Doctor of Philosophy, The Ohio State University, 2005, Veterinary Biosciences

The role of human immunodeficiency virus-1 (HIV-1) accessory proteins in vectors is poorly understood. The impact of HIV-1 accessory proteins was analyzed in a murine in utero gene transfer model. PCR analysis revealed comparable levels of gene transfer by vectors, prepared in the presence or absence of HIV-1 accessory proteins, in multiple organs. Analysis of bone marrow-derived hematopoietic progenitor cells revealed successful gene transfer in both vector groups. However, two months after gene transfer, the percentage of transduced hematopoietic progenitor colonies was significantly reduced in animals that received vectors without accessory proteins. These findings suggested that in utero gene transfer to multiple organs were independent of HIV-1 accessory proteins, but required them for sustained gene transfer to hematopoietic progenitor cells. Human T-lymphotropic virus type 1 (HTLV-1) is the etiological agent of adult T cell leukemia. HTLV-1 encodes accessory proteins, including an open reading frame II product p13II. p13II localizes to mitochondria and reduce cell growth in vitro and tumorgenicity in mice, but its function in lymphocytes remains undetermined. Herein, we analyzed functional properties of Jurkat T cells expressing p13II. Our data indicated that p13II expressing Jurkat T cells were sensitive to ceramide- and FasL-induced apoptosis. Furthermore, pre-incubation of the p13II expressing cell with a farnesyl transferase inhibitor, chemical inhibitor of Ras, markedly reduced FasL induced apoptosis, indicating participation of Ras pathway in survival of p13II-expressing lymphocytes. Our data is the first to demonstrate that p13II alters Ras-mediated apoptosis in T-lymphocytes.HTLV-1 p13II's role in the virus infection in vivo remains undetermined. Herein, we analyzed the functional significance of p13II in HTLV-1 infection, using a cell line that expresses an infectious molecular clone of HTLV-1 with a selective ablation of p13II expression (ACH.p13). (open full item for complete abstract)

Committee: Michael Lairmore (Advisor) Subjects: Biology, Microbiology

Doctor of Philosophy, The Ohio State University, 2024, Pharmaceutical Sciences

Cancers and leishmaniasis are distinct diseases, but the effects of each on people and communities are similarly devastating. Cancers cause over 10 million deaths worldwide each year, and are so widespread that nearly every person has lost a loved one to them, myself included. Leishmaniasis primarily affects tropical countries and in many places where access to medical care is limited, and the visceral form of the disease requires medical treatment to increase chances of survival above 5%. Both cancers and visceral leishmaniasis are diseases that the human immune system alone often cannot overcome, so the continued research into treatments is crucial to develop new and better tools to fight against these diseases. This dissertation details drug discovery efforts for two different projects, one against each disease; chapter 1 introduces readers to each disease state, chapter 2 describes the synthesis and biological evaluation of anticancer compounds, and chapter 3 describes the synthesis and biological evaluation of antileishmanial compounds. Following the serendipitous discovery of an antileukemia hit compound with an arylimidamide-azole scaffold, a series of analogs was synthesized to evaluate modifications to the scaffold. A robust structure-activity relationship (SAR) was developed through the synthesis of these compounds, and analysis of this relationship pointed to specific chemical modifications to the scaffold which improved their anticancer potency. Combining these favorable modifications led to compounds with >4-fold improved potency compared to the parent compound. Among the most potent compounds in this iv series was 2.9k, which displayed an IC50 value of 100 nM against the acute myeloid leukemia (AML) cell line OCI-AML3. Promising compounds in this series were then further evaluated for broad anticancer activity, pharmacokinetic properties, and mechanism of action as described in chapter 2. The antileishmanial compounds described in this dissertation (open full item for complete abstract)

Committee: Karl Werbovetz (Advisor); Xiaolin Cheng (Committee Member); James Fuchs (Committee Member) Subjects: Biology; Chemistry; Organic Chemistry; Pharmaceuticals; Pharmacology; Pharmacy Sciences

Doctor of Philosophy, The Ohio State University, 2020, Molecular, Cellular and Developmental Biology

