nci ccr liver cancer program: special conference on tumor ...anna mae diehl, m.d., duke university...

U.S. Department of Health & Human Services | National Institutes of Health

October 28 – 29, 2019

Building 10, Masur Auditorium

NCI CCR Liver Cancer Program: Special Conference on Tumor Metabolism

Dear colleagues,

Welcome to the inaugural meeting of the National Cancer Institute (NCI) Center for Cancer Research (CCR) Liver Cancer Program (LCP). The NCI-CCR-LCP was launched in 2018 with the mission of developing a multi-disciplinary program focused on the prevention, early detection and improved diagnosis and treatment of liver cancer. The NCI-CCR-LCP will consolidate expertise and create an interactive and collaborative environment to foster liver cancer care and research through innovative laboratory studies and promising clinical applications, such as molecular subgrouping of patients and biomarker-guided molecularly-targeted therapies.

On behalf of our NCI-CCR-LCP Steering Committee, Members and Advisory Board, it is our pleasure to present a meeting program focused on Tumor Metabolism. Distinguished speakers from the United States and across the globe will provide you with the latest updates and future directions in this growing field and related disciplines including:

• Risk Factors• Microbiome and Immune Biology• Genetics and Genomics

The meeting will promote networking during breaks and a poster session as well as foster collaboration and team science among various disciplines of liver cancer research including epidemiology and translational science.

We look forward to sharing these two days of highly stimulating science with you and wish you a wonderful stay in Bethesda as you enjoy the opportunities for advancing liver cancer science and care.

Tim Greten & Xin Wei WangNCI CCR LCP Co-Directors

Anuradha Budhu, Bin Gao, Mitchell Ho & Katherine McGlynnOrganizing Committee

Monday, October 28, 2019

8:00 a.m. – 8:05 a.m. Welcome Remarks: Tim Greten, M.D., NIH, NCI, CCR

8:05 a.m. – 8:10 a.m. Opening Remarks: Doug Lowy, M.D., NIH, NCI

8:10 a.m. – 9:00 a.m. Keynote Lecture: Craig Thompson, M.D. Memorial Sloan Kettering Cancer Center “The role of cancer cell metabolism in shaping the tumor microenvironment”

Session 1 RISK FACTORS AND METABOLISM Co-Chairs: Tim Greten, M.D. & Mitchell Ho, Ph.D.

9:00 a.m. – 9:25 a.m. Katherine McGlynn, Ph.D., M.P.H., NIH, NCI, DCEG “Bacterial translocation and risk of liver cancer in two populations”

9:25 a.m. – 9:50 a.m. Bin Gao, M.D., Ph.D., NIH, NIAAA “Alcohol metabolism, alcoholic liver disease and cancer”

9:50 a.m. – 10:15 a.m. Anna Mae Diehl, M.D., Duke University “Nonalcoholic fatty liver disease and liver cancer”

10:15 a.m. – 10:30 a.m. Bernd Heinrich, M.D., NIH, NCI, CCR “Nonalcoholic fatty liver disease impairs efficacy of T cell-based immunotherapies for liver metastases in mice”

10:30 a.m. – 10:45 a.m. Break

10:45 a.m. – 11:10 a.m. Jake Liang, M.D., NIH, NIDDK “Hepatitis C virus: The bogeyman, a metabolic rubicon and liver cancer”

11:10 a.m. – 11:35 a.m. Anuradha Budhu, Ph.D., NIH, NCI, CCR “Discerning liver tumor subtypes with altered obesity-related metabolism”

11:35 a.m. – 12:00 p.m. Guadalupe Sabio Buzo, Ph.D., Spanish National Center for Cardiovascular Disease “Liver stress kinases—crossroad between obesity and liver cancer”

12:00 p.m. – 1:00 p.m. Lunch (on your own)

1:00 p.m. – 1:10 p.m. Melinda Bachini, Cholangiocarcinoma Foundation

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Session 2 MICROBIOME, IMMUNE BIOLOGY AND METABOLISM Co-Chairs: Frank Gonzalez, Ph.D. & Giorgio Trinchieri, M.D.

1:10 p.m. – 1:35 p.m. Tim Greten, M.D., NIH, NCI, CCR “How does the microbiome and fatty liver disease control anti-tumor immunity?”

1:35 p.m. – 2:00 p.m. Theo Heller, M.D., NIH, NCI, CCR “The missing link? To the portal vein and beyond”

2:00 p.m. – 2:25 p.m. Matam Vijay-Kumar, Ph.D., University of Toledo “Interplay between dietary fiber and gut microbiota in liver cancer: The dark side of fermentation”

2:25 p.m. – 2:40 p.m. Yong He, Ph.D., NIH, NIAAA “MicroRNA-223 in nonalcoholic steatohepatitis and cancer”

2:40 p.m. – 3:40 p.m. Poster Session (FAES Terrace)

3:40 p.m. – 4:05 p.m. Robert Schwabe, M.D., Columbia University “The commensal gut microbiota restrict metastatic tumor growth in the liver”

4:05 p.m. – 4:30 p.m. Grace Guo, MBBS, Ph.D., Rutgers University “Role of FGF15 in promoting liver regeneration”

4:30 p.m. – 4:55 p.m. Dan McVicar, Ph.D., NIH, NCI, CCR “Metabolic niches in cancer and inflammation”

4:55 p.m. – 5:20 p.m. Wanjun Chen, M.D., NIH, NIDCR “Sugar, Th17 cell and mucosal inflammation”

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Tuesday, October 29, 2019

Session 3 GENETICS, GENOMICS AND METABOLISM Co-Chairs: Xin Wang, Ph.D. & Natalie Porat-Shliom, Ph.D.

8:30 a.m. – 8:55 a.m. Lars Zender, Prof. Dr.med, University of Tübingen “Translating liver cancer biology: From functional target discovery to academic drug development”

8:55 a.m. – 9:20 a.m. Wei Guo, Ph.D., University of Pennsylvania “The exosomes in immune suppression and tumor progression”

9:20 a.m. – 9:45 a.m. Satdarshan (Paul) Singh Monga, MD, FAASLD, University of Pittsburgh, School of Medicine “Beta-catenin mutations in hepatocellular cancer: Implications in tumor metabolism and therapy”

9:45 a.m. – 10:00 a.m. Man Hsin Hung, M.D., Ph.D., NIH, NCI, CCR “Tumor methionine metabolism drives T cell dysfunction in hepatocellular carcinoma”

10:00 a.m. – 10:15 a.m. Break

10:15 a.m. – 10:30 a.m. Fuming Li, Ph.D., University of Pennsylvania “FBP1 as a metabolic tumor suppressor in liver cancer”

10:30 a.m. – 10:55 a.m. Jessica Zucman-Rossi, M.D., Ph.D., University Paris Descartes “Natural history of liver tumors revisited by genomics”

10:55 a.m. – 11:20 a.m. Nabeel Bardeesy, Ph.D., Harvard University “Preclinical modeling of targeted therapies for cholangiocarcinoma”

11:20 a.m. – 11:45 a.m. Kevin Brindle, DPhil., University of Cambridge “Magnetic resonance imaging of tumor metabolism”

11:45 a.m. – 12:00 p.m. Closing Remarks: Xin Wang, Ph.D., NIH, NCI, CCR

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NCI CCR Liver Cancer Program: Special Conference

on Tumor Metabolism

Poster Abstracts

Poster # First Author 1 C Alvarez 2 Y Aydin 3 C-W Chang 4 S Chidambaranathn-Reghupaty 5 L Diggs 6 P Farazi 7 B Fleming 8 A Florio 9 J Godfrey 10 Y He 11 B Heinrich 12 M Hung 13 L Kennedy 14 M Khatun 15 W Lai

Poster # First Author 16 JS Lee 17 D Li 18 F Li 19 A Lujambio 20 C Ma 21 L Ma 22 Y Ma 23 R Missiaen 24 M Perez 25 K Sato 26 D Sengupta 27 W Seo 28 S Sinha 29 L Sun 30 A Yu 31 Q Zhang

NCI CCR Liver Cancer Program: Special Conference on Tumor Metabolism October 28-29, 2019

Poster 1

Analysis of Tp53 Gene Mutations Demonstrates that Aflatoxin is a Risk Factor for Hepatocellular Carcinoma in Guatemala

Christian S. Alvarez,1 Jeremy Ortiz,2 Giovanna Bendfeldt Avila,3 Rodrigo E. Hernández Moya,3 Yi Xie,1 Kristie Jones,4 Joaquin Barnoya,5 John Groopman,6,7 Eduardo Gharzouzi,8 Roberto Orozco,9 Katherine A. McGlynn1, Michael Dean1 1Division of Cancer Epidemiology and Genetics, National Cancer Institute, Bethesda, Maryland, USA; 2Instituto de Cancerología/INCAN, Guatemala City, Guatemala; 3Hospital Centro Médico Militar, Guatemala City, Guatemala; 4Cancer Genetics Research Laboratory, Division of Cancer Epidemiology and Genetics, Leidos Biomedical Research Inc., Frederick National Laboratory for Cancer Research, Gaithersburg, MD, USA; 5Unidad de Cirugía Cardiovascular de Guatemala, Guatemala City, Guatemala; 6Department of Environmental Health and Engineering, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, MD, 21205, USA; 7Department of Epidemiology, Bloomberg School of Public Health, Johns Hopkins, University, Baltimore, MD, 21205, USA; 8Integra Cancer Institute, Guatemala City, Guatemala; 9Department of Pathology, Hospital San Juan de Dios, Guatemala City, Guatemala.

Background: Liver cancer, the most common type of which is hepatocellular carcinoma (HCC), is one of the most frequent lethal cancers worldwide with wide sex- and regional- variation. Guatemala has the highest estimated incidence (14.9 per 100,000) and mortality (14.5 per 100,000) rates in the Western Hemisphere. The major risk factors for HCC in Guatemala are not well-characterized as the prevalence of hepatitis B virus (HBV) and hepatitis C virus (HCV) are both low. Aflatoxin B1 (AFB) exposure appears to be common, however, suggesting it could be an important risk factor. As there is a signature AFB1 mutation in codon 249 of the TP53 gene, this study sought to determine the frequency of the codon 249 mutation, as well as other somatic mutations in HCC cases in Guatemala.

Methods: Eighty-three formalin-fixed, paraffin embedded (FFPE) tissues from patients with HCC were obtained from three hospitals in Guatemala City between 2016 and 2017. In addition to the FFPE samples, 40 of the 83 patients also had a fresh frozen HCC biopsy specimen available. Targeted sequencing of TP53 was performed in the 83 FFPE samples using Ion Torent Ampliseq amplicon sequencing technology. Fisher’s exact test was used to examine whether the prevalence of TP53 mutations varied significantly by sex. Using the 40 fresh frozen samples, 245 cancer-related genes were sequenced, including TP53, TERT, CTNNB1, ARID1A, ARID2, AXIN1, NFE2L2, CDKN2A, APC, ATM, FLT3, KRAS, MAP3K1, FGFR3, and others.

Results: Of the 83 FFPE samples, 48 were from men and 35 were from women. The median age at diagnosis was 60 years (Interquartile range (IQR): 52, 68) among men and 62 years (IQR: 42, 69) among women. Among the persons with recorded HBV and HCV status, only 3 were HBV positive and 3 were HCV positive. Overall, 46% of the HCC cases had any TP53 mutation. The mutation prevalence was higher in the tumors from women (57.1%) than in the tumors from men (37.5%), but the difference was not statistically significant. The AFB1 signature TP53 codon 249 mutation was present in 21.7% of the tumors, with no difference in the prevalence among women (22.9%) and men (20.8%) (p=0.99). Among the 40 cases from whom both FFPE and fresh frozen tumor was available, there were four p53 codon 249 mutations present in fresh frozen material but not in FFPE material. The proportion of tumors with any TP53 mutation was 35%. Other common mutations identified were in ARID2 (15%) and APC (13%).

Conclusions: The presence of the TP53 codon 249 mutation indicates that aflatoxin is a risk factor for HCC in Guatemala. The prevalence of HBV was low, suggesting that AFB1 is associated with HCC in the absence of concomitant HBV infection. Further research is warranted to identify other risk factors in Guatemala in order to reduce the high burden of HCC.


Poster 2

Robust Hepatocellular Carcinoma Development in Humanized Liver Mice with Chronic Hepatitis C Virus Infection: A Model to Study Hepatitis C-Induced Liver Cancer

Aydin Y,1 Lin D,1 Song K,1 Lassak A,2 Del Valle L,3 Moroz K,1 Wu T,1 Reiss K,2 Brown TC,4 Guidry JJ,5 Dobek GL,6 Dupepe LM,6 Dash S1 1Department of Pathology and Laboratory Medicine, Tulane University Health Sciences Center, New Orleans, LA, USA; 2Department of Medicine, Louisiana State University Health Sciences Center, New Orleans, LA, USA; 3Department of Pathology, Louisiana State University Health Sciences Center, New Orleans, LA, USA; 4Hayward Genetics Center, Tulane University Medical School, New Orleans, LA, USA; 5Louisiana State University Health Sciences Center Proteomics Core Facility, New Orleans, LA, USA; 6Department of Comparative Medicine, Tulane University School of Medicine, New Orleans, LA, USA.

