Small molecules are a powerful tool to illuminate biological mechanisms and assist in the identification and validation of therapeutic targets. KRAS is the single most frequently mutated oncogene in human cancer, with particularly high mutation frequencies observed in pancreas (95%), colon (45%), and lung (35%) cancer. However, despite three decades of effort, there is no clinical viable KRAS cancer therapy. The first part of this thesis focuses on exploring the potential of directly targeting the KRAS nucleotide binding site. Directly targeting oncogenic KRAS with small molecules in the nucleotide-binding site has had limited success due to the high affinity of KRAS for nucleotide GTP and the high cellular concentration of GTP. The strategy of generating engineered KRAS allele based on shape and covalent complementarity was exploited herein to address this challenge. Using fragment-based small molecule design, a cell-membrane-permeable covalent inhibitor able to irreversibly modify the engineered nucleotide-binding site of KRAS was developed. The second part of this thesis describes the investigation of the therapeutic potential of imidazole ketone erastin (IKE), a small molecule inhibitor of the cystine/glutamate antiporter system xc–, in a subcutaneous xenograft model of Diffuse Large B Cell Lymphoma (DLBCL). A biodegradable polyethylene glycol-poly(lactic-co-glycolic acid) nanoparticle formulation was employed to aid in the delivery of IKE to cancer cells in vivo. This IKE nanoparticle system showed improved tumor accumulation and therapeutic index relative to free IKE, indicating its potential for treating DLBCL. The final part of this thesis describes the study of lipid metabolism features of ferroptotic cell death using quantitative reverse transcription PCR (RT-qPCR) and mass spectrometry-based lipidomic analysis. In summary, this work illustrates how chemistry and chemical biology approaches can supplement existing efforts towards the design and discovery of new drugs for challenging targets, as well as aid in the study of therapeutic mechanisms.

Bibliography: leaves 170-196. / xix, 199 leaves, [84] leaves of plates : ill. (some col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / The results presented in this thesis establish the growth-suppressive activity of the human GAS1 gene and provide the first direct evidence that GAS1 can inhibit the growth of tumours. In addition, this study demonstrates that the antiproliferative effect of GAS1 are mediated by a p53 dependent pathway and that functional inactivation of p53 by either mutation and/or overexpression of the MDM2 oncogene product inhibits the GAS1 mediated growth-suppression. / Thesis (Ph.D.)--University of Adelaide, Dept. of Physiology, 1997?

Thesis (Ph. D.)--Ohio State University, 2006. / Available online via OhioLINK's ETD Center; full text release delayed at author's request until 2007 Aug 10

Thesis (Ph.D.)--Ohio University, August, 2009. / Release of full electronic text on OhioLINK has been delayed until September 1, 2012. Includes bibliographical references (leaves 89-107)

Thesis (Ph.D.)--Ohio University, August, 2009. / Title from PDF t.p. Release of full electronic text on OhioLINK has been delayed until September 1, 2012. Includes bibliographical references (leaves 89-107)

Thesis (doctoral)--University of Wisconsin-Madison, 2000. / Includes bibliographical references.

published_or_final_version / Clinical Oncology / Doctoral / Doctor of Philosophy

Glioblastoma multiforme (GBMs) are the most common and severe form of malignant brain tumors. Despite recent advancement in the fields of surgical resection, radiotherapy and chemotherapy, the prognosis for patients with GBMs remains poor with the median survival rate of approximately a year. Recently, our laboratory has demonstrated the oncogenic role of cell cycle-related kinase (CCRK), a 42-kD protein kinase responsible for regulating cell growth in GBM carcinogenesis, suggesting that CCRK is a candidate oncogene in GBMs. Nevertheless, the regulation of CCRK expression and the cellular mechanism for its overexpression in GBMs remain elusive. Understanding the regulation of human CCRK expression in GBMs should therefore shed light on the development of better prognostic and therapeutic methods for this deadly disease. This study aims to characterize the human CCRK gene and the regulation of its expression in GBMs. We first characterized the 5’ upstream sequence of CCRK by in silico analysis, which revealed the absence of TATA box but the presence of three potential transcription factor binding sites for Sp1, c-Myc and CREB, and identified the transcription start site by 5’-RACE at 240 bp upstream of the start codon. In vitro analysis of the CCRK promoters revealed the presence of nucleotide polymorphisms in three high-grade glioblastoma cell lines U-87 MG, U-138 MG and U-373 MG, and the control fibroblast cell line CCD19Lu. Furthermore, three CpG islands within the CCRK promoter were identified and the CCRK promoter was hypomethylated in these cell lines. Sp1, c-Myc and CREB binding sites as well as the nucleotide polymorphisms on the CCRK promoter were further investigated. The results from electrophoretic mobility shift assay showed that these transcription factors interacted with the corresponding cis-regulatory elements on the CCRK promoter, removal of the potential Sp1 and c-Myc binding sites lowered the CCRK promoter activity by 46 – 66 % in vitro. In addition, mutations introduced to the nucleotide polymorphisms reduced the CCRK promoter activity by 62 – 81 % in U-87 MG cells and enhanced the CCRK promoter activity by 1.35-fold in U-138 MG cells, suggesting their importance in regulating CCRK expression. c-Myc is a proto-oncogene with its overexpression associated with a large variety of tumors. As c-Myc binding site was identified in the CCRK promoter, therefore, the effect of c-Myc on CCRK expression was examined. c-Myc overexpression resulted in significant enhancements in the CCRK promoter activity by 30.74-fold in U-87 MG cells, 26.5-fold in U-373 MG cells and 6.09-fold in U-138 MG cells. On the contrary, c-Myc knockdown reduced the CCRK promoter activity by 49 % in U-87 MG cells, 35 % in U-373 MG cells and 17% in U-138 MG cells. In summary, this study represents the first molecular characterization of the human CCRK gene and findings of this study would prime others for future research on the molecular pathogenesis of CCRK-mediated GBMs and for developing CCRK as a potential therapeutic and diagnostic target for GBMs and possibly other cancers. / published_or_final_version / Biological Sciences / Doctoral / Doctor of Philosophy

