Sir Peter MacCallum Department of Oncology - Theses
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ItemInvestigating the regulation of metabolism by oncogenic transcription factors in liver cancer( 2022)Liver cancer remains one of the most lethal cancers worldwide. Therefore, understanding the pathogenesis of liver cancer is imperative to develop appropriate interventions to combat the disease. Although liver cancer is a heterogeneous disease at the genetic level, one of the unifying features is the deregulation of transcription factors. These transcription factors reprogram metabolism to promote liver tumourigenesis by mechanisms that are still poorly understood. In this thesis, I examined the role that two oncogenic transcription factors play in regulating metabolism and promoting liver growth. Firstly, I investigated the molecular mechanism by which the Hippo pathway nuclear effector, Yes-associated protein (Yap) reprograms lipid metabolism to promote hyperplastic liver growth. Secondly, I studied the effects of commonly amplified oncogene Myc, factor in regulating nucleotide metabolism to promote dysplastic liver growth. Thirdly, I examined the consequence of simultaneous expression of Yap and Myc in the liver to promote tumourigenesis. The central aim of my PhD project was to determine the role that oncogenic transcription factors, namely Yap and Myc play in regulating metabolism during pre-malignant growth and tumorigenesis. Liver cancer occurs in the context of chronic liver disease, where several stages of liver disease eventually develop into liver cancer. In this thesis, I examined the pre-malignant and tumour initiation stages of liver cancer using the zebrafish as an in vivo model. I asked several fundamental questions: 1) What are the key features of the liver tissue following activation of oncogenic transcription factors? 2) What metabolic pathways are regulated by oncogenic transcription factors to promote liver growth? 3) Can these metabolic pathways be targeted to suppress oncogenic liver growth? Using the zebrafish as an in vivo model, I found that: 1) Yap transcriptionally activates serum and glucocorticoid-regulated kinase 1 (SGK1), a regulator of the phosphoinositide 3-kinase (PI3K)- mechanistic target of rapamycin target complex 1 (mTORC1) pathway to promote activation of sterol regulatory-element binding protein (SREBP) lipogenic program. Consequently, this leads to hyperplastic liver growth, which was also characterised by lipid droplet (LD) accumulation in the hepatocytes. 2) Myc transcriptionally activates inosine monophosphate dehydrogenase (IMPDH) to promote de novo guanosine triphosphate (GTP) biosynthesis. This metabolic change promoted dysplastic liver growth, characterised by nucleolus expansion and hepatocyte dedifferentiation. 3) Simultaneous activation of Yap and Myc promote rapid liver tumourigenesis. Classical Yap and Myc gene signatures are upregulated in the liver tumour, which is sensitive to the standard of care chemotherapy, sorafenib. While activation of a single oncogene was not sufficient to induce tumourigenesis, activation of Yap or Myc was able to recapitulate aspects of pre-malignant liver growth. Specifically, hepatocyte-specific Yap activation promoted a fatty liver-like phenotype (steatosis). On the other hand, hepatocyte-specific Myc activation promoted dysplasia, similar to that typically found in the dysplastic nodules of cirrhotic livers. Simultaneous Yap and Myc activation caused rapid liver tumour development that models the initiation of liver tumorigenesis. All in all, I showed that oncogenic transcription factors reprogram metabolism to promote liver growth and tumourigenesis. I revealed that de novo lipogenesis (DNL) and de novo GTP biosynthesis are metabolic vulnerabilities in Yap or Myc driven cancer, respectively.
