently thought to occur due to the inability of hepatocytes to meet the transiently increased demand for heme that is brought on by porphyrinogenic factors.

TET2 is among the most frequently mutated genes in blood cancers with inactivating mutations found in 􀀟30% of myeloid dysplastic syndrome (MDS) patients, as well as in a subset of individuals over 50 years of age with clonal hematopoiesis of indeterminate potential (CHIP), a condition that predisposes affected individuals to progression to myeloid malignancy and atherosclerotic heart disease with heart attack or stroke. TET2 mutations represent an early genetic lesion in hematopoietic stem and progenitor cells (HSPCs), inducing a premalignant state of clonal dominance that predisposes to the acquisition of additional mutations. Our central working hypothesis is that loss of TET2 function leads to dependencies unique to the mutant HSPCs, such that they are killed by drugs that are not toxic to normal HSPCs. Our preliminary data in zebrafish implicates selinexor, eltanexor and sunitinib as drugs that selectively kill Tet2-mutatnt HSPCs at dosages that do not affect normal HSPCs or Dnmt3a-mutant HSPCs. Here we will test these agents against TET2-mutant MDS using the faithful MISTRG MDS-PDX model developed by Dr. Stephanie Halene. Specific Aim: In this proposed project, we will work in collaboration with Dr. Stephanie Halene who is co-leader at the Yale CCEH Animal modeling core to test the in vivo efficacy of selinexor, eltanexor and sunitinib against TET2-mutant human MDS cells in vivo using her unique patient-derived xenotransplantation model (MISTRG MDS-PDX). MISTRG mice express human hematopoietic cytokines and provide disease representation across all MDS subtypes. Research Design and Methods: To test the in vivo efficacy of selinexor, eltanexor and sunitinib against human TET2-mutant MDS cells as compared to DNMT3-mutant MDS cells, we will transplant equal numbers of cryopreserved normal human CD34+ mobilized HSPCs and TET2 mutant MDS CD34+ HSPCs or DNMT3A mutant MDS CD34+ HSPCs into recipient human cytokine producing MISTRG mice and treat them with selinexor, eltanexor, sunitinib or vehicle. We will monitor the effects of each drug and use the TET2 (or DNMT3A) mutation itself in each patient to compare the response to treatment of TET2-mutant or DNMT3A-MDS blood and bone marrow leukocytes vs. normal competitor cells within each cell lineage.

Through regulated self-renewal and differentiation, hematopoietic stem cells (HSCs) replenish shortlived blood cells throughout life. One consequence of HSC dysregulation is their functional exhaustion, such as what occurs during physiological ageing or serial transplantation. Approaches to antagonize or reverse the functional deterioration of HSCs would lead to better therapy and deeper mechanistic understanding of HSC biology. Binding to nucleosome entry/exit site, the linker histone H1 stabilizes nucleosomes, increases chromatin folding and is generally associated with a transcriptionally silent state. High mobility group nucleosome binding domain (HMGN) family of proteins can displace linker histones and promote decompaction of chromatin thereby allowing transcription. Insufficient histone proteins have been implicated in the senescence/aging of several model systems. Aged HSCs have been shown to exhibit compromised chromatin demarcation and transcriptional dysregulation, yielding diminished reconstitution potential with myeloid bias. Therefore, preventing the changes in nucleosome integrity could potentially counter the changes during HSC functional decline. Given the importance of linker histones in nucleosome organization and stability, this proposal aims to test the hypothesis that supplying cells with additional pool of linker histones could promote HSC function. Using two newly generated mouse alleles which drives H1.0 or HMGN1 expression under a doxycycline inducible promoter (iH1.0 or iHMGN1), my preliminary results demonstrate that sustained expression of H1.0, a linker histone isoform, enables superior HSC function as compared to when HMGN1 is overexpressed, a condition that has been recently demonstrated by others to promote HSC activity. Therefore, I hypothesize that sustained H1.0 expression promotes HSC activity by slowing HSC cell cycle and reinstating proper chromatin accessibility. I will first compare iH1.0 and iHMGN1 HSC activity with wild-type HSCs in competitive transplantation in vivo. Then, I will define the molecular mechanisms by which H1.0 regulates HSC function by examining HSC cell cycle. I will then identify genomic regions impacted by H1.0 overexpression using ATAC-seq and ChIP-seq. Global chromatin states as well as those regulating known HSC fate decision genes will be examined, with a focus on myeloid vs. lymphoid commitment.

Lack of adequate iron during critical periods of neurodevelopment can result in poor cognitive and motor capacities of premature neonates. Because iron acquisition is greatest during the third trimester of pregnancy, fetuses born prematurely, or with certain conditions that limit maternal-to-fetal iron transfer, can lack sufficient iron at critical times during brain development. The prevalence of iron deficiency among preterm neonates, and its range of severity, have not been well defined. We have observed that iron deficiency in preterm neonates is sometimes present at birth, and in other cases it occurs during the NICU hospitalization. Little evidence exists to inform best practices for screening, diagnosing, and treating iron deficiency in this population. As an added complexity, certain iron deficient preterm infants remain iron deficient after weeks of oral iron supplementation, suggesting they have poor enteral iron absorption. The long-term goal of our studies is to determine the prevalence of and factors associated with iron deficiency at birth and throughout the NICU hospitalization in preterm neonates. We hypothesize that a proportion of preterm neonates found to be iron deficient during their NICU hospitalization have not yet recovered from an unrecognized congenital iron deficiency. We also hypothesize that among some iron deficient preterm infants, elevated hepcidin levels in their mothers is a mechanistic explanation. In other cases, elevated hepcidin levels in the neonates themselves may account for failure to accrete enterally-administered iron. In a cohort of preterm neonates in the University of Utah NICU, we propose; (1) to serially identify preterm neonates with biochemical iron deficiency and iron-limited erythropoiesis, at birth and during their NICU hospitalization; (2) to measure maternal serum hepcidin levels in this preterm cohort in order to test for associations with iron deficiency at birth; and to measure neonatal serum hepcidin levels as a potential factor in failure to correct iron deficiency with oral supplementation; (3) to evaluate urine ferritin (corrected for urine creatinine) and urinary hepcidin as valid, non-invasive biomarkers for iron deficiency, thus allowing surveillance for iron deficiency without removing blood (and iron). Determining the prevalence of and factors associated with iron deficiency in premature neonates is innovative because the burden of iron deficiency in this population is unclear. The proposed research is significant because it will test factors potentially contributing to iron deficiency and to the response to enteral iron treatment. Optimizing identification and treatment of iron deficiency in preterm neonates will decrease the risk of long-term neurocognitive dysfunction due to this cause.

