Gores, Gregory J.

Background and Aims Interleukin‐22 has beneficial effects on inflammation and impaired hepatic regeneration that characterize alcohol‐associated hepatitis (AH). F‐652 is a recombinant fusion protein of human interleukin‐22 and immunoglobulin G2 fragment crystallizable. This study aims to assess the safety and efficacy signals of F‐652 in patients with moderate and severe AH. Approach and Results A phase‐2 dose‐escalating study was carried out. F‐652 (10 μg/kg, 30 μg/kg, or 45 μg/kg) administered on days 1 and 7 was tested in 3 patients each with moderate (Model for End‐Stage Liver Disease [MELD] scores: 11‐20) and severe AH (MELD scores: 21‐28). Safety was defined by absence of serious adverse events and efficacy was assessed by Lille score, changes in MELD score, and serum bilirubin and aminotransferases at days 28 and 42. Three independent propensity‐matched comparator patient cohorts were used. Plasma extracellular vesicles and multiplex serum cytokines were measured to assess inflammation and hepatic regeneration. Eighteen patients (9 moderate and 9 severe AH) were enrolled, 66% were male, and the mean age was 48 years. The half‐life of F‐652 following the first dose was 61‐85 hours. There were no serious adverse events leading to discontinuation. The MELD score and serum aminotransferases decreased significantly at days 28 and 42 from baseline (P < 0.05). Day‐7 Lille score was 0.45 or less in 83% patients as compared with 6%, 12%, and 56% among the comparator cohorts. Extracellular vesicle counts decreased significantly at day 28 (P < 0.013). Cytokine inflammatory markers were down‐regulated, and regeneration markers were up‐regulated at days 28 and 42. Conclusions F‐652 is safe in doses up to 45 μg/kg and associated with a high rate of improvement as determined by Lille and MELD scores, reductions in markers of inflammation and increases in markers of hepatic regeneration. This study supports the need for randomized placebo‐controlled trials to test the efficacy of F‐652 in AH.

INTRODUCTION: Primary sclerosing cholangitis (PSC) is a chronic, fibroinflammatory, cholestatic liver disease of unknown etiopathogenesis which is recognized as a major risk factor for cholangiocarcinoma (CCA). We aimed to investigate the roles of the biliary and intestinal microflora in the inflammatory process of PSC and to determine if the microflora is associated with progression to CCA. METHODS: We studied the bile and stool microbiomes in samples obtained from patients with PSC (31 stool and 35 bile samples), sporadic CCA (11 stool and 26 bile samples), and PSC coexisting with CCA (16 stool and bile 17 samples). Control bile was obtained from patients with cholelithiasis or choledocholithiasis (n = 17). DNA extraction was followed by amplification and high-throughput next generation sequencing (MiSeq) of the 16S rDNA (V3-V5) region to identify the microbiota present. We performed: 1. analysis of the stool microbiome between PSC and CCA patients; 2. analysis of the biliary microbiome in PSC and CCA patients; 3. comparison of the biliary microbiome collected before and after biliary stent placement. RESULTS: Although bile samples from study patients had significantly lower sequencing depths than stool, their sequencing depths were much higher than bile from negative controls. The microbiomes of stool and bile samples from the same patients were significantly correlated ( P = 0.022). CCA bile samples had significant difference of alpha diversity from control bile samples with less Streptococcaceae and Desulfovibrionaceae (FDR < 10%) than PSC whereas no significant change of stool microbiome was identified between CCA and PSC. Fusobacterium and Gemella were correlated to PSC duration. An increased abundance of Fusobacteria after stent placement was revealed. Fusobacteria, which is inflammation-promoting, was also increased with the number of stents in the bile duct. CONCLUSION: Our study suggests that the bile microbiome is potentially a more distinctive biomarker than the stool microbiome for PSC and CCA development. There is increased inhabitance of inflammation-promoting bacteria in the bile of CCA patients and after biliary stent placement. Increased PSC duration also increases the microbial burden in the bile, which may also promote CCA development. We further detected specific bile microbes statistically associated with CCA or stent presence in the bile ducts, which raises the possibility of their role in the CCA progression or stent obstruction. Additional analyses with more samples are needed.