Background and Hypothesis: Chronic hepatitis C virus (HCV) infection-associated hepatocellular carcinoma (HCC) is the fastest rising type of cancer in the United States. Although most HCC cases develop on the background of cirrhosis, the HALT-C study showed HCC could also develop without cirrhosis. The mechanism of HCV-associated HCC has been challenging to study due to the lack of a small animal model. We hypothesized that high-level HCV replication in mice with humanized liver leads to hepatocellular transformation and HCC development.

Study design and Methods: Five FRG mice (Yecuris) were inoculated with 100 μL - 106 copies/ml HCV JFH-ΔV3-Rluc virus (HCV genotype 2a) through the tail vein. Five FRG mice inoculated with saline served as control. The success of HCV replication in the mice model was confirmed by measuring HCV-RNA levels by real-time RT-PCR. Signs of weight loss and sickness considered as characteristics of HCC development due to HCV infection. Mice with more than % 20-weight reduction were sacrificed. Immunohistochemical staining of FAH, HepPar-1, Human Albumin, HCV Core, BiP, p62, Hsc70, and LAMP2A was performed in liver tissues for verification of humanization, HCV core protein expression and cellular origin of HCC.

Results and Conclusion: High-level human serum albumin was detected in all the mice before and after HCV infection (> 4500 μg/mL) by Elisa indicating efficient human hepatocyte repopulation in all mice. All five mice infected with HCV showed a gradual increase in viral RNA levels up to eight weeks, and the viral titer becomes stable afterward. None of the control mice showed HCV-RNA positive. All HCV infected mice (100%) were become sick and showed weight reduction (>20%) due to HCC development after 16 to 24 weeks. Histological evaluation of H&E sections revealed the presence of multicentric HCC tumor nodules in all infected mice. Most of HCC showed robust expression of FAH, human albumin, and Hep Par 1 suggesting that the tumors were originating from human hepatocytes. IHC staining confirmed HCV core protein expression. Furthermore, the expression of endoplasmic reticulum stress (BIP, HSC70) and autophagy markers (p62) revealed that HCV-induced HCC mechanism involved stress-mediated autophagy switching (from macroautophagy to chaperone-mediated autophagy). In conclusion, our study demonstrated that a high-level of HCV replication in the humanized liver mice model promoted hepatocellular transformation and HCC. This model can be used to study reversible and irreversible oncogenic mechanisms related to HCV infection after the viral cure.

Acknowledgments: This work was supported by NIH grant 1P20GM121288-01.


Poster 3

Global Analysis of Genomic Alterations and Gene Expression of Mitochondrial Ribosomal Proteins in Human Liver Cancer Development and Progression

Alternative title: Reprograming of ribosomal biogenesis may drive hepatocarcinogenesis

Ching-Wen Chang1, Xin Wei Wang1,2 1Laboratory of Human Carcinogenesis, Center for Cancer Research, National Cancer Institute, Bethesda, Maryland, USA; 2Liver Cancer Program, Center for Cancer Research, National Cancer Institute, Bethesda, Maryland, USA.

Background and Hypothesis Hepatocellular carcinoma (HCC) is a heterogeneous and aggressive liver cancer with a poor prognosis. Current evidence suggests that HCC is driven by rare subpopulations of cancer stem cells (CSCs), which contributes to tumor initiation, progression, and therapy resistance. However, mechanisms as to how CSC contributes to hepatocarcinogenesis are vague. Ample evidence indicates a key role of mitochondria in the regulation of cancer stemness as these cellular organelles may serve as central hubs in cell growth and survival. While ribosomes are the protein factories responsible for translating mRNAs into functional proteins, they mainly consist of cytosolic and mitochondrial ribosome complexes, each containing a unique set of ribosomal proteins encoded by nuclear genes. Cytoplasmic ribosomal proteins (CRPs) are responsible for making cytosolic proteins and mitochondrial ribosomal proteins (MRPs) are main players for translating mitochondrial proteins. We hypothesize that somatic deletions of MRPs are key drivers of HCC initiation, progression, and therapy resistance.

Study Design and Methods We use integrated genomics approaches to characterize genomic and transcriptomic profiles of all known MRPs and CRPs in four independent cohorts of HCC patients from Asia, Europe, and North America. We characterized genetic and functional heterogeneity of MRPs linked to HCC prognosis by using single-cell RNA sequencing. Moreover, we investigated genomic and metabolomic profiles to determine the clinical impacts of MRPs deletion and consequently mitochondria-related metabolic alterations associated with HCC progression.

Results and Conclusion We found that heterozygous deletions of a set of core MRPs are common in HCC patients with poor overall survival and disease-free survival. Interestingly, somatic copy number alterations of MRP, but not CRP, impact their transcription. Moreover, we found a dynamic balance between MRP and CRP expression associated with tumor stemness. We also found evidence of heterogeneous expression of MRP genes, but not CRP genes, at the single-cell level, which may be linked to poor clinical outcomes. Furthermore, somatic mutations of TP53 were enriched in HCC patients with inactivation of MRPs. Gene Set Enrichment Analysis revealed that inactivation of MRPs is associated with down-regulation of fatty acid and mitochondrial metabolism while cell proliferation-associated signaling is activated. Metabolomics analysis confirmed that inactivation of MRPs is linked to decreased mitochondrial-associated metabolic pathways and increased glycolytic pathway. Our results are consistent with the hypothesis that inactivation of MRPs by somatic deletions may drive an imbalance of CRP and MRP ribosomal biogenesis, which contributes to HCC stemness and progression.


Poster 4

Interrogating Chromosome 8q Amplification in Hepatocellular Carcinoma (HCC): Potential Oncogenic Function of TATA-box Binding Protein Associated Factor 2 (TAF2)

Saranya Chidambaranathan-Reghupaty1, Mikhail Dozmorov2, Rachel Mendoza1, Devanand Sarkar1,3,4

From the Departments of 1Human and Molecular Genetics and 2Biostatistics, 3Massey Cancer Center, 4VCU Institute of Molecular Medicine (VIMM), Virginia Commonwealth University, Richmond, VA 23298, USA.

BACKGROUND AND HYPOTHESIS: Chromosome 8q amplification is a common event in HCC. This region contains oncogenes like c-Myc and AEG-1/MTDH the co-amplification and functional cooperation of which have been previously demonstrated. The aims of the present study are to identify additional genes that reside in chromosome 8q and are co-amplified with AEG-1 and interrogate their potential oncogenic role.

STUDY DESIGN AND METHODS: Pan-cancer analysis of gene expression data in 38 cancer types in multiple databases was done to identify genes that correlate with AEG-1 expression. Liver cancer samples (n = 371) from TCGA database were analyzed to determine genomic alterations in the identified genes, with focus on TATA-box binding protein associated factor 2 (TAF2). The R2: Genomics Analysis and Visualization Platform was used to determine the correlation between expression levels of AEG-1 and TAF2 genes in two independent datasets. Oncomine database was queried to compare TAF2 copy number differences between HCC samples and normal liver in two independent datasets, TCGA and Guichard, and RNA expression levels in three independent datasets, Roessler Liver 2, Wurmbach Liver, and Chen Liver. A Kaplan-Meier plot based on TAF2 expression levels was generated using TCGA HCC database. R program was used to identify differentially expressed genes between patients expressing high and low TAF2, which were subsequently analyzed using Ingenuity pathway analysis software. Western blotting and immunofluorescence were used to compare TAF2 expression in HCC cell lines. Immunohistochemistry was used to compare TAF2 expression in FFPE tissue sections. shRNA mediated stable TAF2 knockdown clones were generated in HCC cell lines using lipofectamine transfection. MTT and colony formation assays were used to determine changes in proliferation. Wound healing and matrigel invasion assays were used to study differences in migration and invasion, respectively.

RESULTS AND CONCLUSIONS: Analysis of liver cancer samples from TCGA database confirmed that TAF2 was co-expressed with AEG-1 (r = 0.569, p<1E-16). A strong positive correlation between expression levels of AEG-1 and TAF2 genes was observed in two additional independent datasets. TAF2 was amplified in approximately 20% of HCC patients in TCGA dataset. Three independent datasets showed highly significant increase in TAF2 mRNA levels in HCC samples versus normal liver. A significantly strong negative correlation (p = 0.0041) between TAF2 expression levels and HCC patient survival was observed in TCGA database. Canonical pathway analysis by ingenuity software identified significant up-regulation of genes of several oncogenic signaling pathways and marked down-regulation of genes in EIF signaling pathway and oxidative phosphorylation in the TAF2high group. Western blotting, immunofluorescence and immunohistochemistry confirmed that HCC cell lines and HCC liver samples overexpress TAF2. Stable TAF2 knockdown clones showed a significant decrease in their ability to invade and migrate without significantly affecting proliferation. The knockdown of TAF2 causes an increase in E-cadherin level and decrease in Snail expression, a known negative regulator of E-cadherin. In-depth characterization studies are being performed to obtain mechanistic insights into the oncogenic function of TAF2.


Poster 5

Intrahepatic Cholangiocarcinoma: Modulating a Hostile Tumor Microenvironment by Stimulating CD40 Improves Response to Anti-PD-1

Diggs LP1,2, Wang S1, Heinrich B1, Cui L1, Greten TF1 1Thoracic and GI Malignancies Branch, National Cancer Institute, NIH, Bethesda, Maryland; 2Surgical Oncology Program, National Cancer Institute, NIH, Bethesda, Maryland

Background and Hypothesis: Cholangiocarcinoma carries a very poor prognosis in great part due to its minimal response to conventional chemotherapy or targeted immunotherapeutic agents. It is widely accepted that this tumor’s microenvironment is hostile and fosters an immunosuppressive phenotype. Tumor analyses have demonstrated that a considerable proportion of these tumors express PD-1 and PD-L1 and yet the response to immune checkpoint inhibitors has been disappointing. This resistance likely stems from tumor signals that inhibit effector cells. We hypothesize that modulating the tumor microenvironment by stimulating antigen presenting cells (APCs) can improve response to immune checkpoint inhibitors. Specifically, stimulating CD40, a known receptor on all APCs, would likely result in increased cytotoxicity of effector cells when combined with anti-PD-1 immune checkpoint blockade.

Study Design and Methods: To test our hypothesis, we treated C57BL/6 mice with a CD40 agonistic antibody combined with anti-PD-1 antibody. Three different mouse models were used including subcutaneous flank injections and orthotopic liver injections of SB1, an established cholangiocarcinoma cell line. The third model consisted of hydrodynamic tail vein injection of the YAP and AKT plasmids. Mice were then treated with a CD40 agonist (2.5ug/g) weekly and anti-PD-1 (10ug/g) antibody on post-inoculation days 5, 8, and 14. Tumors were measured, weighed and evaluated histologically to evaluate for difference between treated and untreated mice. All tumors were histologically proven to be cholangiocarcinoma. To confirm the histology (CK7 +, CK19+, and HNF4α-) and characterize the tumor microenvironment, we performed immunohistochemical staining of lymphocytes (CD4 and CD8 T cells), myeloid cells (macrophages, MDSCs, dendritic cells), as well as PD-1 and CD-40 expression. Flow cytometric analysis of lymphoid, myeloid, and T cell subpopulation was then performed. Finally, qPCR and cytokine assays were performed to compare the cytokine profile of tumor and peritumoral tissue in treated and untreated mice.

Results and Conclusion: In both flank and intrahepatic models, mice treated with PD-1 alone or CD-40 alone did not demonstrate significantly (p<0.05) smaller tumors. However, when these agents were combined, significantly smaller tumors were noted. Preliminary immunohistochemistry confirmed cholangiocarcinoma with increased areas of necrosis and calcification in the treated as well as increased PD-1 staining in the liver tumor tissue of treated mice. Flow cytometry demonstrated increased PD-1 expression on CD8+ cytotoxic T cells in treated mice. qPCR revealed increased presence of IFNγ on the tumors of mice treated with the drug combination. In conclusion, stimulating CD40 in cholangiocarcinoma significantly enhances response to checkpoint blockade. This process appears to be mediated by CD8+ T cells. This regimen represents a potential alternative to current cytotoxic chemotherapy regimens.


Poster 6

Changes in Fatty Acid Metabolism are Associated with Progression of Lean Nonalcoholic Steatohepatitis-Induced Hepatocellular Carcinoma in the Mouse

Vlock E1,2 and Farazi P1,2 1Department of Epidemiology, College of Public Health, University of Nebraska Medical Center, Omaha, NE; 2Nebraska Center for the Prevention of Obesity Diseases through Dietary Molecules, College of Education and Human Sciences, University of Nebraska-Lincoln, Lincoln, NE.

