This is a repository copy of Macrophage-Derived IL1β and TNFα Regulate Arginine Metabolism in Neuroblastoma. White Rose Research Online URL for this paper: http://eprints.whiterose.ac.uk/145809/ Version: Accepted Version Article: Fultang, L, Gamble, LD, Gneo, L et al. (20 more authors) (2019) Macrophage-Derived IL1β and TNFα Regulate Arginine Metabolism in Neuroblastoma. Cancer Research, 79 (3). pp. 611-624. ISSN 0008-5472 https://doi.org/10.1158/0008-5472.CAN-18-2139 © 2018 American Association for Cancer Research. This is an author produced version of a paper published in Cancer Research. 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. They may be downloaded and/or printed for private study, or other acts as permitted by national copyright laws. 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[email protected] https://eprints.whiterose.ac.uk/ IL-1ß and TNF- create an immune-metabolic loop regulating Arginase 2 via p38-ERK in neuroblastoma Livingstone Fultang1, Laura Gamble2, Luciana Gneo1, Andrea Berry3, Sharon Egan4, Fenna De Bie1, Orli Yogev5, Georgina Eden2, Sarah Booth1, Samantha Brownhill3, Ashley Vardon1, Boris Noyvert6, Carmel McConville6, Andrew Beggs6,Paul Cheng7, Murray Norris2, Heather Etchevers8, Jayne Murray2, David Ziegler2, Louis Chesler5, Ronny Schmidt9, Susan Burchill3, Michelle Haber2, Carmela De Santo1, Francis Mussai1* C De Santo and F Mussai contributed equally to this manuscript. * Corresponding author: Francis Mussai, Institute of Immunology and Immunotherapy, University of Birmingham, Birmingham, B15 2TT, United Kingdom. e-mail: [email protected] 1 Institute of Immunology and Immunotherapy, University of Birmingham, Birmingham, B15 2TT, UK 2 C C I U N S W S A 3 C C ‘ G L I C P U L L LS 7TF, UK 4 School of Veterinary Medicine and Science, Sutton Bonington Campus, University of Nottingham, Nottingham, LE12 5RD, UK 5 The Institute of Cancer Research, London SM2 5MG, UK 6 Institute of Cancer Genomic Sciences, University of Birmingham, Birmingham, Birmingham, B15 2TT, UK 7 Bio-Cancer Treatment International, Hong Kong 8 GMGF, Aix Marseille University, INSERM, UMR_S910, Marseille, France 9 Sciomics GmbH, Heidelberg, Germany 1 Summary Neuroblastoma is the most common solid tumour of childhood, yet the prognosis for high risk disease remains poor. We demonstrate that arginine metabolism via Arginase 2 (ARG2) drives neuroblastoma cell proliferation. Proteomic analysis reveals Stage IV human tumours have a Th1-skewed intra-tumoural microenvironment and tumour cell arginase infiltrating- monocytes to a CD68+ phenotype, which release IL-1 and TNF- in a AKT dependent manner. Il-1 and TNF- signal-back to upregulate ARG2 expression via p38 and ERK1/ signalling in neuroblastoma and neural crest. Tumour cells express ARG2 mis-localised in the cell cytoplasm, whilst deficiencies in Argininosuccinate Synthase (ASS) and Ornithine Transcarbamylase (OTC) enzymes result in tumour cells reliant on extracellular arginine. Therapeutic depletion of arginine with PEGylated-recombinant Arginase is cytotoxic to neuroblastoma and leads to a significant extension in transgenic murine survival. Thus we describe an immune-metabolic regulatory loop between tumour cells and infiltrating myeloid cells regulating ARG2, which can be clinically exploited. 2 Introduction The consumption and metabolism of diverse nutrients by cancer cells is recognised as a key regulator of immunity. Glucose metabolism by cancer cells generates a tumour microenvironment that has low levels of glucose, leading to inhibition of T cell cytotoxicity through the accumulation of lactate, microenvironment acidification, and reduced aerobic glycolysis (Angelin et al., 2017, Calcinotto et al., 2012, Ho et al., 2015). Tumour infiltrating monocyte differentiation and cytokine release may be similarly affected, leading to perturbation of their role in coordinating the surrounding immune response (Dietl et al., 2010, Wei et al., 2006). Amino acid metabolism also plays a critical role in the function of both normal and malignant cells. Although whole body amino acid homeostasis is regulated through restricted inter-organ enzyme expression, at the cellular level enzyme expression is controlled in the intracellular compartment to maintain metabolic precursor supplies and regulate the wider tissue microenvironment (Fultang et al., 2016). Arginine is a semi-essential amino acid, that is metabolised into ornithine and urea by the expression of cytoplasmic Arginase 1 and mitochondrial