Codanin-1 is a ubiquitously expressed protein encoded by Cdan1, mutations in which have been implicated in causing Congenital Dyserythropoietic Anemia Type-1 (CDA-1). Of the several mutations in the human Cdan1 gene, the missense mutation R1042W identified in the Israeli Bedoiun tribe was the first identified. Cells expressing mutant codanin- (CDAN1) exhibit typical morphologically identifiable features in erythroblasts, especially binuclearity and internuclear chromatin bridges. The mechanism of how CDAN1 functions under normal and pathophysiological conditions is yet to be fully realized. We showed that human primary cells undergoing erythroid differentiation display constitutive levels of CDAN1 mRNA throughout while those undergoing megakaryopoiesis show diminished expression of CDAN1 mRNA. We have seen that CDAN1 knockdown inhibits erythroid differentiation in human primary cells and that CDAN1 binds to genomic loci of important erythroid genes while regulating expression of some of these. Our preliminary findings in vitro suggest a role for CDAN1 in chromatin remodeling and transcriptional regulation during hematopoiesis. A complete knockout of CDAN1 resulted in embryonic lethality in mice, suggesting a deeper role for CDAN1 in overall development. We thus propose to produce a viable animal model for studying the role of CDAN1 in hematopoiesis in order to study CDA-1. Due to its ease in genetic manipulation, we aim to generate a stable mutant knock-in zebrafish to investigate the mechanism of CDAN1 function in CDA1.

Codanin-1 is a ubiquitously expressed protein encoded by Cdan1, mutations in which have been implicated in causing Congenital Dyserythropoietic Anemia Type-1 (CDA-1). Of the several mutations in the human Cdan1 gene, the missense mutation R1042W identified in the Israeli Bedoiun tribe was the first identified. Cells expressing mutant codanin- (CDAN1) exhibit typical morphologically identifiable features in erythroblasts, especially binuclearity and internuclear chromatin bridges. The mechanism of how CDAN1 functions under normal and pathophysiological conditions is yet to be fully realized. We showed that human primary cells undergoing erythroid differentiation display constitutive levels of CDAN1 mRNA throughout while those undergoing megakaryopoiesis show diminished expression of CDAN1 mRNA. We have seen that CDAN1 knockdown inhibits erythroid differentiation in human primary cells and that CDAN1 binds to genomic loci of important erythroid genes while regulating expression of some of these. Our preliminary findings in vitro suggest a role for CDAN1 in chromatin remodeling and transcriptional regulation during hematopoiesis. A complete knockout of CDAN1 resulted in embryonic lethality in mice, suggesting a deeper role for CDAN1 in overall development. We thus propose to produce a viable animal model for studying the role of CDAN1 in hematopoiesis in order to study CDA-1. Due to its ease in genetic manipulation, we aim to generate a stable mutant knock-in zebrafish to investigate the mechanism of CDAN1 function in CDA1.

The hematopoietic niche is a dynamic microenvironment made up of multiple cell types and extracellular matrix proteins that interact together to modulate the hematopoietic activity of stem cells (HSC). Osteal macrophages (OM), osteoblasts (OB), and megakaryocytes (MK) are some of the cellular components of this structure. We have recently demonstrated that OMs interact with OBs and MKs and play a major role in HSCs maintenance. We have also established that the cross talk between these different cell types requires direct contact (unpublished data). However, all our previous work has been performed in vitro or under transplantation conditions. The nature of such experimental setup entails the perturbation of the niche microenvironment, losing the spatial information about the niche’s components. Due to this fact, imaging studies are our best alternative to study the functional relationship between the niche’s components and accurately describe its architecture. We intend to develop a tissue clearing and staining protocol that will allow us to visualize the unperturbed hematopoietic niche and reveal crucial spatial information about its components. We will use long bones (femurs) from Fgd5 reporter mice for this purpose. Fgd5 mice express GFP under the control of the Fgd5 gene, that is exclusively expressed in HSCs and endothelial cells. We will employ primary and secondary antibodies, as well as biotin-streptavidin systems, to identify as many different components of the niche as possible. The bones will be imaged using 2-photon and confocal microscopy and analyzed using Imaris and the Volumetric Tissue Exploration and Analysis (VTEA) software, developed in-house by the group of Dr. Kenneth Dunn.

There is an urgent need to identify treatments to reduce the mortality and morbidity associated with COVID-19. Recent autopsy reports have shown up to a 3-fold higher megakaryocyte (MK) number in multiple organs, including the lungs and the heart, in COVID-19 positive (+) patients as compared to COVID-19 negative (-) patients with severe acute respiratory distress syndrome (ARDS).1-3 As many of the serious complications of COVID-19 leading to death include complications associated with hypercoagulability within the pulmonary and cardiac systems, the finding of increased MKs in these organs may be contributing to the mortality, especially in light of the high rate of platelet-rich thrombi observed in multiple organ systems on autopsy reports. Further, a multicenter retrospective study showed that inflammatory cytokines including interleukin-6 and platelet counts >450 x109/L were predictive of thrombosis in COVID-19+ patients.4 Thus, it appears that many patients with COVID-19 have significant pathologic MK and platelet manifestations. COVID-associated cytokine storm is well established among patients,5 and although not reported, thrombopoietin (TPO) may be one of those increased cytokines, such as IL-6 and others. Indeed, TPO is the main MK proliferation and differentiation factor, and we recently found that COVID-19+ patients that died had 523% higher circulating TPO levels compared to healthy controls. Further, although postnatal megakaryopoiesis largely takes place in the bone marrow, it can also take place in mice in several organs including the spleen, liver, and lungs.6 As SDF-1/CXCL12 (stromal cell-derived factor 1) is the most potent physiological chemoattractant for MKs7-9 (MKs express the SDF-1 receptor, CXCR4), we sought to determine whether COVID-19+ patients had elevated SDF-1 levels. Indeed, we found that COVID-19+ patients that died from complications related to the disease had 144% higher circulating SDF-1 levels than healthy controls. Based on these observations, we hypothesize that a “hematopoietic element” consisting of SDF-1 and TPO accompanies the illustrated cytokine storm in COVID and leads to increased MKs and platelets and contributes to morbidity and mortality.