Background and Hypothesis: Lean NAFLD shows a prevalence of 7% in the US and up to 30% in some Asian countries. The most common etiologies of lean NAFLD are a diet high in fructose/fat/cholesterol and genetic predisposition that affects lipid export from the liver. The first phenotypic alterations of this disease involve the accumulation of fat in the liver that can progress to non-alcoholic steatohepatitis (NASH) and ultimately hepatocellular carcinoma (HCC). Little is known about the molecular pathogenesis of lean NASH-HCC. Polyunsaturated fatty acids (PUFAs) were shown to exhibit lower levels in HCC of other etiologies and suppress liver tumorigenesis in vivo and in vitro. However, the potential impact of PUFAs in lean NASH-HCC has not been investigated. We hypothesize that PUFAs have a tumor suppressive role in lean NASH HCC and consequently they would be present at lower levels in plasma and lean NASH-HCC tissue.

Study Design and Methods: Lean NASH-HCC was induced in mice fed a choline deficient, high trans-fat, sucrose, cholesterol (CD-HFSC) diet starting at 4 weeks of age for 51-52 weeks. Control mice were fed an isocaloric low fat diet for the same duration. Liver tissue was analyzed post mortem. The presence of NAFL, NASH, and HCC was assessed in tissue sections stained with hematoxylin and eosin, trichrome, and reticulin. Plasma was obtained from 5 mice with NAFL fed the low-fat diet, 5 mice with pre-malignant lesions (regenerative and dysplastic nodules) fed the CD-HFSC diet, and 5 mice with NASH-HCC fed the CD-HFSC diet at 32 and 55-56 weeks of age. In addition, HCC tissue was taken from the same mice at the endpoint (55-56 weeks of age). Metabolomics for 30 fatty acids (C14-C26) was conducted on these plasma and HCC tissue samples.

Results and Conclusion: Seven fatty acids (5 PUFAs: ω3 and ω6 linolenic α and γ acids, ω3 eicosapentanoic acid, ω3 docosahexanoic acid, and ω6 linoleic acid; 1 saturated fatty acid: arachidic acid, and 1 monounsaturated fatty acid: palmitoleic acid) showed significantly lower plasma levels in mice fed the CD-HFSC diet with pre-malignant lesions and HCC compared to mice with NAFL fed the control diet at both 32 and 55-56 weeks (one-way ANOVA test; P<0.05). The levels of these fatty acids were also low in HCC tissue at 55-56 weeks. The 5 PUFAs showed changes in their levels over time, i.e. they exhibited significantly increased levels at 55-56 weeks compared to 32 weeks in mice with pre-malignant lesions. In conclusion, the levels of certain PUFAs (ω3 and ω6 linolenic α and γ acids, ω3 eicosapentanoic acid, ω3 docosahexanoic acid, and ω6 linoleic acid) decrease with lean NASH-HCC progression and increase over time in mice that develop only pre-malignant lesions. These results suggest that these PUFAs may have tumor suppressive and chemopreventive properties in lean NASH-HCC that should be explored further.


Poster 7

The Engineered Anti-Gpc3 Immunotoxin, HN3-ABD-T20, has Prolonged Serum Retention and Produces Regression in Mouse Liver Cancer Xenograft Models

Fleming BD1, Urban DJ2, Hall M2, Longerich T3, Greten T4, Pastan I1, Ho M1# 1Laboratory of Molecular Biology, National Cancer Institute, Bethesda, Maryland, 20892; 2Chemical Genomics Center, National Center for Advancing Translational Sciences, Rockville, Maryland, 20850; 3Institute of Pathology, University Hospital, Heidelberg, Germany, 69120; 4Thoracic and Gastrointestinal Oncology Branch, National Cancer Institute, Bethesda, Maryland, 20892. #Corresponding Author: Mitchell Ho, National Cancer Institute, Bldg. 37, Room 5002C, Bethesda, MD. 20892, phone: 240-760-7848, fax: 240-541-4501, email: [email protected]

Background and Hypothesis: Treatment of glypican-3 (GPC3) expressing hepatocellular carcinomas (HCC) with HN3-based immunotoxins has been shown to cause potent tumor regression by blocking protein synthesis and by down-regulating the Wnt signaling pathway. However, the immunogenicity and short serum half-life may limit their transition to the clinic.

Study Design and Methods: To address these concerns, we constructed HN3-based immunotoxins with various deimmunized Pseudomonas exotoxin domains. These included HN3-T20 which contains a domain II truncation and mutations in domain III to remove T-cell epitopes. We compared them to the previously reported B cell deimmunized HN3 immunotoxin and the original HN3-PE38 with wild-type Pseudomonas exotoxin.

Results and Conclusion: All of our HN3-based immunotoxins had high affinity, with HN3-T20 having a KD value of 7.4 nM. HN3-T20 retained 73% enzymatic activity when compared to the wild-type immunotoxin in ADP-ribosylation assay. Interestingly, a real-time cell growth inhibition assay demonstrated that a single dose of HN3-T20 at 62.5 ng/ml (1.6 nM) was capable of inhibiting nearly all cell proliferation during the 10-day experiment. To enhance HN3-T20’s serum retention, we tested the effect of adding a streptococcal domain (ABD) and a llama heavy chain antibody domain (ALB1). We developed a highly sensitive ELISA for the detection of Pseudomonas exotoxin in mouse serum and found that HN3-ABD-T20 had a 45-fold higher serum half-life than HN3-T20 (326 min vs 7.3 minutes); consequently, addition of an ABD resulted in HN3-ABD-T20 mediated tumor regression at 1 mg/kg. These data suggest that the deimmunized HN3-ABD-T20 immunotoxin is a high potency therapeutic that can be beneficial to HCC patients.


Poster 8

Global Incidence and Trends in Intrahepatic and Extrahepatic Cholangiocarcinoma between 1993 and 2012

Florio AA1, Ferlay J2, Znaor A2, Ruggieri D3, Alvarez CS1, Laversanne M2, Bray F2, McGlynn KA1, and Petrick JL1 1Division of Cancer Epidemiology and Genetics, National Cancer Institute, Bethesda, MD, USA; 2Cancer Surveillance Section, International Agency for Research on Cancer, Lyon, France; 3Information Management Services, Inc., Rockville, MD, USA

Background: Intrahepatic and extrahepatic cholangiocarcinomas (ICCs and ECCs) are highly fatal tumors that originate in the bile ducts. ICCs comprise 10-12% of primary liver tumors and ECCs comprise a third of biliary tract tumors. Common risk factors include primary sclerosing cholangitis, Caroli’s disease, liver flukes, and metabolic conditions such as obesity and diabetes. Incidence can be difficult to estimate due to changes in cancer registry coding over time.

Recent studies have reported that ICC incidence and mortality rates have increased and that ECC incidence and mortality rates have either decreased or plateaued worldwide. However, prior reports have lacked histology, been limited to single-country incidence, or have been based on mortality data. No studies to date have examined global incidence and trends using high-quality topography- and histology-specific cancer registry data. Therefore, we examined ICC and ECC incidence using data from the International Agency for Research on Cancer’s Cancer Incidence in Five Continents plus (CI5plus) database.

Study Design and Methods: Countries were eligible for inclusion if they were included in CI5plus between 1993 and 2012, provided histology data, and, for ECC analyses, gave authorization for data use, as histology codes are not publicly available. In total, 38 countries contributed ICC data and 33 contributed ECC data. Regional and national cancer registry data were pooled to estimate age-standardized incidence rates per 100,000 person-years (ASR), 95% confidence intervals, and average annual percent changes (AAPC). ICC and ECC trends were tabulated and plotted by country and region. Age-period-cohort models were fit and plotted for selected countries.

Results and Conclusion: ICC incidence was greater than ECC incidence in the majority of countries. The highest rates remained in Asian countries, specifically South Korea (ICC ASR=2.80, ECC ASR=2.24), Thailand (ICC ASR=2.19, ECC ASR=0.71), and Japan (ICC ASR=0.95, ECC ASR=0.83). Results were similar by sex. Males had higher rates of ICC and ECC than females in almost all countries examined. Incidence rates of both ICC and ECC increased between 1993 and 2012 in most countries. Large, significant increases in incidence occurred in Latvia (AAPC=20.1%) and Ireland (AAPC=10.5%) for ICC and in Colombia (AAPC=8.5%) and Ireland (AAPC=6.4%) for ECC. Though there was evidence of decreasing or plateauing incidence in several countries, the only significant decrease between 1993 and 2012 was in Costa Rica for ECC (AAPC=-6.2%).

In the 20 years examined, incidence of ICC and ECC increased in the majority of countries worldwide. The current findings are supported by several prior studies that reported increases in ICC and ECC incidence in the United States. ICC and ECC incidence may continue to increase due to metabolic and infectious etiologic factors. Efforts to further elucidate risk factors contributing to the increase in incidence are warranted.


Poster 9

Restoration of Gluconeogenesis in Liver Cancer

Godfrey J, Skuli N, Riscal R, Li F, and Simon MC

Department of Cell Development and Biology, University of Pennsylvania, Philadelphia, Pennsylvania, USA

Background and Hypothesis: Mortality rates for hepatocellular carcinoma (HCC) have steadily increased by approximately 3% per year over the past two decades. Unfortunately, due to late diagnosis and pathological complexity, treatment options are limited and lack efficacy, especially in advanced disease. Druggable mutations, such as those found in multiple growth signaling kinases, are uncommon in HCC, further restricting options for targeted therapies. However, despite genetic heterogeneity, many of these molecular alterations converge on critical biological pathways, particularly cellular metabolism. Aberrant glucose metabolism via elevated glycolysis is an established hallmark of many cancers, including HCC. This phenomenon is especially curious in HCC because a major function of the normal liver is to generate systemic glucose through the process of “gluconeogenesis.” Therefore, the relationship between gluconeogenesis and glycolysis is an attractive area of research in HCC.

All major gluconeogenic enzymes are downregulated in HCC, suggesting a defective upstream component of the pathway. The gluconeogenic pathway is physiologically stimulated in hepatocytes upon glucagon binding to its target receptor, G-coupled glucagon receptor (GCGR). Following glucagon binding, a conformational change occurs in GCGR to transduce signaling through the second messenger, cyclic AMP (cAMP), which induces transcription of gluconeogenic enzymes. We hypothesize that diminished glucagon-GCGR signaling is partly responsible for the decrease in gluconeogenic gene expression in HCC. Functionally, we hypothesize that re-expression of GCGR will diminish HCC cell proliferation when treated with glucagon through direct antagonism of glycolysis.

Study Design and Methods: We accessed the expression of GCGR in human liver cancer patients by TCGA mRNA data, followed by qPCR validation in HCC cell lines. Using multiple HCC cell lines, we overexpressed GCGR by lentiviral infection and confirmed expression by qPCR and western blot. Cell viability was determined over time by quantifying relative ATP levels using the Cell Titer Glo assay. We plan to validate GCGR activation with glucagon treatment by measuring cAMP, glucose production, and glucose/pyruvate tracing experiments. We will determine the mechanism by using shRNA to knockdown various genes in the pathway and accessing rescue by cell proliferation assays. Finally, we will determine in vivo reproducibility by performing xenograft experiments on HCC cell lines overexpressing GCGR and treating the mice with daily injections of glucagon.

Results and Conclusion: Preliminary data suggest that GCGR is significantly downregulated in both patient HCC tumors and cell lines. Furthermore, HCC tumors expressing higher GCGR levels correlate with more favorable patient overall survival. The HCC cell line, SNU398, overexpressing GCGR displays reduced growth rate following glucagon, but not vehicle, exposure. We predict that future experiments will show that GCGR activation by glucagon decreases glycolytic flux and consequently reduces ATP levels. Moreover, we predict that mice treated with glucagon will display smaller tumors in xenograft experiments.

Overall, the impact of this project could lead towards nutritional-based therapies that promote glucagon signaling and cooperate with chemotherapy to slow HCC tumor progression.


Poster 10

Microrna-223 Ameliorates Nonalcoholic Steatohepatitis and Cancer by Targeting Multiple Inflammatory and Oncogenic Genes in Hepatocytes

Yong He1, Seonghwan Hwang1, Yan Cai1, Seung-Jin Kim1, Mingjiang Xu1, Dingcheng Yang1, Adrien Guillot1, Dechun Feng1, Wonhyo Seo1, Xin Hou1, Bin Gao1 1Laboratory of Liver Diseases, National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health, Bethesda, MD, 20892, USA

Background and Hypothesis: Nonalcoholic fatty liver disease (NAFLD) represents a spectrum of disease ranging from simple steatosis to more severe forms of liver injury including nonalcoholic steatohepatitis (NASH), fibrosis, and hepatocellular carcinoma (HCC). In humans, only 20-40% of patients with fatty liver progress to NASH, and mice fed a high-fed diet (HFD) develop fatty liver but are resistant to NASH development. Neutrophil specific-microRNA-223 (miR-223) was previously reported to act as a fine-tuner of the generation and function of neutrophils and played a crucial role in alcoholic liver disease and drug-induced liver injury. However, the underlying pathogenesis and role of anti-inflammatory miR-223 in modulating NASH and HCC development remain unknown.

