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Purpose: Gene fusions are drivers of many pediatric tumors. In fibrolamellar hepatocellular carcinoma (FLC), a fusion of DNAJB1 and PRKACA is the dominant recurrent mutation. Expression of the DNAJB1-PRKACA fusion gene in mice results in a tumor that recapitulates FLC. However, it is not known whether transient expression of DNAJB1-PRKACA is sufficient only to trigger tumor formation or whether ongoing expression is necessary for maintenance and progression. Experimental Design: We screened short hairpin RNAs (shRNA) tiled over the fusion junction and identified several potent and specific candidates in vitro and two independent FLC patient-derived xenografts (PDX). Results: We show that continued DNAJB1-PRKACA expression is not only required for continued tumor growth, but additionally its inhibition results in cell death. Inhibition of DNAJB1-PRKACA by an inducible shRNA in cells of PDX of FLC resulted in cell death in vitro. Induction of the shRNA inhibits FLC tumors growing in mice with no effect on xenografts from a hepatocellular carcinoma cell line engineered to express DNAJB1-PRKACA. Conclusions: Our results validate DNAJB1-PRKACA as the oncogene in FLC and demonstrate both a continued requirement for the oncogene for tumor growth as well as an oncogenic addiction that can be exploited for targeted therapies. We anticipate our approach will be useful for investigations of other fusion genes in pediatric cancers and spur development of precision therapies.

To repurpose therapeutics for fibrolamellar carcinoma (FLC), we developed and validated patient-derived xenografts (PDX) from surgical resections. Most agents used clinically and inhibitors of oncogenes overexpressed in FLC showed little efficacy on PDX. A high-throughput functional drug screen found primary and metastatic FLC were vulnerable to clinically available inhibitors of TOPO1 and HDAC and to napabucasin. Napabucasin's efficacy was mediated through reactive oxygen species and inhibition of translation initiation, and specific inhibition of eIF4A was effective. The sensitivity of each PDX line inversely correlated with expression of the antiapoptotic protein Bcl-xL, and inhibition of Bcl-xL synergized with other drugs. Screening directly on cells dissociated from patient resections validated these results. This demonstrates that a direct functional screen on patient tumors provides therapeutically informative data within a clinically useful time frame. Identifying these novel therapeutic targets and combination therapies is an urgent need, as effective therapeutics for FLC are currently unavailable.Significance:Therapeutics informed by genomics have not yielded effective therapies for FLC. A functional screen identified TOPO1, HDAC inhibitors, and napabucasin as efficacious and synergistic with inhibition of Bcl-xL. Validation on cells dissociated directly from patient tumors demonstrates the ability for functional precision medicine in a solid tumor.This article is highlighted in the In This Issue feature, p. 2355

Supplementary Figure 1 - 6 and Supplementary Tables 1, 2, and 4

List of compounds in the primary screen

Background Hepatocellular carcinoma (HCC) patient‐derived xenograft (PDX) models hold potential to advance knowledge in HCC biology to help improve systemic therapies. Beside hepatitis B virus‐associated tumors, HCC is poorly established in PDX. Methods PDX formation from fresh HCC biopsies were obtained and implanted intrahepatically or in subrenal capsule (SRC). Mouse liver injury was induced in immunodeficient Fah−/− mice through cycling off nitisinone after HCC biopsy implantation, versus continuous nitisinone as non‐liver injury controls. Mice with macroscopically detectable PDX showed rising human alpha1‐antitrypsin (hAAT) serum levels, and conversely, no PDX was observed in mice with undetectable hAAT. Results Using rising hAAT as a marker for PDX formation, 20 PDX were established out of 45 HCC biopsy specimens (44%) reflecting the four major HCC etiologies most commonly identified at Memorial SloanKettering similar to many other institutions in the United States. PDX was established only in severely immunodeficient mice lacking lymphocytes and NK cells. Implantation under the renal capsule improved PDX formation two‐fold compared to intrahepatic implantation. Two out of 18 biopsies required murine liver injury to establish PDX, one associated with hepatitis C virus and one with alcoholic liver disease. PDX tumors were histologically comparable to biopsy specimens and 75% of PDX lines could be passaged. Conclusions Using cycling off nitisinone‐induced liver injury, HCC biopsies implanted under the renal capsule of severely immunodeficient mice formed PDX with 57% efficiency as determined by rising hAAT levels. These findings facilitate a more efficient make‐up of PDX for research into subset‐specific HCC.