The runt family transcription factor RUNX1 plays a critical role in the emergence of hematopoietic stem cells (HSCs) as well as in the maintenance of normal hematopoiesis. RUNX1 binds DNA through the runt homology domain (RHD) and forms a heterodimeric complex with core binding factor-􀁅 (CBFB), which leads to the recruitment of downstream binding partners to produce epigenetic and transcriptional changes. Despite two decades of study, several limitations remain in our understanding of the role of RUNX1 in adult hematopoiesis, especially in the regulation of hematopoietic stem cell (HSC) self-renewal. Conditionalhematopoietic deletion of mouse Runx1 has been shown to cause impaired lymphopoiesis, erythropoiesis, and megakaryopoiesis, without increasing HSC number or self-renewal. In contrast, somatic missense mutations in the RHD or the dimerization domains of RUNX1 in humans are seen in clonal disorders like myelodysplastic syndrome (MDS), strongly indicating that they promote HSC expansion. In vitro studies indicate that missense mutations produce dominant-negative or neo-functional RUNX1 protein, whose transcriptional consequences may be distinct from those of knockout. However, in vivo studies of Runx1 point mutations have been limited to the generation of mice with germline missense mutations, which cannot be bred to homozygosity due to embryonic lethality. As a consequence, the effects of bi-allelic Runx1 point mutations on adult hematopoiesis have not been studied in vivo. We hypothesize that conditional hematopoietic point mutations in Runx1 will produce phenotypes distinct from those of conditional hematopoietic Runx1 deletion, and will lead to HSC expansion and increase in HSC self-renewal. In this proposal, we aim to generate conditional DNA-binding-defective and heterodimerization-defective Runx1 mutant mice. Using inversion Cre-lox system, we will work with the Boston Children’s Hospital Mouse Embryonic Stem Cell and Gene Targeting Core to generate two mouse strains with conditional hematopoietic Runx1R188Q/R188Q and Runx1G122R/G122R genotypes, which will impair RUNX1 DNA binding and dimerization, respectively. To determine the consequence of these mutations on hematopoiesis, we will study survival, blood counts, bone marrow composition, HSC function, and perform epigenetic and transcriptional profiling studies on selected hematopoietic subpopulations. Our work will shed light on dominant-negative or neo-functional roles of point mutant RUNX1 in hematopoiesis, and especially on the role of RUNX1 in HSC self-renewal.

Heme, an iron-containing organic ring, is a vital cofactor responsible for diverse biological functions and is the major source of bioavailable iron in the human diet. The current dogma states that mammalian heme levels within cells are controlled by balance between heme biosynthesis and heme catabolism. In the past few years, the Hamza lab has led the field by contributing to the discovery of eukaryotic heme transporters and heme trafficking pathways. Our overarching, paradigm-shifting hypothesis is that cellular heme levels are not only maintained by internal heme synthesis (cell-autonomous), but also by distally located proteins which signal systemic heme requirements to a heme trafficking network (cell-nonautonomous). Although emerging evidence supports the existence of a cell-nonautonomous heme communication system in mammals, this concept has remained unexplored. Our recent preliminary studies have revealed an unexpected and novel finding that adult mice under heme depletion and acute need for red blood cells production upregulate erythroid HRG1 expression. This strongly suggests that heme homeostasis during erythropoiesis involves previously unappreciated mechanism(s). Thus, the overall goal of the proposed studies is to use stress erythropoiesis condition (chronic phlebotomies) in different mouse model (WT, HRG1 KO, HRG1EpoR/EpoR mice) to elucidate whether HRG1 heme importer could participate in hemoglobinization of red blood cells. We plan to (a) elucidate the role of HRG1 in erythroid progenitors, and (b) identify the heme-responsive cell-nonautonomous pathway using RNA-seq in WT and HRG1-KO mice.

Iron is an essential micronutrient required for many biological processes, including erythropoiesis, but excess iron is toxic due to its ability to generate reactive oxygen species. Aberrant regulation of the master iron hormone hepcidin is a major underlying cause of most iron disorders including anemia of chronic disease, iron refractory iron deficiency anemia, beta-thalassemia, and hemochromatosis. Hepcidin functions by binding and inducing degradation of the sole iron exporter ferroportin to reduce iron influx into the circulation from dietary sources and body stores. Liver hepcidin expression is regulated by several different signals that indicate whether the body needs more or less iron, including serum and tissue iron levels, erythropoietic drive, and inflammation. Previous studies have discovered that the bone morphogenetic protein (BMP)-SMAD signaling pathway is a central regulator of hepcidin transcription in response to most of its known signals. However, there are many facets of hepcidin regulation that are still not understood, including how hepcidin is regulated by homeostatic iron regulator HFE. Mutations in HFE are the most common cause of hereditary hemochromatosis, an autosomal recessive iron overload disorder with a prevalence of 1 in 300 individuals in the United States. Although HFE mutations were linked to hereditary hemochromatosis over 20 years ago, the precise mechanisms of action of HFE in hepcidin regulation are still unclear. The current working model is that HFE deficiency leads to impaired SMAD signaling responses to BMP ligands. In the current proposal, preliminary data will be presented using a novel mouse model demonstrating that HFE also regulates hepcidin through a SMAD-independent pathway. Herein, I propose to perform bulk RNA sequencing (Seq) and single cell RNA-seq in this mouse model as a discovery approach to identify the unknown SMAD-independent signaling pathway(s) governing hepcidin regulation by HFE. This work holds the promise to shift the paradigm in understanding the mechanism of action of HFE and to discover a novel signaling pathway governing hepcidin and iron homeostasis regulation that may ultimately identify new therapeutic targets for treating iron disorders.

Diabetes increases risk for cardiovascular diseases. Chronic inflammation and oxidative stress appear to be underlying mechanisms of diabetic dysfunction in cardiovascular tissues. Systemic inflammation is due to myelopoietic bias in the stem/progenitor cells and the resulting increased generation of pro-inflammatory monocyte-macrophages. TERT is a subunit of telomerase that is responsible for telomere maintenance and chromosomal stability. Independent of telomerase, TERT regulates hematopoiesis and mitochondrial functions. Our preliminary studies discovered that diabetic CD34+ hematopoietic stem/progenitor cells (HSPCs) express one or more deletion variants of TERT, α-, β- or αβ-variants, while nondiabetic cells do not. Deletion variants of TERT are known to oppose full-length TERT function. The presence of variants correlated with decreased telomerase activity and elevated mitochondrial reactive oxygen species (mitoROS) in diabetic CD34+ cells compared to the nondiabetic. Furthermore, we found that silencing of TGF1 blocked TERT-splicing that accompanied decreased mitoROS and reversed myelopoiesis. This pilot proposal tests the hypothesis that splice variants of TERT mediate increased myelopoiesis and mitochondrial oxidative stress in diabetic CD34+ HSPCs. Splice variants will be silenced by using morpholino-oligonucleotide sequences complementary to specific intron-exon or exon-intron boundaries of the TERT pre-mRNA sequence of TERT, which block the alternate splice sites α- (intron 5/exon 6) and β- (intron 6/exon 7). We will test the impact of this molecular modification on myelopoietic potential in ex vivo and in vivo. A novel mouse model NSGS mouse that support myelopoietic differentiation of human CD34+ cells will be used. Beneficial effects of the TERT variants’ knockdown on mitochondrial functions including oxidative stress, metabolism – glycolysis and oxidative phosphorylation, and mitoDNA damage will be evaluated. This pilot study will enhance our understanding of TERT physiology in diabetic HSPC.

