e16303 Background: Dipeptidyl peptidase 9 (DPP9) is one of four enzymes targeted by a novel compound class that includes talabostat and BXCL701, which have reached clinical oncology trials. DPP9 supresses NLRP1 inflammasome activation and BRCA2 activity. Global DPP9 gene inactivation is lethal in mice and humans. Therefore, to study primary hepatocellular carcinoma, we examined mice in which DPP9 expression is depleted only from hepatocytes. Methods: A hepatocyte-specific DPP9 knockout mouse (Alb-DPP9-KO) was achieved by floxing exons 5 to 7 of full-length DPP9 and crossing this DPP9fl/fl mouse strain with a mouse strain that expresses Cre recombinase under an albumin promoter. Primary liver cancer was induced by treating with diethylnitrosamine (DEN; once), then thioacetamide (TAA) and a high fat diet (HFD) from 4-28 weeks of age. Measurements included tumour burden and total liver qPCR of immunological and autophagy markers. Results: Alb-DPP9-KO mice were healthy. Compared to littermate controls, Alb-DPP9-KO mice had reduced liver mass and subcutaneous adipose tissue mass and had lower fasting plasma glucose. Alb-DPP9-KO mice had fewer small macroscopic liver nodules (< 3 mm diameter) compared to littermate controls. There were no differences in the total number of macroscopic liver nodules, tumour burden, inflammation, fibrosis and steatosis. However, Alb-DPP9-KO livers had increased levels of active caspase-1 protein, indicative of increased inflammasome activity, and increased levels of Nfkbib, Cxcl10 and Ccl5. The Alb-DPP9-KO livers contained increased protein levels of the tumour suppressor p53, and of autophagy markers LC3B, p62 and beclin1. Conclusions: Lifelong intrahepatic DPP9 depletion reduced tumour sizes at 28 weeks of age in this DEN/TAA/HFD experimental model. Mechanisms might include increased caspase-1 activation following NLRP1 activation, increased autophagy and p53 and improved energy metabolism in epithelial cells. DPP9 inhibition may contribute to efficacy in therapies that target DPP4 protease family.

INTRODUCTION:Long-term normothermic machine perfusion (LT-NMP) enables the assessment and optimization of livers for days and, potentially, weeks. However, models of LT-NMP have only been described for human and pig livers, which are resource intensive and impractical for laboratory research. Cost-effective small animal models of LT-NMP are needed for future research. This study aimed to develop a system for LT-NMP of rat livers for up to 72 h. METHODS:This study was performed in two stages: the development phase (n = 20) and validation phase (n = 5). The perfusion system included an organ reservoir, pump, heat exchanger, long-term oxygenator, and dialysis. Hormonal and nutritional support were continuously infused. During the validation phase, five consecutive grafts were perfused using our protocol. At 72 h postreperfusion, grafts were assessed for viability, which was based on hemodynamic stability, mitochondrial function, bile production, and metabolic activity. RESULTS:Rodent livers were supported up to 107 h using our LT-NMP protocol. All grafts in the validation phase remained viable at 72 h (n = 5/5). The median oxygen consumption and bile production at 72 h were 0.079 mLO2/min/g-liver and 8.6 uL/h/g-liver, respectively. All grafts had a systemic vascular resistance less than 0.25 mmHg/mL/min. Metabolic activity, defined as lactate clearance, glucose production, or response to glucagon, was observed in all grafts (5/5). CONCLUSIONS:This is the first study to report LT-NMP of rodent livers up to 5 days. Using our protocol, rat livers could reliably be supported until 72 h. This model provides a greater opportunity to investigate novel therapeutics to assess, optimize, and regenerate liver grafts for transplantation.

