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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ItemDual-specific Chimeric Antigen Receptor T Cells and an Indirect Vaccine against Pancreatic Cancer( 2020)Pancreatic cancer is one of the most aggressive malignancies with an overall 5-year survival rate of <7%. Pancreatic cancer is highly resistant to radiotherapy and chemotherapy, and surgery is not feasible in most patients. In this thesis, I developed a new form of treatment for pancreatic cancer, based on immunotherapy. Adoptive cell transfer (ACT) is a promising form of cancer immunotherapy, which involves the isolation and reinfusion of tumour specific T lymphocytes into patients. While ACT can eliminate substantial burdens of some leukaemia, the ultimate challenge remains the eradication of large solid tumours and metastases for most cancers, including pancreatic cancer. In this thesis, an enhanced ACT treatment strategy for pancreatic cancer was developed, which was termed ‘ACTIV: Adoptive Cell Transfer Incorporating Vaccination’. This treatment included dual-specific T cells that expressed a chimeric antigen receptor (CAR) specific for the tumour antigen Her2, and a TCR specific for the melanocyte protein (pMEL, gp100). These dual specific T cells were termed ‘CARaMEL T cells’. CARaMEL T cells were administered together with an injection of a recombinant vaccinia virus vaccine expressing gp100 (VV-gp100). We hypothesized that adoptively transferred CARaMEL T cells would proliferate mediated by their gp100 TCR, in response to the VV-gp100 vaccine, and kill Her2+ tumours through their anti-Her2 CAR. Functional assays performed in vitro indicated that murine CARaMEL T cells mediated antigen-specific cytokine secretion and killing abilities against pancreatic cancer cells, and demonstrated potent proliferative ability in response to gp100 antigen, confirming our hypothesis. In addition, I found that ACTIV therapy inhibited tumour growth and prolonged the survival of mice bearing Her2+ subcutaneous murine pancreatic tumour. However, tumours usually relapsed after ACTIV therapy administration. Therefore, I directed my study to augment the anti-tumour activity of ACTIV therapy by the administration of either a histone deacetylase inhibitor (Panobinostat) or an immune agonist monoclonal antibody specific for CD40. Panobinostat significantly suppressed the growth of pancreatic cancer cells in vitro through apoptosis and cell cycle arrest. Also, Panobinostat significantly increased the growth suppression of pancreatic cancer cells mediated by CARaMEL T cells. In addition, I found that the combination of ACTIV therapy and Panobinostat significantly reduced the tumour growth and prolonged the survival of mice bearing Her2+ subcutaneous murine pancreatic tumours. In addition, administration of an agonist CD40 monoclonal antibody with ACTIV therapy significantly reduced the tumour growth and prolonged survival of mice bearing subcutaneous Her2+ pancreatic tumours through a T-cell-dependent immune mechanism. Finally, I explored the clinical translational potential for ACTIV therapy through the generation of human CARaMEL T cells expressing both a Her2-specific CAR and a gp100-TCR. In vitro functional assays indicated that human CARaMEL T cells mediated powerful and antigen-specific killing and cytokine secretion against Her2, together with a strong proliferative ability in response to gp100 antigen. In addition, I found that the administration of both human CARaMEL T cells and an adenovirus vaccine expressing gp100 led to potent anti-tumour activity against subcutaneous human Her2+ pancreatic tumours in immunodeficient mice.