P&F Projects 2023-2024

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