Study Design and Methods: Wild-type (WT) mice and miR-223 knockout (miR-223KO) mice were fed with HFD for 3 months. To address whether miR-223 was involved in long-term HFD induced liver cancer development, WT mice and miR-223KO were fed with HFD for one year. Liver tissues were collected and subjected to RT-qPCR and immunochemistry staining.

Results and Conclusion: MiR-223 was highly elevated in hepatocytes in HFD-fed mice and in human NASH samples. Compared with WT mice, miR-223 knockout (miR-223KO) mice showed greater steatosis, liver inflammatory response, liver neutrophil infiltration, liver injury and fibrosis after HFD feeding for 3 months. Ingenuity pathway analysis (IPA) of top disorders and hepatotoxicity revealed that genes involved in cancer and liver hyperplasia/hyperproliferation were highly represented within these networks by microarray analysis to examine hepatic gene expression in WT and miR-223KO mice after HFD feeding for 3 months. Next, after HFD feeding for one year, miR-223KO mice had higher degree of liver fibrosis and tumor incidence than WT mice. Furthermore, microarray analyses revealed that compared to WT mice, HFD-fed miR-223KO mice had greater hepatic expression of many inflammatory genes and cancer-related genes, including (C-X-C motif) chemokine 10 (Cxcl10) and transcriptional co-activator with PDZ-binding motif (Taz), two well-known factors to promote NASH development. In vitro experiments demonstrated that Cxcl10 and Taz are two downstream targets of miR-223 and overexpression of miR-223 reduced their expression in cultured hepatocytes. Hepatic levels of miR-223, CXCL10 and TAZ mRNA were elevated in human NASH samples, which positively correlated with hepatic levels of several miR-223 targeted genes as well as several pro-inflammatory, cancer-related and fibrogenic genes. Conclusion, HFD-fed miR-223KO mice develop a full spectrum of NAFLD, representing a clinically relevant mouse NAFLD model. MiR-223 plays a key role in controlling steatosis-to-NASH progression by inhibiting hepatic Cxcl10 and Taz expression. MiR-223 may be a novel therapeutic target for the treatment of NASH and liver cancer.


Poster 11

Nonalcoholic Fatty Liver Disease Impairs Efficacy of T Cell-Based Immunotherapies for Liver Metastases

Heinrich B1, Brown ZJ1,2, Diggs LP1,3, Vormehr M4,5, Ma C1, Subramanyam V1, Rosato U1, Zheng Q1, Lai CW1,6, Ruf B1, Walz JS1, Korangy F1, Sahin U4,5,7, Greten TF1,8 1Thoracic and GI Malignancies Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA; 2Rutgers Robert Wood Johnson Medical School, Department of Surgery, New Brunswick, NJ; 3Surgical Oncology Branch, National Institutes of Health, Bethesda, MD 20892, USA; 4Biopharmaceutical New Technologies (BioNTech) Corporation, An der Goldgrube 12, 55131 Mainz, Germany; 5University Medical Center of the Johannes Gutenberg University, Langenbeckstraße 1, 55131 Mainz, Germany; 6Liver Diseases Branch, National Institute of Diabetes and Digestive and Kidney Disease, National Institutes of Health, Bethesda, MD 20892, USA; 7TRON – Translational Oncology at the University Medical Center of Johannes Gutenberg University gGmbH, Freiligrathstraße 12, 55131 Mainz, Germany; 8NCI CCR Liver Cancer Program.

Background and Hypothesis The prevalence of non-alcoholic fatty liver disease (NAFLD) is increasing worldwide and current estimates suggest its incidence to be 20%-30% in Western countries and 5%-18% in Asia. Tumor immunotherapy has shown promising efficacy for many types of cancer including melanoma, lung cancer and HCC, but liver metastases were associated with shortened progression-free survival. Although more cancer patients will have NAFLD, the effect of NAFLD on response to immunotherapy has yet to be highlighted. Our group has previously reported that NAFLD causes a selective loss of hepatic CD4+ T cells which impairs antitumor surveillance and promotes HCC. However, it is not known whether NAFLD will also impair T cell responses in a therapeutic immunotherapy setting.

Study Design and Methods B16-M30 RNA vaccine (M30) has been proven to cause a strong CD4+ T cell-dependent antitumor immunity. In this study, we applied M30-vaccination in a mouse model of intrahepatic B16 melanoma tumors and investigated the effect of NAFLD on therapeutic efficacy. Besides this individualized approach, a popular target to boost T cell anti-tumor responses is the activation of OX40 (CD134) receptors. We explored whether the anti-tumor effect of agonistic antibodies against OX40 (aOX40) is impaired in a model of colorectal cancer metastases with underlying NAFLD. Liver tumors were established by intrahepatic injection of B16-F10 melanoma or CT26 colorectal cancer cells. Tumor-bearing mice were treated with M30 or non-immunogenic RNA, or aOX40 or IgG control, respectively.

Results and Conclusion M30-treated mice showed low and stable tumor growth while tumor growth of control RNA-treated mice increased. Similarly, mice fed regular diet and treated with aOX40 showed a lower tumor burden. The efficacy of M30 against liver tumors was tested in NAFLD mice fed MCD diet. Tumor growth kinetics showed that M30 effectively controlled liver tumor growth in mice fed regular diet. In contrast, an increase in tumor growth was measured in NAFLD mice which received M30. Under NAFLD condition, also aOX40 barely showed any effect on tumor growth in the liver. Our results demonstrate that two independent T cell-based immunotherapies effectively control tumor growth in the normal liver but fail to control and reduce tumor burden in NAFLD mice. This was specific to the liver, since treatment of subcutaneous tumors was not affected by NAFLD conditions.


Therapeutic efficacy of both immunotherapy strategies could be restored in NAFLD livers by adding the ROS-inhibitor N-acetylcysteine (NAC) to the drinking water. Mice fed regular diet and treated with M30 showed an increase in CD4+ T cells but decreased regulatory T cells (Treg) and MDSCs. M30 failed to increase CD4+ T cells in mice with NAFLD. This effect could only be seen in the liver whereas spleen CD4+ T cells were not different between groups. NAC can restore efficacy of M30 vaccine through rescue of CD4+ T cells. This is the first study showing that metabolic liver disease can result in immunotherapy failure due to diet induced effects on the tumor immune environment.


Poster 12

Tumor Methionine Metabolism Drives T Cell Dysfunction in Hepatocellular Carcinoma

Hung MH1,2,3, Lee JS4, Franck S1, Ma L1, Chang CW1, Ruchirawat M5, Ruppin E4, Wang XW1,6 1Laboratory of Human Carcinogenesis, Center for Cancer Research, National Cancer Institute, Bethesda, Maryland, USA; 2School of Medicine, National Yang-Ming University, Taipei, Taiwan; 3Division of Medical Oncology, Department of Oncology, Taipei Veterans General Hospital, Taipei, Taiwan; 4Cancer Data Science Lab, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, USA; 5Laboratory of Chemical Carcinogenesis, Chulabhorn Research Institute, Bangkok, Thailand; 6Liver Cancer Program, Center for Cancer Research, National Cancer Institute, Bethesda, Maryland, USA

Background and Hypothesis Exhausted T cells (ETC) refers to a hypofunctional state of T lymphocyte that are often seen in cancer. Chronic T-cell receptor stimulation and recruitment of immune suppressive cells from the tumor microenvironment have been implicated as the mechanisms driving ETC. However, the potential role of tumor in promoting the establishment and progression of ETC remains elusive. Tumor metabolism is a core cancer hallmark. While metabolic alterations of cancer cells are required to support the anabolic requirement of cellular growth and proliferation, they may also affect the composition and function of the non-cancer cells residing in the same microenvironment. Bioinformatically searching the interaction between ETC and tumor metabolism may facilitate the identification of tumor drivers and novel mechanisms attributing ETC in hepatocellular carcinoma (HCC).

Study Design and Methods HCC-specific ETC genes were identified from single cell RNA sequencing of HCC-infiltrating lymphocytes, and global transcriptome of tumor and non-tumor tissues from 735 patients derived from five ancestry-independent HCC cohorts were used to link ETC to HCC prognosis. Correlation of ETC and tumor metabolome was carried out in tumor samples analyzed by both transcriptomics and metabolomics consisting of 718 untargeted metabolites. The clinical impacts of ETC and ETC-associated metabolic alterations were investigated. Potential roles of tumor metabolites on ETC were assessed using an in vitro assay with human CD8+ T cells isolated from health donors.

Results and Conclusion By exploring the single-cell transcriptome profiles of 5,063 HCC-derived lymphocytes, we identified 82 ETC-specific genes and developed a robust exhaustion score with feature selection. We found that high exhaustion score was universally linked to worse patient survival. While the exhaustion score is negatively correlated with the activity of effector T cell in HCC patients with chronic inflammatory liver diseases, such relationship was not observed in non-cancerous patients with acute inflammatory conditions. A correlation analysis between transcriptome and metabolome revealed that two methionine-related metabolites, i.e., 5-methylthioadenosine (MTA) and S-adenosylmethionine (SAM), were the most significantly enriched tumor metabolites linked to ETC. MTA and SAM belong to the salvage recycling pathway of methionine. We found that a preferential activation of salvage pathway to the other methionine recycling mechanism, the de novo pathway, is significantly associated with accumulation of SAM and MTA, and at the phenotypic level, with ETC and poor patient’s prognosis. In vitro experiments revealed that both SAM and MTA inhibit the proliferation potential of CD8+ T cells and attenuate CD2/3/28-mediated T cell activation. These results suggest that reprograming of tumoral methionine metabolism may lead to an accumulation of MTA/SAM that drives ETC in HCC.


Poster 13

Antihistamine Treatment or Depletion of the HDC/Histamine Axis Inhibits MC Migration and Subsequent Tumor Formation, Angiogenesis and EMT

Lindsey Kennedy, Vik Meadows, Gianfranco Alpini, Fanyin Meng, Heather Francis

Division of Gastroenterology and Hepatology, Indiana University, Bloomington, Indiana

Background: Primary sclerosing cholangitis (PSC) is characterized by biliary damage, fibrosis and angiogenesis, and can progress to cholangiocarcinoma (CCA). The multidrug resistance-2 gene knockout (Mdr2-/-) mouse model has increased mast cell (MC) migration and histamine (HA) signaling, and mimics PSC and develops hepatocellular carcinoma (HCC) within 1 year. We have found that depletion of the l-histidine decarboxylase (HDC)/HA axis using 12 wk old HDC/Mdr2 double knockout (DKO) mice reduced MC migration, hepatic damage, biliary proliferation and liver fibrosis; furthermore, treatment with H1 or H2 HA receptor (HR) antagonists decreases these parameters in Mdr2-/- mice. We aimed to understand the impact of HDC/HA axis and H1/H2 HR signaling on the progression of CCA in DKO mice and xenograft models. Methods: We utilized 52 wk old DKO and age-matched Mdr2-/- mice. We evaluated tumor formation by H&E staining. In total liver we assessed: (i) HDC/HR expression by qPCR, (ii) MC presence by mouse MC protease-1 staining, and (iii) ductular reaction by CK-19. We measured liver fibrosis by Sirius Red staining and quantification. Angiogenesis was visualized by staining for CD31 and vWF. Epithelial-mesenchymal transition (EMT) was determined by staining for vimentin and E-cadherin. In a separate set of studies, nu/nu mice were implanted with Mz-Cha-1 CCA cells into their hind flanks. After tumor development, mice were treated with saline, mepyramine (H1HR antagonist, 10 mg/kg/BW) or ranitidine (H2HR antagonist, 10 mg/kg/BW) by IP injection 3x/week for 35 days. Tumor growth was measured 3x/week and tumors were evaluated for proliferation by qPCR for PCNA. MC presence, HDC/HR expression, angiogenesis and EMT were measured with similar parameters described above. In human CCA we evaluated the expression of the HDC/HA/HR axis along with MC presence as described above. Results: Depletion of the HDC/HA axis in DKO mice resulted in little to no tumor formation compared to age-matched Mdr2-/- mice. The HDC/HA/HR axis and MC activation were reduced in DKO mice compared to controls. Additionally, DKO mice had a reduction in ductular reaction, liver fibrosis, angiogenesis and EMT. Similar to our DKO studies, treatment with H1 or H2 HR antagonists decreased HDC/HA/HR signaling and MC migration and activation in nu/nu mice. Furthermore, H1 and H2 HR antagonism reduced tumor growth, angiogenesis and EMT. We found an upregulation of MC presence and the HDC/HA/HR axis in human CCA compared to non-malignant livers. Conclusion: Depletion of the HDC/HA axis blunts tumor growth and progression in Mdr2-/- mice. In xenograft models, inhibition of H1HR or H2HR decreases CCA growth and progression. Combined, our data demonstrate that the HDC/HA/HR axis is critical to CCA progression, and drugs targeting this axis may act as potential therapeutics.