Abstract Background: Cancer of the biliary tract, cholangiocarcinoma (CCA), is increasing in incidence and has limited treatment options. In an attempt to further understand signaling pathways driving oncogenesis and impacting response to therapy we, and others, have identified altered activation of the transcriptional co-activator, Yes- associated protein (YAP) in CCA. Canonical regulatory pathways impacting YAP consist of serine kinases; however, more recently we have demonstrated a central role for tyrosine phosphorylation in regulating YAP function in CCA. Herein we explore the role of tyrosine phosphatases in regulating YAP tyrosine phosphorylation. Methods: In silico analysis of the TCGA cholangiocarcinoma dataset was undertaken specifically evaluating tyrosine phosphatase levels. Molecular studies utilized the human CCA cell lines HuCCT-1 and KMCH as well as the murine CCA cell line SB1 (expressing a flag-tagged YAP). Baseline tyrosine phosphatase levels were assessed by RT-PCR and immunoblot. YAP-interacting phosphatases were identified by co-immunoprecipitation. Tyrosine phosphorylation was inhibited utilizing the multi-kinase inhibitor dasatinib. The pan-tyrosine phosphatase inhibitor sodium orthovanadate (Na3VO4) and the selective SHP1/2 inhibitor NSC87877 were utilized. Tyrosine phosphorylation was assessed by immunoblot. YAP transcriptional activity was evaluated by RT-PCR. Apoptosis was evaluated by caspase 3/7 assay. Results: Multiple tyrosine phosphatases were noted to be expressed at lower levels in tumor versus normal adjacent liver in the TCGA RNAseq dataset. YAP-interacting tyrosine phosphatases identified by co-IP included PTPN1, 11, 23, and PTPRK. Profiling of tyrosine phosphatase levels by RT-PCR and immunoblot demonstrated higher levels in KMCH cells compared to HuCCT-1; notably the YAP-interacting phosphatase PTPN11 (SHP2) was elevated. Consistent with the anticipated function of the phosphatases, immunoblot demonstrated lower levels of tyrosine phosphorylated YAP (p-YAPY357) in KMCH cells compared to HuCCT-1. The role of SHP2 was further probed by incubation of KMCH and HuCCT-1 cells with NSC87877 which was associated with an increase in p-YAPY357 levels and YAP co-transcriptional activity. Conversely, incubation of HuCCT-1 cells with dasatinib rapidly decreased p-YAPY357 levels; and this effect was diminished by pre-incubation with either Na3VO4or NSC87877. The effect on YAP tyrosine phosphorylation paralleled effects on apoptosis; incubation of HuCCT-1 cells with dasatinib lead to an apoptotic response as measured by caspase 3/7 assay, which was eliminated by pre-incubation with Na3VO4 or NSC87877. Conclusions: Inhibition of the tyrosine phosphatase SHP2 is associated with elevated p-YAPY357 levels, elevated YAP co-transcriptional activity, and a blunted therapeutic response to dasatinib in cholangiocarcinoma cell lines. Citation Format: EeeLN Buckarma, Nathan Werneburg, Ayano Niibe, Gregory Gores, Rory Smoot. Tyrosine phosphatase activity is required for response to Src kinase inhibition in cholangiocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3449.

