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

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

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

Master of Science, The Ohio State University, 2022, Genetic Counseling

Acute Myeloid Leukemia (AML) affects approximately 3,200 adolescents and young adults (AYA's) annually, a population with unique challenges given their age at diagnosis. Clinicians are becoming increasingly aware that underlying germline mutations in cancer susceptibility genes contribute to familial MDS/AML. Referral for germline testing is contingent on personal or family history, suspicious findings on somatic tumor testing, or a combination of these. Identification of germline mutations may direct treatment or therapies, inform screening or surveillance, and could initiate cascade testing for at-risk family members. Discovery of MDS/AML predisposition genes began in 1999 and proposed referral guidelines for germline assessment were published by experts in 2013. In 2015, somatic sequencing was universally implemented for all AML patients at the Ohio State University (OSU) James Cancer Center. Despite available referral guidelines, knowledge of familial MDS/AML, and cancer genetic counseling services at OSU, patients at the Ohio State University were largely not referred to genetic counseling for discussion of germline testing. Additionally, AYA AML patient somatic and cytogenetic landscapes at OSU are reflective of those reported so far in the literature, findings that contribute to burgeoning research that AYA AML is biologically distinct from AML in children and adults.

Committee: Julia Cooper MS CGC (Advisor); Bhavana Bhatnagar DO (Committee Member); Ann-Kathrin Eisfeld MD (Committee Member) Subjects: Genetics; Health Care; Oncology

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

Acute myeloid leukemia (AML) is a hematological malignancy that is considered heterogeneous and clonal in nature with many genomic abnormalities. One of the most common mutations in AML is internal tandem duplication (ITD) mutation in the Fms-like tyrosine kinase 3 (FLT3) gene (FLT3-ITD). Approximately 30% and 15% of adult and pediatric AML patients, respectively, harbor this mutation, and these patients with such activating mutations have poor outcomes with lower overall survival (OS) and shorter remission. Small molecule FLT3 inhibitors have been underway for clinical development, and two agents, midostaruin and gilteritinib, have been approved by the Food and Drug Administration in the U.S. However, intrinsic and acquired drug resistance, such as the emergence of secondary tyrosine kinase domain mutations (TKD), resistance mediated by stromal cell and cytokine support, and resistance-conferring co-occurring mutations such as in the gene NRAS, remain a significant area of unmet need for patients who are receiving FLT3 inhibitor therapy. Herein, we utilize different novel agents to address this unmet need in FLT3-ITD positive (+) AML. Chapter 2 describes our efforts for the discovery of FLT3 inhibitors that have activity against both de novo and resistance-conferring TKD mutant FLT3-ITD+ AML. From a high-throughput screen, a hit compound with FLT3 activity was discovered based on a kinase-binding profile. A series of analogs based on the hit has been synthesized, and a subset of compounds were selected for further evaluation. In chapter 3, we explore repurposing of the JAK2/FLT3 inhibitor pacritinib, which is being developed in myelofibrosis, in a phase I clinical trial for FLT3-ITD+ AML. We describe not only its preclinical activity against drug resistant models of FLT3-ITD+ AML but also the preliminary clinical efficacy, safety, and pharmacokinetic profiles of pacritinib in patients. Chapter 4 describes the translational development of a novel compound, TP-0903, (open full item for complete abstract)

Committee: Sharyn Baker PharmD, PhD (Advisor); Alex Sparreboom PhD (Committee Member); Kari Hoyt PhD (Committee Member); Lara Sucheston-Campbell PhD (Committee Member) Subjects: Oncology; Pharmacy Sciences

