P&F Projects 2023-2024

Emanuela Bruscia Abstract Type A 2024

Cystic Fibrosis (CF) is a chronic multisystem disease caused by mutations in the CFTR gene, leading to severe lung damage and hyperinflammation. This study explores the role of pro-inflammatory monocytes and hematopoietic stem and progenitor cells (HSPCs) in sustaining chronic inflammation in CF. Using CFTR knockout mice, we demonstrate that monocytes contribute to persistent neutrophilic lung inflammation and irreversible lung damage, with CF HSPCs exhibiting epigenetic and transcriptional alterations that predispose them to a myeloid-biased and hyper-inflammatory state. We also investigate the potential link between CFrelated

bone abnormalities and altered bone marrow environments, hypothesizing that these abnormalities contribute to the hyper-inflammatory phenotype of HSPCs. Utilizing CODEX technology, we aim to characterize cellular distributions within CF bones and understand how chronic lung inflammation impacts HSPCs and stromal cells. This research provides critical insights into the bone-lung axis in CF, potentially leading to novel therapeutic interventions to improve outcomes for people with CF.

Lab link: https://medicine.yale.edu/profile/emanuela-bruscia/

Rachel Bakyayita Kyeyune Abstract Type A 2024

Hematopoietic Stem and Progenitor Cells (HSPCs; CD34+) are ideal targets for cell and gene therapies due to their capacity for producing blood cells over a lifetime. However, achieving effective transduction in HSPCs is challenging, often requiring high vector doses and extended ex vivo culture times. Efficient transduction involves three key steps: entry of the viral particle into the cell mediated by the envelope protein interacting with cell surface receptors, unloading of viral particle genomic cargo, and integration of this cargo into the host genome. Our goal is to simplify the manufacturing process for efficient LV transduction by reducing vector dose and culture time, thereby lowering costs and increasing accessibility. By optimizing existing technologies, our research aims to make gene therapies more feasible in low-resource settings and advance the development of cost-effective treatments. Traditionally, lentiviral vectors (LVs) pseudotyped with vesicular stomatitis virus glycoprotein (VSV-G) are used for gene therapy. However, VSV-G-pseudotyped vectors interact with low-density lipoprotein receptors (LDL-R),

which are not expressed on resting CD34+ cells, necessitating cell stimulation for receptor expression. Alternative viral envelope glycoproteins such as those from cocal, measles and nipah viruses might facilitate more efficient transduction in quiescent blood cells via alternative receptors. Our preliminary data demonstrate that cocal-pseudotyped LVs outperform VSV-G-pseudotyped LVs, especially when combined with magnetically assisted transduction (MAT), a technique that has been used to enhance gene delivery into cells. This study will investigate hybrid cocal paramyxovirus pseudotyped vector variants that could use the LDL-R as well as receptors like SLAM and CD46, which have been reported to be expressed on unstimulated CD34+ cells. Our preliminary data show that while SLAM is modestly expressed by unstimulated CD34+ cells, CD46 expression is more robust and stable but requires at least 1 day of culture (similar to LDL-R). Here we will compare the transduction efficiency of these hybrid vector variants, with and without MAT, in vitro and in vivo. These results will identify novel LV pseudotypes which can reduce the time and cost of manufacturing CD34+ HSPCs for gene therapy applications amenable in low resource settings.

Lab link: https://www.seattlechildrens.org/research/research-institute/careers/invent-at-seattle-childrens/scholars/

Phillip A Doerfler Abstract Type A 2024

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.

Lab link: https://versiti.org/versiti-blood-research-institute/our-investigators/phillip-a-doerfler

Azad Abstract Type A 2024

Excessive erythrocytosis (EE) is a predominant trait in some high-altitude dwellers suffering from Monge’s disease (or Chronic Mountain Sickness, CMS) but not in other subjects living at the same altitude in the Andes. We took advantage of this human “experiment in nature” and studied both populations (with CMS and without, non-CMS). Subjects with Monge’s disease or CMS constitute a unique population that allows us to study how mechanisms of erythropoiesis can go awry due to high altitude chronic hypoxic conditions. Although EE could be advantageous at high altitude because it increases O2-carrying capacity, this adaptive pattern to high altitude has deleterious effects since blood increases its viscosity and induces serious morbidities, such as myocardial infarction and stroke in young adults. Using the iPS-derived cells from this unique Andean population, we have built an in-vitro model that mimics the hypoxia-induced polycythemia in CMS subjects. We have validated our finding in native CD34+ve cells as well. From whole genome sequencing of over one hundred subjects of CMS and non-CMS subjects, we extracted important candidate genes that play an important role in erythropoiesis at high altitude. One such gene is SENP1, a desumoylase. SENP1 plays an important role in definitive erythropoiesis and SENP1KO mouse are embryonic lethal and die due to severe anemia. We have recently gathered evidence showing that the increase in SENP1 in hypoxia in CMS is critical for the hypoxia-induced polycythemia in CMS. However, there are a number of questions related to SENP1 that are not understood. For example, we do not know how or why SENP1 is up-regulated in hypoxia in CMS but not in non-CMS. How the SNPs (single nucleotide polymorphisms) in the SENP1 region change the interaction with critical transcription factors such as GATA1 and functionally alter the erythropoietic response in CMS subjects. Furthermore, by using bioinformatics fine mapping tool iSAFE (integrated Selection of Allele Favored by Evolution) we have recently identified the likely causal SNP(s) (out of the 95 differential SNPs) that are responsible for upregulation of SENP1 in the CMS cells under hypoxia. Using this method, we will focus on the top ranked differential SNP (rs7959755) in the SENP1 region that could alter its expression. It is interesting to note that these differential SNPs coincide with binding sites of transcriptional factors involved in erythropoiesis such as RUNX2, CTCF, GATA1 and PAX5. Understanding the genetic mechanisms underlying erythropoiesis in both groups of subjects can provide opportunities to study its regulation at the molecular level that in turn can help to develop novel drug targets for red blood cells related 

Lab link: https://pediatrics.ucsd.edu/research/faculty-labs/haddad-lab/team/priti-azad.html

Pajcin Abstract Type A 2024

 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.

