This document only includes an excerpt of the corresponding thesis or dissertation. To request a digital scan of the full text, please contact the Ruth Lilly Medical Library's Interlibrary Loan Department (rlmlill@iu.edu).

Call number: LD2668 .T4 1963 L54 / Master of Science

No description available.

No description available.

Haematopoiesis requires strict regulation in order to maintain a balanced production of the various blood cell components. Escape from this regulation contributes to the development of cancers such as leukemia. SOCS2 is a member of the Suppressor of cytokine signalling (SOCS) family, and normally functions as a negative regulator of the JAK/STAT pathway. I examined gene expression and promoter methylation in acute myeloid leukemia (AML) cell lines and patient samples. SOCS2 expression was quite variable in AML patients, and very low in acute promyelocytic leukemia (APL) patients. Promoter hyper-methylation was found in these patients, particularly those with high white blood cell count and a FLT3-ITD. I speculate that SOCS2 interacts with an aspect of the signalling complex to inhibit cell growth in these patients, and silencing SOCS2 is necessary for leukemia progression. Treating these patients with a de-methylating agent, such as decitabine, may show promise in the clinic.

E2A is a transcription factor that plays a particularly critical role in lymphopoiesis. The chromosomal translocation 1;19, disrupts the E2A gene and results in the expression of the fusion oncoprotein E2A-PBX1, which is implicated in acute lymphoblastic leukemia. Both E2A and E2A-PBX1 contain two activation domains, AD1 and AD2, which comprise conserved ΦxxΦΦ motifs where Φ denotes a hydrophobic amino acid. These domains function to recruit transcriptional co-activators and repressors, including the histone acetyl transferase CREB binding protein (CBP) and its paralog p300. The PCET motif within E2A AD1 interacts with the KIX domain of CBP/p300, the disruption of which abrogates the transcriptional activation by E2A and the transformative properties of E2A-PBX1. The generation of a peptide-based inhibitor targeting the PCET:KIX interaction would serve useful in further assessing the role of E2A and E2A-PBX1 in lymphopoiesis and leukemogenesis. An interaction between E2A AD2 and the KIX domain has also been recently identified, and the TAZ domains of CBP/p300 have been shown to interact with several transcription factors that contain ΦxxΦΦ motifs. Thus the design of an inhibitor of the E2A:CBP/p300 interaction requires the full complement of interactions between E2A and the various domains of CBP/p300 to be elucidated. Here, we have used nuclear magnetic resonance (NMR) spectroscopy to determine that AD2 interacts with KIX at the same site as PCET, which indicates that the E2A:KIX interaction can be disrupted by targeting a single binding site. Using an iterative synthetic peptide microarray approach, a peptide with the sequence DKELQDLLDFSLQY was derived from PCET to interact with KIX with higher affinity than the wild type sequence. This peptide now serves as a lead molecule for further development as an inhibitor of the E2A:CBP/p300 interaction. Fluorescence anisotropy, peptide microarray technology, and isothermal titration calorimetry were employed to characterize interactions between both TAZ domains of CBP/p300 and the PCET motif and AD2 of E2A. Alanine substitution of residues within PCET demonstrated that the ΦxxΦΦ motif is a key mediator of these interactions, analogous to the PCET:KIX interaction. These findings now inform future work to establish possible physiological roles for the E2A:TAZ1 and E2A:TAZ2 interactions. / Thesis (Master, Biochemistry) -- Queen's University, 2013-08-06 13:52:28.248

