Sir Peter MacCallum Department of Oncology - Theses
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ItemElucidating epigenetic mechanisms of cancer immune evasion( 2023-03)Cancer immunotherapies have revolutionised the management of a wide range of haematological and solid organ malignancies, due to their potential to induce durable remissions in a proportion of responders. However, primary or acquired resistance remains problematic for the majority of patients, and typically arises from tumour-intrinsic properties that reduce immunogenicity or extrinsic factors promoting an immunosuppressive tumour microenvironment. Effective tumour antigen presentation via major histocompatibility proteins (MHC-I and/or MHC-II) to immune effector cells is a critical component of the adaptive anti-cancer response and genetic disruption of the MHC-I and/or MHC-II antigen presentation pathways, either through inactivating mutations or transcriptional silencing, is a well-recognised cause of resistance to both pharmacological and cellular immunotherapies. In this thesis, I explore epigenetic mechanisms of MHC-I and MHC-II repression in cancer and identify an evolutionarily conserved role for Polycomb repressive complex 2 (PRC2) in MHC-I silencing. I also discover two key mechanisms of MHC-II regulation in acute myeloid leukaemia and melanoma: transcriptional repression of MHC-II pathway genes by the C-terminal binding protein (CtBP) co-repressor complex, and post-translational regulation of CIITA, the master regulator of MHC-II expression, by the FBXO11-containing E3 ubiquitin ligase complex. Targeting of these repressive pathways efficiently upregulates cell surface MHC expression and augments in vitro and in vivo adaptive immune responses. These findings provide valuable biological insights into mechanisms of cancer immune evasion and establish the scientific rationale for further pre-clinical and translational studies of these novel therapeutic strategies to overcome immunotherapy resistance via restoration of tumour antigen presentation.
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ItemInvestigating oncogenic FMS-like tyrosine kinase 3 (FLT3)-induced metabolic reprogramming in acute myeloid leukaemia( 2021)Mutations in the FMS-like tyrosine kinase 3 (FLT3) gene are among the most frequently occurring somatic mutations in acute myeloid leukaemia (AML), a disease that presents with devastating prognosis. FLT3 is primarily expressed on hematopoietic progenitor cells, and during early haematopoiesis coordinates a ligand-dependent signalling cascade that regulates the proliferation and maturation of the progenitor pool. FLT3 internal tandem duplication (FLT3-ITD), the most common type of FLT3 mutation, promotes constitutive FLT3 kinase activity and hyperactivation of downstream signalling pathways including STAT5, PI3K/mTOR and MAPK. Though our understanding of the molecular pathways downstream of mutant FLT3 have greatly improved in the modern genomic sequencing era, the repertoire of molecular signalling events induced by mutant-FLT3 to drive leukaemogenesis has not been fully characterised. In this thesis, a murine model of MLL-rearranged AML harbouring inducible FLT3-ITD expression was developed and used, along with human AML cell lines, to demonstrate that FLT3-ITD promotes serine uptake and serine biosynthesis via transcriptional regulation of neutral amino acid transporters (SLC1A4 and SLC1A5) and genes in the de novo serine biosynthesis pathway (PHGDH and PSAT1). Mechanistically, dysregulation of serine metabolism in FLT3-ITD-driven AML is dependent on the mTORC1-ATF4 axis, which drives RNA-Pol II occupancy at PHGDH, PSAT1, SLC1A4 and SLC1A5. Genetic or pharmacological inhibition of the de novo serine biosynthesis pathway, in vitro and in vivo, selectively inhibited the proliferation of FLT3-ITD-driven AML cells. Pharmacological inhibition of the de novo serine biosynthesis pathway using WQ-2101, an inhibitor of PHGDH, the first rate-limiting enzyme of the de novo serine biosynthesis pathway, sensitises FLT3-ITD-driven AML cells, including primary patient samples, to the standard of care chemotherapy agent cytarabine via exacerbation of DNA damage. Given that transcriptional activation of de novo serine biosynthesis and serine uptake was mediated by mTORC1 (a master regulator of biomass production and cellular metabolism), and that therapeutic responses in vitro and in vivo to mTORC1 inhibitors are poor, a small molecule compound screen utilising 181 epigenetic inhibitory compounds to determine novel synthetic lethal interactions between epigenetic regulators and mTORC1 inhibition was performed. This analysis revealed that inhibition of lysine-specific methyltransferase SETD8, the only known enzyme that catalyses H4K20 monomethylation (H4K20me1), synergised with mTORC1 inhibition. Transcriptional profiling suggested dual SETD8/mTORC1 inhibition preferentially suppressed mTORC1 target genes mediating amino acid biosynthesis and transamination (including de novo serine biosynthesis) to a greater extent than either single agent SETD8 or mTORC1 inhibition alone. Importantly, these observations were independent of global transcriptional repression induced by impaired cell viability or suppression of global transcription. Thus, this preliminary work suggests mTORC1 and/or its target genes and pathways may be dependent on SETD8 and/or H4K20me1 or, alternatively, mTORC1 functionally regulates SETD8. Collectively, the results presented herein provide novel insights into FLT3-ITD-induced metabolic reprogramming events in AML and identify a targetable metabolic dependency in this poor prognosis subtype of disease. In addition, these results provide the preliminary basis of a SETD8/mTORC1 synthetic dependency that can be exploited in FLT3-ITD-driven AML.