Lab link: https://mcph.uic.edu/pajcini-lab/

Ropa Abstract Type B 2024

Hematopoietic cell therapies are life-saving treatments for hematologic disorders. Umbilical cord blood is an important source of donor cells for these treatments, especially for racial or ethnic minority patients who are underrepresented on other allogeneic donor registries. However, its utility is limited by low number of cells found in a single unit. At many transplant centers, high cellularity is a primary criteria for unit selection for use in therapy. However, cord blood units with low total cellularity can be rich in functionally potent hematopoietic stem and progenitor cells, the populations responsible for engraftment and immune reconstitution. Thus, to enhance outcomes for cord blood derived cell therapy, it is critical to 1) identify the most potent cord blood units and 2) find molecular pathways that can be targeted to improve hematopoietic cell potency. Here we will use transplantation outcomes from patients as a direct measure of human cord blood hematopoietic cell potency. We will test if expression of 25 candidate genes identified through mouse model screens are retrospectively correlated with cord blood transplantation outcomes in patients. We will do this by performing transcriptomic and targeted gene expression analyses on small segments of cryopreserved cord blood units that have been retained from units with known clinical transplantation patient outcomes and/or clinical potency assay metrics. This will allow us to examine whether the expression level of our candidate gene panel as well as the global transcriptomic profile of hematopoietic cells can be used to model clinical outcomes. We will further validate the importance of genes that accurately predict positive transplantation outcomes or potency in regulating hematopoietic cell growth and function ex vivo to lay the foundation for future full-scale in vivo and mechanistic studies focused on these genes. This work should reveal a targeted gene panel that can be used to accurately predict the optimal cord blood units for use in treatment and will elucidate genes and gene programs that can be targeted to improve the functional potency of hematopoietic stem and progenitor cells for the overall enhancement of hematopoietic cell therapies and improved patient outcomes. While this work focuses on cord blood transplantation, this approach is broadly applicable to hematopoietic cell therapies from varied donor sources.

Lab link: https://medicine.iu.edu/faculty/43997/ropa-jim

Chavez Stolla Abstract Type B 2024

Autophagy is a cellular recycling pathway that is essential for the maintenance and differentiation of hematopoietic cells. Recently, the selective autophagy receptor Optineurin (OPTN) was shown to regulate mitophagy in human and murine models of Acute Myeloid Leukemia. Thus, selective autophagy receptors represent a new therapeutic target for modulation in hematopoietic disease. Selective autophagy receptors link damaged/dysfunctional organelles or macromolecules to autophagosomes for degradation by the lysosome. Although Optineurin has been implicated in AML, its role in normal hematopoiesis is unknown. OPTN is expressed throughout the hematopoietic hierarchy and is most abundant during erythroid differentiation. Autophagy is essential for erythropoiesis and contributes to the clearance of organelles during the final stages if maturation. Since Optineurin is known to promote mitophagy by linking ubiquitinated mitochondria to autophagosomes, we hypothesize that Optineurin facilitates ubiquitin-dependent mitophagy in erythropoiesis. To date only ubiquitin-independent mitophagy has been identified during erythropoiesis. To test this hypothesis, we seek to characterize the contribution of Optineurin to erythropoiesis (Aim1) and determine the contribution of ubiquitination to mitochondrial clearance (Aim 2). Completing this project will delineate the contribution of Optineurin to normal erythropoiesis. Furthermore, these studies will provide the groundwork for further investigation of Optineurin more broadly in other hematopoietic lineages.

Lab link: https://hemonc.uw.edu/people/massiel-stolla

Vantuytsel Abstract Type B 2024

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.

Lab link: https://sites.bu.edu/vanuytsellab/people/

Mancuso Abstract Type A P&F 2024

Although metabolism is required to sustain the basic needs of all cells, specific metabolic changes also strongly influence stem cell fate and function. Hematopoietic stem cells (HSCs) maintain a low mitochondrial metabolic activity and rely on anaerobic glycolysis to support ATP production during quiescence and depend on oxidative phosphorylation (OXPHOS) for activation and differentiation. Although much has been determined regarding the metabolism of HSC, little is known regarding the relevance of the metabolism in megakaryocyte-erythroid progenitors (MEP) specification. My preliminary data reveal significant metabolic differences between MEP and their downstream lineage committed progenitors. With the goal of elucidating how metabolism regulates/affects MEP fate, I propose to use LC-MS to determine specific metabolic shifts as primary human MEP undergo fate specification to the megakaryocytic and erythroid lineages.

Lab link: https://krauselab.net/members/rubia/

Leibold Abstract Type A P&F 2024

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.

Lab link: https://medicine.utah.edu/internal-medicine/hematology/labs/leibold