Leung Po-Ki. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2002. / Includes bibliographical references (leaves 204-235). / Abstracts in English and Chinese. / ACKNOWLEDGEMENTS --- p.i / ABBREVIATIONS --- p.ii / ABSTRACT --- p.vi / 撮要 --- p.x / TABLE OF CONTENTS --- p.xiii / Chapter CHAPTER 1: --- GENERAL INTRODUCTION / Chapter 1.1 --- Hematopoiesis & Leukemia --- p.1 / Chapter 1.1.1 --- An Overview on Hematopoiesis --- p.1 / Chapter 1.1.2 --- Leukemia - Aberrant Hematopoiesis --- p.4 / Chapter 1.1.2.1 --- Classification and Epidemiology of Leukemia --- p.4 / Chapter 1.1.2.2 --- Pathophysiology and Etiology of Leukemia --- p.7 / Chapter 1.1.2.3 --- Conventional Treatments for Leukemia --- p.9 / Chapter 1.1.2.4 --- New Avenues for Leukemia Therapy --- p.11 / Chapter 1.2 --- Coumarins: General Properties and Pharmacological Activities --- p.13 / Chapter 1.2.1 --- Introduction to Coumarins --- p.13 / Chapter 1.2.1.1 --- Historical Development of Coumarins --- p.13 / Chapter 1.2.1.2 --- Occurrence and Functions of Coumarins in Plants --- p.13 / Chapter 1.2.2 --- Phytochemistry and Metabolism of Coumarins --- p.14 / Chapter 1.2.2.1 --- Chemical Structures of Coumarins --- p.14 / Chapter 1.2.2.2 --- Biosynthesis of Coumarins --- p.18 / Chapter 1.2.2.3 --- Toxicology of Coumarins --- p.18 / Chapter 1.2.2.4 --- Metabolic Pathways and Pharmacokinetics of Coumarins --- p.19 / Chapter 1.2.3 --- Pharmacological Activities of Coumarins --- p.22 / Chapter 1.2.3.1 --- Anti-edema and Anti-inflammatory Activities --- p.22 / Chapter 1.2.3.2 --- Immunomodulatory Activity --- p.23 / Chapter 1.2.3.3 --- Anti-tumor Activity --- p.23 / Chapter 1.2.3.3.1 --- Mode of Entry of Coumarins into Tumor Cells --- p.23 / Chapter 1.2.3.3.2 --- Anti-carcinogenic Effect --- p.24 / Chapter 1.2.3.3.3 --- Anti-proliferative Activity --- p.25 / Chapter 1.2.3.3.4 --- Induction of Cell Differentiation --- p.26 / Chapter 1.2.3.3.5 --- Other Biological Activities --- p.26 / Chapter 1.2.4 --- Clinical Applications of Coumarins --- p.27 / Chapter 1.2.4.1 --- Treatment of Lymphoedema and Other High-protein Edemas --- p.27 / Chapter 1.2.4.2 --- Treatment of Thermal Injuries --- p.27 / Chapter 1.2.4.3 --- Therapeutic Agent for Renal Cell Carcinoma --- p.28 / Chapter 1.2.4.4 --- Therapy of Prostate Cancer --- p.29 / Chapter 1.3 --- Tumor Models Used in This Study --- p.30 / Chapter 1.3.1 --- Myeloid Leukemias --- p.30 / Chapter 1.3.1.1 --- HL-60 --- p.30 / Chapter 1.3.1.2 --- K562 --- p.30 / Chapter 1.3.1.3 --- EoL-1 --- p.30 / Chapter 1.3.1.4 --- WEHI-3B JCS --- p.31 / Chapter 1.3.2 --- Neuroblastoma - Neuro-2a BU-1 --- p.31 / Chapter 1.4 --- Aims and Scopes of This Investigation --- p.33 / Chapter CHAPTER 2: --- MATERIALS AND METHODS / Chapter 2.1 --- Materials --- p.36 / Chapter 2.1.1 --- Animals --- p.36 / Chapter 2.1.2 --- Cell Lines --- p.36 / Chapter 2.1.3 --- "Cell Culture Medium, Buffers and Other Reagents" --- p.37 / Chapter 2.1.4 --- [methyl-3H] Thymidine (3H-TdR) --- p.40 / Chapter 2.1.5 --- Methylthiazoletetrazolium (MTT) --- p.41 / Chapter 2.1.6 --- Nitro Blue Tetrazolium (NBT) --- p.41 / Chapter 2.1.7 --- Liquid Scintillation Cocktail --- p.42 / Chapter 2.1.8 --- Reagents and Buffers for Flow Cytometry --- p.42 / Chapter 2.1.9 --- Mouse Anti-MAP-2 Monoclonal Antibody --- p.44 / Chapter 2.1.10 --- Reagents for DNA Extraction --- p.44 / Chapter 2.1.11 --- Reagents for Total RNA Isolation --- p.45 / Chapter 2.1.12 --- Reagents and Buffers for RT-PCR --- p.46 / Chapter 2.1.13 --- Reagents and Buffers for Gel Electrophoresis --- p.49 / Chapter 2.1.14 --- Reagents and Buffers for Western Blot Analysis --- p.50 / Chapter 2.1.15 --- Reagents for Measuring Caspase Activity --- p.58 / Chapter 2.2 --- Methods / Chapter 2.2.1 --- Culture of the Tumor Cell Lines --- p.61 / Chapter 2.2.2 --- "Isolation, Preparation and Culture of Mouse Peritoneal Macrophages" --- p.61 / Chapter 2.2.3 --- Determination of Cell Proliferation by [3H]-TdR Incorporation Assay --- p.62 / Chapter 2.2.4 --- Determination of Cell Viability --- p.62 / Chapter 2.2.5 --- Cell Morphology Study --- p.63 / Chapter 2.2.6 --- Immunocytochemistry --- p.64 / Chapter 2.2.7 --- Confocal Microscopy --- p.64 / Chapter 2.2.8 --- NBT Reduction Assay --- p.65 / Chapter 2.2.9 --- In vivo Tumorigenicity Assay --- p.65 / Chapter 2.2.10 --- In