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

PhD, University of Cincinnati, 2019, Arts and Sciences: Chemistry

Chemotherapy for cancer always has many off-target effects that damage healthy cells. Many chemotherapeutic drugs can affect the function of normal cells in the heart, kidneys or nervous system cause another health problem. Reactive Oxygen Species (ROS) are molecules produced in cell metabolism. ROS are important for proliferation, differentiation, senescence, apoptosis, and chemoresistance of cancer cells. Compared to normal cells, it has been found that ROS concentration is elevated in lots of cancers. These differences between cancer and normal cells are the basis for the development of new chemotherapy agents that selectively target the cancer cell but not impair the function of normal cells. Based on these observations, we made several ROS activated compounds and found a lead compound, MA14, which has an IC50 of 2 µm against AML cancer cells. I hypothesized that the high selectivity of MA14 is due to its unique oxidation mechanism in the presence of H2O2. Herein, the oxidation mechanism of MA14 was investigated using MS and NMR spectroscopy. In the presence of ROS, MA14 goes through oxidative-cyclizing to form two isomers. These isomers could be further oxidized to non-toxic products. I also synthesized several molecules to study the relationship between structure and activity. It is shown that the a-carbon of the side chain is essential for cytotoxicity. The size of the cyclic amine has a moderate influence on activity. I took advantage of the finding that the phenol group to the MA14 could be modified. In particular, I designed and synthesized several compounds based on the MA14 structure that released substituted phenols in the presence of ROS. It is shown that the oxidation mechanism is influenced by the electron density of the phenol. Further, we found that these compounds could selectively target AML cells while the normal cell can be surviving. To further develop the concept, we designed and synthesized prodrugs, termed self-cyclizing reagents, w (open full item for complete abstract)

Committee: Edward Merino Ph.D. (Committee Chair); Patrick Limbach Ph.D. (Committee Member); David Smithrud Ph.D. (Committee Member) Subjects: Biochemistry

PhD, University of Cincinnati, 2019, Medicine: Immunology

Targeted inhibitors to oncogenic kinases demonstrate encouraging clinical responses early in the treatment course, however most patients will relapse due to target-dependent mechanisms that mitigate enzyme-inhibitor binding, or through target-independent mechanisms, such as alternate activation of survival and proliferation pathways, known as adaptive resistance. Here we describe mechanisms of adaptive resistance in FLT3 mutant acute myeloid leukemia (AML) by examining integrative in-cell kinase and gene regulatory network responses after oncogenic signaling blockade by FLT3 inhibitors (FLT3i). We identified activation of innate immune stress response pathways after treatment of FLT3-mutant AML cells with FLT3i and showed that innate immune pathway activation via the IRAK1/4 kinase complex contributes to adaptive resistance in FLT3-mutant AML cells. To overcome this acute adaptive resistance mechanism, we developed a small molecule that simultaneously inhibits FLT3 and IRAK1/4 kinases. The multi-kinase FLT3-IRAK1/4 inhibitor eliminated adaptively resistant FLT3-ITD AML cells in vitro and in vivo, and displayed superior efficacy as compared to current targeted FLT3 therapies. These findings uncover a polypharmacologic strategy for overcoming adaptive resistance to therapy in AML by targeting immune stress response pathways.

Committee: Daniel Starczynowski Ph.D. (Committee Chair); H. Leighton Grimes Ph.D. (Committee Member); Ashish Kumar M.D. (Committee Member); Chandrashekhar Pasare (Committee Member); William Seibel Ph.D. (Committee Member) Subjects: Oncology

PhD, University of Cincinnati, 2019, Medicine: Cancer and Cell Biology

The innate immune system is a complex network that recognizes and responds to foreign particles. Innate immune signaling is fundamentally involved in inflammation and increasing evidence implicates chronic innate and inflammatory signaling as a risk factor in cancer and hematologic malignancies. Recent studies have implicated the dysregulation of innate immune signaling in the pathogenesis of Myelodysplastic Syndrome (MDS) and Acute Myeloid Leukemia (AML). However, the precise genetic alterations that cause innate immune signaling activation in hematologic malignancies are not fully defined. Hematologic malignancies have a particularly high frequency of mutations in RNA splicing factors. How these mutations contribute to disease is not fully understood. A global analysis of exon usage in AML samples revealed a subset of genes is regulated exclusively at the isoform level in leukemia, resulting in anticorrelated expression of individual RNA isoforms. Many of these genes regulated by RNA isoform changes are associated with inflammatory and immune pathways. IRAK4 was the most significant immune pathway gene that undergoes isoform switching. Increased expression of the long isoform of IRAK4 (IRAK4-L), which includes exon 4, was found in MDS and AML cell lines and primary patient samples and results in maximal activation of NF-kB. Elevated IRAK4-L isoform expression is associated with poor prognosis in MDS and AML and is significantly associated with mutations in splicing factor U2AF1. Further, U2AF1 directly regulates the splicing of IRAK4 to increase the expression of IRAK4-L. Inhibition of IRAK4 abrogates leukemic growth in vitro and in vivo and is more efficacious in AML cells with U2AF1 mutations and/or higher expression of the IRAK4-L isoform. Thus, mutations in U2AF1 splicing factor induce expression of therapeutically targetable "active" IRAK4 isoforms and provide the first genetic link to activation of chronic innate immune signaling in MDS and AML. The conseq (open full item for complete abstract)

