Saudi Cultural Missions Theses & Dissertations
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Item Restricted Evaluation of DNA Repair Biomarkers in Epithelial Ovarian and Breast Cancers(Saudi Digital Library, 2025-01-02) (Shoqafi, Ahmed); Madhusudan, SrinivasanDNA damage signalling and repair pathways play a crucial role in the regulation of genomic stability. Impaired DNA repair promotes genomic instability, a key route to the development of cancers. Cells have developed signalling pathways that are activated in response to DNA damage to detect and repair the damage that has occurred. Ataxia-Telangiectasia Mutated (ATM) and Ataxia-Telangiectasia and Rad3-related (ATR) kinases are activated in the presence of DNA damage and initiate a series of phosphorylation events that lead to the activation of downstream effectors. Some of these effectors include p53 and BRCA1, which regulate cell cycle, DNA repair, and apoptosis. The major DNA repair pathways are nucleotide excision repair (NER), which removes bulky DNA adducts; base excision repair (BER), which is involved in repairing small, non-helix-distorting base lesions; mismatch repair (MMR), which corrects replication errors and double-strand break repair which include homologous recombination (HR) and non-homologous end joining (NHEJ). All these pathways are regulated in a manner that is specific to the type of damage that needs to be repaired so that repair is as accurate as possible. Since conventional therapies for advanced epithelial ovarian cancer (EOC) and breast cancers have been found to be less effective, there has been a shift to search for new therapeutic approaches that aim at targeting the DNA repair pathways. The host laboratory, using whole-exome sequencing in platinum-sensitive (PEO1, A2780) and platinum-resistant (PEO4, A2780cis) ovarian cancer cell lines, identified TP73 and POLE as potential predictors of platinum resistance. In the current study, I have investigated the role of TP73 and POLE in EOC and breast cancers in detail. TP73 is a member of the TP53 family of transcription factors that are involved in DNA repair, cell growth, migration, and death. In 331 EOC samples, I observed that high protein expression of TP73 was associated with higher tumour grade, late-stage disease and shorter progression free survival (PFS). In the large publicly available clinical cohort (n=522) and the cancer genome atlas (TCGA) ovarian cohort (n=182), TP73 transcript was upregulated in tumours compared to normal tissues and associated with shorter PFS. Preclinically, I have shown that overexpression of TP73 in A2780 platinum-sensitive ovarian cancer cells enhanced cell proliferation, invasion and increased DNA repair capacity. In clinical breast cancers, analysis of TP73 expression in 1,369 invasive breast cancers and 317 DCIS cases revealed that high cytoplasmic TP73 expression is significantly associated with aggressive disease features, including high tumour grade, ER negativity, triple-negative phenotype, and poor breast cancer-specific survival, particularly in the TP53 mutant subgroup. These findings highlight the prognostic and predictive significance of TP73 in EOC and breast cancers. POLE has roles during DNA replication and repair pathways. I have investigated POLE expression in EOC and breast cancers. Immunohistochemical analysis of 331 EOC samples revealed that 75% exhibited low nuclear POLE expression, while 25% showed high expression. High POLE levels were significantly associated with higher tumour grade, poor progression free survival (PFS) and overall survival (OS). The transcriptomic levels of POLE were analysed in patients with EOC, revealing that high POLE mRNA expression was significantly associated with poor progression free survival (PFS) and overall survival (OS) (All p=<0,05). Functional studies in platinum-resistant OVCAR 4 cells demonstrated that POLE knockdown increased cisplatin sensitivity, which was associated with double-strand break (DSB) accumulation, S-phase cell cycle arrest and increased apoptosis. The data supports the role of POLE in predicting response to platinum chemotherapy in EOC. The study of 1,480 invasive breast cancer cases revealed that exhibited low nuclear POLE expression, which was associated with aggressive tumour features, poorer breast cancer specific survival (BCSS), and reduced response to endocrine therapy in ER+ and luminal subtypes. These findings suggest that POLE may be a predictive factor in ER+ breast cancers. Taken together, the data provides evidence for the role of TP73 and POLE as potential biomarkers in EOC and breast cancers.82 0Item Restricted Development of a novel therapeutic intervention for p53-mutant advanced cancers leveraging their DNA damage response liabilities(University at Buffalo, 2025-05) Alruwaili, Mohammed Muharrab; Andrei, BakinColorectal Cancer (CRC) and Pancreatic Ductal Adenocarcinoma (PDAC) are among most lethal cancers worldwide. Despite initial response to standard-of-care therapy, a significant proportion of CRC/PDAC cancers relapse and progress to metastatic disease with poor overall survival (OS). Thus, better treatment options are urgently needed. Genetic alterations in the tumor suppressor p53 gene (TP53) are found in most CRC and PDAC cases and contribute to cancer relapse, progression, and metastasis. Even though the functional consequences of p53 mutations have been extensively studied, there are no