vivo Anti-tumor Study --- p.65 / Chapter 2.2.11 --- Measurement of In vivo Macrophage Migration --- p.66 / Chapter 2.2.12 --- Measurement of Cytokine Production by ELISA --- p.66 / Chapter 2.2.13 --- Measurement of Apoptosis by DNA Fragmentation Analysis --- p.67 / Chapter 2.2.14 --- Determination of the Mitochondrial Membrane Potential --- p.67 / Chapter 2.2.15 --- Cell Cycle/DNA Content Evaluation --- p.68 / Chapter 2.2.16 --- Nitric oxide/Annexin V-PE Dual Sensor Assay --- p.68 / Chapter 2.2.17 --- Gene Expression Study --- p.68 / Chapter 2.2.18 --- Protein Expression Study --- p.71 / Chapter 2.2.19 --- Measurement of Caspase Activity --- p.74 / Chapter 2.2.20 --- Statistical Analysis --- p.75 / Chapter CHAPTER 3: --- STUDIES ON THE ANTI-TUMOR ACTIVITIES OF COUMARINS ON MYELOID LEUKEMIA CELLS / Chapter 3.1 --- Introduction --- p.76 / Chapter 3.2 --- Results --- p.18 / Chapter 3.2.1 --- Differential Anti-proliferative Effect of Coumarins on Various Leukemic Cell Lines In Vitro --- p.78 / Chapter 3.2.2 --- Cytotoxic Effect of Coumarins on Various Leukemic Cell Lines In Vitro --- p.91 / Chapter 3.2.3 --- "Kinetic, Reversibility and Stability Studies of the Anti- proliferative Effect of Coumarins on the Leukemia JCS cells" --- p.94 / Chapter 3.2.4 --- Induction of DNA Fragmentation in Myeloid Leukemia Cells by Coumarins --- p.100 / Chapter 3.2.5 --- Effect of Coumarins on the Cell Cycle Kinetics of the Leukemia JCS Cells In Vitro --- p.107 / Chapter 3.2.6 --- Effect of Coumarins on the In Vivo Tumorigenicity of the Leukemia JCS Cells --- p.112 / Chapter 3.2.7 --- Effect of Esculetin on the In Vivo Growth of the Leukemia JCS cells in Syngeneic Mice --- p.115 / Chapter 3.3 --- Discussion --- p.117 / Chapter CHAPTER 4: --- AN INVESTIGATION ON THE DIFFERENTIATION- INDUCING EFFECT OF COUMARINS / Chapter 4.1 --- Introduction --- p.122 / Chapter 4.2 --- Results --- p.124 / Chapter 4.2.1 --- The Differentiation-inducing Effect of Coumarins on Myeloid Leukemia Cells --- p.124 / Chapter 4.2.1.1 --- Morphological Changes in Coumarin-treated HL-60 Cells --- p.124 / Chapter 4.2.1.2 --- NBT Reduction of HL-60 Cells --- p.127 / Chapter 4.2.1.3 --- Effects of Coumarins on the Cell Size and Granularity of HL-60 Cells --- p.129 / Chapter 4.2.2 --- The Anti-proliferative and Differentiation-inducing Effects of Coumarins on Neuroblastoma Cells --- p.131 / Chapter 4.2.2.1 --- Anti-proliferative Effect of Coumarins on the BU-1 Cell Line In Vitro --- p.131 / Chapter 4.2.2.2 --- Morphological Changes in Coumarin-treated BU-1 Cells --- p.134 / Chapter 4.2.2.3 --- Immunocytochemistry of Coumarin-treated BU-1 Cells --- p.137 / Chapter 4.3 --- Discussion --- p.139 / Chapter CHAPTER 5: --- MECHANISTIC STUDIES ON THE ANTI-LEUKEMIC ACTIVITIES OF COUMARINS / Chapter 5.1 --- Introduction --- p.142 / Chapter 5.2 --- Results --- p.147 / Chapter 5.2.1 --- Modulatory Effects of Coumarins on the Expression of Apoptosis-regulatory Genes in the Leukemia JCS Cells --- p.147 / Chapter 5.2.2 --- Modulatory Effects of Coumarins on the Expression of Growth-related Genes in the Leukemia JCS Cells --- p.151 / Chapter 5.2.3 --- Modulatory Effects of Coumarins on the Expression of Apoptosis-regulatory Proteins in Leukemia JCS Cells --- p.157 / Chapter 5.2.4 --- Modulatory Effects of Coumarins on the Expression of Growth-related Proteins in Leukemia JCS Cells --- p.162 / Chapter 5.2.5 --- Effect of Coumarins on the Mitochondrial Membrane Depolarization of the Leukemia JCS cells --- p.165 / Chapter 5.2.6 --- Induction of Apoptosis and Nitric Oxide Production in Leukemia JCS Cells by Coumarins --- p.168 / Chapter 5.2.7 --- Effects of Coumarins on the Caspase Activity in the Leukemia JCS cells --- p.172 / Chapter 5.3 --- Discussion --- p.177 / Chapter CHAPTER 6: --- STUDIES ON THE IMMUNOMODULATORY EFFECT OF COUMARINS ON MURINE MACROPHAGES / Chapter 6.1 --- Introduction --- p.185 / Chapter 6.2 --- Results --- p.188 / Chapter 6.2.1 --- Effect of Coumarins on the Viability of Macrophages In vitro --- p.188 / Chapter 6.2.2 --- Effect of Coumarins on the In vivo Migration of Macrophages --- p.190 / Chapter 6.2.3 --- Effect of Coumarins on Cytokine Production by Macrophages --- p.192 / Chapter 6.3 --- Discussion --- p.194 / Chapter CHAPTER 7: --- CONCLUSIONS AND FUTURE PERSPECTIVES --- p.197 / REFERENCES --- p.204