To repurpose therapeutics for fibrolamellar carcinoma (FLC), we developed and validated patient-derived xenografts (PDX) from surgical resections. Most agents used clinically and inhibitors of oncogenes overexpressed in FLC showed little efficacy on PDX. A high-throughput functional drug screen found primary and metastatic FLC were vulnerable to clinically available inhibitors of TOPO1 and HDAC and to napabucasin. Napabucasin's efficacy was mediated through reactive oxygen species and inhibition of translation initiation, and specific inhibition of eIF4A was effective. The sensitivity of each PDX line inversely correlated with expression of the antiapoptotic protein Bcl-xL, and inhibition of Bcl-xL synergized with other drugs. Screening directly on cells dissociated from patient resections validated these results. This demonstrates that a direct functional screen on patient tumors provides therapeutically informative data within a clinically useful time frame. Identifying these novel therapeutic targets and combination therapies is an urgent need, as effective therapeutics for FLC are currently unavailable. Significance: Therapeutics informed by genomics have not yielded effective therapies for FLC. A functional screen identified TOPO1, HDAC inhibitors, and napabucasin as efficacious and synergistic with inhibition of Bcl-xL. Validation on cells dissociated directly from patient tumors demonstrates the ability for functional precision medicine in a solid tumor. This article is highlighted in the In This Issue feature, p. 2355

Fibrolamellar Hepatocellular Carcinoma (FLC) is a rare liver cancer affecting adolescents and young adults, with no gender or ethnicity predilection and without history of underlying viral hepatitis, cirrhosis, or other known risk factors. Almost all FLC patients present a somatic heterozygous deletion in chromosome 19p13.12, DNAJB1::PRKACA, which is sufficient to drive FLC in mice. A few studies comparing FLC tumors with adjacent non-transformed liver (normal) samples revealed many transcriptional differences. However, there were done in very small datasets and analyzed using different bioinformatic methods, resulting in just 18-47% agreement between them. This study aims to comprehensively characterize the transcriptome of FLC at bulk and spatial single-cell resolution. The whole transcriptome of 109 FLC frozen patient samples, the largest RNA-seq dataset of FLC to date, was sequenced using different library preparation and ribosomal depletion methods. Only paired tumor and normal tissue samples resected from the same patient were used and divided into two groups: exploration (3 datasets, 67 samples) and testing (2 datasets, 17 samples). Additionally, as external validation datasets, RNA-seq samples from previously published studies were collected, including Sorenson et al. (FLC: 26, normal: 9, paired: 8), the TCGA-LIHC study (FLC: 6, normal: 1, paired: 1), Hirsch et al. (FLC: 15, normal: 3, paired: 0) and Francisco et al. (FLC: 27, normal: 10, paired: 9). All were reanalyzed using state-of-the-art bioinformatic methods: mapped to the Human Genome GRCh38.103 and transcripts quantified using Salmon 1.6.0, unsupervised clustering exploration using PCA, tSNE and UMAP, differential expression calculated using DESeq2 1.28.1, and checking for detectability and consistency among datasets. We found 857 up- and 988 down-regulated genes presenting the same dysregulation in the exploration datasets and confirmed in the testing and external datasets. We call these genes the transcriptional FLC signature. The FLC signature was further characterized by comparing it with the genes differentially expressed in other liver cancers: hepatocellular carcinoma (41 paired samples), hepatoblastoma (22 paired samples), and cholangiocarcinoma (27 paired samples). We found 276 up- and 352 down-regulated genes altered in other liver cancers as well as FLC, but 156 up- and 68 down-regulated only in FLC. The 112 genes with the strongest dysregulation (56 up and 56 down) were used for a MERFISH screening, providing for the first time a single-cell spatial transcriptomic characterization of FLC. This showed clear differential expression patterns in tumor, normal, stromal, and infiltrating immune cells, allowing the identification of how different cell types contribute to the transcriptional FLC signature. Citation Format: David Requena, Aldhair Medico, Luis F. Soto, Mahsa Shirani, James A. Saltsman, Gadi Lalazar, Michael P. LaQuaglia, Sanford M. Simon. Bulk and spatial single-cell transcriptomic characterization of fibrolamellar hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1516.