FDA-approved drug or their combination targeting p53-mutant cancers. This thesis work is aimed at the development of effective therapeutic approaches for p53-mutant cancers. The initial concept of the two-drug strategy, working as an inducer-amplifier pair, was suggested for selectively targeting p53-mutant triple-negative breast cancer (Zonneville at al., Commun. Biology, 2021). The current thesis research investigated the mechanisms underlying the efficacy and selectivity of our two-drug strategy using p53-mutant CRC and PDAC models. Our two-drug strategy utilizes thymidine analogue TAS102, acting as an inducer of single-strand DNA breaks through a post-replicative DNA repair, and poly (ADP-ribose) polymerase (PARP) inhibitor, acting by delaying repair of single-strand breaks in DNA. This two-drug concept is based on the following rationale. Our analysis of the genomic data from the Cancer Genome Atlas (TCGA) showed high expression levels of Base-Excision Repair (BER) and elevated tumor mutational burden (TMB) in p53-mutant CRC and PDAC, suggesting impairment in BER. The BER activity in CRC and PADC cells was examined by a new methodology with thymidine analogues (ethynyl-deoxyuridine (EdU) or trifluorothymidine (TFT, a component of TAS102)). The analysis revealed a significant delay in the removal of genomic EdU or TFT in p53-mutant cells compared to isogenic p53 wildtype (p53WT) cells. We also noted that p53-mutant cells accumulated in the late S/G2 phase while p53WT accumulated in G1. Thymidine analogues (EdU, TFT) induced buildup of DNA breaks in p53-mutant cancer cells, while non-tumor p53WT cells showed only a transient DNA damage. Further, addition of a PARP inhibitor (PARPi) increased DNA double-strand breaks (DSBs) and cell death selectively in p53-mutant cells, while PARPi alone did not induce DNA damage. Cytotoxicity analysis showed that thymidine analogues synergized with PARP inhibitors selectively in p53-mutant cells. In preclinical vivo models, the TAS102-PARPi combination was far more effective than either drug alone in p53-mutant Cell-Derived Xenograft (CDX) and Patient-Derived Xenograft (PDX) models. Immunohistochemistry data showed that the two-drug treatment increased DNA damage and cell death while decreasing cell proliferation. The two-drug combination and TAS102 exhibited comparable tumor control in the p53WT PDX model. Importantly, the TAS102-PARPi therapy did not exhibit major side toxic effects in mice even after prolonged drug administration. Our two-drug TAS102-PARPi strategy is now being tested in the first-in-human phase I study with TAS102 and talazoparib in refractory CRC (NCT04514497, PI Dr. Fountzilas). The dose-escalation part of the study showed that the TAS102-talazoparib treatment is well-tolerated, and no major dose-limiting toxicities were observed. The analysis showed that the addition of talazoparib to TAS102 increased TFT-positive cells in peripheral blood mononuclear cells (PBMCs) and in tumor tissues. The median progression-free survival (mPFS) was 5.7 months in the DL3 group compared to 1.9 months for historical TAS102 alone. Investigation of the mechanism behind the two-drug strategy showed that incorporation of thymidine analogues into DNA provoked post-replicative BER-mediated repair, generating DNA single-strand breaks (SSBs). As SSB repair is assisted by PARP, the inhibition of PARP increased more lethal DSBs. Non-tumor p53WT cells responded with activation of the p53-p21 axis, leading to G1-arrest and efficient removal of thymidine analogues. In contrast, p53-mutant cells, lacking the G1 checkpoint, accumulated DNA damage and were arrested in G2 for a prolonged time. Investigation of DNA damage response (DDR) revealed that TAS102-PARPi induced signaling pathways mediated by ATM (Ataxia-Telangiectasia Mutated) and ATR (Ataxia-Telangiectasia Rad3-related) kinases. Our study showed that ATM kinase-controlled activation of the p53-p21 axis, whereas ATR kinase-controlled activation of CHK1 and WEE1 kinases, which mediate induction of the G2-checkpoint, blocking the entrance to mitosis. Furthermore, the cell-cycle analysis demonstrated that the TAS102-PARPi treatment induced G2-arrest and high levels of DSBs selectively in p53-mutant cells while p53WT cells were transiently arrested in G1 and resumed the cell cycle after drug withdrawal. This finding suggested that blockade of ATR kinase or its downstream effectors may release p53-mutant cells with unrepaired DNA into mitosis, leading to cell death. This hypothesis was examined using several approaches, including cytotoxicity, immunofluorescence microscopy, and flow cytometry. We found that the blockade of ATM reduced sensitivity to TAS102-talazoparib in all tested cell lines. In contrast, blockade of ATR kinase markedly enhanced the cytotoxicity of TAS102-talazoparib in p53-mutant cells and increased levels of γH2AX and phospho-H3 in cells treated with TAS102-talazoparib, indicating increased DNA damage and entrance to mitosis. Similar effects were observed by blockade of downstream effector kinase WEE1. Furthermore, blockade of WEE1 markedly increased cell death in p53-mutant cells treated with TAS102-talazoparib. Based on these data, we developed a triple-drug strategy combining the TAS102-PARPi treatment with a sequential delayed application of the G2 checkpoint kinase inhibitors. This sequential triple-drug combination works as an inducer–amplifier–terminator trio