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ItemIdentification of cooperating oncogenic lesions in Myc-driven lymphoma( 2017)MYC is a potent oncogene that is deregulated in nearly 50% of all human malignancies and as such, is considered an attractive molecular target for inhibition. However, MYC is rarely mutated, has no enzymatic activity that can be pharmacologically exploited and is expressed by normal cells leading to the current view that MYC is “undruggable” and indeed, its pharmacological inhibition has proved elusive. Therefore, discovering genes and pathways that interact with oncogenic MYC signalling and identifying them as potential therapeutic targets in cancers with ectopic MYC expression is of high clinical importance. The Eμ-Myc mouse has been utilised extensively as a faithful model of MYC-driven B cell lymphomagenesis. The Eμ-Myc transgene mimics the t(8;14) translocation apparent in Burkitt’s lymphoma, where ectopic Myc expression is driven by the Eμ- (IGH) promoter elements. Despite being driven by a single oncogene, Eμ-Myc lymphomas demonstrate remarkable heterogeneity indicating that the pathway to frank clonal neoplasia relies on oncogenic Myc signalling and the acquisition of at least one other mutation that cooperates with Myc. Hence, the Eμ-Myc model is a powerful tool in identifying MYC-cooperative genes and pathways. However, to date there has only been partial characterisation of the secondary, tertiary and quaternary mutations that can cooperate with Myc in driving Eμ-Myc lymphomagenesis. To identify somatically acquired Myc-cooperative lesions, massive-parallel sequencing was applied to spontaneous Eμ-Myc B cell lymphomas. Whole genome sequencing was used to map three copies of the Eμ-Myc transgene to chromosome 19 in the germline corresponding with an adjacent chromosome 19 segmental copy number gain. The chromosome 19 amplicon is in a region syntenic to an oncogenic region frequently amplified in human B cell malignancies. The chromosome 19 amplicon was demonstrated to undergo additional somatic gain in 50% of Eμ-Myc lymphoma. In addition to the identification of mutations in genes already implicated in Eμ-Myc lymphoma (Trp53, Cdkn2a, Nras, Kras), whole exome sequencing identified high frequency protein truncating mutations in Bcl6-co-repressor (Bcor). Furthermore, co-occurring tertiary driver lesions involving Cdkn2a (p19ARF) deletion and either Bcor or Ras mutations were identified in clonal Eμ-Myc lymphomas. RNAi and CRISPR-Cas9 mediated knockdown/knockout of Bcor in Eμ-Myc foetal liver hematopoietic stem cells reconstituted into recipient mice demonstrated significantly reduced latency of disease onset, validating Bcor as a tumor suppressor gene in the Eμ-Myc model. Gene-expression profiling of these Eμ-Myc tumours with forced Bcor-loss identified a reliable signature of Bcor loss that was distinct to Trp53 mutation signatures and was redolent of Tgfβ-pathway activation signature. The Eμ-Myc model of lymphoma has been heavily utilised but never fully genomically characterised until now. By applying next generation sequencing technology to a first generation animal model of cancer, this thesis challenges several persisting assumptions made about Eμ-Myc lymphoma. Firstly, data herein suggests that both oncogenic Myc expression along with the chromosome 19 amplification is the initiating driver event in Eμ-Myc lymphoma. This has obvious implications for the conclusions drawn in many publications predicated on the assumption that ectopic Myc expression is the exclusive initiating oncogenic lesion in Eμ-Myc lymphoma. Secondly, the discovery that homozygous deletion of Cdkn2a does not totally attenuate selective pressure for the acquisition of tertiary driver mutations indicates the significance of Cdkn2a deletion in Eμ-Myc lymphoma is overestimated. Thus, deductions made about the cooperative mechanism between CDKN2A and how it opposes proliferative MYC-signalling in human neoplastic transformation may need to be revisited. Finally, the identification of biologically functional high frequency Bcor mutations in Eμ-Myc lymphoma has defined a novel pathway that is potentially capable of restraining oncogenic MYC activity. That Bcor inactivation is reminiscent of Tgfβ-pathway enhancement is suggestive of perturbation of the oncogene induced senescence pathway. If this is the pathway through which Bcor exerts its tumour suppressive activity then it is feasible that dissection of this pathway will lead to the identification of novel therapeutic targets that can be selectively exploited in human malignancies in which MYC is oncogenically deregulated.