P&F Projects 2021

The abstracts for the 2021 P&F projects can be found below. A table summarizing all the P&F projects for 2021 can be found here.




Scalf Abstract Type B P&F 2022


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.




Klein Abstract Type B P&F 2022


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.




Karnik Abstract Type A P&F 2022


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.




Leibold Abstract Type A P&F 2022


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.




Paralkar Abstract Type A P&F 2021


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.





Dadwal Abstract Type A P&F 2021


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.





Srour Abstract Type A P&F 2021


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.





Kupfer Abstract Type A P&F 2021


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.





Kim Abstract Type A P&F 2021


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 <em>Cdan1 </em>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 <em>in vitro </em>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.





Bahr Abstract Type B P&F 2021


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.
 




Karatepe Abstract Type B P&F 2021


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.




Prutsch Abstract Type B P&F 2021


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.





Yasuda Abstract Type B P&F 2021


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.





Boddu Abstract Type B P&F 2021


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.





Paddison Abstract Type B P&F 2021


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&amp;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.





Ghelier Abstract Type B P&F 2021


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.





Montgomery Abstract Type A P&F 2021


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.





Heck Abstract Type A P&F 2021


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.





Haase Abstract Type B P&F 2021


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.
 




Medlock Abstract Type B P&F 2021


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.





DuCamp Abstract Type A P&F 2021


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.
 




Sangkhae Abstract Type B P&F 2021


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.





Perovanovic Abstract Type A P&F 2021


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.





Bonner Abstract Type A P&F 2021


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.





Biancon Abstract Type B P&F 2021


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.
 




Sue Abstract Type B P&F 2021


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.





Zhao Abstract Type B P&F 2021


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.





DuCamp Abstract Type B P&F 2021


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.
 




Seu Abstract Type B P&F 2021


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.





Mayday Abstract Type B P&F 2022


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.





Prasada Rao Jarajapu Abstract Type B P&F 2022


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





Xiao Abstract Type B P&F 2022


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.





Belot Abstract Type B P&F 2022


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.