Arginase 2 (ARG2), or Nitric Oxide Synthase (NOS) enzymes into reactive nitric oxide species (Morris, 2016). These metabolites feed forward into diverse roles in cell signalling, proliferation and protein synthesis. Cellular breakdown of arginine also plays a critical role in regulating the immune response, a process which has been capitalised on by malignant cells to contribute to their immune escape (De Santo et al., 2010). We recently identified that Acute Myeloid Leukaemias (AML) and neuroblastoma, two of the most common and devastating cancers of childhood create a 3 potent immunosuppressive microenvironment through the expression of ARG2 enzyme which suppresses T-cell immunity (Mussai et al., 2015b, Mussai et al., 2013). Although the metabolic effect of cancer cells on shaping the responsiveness of surrounding immune populations is increasingly well described, the reciprocal effects of immune cell populations on modulating cancer cell amino acid metabolism have not previously been reported. In particular the role of arginine metabolism in this process is unknown and the signals which regulate ARG2 in cancer are unknown. Here we demonstrate how myeloid cells within the tumour microenvironment and tumour cells engage in reciprocal cross-talk to regulate the expression of ARG2 in neuroblastoma cells, and how this arginine metabolism plays a central role in neuroblastoma pathogenesis. Importantly the study identifies arginine metabolism as a clinically relevant therapeutic target. 4 Results Stage IV neuroblastomas have a Th1 cytokine enriched microenvironment We established that ARG2 expression by neuroblastoma cells creates an immunosuppressive microenvironment and ne responses and immunotherapy (Mussai et al., 2015b). However, the role of ARG2 in neuroblastoma development and more widely in human cancers has only received limited study. Expression of ARG2 in human GD2+ neuroblastoma cells was confirmed by immunohistochemistry of tumour biopsies at diagnosis (Figure S1A). To understand if arginine metabolism is central to tumour cell growth we first blocked uptake of arginine from the microenvironment via Cationic Amino Acid Transporter-1 (CAT1). N-nitro-L-arginine (L-NAME) led to a significant decrease in tumour cell proliferation (Figure 1A). Culture of tumour cells in the absence of arginine similarly inhibited tumour cell metabolic activity (Figure 1B). shRNA knock-down of ARG2 led to a significant reduction in cell proliferation in the highest expressing cells (SKNMC>KELLY) (Figure 1C, and Figure S1B). Thus arginine metabolism by ARG2 in neuroblastoma cells contributes to tumour cell proliferation. The pathways which regulate ARG2 expression in cancer cells have not been reported to date. We showed that ARG2 expression is highest in Stage IV tumours and is associated with a worse overall survival (Mussai et al., 2015b). To gain a deeper understanding of the immune microenvironment within these tumours, we investigated the proteomic profile inside 23 human neuroblastomas at diagnosis (9 Stage I and 14 Stage IV) using a novel antibody proteomic platform that characterises the expression of 900-cancer-related proteins (Ruiz- Babot et al., 2018). Non-metric multidimensional scaling for all analysed samples based on 5 the complete protein expression data revealed separate clustering of Stage I and Stage IV tumours (Figure 1D). Heat-map representation of protein signals reveal that Stage I and Stage IV tumours show distinct molecular proteomic subgroups, with 7 Stage IV tumours (P21-27) forming a distinct group, while 3 others (P10,P15,P16), had proteomes closer to Stage I tumours (Figure S1C; Related to Figure 1D). Characterisation of the Stage IV tumours identified significantly higher levels of the Th1 cytokines IL-1IFN-and TNF- than Stage I tumours (Figure 1E). In contrast, Stage I tumours had increased expression of the Th2-related cytokines TGF-, IL-10, and IL-4 (Figure 1F). No significant differences in IL-6 and IL-13 expression were identified. The finding of high levels of IL-1IFN-and TNF-identifies that Stage IV tumours are skewed towards a Th1 microenvironment. Tissue micro-arrays of 27 human neuroblastomas at diagnosis validated the expression of IL-1TNF- and IFN- within tumour samples compared to healthy peripheral nerve tissueFigure 1G). No association of cytokine expression with anatomical tumour site was identified. We hypothesised the cytokine microenvironment may help drive tumour development. Cytokines may be functional either within