Epigenetic gene regulation enables multicellular organisms to develop from a single cell. Epigenetic modifications refer to stable, yet reversable, changes to the genome that do not alter the DNA sequence. These function to control the accessibility of the genome to transcriptional machinery. DNA methylation is an epigenetic modification critical for control of development and defects are common in diseases such as cancer. Chronic lymphocytic leukemia (CLL) and acute myeloid leukemia (AML) are two of the most common leukemias in adults. Both display a high degree of clinical heterogeneity, and global DNA methylation patterns have identified distinct biological subtypes in each disease. Identification of these patterns requires methods that interrogate the methylation from across the genome. However, because these methods are often too costly and require complex data analysis to process and interpret the results making it difficult to analyze large sample cohorts, I developed a novel method, the Methylation-iPLEX (Me-iPLEX), for analyzing DNA methylation from multiple regions of the genome in a high-resolution and high throughput manner. The epigenetic subtypes observed in CLL largely reflect the natural history of the cell of origin. The efficient nature of the Me-iPLEX enabled me to interrogate the epigenetic subtypes of 1286 CLL patients and examine the prognostic significance of the epigenetic subtype across disease stages and with multiple therapies. The large sample size also enabled me to identify several biological traits associated with the subtypes as well as determining that epigenetic subtypes retained prognostic significance after stratifying by biologically related biomarkers. Past studies analyzing DNA methylation patterns in AML identified patterns associated with common genetic aberrations. These aberrations currently form the basis of our understanding of disease mechanisms and are used to predict treatment response. I analyzed Illlumina genom (open full item for complete abstract)

Committee: Christopher Oakes PhD (Advisor); John Byrd MD (Advisor); Ramiro Garzon MD (Committee Member); Kevin Coombes PhD (Committee Member) Subjects: Bioinformatics; Biology; Genetics; Molecular Biology; Oncology

Doctor of Philosophy, The Ohio State University, 2020, Pharmaceutical Sciences

Despite novel therapeutic agents and approaches, hematopoietic stem cell transplant (HSCT) is currently the only potential curative therapy for those with advanced blood malignancies, including but not limited to acute myeloblastic and lymphoblastic leukemia, and myelodysplastic syndrome (MDS). HSCT involves the transfer of hematopoietic stem cells from a healthy donor to the patient with compromised bone marrow to reestablish healthy bone marrow function. However, overall survival after HSCT remains to be low, despite improvements in human leukocyte antigen (HLA) matching algorithms, treatment protocols and patient care, hence hindering a wider application of HSCT. Therefore, exploring and understanding the factors that contribute to such toxicities is required to improve outcomes. We explored the contribution of common germline non-HLA genetic variants to post-HSCT survival outcomes herein. We took advantage of Determining the Influence of Susceptibility COnveying Variants Related to one-Year mortality af- ter BMT (DISCOVeRY-BMT) study. DISCOVeRY-BMT study is the first genome- wide study of 3,532 donor-recipient pairs, investigating associations with 1-year sur- vival outcomes after matched unrelated donor HSCT. DISCOVeRY-BMT allowed us to work with a unique dataset that consists of adjudicated survival outcomes, many relevant clinical variables and extensive genome-wide genetic data obtained from SNP and Exome arrays. We took three different approaches to explore such associations: (1) candidate gene, (2) endophenotype and (3) genome-wide. As discussed in Chapter 2, we were unable to validate or replicate previous findings that followed candidate gene approach. We showed that these studies were conducted with much smaller sample sizes and heterogeneous patient populations that possibly contributed to false positive findings. However, we also showed that variants selected for exploring such associations did not have an impact on the gene of interest and hen (open full item for complete abstract)

Committee: Lara Sucheston-Campbell PhD (Advisor); Kellie Archer PhD (Committee Member); Moray Campbell PhD (Committee Member); Daniel Kinnamon PhD (Committee Member) Subjects: Bioinformatics; Biomedical Research; Biostatistics; Epidemiology; Genetics; Pharmacology

Doctor of Philosophy, The Ohio State University, 2019, Molecular, Cellular and Developmental Biology