to induce cell death in p53-mutant cells selectively. The in vivo studies showed that this sequential triple-drug strategy was more effective in tumor growth control compared to the TAS102-PARPi treatment or WEE1i alone. In the CRC HT29 tumor xenograft model, the triple-drug treatment reduced tumor growth by 63%, compared to 18% by WEE1i or 43% by TAS102-talazoparib alone. Likewise, the triple-drug regimen showed greater tumor control in the CRC-PDX-03-26-RP model carrying p53H178T/Ter67fs and KRAS-G12D. In the PDAC PDX-14312 model, carrying p53R175H, a triple-drug treatment reduced tumor growth by ~80%, whereas WEE1i monotherapy by ~30% or TAS102-talazoparib by ~43%. Importantly, neither of the treatments exhibited major side toxic effects or caused changes in major organs (kidney, liver, lungs, heart) based on the pathology analysis of mice. These preclinical data provide strong support for a sequential triple-drug treatment strategy that works as an inducer–amplifier–terminator trio to induce cell death in p53-mutant cancers. This novel triple-drug therapeutic strategy for p53-mutant cancers has strong potential to improve the management of CRC and PDAC and meaningfully impact the health of cancer patients.16 0Item Restricted Role of Retinoic Acid Receptor β Signalling in Alzheimer’s Disease(Saudi Digital Library, 2023-08-27) Almuallim, Hassan Yousef Othman; Corcoran, JonathanAlzheimer's disease, the most common form of Dementia, is characterized by a debilitating progression of memory impairment and cognitive decline. The distinct neuropathological hallmarks characterizing Alzheimer's disease revolve around the existence of senile plaques containing amyloid-β, as well as neurofibrillary tangles composed of tau protein, precipitating oxidative stress and neuroinflammation. The therapeutic options for Alzheimer’s disease are either symptomatic treatments or disease-modifying drugs, with the latter currently showing significant investment in targeting amyloid-β. Interestingly, DNA double-strand breaks play a role in increasing the secretion of amyloid-β, consequently triggering the aggregation of amyloid-β plaques. However, there are currently no therapeutic options available for Alzheimer’s disease that specifically target the induction of DNA repair. KCL286, a synthetic retinoic acid receptor beta agonist with promising results from a phase I clinical trial, has demonstrated its capacity to initiate DNA repair mechanisms in a nerve injury model. In this context, we embarked upon an investigation into the effects of KCL286 administration in Tg2576 mice, a transgenic strain characterized by human amyloid precursor protein overexpression and consequently amyloid-β accumulation. A three-month treatment significantly reduced the DNA damage response protein γH2AX, in particular the gene expression of γH2AX within the hippocampus. The inducing of DNA double-strand break repair corresponded with the clearance of amyloid-β deposits and reduction in inflammation. The study underscores the potential of stimulating the RARβ signalling pathway via the RARβ agonist KCL286, as it appears to induce DNA repair mechanism and amyloid-β clearance. This reinforces the drug’s promise as a prospective therapeutic candidate for Alzheimer's disease.22 0Item Restricted Effect of the inflammatory mediator TNF-α on colorectal cancer epithelial cells development and metastasis, role of dietary carcinogens and miRNA(ICL, 2023-05-01) Alotaibi, Aminah Ghazi; Gooderham, Nigel; Li, JiaColorectal cancer (CRC) is the third most common cancer world-wide and second leading cause of mortality. The majority of CRC cancer cases result from epigenetic and genetic alterations that promote development and metastasis of the disease. Exposure to environmental and dietary carcinogens are strongly associated with CRC. Also, inflammatory mediators are known as a major risk factor for CRC, however the underlying mechanisms are still understudied. Upregulation of pro-inflammatory mediators and dysregulation of miRNAs in the tumour microenvironment (TME) have been observed in CRC. Tumour necrosis factor alpha (TNF-α) is a pleiotropic cytokine thought to play numerous roles in tumour progression including epigenetic gene regulation, activation of tumour promoting signalling pathways, thus presence of TNF- in CRC tumour microenvironment may be key to promoting CRC progression. I hypothesise that the presence of TNF- α in the TME could regulate miRNAs and enzyme expression to induce DNA damage caused by dietary carcinogens, thereby stimulating changes that promote CRC development and metastasis. The present study investigated this hypothesis through a mechanistic approach with in vitro cell line culture. Effects of TNF-α on phenotypic changes were observed and the potential involvement of miRNAs were determined. The results showed that TNF-α enhances dietary carcinogen-induced DNA damage through activation of JNK signalling pathway. Also, TNF-α induced metastatic phenotype cell proliferation and migration through miRNA regulation. Moreover TNF-α regulated expression of CYP450 enzymes through miRNA regulation, which can promote chemical carcinogen genotoxicity. Taken together, the data indicated that CRC progression and metastasis may be related to epigenetic and inflammatory mediators active at the tumour site. Understanding these molecular mechanisms could provide better prevention and therapeutic strategies.27 0