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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

Mammalian target of rapamycin (mTOR) is a central regulator of mammalian metabolism and physiology. Aberrant hyperactivation of the mTOR pathway promotes tumor growth and metastasis, and can also promote tumor resistance to chemotherapy and cancer drugs; this makes mTOR an attractive cancer therapeutic target. mTOR inhibitors have been approved to treat cancer; however, the mechanisms underlying drug sensitivity remain poorly understood. Here, whole exome sequencing of three chromophobe renal cell carcinoma (chRCC) patients with exceptional mTOR inhibitor sensitivity revealed that all three patients shared somatic mutations in the deubiquitinase gene USP9X. The clonal characteristics of the mutations, which were amassed by studying multiple patients' primary and metastatic samples from various years, together with the low USP9X mutation rate in unselected chRCC series, reinforced a causal link between USP9X and mTOR inhibitor sensitivity. Rapamycin treatment of USP9X-depleted HeLa and renal cancer 786-O cells, along with the pharmacological inhibition of USP9X, confirmed that this protein plays a role in patients' sensitivity to mTOR inhibitors. USP9X was not found to exert a direct effect on mTORC1, but subsequent ubiquitylome analyses identified p62 as a direct USP9X target. Increased p62 ubiquitination and the augmented rapamycin effect upon bortezomib treatment, together with the results of p62 and LC3 immunofluorescence assays, suggested that dysregulated autophagy in USP9X-depleted cells can have a synergistic effect with mTOR inhibitors. In summary, we show that USP9X constitutes a potential novel marker of sensitivity to mTOR inhibitors in chRCC patients, and represents a clinical strategy for increasing the sensitivity to these drugs.

Congenital diaphragmatic hernia (CDH) can occur in isolation or in conjunction with other birth defects (CDH+). A molecular etiology can only be identified in a subset of CDH cases. This is due, in part, to an incomplete understanding of the genes that contribute to diaphragm development. Here, we used clinical and molecular data from 36 individuals with CDH+ who are cataloged in the DECIPHER database to identify genes that may play a role in diaphragm development and to discover new phenotypic expansions. Among this group, we identified individuals who carried putatively deleterious sequence or copy number variants affecting CREBBP, SMARCA4, UBA2, and USP9X. The role of these genes in diaphragm development was supported by their expression in the developing mouse diaphragm, their similarity to known CDH genes using data from a previously published and validated machine learning algorithm, and/or the presence of CDH in other individuals with their associated genetic disorders. Our results demonstrate how data from DECIPHER, and other public databases, can be used to identify new phenotypic expansions and suggest that CREBBP, SMARCA4, UBA2, and USP9X play a role in diaphragm development.

Full Text, Thesis (PhD Doctorate), Doctor of Philosophy (PhD), School of Environment and Sc, Science, Environment, Engineering and Technology, Autism Spectrum Disorder (ASD) and Intellectual Disability (ID) are two of the most common neurodevelopmental disorders classified by the Diagnostic and Statistical Manual of Mental disorders 5th Edition. Both are often comorbid, meaning that they often exist in the same patient. Due to this, it has been hypothesised that there may be a common cause for ID and ASD, a common disruption in the same neurodevelopmental pathway. A gene that has been implicated in both the pathogenesis of ID and ASD is the Ubiquitin-Specific Protease 9 XLinked (USP9X). Targets of USP9X and USP9X itself have been found to be part of mTOR, TGFb, and WNT signalling pathways, all equally important pathways in the developing brain. In addition, deletion of Usp9x in mice has been found to have immense effects on the postnatal development of the hippocampus. Recently, a study reported that Usp9x-null mice had learning and memory deficits, similar to that seen in ID patients with USP9X mutations. However, to date, there has been no extensive study into how the deletion of Usp9x in mice affects cortical development nor investigated if any ASD-related behaviour manifested from the deletion. Therefore, this study set out to address those questions by deleting Usp9x from the dorsal telencephalon of mice and used them as a model system to investigate the role of Usp9x in cortical development and behaviour. Adult male mice where Usp9x has been deleted (Usp9x-/y) and their control littermates (Usp9x+/y) were used in this project. Firstly, the adult neocortex was characterised using haematoxylin staining, where measurements were made in several areas of the neocortex. Results indicate that the neocortex were minimally affected with the deletion of Usp9x; the thickness of most areas remained not significantly different to controls, barring the retrosplenial cortex being thicker in Usp9x-/y mice compared to Usp9x+/y mice. However, the deletion of Usp9x lead to a significantly smaller corpus callosum compar