RNA binding motif protein 15 (RBM15) is a key regulator of N6-methyladenosine (m6A) epitranscriptome modification and is essential for recruitment of the m6A writer protein complex to target RNAs. It has been shown that RBM15 is important for hematopoietic stem cell (HSC) maintenance and quiescence, but its role in the fate specification of hematopoietic progenitor cells (HPCs) remains poorly understood. RBM15 is part of the recurrent t(1;22) translocation associated with infantile acute megakaryoblastic leukemia; therefore, investigation into its role in megakaryocyte differentiation and maturation is warranted. Understanding the mechanistic role of RBM15 in the process of megakaryocyte maturation will not only provide potential avenues of investigation for treatment of AMKL but will also shed light on the role of this protein and the m6A epitranscriptome in megakaryopoiesis. This proposal aims to understand the transcriptomic targets of RBM15 (Aim 1) and the m6A epitranscriptomic modifications regulated by RBM15 (Aim 2) in a model of megakaryopoiesis. Using enhanced crosslinking and immunoprecipitation sequencing techniques, we will characterize these targets at single nucleotide resolution. Comparison to RNA-seq will provide insight into the consequences of RBM15 binding and modification on target transcripts. By determining the exact nucleotide bound by RBM15 and the exact adenosine modified nearby, we will gain detailed insight into the epitranscriptome dynamics involved in hematopoietic differentiation. Completion of this project will illuminate the transcriptome and epitranscriptome interactions of RBM15 and will provide candidate genes that may be critical for downstream mechanistic effects driving megakaryopoiesis. By filling gaps in the field, this work will lay the foundation for further investigation of the epitranscriptome in hematopoietic fate decision.

Due to its presence in proteins involved in hemoglobin synthesis, DNA synthesis and mitochondrial respiration, eukaryotic cells require iron for growth and proliferation. Regulation of cellular iron content is crucial: excess cellular iron catalyzes the generation of reactive oxygen species that damage DNA and proteins, while cellular iron deficiency causes cell cycle arrest and cell death. Dysregulation of iron homeostasis caused by iron deficiency or iron excess leads to hematological, neurodegenerative and metabolic disorders. Vertebrate iron metabolism is controlled post-transcriptionally by iron-regulatory protein 2 (Irp2). Irp2 binds to iron-responsive elements (IREs) in the mRNAs of proteins involved in iron uptake (transferrin receptor 1), sequestration (ferritin) and export (ferroportin), and regulates the translation or stability of these mRNAs. Our previous work show that Irp2 is regulated by iron-dependent proteolysis by the FBXL5 SCF-ubiquitin ligase. We also discovered a novel iron-independent mechanism for regulating Irp2 RNA-binding activity during the cell cycle. Irp2 is phosphorylated at S157 by Cdk1/cyclin B during G2/M and dephosphorylated by Cdc14A during mitotic exit. Irp2-S157 phosphorylation blocks its interaction with RNA to increase ferritin synthesis and decrease TfR1 mRNA stability during mitosis. The significance of S157 phosphorylation was investigated in mice where Ser157 was mutated to Ala157 (Irp2A/A)). Irp2A/A mice display anemia, defective erythroid terminal differentiation and dysregulated systemic iron metabolism. Our overall objective is to determine the role of Irp2-S157 phosphorylation in hematopoiesis. Our goal here is characterize the anemia in WT and Irp2A/A mice. For these studies, we propose to utilize the University of Utah Center for Iron & Heme Disorders Core for quantification of iron and heme content in tissue samples from WT and Irp2A/A mice.

Bone marrow (BM) is the primary site of hematopoiesis in mammals and where hematopoietic stem cells (HSCs) reside. As the age advances, the BM architecture changes and the hematopoietic potential of HSCs declines. The pathways involved with this decline in HSC regenerative potential are not fully understood. However, we know that from previous studies by our group and others on dissociated bone marrow, the cells of the hematopoietic niche (HN) such as osteomacs (OMs), megakaryocytes (MKs), and osteoblasts (OBs) are involved in maintaining the HSC function. These studies use established techniques such as flow cytometry and immunofluorescence which use dissociated BM either to quantify the cells of the HN or the tissue to visualize up to three cell markers at a time. However, it is important to understand the spatial relationships of the cells of the HN with each other in an unperturbed BM to better comprehend the interactions of these cells in the BM niche and how they change as the age advances. A multiplex high-resolution imaging technology, CODEX, that can visualize up to 60 markers simultaneously can overcome the limitations of traditional techniques to visualize the entire niche. We propose to use the CODEX to image the HN to study the spatial relationships between the cells of HN in young and old mice to better understand the HN architectural landscape and to develop the methodology for CODEX use so it can become a research and investigative tool for other hematology investigators. Plotting the architectural landscape of the BM of both young and old mice will enhance our understanding of the relationship between these cells in the BM niche and to help us develop the methodology and protocols of the CODEX so it can become a research and investigative tool for non-malignant hematology investigators.