Committee: Daniel Starczynowski Ph.D. (Committee Chair); Jose Cancelas-Perez M.D. (Committee Member); Matthew Flick Ph.D. (Committee Member); Kakajan Komurov Ph.D. (Committee Member); Nathan Salomonis M.D. (Committee Member) Subjects: Oncology

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

PhD, University of Cincinnati, 2018, Arts and Sciences: Chemistry

Anticancer agents that modify DNA are a mainstay of chemotherapy regimens, but development of new classes of these agents has slowed because of the modifications of DNA in non-cancerous cells. This is what gives rise to serious side effects via poor selectivity. The Merino Lab has developed a pro-drug strategy to achieve specificity by translating the finding that levels of reactive oxygen species (ROS) are elevated in cancers, such as Acute Myeloid Leukemia (AML). This pro-drug approach allows cellular ROS to oxidize the pro-drug into its active form to achieve selective cytotoxicity. Our current lead agent (A100) is shown to have 10-fold selectivity between AML cells over normal CD34+ blood cells in vitro and showed some efficacy in the in vivo AML mouse model. This work started with computational analysis to determine what parts of the molecule were a target for metabolic enzymes. The first step taken to improve the molecule was to add polyethylene glycol (PEG) to the free phenol, increasing both its solubility and metabolic stability. To prove metabolic stability, binding assays against CYP1A2 (Cytochrome P450, Isoform 1A2) were done, as CYP1A2 is known to attack alcohols. The synthetic addition of the PEG increased stability against CYP1A2 by almost 50%. To prove stability in a more complex matrix, total stability was measured via half-life in pooled human liver microsomes. The PEGylated compound (A100-PEG) showed a 7-fold increase in the half-life of A100, as compared to A100 alone. A100 alone was not detectable in mouse blood samples after 15 minutes; however, in all three mouse models, A100-PEG was detectable even after 100 minutes and was calculated to have a 6-fold increase in half-life, as compared to A100. Though stable, A100-PEG only showed similar efficacy in the in vivo AML mouse model; however, an IV injection was used instead of the previous IP injection. Having improved its solubility and metabolic stability, the next step was to improve agen (open full item for complete abstract)

Committee: Edward Merino Ph.D. (Committee Chair); Patrick Limbach Ph.D. (Committee Member); Laura Sagle Ph.D. (Committee Member) Subjects: Pharmaceuticals

PhD, University of Cincinnati, 2018, Medicine: Cancer and Cell Biology

Granulocyte-colony stimulating factor receptor (G-CSFR) controls myeloid progenitor proliferation and differentiation to neutrophils. Mutations in CSF3R (encoding G-CSFR) have been reported in patients with chronic neutrophilic leukemia (CNL) and acute myeloid leukemia (AML); however, despite years of research, the malignant downstream signaling of the mutated G-CSFRs is not well understood. To have a system level understanding of the normal and malignant G-CSFR signaling, a global quantitative phosphoproteomics approach was utilized. BaF3 murine cell line based in vitro model system was developed expressing wild type (WT), proximal mutation (T618I), and distal truncation mutation (Q741x). SILAC-based quantitative phosphoproteomics studies were performed with WT and mutant receptors by sequential enrichment for phosphotyrosine (pTyr) followed by nano-LC-MS/MS analyses. The flow-through of pTyr enrichment was further processed for phospho-serine/threonine using titanium dioxide columns. Based on computational, bioinformatics and kinase enrichment analysis of the pTyr data, Bruton's tyrosine kinase (Btk) was found to be activated (increase in phosphorylation) in the mutated receptors but not in WT. The aberrant activation of Btk was further validated using immunoblots in the BaF3 and 32D cell line lysates (murine myeloid 32D cell line based in vitro model system was generated expressing WT, and mutant receptors similar to BaF3 system). Furthermore, ibrutinib based chemical inhibition of Btk was performed in the 32D as well as primary bone marrow cells from WT and truncation G-CSFR knock-in mouse and the readout was measured by cell death (for IC50 analysis) and colony forming unit (CFU) assays. Chemical inhibition of Btk led to increased sensitivity (lower IC50) in both 32D and primary cells and significantly lower clonogenicity in the mutant cells compared to WT primary cells. To further validate the findings of the in vitro and mouse in vivo studies in human sam (open full item for complete abstract)

