This is a repository copy of Dysregulation at multiple points of the kynurenine pathway is a ubiquitous feature of renal cancer: implications for tumour immune evasion. White Rose Research Online URL for this paper: http://eprints.whiterose.ac.uk/158462/ Version: Accepted Version Article: Hornigold, N, Dunn, KR, Craven, RA et al. (22 more authors) (2020) Dysregulation at multiple points of the kynurenine pathway is a ubiquitous feature of renal cancer: implications for tumour immune evasion. British Journal of Cancer. ISSN 0007-0920 https://doi.org/10.1038/s41416-020-0874-y © The Author(s), under exclusive licence to Cancer Research UK 2020. This is an author produced version of an article published in British Journal of Cancer. Uploaded in accordance with the publisher's self-archiving policy. Reuse Items deposited in White Rose Research Online are protected by copyright, with all rights reserved unless indicated otherwise. 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[email protected] https://eprints.whiterose.ac.uk/ 1 Dysregulation at multiple points of the kynurenine pathway is a 2 ubiquitous feature of renal cancer: implications for tumour 3 immune evasion 4 Nick Hornigold*1, Karen R Dunn*1, Rachel A Craven*1, Alexandre Zougman1, Sebastian 5 Trainor1, Rebecca Shreeve1, Joanne Brown1, Helen Sewell1, Michael Shires2, Margaret 6 Knowles3, Tsutomu Fukuwatari4, Eamonn R Maher5, Julie Burns3, Selina Bhattarai6, Mini 7 Menon6, Alvis Brazma7, Ghislaine Scelo8, Lara Feulner9, Yasser Riazalhosseini9, Mark 8 Lathrop9, Adrian Harris10, Peter J Selby1, Rosamonde E Banks1¡, Naveen S Vasudev1¡ 9 *these authors contributed equally to this work 10 ¡equal senior authors 11 1Clinical and Biomedical Proteomics Group, University of Leeds, St. James's University 12 Hospital, Beckett Street, Leeds LS9 7TF, UK; 2Leeds Institute of Medical Research at St 13 James’s, University of Leeds, St. James's University Hospital, Beckett Street, Leeds LS9 14 7TF, UK; 3Molecular Genetics Group, University of Leeds, St. James's University 15 Hospital, Beckett Street, Leeds LS9 7TF; 4Department of Nutrition, The University of Shiga 16 Prefecture, 2500 Hassaka, Hikone 5228533, Japan; 5Department of Medical Genetics, 17 University of Cambridge and NIHR Cambridge Biomedical Research Centre, and Cancer 18 Research UK Cambridge Centre, Cambridge Biomedical Campus, Cambridge CB2 0QQ, 19 UK; 6Department of Pathology, St James’s University Hospital, Beckett Street, Leeds LS9 20 7TF UK; 7European Molecular Biology Laboratory, European Bioinformatics Institute, 8 21 EMBL-EBI, Wellcome Trust Genome Campus, Hinxton CB10 1SD, UK;. International 22 Agency for Research on Cancer (IARC), Genetic Epidemiology Group, 150 cours Albert 23 Thomas, 69372, Lyon, France; 9McGill University and Genome Quebec Innovation 24 Centre, 740 Doctor Penfield Avenue, Montreal, QC, H3A 0G1, Canada;10Cancer 25 Research UK Clinical Centre, Weatherall Institute of Molecular Medicine, John Radcliffe 26 Hospital, Headington, Oxford OX3 9DS, UK 27 28 29 Corresponding author: Dr N Vasudev, St. James's University Hospital, Beckett Street, 30 Leeds LS9 7TF, UK. Tel: +44 113 2067680; Fax: +44 113 2429886; e-mail: 31 [email protected] 32 33 Running Title: Dysregulation of kynurenine pathway in RCC 34 Keywords: IDO1; kynurenine pathway; proteomics; QPRT; renal cell carcinoma 35 36 37 38 39 40 41 42 43 1 44 Abstract 45 46 Background: Indoleamine 2,3-dioxygenase (IDO), the first step in the kynurenine 47 pathway (KP), is upregulated in some cancers and represents an attractive therapeutic 48 target given its role in tumour immune evasion. However, the recent failure of an IDO 49 inhibitor in a late phase trial raises questions about this strategy. 50 Methods: Matched renal cell carcinoma (RCC) and normal kidney tissues were subject 51 to proteomic profiling. Tissue immunohistochemistry and gene expression data were used 52 to validate findings. Phenotypic effects of loss/gain of expression were examined in vitro. 53 Results: Quinolate phosphoribosyltransferase (QPRT), the final and rate-limiting enzyme 54 in the KP, was identified as being down-regulated in RCC. Loss of QPRT expression led 55 to increased potential for anchorage-independent growth. Gene expression, mass 56 spectrometry (clear cell and chromophobe RCC) and tissue immunohistochemistry (clear 57 cell, papillary and chromophobe), confirmed loss or decreased expression of QPRT, and 58 showed down-regulation of other KP enzymes including kynurenine 3-monoxygenase 59 (KMO) and 3-hydroxyanthranilate-3,4-dioxygenase (HAAO), with a concomitant 60 maintenance or up-regulation of nicotinamide phosphoribosyltransferase (NAMPT), the 61 key enzyme in the NAD+ salvage pathway. 