In mouse models of biliary tract diseases, macrophages are recruited to the periductal milieu and promote injury and cholestasis. Although cell necrosis with release of biomolecules termed damage-associated molecular patterns (DAMPs) promotes recruitment and activation of macrophages, necrosis was not observed in these studies. Because extracellular vesicles (EVs) are important in cell-to-cell communication, we postulated that activated cholangiocytes may release EVs containing DAMPs as cargo. Both the human (NHC) and mouse cholangiocyte (603B) cell lines display constitutive activation with mRNA expression of chemokines. Proteomic analysis revealed that EVs from both cell lines contained the DAMP S100A11, a ligand for the receptor for advanced glycation end products (RAGE). Bone marrow-derived macrophages (BMDM) incubated with EVs derived from the mouse 603B cell line increased mRNA expression of proinflammatory cytokines. Genetic or pharmacologic inhibition of RAGE reduced BMDM expression of proinflammatory cytokines treated with EVs. RAGE signaling resulted in activation of the canonical NF-κB pathway, and consistently, proinflammatory cytokine expression was blunted by the IKKα/β inhibitor TPCA-1 in BMDM incubated with EVs. We also demonstrated that primary mouse cholangiocyte-derived organoids express chemokines indicating cholangiocyte activation, release EVs containing S100A11, and stimulate proinflammatory cytokine expression in BMDM by a RAGE-dependent pathway. In conclusion, these observations identify a non-cell death mechanism for cellular release of DAMPs by activated cholangiocytes, namely by releasing DAMPs as EV cargo. These data also suggest RAGE inhibitors may be salutary in macrophage-associated inflammatory diseases of the bile ducts.

Diabetes increases the risk of liver disease progression and cirrhosis development in patients with nonalcoholic steatohepatitis (NASH). The association between diabetes and the risk of hepatocellular carcinoma (HCC) in NASH patients with cirrhosis is not well quantified. All patients with the diagnosis of NASH cirrhosis seen at Mayo Clinic Rochester between January 2006 and December 2015 were identified. All adult liver transplant registrants with NASH between 2004 and 2017 were identified using the United Network for Organ Sharing (UNOS)/Organ Procurement and Transplantation registry for external validation. Cox proportional hazard analysis was performed to investigate the association between diabetes and HCC risk. Among 354 Mayo Clinic patients with NASH cirrhosis, 253 (71%) had diabetes and 145 (41%) were male. Mean age at cirrhosis evaluation was 62. During a median follow‐up of 47 months, 30 patients developed HCC. Diabetes was associated with an increased risk of developing HCC in univariate (hazard ratio [HR] = 3.6; 95% confidence interval [CI] = 1.1‐11.9; P = 0.04) and multivariable analysis (HR = 4.2; 95% CI = 1.2‐14.2; P = 0.02). In addition, age (per decade, HR = 1.8; 95% CI = 1.2‐2.6; P < 0.01) and low serum albumin (HR = 2.1; 95% CI = 1.5‐2.9; P < 0.01) were significantly associated with an increased risk of developing HCC in multivariable analysis. Other metabolic risk factors, including body mass index, hyperlipidemia, and hypertension, were not associated with HCC risk. Among UNOS NASH registrants (N = 6,630), 58% had diabetes. Diabetes was associated with an increased risk of developing HCC in univariate (HR = 1.4; 95% CI = 1.1‐1.8; P < 0.01) and multivariable (HR = 1.3; 95% CI = 1.0‐1.7; P = 0.03) analysis. Conclusion: Diabetes is associated with an increased risk of HCC in patients with NASH cirrhosis.

The Hippo pathway and its effector protein YAP (a transcriptional coactivator) have been identified as important in the biology of both hepatocellular carcinoma and cholangiocarcinoma. First identified as a tumor suppressor pathway in Drosophila , the understanding of the mammalian YAP signaling and its regulation continues to expand. In its “on” function, the canonical regulatory Hippo pathway, a well-described serine/threonine kinase module, regulates YAP function by restricting its subcellular localization to the cytoplasm. In contrast, when the Hippo pathway is “off,” YAP translocates to the nucleus and drives cotranscriptional activity. Given the role of Hippo/YAP signaling in hepatic malignancies, investigators have sought to target these molecules; however, standard approaches have not been successful based on the pathways’ negative regulatory role. More recently, additional regulatory mechanisms, such as tyrosine phosphorylation, of YAP have been described. These represent positive regulatory events that may be targetable. Additionally, several groups have identified potentiating feed-forward signaling for YAP in multiple contexts, suggesting other experimental therapeutic approaches to interrupt these signaling loops. Herein we explore the current data supporting alternative YAP regulatory pathways, review the described feed-forward signaling cascades that are YAP dependent, and explore targeting strategies that have been employed in preclinical models of hepatic malignancies.

