Sir Peter MacCallum Department of Oncology - Theses
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ItemNovel approaches to harness the anti-tumour activity of natural killer cells( 2022)Despite revolutionary advances in cancer treatment with immunotherapy, durable clinical benefit is limited to a subset of patients. Current forms of immunotherapy, including checkpoint inhibition and chimeric antigen receptor T cells, are primarily restricted to targeting cytotoxic CD8+ T cells of the adaptive immune system. Natural killer (NK) cells are the cytotoxic effector cells of the innate immune response and are emerging as a promising and alternative target for cancer immunotherapy, however, comprehensive functional genetic studies examining anti-tumour NK cell activity are limited. Using genome-scale clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR associated protein 9 screening technology, we sought to comprehensively identify and validate tumour genes that potently influence sensitivity to primary NK cells. We first demonstrate that B16-F10 mouse melanoma cells lacking genes encoding proteins involved in interferon-gamma (IFN-gamma) signaling and antigen processing/presentation are highly sensitive to killing by NK cells, a process dependent on the absence of major histocompatibility complex class I expression. As checkpoint inhibition-resistant melanoma patients present with loss-of-function mutations within these pathways, our findings highlight intratumoural NK cells as a potent strategy to limit and/or overcome resistance to CD8+ T cell attack during conventional checkpoint inhibition immunotherapy. We additionally identify Rnf31 as a novel negative regulator of tumour cell sensitivity to NK cell killing. Rnf31 encodes for HOIP, which has an established role in driving tumour necrosis factor (TNF)-mediated gene induction and inhibition of TNF-induced cell death in certain cell types. HOIP-deficient melanoma cells not only exhibited increased sensitivity to NK cells, but also to antigen-specific CD8+ T cells. We surprisingly demonstrate that HOIP protects tumour cells from apoptosis induced by combined IFN-gamma and TNF, rather than TNF alone. We provide valuable mechanistic insight into the transcription-dependent form of apoptosis induced by combined IFN-gamma and TNF limited by HOIP, pharmacological validation using a HOIP inhibitor, and in vivo validation using HOIP-deficient B16-F10 ovalbumin-expressing tumours that exhibit enhanced CD8+ T cell-mediated control. Inhibition of tumour HOIP activity may therefore enhance NK and CD8+ T cell anti-tumour responses through unlocking the apoptotic potential of combined IFN-gamma and TNF secreted by these immune cells, representing a potential new combinatorial target for current and emerging immunotherapies. Collectively, we impartially identify tumour-specific genes that powerfully modulate tumour cell sensitivity to primary NK cells. We provide validation of established yet clinically relevant tumour genes associated with evasion from CD8+ T cells that mediate resistance to checkpoint inhibition immunotherapy, and additionally validate Rnf31 as a novel tumour regulator of not only sensitivity to NK cells, but also CD8+ T cells. We lastly present appropriate methodology and molecular tools that may facilitate analogous screening within NK cell lines to identify targetable regulators of tumour cell-induced IFN-gamma production by NK cells. Taken together, we advocate that NK cells may play an important role in combating resistance to checkpoint inhibition therapy in melanoma and identify HOIP as a potential combinatorial tumour target that may enhance future NK cell-based immunotherapy endeavours through secreted IFN-gamma and TNF, which may form the basis of a future immunotherapeutic strategy.
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ItemInvestigating mechanisms for Elotuzumab and Lenalidomide therapy in Multiple Myeloma( 2021)Multiple myeloma (MM) is a haematological malignancy of plasma cells with disease present in the bone marrow. Despite the success of new therapies in extending life expectancy, MM is still considered incurable and requires further investigation into new treatments. In the context of refractory/relapsed MM (RRMM), the combination of the two immunotherapies Elotuzumab (Elo, anti-SLAMF7 mAb) and Lenalidomide (Len, IMiD) has resulted in an 82% objective response rate and 16% complete remission in patients. This success led to FDA approval of Elo plus Len treatment for patients with RRMM and has warranted further investigations into the exact mechanism of action. Elo targets SLAMF7 antigen which is highly expressed on MM tumour cells and activates NK cells for antibody dependent cell-mediated cytotoxicity (ADCC) resulting in MM tumour cell death. Len is an immunomodulatory agent commonly used to treat a wide range of haematological cancers. Len has been shown to activate various cells of the immune system as well as directly suppress tumour progression. This thesis aims to identify how the mechanisms of Len intersect with, and enhance, Elo activated NK cell control of tumours. The work from this thesis revealed that to induce ADCC, Elo required both expression of SLAMF7 on MM target cells and NK cell CD16 engagement. Elo-activated NK cells had increased expression of CD107a and CD69, as well as loss of CD16 expression which was a result of ADAM17 induced cleavage. Elo activation of NK cells also secreted increased levels of cytokines and chemokines associated with recruitment of effector immune cells. In both in-vitro and in-vivo studies, Len significantly enhanced Elo-induced healthy donor NK cell killing of MM tumour cells. In contrast, only 4/12 RRMM patient (responders) NK cells induced increased MM cell killing in the context of Elo plus Len. Important for this increased cytotoxicity was the expression of CD54 on NK cells that allowed differentiation between these responder and non-responder patients. CD54 was also more highly expressed on NK cells activated by Elo plus Len compared to Elo activation alone. However, further phenotypic studies and RNA sequencing did not reveal any further mechanisms in NK cells by which Len enhances Elo activation. Further studies revealed that MM tumour (myeloma) cells specifically upregulated CD54 and CD11a expression in response to Elo plus Len activation of NK cells and this did not occur with Elo treatment alone. This increase in CD54/CD11a expression on myeloma cells was also dependent on the presence of CD14+ monocytes. RNA sequencing also revealed enrichment of cytokine and chemokine signalling/secretion pathways in MM cells specifically in the context of Elo and Len activated NK cells and monocytes. In conclusion, these data indicate that a complex network of direct and indirect mechanisms between NK cells, monocytes and myeloma cells contribute to the Elo plus Len treatment induced response in RRMM patients. Elo alone activates NK cells and monocytes, whereas the Len effect is largely on myeloma cells. Collectively the two treatments led to robust myeloma antibody dependent cellular cytotoxicity (ADCC). There was a strong correlation between increased myeloma cell CD54/CD11a expression and myeloma cell ADCC suggesting that stabilisation of the immune synapse with strong adhesion may be a key factor for this improved myeloma cell killing. Finally, this study revealed that upregulation of MM cell CD54 expression on MM cells may be a useful predictive biomarker to stratify RRMM patients for Elo plus Len therapy.