Clonal diversity in cancer has been proposed as a mechanism underlying patient-to-patient variability in therapeutic response, as well as the variability in the likelihood and the time to relapse of acute myeloid leukemia (AML) and other cancers. As a neoplasm develops it often continues to mutate, diversifying into differing clonal populations. Darwinian evolutionary pressures such as inherent fitness imbalances, immune system interactions, and chemotherapy treatments target sensitive clones and drive competition between the clonal populations; selecting for dynamic and resistant cell lines. In this way clonal diversity is conceivable as an impediment to a complete remission with more populations offering more opportunities for therapy resistance. Bulk next generation sequencing (NGS) is currently used to assess clonal composition in leukemia but requires several broad assumptions be made, which can result in incorrect assessments of diversity. Factors such as differences in zygosity of mutations, convergent evolution, or contamination with wild-type/non-cancerous cells can artificially raise or lower reported variable allele frequencies (VAF), leading to errors in clonal assessments. To examine discrepancies between the actual clonal structure and the clonal structures determined through bulk sequencing we developed a novel method of sampling the cell population to identify concurrent mutations. We first created an in silico model which randomly draws cell samples from a simulated tumor multiple times and calculates the VAF for each mutant allele in each sample. By tracking the correlation of mutations between sample replicates, a clonal composition that is not observable from the bulk NGS VAF becomes apparent. We then created in vitro model tumors from AML cell lines, isolated low cell number samples via flow cytometry, and applied a multiplex/nested PCR protocol with pyrosequencing to quantify VAFs in each sample. Again, by calculating the correlation of mutant alleles between replicates, previously unseen with NGS characteristics of the clonal structure becomes evident. Population sampling analysis may potentially offer a solution for clarifying how we can interpret NGS clonal analyses.