355 Background: In this study, we sought to determine the prognostic factors impacting survival of patients with NELM who underwent LT at Mayo Clinic. Methods: We performed a three-site retrospective analysis of patients with unresectable NELM treated with LT from 1990 -2017. Cases were identified from clinical databases at Mayo Clinic. Data on demographics, timing of diagnosis and transplant, extent of liver involvement, and tumor grade, were extracted. Extent of liver involvement was calculated using previously validated standardized volumetry with high defined as > 25% increase in the weight of the extracted liver at the time of the transplant as compared to the standard liver volume. Survival was calculated using the Kaplan–Meier method. Results: We identified 37 patients. Median age at LT was 47 (range 17.0-64.0), male: female (73% vs. 27%, respectively). Primary tumor site was in the pancreas in 51.3% and in the small bowels in 43.2%; 78.4% had NELM at presentation. While primary tumor resection was done at the time of LT in 5 (13.9%) cases, most patients (80.6%) had ˃6-months interval between primary tumor resection and LT surgery. Prior to LT, 25 (74.3%) patients received somatostatin analogues and 18 (61.3%) had liver-directed therapy. Median time from diagnosis to transplant was 33.6 months (0-246). High extent of liver involvement was identified in 12 patients (33.3%). Disease-Free Survival (DFS) at 3, 5, and 10 years was 56.8%, 43.1%, and 22.4% respectively. Overall-survival (OS) at 3, 5, and 10 years was 86.9%, 79.6%, and 52.6% respectively. High extent of liver involvement at the time of transplantation was associated with inferior DFS and OS and patients˃45 years-old at the time of transplantation had worse DFS. Patients whose disease met the OPTN (Organ Procurement and Transplantation Network) criteria for NELM transplantation had significantly better DFS and OS with median 10-year OS of 80.7 % vs. 17%. Conclusions: LT is a reasonable therapeutic option for selected patients with unresectable NELM with 10-year OS of 52.6%. High extent of liver involvement was associated with worse long-term outcomes while meeting OPTN criteria resulted in best outcomes.

Hedgehog (HH) signaling participates in hepatobiliary repair after injury and is activated in patients with cholangiopathies. Cholangiopathies are associated with bile duct (BD) hyperplasia, including expansion of peribiliary glands, the niche for biliary progenitor cells. The inflammation‐associated cytokine interleukin (IL)‐33 is also up‐regulated in cholangiopathies, including cholangiocarcinoma. We hypothesized that HH signaling synergizes with IL‐33 in acute inflammation‐induced BD hyperplasia. We measured extrahepatic BD (EHBD) thickness and cell proliferation with and without an IL‐33 challenge in wild‐type mice, mice overexpressing Sonic HH ( pCMV‐Shh ), and mice with loss of the HH pathway effector glioma‐associated oncogene 1 ( Gli1lacZ/lacZ ). LacZ reporter mice were used to map the expression of HH effector genes in mouse EHBDs. An EHBD organoid (BDO) system was developed to study biliary progenitor cells in vitro . EHBDs from the HH overexpressing pCMV‐Shh mice showed increased epithelial cell proliferation and hyperplasia when challenged with IL‐33. In Gli1lacZ/lacZ mice, we observed a decreased proliferative response to IL‐33 and decreased expression of Il6 . The HH ligands Shh and Indian HH ( Ihh ) were expressed in epithelial cells, whereas the transcriptional effectors Gli1 , Gli2 , and Gli3 and the HH receptor Patched1 ( Ptch1 ) were expressed in stromal cells, as assessed by in situ hybridization and lacZ reporter mice. Although BDO cells lacked canonical HH signaling, they expressed the IL‐33 receptor suppression of tumorigenicity 2. Accordingly, IL‐33 treatment directly induced BDO cell proliferation in a nuclear factor κB‐dependent manner. Conclusion: HH ligand overexpression enhances EHBD epithelial cell proliferation induced by IL‐33. This proproliferative synergism of HH and IL‐33 involves crosstalk between HH ligand‐producing epithelial cells and HH‐responding stromal cells.