Dipeptidyl peptidase 9 (DPP9) is an indispensable intracellular protease. Among its many molecular functions is suppression of the NLRP1 inflammasome. Inhibitors targeting all four proteases of the DPP4 family, including DPP9, can reduce tumour burden, including in mouse liver. To explore hepatocyte DPP9 in experimental hepatocellular carcinoma (HCC), we generated hepatocyte-specific DPP9-KO mice by crossing albumin-Cre mice with DPP9 floxed mice and treated sequentially with diethylnitrosamine, then with thioacetamide combined with an atherogenic high-fat diet until 28 weeks of age. DPP9-KO mice had less body, liver and subcutaneous adipose tissue mass, lower fasting plasma glucose and fewer small macroscopic liver nodules compared to DPP9-WT control mice. However, there were no differences in the total number of macroscopic liver nodules, or of microscopic tumour burden, inflammation, fibrosis or steatosis. Consistent with the known function of DPP9 to suppress NLRP1 activation, activated caspase-1 protein and inflammation markers Nfkbib, Cxcl10 and Ccl5 were elevated in DPP9-KO liver. The tumour suppressor protein p53 was increased and the autophagy proteins beclin1, LC3B and p62 were altered. In conclusion, hepatocyte-specific DPP9 gene deletion in experimental primary liver cancer improved energy metabolism and may reduce liver cancer initiation, via mechanisms that may include increased autophagy and tumour suppression.

Bile duct regeneration is hypothesized to prevent biliary strictures, a leading cause of morbidity after liver transplantation. Assessing the capacity for biliary regeneration may identify grafts as suitable for transplantation that are currently declined, but this has been unfeasible until now. This study used long-term ex situ normothermic machine perfusion (LT-NMP) to assess biliary regeneration. Human livers that were declined for transplantation were perfused at 36 °C for up to 13.5 days. Bile duct biopsies, bile, and perfusate were collected throughout perfusion, which were examined for features of injury and regeneration. Biliary regeneration was defined as new Ki-67-positive biliary epithelium following severe injury. Ten livers were perfused for a median duration of 7.5 days. Severe bile duct injury occurred in all grafts, and biliary regeneration occurred in 70% of grafts. Traditional biomarkers of biliary viability such as bile glucose improved during perfusion but this was not associated with biliary regeneration (P > .05). In contrast, the maintenance of interleukin-6 and vascular endothelial growth factor-A levels in bile was associated with biliary regeneration (P = .017 for both cytokines). This is the first study to demonstrate biliary regeneration during LT-NMP and identify a cytokine signature in bile as a novel biomarker for biliary regeneration during LT-NMP.

There are no ideal non-invasive tests for assessing the severity of liver fibrosis in people with metabolic dysfunction-associated steatotic liver disease (MASLD) and class 3 obesity, where body habitus often makes imaging technically challenging. This study aimed to assess the applicability and diagnostic performance of two-dimensional shear wave elastography (2D-SWE), alongside several serum-based liver fibrosis scoring methods, in individuals with class 3 obesity. A cross-sectional study was conducted in patients aged ≥18 years and with a body mass index (BMI) ≥ 40 kg/m2 who were participants in a publicly funded multidisciplinary weight management program in South Western Sydney. The 2D-SWE was performed using the ElastQ Imaging (EQI) procedure with the Phillips EPIQ Elite series ultrasound. An EQI Median value of ≥6.43 kPa was taken as a cutoff score for significant fibrosis, and the scan was considered valid when the liver EQI IQR/Med value was <30%. The Fibrosis-4 (FIB-4) index, AST-to-platelet ratio index (APRI), NAFLD fibrosis score (NFS), and circulating fibroblast activation protein index (FAP index) were calculated from fasting blood samples. The participants (n = 116; 67.2% female) were aged 47.2 ± 12.9 years, with BMI 54.5 ± 11.0 kg/m2. EQI Median values were obtained for 97.4% (113/116) of the 2D-SWE scans, and 91.4% (106/116) of the scans were considered valid. The EQI Median values exhibited a moderately positive correlation with the FIB-4 index (r = 0.438; p < 0.001) and a weakly positive correlation with the APRI (r = 0.388; p < 0.001), NFS (r = 0.210; p = 0.036) and FAP index (r = 0.226; p = 0.020). All liver fibrosis scores were positively correlated with one another. Among those referred for a liver biopsy based on the 2D-SWE and serum scores, half (11/22) underwent liver biopsy, and their 2D-SWE scores exhibited 72.7% accuracy (sensitivity: 71.4%; specificity: 75%) in detecting significant fibrosis. Our results show that 2D-SWE is a feasible, non-invasive test to assess liver fibrosis among people with class 3 obesity. Further research is needed to assess how 2D-SWE can be used alongside existing serum-based risk scores to reliably detect significant fibrosis, which would potentially reduce the need for invasive liver biopsy.