Fibrolamellar carcinoma (FLC) is a rare, often lethal, liver cancer affecting adolescents and young adults, for which there are no approved therapeutics. The development of therapeutics is hampered by a lack of in vitro models. Organoids have shown utility as a model system for studying many diseases. In this study, tumor tissue and the adjacent non-tumor liver were obtained at the time of surgery. The tissue was dissociated and grown as organoids. We developed 21 patient-derived organoid lines: 12 from metastases, three from the liver tumor and six from adjacent non-tumor liver. These patient-derived FLC organoids recapitulate the histologic morphology, immunohistochemistry, and transcriptome of the patient tumor. Patient-derived FLC organoids were used in a preliminary high-throughput drug screen to show proof of concept for the identification of therapeutics. This model system has the potential to improve our understanding of this rare cancer and holds significant promise for drug testing and development., Stem Cell Reports, 17 (8), ISSN:2213-6711

Fibrolamellar hepatocellular carcinoma (FLC) is a lethal pediatric cancer affecting adolescents and young adults. Besides surgical resection, there is no approved and effective therapy. FLC is driven by a fusion oncokinase, DNAJB1-PRKACA, that arises from a deletion fusing exon 1 of DNAJB1 and exons 2-10 of PRKACA, the catalytic subunit of protein kinase A. Expression of this chimeric oncokinase is capable of recapitulating the key histologic and transcriptomic features of FLC. Using PDXs, we tested therapeutics that have been used clinically for FLC or that target pathways identified via the transcriptomic signature of FLC, in addition to an agnostic library of clinical-stage drugs. Several compounds were identified with a varying degree of efficacy in different PDX lines as compared to our control line, primary human hepatocytes. One of the top hits was irinotecan, and its active metabolite SN38. Irinotecan is a topoisomerase I inhibitor and is clinically utilized in a wide variety of adult and pediatric cancers. A second top hit was Navitoclax, an inhibitor of the anti-apoptotic proteins Bcl-2 and Bcl-xL. Tool compounds that targeted Bcl-xL, but not bcl-2 were efficacious. This suggested a possible link between Bcl-xL, which is overexpressed in some PDX lines, and the resistance profile they showed in our screen. Unfortunately, Navitoclax has an on-target and dose-limiting toxicity of thrombocytopenia, which limits its clinical application. DT2216 is a VHL-based PROTAC that degrades Bcl-xL. DT2216 has two potential benefits over Navitoclax: 1) DT2216 is a more potent than Navitoclax against various Bcl-xL dependent tumor cells; and 2) it is less toxic to platelets than Navitoclax by targeting Bcl-xl to VHL that is poorly expressed in platelets to avoid induction of severe thrombocytopenia. A phase 1 clinical trial of DT2216 in relapsed/refractory malignancies is now ongoing (NCT04886622). We tested if the combination of DT2216 and irinotecan might have a therapeutic potential in FLC. Indeed, in vitro screening of the proposed combo, DT2216 and SN38, showed an additive or a synergistical effect in 4 validated FLC cell lines. We tested both the efficacy of DT2216 on Bcl-xL levels in FLC as well as platelet counts to identify any developing thrombocytopenia. DT2216 was less disruptive to platelets than Navitoclax at therapeutically relevant pre-clinical doses. Next, we tested 4 of our PDX models for FLC using various dosing regimens to identify the most efficacious regimen with the minimum side effects. Mice cohorts were evaluated for weight loss and tumor volume. Mice treated with DT2216 and SN38 showed consistent therapeutic benefit which was not evident in controls and less notable as monotherapy. In conclusion, TOPO1 and Bcl-xL prove to be promising targets of interest in FLC. Targeting both with DT2216 and irinotecan leads to tumor shrinkage in pre-clinical models and offer a potential therapeutic/pharmacological intervention for FLC. Citation Format: Bassem Shebl, Gadi Lalazar, Denise Ng, Tova Finkelstein, Guangrong Zheng, Peiyi Zhang, Carly Rosemore, Michael Ortiz, Daohong Zhou, Sanford Simon, Philip Coffino. Targeting Bcl-xL in fibrolamellar hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3888.

Advanced non-alcoholic fatty liver disease (NAFLD) is a rapidly emerging global health problem associated with pre-disposing genetic polymorphisms, most strikingly an isoleucine to methionine substitution in patatin-like phospholipase domain-containing protein 3 (PNPLA3-I148M). Here, we study how human hepatocytes with PNPLA3 148I and 148M variants engrafted in the livers of chimeric mice respond to a hypercaloric Western-style diet. As early as 4 weeks, mice developed dyslipidemia, impaired glucose tolerance, and steatohepatitis selectively in the human graft, followed by pericellular fibrosis after 8 weeks of hypercaloric feeding. The PNPLA3 148M variant, either from a homozygous 148M human donor or overexpressed in a homozygous 148I donor background, caused widespread microvesicular steatosis and even more severe steatohepatitis. We conclude that PNPLA3 148M in human hepatocytes exacerbates NAFLD. These models will facilitate mechanistic studies into human genetic variants associated with advanced fatty liver diseases.