Poster 14

Exogenous Expression of miR-30e Inhibits Autophagy and Fatty Acid Synthesis in Hepatocellular Carcinoma Cells

Khatun M, Sasaki R, Sur S, and Ray RB

Department of Pathology, and Liver Center, Saint Louis University, Missouri, USA

Background and Hypothesis: Hepatocellular carcinoma (HCC), a common form of liver cancer, is among the deadliest cancers worldwide. The overall incidence of liver cancer and mortality are increasing frighteningly. The main risk factors for HCC include, infection with HBV and HCV, obesity, excessive alcohol consumption and metabolic diseases. We have previously observed a reduced expression of microRNA-30e (miR-30e) in HCC patient sera. In silico prediction suggested that autophagy related gene 5 (ATG5) is a direct target of miR-30e. ATG5 is involved in autophagy biogenesis; and HBV and HCV infection in hepatocytes induces autophagy. Further, autophagy has been implicated in de novo lipogenesis. In this study, we investigated whether miR-30e inhibition alters autophagy and lipid metabolism in hepatocellular carcinoma (Huh7.5) cells and in liver specimens from HCC patients.

Study Design and Methods: We investigated the status of miR-30e-ATG5 axis in Huh7.5 cells. For this, we immunoprecipitated of miR-30e RISC complex to examine the association of ATG5 and examined by qRT-PCR. Ectopic expression of miR-30e in Huh7.5 cells was examined by qRT-PCR and Western blot analysis. Downstream signaling pathways of ATG5 were analyzed by Western blot. Inhibition of autophagy was examined by confocal microscopy. Work is in progress for correlation of expression levels of miR-30e and ATG5 in liver tissues from HCC patients.

Results and Conclusion: We observed the presence of ATG5 in miR-30e-AGO2 RISC complex. Exogenous expression of miR-30e reduced ATG5 expression in Huh7.5 cells. Overexpression of miR-30e also inhibited autophagy in HCV infected hepatocytes. We further observed that miR-30e expression is downregulated in HCV infected hepatocytes. Subsequent studies suggested that ATG5 knockdown in Huh7.5 cells resulted in the remarkable inhibition of SREBP-1c and FASN levels. Further, overexpression of miR-30e decreased lipid synthesis related proteins, SREBP-1c and FASN, in hepatocytes. The status of miR-30e and ATG5 in liver specimens from HCC patients should further clarify these observations. Together our results provide a new mechanistic insight into the interactions between autophagy and lipid synthesis via inhibition of miR-30e.


Poster 15

Where Did the NKT Go? A Characterization of Tumor-Infiltrating NK-Like T Cells in Murine Liver Tumor Models

Chunwei Walter Lai1,2, Laurence Diggs2,3, Chi Ma2, Zack Brown2,3,4, Bernd Heinrich2, Qianfei Zhang2, Varun Subramanyam2, Umberto Rosato2, Benjamin Ruf2, Linda Cui2, Simon Wabitsch2, Firouzeh Korangy2 and Tim F. Greten2 1Liver Disease Branch, National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, Maryland, USA; 2Thoracic and GI Malignancies Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, USA; 3Surgical Oncology Branch, National Institutes of Health, Bethesda, Maryland, USA; 4Rutgers Robert Wood Johnson Medical School, Department of Surgery, New Brunswick, New Jersey, USA

Invariant NKT cells (iNKT) function as a bridge between innate and adoptive immune responses and have been an attractive target for antitumor therapy; however, their anti-cancer application has historically been limited by immune evasion/suppression of the tumor microenvironment (TME). The immunotolerogenic nature of the liver could further hinder hepatic NKT in tumor surveillance / clearance, but it has not been studied how hepatic iNKTs behave in such unique TME.

We hypothesize that iNKT infiltrates the tumor and coordinate antitumor attacks. Tumor-infiltrating lymphocytes (TIL) and hepatic lymphocytes from three orthotopic models (intrahepatic injection of B16 melanoma, RIL-175 hepatoma or CT26 colorectal carcinoma) and one inflammatory carcinogenic model (Diethylnitrosamine (DEN)-induced liver injury) were isolated and analyzed using flow cytometry. CD1d surface expression of the tumors was also characterized using flow cytometry. CXCL16 mRNA levels were quantified by qPCR to correlate its level with iNKT distribution. We found a paucity of TIL-iNKT compared to peri-tumoral iNKT in all four liver models (peri-tumor to TIL: RIL-175, 8.0% vs. 0.3%, p<0.001; B16, 10.7% vs 0.39, p = 0.0079; CT26, 11.1% vs 2.4%, n=2; DEN, 9.34% vs 5.51%, p = 0.429). The tumors represent a range of CD1d expression. CXCL16 mRNA level, a chemokine that attracts iNKT, is paradoxically higher in the tumor than the adjacent nontumor tissue. Interestingly, the decrease in TIL-iNKT appears to correlate inversely with tumor burden in the DEN model. Oral vancomycin-mediated gut microbiome modification (Ma et al, Science 2018) did not improve TIL-iNKT frequency.

Conclusion: The low frequency of TIL-iNKT is not due to tumor CD1d expression or CXCL16 level, but the degrees of deficiency appears to correlate with tumor burden. The underlying mechanism causing low TIL-iNKT frequency requires further investigation.


Poster 16

Blocking Purine Synthesis in High Urea Cycle Tumors Promotes Response to Immunotherapy

Keshet R2,*, Lee JS1,*, Ruppin E1,** ,Erez A2,** 1Cancer Data Science Lab, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, USA; 2Department of Biological Regulation, Weizmann Institute of Science, 7610001 Rehovot, Israel. *Equally contributing first authors, **Equally contributing last authors

Background and Hypothesis Recent clinical trials in advanced hepatocellular carcinoma (HCC) indicate that programmed cell death protein-1 (PD-1) immune checkpoint inhibitors (ICI) have a potential to provide more effective treatments. Recently we have shown that the urea cycle (UC), with which the mammalian liver detoxifies nitrogen metabolites such as ammonia is a strong determinant of the response to ICI (Lee et al., 2018). To recapitulate, we have shown that: (i) The dysregulation of the UC is a frequent event in main tumors, which in turn activates pyrimidine biosynthesis, resulting in nucleotide imbalance with excessive pyrimidine over purine nucleotides. (ii) This imbalance creates a novel genomic signature consisting of pyrimidine-rich transversion mutations, termed PTMB. (iii) This signature leads to a better response to ICI via the presentation of more hydrophobic and immunogenic neo-peptides. Building upon this observation, we next hypothesized that inhibition of purine nucleotide synthesis may further increase the pyrimidine-to-purine ratio and expand the number of patients that could benefit from ICI.

Study Design and Methods To test this hypothesis, we first analyzed the landscape of UC enzymes’ activity and the level of the PTMB bias in liver cancer across CCLE cell lines and TCGA data. We studied those samples to further understand their biology, with particular focus on those samples with high UC enzyme activities (complementing (Lee et al., 2018) where we have focused primarily on studying tumors with low UC activities). We studied the consequences of the activation of UC enzymes on nucleotide imbalance, mutational bias, and the response to immunotherapy across in vitro and patient data. Subsequently, we performed in vivo experiments to test whether treating mouse models with a purine synthesis inhibitor that reverts the purine-to-pyrimidine ratio and increases PTMB levels can sensitize resistant tumors to ICI.

Results and Conclusion Our analysis of TCGA liver cancer patient data and the CCLE liver cell line panel shows that UC enzyme activities and purine-to-pyrimidine mutational bias levels are highly variable in liver cancer. Our current experiments suggest that, while low expression of UC enzymes is associated with better response to immunotherapy as was reported in (Lee et al., 2018), the heightened expression of UC enzymes in tumors lead to: (i) increased nitrogen oxide synthesis, activating the gluconeogenesis by S-nitrosylation of the enzymes, (ii) enhanced purine synthesis causing a nucleotide imbalance favoring purines. (iii) The resulting purine-rich mutational bias found in these cells associates with poor response to ICI in murine models. However, a purine synthase inhibitor treatment successfully reverts the purine rich bias, increases PTMB levels, and markedly enhances the response to immunotherapy. Our results demonstrate for the first time that inhibiting purine synthesis can improve tumor response to immunotherapy, potentially increasing the fraction of patients that may benefit from immunotherapy. Given the central role of the UC in liver, the broad spectrum of UC activity observed in HCC, and the emerging role of immunotherapy in this cancer type, we postulate that these findings may be of particular translational importance for further advancing liver cancer treatment.


References Lee, J.S., Adler, L., Karathia, H., Carmel, N., Rabinovich, S., Auslander, N., Keshet, R., Stettner, N., Silberman, A., Agemy, L., et al. (2018). Urea Cycle Dysregulation Generates Clinically Relevant Genomic and Biochemical Signatures. Cell 174, 1559-1570 e1522.


Poster 17

Long Persistent Chimeric Antigen Receptor T Cells That Target Glypican-3 Eradicate Orthotopic Hepatocellular Carcinoma in Mice

Li D1,2#, Li N1#, Zhang YF1,3, Fu HY1,4, Feng MQ1, Duan ZJ1, Hummer AM1, Yang HJ1, Torres MB1, Luo XL5, Su L6, Zhu H7, Kramer J8, Zhou J9, Mackay S9, Chen JQ5, Wu XL6, Hall MD7, Wang Q2, Abate-Daga D10, Hewitt SM11, Orentas RJ12, Greten TF13, Ho M1* 1Laboratory of Molecular Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892, USA; 2School of Life Sciences, East China Normal University, Shanghai 200241, China; 3Department of Microbial Pathogenesis, School of Dentistry, University of Maryland, Baltimore, MD 21201, USA; 4Department of Immunology, Norman Bethune College of Medicine, Jilin University, Changchun 130021, China; 5Collaborative Protein Technology Resource, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892, USA; 6Cancer Research Technology Program, Leidos Biomedical Research, Inc., Frederick National Laboratory for Cancer Research, Frederick, Maryland 21702, USA; 7Chemical Genomics Center, National Center for Advancing Translational Sciences, National Institutes of Health, Rockville, Maryland 20850, USA; 8Animal Facility, Leidos Biomedical Research, Inc., National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892, USA; 9IsoPlexis, Branford, Connecticut 06405, USA; 10Departments of Immunology, Cutaneous Oncology, and Gastrointestinal Oncology, H. Lee Moffitt Cancer Center and Research Institute, Tampa, FL 33612; 11Laboratory of Pathology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892, USA; 12Seattle Children’s Research Institute, Seattle, Washington 98101, USA; 13Thoracic and Gastrointestinal Oncology Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892, USA #Both authors contribute equally to this work. *Correspondence author

Background and hypothesis Glypican-3 (GPC3) is an oncofetal antigen involved in Wnt-dependent cell proliferation and highly expressed in hepatocellular carcinoma (HCC). One of the major obstacles in chimeric antigen receptor (CAR) generation is the optimization of antibody domains. Function of CARs is affected by their tumor specificity and epitopes for antitumor activity.

Study Design and Methods In this study, we used T cells from healthy donors and HCC patients to create the CAR T cells based on the hYP7 and HN3 antibodies that are specific for the C-lobe and N-lobe of GPC3, respectively, and tested them in HCC xenograft models including an orthotopic model in mice. To analyze the persistence of CAR T cells, we used droplet digital PCR and genomic sequencing to measure CAR expression in tissues and determine their integration sites in the host genome.

Results and Conclusion CAR (hYP7) T cells demonstrate superior anti-tumor activity in mice as compared to CAR (HN3) T cells. The CAR T-cell treatment not only induces the perforin/granzyme-mediated apoptosis but also reduces the expression of active β-catenin in HCC cells, indicating the role of Wnt signaling in efficacy of GPC3-targeted CAR-T cell therapy. The mice treated with CAR (hYP7) T cells, through a small subset of polyfunctional CD8+ T cells, exhibited highly persistent T cell expansion in the tumor microenvironment and the spleen for over 5 weeks after a single treatment. Furthermore, a subset of integration sites, including DAD1, may contribute to the persistence of CAR T cells. The hYP7 antibody-derived GPC3-specific CAR is a promising therapeutic that is ready for a clinical trial treating advanced stage liver cancer.


Poster 18

FBP1 Loss Disrupts Hepatic Metabolism and Promotes Liver Cancer Via a Senescence Secretome

Li F1,2, Huang P1,2, Spata M1,2, Guo K1,2, Riscal R1,2, Godfrey J1,2, Lee K1,2, Lin N1,2, Lee P1,2, Blair I3, Keith B 1,2,4, Li B5,6, Simon MC*1,2,7 1Abramson Family Cancer Research Institute, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104; 2Department of Cancer Biology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104; 3Department of Pharmacology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104; 4The Wistar Institute, Philadelphia, Pennsylvania, 19104; 5Department of Biochemistry, Zhongshan School of Medicine, Sun Yat-Sen University, Guangzhou 510080, China; 6RNA Biomedical Institute, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou 510120, China; 7Department of Cell and Developmental Biology, University of Pennsylvania, Philadelphia, Pennsylvania 19104 *Correspondence: [email protected] Abramson Family Cancer Research Institute Perelman School of Medicine 456 BRB II/III 421 Curie Boulevard Philadelphia, PA 19104

Background and Hypothesis: Primary liver cancer is a leading cause of cancer-related mortality worldwide. How crosstalk between deregulated hepatocyte metabolism and cells within tumor microenvironment (TME) regulates primary liver tumorigenesis remains unclear. The gluconeogenic enzyme fructose bisphosphatase 1 (FBP1) has been implicated as a potential metabolic tumor suppressor that functions by antagonizing glycolysis and/or hypoxia inducible factors (HIF), but the genetic evidence has been lacking. We hypothesized that FBP1 loss may function through HIF-independent mechanisms in liver tumorigenesis.

