Sir Peter MacCallum Department of Oncology - Theses
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ItemDefining the immune landscape in rectal cancer and its relationship to neo-adjuvant chemoradiotherapy and immunotherapy responses( 2023-07)Colorectal cancer (CRC) is the second leading cause of cancer-related mortality worldwide. Approximately 25% of these tumours are in the rectum. Rectal cancers that are locally advanced or have poor prognostic features on staging are typically treated with neo-adjuvant chemoradiotherapy prior to surgical resection. However, despite intensive efforts to improve the response rates to neo-adjuvant therapy, most patients do not achieve a complete pathological response. Patients that do achieve a complete response to neo-adjuvant therapy may be candidates for organ preservation. There has been limited progress in identifying treatment protocols with improved therapeutic efficiency, or in extending the treatment options for patients that have an incomplete response. Additional complexity is added by the inability to accurately identify which patients will respond to treatment, and deficiencies in understanding the mechanisms that drive treatment response. This thesis focuses on first reviewing the novel treatment options that have been investigated for patients with rectal cancer to date and assessing the ongoing inclination of surgeons to pursue novel therapies for their patients. Additionally personalised tumour models (tumouroids) and differentially expressed genes were assessed for the utility in predicting which patients are likely to respond to neo-adjuvant therapy. There has been recent attention on the ability of immune checkpoint blockade inhibitors to “rescue” cytotoxic mediated immune responses and extend treatment responses for patients with tumours including melanoma, non-small cell lung carcinoma and renal cell carcinoma. The use of these agents in the setting of neo-adjuvant therapy for rectal cancer remains undefined. Thus, a phase II clinical trial was established to assess the role of immune checkpoint blockade inhibition in the neo-adjuvant setting for rectal cancer, with a particular focus on the effect of this therapy on tumour response and the tumour immune landscape. Finally, the tumour microenvironment has an accepted and essential role in response to neo-adjuvant therapy. However, newly identified cells and cellular structures such as tissue resident memory cells and tertiary lymphoid structures are altering our understanding of anti-tumour immunity but remain under investigated in the setting of rectal cancer. To address this deficiency multiplex immunohistochemistry was employed to define the immune landscape of patients receiving both chemoradiotherapy and immune checkpoint blockade inhibition, with a particular focus on cell types previously under appreciated. Defining the immune landscape of these tumours in such a manner may allow a deeper understanding of the mechanisms driving therapy response and facilitate targeted patient therapy.
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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.