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ItemDeciphering tumour heterogeneity in acute myeloid leukaemia at the single cell level( 2020)The advent of next-generation sequencing (NGS) has allowed researchers to appreciate the enormous heterogeneity that exists between cells within a single tumour. This intratumour heterogeneity leads to diverse phenotypic outcomes, resulting in functionally distinct subpopulations of cancer cells. This functional heterogeneity fuels tumour evolution and therapeutic resistance and is thus a major barrier to producing cures in cancer. Acute myeloid leukaemia (AML) is an aggressive and heterogeneous malignancy with a high relapse rate. The prevailing paradigm to explain relapse in AML posits that genetic heterogeneity leads to pre-existing or acquired mutations that render certain cells refractory to therapy, resulting in the outgrowth of a resistant clone. Large-scale sequencing studies aimed at cataloguing genetic heterogeneity in AML have revealed several important observations. Firstly, AML has one of the lowest mutational burdens of any cancer. Secondly, a significant proportion of clinical relapse events cannot be attributed to an underlying genetic change. These important findings raise the possibility that mutations alone are insufficient to fully explain therapeutic resistance in AML. Indeed, we are now beginning to appreciate that both tumour evolution and clinical relapse can be driven by non-genetic processes. However, characterising the full extent of non-genetic heterogeneity and its relative contribution to both the evolutionary trajectory of the disease and therapeutic resistance requires innovative single cell methodologies. Single-cell RNA sequencing (scRNA-seq) has been instrumental in revealing the phenotypic heterogeneity of rare subpopulations of cells within a complex tumour. However, it is difficult to infer clonal relationships from scRNA-seq alone and this has hampered our ability to understand how individual malignant cells evolve over time. To overcome some of these challenges, we present a lentiviral method of tagging cells with unique heritable barcodes that are stably transcribed into RNA molecules in cells and therefore highly detected in microfluidic scRNA-seq workflows. This strategy, termed Single-cell Profiling and LINeage TRacking with expressed barcodes (SPLINTR), offers the ability to match the gene expression programmes of individual cells to their clonal lineage, in order to establish how initial transcriptional differences amongst heterogeneous malignant cells can shape thier future clonal behaviour during cancer progression. We apply our SPLINTR barcoding system to an in vivo model of clonal competition in order to determine the early transcriptional signatures that are associated with future clonal dominance in AML. We discover that clonal dominance is largely an intrinsic property amongst genetically identical clones. However, we find the deterministic nature of dominance is altered by the presence of other distinct competing mutational clones. Furthermore, SPLINTR enabled us to retrospectively identify a novel set of differentially expressed genes contained within certain clones prior to transplantation, which distinguished them from losing clones and was associated with their future dominance during disease progression. Finally, we find that resistance occurs to BET inhibitor therapy in the clinic in the absence of a clear genetic event. scRNA-seq of paired baseline and relapse AML patient bone marrow samples revealed than non-genetic resistance originates from either a population of pre-existing cells that phenotypically resemble LSCs, or through transcriptional adaptation as a result of therapeutic pressure. We then use SPLINTR coupled with scRNA-seq to interrogate our previously published in vitro model of non-genetic resistance to BET bromodomain inhibition. This provided further evidence that Lamarckian evolution in the form of gradual transcriptional adaptation drives non-genetic resistance. Future work aims to unravel the epigenetic states that mediate non-genetic transcriptional adaptation in a broader therapeutic context in AML. Collectively, the research presented in this thesis demonstrates the importance of applying novel single cell technologies to investigations of cellular diversity in cancer and highlights the underappreciated role of non-genetic heterogeneity in driving both disease evolution and therapeutic resistance in AML. These studies provide the molecular tools and rationale to further define the mechanisms by which non-genetic heterogeneity shapes cellular behaviour in cancer.