Sir Peter MacCallum Department of Oncology - Theses
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ItemThe role of USP9X in Low-Grade Serous Ovarian Cancer( 2023)Low-grade serous ovarian carcinoma (LGSOC) is a rare histotype of epithelial ovarian cancer (EOC), and accounts for approximately 3-5% of diagnosed EOC cases. LGSOC is characterised by wildtype TP53 expression, frequent aberrance in the RAS/RAF signalling pathway, and relative genomic stability which in part explains LGSOC resistance to current standard-of-care platinum-based chemotherapeutics. Current chemotherapy strategies for LGSOC have predominantly been driven by that of the far more common high-grade serous subtype. Optimal cytoreductive surgery is challenging, given that the majority of LGSOC diagnoses are at a late stage where the cancer has metastasised from the primary site. Recently, alternative therapy strategies including targeted therapy of the RAS/RAF pathway have shown efficacy against tumours, but further characterisation into potential novel drivers of this disease is required to expand the treatment repertoire for patients suffering from this disease. Previous sequencing studies elucidated USP9X as one such potential driver of LGSOC. USP9X is a deubiquitinase involved in protein turnover. The gene has been implicated as both oncogenic and tumour-suppressive depending on the cancer type being investigated. In the context of LGSOC, little is known as to the role that this gene has in disease development. This thesis evaluated 121 LGSOC cases, 71 sequenced via targeted sequencing, 49 through whole exome sequencing, and 1 by whole genome sequencing. Sequencing results identified USP9X mutations at a frequency of 14%, and as the most frequently mutated non-RAS/RAF gene in the assessed cohort. Interrogation into the allelic status of these mutations revealed more than half of the mutations were inactivating, suggesting a tumour-suppressive function; USP9X was elucidated to follow a classical two-hit tumour suppressor model. Gene knockdown and knockout experiments on LGSOC cell lines highlighted a potential perturbance to clonogenic survival, but not to migration and proliferation. Mass spectrometry analysis on USP9Xnull LGSOC cell lines identified the molecular chaperone BAG3 as a likely direct substrate of USP9X, and the deubiquitinase as a potential regulator of the mTORC signalling pathway. Assessment of the global proteomic perturbations as a result of USP9X downregulation suggested the downstream consequences of USP9X suppression are likely to be decreased cell adhesion, and potentially increased cell migration and invasion.
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ItemOvercoming resistance to PI3K inhibitors in colorectal cancer( 2021)Colorectal cancer (CRC) is responsible for the second highest number of cancer deaths worldwide, with stage IV patients having a 5-year survival rate of only 14%. One of the treatments in development involves a targeted therapy directed at PI3K alpha, a protein mutated in 18% of CRC patients. Particularly, PI3K alpha is involved in several cancer hallmarks including survival, metabolism and migration. The introduction of PI3K inhibitors as targeted therapy in clinical trials caused an increase in overall survival and a decrease in disease progression. However, ultimately, treatment failure and tumour progression still occur due to development of drug resistance. Therefore, the aim of this project was to identify mechanisms of resistance to PI3K targeted therapy that can be exploited to overcome treatment failure. Specifically, the focus in this study is on BYL719, a PI3K alpha specific inhibitor. Firstly, a novel subcutaneous syngeneic mouse model of CRC was generated by the syngeneic transplant of Pik3ca mutated and Apc deleted gastrointestinal tumours from a mouse model of CRC. Tumour growth in this model was reduced upon treatment with BYL719. Mice were then chronically treated with BYL719 to induce resistance. Although the development of resistance was not confirmed within the time-frame of this thesis, this model proved to be a useful tool for pharmacological studies. Secondly, in silico analyses were performed, correlating the sensitivity of solid cancer cell lines to PI3K inhibition with CRISPR KO/mRNA/protein/metabolite data from online pharmacogenomic datasets. These analyses identified multiple pathways potentially involved with sensitivity to PI3K inhibition, including proteins involved in the regulation of the cytoskeleton, the PI3K/MAPK pathway, the endomembrane system and lipid and glutamine metabolism. Finally, an in vitro approach was performed using genome-wide CRISPR KO screen techniques, to identify additional functional pathways involved with sensitivity to PI3K inhibition. The results identified potential involvement of PI3K/Akt/MAPK signaling, mTOR/protein synthesis pathways, the Wnt pathway, TGF-beta pathway and metabolism, including lipids, glycolysis and the mitochondrial respiratory chain. Ultimately, these findings should enable the rational design of novel combination treatments with PI3K inhibitors to prevent or overcome resistance. Moreover, the findings might also be used to screen patients to predict their response to PI3K inhibitors.