Immune cells of myeloid origin have a unique role in the body's response to non-self entities. The cells, including monocytes and macrophages, carry out a diverse array of functions including phagocytosis, the uptake and presentation of foreign antigens, environmental debris, and damaged cells; the release of cytokines that coordinate acute inflammatory responses; and cytotoxic effector functions that result in the destruction of targets. In acute myeloid leukemia (AML), a differentiation block in the myeloid cell lineage prevents proper maturation of monocytes and macrophages. Instead, leukemic blasts rapidly accumulate and proliferate in the bone marrow, blood, and organs preventing proper hemocytic development. Patient death is caused mainly by infection, followed by hemorrhage and organ failure. The most common form of adult leukemia, AML has a low five year survival rate of 26.6% and a high rate of patient relapse. Taken with the high average age of diagnosis and the fact that certain elderly patients are unable to participate in the standard treatment of high-intensity chemotherapy, it is clear that there is a need for innovative, less toxic therapeutic approaches to the disease. One such approach is the re-invigoration of the patient's own immune system, typically suppressed in a myriad of ways due to the disease. This is explored in two novel studies presented here. The first, detailed in Chapter 2, takes advantage of the effector function that myeloid cells naturally possess; expression of Fcγ receptors on the cell surface allow for interaction with antibody opsonized targets. By eliciting expression of the antigen for the α-CD38 antibody daratumumab on the surface of AML blasts with all-trans retinoic acid (ATRA), we demonstrated it was possible to induce antibody-dependent blast-to-blast killing amongst the cancer itself, with blasts functioning as both targets and effectors, a phenomenon we termed fratricide. This antibody-induced fratricide (open full item for complete abstract)

Committee: Susheela Tridandapani PhD (Advisor); James Blachly MD (Committee Member); John Byrd MD (Committee Member); Amanda Toland PhD (Committee Member) Subjects: Molecular Biology

Doctor of Philosophy, The Ohio State University, 2019, Biomedical Sciences

Acute Myeloid Leukemia (AML) is the most common acute leukemia in adults affecting almost 12,000 people each year in the US. This disease is collectively characterized by an accumulation of rapidly proliferating neoplastic cells of the myeloid lineage with differentiation defects. In spite of the vast amount of information known about AML and the identification of favorable prognosis factors, a large percentage of patients relapse and succumb to this disease. In addition, the inter- and intra-tumor heterogeneity of AML makes the identification of therapeutic targets for this disease particularly challenging. Future studies are warranted to identify multi-targeted agents that could influence AML as a composite disease. A target that shows promise in targeting the bulk AML leukemic cell population is nicotinamide phosphoribosyltransferase (NAMPT). NAMPT is a protein involved in the generation of NAD+ in tumor cells, an important mediator of enzymatic reactions involved in various functions of leukemic disease progression. Leukemic blasts show a higher NAD+ turnover rate than normal cells, suggesting that NAD+ biosynthesis could be critically required in hematologic malignancies and therefore targeting the regeneration of NAD+ offers an attractive alternative strategy in AML. Inhibitors of NAMPT that have been described by others have shown potent anti-tumor activity and selectivity of several tumor models, including AML, while preserving the viability and functionality of normal tissues. While two agents targeting NAMPT have been tested in Phase I clinical trials, dose-limiting toxicities including thrombocytopenia and gastrointestinal toxicities led to their clinical discontinuation. Novel compounds with improved tolerability are needed. We sought to determine the mechanism of anti-tumor activity on AML leukemic cell population using a novel compound, KPT-9274, targeting NAMPT. We will also highlight several mechanisms used to antagonize AML disease progression v (open full item for complete abstract)

Committee: John Byrd (Advisor); Rosa Lapalombella (Advisor); Sameek Roychowdhury (Committee Chair); Vinay Puduvalli (Committee Member) Subjects: Biomedical Research; Oncology; Pharmacology

Doctor of Philosophy, The Ohio State University, 1983, Graduate School

Committee: Not Provided (Other) Subjects: Biology

Doctor of Philosophy, The Ohio State University, 1980, Graduate School

Committee: Not Provided (Other) Subjects: Health Sciences

PhD, University of Cincinnati, 2007, Medicine : Molecular Genetics, Biochemistry, and Microbiology