This collaborative proposal targets fatty acid oxidation (FAO) to expand human hematopoietic stem cells (HSCs) in chemically defined culture conditions. Independent research paths in the Tong and Klein labs have converged on the unexpected finding that FAO activation enhances HSC function during ex vivo expansion. The Tong lab has found that genetic disruption of the adaptor protein LNK, which expands long-term HSCs >10-fold, activates a metabolic program that increases FAO and reduces oxidative stress. Independently, a high throughput screen in the Klein lab identified drugs that induce FAO through activation of PPARa as potent enhancers of ex vivo HSC expansion. Here we propose to test the hypothesis that FAO activation is required for HSC expansion and that PPARa agonists can be used for therapeutic HSC expansion ex vivo. Investigation of metabolic regulators of hematopoiesis is a new area of research for both the Tong and Klein labs that could have substantial therapeutic impact. The significance of this proposal includes: 1. The ability to expand human HSCs in umbilical cord blood units will substantially increase the number of available HLA matches for patients needing HSC transplant, notably patients with myelodysplastia, advanced myeloproliferative disorders, and bone marrow failure. 2. Gene editing techniques for the treatment of inherited blood disorders are limited by the loss of long term (LT)-HSCs during ex vivo manipulations; our low cytokine culture conditions maintain LT-HSCs ex vivo without loss of reconstituting activity, providing an ideal platform for therapeutic gene editing. At a basic science level, our preliminary data and published work from others identify a role for FAO in the maintenance of LT-HSCs. However, the mechanisms by which FAO enhances HSC function have not been explored. Furthermore, while PPARa agonists are widely used clinically to treat hyperlipidemia (e.g. gemfibrozil/lopid), these drugs have not previously been applied to therapeutic HSC expansion. Our data are too preliminary for an R01 submission; these high risk/high gain experiments will require transplantation studies in mice and additional metabolic analyses to support a collaborative R01 submission to the NIDDK in the future.

Proliferation followed by differentiation of the granulocyte and macrophage progenitors (GMPs) sustains the rapid cellular turnover of the myeloid lineages. Their essentiality is highlighted by the side effects of many chemotherapy drugs designed to target the proliferating cancer cells. This highly proliferative state, however, is transient for a given GMP cell, which exits from the proliferative state upon differentiation. My lab previously found that GMPs divide every 6-8 hours at their peak proliferative state, and this state lasts for ~1-2 days, at least in vitro, when cell cycle slows upon differentiation. These proliferative progenitors possess enormous regenerative potential; agents that can tap into this regenerative power without transformation would provide ample therapeutic opportunities. In our search for such agents, I discovered a small molecule to display such activity, yielding orders of magnitude more expansion without impairing subsequent differentiation. My proposal is to identify the molecular target(s) for the small molecule and to test whether it can promote hematopoietic recovery following injury in vivo. We have teamed up with renowned Yale experts in medicinal chemistry and target identification, Dr. Craig Crews, and have obtained critical evidence to support feasibility using a targeted proteomic approach to unveil the molecular target(s)/mechanism for the small molecule. In a second aim as supported by encouraging preliminary results, I will administer the small molecule to mice post irradiation and 5’ fluorouracil to test whether it promotes hematopoietic/myeloid recovery post injury. Overall, the experiments in this proposal could unveil a novel class of chemical agents with pharmacologic potential for hematopoietic regeneration.

The bone marrow (BM) microenvironment directs the self-renewal, differentiation, and homeostasis of hematopoietic stem cells and progenitors. Aging-associated perturbations in the BM niche, attributed in large part to alterations in regulatory cues within the local extracellular matrix (ECM), contribute to declining hematopoietic function. However, interactions between hematopoietic cells and the local ECM remain largely uncharacterized, and the contribution of changes in the spatial organization of cell-matrix crosstalk to diminished hematopoiesis is not understood. Our recent studies have indicated that the ECM produced by elderly BM stromal cells negatively affects the function of the BM niche and entails significant alterations in the matrix proteome relative to that in young BM. The study intends to identify aging-related changes in the spatial organization of key matrix proteins and their relationship with BM cell types. This will be achieved through high resolution mapping of cell-matrix interactions in BM of young and elderly mice using PhenoCycler multiplex imaging (CODEX). Ultimately, this research seeks to unravel the fundamental mechanisms by which aging related alterations in matrix architecture impair the function of the BM niche, with potential implications for addressing aging-associated hematological disorders and repairing the damaged or dysregulated BM niche.

The purpose of this Pilot & Feasibility Grant is to elucidate the role of High Mobility Group A1 (HMGA1) chromatin regulators in clonal hematopoiesis (CH) and associated cardiovascular disease (CVD). CH results from expansion of hematopoietic stem cells (HSCs) harboring a mutation that provides a fitness advantage. In addition to myeloid malignancies, CH mutations are associated with increased risk of diverse cardiovascular diseases (CVD), including venous thromboembolism (VTE), atherosclerosis (AS), coronary artery disease (CAD), myocardial infarction (MI), and stroke, all independent of typical CVD risk factors such as hyperlipidemia, obesity, and diabetes. Importantly, incidence of CH is rising as our population ages and there is an unmet need for treatments to prevent CVD associated with CH. Indeed, individuals harboring CH mutations in TET2, DNMT3A, ASXL1, or JAK2 have an almost 2-fold higher risk for CVD; however, the underlying mechanisms are only beginning to emerge and further studies to identify therapeutic targets are needed. Here, we take a novel approach by focusing on the HMGA1 chromatin regulators in CVD associated with CH. Our premise that HMGA1 plays a critical role in CVD associated with CH is based on the following preliminary data: 1) HMGA1 overexpression induces clonal expansion in adult stem cells by amplifying genes involved in inflammation and proliferation1. 2) In JAK2V617F transgenic mouse models of CH and myeloproliferative neoplasms (MPN), loss of just a single Hmga1 allele in hematopoietic stem cells (HSCs) dampens thrombocytosis, erythrocytosis, and neutrophilia, all of which are linked to CVD. 3) Moreover, HMGA1 deficiency prevents splenomegaly, megakaryocyte hyperplasia, and progression to myelofibrosis (MF). 4) Importantly, MF is characterized by excessive inflammatory signals from megakaryocytes and other progenitors, all of which could fuel CVD1. 5) HMGA1 also expands HSC with a monocyte-bias and increases IL6 expression, which are also linked to atherosclerosis (AS) and CVD. Together, these exciting results led us to the following hypotheses: 1) HMGA1 is required for the development of AS and other manifestations of CVD in CH through specific transcriptional networks, and, 2) HMGA1 networks expand specific stem and progenitor populations that increase pro-inflammatory signals. To begin to test this, we propose a strategic collaboration with Dr. Krause at YCCEH to elucidate aberrant inflammatory cytokine/chemokine networks induced by HMGA1 in well-established Tet2 mutant mouse models of atherosclerosis (AS) with the following Aims:

A) To identify HMGA1 transcriptional networks and the cell(s) of origin for Hmga1-driven, aberrant inflammatory signaling in Tet2 mutant CH using single cell RNA sequencing (scRNAseq) and,

B) To define HMGA1-dependent cytokines and chemokine pathways in Tet2 mutant mice with AS. Our focus will be on cytokines and networks that could be targeted in therapy. Thus, our collaborative studies with Dr. Diane Krause (Director of the Yale CCEH and expert in hematopoiesis), will not only reveal mechanistic insight into disease-related cytokine pathways in CH, but we also expect to discover new actionable mechanisms to prevent progression.

