bioRxiv preprint doi: https://doi.org/10.1101/2021.06.12.448188; this version posted June 14, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 1 Title 2 Pharmacologic and genetic inhibition of cholesterol esterification reduces tumour burden: a 3 pan-cancer systematic review and meta-analysis of preclinical models 4 Authors 5 Alex Websdale1, Yi Kiew1,$, Philip Chalmers1,$, Xinyu Chen1, Giorgia Cioccoloni1, Thomas A 6 Hughes2, Xinyu Luo1, Rufaro Mwarzi1, Marc Poirot3, Hanne Røberg-Larsen4, Ruoying Wu1, 7 Mengfan Xu1, Michael A. Zulyniak1 and James L Thorne1,€. 8 Affiliations 9 1School of Food Science and Nutrition, University of Leeds, Leeds, LS2 9JT, UK. 10 2School of Medicine, University of Leeds, Leeds, LS9 7TF, UK 11 3Cancer Research Center of Toulouse, Inserm, CNRS, University of Toulouse, Toulouse, France 12 4Department of Chemistry, University of Oslo, Norway 13 $These authors contributed equally 14 €Corresponding author [email protected] 15 16 Abstract 17 Cholesterol esterification proteins Sterol-O acyltransferases (SOAT) 1 and 2 are emerging 18 prognostic markers in many cancers. These enzymes utilise fatty acids conjugated to 19 coenzyme A to esterify cholesterol. Cholesterol esterification is tightly regulated and enables 20 formation of lipid droplets that act as storage organelles for lipid soluble vitamins and 21 minerals, and as cholesterol reservoirs. In cancer, this provides rapid access to cholesterol to 22 maintain continual synthesis of the plasma membrane. In this systematic review and meta- bioRxiv preprint doi: https://doi.org/10.1101/2021.06.12.448188; this version posted June 14, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 23 analysis, we summarise the current depth of understanding of the role of this metabolic 24 pathway in pan-cancer development. A systematic search of PubMed, Scopus, and Web of 25 Science for preclinical studies identified eight studies where cholesteryl ester concentrations 26 were compared between tumour and adjacent-normal tissue, and 24 studies where 27 cholesterol esterification was blocked by pharmacological or genetic approaches. Tumour 28 tissue had a significantly greater concentration of cholesteryl esters than non-tumour tissue 29 (p<0.0001). Pharmacological or genetic inhibition of SOAT was associated with significantly 30 smaller tumours of all types (p≤0.002). SOAT inhibition increased tumour apoptosis 31 (p=0.007), CD8+ lymphocyte infiltration and cytotoxicity (p≤0.05), and reduced proliferation 32 (p=0.0003) and metastasis (p<0.0001). Significant risk of publication bias was found and may 33 have contributed to a 32% overestimation of the meta-analysed effect size was 34 overestimated. Avasimibe, the most frequently used SOAT inhibitor, was effective at doses 35 equivalent to those previously reported to be safe and tolerable in humans. This work 36 indicates that SOAT inhibition should be explored in clinical trials as an adjunct to existing 37 anti-neoplastic agents. 38 1. Introduction 39 Esterification is a tightly regulated component of cholesterol homeostasis and enables 40 cholesterol packaging into lipid droplets. Intra-cellular storage of cholesterol allows ready 41 access to meet the high demand for de novo plasma membrane synthesis during the rapid 42 proliferation of cells, for example during tumour growth. Several diseases are linked to 43 cholesterol esterification including a range of neurological conditions, lipid disorders, and 44 cancer. The synthesis of cholesteryl esters (CE) is catalysed by Sterol O-acyltransferase 1 and 45 2 (SOAT1 and SOAT2), and Lecithin cholesterol acyltransferase (LCAT). SOAT1 and SOAT2, 46 utilise fatty acid-coenzyme A conjugates to preferentially generate oleoyl (Fig1A), and 47 linoleoyl or palmitoyl CEs (Fig1B), respectively, and coenzyme A as a biproduct [1]. LCAT 48 utilises phosphatidylcholine (lecithin) to produce oleoyl CEs [2] but instead of producing 49 coenzyme A as a biproduct, as is the case for SOAT1 and SOAT2, lysophosphatidylcholine is 50 the biproduct (Fig1C). SOAT1 is ubiquitously expressed in tissues while SOAT2 is restricted to 51 small intestines and the liver [3] and LCAT is expressed in the liver and secreted into 52 circulation in lipoprotein complexes [4, 5]. bioRxiv preprint doi: https://doi.org/10.1101/2021.06.12.448188; this version posted June 14, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 