Sir Peter MacCallum Department of Oncology - Theses

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    Examining the effects of BRAF, MEK and CDK4/6 inhibition on anti-tumor immunity in BRAFV600 melanoma
    Lelliott, Emily Jane ( 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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    Study of the immunomodulatory effects of radiation therapy in solid cancers
    Sia, Joseph Ikin ( 2020)
    Radiation therapy (RT) has evolved over more than a century into a well-established, highly sophisticated and major cancer treatment modality today. A paradigm shift that has occurred within the last two decades is the growing understanding that RT can induce host immune responses that contribute to tumour control, beyond direct radiation-induced cytotoxicity. Contemporaneously, the advent of modern cancer immunotherapy such as immune checkpoint inhibitors has revolutionised the field of oncology and highlighted the potential of harnessing the immune system to suppress and eradicate tumours. Inevitably, resistance to cancer immunotherapy has also brought into focus immunological barriers that preclude cancer immunity. In this context, an increasing body of pre-clinical and clinical studies substantiate the use of RT as a unique candidate to complement cancer immunotherapy in non-overlapping mechanisms to overcome such barriers. However, instruction on the optimal integration of RT and cancer immunotherapy is scarce. For the radiation oncologist, an outstanding gap in knowledge is how radiation dose-fractionation influences the immunomodulatory effects of RT and its synergy with cancer immunotherapy. In this PhD project, mouse models of solid cancer were used to systematically interrogate this question by employing a series of rationally selected radiation dose-fractionation regimens to dissect the immunological impact of dose per fraction (DPF) from that of total dose, as represented by biological effective dose (BED). In orthotopic AT3-OVA mammary carcinomas, radiation-induced CD8+ T cell responses were found to be regulated by radiation DPF, rather than BED. By contrast, radiation-induced natural killer (NK) cell responses in the same tumours were independent of radiation DPF but required a sufficient BED. Mechanistic investigations examining the cellular and transcriptional changes in AT3-OVA tumours evoked by radiation demonstrated that the differential regulation of anti-tumour immune responses by radiation DPF and BED was not primarily dictated by differences in tumour cell-intrinsic immunogenicity, but rather by the effector and suppressor dynamics in the tumour immune microenvironment, of which regulatory T cells played a central role. Furthermore, cross-examination of subcutaneously implanted MC38 colon carcinomas and publicly available transcriptomic data of human cancers pre- and post-RT suggested that radiation-induced immune responses are also significantly shaped by the tumour type. Lastly, the impact of radiation dose-fractionation on the anti-tumour activity of immune checkpoint inhibitors targeting the adaptive and innate immune arms was examined in AT3-OVA tumours, confirming the corollary that RT and immune checkpoint inhibitors do not universally synergise, but require selection of radiation regimens and checkpoint targets that are predicated on biological rationale. Overall, this PhD project represents a comprehensive side-by-side pre-clinical study of the effects of radiation dose-fractionation on host anti-tumour immune responses. Results presented herein contribute towards a clearer understanding of this complex and clinically urgent question. More broadly, insights from this project will help guide the refashioning of RT into an exciting key adjunct in the immuno-oncology era.