Coordinated checks and balances maintain steady rates of red blood cell production and allow for the rate of production to accelerate in contexts of acute anemia. The mechanisms driving this accelerated rate and phenotypic diversity of anemia responses in human populations are poorly understood. The aims of this proposal will develop new tools and methods to interrogate DNA elements controlling acceleration of stem cell activities in anemia model systems. We previously implemented a multifactor prioritization strategy to identify and functionally interrogate cis-regulatory elements involved in recovery from acute anemia. This analysis revealed important transcriptional control of newly identified anemia induced genes, including the Ssx-2 interacting protein (Ssx2ip). Over a week-long time course of acute anemia recovery, we observed erythroid gene activation mediated by GATA factor occupied cis-elements occurs during a phase coincident with low hematocrit and high progenitor activities. However, this analysis also revealed distinct transcriptional programs activated at earlier and later time points. Specifically, our analysis identified AP-1 transcription factor activity is transiently increased at 24 hours post-anemia, but then rapidly turned off. Despite established roles for AP-1 in inflammatory responses, the role of this crucial transcription factor in anemia response mechanisms has not been studied. We hypothesize that AP-1 transcription factors are required to initiate transcriptional programs in HSPCs and erythroid precursors to mediate effective anemia recovery. This pilot project will explore and develop preliminary data to test mechanistic models for dynamic transcriptional control in anemia recovery. We will use multiple facilities sponsored by the Cooperative Centers of Excellence in Hematology (CCEH) to acquire quality controlled human hematopoietic stem/progenitor cells (HSPCs) and for next-generation sequencing of ATACseq samples. We will also initiate a new collaboration with Dr. Kellie Machlus to investigate erythroid precursor function in bone marrow-like organoid systems. These aims will test mechanisms of AP-1 mediated transcriptional activation in anemia and develop better tools for studying dynamic changes throughout the anemia recovery timeline. This research direction represents a new avenue for our lab to study AP-1 activity. By testing the role of enhancer mediated mechanisms required for anemia recovery, these findings will have important implications for understanding the genetic requirements for erythropoietic progenitors’ activities in red blood cell disorders. The proposed experiments are expected to reveal unique enhancer-responsive mechanisms with broad biomedical implications in hematologic disease, including myelodysplasia, bone marrow transplantation, and chronic anemias.

Regulation of cellular iron content is crucial: excess cellular iron catalyzes the generation of reactive oxygen species (ROS) that damage DNA and proteins, while cellular iron deficiency causes cell cycle arrest and cell death. Dysregulation of iron homeostasis caused by iron deficiency or iron excess leads to common hematological, neurodegenerative and metabolic disorders. Vertebrate iron metabolism is controlled post-transcriptionally by iron-regulatory protein 2 (Irp2). Irp2 binds to iron-responsive elements (IREs) in the mRNAs of proteins involved in iron uptake (transferrin receptor 1, TfR1), sequestration (ferritin) and export (ferroportin), and regulates the translation or stability of these mRNAs. Our previous work show that Irp2 is regulated by iron-dependent proteolysis by the FBXL5 SCF-ubiquitin ligase. We also discovered a novel iron-independent mechanism for regulating Irp2 RNA-binding activity during the cell cycle. Irp2 is phosphorylated at serine157 by Cdk1/cyclin B during G2/M and dephosphorylated by Cdc14A at mitotic exit. S157 phosphorylation inhibits Irp2 RNA-binding activity during mitosis to increase ferritin and decrease TfR1 expression. Our studies show that expression of a Irp2-S157A mutant in Irp2-deficient mouse embryonic fibroblasts causes a G2/M delay and slows proliferation. The physiological significance of S157 phosphorylation was investigated in mice where S157 was mutated to Ala157 (Irp2A/A)). Irp2A/A mice display normocytic normochromic anemia, dysregulated systemic iron metabolism, defective erythroid terminal differentiation and splenomegaly. Analysis of the proteome in WT and Ter119+ cells reveals significant changes in metabolic enzymes and other proteins in Irp2A/A Ter119+ cells, suggesting that metabolites are altered in Irp2A/A Ter119+ cells. Our objective here is to characterize the metabolomes of WT and Irp2A/A Ter119+ erythroblasts. These studies will provide a comprehensive view of the proteome and metabolome of erythropoiesis in WT and Irp2A/A mice. For these studies, we propose to utilize the University of Utah Center for Iron & Heme Disorders Core (CIHD) Metabolomics Core for metabolomic analyses.

Recent advances in gene therapy and editing approaches allow for sickle (SCD) patients to be treated withtheir own therapeutically modified stem cells. To ensure that these emerging therapies are maximally effective, a better understanding of SCD hematopoietic stem and progenitor cells (HSPC) and how their unique expression profile relates to stem cell function, will be instrumental. CD34 is a marker expressed on a broad spectrum of HSPCs and is used in transplantation settings as an indicator of transplantation success due to its positive correlation with stem cell engraftment. In a SCD setting however, CD34 is an unreliable marker for HSC frequency and transplantation success as SCD CD34+ cells show unexpected coexpression of lineage markers, indicating massively expanded progenitor compartments with often unknown functional relevance. This complicates quantification of HSCs based on CD34 expression and could lead to overestimation of the HSCs present, resulting in delayed engraftment and prolonged cytopenia. These findings highlight the need for additional characterization of the SCD HSPC fraction in a way that supersedes standard immunophenotyping efforts and allows for a more accurate quantification of functional HSCs. In addition, whereas existing profiling efforts have focused on peripheral blood or bone marrow, current gene therapy and editing approaches rely on plerixafor-mobilized HSPCs and this stem cell source remains understudied. We hypothesize that in SCD, a chronically inflamed bone marrow niche impacts HSC function, resulting in a unique signature. By connecting functional engraftment data to RNA and cell surface marker expression profiles of plerixafor-mobilized SCD HSPCs, we aim to further define this SCD-specific HSPC signature and how it intersects with stem cell functionality, to improve clinical HSC quantification. Here, we will harness the tools and expertise that we recently built when establishing a molecular blueprint of the most functional HSCs. Applying these in a SCD context, we will map the cell surface marker co-expression profile of SCD HSPCs versus healthy control HSPCs using a comprehensive spectral flow cytometry antibody panel, including EPCR, which we recently identified as a marker for the most functional HSCs. To acquire further resolution of the HSPC fraction, we will connect cell surface marker co-expression patterns to RNA expression via CITE-Seq. The resulting integrated multi-modal data set will then be linked to engraftment potential via xenotransplantation assays in NSG mice to understand how the unique SCD HSPC signature intersects with HSC functionality. Moreover, as SCD patients with advanced disease are less successful at mobilizing sufficient CD34+ cells for cell therapy, finding ways to expand the number of functional HSCs collected will be important to make gene therapy and editing approaches accessible to the patients most in need of such therapies. To aid in advancing this goal, we will extend the functional characterization of SCD HSPCs and assess their response to ex vivo culture conditions aimed at stimulating HSC expansion.