Committee: Kenneth Greis Ph.D. (Committee Chair); Mohammad Azam Ph.D. (Committee Member); H. Grimes Ph.D. (Committee Member); Patrick Limbach Ph.D. (Committee Member); Daniel Starczynowski Ph.D. (Committee Member); Xiaoting Zhang Ph.D. (Committee Member) Subjects: Health Sciences

PhD, University of Cincinnati, 2017, Medicine: Cancer and Cell Biology

In this thesis work, we investigate the role of autophagy in normal and malignant hematopoiesis. In normal hematopoiesis, we study the mechanism of autophagy regulation by mTOR in hematopoietic stem and progenitor cells (HSPCs) using genetic mTOR knockout and knock-in mouse models. We find that HSPCs have varied basal autophagy activity in different subpopulations, higher in more primitive hematopoietic stem cells (HSC) and lower in more differentiated progenitor cells, suggesting varied dependence on autophagy in these cells. We also observe that the autophagy activity responds differently to mTOR deletion in HSPCs subpopulations. HSC and GMP subpopulations show mTOR independent autophagy regulation, while CMP has increased autophagy activity upon mTOR deletion. We speculate that a compensatory kinase pathway in HSC and GMP exists to negatively regulate autophagy activity upon mTOR loss in HSC population based on our kinase inhibitor data. We also find that the autophagy response in mTOR knock-in cells is similar to mTOR knockout, suggesting that mTOR regulates autophagy through its kinase function, not a protein scaffolding effect. The autophagy response in Raptor knockout cells mimics that of the mTOR knockout, indicating that mTORC1 regulates autophagy in HSPCs. This project is progressing and more studies are needed to validate our current observations and conclusions. In malignant hematopoiesis, we investigate the therapeutic potential of inhibiting autophagy for AML treatment. We show that Kmt2a/Mll-Mllt3/Af9 AML (MA9-AML) cells have high autophagy flux compared to normal bone marrow cells, but autophagy-specific targeting, either through Rb1cc1-disruption to abolish autophagy initiation, or via Atg5-disruption to prevent autophagosome membrane elongation, does not affect the growth or survival of MA9-AML cells, either in vitro or in vivo. Mechanistically, neither Atg5 nor Rb1cc1 disruption impairs the endolysosome formation or survival signaling pathways (open full item for complete abstract)

Committee: Yi Zheng Ph.D. (Committee Chair); Maria Czyzyk-Krzeska M.D. Ph.D. (Committee Member); Marie-Dominique Filippi Ph.D. (Committee Member); Gang Huang Ph.D. (Committee Member); Daniel Starczynowski Ph.D. (Committee Member) Subjects: Molecular Biology

Doctor of Philosophy, Case Western Reserve University, 2017, Pathology

Acute myeloid leukemia (AML) is an aggressive disease with a poor 5-year survival of 21% that is characterized by a differentiation arrest of immature myeloid cells. For a rare subtype of AML (Acute promyeloctyic leukemia, 10% of cases) all-trans retinoic acid therapy removes the differentiation block, yielding over a 90% cure rate. However, this treatment is not effective for the other 90% of AML patients, suggesting new differentiation strategies are needed. Using an NBT differentiation screening assay, our lab has defined TLR8 and GSK3ß as novel AML differentiation therapy targets. We show that the TLR7/8 ligand, R848, promotes AML differentiation and growth inhibition in vitro and in vivo in a TLR8/MyD88/p38 dependent manner, exhibiting direct anti-leukemic effects independent of its immunomodulating properties that are currently under investigation in cellular cancer therapies. We show that AML cells exhibit an aberrant nuclear GSK3ß and this nuclear fraction drives AML growth and drug resistance in vivo and in vitro. Nuclear GSK3ß promotes drug resistance partially through activation of the pro-survival NF-¿B signaling pathway. Finally, we show that nuclear GSK3ß localization strongly correlates to poorer patient survival (N=86 HR= 2.2 p=0.006). Nuclear localization of GSK3ß may define a novel oncogenic mechanism as well as a new therapeutic target in AML.