Autism Spectrum Disorder (ASD) and Intellectual Disability (ID) are two of the most common neurodevelopmental disorders classified by the Diagnostic and Statistical Manual of Mental disorders 5th Edition. Both are often comorbid, meaning that they often exist in the same patient. Due to this, it has been hypothesised that there may be a common cause for ID and ASD, a common disruption in the same neurodevelopmental pathway. A gene that has been implicated in both the pathogenesis of ID and ASD is the Ubiquitin-Specific Protease 9 XLinked (USP9X). Targets of USP9X and USP9X itself have been found to be part of mTOR, TGFb, and WNT signalling pathways, all equally important pathways in the developing brain. In addition, deletion of Usp9x in mice has been found to have immense effects on the postnatal development of the hippocampus. Recently, a study reported that Usp9x-null mice had learning and memory deficits, similar to that seen in ID patients with USP9X mutations. However, to date, there has been no extensive study into how the deletion of Usp9x in mice affects cortical development nor investigated if any ASD-related behaviour manifested from the deletion. Therefore, this study set out to address those questions by deleting Usp9x from the dorsal telencephalon of mice and used them as a model system to investigate the role of Usp9x in cortical development and behaviour. Adult male mice where Usp9x has been deleted (Usp9x-/y) and their control littermates (Usp9x+/y) were used in this project. Firstly, the adult neocortex was characterised using haematoxylin staining, where measurements were made in several areas of the neocortex. Results indicate that the neocortex were minimally affected with the deletion of Usp9x; the thickness of most areas remained not significantly different to controls, barring the retrosplenial cortex being thicker in Usp9x-/y mice compared to Usp9x+/y mice. However, the deletion of Usp9x lead to a significantly smaller corpus callosum compar, Thesis (PhD Doctorate), Doctor of Philosophy (PhD), School of Environment and Sc, Science, Environment, Engineering and Technology, Full Text

BackgroundThe RAS/MAPK pathway has been intensively studied in cancer. Constitutive activation of ERK1 and ERK2 is frequently found in cancer cells from a variety of tissues. In clinical practice and clinical trials, small molecules targeting receptor tyrosine kinases or components in the MAPK cascade are used for treatment. MEK1 and MEK2 are ideal targets because these enzymes are physiologically important and have narrow substrate specificities and distinctive structural characteristics. Despite success in pre-clinical testing, only two MEK inhibitors, trametinib and cobimetinib, have been approved, both for treatment of BRAF-mutant melanoma. Surprisingly, the efficacy of MEK inhibitors in other tumors has been disappointing. These facts suggest the need for a different approach. We here consider transcription factor ETS1 and ETS2 as alternate therapeutic targets because they are major MAPK downstream effectors.Main textThe lack of clinical efficacy of MEK inhibitors is attributed mostly to a subsequent loss of negative feedback regulation in the MAPK pathway. To overcome this obstacle, second-generation MEK inhibitors, so-called "feedback busters," have been developed. However, their efficacy is still unsatisfactory in the majority of cancers. To substitute ETS-targeted therapy, therapeutic strategies to modulate the transcription factor in cancer must be considered. Chemical targeting of ETS1 for proteolysis is a promising strategy; Src and USP9X inhibitors might achieve this by accelerating ETS1 protein turnover. Targeting the ETS1 interface might have great therapeutic value because ETS1 dimerizes itself or with other transcription factors to regulate target genes. In addition, transcriptional cofactors, including CBP/p300 and BRD4, represent intriguing targets for both ETS1 and ETS2.ConclusionsETS-targeted therapy appears to be promising. However, it may have a potential problem. It might inhibit autoregulatory negative feedback loops in the MAPK pathway, wit

Children with Down syndrome (DS) are prone to development of high-risk B-cell precursor ALL (DS-ALL), which differs genetically from most sporadic pediatric ALLs. Increased expression of cytokine receptor-like factor 2 (CRLF2), the receptor to thymic stromal lymphopoietin (TSLP), characterizes about half of DS-ALLs and also a subgroup of sporadic "Philadelphia-like" ALLs. To understand the pathogenesis of relapsed DS-ALL, we performed integrative genomic analysis of 25 matched diagnosis-remission and -relapse DS-ALLs. We found that the CRLF2 rearrangements are early events during DS-ALL evolution and generally stable between diagnoses and relapse. Secondary activating signaling events in the JAK-STAT/RAS pathway were ubiquitous but highly redundant between diagnosis and relapse, suggesting that signaling is essential but that no specific mutations are "relapse driving." We further found that activated JAK2 may be naturally suppressed in 25% of CRLF2(pos) DS-ALLs by loss-of-function aberrations in USP9X, a deubiquitinase previously shown to stabilize the activated phosphorylated JAK2. Interrogation of large ALL genomic databases extended our findings up to 25% of CRLF2(pos), Philadelphia-like ALLs. Pharmacological or genetic inhibition of USP9X, as well as treatment with low-dose ruxolitinib, enhanced the survival of pre-B ALL cells overexpressing mutated JAK2. Thus, somehow counterintuitive, we found that suppression of JAK-STAT "hypersignaling" may be beneficial to leukemic B-cell precursors. This finding and the reduction of JAK mutated clones at relapse suggest that the therapeutic effect of JAK specific inhibitors may be limited. Rather, combined signaling inhibitors or direct targeting of the TSLP receptor may be a useful therapeutic strategy for DS-ALL