In a recent published study (Shao L et al PNAS 2023), we established that the Jagged1-driven hematopoietic-to-hematopoietic Notch signaling is critical for survival and maturation of fetal liver (FL) hematopoietic stem cells (HSCs). Our transcriptomic analysis of FL HSCs identified several cell fate identity genes, several of which are well-known hematopoietic factors (GATA2, Mllt3 ect.) that are negatively affected by loss of hematopoietic Jag1 in FL HSCs. We are now embarking on a new study to determine what are the FL niche-specific factors that drive HSC expansion. For this, we specifically focused on secreted factors that can impact extracellular aspects of the FL microenvironment. We identified neutrophilic granule protein (NGP), a Cathelicidin-family, anti-microbial peptide (CAMP) family member, as a direct Notch target gene that is highly expressed by FL HSCs, multipotent and myeloid progenitors during FL development, but not expressed by endothelial, stromal and hepatic cells. We propose to determine their functional role by generating a conditional transgenic deletion of NGP and in combination with CAMP in hematopoietic FL cells. For this we will generate an NGP conditional knockout mouse in the C57Bl/6 background. We will then assess the developmental or post-natal requirement during hematopoietic development survival and function in the fetal liver, neonate liver and in a transplant setting to irradiated adult mice.

The knowledge concerning long-term genotoxic effects of CRISPR/Cas9 genome editing in hematopoietic stem and progenitor cells (HSPCs) is limited. With a focus on therapeutically relevant targets for sickle cell disease and other blood disorders, I plan to address critical knowledge gaps regarding the persistence and consequences of chromosomal abnormalities induced by intentional DNA damage during genome editing. Our published data have demonstrated that genome editing can lead to micronucleus formation in HSPCs, indicating chromosomal instability. In our preliminary studies, we’ve made several key observations. We’ve successfully optimized genome editing approaches for pharmacologic and FACS enrichment of HSPCs in the event of segmental chromosome loss. We also observed that HSPCs with micronuclei can undergo mitosis. This indicates both before and after genome editing, cell cycle checkpoints to prevent persistent chromosomal abnormalities are failing. These preliminary findings suggest complex interactions between DNA repair pathways, cell cycle regulation, and apoptosis in maintaining genomic stability after genome editing. Drawing from these observations, we hypothesize that unresolved DNA damage from genome editing can lead to persistent chromosomal abnormalities, including complex events like chromothripsis, and that the mechanisms preventing such abnormalities involve intricate regulation of DNA repair and cell death pathways. Our overall goals are two-fold: first, comprehensively characterize the spectrum and persistence of chromosomal abnormalities induced by therapeutic genome editing in HSPCs, both in vitro and in vivo; and second, to study the mechanisms underlying micronucleus formation and resolution after genome editing. By achieving these goals, we aim to enhance our understanding of the long-term safety of CRISPR-based therapies and potentially inform strategies to improve the genomic stability of edited cells for clinical applications.

As primitive erythroid precursors proliferate and differentiate, they come into contact with various cell types and their secreted factors. However, it is poorly understood how these erythroblast-microenvironment interactions promote the first wave of erythroid differentiation in the embryo. A detailed knowledge of contributions by the erythroid microenvironment will not only help identify factors that could ameliorate anemias of different etiologies but also inform approaches to improve the efficiency of generating red blood cells in vitro for cell-based transfusion therapies. We have used zebrafish as a powerful in-vivo model with the overall goal to understand the role of metabolism in erythropoiesis. Our studies have revealed the essential mitochondrial de novo pyrimidine synthesis enzyme dihydroorotate dehydrogenase (DHODH) as a critical driver for the differentiation of primitive erythroid precursors. Inhibition of DHODH leads to an erythroid differentiation block that we found can be rescued by selectively modulating other central metabolic pathways. Importantly, multiple lines of evidence from our laboratory suggest that DHODH’s function in erythroid lineage progression is partially dependent on the embryonic microenvironment. We hypothesize that compromised de novo pyrimidine synthesis and the resulting metabolic and potentially cellular alterations in the erythroid microenvironment contribute to the block in primitive erythropoiesis and thus anemia. To test our hypothesis, we propose to combine single-cell RNA-sequencing (scRNA-seq) and spatial transcriptomics to interrogate the erythroid microenvironment in conditions of inhibited and normal de novo pyrimidine synthesis and additionally modulated metabolic pathways. While scRNA-seq yields deeper-level information about cellular transcriptomes in individual cells, spatial transcriptomics, despite lower depth and resolution, avoids tissue dissociation and maintains spatial transcript information. We will perform targeted scRNA-seq experiments at two developmental timepoints (6- and 18-somite stage) upon simultaneously inhibiting DHODH and interfering with specific other central metabolic pathways compared to single or no perturbations. Complementary to these studies, we will define specific spatial gene expression changes in the erythroid microenvironment in zebrafish embryo sections at the same developmental stages and in the same conditions. Integrating these two datasets will yield information about altered cell type dynamics resulting from changes in pyrimidine metabolism and its intersection with key central metabolic pathways and will allow us to deduce localized metabolic and related cellular changes from transcriptional changes. Experimental design and data analysis will be facilitated by the Cooperative Centers of Excellence in Hematology (CCEH) Core at Cincinnati Children’s Hospital Medical Center. Our studies will, for the first time, identify critical cell types and infer their transcriptional-metabolic signatures in the erythroid microenvironment that support erythroid differentiation. This research direction constitutes a new avenue for our lab that will strongly support our long-term goal to elucidate the role of metabolism in erythropoiesis. Moreover, the proposed project will deliver critical preliminary data for a competitive R01 grant application.