The Ets family transcription factor PU.1 (encoded by Sfpi1) is essential for both myeloid and lymphoid development. Previous data has demonstrated that high concentrations of PU.1 protein promote macrophage development in vitro, whereas low concentrations permit B cell development in vitro. Additionally, enforced expression of PU.1 assists in erythroblast immortalization and murine erythroleukemia. These data demonstrate that alteration of PU.1 expression levels directly affects hematopoietic development. We hypothesized that high level expression of PU.1 in developing progenitors correlates with myeloid lineage commitment. We tested this hypothesis by several methods. First, we immortalized murine fetal liver hematopoietic progenitors by retroviral expression of PU.1 in combination with granulocyte-monocyte colony stimulating factor stimulation. Immortalized cells possessed characteristics of multiple lineages and were demonstrated to be bipotential upon removal of exogenous PU.1. Second, we utilized an allele of Sfpi1 (termed BN), and an allele in which neomycin was removed (Sfpi1Blac), which results in hypomorphic expression of PU.1. Sfpi1BN/BN mice showed an intrinsic block to early B cell development. In addition, myeloid development was impaired in Sfpi1BN/BN fetal liver whereas neonatal Sfpi1BN/BN mice have a dramatic expansion of immature myeloid cells. These results demonstrate that over-expression of PU.1 can block differentiation prior to myeloid or erythroid commitment. Additionally, high levels of PU.1 are required for B cell specification, but not myeloid. These results demonstrate the critical importance of an appropriate threshold of PU.1 activity for normal hematopoiesis.

Committee: Dr. Rodney DeKoter (Committee Chair); Dr. Thomas Doetschman (Other); Dr. Jerry Lingrel (Other); Dr. Christopher Karp (Other); Dr. James Mulloy (Other) Subjects:

Doctor of Philosophy, The Ohio State University, 2012, Pharmacy

Protein metabolism comprises the biochemical processes that regulate the synthesis of proteins, their post-translational modification and degradation. In cancer cells, pathways targeting protein metabolism are often deregulated to accumulate signals that promote survival. These changes are manifested in malignant cells by way of upregulation of protein synthesis, suppression of protein degradation and selective expression of anti-apoptotic, pro-proliferative and pro-survival proteins. B-cell malignancies such as chronic lymphocytic leukemia (CLL) and acute lymphocytic leukemia (ALL) are caused by the accumulation of B cells that are resistant to apoptosis. Both diseases are characterized by imbalanced expression of pro-apoptotic and anti-apoptotic Bcl-2 family members, constitutive expression of the nuclear factor NF-κB and mutations or deletions in the tumor suppressor p53 - signals that support the survival of leukemic B cells. Current therapies in CLL and ALL target normal immune cells in addition to malignant cells, causing profound immune suppression with an accompanying risk of lethal secondary infections. Furthermore, a common problem with most therapies is that patients who initially responded favorably relapse over time. To a large extent, this occurs in patients with advanced diseases that have p53 mutations in B cells, which become resistant to therapies dependent on wild-type p53 function. A major focus of therapeutic development has therefore been the investigation of agents that have minimal effects on T cells and are cytotoxic to B cells independent of p53 or overexpression of anti-apoptotic Bcl-2 family proteins. Two such therapeutic agents: silvestrol, a translation initiation inhibitor, and carfilzomib, a proteasome inhibitor, are introduced for the treatments of ALL and CLL in this thesis. The work presented herein provides proof of concept that drugs that affect protein metabolism pathways can selectively and potently target B cells over T cells (open full item for complete abstract)

Committee: John Byrd (Advisor); David Lucas (Advisor); Mitch Phelps (Committee Member); Kristy Ainslie (Committee Member) Subjects:

Master of Science, The Ohio State University, 1970, Graduate School

Committee: Not Provided (Other) Subjects:

Master of Science, The Ohio State University, 1960, Graduate School

Committee: Not Provided (Other) Subjects:

Master of Science, The Ohio State University, 1961, Graduate School

Committee: Not Provided (Other) Subjects:

Master of Science, The Ohio State University, 1968, Graduate School

Committee: Not Provided (Other) Subjects:

PhD, University of Cincinnati, 2024, Medicine: Cancer and Cell Biology

Altered protein homeostasis is a key feature of many human cancers, with regulation primarily governed by the proteasomal degradation of proteins modified with Lysine-48-linked polyubiquitin chains. The ubiquitination process involves three key enzymes: E1, E2, and E3. In humans, there are up to 40 E2 conjugating enzymes, crucial for specifying substrate and ubiquitin linkage specificity. Our recent study identified UBE2N as a target in myeloid malignancies, including acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS), through the inhibition of its catalytic domain using novel small molecule inhibitors. In this study, we aimed to understand the exquisite dependencies of MDS/AML cells on UBE2N function. To achieve this, we developed and characterized a mouse model with impaired catalytic activity. We found that the catalytic active site cysteine 87 (C87) of UBE2N is indispensable for AML development. In contrast, normal hematopoiesis remained largely unaffected by the loss of UBE2N catalytic function, thus confirming UBE2N as a critical dependency and a druggable target in AML. UBE2N stands out among E2 enzymes as it exclusively synthesizes Lysine-63 (K63)-linked polyubiquitin chains on target proteins in collaboration with specific E3 ligases, leading to the stabilization or activation of protein substrates, rather than their degradation. Through a ubiquitin enrichment followed by proteomic analysis in isogenic UBE2N-deficient AML cells, we identified a network of leukemia-associated proto-oncoproteins regulated by UBE2N. Pharmacological or genetic inhibition of UBE2N in AML cells significantly reduced the protein levels of these targets, suggesting that UBE2N-mediated K63-linked ubiquitination of select proteins is essential for their stabilization. Interestingly, inhibition of UBE2N led to the degradation of these proto-oncoproteins by the immunoproteasome. Treatment with the immunoproteasome inhibitor ONX-0914 rescued the decreased protein e (open full item for complete abstract)

Committee: Daniel Starczynowski Ph.D. (Committee Chair); John Cunningham Ph.D. (Committee Member); Nicolas Nassar Ph.D M.A B.A. (Committee Member); Susanne Wells Ph.D. (Committee Member); Andrew Volk Ph.D. (Committee Member) Subjects: Cellular Biology

Doctor of Philosophy, Case Western Reserve University, 2024, Pathology

AML is the most common acute leukemia in adults with an overall poor prognosis and high relapse rate. Multiple factors including genetic abnormalities, differentiation defects and altered cellular metabolism contribute to AML development and progression. Though the roles of oxidative phosphorylation and glycolysis are defined in AML, the role of the HBP, which regulates the O-GlcNAcylation of cytoplasmic and nuclear proteins, remains poorly defined. We studied the expression of the key enzymes involved in the HBP in AML blasts and stem cells at the single-cell and bulk level. We found higher expression levels of the key enzymes in the HBP in AML as compared to healthy donors in whole blood. We also observed elevated OGT and OGA expression in AML stem and bulk cells as compared to normal HSPCs. Gene set analysis showed substantial enrichment of the NF-κB pathway in AML cells expressing high OGT levels. We found AML bulk cells and stem cells show enhanced OGT protein expression and global O-GlcNAcylation compared to normal HSPCs, validating our in-silico findings. Our study suggests the HBP may prove a potential target, alone or in combination with other therapeutic approaches, to impact both AML blasts and stem cells. Moreover, as insufficient targeting of AML stem cells by traditional chemotherapy is thought to lead to relapse, blocking HBP and O-GlcNAcylation in AML stem cells may represent a novel promising target to control relapse. Additionally, prognostic biomarker discovery approaches based upon bulk analysis are unable to capture key attributes of rare subsets of cells that play a critical role in patient outcomes. Single-cell RNA sequencing is a powerful technique that enables the assessment of rare subsets of cells, but this technique is not amenable to clinical diagnostics. One area where improved prognostic biomarkers are important is for the management of pediatric AML patients with a FLT3-ITD genetic abnormality. We utilized single-cell data from the ra (open full item for complete abstract)

Committee: Brian Cobb (Committee Chair); David Wald (Advisor); Tae Hyun Hwang (Advisor); Stanley Huang (Committee Member); Li Lily Wang (Committee Member); Clive Hamlin (Committee Member) Subjects: Biostatistics; Immunology; Oncology