Study Design and Methods: The expression change of FBP1 was evaluated in human TCGA dataset and tissuesrray, as well as in murine models. A Fbp1 conditional allele was generated to study the effects of FBP1 loss on liver metabolism and tumorigenesis using the DEN model and p53 model. RNA-sequencing, secretome profiling and lipiomics were performed to characterize the phenotypes. In vitro cell culture and in vivo drug treatment were performed to study the crosstalk between FBP1-deficient hepatocytes and hepatic stellate cells, and the impact on tumorigenesis.

Results and Conclusion: Hepatocyte-specific loss of gluconeogenic enzyme fructose bisphosphatase 1 (FBP1) disrupts liver metabolic homeostasis and promotes tumour progression via a senescence secretome in TME. FBP1 is universally silenced in both human and murine liver tumors, and hepatic Fbp1 deletion leads to mild steatosis, fibrosis, and senescence in hepatic stellate cells (HSCs). Senescent HSCs promote tumor growth through a senescence-associated secretory phenotype (SASP), and can be inhibited by the “senolytic” drugs dasatinib and quercetin. Furthermore, FBP1-deficient hepatocytes signal to HSCs through HMGB1, and blocking its release with the small molecule inflachromene limits HSC activation, SASP and tumor progression driven by FBP1 loss. Collectively, these findings provide the genetic evidence for FBP1 as a metabolic tumor supressor, and establish a critical link between metabolism and senescence that contributes to liver tumor growth.


Poster 19

β-Catenin Activation Promotes Immune Escape and Resistance to Anti-PD-1 Therapy in Hepatocellular Carcinoma

Ruiz De Galarreta M1,2,3, Bresnahan E1,2,3, Molina-Sánchez P1,2,3, Lindblad KE1,2,3, Maier B1,3, Sia D2, Puigvehi M2, Miguela V1,2,3, Casanova-Acebes M1,3, Dhainaut M1,3, Villacorta-Martin C2, Singhi AD4, Moghe A4, Von Felden J2, Tal Grinspan L1,2,3, Wang S2, Kamphorst AO1,3, Monga SP4, Brown BD3, Villanueva A2, Llovet JM2, Merad M1,3, Lujambio A1,2,3 1Department of Oncological Sciences, Icahn School of Medicine at Mount Sinai, New York, USA; 2Liver Cancer Program, Division of Liver Diseases, Department of Medicine, Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, New York, USA; 3The Precision Immunology Institute, Icahn School of Medicine at Mount Sinai, New York, USA; 4Division of Experimental Pathology, Department of Pathology, Division of Gastroenterology, Hepatology and Nutrition, Department of Medicine, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA.

Background and hypothesis Hepatocellular carcinoma (HCC) represents a major health problem, causing more than 700,000 deaths annually worldwide. Recently, nivolumab and pembrolizumab, two PD-1 (programmed cell death 1) immune checkpoint inhibitors, were approved by the FDA for second line HCC treatment, after showing unprecedented results in phase II clinical trials. However, not all HCC patients are sensitive, indicating the existence of mechanisms of resistance to anti-PD-1 therapy and highlighting the urgent need to identify biomarkers for optimal patient selection. Our hypothesis is that the identification of novel mechanisms of immune escape will also unveil mechanisms of resistance to anti-PD-1.

Study design and methods In order to identify oncogenic signaling pathways in HCC that promote immune escape and affect response to anti-PD-1 therapy, we created a novel non-germline genetically-engineered mouse model of HCC that allows interrogating how genetic alterations relevant to human disease affect immune surveillance and response to immunotherapies. The model is based on the hydrodynamic tail vein delivery of genetic elements to overexpress oncogenes (with transposon-based vectors), delete tumor suppressor genes (with CRISPR vectors), and modulate immunogenicity (with model tumor antigens) specifically in hepatocytes. The effect of the expression of model tumor antigens in liver tumor formation was tested in several independent models of HCC. Transcriptional analysis and immune profiling of the resulting tumors was performed. Finally, response to anti-PD-1 therapy has been assessed in all the models.

Results and conclusions Expression of tumor antigens in the context of MYC overexpression and loss of p53 (MYC;p53-/-) led to immune surveillance, with a decrease in tumor formation and increased survival, an effect mediated by dendritic and T cells. In contrast, expression of tumor antigens in the context of MYC overexpression and β-Catenin activation (MYC;CTNNB1) led to immune escape and immune cell exclusion, with an increase in tumorigenesis and decreased survival, demonstrating that CTNNB1 activation in HCC cells promotes immune escape. Accordingly, mice harboring MYC;p53-/- tumors responded to anti-PD-1 therapy while mice harboring MYC;CTNNB1 tumors did not, indicating that CTNNB1 activation confers resistance to anti-PD-1 therapy in HCC, a results that was validated in HCC patients. Mechanistically, expression of chemokine Ccl5 was able to restore immune surveillance of CTNNB1-driven tumors (Ruiz de Galarreta, Cancer Discovery, 2019). We are currently using our model to screen for additional genes involved in immune escape in HCC and to test novel combination immunotherapies to restore sensitivity to anti-PD-1. Together, β-catenin activation promotes immune escape and resistance to anti-PD-1 therapy in our murine model of HCC and could represent a biomarker for HCC patient exclusion.


Poster 20

Platelets Inhibits HCC in Non-Alcoholic Fatty Liver Disease

Qiong FU1, Chi MA1, Laurenc Diggs1, Varun K Subramanyam1,Tim F Greten1 1Thoracic and Gastrointestinal Oncology Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA.

Non-alcoholic fatty liver disease (NAFLD), a common condition in obese patients and a leading cause of liver-related morbidity in US and globally, has been established as a risk factor for HCC. NAFLD patients have bigger mean platelet volume(MPV), a surrogate of platelet activation. Most recently, in both mice and humans with NAFLD, platelets have been found to aggerate in liver and become activated. Platelets are known to interact with immune cells. However, the role of platelets on HCC disease progression in NAFLD is still unknown. In this study we tried to address this question using established murine NAFLD-HCC models. Liver HCC tumors were induced by DEN carcinogen, hepatocyte-selective overexpression of MYC oncogene, or using established mouse HCC tumor line Hep55.1c. NFALD was induced by feeding mice with choline-deficient L-amino acid-defined diet (CDAA) or methionine-choline deficient (MCD) diet. To block platelet function, clinically used anti-platelet medications clopidogrel or ticagrelor was used. Platelet depletion was performed by injecting an antibody targeting GPIB�. Clopidogrel treatment caused a significant increase of liver tumor in DEN-CDAA mice. While, no change of liver tumor was found in mice without NAFLD. Similarly, clopidogrel increased liver tumors of MYC mice fed MCD diet. Next, the effect of platelets on established HCC was investigated, using orthotopic implantation of Hep55.1c tumor cells into livers of congenic C57BL/6 mice fed MCD diet. As expected, clopidogrel group presented bigger Hep55.1c tumors. Consistently, treatment of ticagrelor, another clinically used anti-platelet medication, increased intrahepatic Hep55.1c tumor burden in MCD-fed mice. To directly prove tumor-modulating function of platelets, platelet depletion was performed using anti-GPIba antibody injection. Indeed, platelet depletion caused a higher live tumor burden in NAFLD mice. Together, using two dietary NAFLD models in all the three genetic, carcinogen-induced, and orthotopic HCC models with different mouse strain background, our results demonstrate that platelets induce an anti-tumor immunity in NAFLD. Anti-platelet therapy is used in NAFLD patients, and clopidogrel usage is significantly higher in NAFLD patients than non-NAFLD patients. Precaution should be used when apply anti-platelet therapy to NAFLD patients with or at the risk of developing liver tumors.


Poster 21

Tumor Cell Biodiversity Drives Microenvironmental Reprogramming in Liver Cancer

Ma L1,8, Hernandez MO1,2,8, Zhao Y2, Mehta M2, Tran B2, Kelly M2, Rae Z2, Hernandez JM3, Davis JL3, Martin SP1,3, Kleiner DE4,5, Hewitt SM4, Ylaya K4, Wood BJ5,6, Greten TF5,7, Wang XW1,5 1Laboratory of Human Carcinogenesis, Center for Cancer Research, National Cancer Institute, Bethesda, Maryland 20892, USA; 2Frederick National Laboratory for Cancer Research, Leidos Biomedical Research, Inc., Frederick, Maryland 20701, USA; 3Surgical Oncology Program, Center for Cancer Research, National Cancer Institute, Bethesda, Maryland 20892, USA; 4Laboratory of Pathology, Center for Cancer Research, National Cancer Institute, Bethesda, Maryland 20892, USA; 5Liver Cancer Program, Center for Cancer Research, National Cancer Institute, Bethesda, Maryland 20892, USA; 6NIH Center for Interventional Oncology, Bethesda, Maryland 20892, USA; 7Thoracic and GI Malignancies Branch, Center for Cancer Research, National Cancer Institute, Bethesda, Maryland 20892, USA 8These authors contributed equally

Background and Hypothesis Cellular diversity in tumors is key factor for therapeutic failures and lethal outcomes of solid malignancies. Primary liver cancer (PLC), the second most lethal malignancy worldwide, is etiologically and biologically heterogeneous. While most of PLC patients are refractory to molecularly-targeted systemic therapeutics, some patients show remarkably durable responses to immune checkpoint inhibitors with a significantly improved overall survival. However, it is unclear mechanistically why some patients respond to immunotherapy favorably whereas others do not. What are the collective behaviors and regulations of a tumor cell community with respect to its survival fitness that consequently impact a patient’s prognosis? We aimed to determine the transcriptomic landscape of tumor cell communities in PLC based on single-cell RNA sequencing (scRNA-seq), and to dissect the impact of tumor transcriptomic diversity on patient outcome.

Study Design and Methods We generated droplet-based scRNA-seq profiles of freshly-isolated primary tumor from 19 PLC patients who were enrolled at the NIH Clinical Center under the liver moonshot program for immune checkpoint inhibition clinical trials. Based on the single-cell transcriptomes, we developed a method to measure tumor cell biodiversity and further investigated the association of tumor transcriptomic diversity and overall survival in PLC patients treated with immune checkpoint inhibitors. We also sought to determine the cellular factors produced by high diversity tumor cells that could induce tumor microenvironment (TME) reprogramming by using a pathway-based strategy. We used bulk genomic and transcriptomic profiles of liver tumors from different cohorts to validate our results.

Results and Conclusion We determined the single-cell transcriptomic landscape of liver cancer biospecimens from 19 patients. We found varying degrees of heterogeneity in malignant cells within and between tumors and diverse landscapes of TME. Strikingly, tumors with higher transcriptomic diversity were associated with patient’s worse overall survival. We found a link between hypoxia-dependent VEGF expression in tumor diversity and TME polarization. Moreover, T cells from higher heterogenous tumors showed lower cytolytic activities. Consistent results were found using bulk genomic and transcriptomic profiles of 765 liver tumors. Our results offer insight into the diverse ecosystem of liver cancer and its impact on patient prognosis.


Poster 22

HSD17B13 is Associated to the Pathogenesis of Non-Alcoholic Fatty Liver Disease

Ma Y1, Belyaeva OV2, Brown PM1, Karki S2, Kedishvili NY2, Rotman Y1

1Liver & Energy Metabolism Unit, Liver Diseases Branch, NIDDK, NIH, Bethesda, MD; 2Department of Biochemistry and Molecular Genetics, University of Alabama – Birmingham, Birmingham, AL

Background and hypothesis: Non-alcoholic fatty liver disease (NAFLD) is the most common liver disorder in developed countries, and has significant heritable components. Previously, our group have found SNPs in HSD17B13 (17-beta hydroxysteroid dehydrogenase 13) that are significantly associated with histological features of NAFLD in a large cohort of NAFLD subjects. We aim to identify the function and potential substrate of HSD17B13 and its role in NAFLD pathogenesis. We hypothesized that HSD17B13 represents a retinol dehydrogenase (RDH), converting retinol (ROL) to retinaldehyde, the rate-limiting step in retinoic acid synthesis, based on structural similarity between HSD17B13 and RDH.

Study Design and Methods: Hepatic gene expression was assessed by qPCR in human NAFLD and healthy controls. Retinol dehydrogenase (RDH) activity was measured by HPLC in HSD17B13-transfected HEK293 cells. Protein mutants were generated using site-directed mutagenesis. Hsd17b13 knock-out (KO) mice and their littermate controlled wild type mice were fed with regular chow or obesogenic diets for 12 weeks.