Hepatocellular carcinoma (HCC) is the most common tumors worldwide. In contrast to other cancers, the prognosis of HCC is extremely poor, with less that 5% of 5-year survival rate worldwide. From our previous studies, we isolated Chromodomain Helicases/ATPase DNA binding protein1-Like (CHD1L) gene from chromosome 1q21, and characterized it as a specific oncogene in HCC. By using 2D-PAGE and MALDI-TOF mass spectrometry approach, Translationally Controlled Tumor Protein (TCTP) was identified as a CHD1L target, which was preferentially expressed in CHD1L-transfected cells. TCTP is a highly conserved protein and expressed in almost all mammalian tissues. It has been reported that TCTP interacts with microtubules in a cell-cycle-dependent manner, and functions as a prosurvival factor and inhibiting apoptosis. To better understand the molecular mechanisms of HCC progression, the effect of TCTP overexpression in HCC and the mechanism by which TCTP regulated cell-cycle progression were elucidated in this study. CHD1L is a unique oncogene belongs to SNF2-like subfamily. Mechanistic studies found that CHD1L protein directly binds to the promoter region (nt -733 to -1,027) of TCTP and activated TCTP transcription. Investigation of clinical HCC specimens found that overexpression of TCTP was not only significantly associated with the advanced tumor stage (P = 0.037) and overall survival time of HCC patients (P = 0.034), but also an independent marker associated with poor prognostic outcomes. Functional studies demonstrated that TCTP has tumorigenic abilities and overexpression of TCTP contributed to the mitotic defects of tumor cells. Further mechanistic studies demonstrated that TCTP promoted the ubiquitin-proteasome degradation of Cdc25c during mitotic progression, which caused the failure in the dephosphorylation of Cdk1 on Tyr 15 and decreased Cdk1 activity. The consequence of chromosome missegregation and mitotic catastrophe results in aneuploidy, which is frequently observed in cancer. In addition, the correlation between TCTP overexpression and metastatic potential of HCC was elucidated by examined the expression levels of TCTP using a tissue microarray (TMA) containing 60 pairs of primary HCCs and their matched metastases. Further studies demonstrated that overexpression of TCTP shows high incidence of extrahepatic metastasis and positive correlation was found between TCTP and MMP-2 or MMP-9 (Spearmen correlation coefficient=0.466, and 0.352, respectively, P<0.001 for both). In vitro functional studies showed that TCTP protein associated with promoter regions of MMP-2 and MMP-9 and activates their transcriptions. Molecular analyses revealed that TCTP served as a JunD coactivator and formed complexes with JunD and bind with consensus AP-1 sites on MMP-2 and MMP-9 promoters to enhance their expression in HCC cells. More importantly, high co-expression of TCTP and MMP-2 or MMP-9 was significantly associated with poor disease-free survival (log rank= 8.146, and 11.677 respectively, P =0.017 and 0.003 respectively). In summary, two novel molecular mechanisms (CDH1L/TCTP/Cdc25C/Cdk1) and (TCTP/JunD/MMP-2, MMP-9) were revealed during HCC progression and metastasis. Also, the prognostic value of TCTP and MMP-2 or MMP-9 coexpression for HCC was highlight in this study. / published_or_final_version / Clinical Oncology / Doctoral / Doctor of Philosophy

published_or_final_version / abstract / toc / Clinical Oncology / Doctoral / Doctor of Philosophy