MS, University of Cincinnati, 2024, Medicine: Cancer and Cell Biology

Acute myeloid leukemia (AML) is a rare hematological malignancy characterized by genetic and epigenetic aberrations in hematopoietic progenitor/stem cells (HPSCs). Molecular profiling studies of AML patients have suggested a sequential acquisition of co-occurring mutations during leukemogenesis, with mutations in epigenetic regulatory genes like TET2 and DNMT3A often serving as the initiating events due to their high variant allele frequencies (VAF). Moreover, these initiating mutations exhibit a key role in clonal hematopoiesis (CH), a state marked by the expansion of dominant mutant HPSC clones without evident disease. Concurrent mutations, notably in genes such as NPM1, NRAS, and FLT3, are known to co-occur with epigenetic modifier mutations and are believed to manifest within a pre-existing mutant clone. While targeted therapies have been established for specific AML mutations like FLT3 and IDH1/2, treating patients lacking these mutations or those who develop resistance remains a critical challenge. Recent strides in single-cell sequencing have revolutionized our comprehension of AML pathogenesis by unveiling the clonal architecture and mutation order, which is not possible through bulk sequencing approaches. Leveraging technologies such as single-cell targeted DNA sequencing, we and others have dissected genetic variations at the single-cell level, unraveling how specific combinations of mutations, such as TET2/NPM1, can synergistically propel leukemogenesis, resulting in the amplification of double mutant clones compared to single mutant clones. Furthermore, single-cell multi-omics investigations have delineated that the co-mutational context exerts a profound impact on the differentiation and immunophenotype of transformed cells. Investigating distinct genotypic states in AML aims to furnish invaluable insights into the intricate web of co-mutational synergy and the influence of mutation chronology on leukemia progression, ultimately directing the developmen (open full item for complete abstract)

Committee: Linde Miles Ph.D. (Committee Chair); Andrew Volk Ph.D. (Committee Member); Erin Hertlein Ph.D. (Committee Member) Subjects: Oncology

Doctor of Philosophy, The Ohio State University, 2023, Molecular, Cellular and Developmental Biology

Chronic lymphocytic leukemia (CLL) is the most prevalent adult leukemia in the western world, accounting for more than 4000 deaths in the United States every year. It is a malignancy of CD5+CD23+CD19+ B lymphocytes, characterized by activation of B cell receptor (BCR) signaling that in turn activates several downstream cell survival pathways, and migration of these B cells (B-CLL cells) to pro-survival niches like the bone marrow, lymph nodes and spleen. The only curative therapy is allogeneic hematopoietic stem cell transplantation, but this therapy is associated with common risks like graft-versus-host-disease and pulmonary complications. BCR signaling inhibitors are effective agents for CLL treatment, but therapeutic resistance and relapse remain problematic, necessitating the identification of new CLL targets that can bypass current disease resistance. Sialic-acid-binding immunoglobulin-like lectins (Siglecs) are a family of cell surface glycoproteins that demonstrate cell-type specific expression and can modulate receptor signaling. Many studies have shown the importance of Siglecs in tumor immunosurveillance including immunosuppression, thereby making them attractive anti-cancer molecular targets. Our study focuses on evaluating a novel Siglec member, Siglec-6, which was recently found to be upregulated on the surface of B cells from CLL patients but not on B cells from healthy donors. CLL-specific upregulation of Siglec-6 thus makes it a valuable therapeutic target and warrants the development of transgenic mouse models and cell line systems to further explore the biological role of Siglec-6. While Siglec-6 has been extensively studied in trophoblastic cells, mast cells and bladder cancer cells, the physiological role of Siglec-6 in immune cells is still poorly understood. In the first part of this dissertation, we utilized our novel Siglec-6 transgenic mouse models to identify a specific role for Siglec-6 in regulating B cell functions. Siglec-6 transgenic (open full item for complete abstract)

Committee: Natarajan Muthusamy (Advisor); Meixiao Long (Committee Member); Lalit Sehgal (Committee Member); Paul Martin (Committee Member) Subjects: Cellular Biology; Molecular Biology