Background Hepatocellular carcinoma (HCC) remains a leading life-threatening health challenge worldwide, with pressing needs for novel therapeutic strategies. Sphingosine kinase 1 (SphK1), a well-established pro-cancer enzyme, is aberrantly overexpressed in a multitude of malignancies, including HCC. Our previous research has shown that genetic ablation of Sphk1 mitigates HCC progression in mice. Therefore, the development of PF-543, a highly selective SphK1 inhibitor, opens a new avenue for HCC treatment. However, the anti-cancer efficacy of PF-543 has not yet been investigated in primary cancer models in vivo, thereby limiting its further translation. Methods Building upon the identification of the active form of SphK1 as a viable therapeutic target in human HCC specimens, we assessed the capacity of PF-543 in suppressing tumor progression using a diethylnitrosamine-induced mouse model of primary HCC. We further delineated its underlying mechanisms in both HCC and endothelial cells. Key findings were validated in Sphk1 knockout mice and lentiviral-mediated SphK1 knockdown cells. Results SphK1 activity was found to be elevated in human HCC tissues. Administration of PF-543 effectively abrogated hepatic SphK1 activity and significantly suppressed HCC progression in diethylnitrosamine-treated mice. The primary mechanism of action was through the inhibition of tumor neovascularization, as PF-543 disrupted endothelial cell angiogenesis even in a pro-angiogenic milieu. Mechanistically, PF-543 induced proteasomal degradation of the critical glycolytic enzyme 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3, thus restricting the energy supply essential for tumor angiogenesis. These effects of PF-543 could be reversed upon S1P supplementation in an S1P receptor-dependent manner. Conclusions This study provides the first in vivo evidence supporting the potential of PF-543 as an effective anti-HCC agent. It also uncovers previously undescribed links between the pro-cancer, pro-angiogenic and pro-glycolytic roles of the SphK1/S1P/S1P receptor axis. Importantly, unlike conventional anti-HCC drugs that target individual pro-angiogenic drivers, PF-543 impairs the PFKFB3-dictated glycolytic energy engine that fuels tumor angiogenesis, representing a novel and potentially safer therapeutic strategy for HCC.