53 Esterification of cholesterol in tumours is beneficial for cancer growth and studies of SOAT1, 54 SOAT2 and LCAT in humans indicate this metabolic process is deregulated in cancer. Elevated 55 SOAT1 and SOAT2 expression in tumours has been linked to higher grade of breast [6] and 56 renal [7] cancer, respectively, and high expression SOAT1 has also been linked to poor 57 prognosis for patients with liver [8], glioma [9], pancreatic [10] and adrenocortical [11] 58 cancers. Furthermore, increased intracellular lipid droplet content, indicative of cholesterol 59 esterification, is associated with reduced overall survival [12] and elevated cholesteryl oleoyl 60 ester levels has been proposed as a prognostic biomarker for prostate cancer [13]. 61 Conversely, high LCAT expression is associated with improved prognosis for liver cancer 62 patients [14] and is often lower in liver cancer than normal tissue in humans [15] and rat 63 models [16-18]. At the molecular level, cholesteryl esters promote cancer proliferation and 64 invasiveness [19] and thus SOATs were considered as promising targets and the anticancer 65 action of natural SOAT inhibitors such as auraptene and bryonolic acid was elucidated [20, 66 21]. Several small molecule inhibitors of cholesterol esterification have been explored in 67 clinical trials for non-cancer related diseases providing an extensive understanding of their 68 tolerability, toxicity and side-effect profiles. Avasimibe, first discovered in 1996 [22], is a dual 69 SOAT1 and SOAT2 inhibitor [23, 24] and has been used in clinical trials for coronary 70 atherosclerosis [25] and homozygous familial hypercholesterolemia [26]. Avasimin is human 71 serum albumin encapsulated avasimibe that was developed to improve avasimibe solubility 72 [27]. K-604 is a SOAT1 specific inhibitor [24] and has been tested for both safety and efficacy 73 as a treatment against atherosclerosis (NCT00851500), however results from the trial have 74 not been published. ATR-101 (Nevanimibe) is a SOAT1 specific inhibitor [28] that has been 75 tested in a clinical trial against adrenocortical carcinoma (NCT01898715) [29] and Cushing’s 76 syndrome (NCT03053271). Pactimibe also inhibits both SOAT1 and SOAT2 [23, 30], but a 77 clinical trial (NCT00151788) administering 100 mg/day was terminated early due to a 78 significant increase in major cardiovascular disease events [31]; pactimibe remains untested 79 in pre-clinical cancer models. Drugs targeting CE synthesis that have been evaluated in clinical 80 trials are summarised in Table 1. 81 There is a significant body of research investigating cholesterol esterification in pre-clinical 82 cancer models. Many of these studies utilise pharmacological or genetic inhibitors of SOAT to 83 provide insight into the cellular and molecular role of the enzyme and propose repurposing bioRxiv preprint doi: https://doi.org/10.1101/2021.06.12.448188; this version posted June 14, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 84 SOAT inhibitors as cancer therapies. However, the pharmacological compounds remain 85 underexplored in the clinical cancer setting and may be suitable for repurposing. This 86 systematic review and meta-analysis summarises the evidence regarding cholesterol 87 esterification enzymes as therapeutic targets in cancer and details the range of 88 pharmacological approaches that are closest to clinical translation. 89 2. Methods 90 2.1 Search strategy 91 The search strategy was applied to PubMed, SCOPUS, Web of Science and the Cochrane 92 Library, and records were retrieved up until April 2021; the strategy is registered in the 93 PROSPERO database (CRD42020202409) with the following modifications: records evaluating 94 sulfation and associated enzymes were excluded from the study. 95 2.2 Study selection 96 Titles and abstracts were screened against inclusion criteria; (i) original research, (ii) 97 investigated cancer, (iii) assessed an in vivo pre-clinical animal model and (iv) modulated 98 cholesterol esterification. Each abstract was assessed by two independent assessors and 99 discrepancies resolved by a third member of the research team. All publications that satisfied 100 the above criteria were included for qualitative assessment. 101 2.3 Data extraction 102 Publication that reported adequate data for quantitative assessment were included in the 103