PhD, University of Cincinnati, 2023, Medicine: Cancer and Cell Biology

IRAKs are a family of related kinases that operate at the nexus of multiple inflammatory pathways implicated in myeloid malignancies. IRAK4 inhibitors have advanced into clinical trials for MDS and AML. Early data from these trials validate IRAK4 as a therapeutic target; however, the overall responses remain modest. Herein, we explored mechanisms of resistance to IRAK4-selective inhibitors in MDS/AML. Consistent with observations from clinical trials, inhibition or deletion of IRAK4 in MDS/AML cell lines and patient samples lines resulted in incomplete suppression of LSPCs and a corresponding activation of innate immune pathways. Given the evolutionary conserved redundancy of IRAK-dependent pathways, we examined the expression of IRAK paralogs. Deficiency or inhibition of IRAK4 in MDS/AML cells induced overexpression and activation of the IRAK4 paralog, IRAK1. To validate the compensation of IRAK1, we deleted IRAK1 in MDS/AML cells and found that concomitant suppression of IRAK1 led to a significant reduction of LSPC function in IRAK4-KO MDS/AML cells. Canonical IRAK signaling depends on recruitment of MyD88 and IRAK4 to activated receptors, which results in the subsequent recruitment and activation of IRAK1. Based on this canonical model, IRAK1 and IRAK4 are independently essential for signaling downstream of MyD88. However, MyD88-KO AML cells did not exhibit a functional defect but did retain sensitivity to deficiency of IRAK1 and IRAK4, indicating canonical MyD88-dependent signaling is not operational in MDS/AML. We next investigated gene expression programs dependent on IRAK1 and/or IRAK4, yet independent of MyD88. IRAK1/4 deletion correlated with dysregulation of transcriptional programs involved in stem cell maintenance and myeloid differentiation. The requirement for IRAK1/4 in preserving an undifferentiated LSPC state was corroborated by morphological and immunophenotypic assessment of IRAK1/4dKO MDS/AML cells. To delineate non-canonical sign (open full item for complete abstract)

Committee: Daniel Starczynowski Ph.D. (Committee Chair); Jose Cancelas-Perez M.D. Ph.D. (Committee Member); Andrew Volk Ph.D. (Committee Member); Chandrashekhar Pasare DVM PhD (Committee Member); H. Leighton Grimes Ph.D. (Committee Member) Subjects: Cellular Biology

Doctor of Philosophy, Case Western Reserve University, 2023, Pathology

T-cells are a central component of the adaptive immune response that promote the clearance of pathogens, immune tolerance, and tissue homeostasis. T-cell development, survival, and function are coordinated by NOTCH1, T-cell receptor (TCR), and cytokine signaling in a stage and location specific manner. The strength/persistence of these signals directly influences thymocyte development and peripheral T-cell maintenance. However, mechanisms that precisely interpret these signals to transcriptional and proteostasis machinery are poorly understood. In this dissertation, I investigate the hypothesis that developmental and homeostatic T-cell signals are translated by “adaptor proteins”. Adaptors modulate expression and activity of this machinery by facilitating protein-protein interactions. Specifically, I describe how two recently discovered adaptor proteins, SEL1L and CHMP5, play critical roles in T-cell development, homeostasis, and malignancy. During thymocyte development, signals from the pre-TCR and NOTCH1 coordinately instruct β-selection to generate mature T-cells. I discovered that SEL1L, an adaptor for the ER-associated degradation complex is induced by NOTCH1 to mitigate ER stress and apoptosis during β-selection. Consequently, deletion of SEL1L abrogated early thymocyte development. This research supports that SEL1L couples developmental signals to proteostasis machinery to enable thymocyte differentiation. In the thymus, CHMP5's adaptor function was discovered to be induced by positive selection TCR signals to ensure post-selection thymocyte survival. Subsequently, I found that CHMP5 is also required for the maintenance of peripheral T-cells. CHMP5 is stabilized downstream of TCR and IL-7 signaling, and loss of CHMP5 dramatically impairs the survival of CD4 and CD8 T-cells. Because dysregulation of developmental signals in thymocytes contributes to T-cell leukemogenesis, I hypothesized that CHMP5 could facilitate thymocyte survival downstream of onco (open full item for complete abstract)

Committee: Stanley Adoro (Advisor) Subjects: Biochemistry; Biology; Cellular Biology; Immunology; Molecular Biology