Advanced non-alcoholic fatty liver disease (NAFLD) is a rapidly emerging global health problem associated with pre-disposing genetic polymorphisms, most strikingly an isoleucine to methionine substitution in patatin-like phospholipase domain-containing protein 3 (PNPLA3-I148M). Here, we study how human hepatocytes with PNPLA3 148I and 148M variants engrafted in the livers of chimeric mice respond to hypercaloric diets. As early as 4 weeks, mice developed dyslipidemia, impaired glucose tolerance, and steatohepatitis selectively in the human graft, followed by pericellular fibrosis after 8 weeks of hypercaloric feeding. The PNPLA3 148M variant, either from a homozygous 148M human donor or overexpressed in a homozygous 148I donor background, caused microvesicular and more severe steatosis. In these livers hepatocytes displayed frequent ballooning degeneration, resulting in more active steatohepatitis than in 148I livers. We conclude that PNPLA3 148M in human hepatocytes exacerbates NAFLD. These models will facilitate mechanistic studies into human genetics associated with advanced fatty liver diseases.

Fibrolamellar hepatocellular carcinoma (FLC) is a rare form of primary liver cancer that affects adolescents and young adults without underlying liver disease. Surgery remains the mainstay of therapy; however, most patients are either not surgical candidates or suffer from recurrence. There is no approved systemic therapy and the overall survival remains poor. Historically classified as a subtype of hepatocellular carcinoma (HCC), FLC has a unique clinical, histological, and molecular presentation. At the genomic level, FLC contains a single 400kB deletion in chromosome 19, leading to a functional DNAJB1-PRKACA fusion protein. In this review, we detail the recent advances in our understanding of the molecular underpinnings of FLC and outline the current knowledge gaps.

Background and Aims: Acetaminophen (APAP) and HMG-CoA reductase inhibitors are common causes of drug-induced liver injury (DILI). This study aimed to determine the ability to reduce APAP- and statins-mediated liver injury by using formulations that combine glycosphingolipids and vitamin E. Methods: Mice were injected with APAP or with statins and treated before and after with β-glucosylceramide (GC), with or without vitamin E. Mice were followed for changes in liver enzymes, liver histology, hepatic expression of JNK, STAT3 and caspase 3, as well as intrahepatic natural killer T cells (NKT) and the serum cytokine levels by flow cytometry. Results: Administration of GC before or after APAP alleviated the liver damage, as noted by a reduction of the liver enzymes, improvement in the liver histology and decreased hepatic caspase 3 expression. Beneficial effect was associated with a reduction of the intrahepatic NKT, JNK expression in the liver, and increased glutathione in the liver, and decreased TNF-α serum levels. Synergistic effect of co-administration of GC with vitamin E was observed. Similar protective effect of GC on statin-mediated liver damage was documented by a reduction in liver enzymes and improved liver histology, which was mediated by reduction of NKT, increased STAT3 expression in the liver, and reduced the TGF-β and IL17 levels. Conclusions: β-glycosphingolipids exert a hepatoprotective effect on APAP- and statins-mediated liver damage. Vitamin E exerted a synergistic effect to that of GC. The generation of “safer drug” formulations, which include an active molecule combined with a hepatoprotective adjuvant, may provide an answer to the real unmet need of DILI.

// Benjamin A. Farber 1, 2 , Gadi Lalazar 1 , Elana P. Simon 1 , William J. Hammond 1, 2 , David Requena 1 , Umesh K. Bhanot 3 , Michael P. La Quaglia 2 and Sanford M. Simon 1 1 Laboratory of Cellular Biophysics, The Rockefeller University, New York, 10065 NY, USA 2 Division of Pediatric Surgery, Department of Surgery, Memorial Sloan-Kettering Cancer Center, New York, 10065 NY, USA 3 Pathology Core Facility Memorial Sloan-Kettering Cancer Center, New York, 10065 NY, USA Correspondence to: Sanford M. Simon, email: simon@rockefeller.edu Keywords: pediatric cancer; fusion protein; microRNA; lncRNAs; liver cancer Received: September 11, 2017 Accepted: December 08, 2017 Published: December 15, 2017 ABSTRACT Fibrolamellar hepatocellular carcinoma (FLC) is a rare primary liver cancer found in adolescents and young adults without underlying liver disease. A deletion of ~400 kD has been found in one copy of chromosome 19 in the tumor tissue of all patients tested. This produces a fusion of the genes DNAJB1 and PRKACA which, in turn, produces a chimeric transcript and protein. Transcriptomic analysis of the tumor has shown upregulation of various oncologically relevant pathways, including EGF/ErbB, Aurora Kinase A, pak21 and wnt. To explore other factors that may contribute to oncogenesis, we examined the microRNA (miRNA) and long non-coding RNA (lncRNA) expression in FLC. The non-coding RNA expression profile in tumor tissue samples is distinctly different from the adjacent normal liver and from other liver tumors. Furthermore, miRZip knock down or over expression of certain miRNAs led to changes in the levels of coding genes that recapitulated changes observed in FLC, suggesting mechanistically that the changes in the cellular levels of miRNA are not merely correlative. Thus, in addition to serving as diagnostic tools for FLC, non-coding RNAs may serve as therapeutic targets.