Committee: George Dubyak PhD (Committee Chair); David Wald MD PhD (Advisor); Xiaoxia Li PhD (Committee Member); Mark Jackson PhD (Committee Member); Parameswaran Ramakrishnan PhD (Committee Member); Howard Meyerson PhD (Committee Member) Subjects: Cellular Biology; Oncology; Pathology

PhD, University of Cincinnati, 2016, Arts and Sciences: Chemistry

Cancer is defined as abnormal growth of cells which tend to proliferate in an uncontrolled way and, in some cases, to metastasize. Cancer can be initiated by various factors working alone or in combination. Some cancers are caused by external factors such as tobacco, diet, certain chemicals, radiation, and viruses while other cancers are caused by internal factors such as hormones, immune conditions, and inherited genetic mutations. Damage to DNA by reactive oxygen species (ROS) is widely accepted as a major cause of cancer. ROS are chemically reactive molecules formed as a natural byproduct of metabolism in cells. These species play an important role in cell signaling and homeostasis. ROS levels are kept under control by cellular antioxidant systems. Whenever there is an imbalance in ROS generation ad its regulation, it leads to oxidative stress. Elevated levels of ROS have been detected in different types of cancer cells where they are known to promote tumor progression and survival including renal cell carcinoma, melanoma, and leukemia. These ROS levels are higher when compared to normal cells. This high ROS levels in cancer cells makes them susceptible to oxidative stress induced cell death and can be exploited for development of selective anticancer therapy. Many chemotherapeutic strategies are designed to increase cellular ROS levels to induce irreparable damages to cell leading to apoptosis. This can be achieved by using compounds that inhibit antioxidant systems or through inhibition of specific signaling pathways that upregulate antioxidants in cancer cells. The resulting increase in ROS may induce tumor cell death either through damaging functions of ROS like DNA oxidation, lipid peroxidation or by specific initiation of apoptosis via death signaling pathways. As basal ROS levels in normal cells are comparatively low, these cells are not significantly affected leading to selectivity in chemotherapy. My work focuses on design and development of novel ant (open full item for complete abstract)

Committee: Edward Merino Ph.D. (Committee Chair); Michael Baldwin Ph.D. (Committee Member); James Mack Ph.D. (Committee Member) Subjects: Organic Chemistry

PhD, University of Cincinnati, 2014, Arts and Sciences: Chemistry

DNA modifying agents are stalwarts of chemotherapeutic cancer treatments, but require vital design improvements to improve selectivity, lower side effects, and continue their widespread use. A key problem for DNA modifying agents is lack of specificity. To address this issue, our lab designs novel molecular scaffolds which are activated by a hallmark of some cancers: increased oxidative stress cause by reactive oxygen species (ROS). Oxidative stress, as measured by levels of ROS, oxidized biomolecules, and enzyme activity, is a hallmark of certain cancer cells. My work focuses on a potential path forward in the design of DNA modifying agents by exploiting the increased ROS into a pro-drug approach. Elevation of ROS has been linked to oncogenesis and has been found in several aggressive cancers, including renal cell carcinoma, melanoma, and leukemia. ROS occurs in four major endogenous forms within the cell: superoxide, hydrogen peroxide, singlet oxygen, and hydroxyl radical. ROS occur in cells via two discrete mechanisms; first, as a byproduct of metabolism. For example, mitochondria generate superoxide via complex I and III during oxidative phosphorylation. Secondly, ROS are known to derive from several enzymes. Especially important are NADPH oxidases that regulate the function of several tyrosine kinases involved in cell growth and survival. Amplified ROS results in increased DNA damage and mutation, tumor heterogeneity, the ability to self-replicate, and angiogenesis. In turn, these mutations cause enhanced activation of oncogenes. Consequently, it is no surprise that levels of ROS-induced DNA damage correlates with cancer prognosis. We utilize a design strategy wherein the pro-drug is stable, but upon ROS activation a reactive molecule is formed. This leads to more reactive forms of the molecule being present in cancer cells. Thus reactivity, and not uptake, is controlled to induce cytotoxicity more specifically in cancer cells and lower off-target reacti (open full item for complete abstract)

Committee: Edward Merino Ph.D. (Committee Chair); David Smithrud Ph.D. (Committee Member); Pearl Tsang Ph.D. (Committee Member) Subjects: Biochemistry