Objective: Metabolic fatty liver disease drives chronic liver injury leading to fibrosis. This study aimed to establish a model utilising serum circulating fibroblast activation protein (cFAP) to diagnose advanced fibrosis in patients with fatty liver disease. Design: Two retrospective cohorts recruited from tertiary hepatology clinics were studied as training (n=160) and external validation cohorts (n=342), with prevalence of histologic advanced fibrosis (F3/F4) of 20% and 11%, respectively. A marker of activated mesenchymal fibrogenic cells, cFAP, was measured using our single-step enzyme assay. A predictive model, FAP Index, containing age, type 2 diabetes, alanine transaminase and ordinal cFAP was developed using logistic regression. Diagnostic accuracy of FAP Index was assessed on a single and then sequential basis. Results: FAP Index AUROC was 0.875 (95% CI 0.813-0.938) in the training cohort and 0.841 (95% CI 0.776-0.906) in the validation cohort. Low cut-off -1.68 (Sensitivity 80.0%, negative predictive value 95.5%) and high cut-off +0.953 values (Specificity 97.7%, positive predictive value 88.9%) excluded and diagnosed advanced fibrosis, respectively. In the validation cohort, FAP Index then FIB-4 reduced indeterminate results by one-third compared to FIB-4 alone. Whereas FAP-Index followed by NFS (NAFLD Fibrosis Score) resulted in a reduction of indeterminate results by 70% compared to NFS alone. Conclusion: FAP Index is a novel, rapid, robust, inexpensive diagnostic tool for advanced fibrosis in metabolic fatty liver disease. Applying FAP Index followed by FIB-4 or NFS facilitates accurate risk-stratification of patients by greatly reducing the frequency of indeterminate results compared to FIB-4 or NFS alone, without compromising negative predictive value. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement This project was funded by National Health and Medical Research Council (NHMRC) of Australia, grants APP1053206, APP1105238, APP1107178, APP1108422, APP1196492, APP2001692, Rebecca L Cooper Medical Research Foundation, Centenary Institute Foundation, a Gilead Fellowship, the Robert W. Storr Bequest to the Sydney Medical Foundation of The University of Sydney, Cancer Institute NSW grant 2021/ATRG2028, the Hollywood Private Hospital Research Foundation and the Sir Charles Gairdner Osborne Parke Health Care Group. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: Human Ethics Committee of Sydney Local Health District gave ethical approval for this work. Approval HREC_X18-0241. Human Ethics Committee of Western Sydney Local Health District gave ethical approval for this work. Approval 2019/ETH02319. Human Ethics Committee of Sir Charles Gairdner Hospital, Perth, gave ethical approval for this work. Approval RGS 01287. Human Ethics Committee of The Alfred Hospital, Melbourne, gave ethical approval for this work. Approval 195/15. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes All data produced in the present study are available upon reasonable request to the authors

High fructose diets are associated with an increased risk of liver cancer. Previous studies in mice suggest increased lipogenesis is a key mechanism linking high fructose diets to liver tumour growth. However, these studies administered fructose to mice at supraphysiological levels. The aim of this study was to determine whether liver tumour growth and lipogenesis were altered in mice fed fructose at physiological levels. To test this, we injected male C57BL/6 mice with the liver carcinogen diethylnitrosamine and then fed them diets without fructose or fructose ranging from 10 to 20 % total calories. Results showed mice fed diets with ≥15 % fructose had significantly increased liver tumour numbers (2-4-fold) and total tumour burden (∼7-fold) vs mice fed no-fructose diets. However, fructose-associated tumour burden was not associated with lipogenesis. Conversely, unbiased metabolomic analyses revealed bile acids were elevated in the sera of mice fed a 15 % fructose diet vs mice fed a no-fructose diet. Using a syngeneic ectopic liver tumour model, we show that ursodeoxycholic acid, which decreases systemic bile acids, significantly reduced liver tumour growth in mice fed the 15 % fructose diet but not mice fed a no-fructose diet. These results point to a novel role for systemic bile acids in mediating liver tumour growth associated with a high fructose diet. Overall, our study shows fructose intake at or above normal human consumption (≥15 %) is associated with increased liver tumour numbers and growth and that modulating systemic bile acids inhibits fructose-associated liver tumour growth in mice.