Acute lymphoblastic leukemia (ALL) is an aggressive hematological cancer which arises from the malignant transformation of B-cell or T-cell progenitors. Despite recent pioneering improvements in intensified combination chemotherapy, 20% of pediatric and 50% of adult ALL patients present with primary drug-resistant leukemia or develop relapse. Treatment of refractory and relapsed ALL has remained a significant clinical challenge with survival rates following relapse of only 40%, highlighting the need to understand the mechanisms which drive drug resistance and relapse of ALL. Through extensive sequencing analyses of matched diagnostic, remission and relapsed DNA samples from patients with B-precursor ALL (B-ALL) and T-cell ALL (T-ALL) we have identified recurrent relapse-specific gain-of-function mutations in the cytosolic 5'-nucleotidase II (NT5C2) gene in 25% of relapsed T-ALLs and 6% of relapsed B-ALLs. NT5C2 is a highly conserved, ubiquitously expressed enzyme which regulates intracellular purine nucleotide levels by dephosphorylating purine monophosphates. NT5C2 also dephosphorylates key metabolites in the activation of purine analog prodrugs such as 6-mercaptopurine and 6-thioguanine which are routinely used in the treatment of ALL, allowing purine analog nucleosides to be readily exported out of the cell. Here we show that mutant NT5C2 proteins have increased 5’-nucleotidase activity and confer resistance to 6-mercaptopurine and 6-thioguanine chemotherapy when expressed in leukemic cells. Consistently, NT5C2 mutations correlate with early relapse and relapse while under therapy. We present a novel T-ALL conditional inducible knock-in mouse model of the highly recurrent NT5C2 R367Q mutation and show that expression of one Nt5c2 R367Q allele from the endogenous locus in primary T-ALL lymphoblasts induces overt resistance and disease progression under therapy with 6-mercaptopurine in vivo, while surprisingly conferring reduced growth and decreased leukemia initiating activity in the absence of chemotherapy. Metabolically we show that the observed loss of fitness in Nt5c2 R367Q tumors can be explained by a severe depletion of endogenous purine monophosphate metabolites as a result of increased Nt5c2 5’-nucleotidase activity. Consistently, using ultra-sensitive mutation analyses we show that relapse-associated NT5C2 mutations are not detectable at initial disease presentation, indicating that NT5C2-mutant tumor cells are negatively selected by clonal competition in the early stages of disease development and only positively selected under prolonged 6-mercaptopruine chemotherapy which is the backbone treatment for ALL following remission. Our findings present the first known example of chemotherapy resistance and disease progression driven by a tumor clone with decreased leukemia initiating activity, highlighting the intense selective pressure of chemotherapy in the clonal evolution of tumors from diagnosis to relapse. Through extensive biochemical and structural characterizations of recombinant NT5C2 mutant proteins, we have grouped relapse-specific NT5C2 activating mutations into 3 different classes, each conferring unique enzymatic behavior in basal conditions and in response to allosteric activation, and each with unique structural features which mediate increased 5’-nucleotidase activity. Moreover, we identify a novel auto-regulatory switch-off mechanism of the NT5C2 enzyme involving movement of an unstructured flexible loop, and present the first crystal structure view of the NT5C2 C-terminal acidic tail, implicating it as an auto-inhibitory brake to the allosteric activation of the enzyme. The presence of multiple mutational mechanisms of activating such a highly conserved enzyme, especially in light of the inherent loss of fitness to the tumor cells, indicates a strong convergent evolution towards activating NT5C2. This is supported by our discovery that patients can harbor multiple leukemic clones with NT5C2 mutations at relapse. Overall our findings highlight NT5C2 as a major driver of drug resistance and relapse of ALL and pinpoint metabolic susceptibilities which could be exploited therapeutically to target NT5C2-mutant tumors in the future. Our in-depth structural and enzymatic knowledge of mutant NT5C2 proteins will serve as an essential tool in the rational drug development of novel NT5C2 inhibitors with increased specificity and selectivity for mutant NT5C2, while our novel Nt5c2 R367Q knock-in mouse model will serve as a platform for the pre-clinical testing of both NT5C2 inhibitors and alternative compounds selective for Nt5c2-mutant leukemias which can be used for prevention and treatment of relapsed ALL.

Thesis (M. Ed.)--Kutztown State College. / Source: Masters Abstracts International, Volume: 45-06, page: 3060. Typescript. Includes bibliographical references (leaves 32-34).