PhD, University of Cincinnati, 2014, Medicine: Cancer and Cell Biology

Innate immune signaling has an essential role in inflammation, and the dysregulation of signaling components within this pathway is increasingly being recognized as a mediator in cancer initiation and progression. The innate immune system is an evolutionarily conserved pathogen pattern recognition apparatus, which defends the host in a non-specific manner. Pathogens and cytokines signal to immune cells through the toll-like receptor (TLR) and interleukin-1 receptor (IL1R) superfamily. In order to mediate an inflammatory response, TLRs and IL1R require interleukin-1 associated receptor kinases (IRAKs). Herein, we demonstrate that IRAK1 is activated and overexpressed in Myelodysplastic Syndrome (MDS) and Acute Myeloid Leukemia (AML); two closely related hematologic malignancies. Furthermore, pharmacological (IRAK-Inh) and RNAi-mediated inhibition of IRAK1 is effective in eliminating disease-propagating cells. Integrated gene expression analysis revealed compensatory BCL2 upregulation following small-molecule IRAK1 inhibition. This proved to be a drugable vulnerability, as BCL2 inhibition potently synergized with IRAK-Inh to induce rapid cell death, even in IRAK-Inh-refractory cell lines. Importantly, suppression of IRAK1 signaling, through either RNAi or small-molecule inhibition, is tolerated in normal CD34+ cells, suggesting a potential therapeutic window for MDS and AML patients. To examine the effect of cancer-modifying therapies, like IRAK inhibition, we developed a novel xenograft model, utilizing an MDS-derived patient cell line, MDSL. Immunocompromised animals receiving MDSL xenografts developed progressive anemia and thrombocytopenia, thus recapitulating clinical features of the disease. These mice displayed rapid morbidity resulting from MDSL engraftment in bone marrow, spleen and peripheral blood. In this setting, both RNAi-mediated and small-molecule IRAK1 inhibition was effective in reducing MDSL cell burden, and provided a significant survival benefit. T (open full item for complete abstract)

Committee: Daniel Starczynowski Ph.D. (Committee Chair); Gang Huang Ph.D. (Committee Member); H. Leighton Grimes Ph.D. (Committee Member); Ashish R. Kumar M.D. Ph.D. (Committee Member); Maria Czyzyk-Krzeska M.D. Ph.D. (Committee Member); James Mulloy Ph.D. (Committee Member) Subjects: Cellular Biology

Doctor of Philosophy, The Ohio State University, 2014, Molecular, Cellular and Developmental Biology

Acute myeloid leukemia (AML) is a biologically complex neoplastic disease of the hematopoietic system, characterized by an uncontrolled proliferation of malignant myeloid precursors leading to bone marrow failure at the clinical level. Today, the majority of AML patients fail to achieve long-term survival. Thus, new therapeutic approaches are needed. MicroRNAs (miRs), short noncoding RNAs that regulate the expression of their target mRNA-encoded proteins, are involved in tumorigenesis. We demonstrated that deregulated miR-29b and miR-181a in AML patients were associated with worse outcome. Moreover, AML patients with a higher pre-treatment level of miR-29b respond better to a hypomethylating agent, decitabine; and patients with higher miR-181a have longer survival under cytarabine/daunorubicine-based chemotherapy. Thus, increasing the levels of these miRs prior to the respective treatments may be beneficial. However, free synthetic miRs are easily degraded in the bio-fluid and have limited cellular uptake. To overcome this problem and explore miR-based therapy, our research focused on three major aims: (1) to develop a novel nanocarrier suitable for delivering miRs into AML cells; (2) to deliver miR-29b and assess the antileukemic activity; and (3) to investigate the role of miR-181a in AML, unravel the mechanism, and perform therapeutic evaluation via nanocarrier-delivered miR-181a. For aim 1, since AML cells overexpress transferrin receptor on their surface, we formulated novel transferrin (Tf) targeted anionic lipid based nanoparticles (NP) encapsulating miR mimic and demonstrated low toxicity and high efficiency. For aim 2, following miR-29b-nanoparticle treatment, we showed a significant increase in intracellular miR-29b levels and downregulation of its known targets. This resulted in decreased leukemia growth and improved survival in an AML mouse model. Furthermore, we showed that pretreatment with miR-29b nanoparticles improved the antileukemic activity of de (open full item for complete abstract)

Committee: L. James Lee (Advisor); Guido Marcucci (Advisor); Robert Lee (Committee Member); Natarajan Muthusamy (Committee Member) Subjects: Biomedical Engineering; Cellular Biology; Molecular Biology