62 Conclusions: Widespread dysregulation of the KP is common in RCC and is likely to 63 contribute to tumour immune evasion, carrying implications for effective therapeutic 64 targeting of this critical pathway 65 66 67 68 69 70 71 72 73 2 74 Introduction 75 Renal cancer is one of the ten most common adult cancers, accounting for over 100,000 76 deaths worldwide each year.1 It is also a cancer with one of the highest projected 77 increases in incidence over the next two decades.2 Almost 90% of these cancers arise 78 within the renal parenchyma and are termed renal cell carcinomas (RCCs). The most 79 common (75%) histological subtype is clear cell RCC (conventional) (ccRCC), which are 80 characterised by loss of the VHL tumour suppressor gene, followed by papillary (10-15%) 81 and chromophobe (5%) RCC. Each is considered to arise from distinct parts of the human 82 nephron, are genetically distinct3 and vary considerably in their clinical behaviour. 83 Treatment options for patients with RCC have burgeoned in recent years but, despite this, 84 it is a cancer that remains incurable for most patients with advanced disease. As in a 85 number of other tumour types, immunotherapy, in the form of checkpoint inhibitors (CPI), 86 has come to the forefront of patient treatment, both in the first- and second-line settings. 87 However, whilst some patients are observed to have deep and durable responses to these 88 agents, many patients fail to respond.4 It appears, therefore, that alone, CPI are not always 89 sufficient to overcome immune evasion and immune tolerance by tumours. Hence, 90 increasing attention is being focused on combining these drugs with other immune- 91 modifying targeted agents.5 92 Alterations in cellular metabolism are a hallmark of cancer,6 most notably perhaps the 93 ‘Warburg effect’, that describes the increased rate of glycolysis with reduced oxidative 94 phosphorylation characteristic of tumours.7 We were amongst the first to demonstrate this 95 comprehensively in renal cancers, using a proteomic approach, showing an up-regulation 96 in the majority of proteins in the glycolytic pathway and a parallel downregulation of 97 mitochondrial enzymes in comparison to normal renal tissues,8 highlighting novel 98 opportunities for therapeutic targeting.9 99 More recently, alteration in the metabolism of the essential amino acid tryptophan in 100 cancer, through the kynurenine pathway, has come to wide attention as a mechanism by 3 101 which tumours may escape immune control and promote disease progression. The 102 enzymes indoleamine 2,3-dioxygenase (IDO1, IDO2) and tryptophan 2,3-dioxygenase 103 (TDO) initiate the first steps in the kynurenine pathway, converting tryptophan to 104 kynurenine, with the TDO-dependent pathway in the liver normally accounting for the 105 majority of tryptophan metabolism and IDO-mediated metabolism predominantly 106 occurring secondary to inflammation and cytokine-induced upregulation.10 In normal 107 physiology, IDO plays an important role in tolerance to non-self antigens, for example fetal 108 antigens, where such immune non-responsiveness may be important.11,12 Up-regulation 109 of IDO leads to tryptophan depletion and kynurenine accumulation, which appear to work 110 in concert to mediate immunosuppression, via T-cell anergy and apoptosis and 111 suppressed T-cell differentiation.13 The harnessing of this phenomenon by tumours has 112 led to the development of inhibitors of IDO1 that have progressed to clinical trials in 113 combination with CPI. Despite much promise, initial results have, however, been 114 disappointing and the future of these agents currently remains uncertain.14,15 This may be 115 because of patient selection and lack of suitable profiling of immuno-regulating 116 metabolism, emphasising the need for a deeper understanding of these pathways. 117 Here, using a proteomic-based approach, we show that the kynurenine pathway is more 118 broadly disrupted than has been previously considered, extending beyond IDO1, that this 119 is a common event in RCC and is not just restricted to the clear cell histological subtype, 120 and suggests possible redundancy in the pathway within the tumour setting. Our findings 121 are of significance in terms of highlighting various aspects of this pathway for potential 122 therapeutic targeting, patient