Sir Peter MacCallum Department of Oncology - Theses
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ItemExamining the effects of BRAF, MEK and CDK4/6 inhibition on anti-tumor immunity in BRAFV600 melanoma( 2020)The recent advent of targeted and immune-based therapies has revolutionized the treatment of melanoma, and transformed outcomes for patients with metastatic disease. However, the mechanisms underpinning the clinical efficacy of these approaches are still being elucidated. The majority of patients develop resistance to the current standard-of-care targeted therapy, dual BRAF and MEK inhibition (BRAFi+MEKi), prompting evaluation of a new combination incorporating a CDK4/6 inhibitor. Based on promising preclinical data, combined BRAF, MEK and CDK4/6 inhibition (triple therapy) has recently entered clinical trials for the treatment of BRAFV600 melanoma. Interestingly, while BRAFi+MEKi therapy was initially developed on the basis of potent tumor-intrinsic effects, it was later discovered to have significant immune-potentiating activity. Recent studies have also identified immune-related impacts of CDK4/6 inhibition, though these are less well defined and appear to be both immune-potentiating and immune-inhibitory. BRAFV600 melanoma patients are also eligible for immunotherapies, and hence the immunomodulatory activity of these targeted inhibitors makes first-line treatment decisions complex. The aim of this thesis was to examine the immunomodulatory effects of BRAF, MEK and CDK4/6 inhibition, with an ultimate goal of providing critical information to aid in the clinical management of BRAFV600 melanoma patients. Examining mechanisms of the immunomodulatory effects of targeted therapies requires preclinical mouse models of melanoma that are both immunogenic, and harbor the oncogenic drivers targeted by the therapies being evaluated. To address this, we developed a novel immunogenic BrafV600ECdkn2a-/-Pten-/- melanoma mouse model, called YOVAL1.1. YOVAL1.1 tumors are transplantable in immunocompetent mice and amenable to standard-of-care melanoma therapies, including BRAFi+MEKi and immune checkpoint blockade. This, coupled with the Cdkn2a status, which infers some sensitivity to CDK4/6 inhibitors, makes this an ideal preclinical model to evaluate the immunomodulatory effects of the triple therapy. Using this model, we demonstrated that triple therapy promotes durable tumor control through tumor-intrinsic mechanisms, while promoting immunogenic cell death and T cell infiltration. However, despite this, tumors treated with triple therapy were unresponsive to immune checkpoint blockade. Flow cytometric and single cell RNA-seq analyses of tumor infiltrating immune populations revealed that triple therapy markedly depleted pro-inflammatory macrophages and cross priming CD103+ dendritic cells, the absence of which correlated with poor overall survival and clinical responses to immune checkpoint blockade in melanoma patients. Indeed, immune populations isolated from tumors of mice treated with triple therapy failed to stimulate T cell responses ex vivo. Hence, while combined BRAF, MEK and CDK4/6 inhibition demonstrated favorable tumor-intrinsic activity, these data suggest that collateral effects on tumor-infiltrating myeloid populations may impact on anti-tumor immunity. Several recent studies have reported immune-potentiating effects of CDK4/6 inhibition, and subsequent synergy with immune checkpoint blockade. However, T cells are the primary target of these immunotherapies, and an understanding of the direct effects of CDK4/6 inhibition on this cellular subset was lacking. In this thesis, using integrated epigenomic, transcriptomic and single cell CITE-seq analyses, we identified a novel role for CDK4/6 in regulating T cell fate. Specifically, we demonstrated that CDK4/6 inhibition promoted the phenotypic and functional acquisition of T cell memory. Genome-wide CRISPR/Cas9 screening and phospho-proteomics revealed that memory formation in response to CDK4/6 inhibition was cell intrinsic and required RB. Pre-conditioning human CAR T cells with a CDK4/6 inhibitor enhanced their persistence and tumor control, and clinical treatment with a CDK4/6 inhibitor promoted expansion of memory T cells in a melanoma patient, priming a response to immune checkpoint blockade. Collectively these findings highlight the multi-faceted immunomodulatory activity of BRAF, MEK and CDK4/6 inhibition. The addition of a CDK4/6 inhibitor to dual BRAFi+MEKi led to the depletion of intratumoral myeloid subsets that may be critical for supporting a therapeutically beneficial T cell response. In contrast, as an individual therapy, CDK4/6 inhibition promoted effector and memory T cell activity, suggesting that, with optimal scheduling to prevent myeloid depletion, CDK4/6 inhibitors may be used to enhance and prolong BRAFi/MEKi-induced anti-tumor T cell immunity. Defining the mechanisms that underpin the clinical efficacy of these available therapies is a critical step forward in optimising novel combination and scheduling approaches to combat melanoma and improve patient outcomes.
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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.