Full Text, Thesis (PhD Doctorate), Doctor of Philosophy (PhD), School of Natural Sciences, Science, Environment, Engineering and Technology, Squamous cell carcinoma is a malignancy of squamous epithelia and affects multiple organs of the body. Investigating the underlying signalling pathways and the molecular mechanisms is essential to gain a profound understanding of the disease pathology, aetiology and to uncover potential drug targets. The deubiquitylating enzyme, USP9X, has been associated with epithelial cancers of breasts, colon, lung, liver, pancreas, mouth and the skin. The current study investigated the functional and molecular role of USP9X in head and neck squamous carcinoma cell lines (HNSCC) and skin keratinocytes. Also, the role of USP9X in DNA damage response of keratinocytes was investigated. USP9X protein expression was altered in four HNSCC cell lines, derived from tongue (SCC15 and CAL27) and pharynx (Fadu and Detroit 562) by treating them with siRNAs (loss of function) or by transfecting them with plasmids (gain of function). CyQuant and Flow cytometry analyses revealed that USP9X promoted proliferation and regulated cell cycle of these cell lines. RT-qPCR analysis of target gene expression revealed that USP9X also regulated Notch activity in these cells. However, at least in one cell line (FaDu), USP9X’s regulation of proliferation did not appear to operate through the Notch pathway but possibly occurs via mTOR pathway. Immunoblot analysis revealed a decrease in downstream targets of mTOR complex 1 namely, total ribosomal protein (S6) and its phosphorylated form (pS6), when USP9X was depleted from FaDu cells. In contrast, in immortalised but non- tumourigenic HaCaT human keratinocytes, USP9X depletion led to an increase in cell proliferation and again, USP9X levels directly correlated with Notch pathway activity.

Squamous cell carcinoma is a malignancy of squamous epithelia and affects multiple organs of the body. Investigating the underlying signalling pathways and the molecular mechanisms is essential to gain a profound understanding of the disease pathology, aetiology and to uncover potential drug targets. The deubiquitylating enzyme, USP9X, has been associated with epithelial cancers of breasts, colon, lung, liver, pancreas, mouth and the skin. The current study investigated the functional and molecular role of USP9X in head and neck squamous carcinoma cell lines (HNSCC) and skin keratinocytes. Also, the role of USP9X in DNA damage response of keratinocytes was investigated. USP9X protein expression was altered in four HNSCC cell lines, derived from tongue (SCC15 and CAL27) and pharynx (Fadu and Detroit 562) by treating them with siRNAs (loss of function) or by transfecting them with plasmids (gain of function). CyQuant and Flow cytometry analyses revealed that USP9X promoted proliferation and regulated cell cycle of these cell lines. RT-qPCR analysis of target gene expression revealed that USP9X also regulated Notch activity in these cells. However, at least in one cell line (FaDu), USP9X’s regulation of proliferation did not appear to operate through the Notch pathway but possibly occurs via mTOR pathway. Immunoblot analysis revealed a decrease in downstream targets of mTOR complex 1 namely, total ribosomal protein (S6) and its phosphorylated form (pS6), when USP9X was depleted from FaDu cells. In contrast, in immortalised but non- tumourigenic HaCaT human keratinocytes, USP9X depletion led to an increase in cell proliferation and again, USP9X levels directly correlated with Notch pathway activity., Thesis (PhD Doctorate), Doctor of Philosophy (PhD), School of Natural Sciences, Science, Environment, Engineering and Technology, Full Text

The three EglN prolyl hydroxylases (EglN1, EglN2, and EglN3) regulate the stability of the HIF transcription factor. We recently showed that loss of EglN2, however, also leads to down-regulation of Cyclin D1 and decreased cell proliferation in a HIF-independent manner. Here we report that EglN2 can hydroxylate FOXO3a on two specific prolyl residues in vitro and in vivo. Hydroxylation of these sites prevents the binding of USP9x deubiquitinase, thereby promoting the proteasomal degradation of FOXO3a. FOXO transcription factors can repress Cyclin D1 transcription. Failure to hydroxylate FOXO3a promotes its accumulation in cells, which in turn suppresses Cyclin D1 expression. These findings provide new insights into post-transcriptional control of FOXO3a and provide a new avenue for pharmacologically altering Cyclin D1 activity.

Deubiquitinating enzymes (or DUBs) attack the ubiquitin-based isopeptide bond, thus counteracting ubiquitinprotein ligase activity in vivo. By disassembling ubiquitin-substrate and ubiquitin-ubiquitin covalent links, deubiquitinating enzymes exert a very powerful control of many signaling processes within the ubiquitin-proteasome system (UPS). Very active research in this field in the last decade shows that deubiquitinating enzymes play important regulatory roles in aspects relevant to cancer, such as proteasome activity, p53 stability, the regulation of fanconi anemia related proteins, tumor cell apoptosis induction, to mention a few. Thus, deubiquitinating enzymes have emerged as interesting drug targets in cancer research. Here, the pharmacological inhibition of DUBs and its potential effect in cancer treatment are reviewed. © 2014 Bentham Science Publishers.