Dipeptidyl peptidase 9 (DPP9) is a multifunctional intracellular protease with roles in tumour growth, inflammation and mitochondrial function. We developed a hepatocyte-specific DPP9 knockout mouse (DPP9-KO) to explore DPP9 in a mouse model of hepatocellular carcinoma (HCC). DPP9-KO mice were generated by crossing Albumin-Cre mice (Wt/Wt Cre+/+) with DPP9 floxed mice (Fl/Fl Cre-/-). Mice were treated with Diethylnitrosamine and Thioacetamide then an atherogenic High Fat Diet until 28 weeks of age. DPP9-KO mice had reduced liver and subcuta-neous adipose tissue mass and lower fasting plasma glucose, fewer small macroscopic liver nod-ules compared to DPP9-WT mice. However, there were no differences in the total number of mac-roscopic liver nodules, tumour burden, inflammation score and steatosis score. Consistent with the known ability of DPP9 to suppress NLRP1 activation, activated caspase-1 protein was ele-vated in DPP9-KO mouse liver. Additionally, Nfkbib, Cxcl10 and Ccl5 mRNA and protein levels of autophagy marker beclin1 and tumour suppressor p53 were increased. In conclusion, DPP9 de-pletion in hepatocytes may reduce liver cancer initiation, via mechanisms that may include in-creased autophagy and innate tumour suppression in this experimental model. Finally, the data supports DPP9 having a role in glucose regulation by the liver.

Supplementary Figures S1-S4 from A Novel Role of Dipeptidyl Peptidase 9 in Epidermal Growth Factor Signaling

Introduction: Ischaemia is inevitable during organ preservation and contributes to graft dysfunction after liver transplantation. Early oxygenation of grafts during organ preservation, such as oxygenated washout (OW), has been shown to reduce ischaemic injury. However, the optimal oxygen concentration for OW is unknown. Hyper-oxygenation can induce reactive oxygen species and graft injury. This study compared non-oxygenated preservation with moderate and high oxygen concentrations during OW in rat livers. Methods: Donation after circulatory death livers were procured from 10 rats. Grafts were randomised to three groups: control (non-oxygenated University of Wisconsin Solution (UW)), moderate concentration OW (Mod-OW) (25-28mg dissolved oxygen/L) and high concentration OW(High-OW) (>50mg dissolved oxygen/L). Livers were flushed and stored at 4℃. After 24 hours of cold storage, livers were placed on isolated liver-reperfusion (ILRP) for 120 minutes. Dissolved oxygen in preservation fluid was measured throughout cold storage. Tissue ATP, as a marker of graft viability, was measured at 0, 6 and 24H cold storage, and after ILRP. Liver biochemistry and graft oxygen consumption were measured during ILRP. Results: The control group had lower dissolved oxygen in the preservation fluid than OW groups at 0H and 6H (all p<0.05) (Figure 1A-B). Tissue ATP was similar between groups at 0H. By 6H, grafts in the Mod-OW group had greater tissue ATP than the High-OW group (15.7 vs 8.4 ug/mg, p = 0.01) (Figure 1C). After 24H cold storage, preservation fluid oxygenation and tissue ATP were similar between all groups. During 120 minutes ILRP, the mean oxygen consumption of the Mod-OW group was greater than the High-OW group (47.2 vs 24.4 uL/min/g-liver, p = 0.048) (Figure 1D). No difference in liver biochemistry was observed (Figure 1E-F). Conclusion: Rat livers receiving Mod-OW had increased tissue viability at 6h and oxygen consumption on ILRP compared to High-OW. Our findings suggest that high oxygen concentrations may lead to reduced graft viability. Although OW may be beneficial for organ preservation, oxygen concentrations should be closely monitored to prevent hyper-oxygenation.

N-terminal sequences are important sites for post-translational modifications that alter protein localization, activity, and stability. Dipeptidyl peptidase 9 (DPP9) is a serine aminopeptidase with the rare ability to cleave off N-terminal dipeptides with imino acid proline in the second position. Here, we identify the tumor-suppressor BRCA2 as a DPP9 substrate and show this interaction to be induced by DNA damage. We present crystallographic structures documenting intracrystalline enzymatic activity of DPP9, with the N-terminal Met1-Pro2 of a BRCA21-40 peptide captured in its active site. Intriguingly, DPP9-depleted cells are hypersensitive to genotoxic agents and are impaired in the repair of DNA double-strand breaks by homologous recombination. Mechanistically, DPP9 targets BRCA2 for degradation and promotes the formation of RAD51 foci, the downstream function of BRCA2. N-terminal truncation mutants of BRCA2 that mimic a DPP9 product phenocopy reduced BRCA2 stability and rescue RAD51 foci formation in DPP9-deficient cells. Taken together, we present DPP9 as a regulator of BRCA2 stability and propose that by fine-tuning the cellular concentrations of BRCA2, DPP9 alters the BRCA2 interactome, providing a possible explanation for DPP9's role in cancer.

