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.