Results and Conclusion: In patients with non-alcoholic steatohepatitis (n=43), hepatic expression of HSD17B13 is increased 5.9-fold (p=0.003) compared to healthy controls (n=14). In cell culture, HSD17B13 is targeted to lipid droplets (LD) depending on conserved sequences. Knock-out or overexpression of HSD17B13 in HepG2 cells does not impact lipid content. As expected, we found it has RDH activity comparable to RDH10, a known RDH. One missense SNP encoding a P260S mutation and a splice site SNP generating two splicing variants are associated with histology features in our human genetic study. We generated a P260S mutant and two splicing mutants, and found them have no RDH activity despite proper LD targeting.

Consistent with the in vitro data, KO mice show 37% decrease in liver RDH activity, 4-fold increase in hepatic ROL and 36% lower serum ROL levels (p<0.05 for all). KO mice on high fat diet have significantly improved insulin sensitivity (by HOMA-IR, glucose and insulin tolerance tests) that correlates with their serum ROL (r=0.73, p=0.03).

In conclusion, using genetic and expression data we demonstrate an association between HSD17B13 and human NAFLD. We show that the gene encodes a RDH and in mice it is involved in insulin sensitivity regulation, likely through retinoid signaling. Retinoids are known to play a critical role in hepatic stellate cells activation and NAFLD progression and our data suggests that HSD17B13 affects the pathogenesis of NAFLD through its hepatic RDH activity.


Poster 23

L-Arginine: A Substrate for Immunosuppressive Nutrient Competition Between Immune and Tumor Cells in Hepatocellular Carcinoma

Missiaen R1,2, Li F1,2, Simon MC1,2,3 1Abramson Family Cancer Research Institute, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA. 2Department of Cancer Biology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA; 3Department of Cell and Developmental Biology, University of Pennsylvania, Philadelphia, PA.

Over recent decades, the number of people diagnosed with hepatocellular carcinoma (HCC), has increased significantly. Unfortunately, the only curative treatment available is tumor resection, successful exclusively in early stages of disease. Although several new therapies have been developed, their effects are limited with low overall survival benefits. Recently, immunotherapy, a promising new line of anti-cancer treatment has entered the clinic. However, tumors employ several mechanisms to suppress the immune system and escape therapy-induced immune responses. Understanding immune suppressive mechanisms is therefore key for optimizing these therapies and increasing the likelihood of immunotherapy success in HCC.

Our data suggest that HCC cells, in contrast to healthy liver cells, lack the metabolic program to synthesize L-arginine (L-Arg). Moreover, depletion of extracellular L-Arg strongly impairs cell cycle progression, but does not affect viability. We aim to determine the fate and function of L-Arg in HCC, and to characterize cellular mechanisms inducing cell cycle arrest upon L-Arg limitation. Similarly, immune cells, such as macrophages and T-cells capable of targeting tumor cells, also rely on exogenous L-Arg for their survival and anti-tumor function. I therefore hypothesize that tumor and immune cells compete for environmental L-Arg. Because tumor cells can adapt more readily to stress conditions than immune cells, depletion of available L-Arg will likely affect immune cells more rapidly than HCC cells, creating an immune suppressive and immunotherapy resistant environment. In this study, I will further characterize L-Arg competition and investigate the therapeutic potential of increasing L-Arg availability for immune cells in this setting.

These data will provide new insights into mechanisms responsible for immune suppression in HCC tumors, and help design novel therapies for HCC patients. These findings will not only be applicable to HCC, but also prove useful for other cancers relying on environmental L-Arg, such as melanoma, lymphoma, and renal cancer.


Poster 24

Exploiting Collateral Vulnerabilities Created by a Covalent Oncometabolite

Perez, Minervo1 Meier, Jordan L1 1Chemical Biology Laboratory, National Cancer Institute-Frederick

Background and hypothesis: Since the discovery of the Warburg effect, many cancers have been found to be fueled by reprogrammed cellular metabolism. A prototypical example occurs in hereditary leiomyomatosis and renal cell carcinoma (HLRCC), a hereditary cancer syndrome in which loss of heterozygosity at the fumarate hydratase (FH) locus causes accumulation of the oncometabolite fumarate. Fumarate is an electrophilic metabolite that can covalently modify cysteines, leading to a rare non-enzymatic posttranslational modification cysteine S-succination.

Study design and methods: The goal of this project is to apply an unbiased screening approach to identify small molecules that selectively disrupt HLRCC cell growth by exploiting collateral vulnerabilities created by S-succination of proteins essential for cell growth. In our initial studies we have assembled a chemically diverse electrophilic fragment library and cell-based screening strategies.

Results and conclusions: These strategies will be applied in concert to identify HLRCC selective fragments, followed by target elucidation using a recently developed chemoproteomic mapping strategy. In addition to providing new optimizable leads for drug discovery efforts, these studies have the potential to provide seminal insights into how metabolism drives malignancy in HLRCC and other metabolically-deranged cancers.


Poster 25

Novel Hydrazone Derivatives Decrease Cholangiocarcinoma Proliferation by Inhibition of Epithelial-Mesenchymal Transition

Keisaku Sato1,2, Fanyin Meng1,2, Heather Francis1,2, Gianfranco Alpini1,2, Praveen Kusumanchi1,2, Suthat Liangpunsakul1,2 1Indiana Center for Liver Research, Division of Gastroenterology & Hepatology, Department of Medicine, Indiana University School of Medicine, Indianapolis, Indiana; 2Richard L. Roudebush VA Medical Center, Indianapolis, Indiana

Background: Cholangiocarcinoma (CCA) is the second most common cancer in primary liver malignances. Chemotherapy can reduce the possibility of recurrence after surgery although this treatment is limited and poorly effective with 5-year survival rate under 30%. Therefore, the development of novel drugs for CCA is needed. Hydrazones are a class of organic compounds and previous studies have demonstrated their anti-cancer effects against colorectal carcinoma. In this study, we developed novel therapeutic drugs and evaluated the in vitro anti-cancer effects for potential in vivo chemotherapeutic approach.

Methods: 23 novel hydrazone derivatives were synthesized and their structures determined by NMR and mass spectrometry. Human intra- and extra-hepatic CCA lines, HUCCT, Mz-ChA-1, CCLP, TFK-1 and Huh28 were incubated for 72 hours with various concentrations (1 µM to 100 µM) of hydrazone derivatives and cell proliferation was analyzed by MTS assay. The human normal immortalized cholangiocyte H69 was served as control. RNAs was harvested from CCA cell lines after 72 hours of treatments and RT-qPCR was performed to evaluate expression levels of cell proliferation (PCNA and Ki67), inflammatory cytokines (IL-1β and IL-6), and epithelial-mesenchymal transition (EMT, vimentin).

Results: Three novel hydrazone derivatives RPC038, RPC042 and RPC046 showed anti-cancer effects and inhibited cell proliferation against 60 cancer cell lines with IC50 ranging from 0.1–1.9 µM. RPC038, RPC042 and RPC046 also inhibited cell proliferation (IC50) in CCA cell lines, SG231 (4 µM, 21 µM and 8 µM), CCLP (7 µM, 5 µM and 8 µM), TFK-1 (13 µM, 6 µM), Huh28 (12.5 µM,6 µM,25 µM) HUCCT (11 µM,11 µM) and Mz-CHA-1(25 µM,40 µM,22 µM) respectively. In H69 cells, the IC50 was higher than all CCA cell lines, indicating limited toxicity on normal cholangiocytes. qPCR of SG231, CCLP and Mz-CHA-1 treated with novel derivatives showed -70% and -60% decreased expression for cell proliferation markers PCNA and Ki67 respectively; -80% and -60% decreased expression for inflammatory cytokines Il-1 β and IL-6; and EMT, vimentin was showed a 70% reduced expression. The expression of apoptotic marker p16 was up by 30%.

Conclusion: The preliminary findings suggest that three novel hydrazone derivatives RPC038, RPC042 and RPC046 have anti-cancer effects against CCA cell lines. Although further studies are required, they can be potential therapeutic drugs for CCA.


Poster 26

miR-122 Regulates Hepatic Glutamine Metabolism by Directly Targeting Mitochondrial Glutaminase

Sengupta D1, Cassel T2, Teng KY1, Hu P1, Barajas J1, Fan T2 and Ghoshal K1 1Department of pathology, Comprehensive Cancer Center, Ohio state University College of Medicine, Columbus, OH; 2University of Kentucky, Lexington, KY.

It is well established that the liver specific miR-122, a bona fide tumor suppressor, plays critical role in lipid homeostasis (1). However, its role, if any, in amino acid metabolism has not been explored.

Since glutamine is an important energy source, we monitored glutamine metabolism in the wild type (WT) and miR-122 knockout (KO) mice by SIRM (Stable Isotope Resolved Metabolomics) studies. To this end, 6-8 weeks old mice were injected with [U-13C,15N]-L-glutamine (gln) (7mg/mouse, 2x at 15 min interval) and sacrificed 15 min after the last injection. Analysis of polar metabolites from snap-frozen liver tissues by NMR and IC-MS showed elevated glutamine, glutamate, α-ketoglutarate, isocitrate and citrate levels without significant changes in succinate, malate or fumarate levels in KO livers suggest enhanced glutaminolysis and a break in the latter half of the Krebs cycle in miR-122 depleted livers. Reduced conversion of [13C,15N-glutamine] to 13C3-glucose-6-phosphate and -fructose-6-phosphate implicates reduced gluconeogenesis from glutamine in KO livers while that to 13C-glutathiones suggests decreased synthesis and/or increased utilization.

To elucidate the underlying mechanism, we focused on HITS-CLIP and RNA-seq data of the WT and KO mice that identified functional miR-122 targets in the liver transcriptome (2). The results showed that Gls is a direct miR-122 target, which was validated by luciferase reporter assay. Importantly, Gls expression was suppressed in glutamine dependent hepatoma cells by ectopic expression of miR-122 mimic. These results explain that the increased conversion of [U-13C,15N]-L-glutamine to glutamate and other TCA cycle metabolites is likely due to the upregulation of Gls in miR-122 depleted livers. TCGA-LIHC database analysis showed that GLS expression in tumors inversely correlated with miR-122 expression. Upregulation of GLS protein level in primary HCCs relative to the matching benign livers suggests enhanced use of glutamine as energy source by the tumor cells.

Collectively, these results show that miR-122 modulates glutamine metabolism both in vitro and in vivo. These results implicate therapeutic potential of glutaminase inhibitors in patients bearing HCCs that exhibit low miR-122 level.

(supported in part, by R01CA193244 grant from NIH and Pelotonia Idea grant)

1. Essential metabolic, anti-inflammatory, and anti-tumorigenic functions of miR-122 in liver. J Clin Invest. 2012 Aug;122(8):2871-83. 2. Argonaute CLIP Defines a Deregulated miR-122-Bound Transcriptome that Correlates with Patient Survival in Human Liver Cancer. Mol Cell. 2017 Aug 3;67(3):400-410.e7.


Poster 27

ALDH2 Deficiency Promotes Alcohol-Associated Liver Cancer by Activating Oncogenic Pathways Via Oxidized DNA Enriched Extracellular Vesicles

Seo W1, Gao Y2, He Y1, Feng D1, Park SH1, Ren T1, Kim SJ1, Hwang S1, Liangpunsakul S3,4,5, Niu J2, and Gao B1 1Laboratory of Liver Diseases, National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health, Bethesda, USA; 2Department of Hepatology, The First Hospital of Jilin University, Jilin University, Changchun, China; 3Division of Gastroenterology and Hepatology, Department of Medicine, Indiana University, Indianapolis, USA; 4Department of Biochemistry and Molecular Biology, Indiana University, Indianapolis, USA; 5Roudebush Veterans Administration Medical Center, Indianapolis, USA

Background and hypothesis: Hepatocellular carcinoma (HCC) is the third-leading cause of cancer-related deaths worldwide and excessive alcohol drinking has been recognized as a major cause of HCC. Acetaldehyde, a main metabolite of alcohol, causes hepatocellular injury and impairs cellular functions by adduct formation, consequently promotes DNA damage and mutation. Aldehyde dehydrogenase 2 (ALDH2) is a key enzyme to detoxify acetaldehyde produced throughout alcohol metabolism, however, 30-40% of Asian population carries an inactive ALDH2 gene and show acetaldehyde accumulation after alcohol drinking. It has been recognized that the association between ALDH2 polymorphism and esophageal/gastric cancer, whereas the effects of ALDH2 in the progression of HCC and the underlying mechanisms remain largely unknown.

Study design and methods: For the study of the association of ALDH2 polymorphisms and disease progression, chronic viral hepatitis B infected patients (n=646) and healthy controls (n=283) were recruited. In animal study, chronic CCl4 administration (fibrosis) and alcohol-associated HCC mouse model was developed, and three strains of ALDH2-deficient mice were used: including Aldh2 global knockout (KO), ALDH2*2 knock-in mutant mice, liver-specific Aldh2 KO mice.