Type 2 diabetes is an independent risk factor for non-alcoholic steatohepatitis (NASH) progression and its mediators have not been resolved. In this study, a pathogenic role of cellular communication network factor 2 (CCN2) protein in NASH pathology, was investigated in an established preclinical NASH model. Male wild type C57BL/6 mice received either Chow or high fat diet (HFD) for 26 weeks, with some mice in each group randomly selected to receive low dose streptozotocin (STZ: 3 i.p. injections, 65 mg/kg) at 15 weeks to induce type 2 diabetes. In the final 10 of the 26 weeks mice from each group were administered i.p. either rabbit anti-CCN2 neutralizing antibody (CCN2Ab) or as control normal rabbit IgG, at a dose of 150 µg per mouse twice/week. NASH developed in the HFD plus diabetes (HFD+DM) group. Administration of CCN2Ab significantly downregulated collagen I and collagen III mRNA induction and prevented pro-inflammatory MCP-1 mRNA induction in HFD+DM mice. At the protein level, CCN2Ab significantly attenuated collagen accumulation by PSR stain and collagen I protein induction in HFD+DM. Phosphorylation of the pro-fibrotic ERK signalling pathway in liver in HFD+DM was attenuated by CCN2Ab treatment. Intrahepatic CCN1 mRNA was induced, whereas CCN3 was downregulated at both the mRNA and protein levels in HFD+DM. CCN3 down-regulation was prevented by CCN2Ab treatment. This in vivo study indicates that CCN2 is a molecular target in NASH with high fat diet and diabetes, and that regulation of ERK signalling is implicated in this process.

INTRODUCTION: Dipeptidyl peptidase (DPP)-4 is part of a larger family of proteases referred to as DPPs. DPP4 has been suggested as a possible biomarker for inflammatory bowel disease (IBD). Circulating DPP4 (cDPP4) enzyme activity was investigated as a potential biomarker for IBD. In addition, DPP enzyme activity and gene expression were quantified in colonic tissue of patients with IBD and non-IBD. METHODS: In study 1, DPP enzyme activity was quantified in plasma samples from 220 patients with IBD (Crohn's disease [CD] n = 130 and ulcerative colitis [UC] n = 90) and non-IBD controls (n = 26) using a colorimetric assay. In study 2, tissue and plasma samples were collected from 26 patients with IBD and 20 non-IBD controls. Plasma C-reactive protein (CRP) was quantified in all patients. Colonic DPP4, DPP8, DPP9, and fibroblast activation protein (FAP) gene expression was determined by quantitative polymerase chain reaction. cDPP and cFAP enzyme activity was also measured. Sensitivity and specificity were determined by receiver operating characteristic curve analysis. RESULTS: In study 1, total cDPP activity was found to differentiate patients with CD with active disease (n = 18) from those in remission (n = 19; sensitivity 78% and specificity 63%). In study 2, total cDPP and cFAP activity was 28% and 48% lower in patients with elevated CRP (>10 mg/L), respectively, compared with patients with normal CRP. Gene expression of DPP4, FAP, and DPP8 was also significantly higher in colonic biopsies from patients with IBD compared with non-IBD patients (P < 0.05). DISCUSSION: Our findings implicate the DPP enzyme family in intestinal inflammation and suggest future biomarker applications to differentiate the pathophysiological aspects of IBD.