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ItemInvestigating mechanisms of sensitivity and resistance to immunomodulatory thalidomide analogues in multiple myeloma( 2021)Multiple myeloma (MM) is an incurable plasma cell neoplasm. The incorporation of immunomodulatory imide drugs (IMiDs) – thalidomide, lenalidomide and pomalidomide - into clinical practice has improved survival and quality of life of MM patients. However, most patients develop therapeutic resistance to IMiDs, representing a significant clinical problem. IMiDs exert anti-MM effects by engaging Cereblon (CRBN), a ligand of the E3 ligase complex CUL4-DDB1-RBX1. By binding CRBN, IMiDs promote ubiquitination and proteasomal degradation of important MM cell transcription factors, IKZF1 (Ikaros) and IKZF3 (Aiolos). Degradation of such neosubstrates has also been shown to augment an anti-cancer immune response by activation of cytotoxic effector cells against MM plasma cells. Finally, it has been demonstrated that IMiDs modulate events occurring within the bone marrow microenvironment with negative impact on the MM-sustaining niche. Despite these recent advances in the characterisation of IMiD mechanism of action, the precise molecular events underpinning the anti-MM activity of IMiDs remain incompletely understood. Further insight is required to improve clinical responses to IMiDs and overcome therapy resistance in MM. In this work, genome-wide approaches such as RNA-sequencing and CRISPR-based functional genomics screening were employed to dissect the molecular mechanisms of IMiD-sensitivity and -resistance in models of acquired resistance to lenalidomide. RNA-sequencing revealed a strong interferon-like transcriptional response and increased transcription of genes downstream the MAPK pathway, together with downregulation of MYC-responsive genes, following IMiD treatment of IMiD-sensitive MM cells. Conversely, IMiD-resistant cells displayed reduced transcriptional changes following IMiD exposure as a likely consequence of CRBN downregulation and subsequent attenuation of neosubstrate degradation. Genome-wide CRISPR screening demonstrated that loss of CRBN and other genes regulating protein degradation, such as subunits of the COP9 signalosome, but also loss of genes that are not implicated in protein turnover, such as NCOR1 and EDC4, is sufficient to cause resistance to IMiDs in IMiD-sensitive cells. CRIPSR screening also demonstrated that resensitisation of IMiD-resistant cells to IMiDs can occur through loss of ATXN7, TOP2B and other genes implicated in several distinct biological processes, such as glucose metabolism, cell cycle progression and RNA processing. Dexrazoxane, a pan-TOP2 inhibitor and TOP2B-selective degrader, was discovered to possess anti-proliferative properties and combinatorial activity with lenalidomide in certain MM cell lines. To develop a deeper understanding of the biology of CRBN and IMiDs, proximity labelling with BioID2 was employed to characterise the interactome of CRBN in the presence and absence of an IMiD. Co-treatment of BioID2-CRBN-expressing OPM2 cells with IMiDs and/or the proteasome inhibitor bortezomib, followed by liquid chromatography mass spectrometry proteomics, allowed identification of known CRBN interactors (COP9 signalosome subunits) and neosubstrates (IKZF1, IKZF3 and CK1alpha). This approach enabled further characterisation of CRBN-associated proteome and identification of new CRBN and CRBN-IMiD interacting partners, such as MEF2C, DVL1, DVL2, MYH9, PRRC2C and EDC4. This data led to the hypothesis that CRBN may be implicated in several novel biological processes, such as RNA processing and protein translation regulation. Additionally, some of the newly identified interacting partners may modulate the MM niche in the bone marrow and may help understand the occurrence of side effects to IMiD therapy. This work has further defined transcriptional signatures of IMiD treatment and of acquired IMiD-resistance. TOP2B was found to be a potential druggable vulnerability of MM cells with acquired IMiD-resistance. The unbiased study of the CRBN-interacting proteome has helped broaden the knowledge on its biological activities both in presence and absence of an IMiD. Collectively, this thesis provides new and important insight into IMiD mechanism of action and further expands knowledge of IMiD biology.