Results and conclusion: ALDH2 polymorphism is not associated with liver disease progression from liver injury to cirrhosis in viral hepatitis patients, but is associated with the increased risk of HCC development in HBV cirrhotic patients who drink chronic alcohol consumption. In animal study, ALDH2 KO mice were more susceptible to chronic CCl4 plus ethanol-induced HCC and liver fibrosis. ALDH2 KO mice also showed higher levels of oxidative stress/lipid peroxidation and increased DNA damage than WT mice after chronic CCl4 plus ethanol challenge. In contrast, without alcohol consumption, WT and ALDH2 KO mice had the similar grade of liver fibrosis and liver cancer after CCl4 treatment alone. Furthermore, chronic CCl4 plus ethanol challenge significantly increased extracellular vesicle (EV) formation in blood serum and in vitro hepatocyte culture. Surprisingly, oxidized DNA was found in hepatocyte-derived EVs and the amount of oxidized DNA was significantly increased in ALDH2 deficient mouse blood serum and cultured medium. In vitro experiments demonstrated that these oxidized DNA enriched EVs can activate several oncogenic signaling pathways in the presence of acetaldehyde. ALDH2 deficiency is associated with an increased risk of alcohol associated-HCC development from cirrhosis/fibrosis both in patients and mice. Mechanistic studies reveal a novel mechanism that ALDH2-deficient hepatocytes promote HCC by transferring oxidized DNA-enriched EVs that activate multiple oncogenic pathways in HCC.


Poster 28

Impact Abrogates Metastatic Outgrowth of Pancreatic Cancer Cells in the Liver Through Inhibition of GNC1-ATF4 Signaling at Physiologic Glutamine

Sinha S1, Ayabe R1, Ranjan A1, Wach M1, Ruff S1, Remmert K1, Beek VE2, Alexander I1, Gupta S1, McDonald J1, Li E2, Hernandez J1 1Surgical Oncology Program, National Cancer Institute, USA; 2Amsterdam University Medical Center, Netherlands

Background and Hypothesis: Although liver metastasis is a major cause of pancreatic cancer mortality, the mechanisms by which KRAS-driven pancreatic tumor cells initiate metastatic outgrowth in the liver are unknown. High-throughput next-generation sequencing studies have revealed that driver mutations and copy number alterations are highly concordant between matched primary tumors and metastases, suggesting that metastatic outgrowth and colonization involves the cooperation of other cellular factors. We hypothesize that in the absence of metastasis-specific driver mutations, the initial adaptation and outgrowth steps of metastasis are governed by metabolic processes that are induced/activated by the physiological conditions of the target organ.

Method: To identify metabolic drivers of liver colonization, a retroviral cDNA library was generated from a highly metastatic pancreatic cell line (M-4964Liv) derived from a KPC mouse which forms macroscopic liver lesions upon splenic injection in mice. The cDNA library was then transduced into a low metastatic-capacity KPC cells (D-4964Liv) cells, which does not give rise to liver lesions upon splenic injection in mice. This methodology allowed for all the cDNA’s from the metastatic cells to express in the low-metastatic-capacity cells but only those cDNAs that can trigger outgrowth will give rise to a metastatic liver lesion.

Results: The screening strategy led to the identification of the translational regulator general control of amino-acid synthesis 1-like 1 (GCN1). We show that activation of GCN1 at levels equivalent to that measurable in the normal liver activates the master regulator of metabolic homeostasis ATF4, oncogenic mTORC1 signaling, and pluripotency factor SOX2 in highly metastatic pancreatic cell line KPC177996. Consistent with this, knockdown of GCN1 inhibits ATF4, mTORC1, and expression of Sox2, and dramatically abrogates liver metastasis upon splenic injection. These findings suggest that GCN1-ATF4 signaling plays an integral role in metastatic outgrowth. Pathway interrogation revealed that the GCN1-ATF4 signaling is governed by a competitive inhibitor, IMPACT, which is induced at glutamine concentrations consistent with those observed in the liver and binds directly to and inhibits GCN1. Interestingly, the expression of IMPACT is lost in highly metastatic pancreatic cell lines leading to activation of the GCN1-ATF4 pathway and mTORC1, while overexpression of IMPACT effectively inhibits the pathway and reduces metastatic outgrowth of pancreatic cancer cells in the liver.

Conclusions: The loss of expression of IMPACT leading to GCN1-dependent activation of ATF4 and mTORC1 provides metabolic plasticity during the initial steps of metastatic outgrowth. Inhibitors targeting the GCN1-ATF4 pathway or mTORC1 may therefore benefit patients with pancreatic adenocarcinoma by inhibiting the outgrowth of micrometastases.


Poster 29

STEAP2 is Upregulated and Necessary for Hepatocellular Carcinoma Progression Via Increased Copper Levels and Stress-Activated Map Kinase Activity

Sun L-Z1, Zeballos C1, Easley A1, Bouamar H1, Chiu Y-C2, Chen Y2,3, and Cigarroa F4 1Department of Cell Systems & Anatomy, 2Department of Epidemiology and Statistics, 3Greehey Children’s Cancer Research Institute, 4Transplant Center, University of Texas Health Science Center at San Antonio, Texas, USA

Background and Hypothesis To understand potential molecular mechanisms that may drive hepatocellular carcinoma (HCC) development and progression, we performed whole genome RNA sequencing using total RNA samples from paired adjacent non-tumor liver and HCC tumor tissues of nine local HCC patients. Analysis of the differentially expressed genes revealed that the most significant alteration of Biological Processes (BPs) is oxidation reduction, which involves a gene coding for Six Transmembrane Epithelial Antigen of Prostate 2 (STEAP2) protein. STEAP2 belongs to the STEAP family of metalloreductases involved in the reduction and uptake of iron and copper ions into a cell. Consistently, we found significantly higher levels of total copper in the HCC tissues than those in the paired adjacent non-tumor tissues. The upregulation of STEAP2 expression in HCC was confirmed at its protein level and in TCGA and other published HCC gene expression datasets. Because hepatic copper overload associated with Wilson’s disease is a known risk factor for HCC, we hypothesize that STEAP2 upregulation and copper accumulation may contribute to HCC progression.

Study Design and Methods Paired HCC and adjacent non-tumor tissues were collected from local HCC patients for RNA sequencing, metal ion measurement, measurements of STEAP2 RNA level with RT-qPCR and protein level with Western blot. Public datasets were queried for STEAP2 expression in HCC and non-tumor tissues. HCC cell lines with knockdown (KD) and overexpression (OE) of STEAP2 were created to perform mechanistic studies including measurements of copper levels and MAP kinase activities, cell proliferation, migration, and anchorage independent growth in vitro and tumor growth in vivo.

Results and Conclusion STEAP2 is significantly upregulated in various HCC gene expression datasets. Its expression is positively associated with tumor grade. STEAP2 KD in HCC cell lines decreased cell growth, migration, invasion, and xenograft tumor growth, while STEAP2 OE showed opposite effects. STEAP2 KD in HCC cells also reduced intracellular copper levels and activating phosphorylation of stress-activated MAP kinases including p38 and JNK and their downstream substrates. On the other hand, treatment with copper rescued the reduced HCC cell migration due to STEATP2 KD and activated p38 and JNK. Furthermore, treatment with p38 or JNK inhibitors significantly inhibited HCC cell migration. Thus, STEAP2 appears to play a malignant-promoting role in HCC cells by driving migration and invasion via increased copper levels and MAP kinase activities. Our study uncovered a novel molecular mechanism contributing to HCC malignancy and a potential therapeutic target for HCC treatment.


Poster 30

Bioengineering of Novel RNA Agents to Dissect HCC Cell Metabolism

Ai-Ming Yu*, Mei-Juan Tu, Joseph L. Jilek, Qian-Yu Zhang

Department of Biochemistry & Molecular Medicine, UC Davis, Sacramento, CA 95817, USA

Background and Hypothesis: Hepatocellular carcinoma (HCC) is the most common malignancy of liver, and the incidence of HCC continues to increase in the US, in contrast to a declining trend of all cancers since 2009. Conversely, the overall median survival of HCC patients is 29.8 month without much improvement over the years. Indeed, HCC cells are reprogrammed and become addicted to continuous supply and metabolism of nutrients (e.g., glucose and amino acids or AAs) to drive the synthesis of biologic molecules for proliferation and growth. However, there is a critical gap in the understanding of important regulatory mechanism such as those controlled by noncoding miRNAs behind nutrient supply and homeostasis. Most importantly, current research is limited to the use of chemo-engineered RNA mimics bearing extensive and various degrees and types of artificial modifications. This is in sharp contrast to protein research and therapy that utilize recombinant or bioengineered proteins produced and folded in living cells, instead of synthetic polypeptides or proteins made in test tubes. Therefore, we hypothesized that bioengineered RNA molecules, produced and folded in living cell, would better capture the structures, biological functions, and safety profiles of cellular RNAs for the study of HCC cell metabolism.

Study Design and Methods: We employed the one-of-a-kind RNA bioengineering technology newly established in our lab to design and produce novel biologic RNAi molecules. We determined their antiproliferative activities against multiple HCC cell lines using MTT assay. We further delineated the mechanistic actions of several miRNAs in the regulation of cell metabolism using a combination of molecular and cellular biological techniques as well as accurate analytical assays. Efficacy of bioengineered RNA therapeutics was defined in clinically-relevant orthotopic HCC xenograft mousemodels by using live animal and ex vivo imaging, as well as biochemical and histopathological analyses.

Results and Conclusion: We produced a panel of recombinant RNAi molecules via bacterial fermentation and purified each to a high degree of homogeneity by FPLC. A number of biologic RNAi molecules exhibited potent antiproliferative activities against HCC cells. Among them, let-7c and miR-148a were revealed to modulate the expression of nutrient transporters and/or metabolizing enzymes (e.g, glucose transporter SLC2A1/GLUT1, AA transporters SLC1A5/ASCT2 and SLC7A5/LAT1, and glutaminase or GLS that converts glutamine to glutamate), and subsequently to alter intracellular AA and glucose homeostasis, as well as glycolytic capacity. In addition, biologic miRNAs were effective to control tumor progression and improve overall survival in HCC animal models. These findings demonstrate that bioengineered or recombinant RNAs represent a novel class of agents to dissect the regulatory mechanisms underlying HCC cell metabolism and assess new therapeutic strategies.

Acknowledgements: This study was supported by grants R01GM133888 and R01CA225958 from NIH.


Poster 31

GUT Microbiome-Dependent Accumulation of Myeloid Cells Promotes Colitis-Associated Intrahepatic Cholangiocarcinoma

Qianfei Zhang1, Chi Ma1, Bernd Heinrich1, Umberto Rosato1, Laurence P. Diggs1, Varun K. Subramanyam1, Tim Greten1,2 1Gastrointestinal Malignancy Section, Thoracic and Gastrointestinal Oncology Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA; 2NCI-CCR Liver Cancer Program, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA.

Background and Hypothesis: Intrahepatic cholangiocarcinoma (iCCA) is the second most common primary hepatic malignancy, and its incidence is rising. Colitis is associated with an increased risk for iCCA, but the mechanism is unknown. The hypothesis is colitis-induced bacterial translocation promotes iCCA. Study Design and Methods: Bacterial translocation into liver in DSS-colitis model was first confirmed. In iCCA mouse model, colitis was induced to explore the effect of colitis on iCCA. Gut microbiome depletion in iCCA mouse model was performed to demonstrate the role of bacteria in iCCA. TLR4 KO mice were used to confirm the interaction of LPS/bacteria with TLR4 during iCCA development. Colitis was induced by DSS in drinking water. Bacterial translocation was detected by culture and fluorescence in situ hybridization (FISH). Oncogene plasmids AKT and YAP, AKT and NICD1 was delivered to induce iCCA by hydrodynamic injection. Immune cells in liver and spleen were determined by flow cytometry and immunochemistry. CXCL1 mRNA expression levels was detected by Real-time PCR. Gut permeability was detected by oral gavage of FITC-dextran. Histology of colon and liver tissues was detected by Hematoxylin Eosin (H&E) staining. Results and Conclusion: Colitis increased bacterial translocation to the liver, and neomycin-sensitive bacteria promoted iCCA growth, accompanied by a robust increase of CXCR2+ polymorphonuclear immature myeloid cells (PMN-iMC). Depleting PMN-iMC reversed colitis-dependent iCCA development. Accumulation of CXCR2+ PMN-iMC in the liver was regulated by TLR4-dependent CXCL1 expression of hepatocytes. PMN-iMC were more frequently found in liver biopsies derived from patients with primary sclerosing cholangitis (PSC) with active colitis than with inactive colitis. Our study provides a novel mechanism of how the gut microbiome modulates liver-antitumor immunity and contributes to colitis-promoted iCCA progression.

nci ccr liver cancer program: special conference on tumor ...anna mae diehl, m.d., duke university...

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