Acute porphyrias are rare metabolic disorders marked by episodes of severe abdominal pain,

neurological issues ranging from anxiety and confusion to seizures or paralysis, and extreme light sensitivity. These disease “attacks” are due to inherited mutations in enzymes of the heme biosynthesis pathway and the consequent accumulation of toxic heme precursors, including cyclic tetrapyrroles called porphyrins that generate free radicals when exposed to light. The basic science of acute porphyrias has primarily been studied in cultured cells and mouse models. However, the deep evolutionary conservation of the heme biosynthesis pathway as well as the photosensitizing effects of porphyrins also present opportunities to explore mechanisms of disease pathogenesis in nontraditional systems. We recently showed planarian flatworms generate porphyrins in the pigment cells of their skin, due to a physiological heme biosynthesis bottleneck. This leads to rapid ablation of the pigment cells in response to prolonged light exposure. In humans, dieting or fasting induces expression of the first and rate-limiting pathway enzyme, 5-aminolevulinic acid synthase (ALAS), thereby exacerbating the buildup of toxic intermediates in the presence of a downstream blockage. Remarkably, we found this effect is conserved in planarians, establishing a novel and experimentally tractable acute porphyria model. Here, we propose to use ultra performance liquid chromatography (UPLC) assays performed by the Center for Iron and Hematology Disorders (CIHD) at the University of Utah to gain further insight into the biochemistry of planarian porphyrins. Specifically, we will: 1) seek to identify the major porphyrin(s) and/or derivative(s) produced in two different planarian species; and 2) utilize defined laboratory diets to examine the impacts of changes in carbohydrate and protein consumption, as well as total caloric intake, on ALAS activity in tissue lysates. Together with another ongoing line of research in our lab, these experiments stand to provide new insight into nutrient-sensing pathways mediating dietary impacts on porphyrin/heme biosynthesis. In turn, this has the potential to advance our understanding of the pathophysiology of acute porphyrias and to reveal new avenues for treating these disorders.

Hematopoietic Stem and Progenitor Cells (HSPCs) are regulated by the combinatorial action of group of master transcriptional regulators. Their coordinated activity dictates the fate of HSCs and multipotent progenitors towards self-renewal vs differentiation. This precise control by transcription factors (TFs) needs optimal TF concentration in each cell. To date, our understanding of TFs is obtained from studies of RNAi, knockout, or overexpression. However, there is a knowledge gap in understanding how dosages of critical transcription factors modulate HSPCs. Targeted protein degradation is an attractive approach to study the role of TF dosages in regulating HSPC cell states, however this approach has largely been deployed thus far in cell lines, which cannot capture all aspects of primary cell biology. The goal of this proposal is to A) Integrate FKBPV degrons into endogenous loci of TFs SPI1, MYB, and GATA2 in human CD34+ cells, and 2) Perform dose-dependent degradation of SPI1, MYB, and GATA2 and study their effect on proliferation and lineage commitment of human CD34+ cells. I am thus proposing to engineer a degron-based model system in human CD34+ cells, which will allow me to study the dosages of endogenous TFs, and establish a valuable kit that can be extended towards broader studies in human hematopoiesis.

My mentor’s research focused on understanding the role of cell-type specific TFs in the rRNA transcription and ribosome biogenesis in hematopoiesis. For this project, my PI received an NIGMS grant and I received an ASH Scholar Award. However, my future independent research will focus on engineering bi-allelic degron systems in human primary hematopoietic cells to study the dosage-dependent roles of key hematopoietic regulators. This necessitates proficiency in HSC culturing and maintenance. I take this award as an opportunity to learn 1) Long-term ex vivo culturing of CD34+ cells and assays related to HSC differentiation and 2) Hands-on training to prepare non-integrating AAV virus. This training will provide key tools to begin my independent research lab.

Also, research data derived from this pilot study will provide preliminary data to lay a strong foundation for my future R01 grant application on developing primary human ex vivo and mouse in vivo HSC degron models to study bone marrow failures.

Hematopoiesis is the continuous process of blood cell production sustained by a rare population of hematopoietic stem cells (HSCs) that reside in the adult bone marrow. HSCs possess the ability to both self-renew and undergo lineage-restricted differentiation upon receiving cues from the microenvironment. To date, most of the studies have focused on the roles of transcription factors and chromatin regulators and how they modulate gene expression programs during hematopoietic differentiation. The role of post-transcriptional pathways has gained significant attention following the discovery that splicing factors are frequent targets of somatic mutations in in age-related blood disorders such as clonal hematopoiesis (CH) and myelodysplastic syndromes (MDS). These discoveries suggest that RNA splicing regulation is a key determinant of hematopoietic homeostasis and pathology. Moreover, we currently do not understand the full repertoire of mRNA isoforms and their functionality in lineage commitment and differentiation, and the identity of pathologic isoforms that drive CH and MDS. In this proposal, we aim to leverage novel sequencing and computational approaches to capture global splicing alterations in normal and dysplastic hematopoiesis. Additionally, we will validate the functionality of newly discovered isoforms using high-throughput assays. Successful completion of this pilot study will provide critical insight on how aberrant isoform usage and heterogeneity fuels the decline of HSC function in age-related blood disorders.

Hematopoietic stem cells (HSCs) maintain life-long hematopoiesis through the balance act between self-renewal and differentiation. Quiescent HSCs have higher self-renewal capacity and increased repopulation potential compared to activated/cycling HSCs. Therefore, a better understanding of HSC quiescence mechanisms will provide critical insights into the development of new strategies to increase self-renewal capacity of HSCs for clinical application. The overarching goal of this project is to find new strategies to increase self-renewal capacity of HSCs ex vivo so we can increase the availability of rare HSC population for transplantation as well as for gene therapy. We found that HSCs can secrete cytokines and their secretion profiles change in a context dependent manner. We also found that a proteoglycan protein is uniquely secreted by steady state HSCs among hematopoietic stem and progenitor cells and a proteoglycan protein can increase quiescent HSCs in culture conditions. Based on these findings, this proposal aims to investigate the function of a proteoglycan protein in HSC quiescence and to test whether a proteoglycan protein can be used to increase self-renewal capacity of HSCs ex vivo. In Aim 1, we will investigate whether a proteoglycan protein promotes HSC quiescence and the underlying mechanism using genetic mouse models and transplantation assays. In Aim 2, we will test whether a proteoglycan protein can increase human HSC self-renewal capacity ex vivo using a 3D bone marrow organoid model and transplantation assays. Collectively, this study will identify a novel self-regulatory mechanism of HSC quiescence whereby HSCs maintain their quiescence pool through autocrine by secreting a proteoglycan protein. This study will also help provide new strategies to expand HSCs ex vivo for therapeutic purposes.