A segmental deletion resulting in DNAJB1-PRKACA gene fusion is now recognized as the signature genetic event of fibrolamellar hepatocellular carcinoma (FL-HCC), a rare but lethal liver cancer that primarily affects adolescents and young adults. Here, we implement CRISPR/Cas9 genome editing and transposon-mediated somatic gene transfer to demonstrate that expression of both the endogenous fusion protein or a chimeric cDNA leads to the formation of indolent liver tumors in mice that closely resemble human FL-HCC. Notably, overexpression of the wild type PRKACA was unable to fully recapitulate the oncogenic activity of DNAJB1-PRKACA, implying that FL-HCC does not simply result from enhanced PRKACA expression. Tumorigenesis was significantly enhanced by genetic activation of β-catenin, an observation supported by evidence of recurrent Wnt pathway mutations in human FL-HCC, as well as treatment with hepatotoxin 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC), which causes tissue injury, inflammation and fibrosis. Our study validates the DNAJB1-PRKACA fusion kinase as an oncogenic driver and candidate drug target for FL-HCC and establishes a practical model for preclinical studies to identify strategies to treat this disease.SignificanceEfforts to understand and treat FL-HCC have been confounded by a lack of models that accurately reflect the genetics and biology of the disease. Here, we demonstrate that the Dnajb1-Prkaca gene fusion drives tumorigenesis in mice, and that fusion to DNAJB1 drives FL-HCC initiation more effectively than wild type PRKACA overexpression. The requirement of the PRKACA kinase domain in tumor initiation establishes the potential utility of kinase inhibitors targeting the fusion. By identifying genetic and environmental factors that can enhance the consistency and aggressiveness of disease progression, we reveal biological characteristics of the disease and advance a robust platform for future pre-clinical studies.

During sepsis, bacterial products, particularly LPS, trigger injury in organs such as the liver. This common condition remains largely untreatable, in part due to a lack of understanding of how high concentrations of LPS cause cellular injury. In the liver, the lysosomal degradative pathway of autophagy performs essential hepatoprotective functions and is induced by LPS. We, therefore, examined whether hepatocyte autophagy protects against liver injury from septic levels of LPS. Mice with an inducible hepatocyte-specific knockout of the critical autophagy gene Atg7 were examined for their sensitivity to high-dose LPS. Increased liver injury occurred in knockout mice, as determined by significantly increased serum alanine aminotransferase levels, histological evidence of liver injury, terminal deoxynucleotide transferase-mediated deoxyuridine triphosphate nick end-labeling, and effector caspase-3 and -7 activation. Hepatic inflammation and proinflammatory cytokine induction were unaffected by the decrease in hepatocyte autophagy. Although knockout mice had normal NF-κB signaling, hepatic levels of Akt1 and Akt2 phosphorylation in response to LPS were decreased. Cultured hepatocytes from knockout mice displayed a generalized defect in Akt signaling in response to multiple stimuli, including LPS, TNF, and IL-1β. Akt activation mediates hepatocyte resistance to TNF cytotoxicity, and anti-TNF antibodies significantly decreased LPS-induced liver injury in knockout mice, indicating that the loss of autophagy sensitized to TNF-dependent liver damage. Hepatocyte autophagy, therefore, protects against LPS-induced liver injury. Conditions such as aging and steatosis that impair hepatic autophagy may predispose to poor outcomes from sepsis through this mechanism.

Hepatoblastoma is the most common childhood liver cancer. Although survival has improved significantly over the past few decades, there remains a group of children with aggressive disease who do not respond to current treatment regimens. There is a critical need for novel models to study aggressive hepatoblastoma as research to find new treatments is hampered by the small number of laboratory models of the disease. Organoids have emerged as robust models for many diseases, including cancer. We have generated and characterized a novel organoid model of aggressive hepatoblastoma directly from freshly resected patient tumors as a proof of concept for this approach. Hepatoblastoma tumor organoids recapitulate the key elements of patient tumors, including tumor architecture, mutational profile, gene expression patterns, and features of Wnt/&beta, catenin signaling that are hallmarks of hepatoblastoma pathophysiology. Tumor organoids were successfully used alongside non-tumor liver organoids from the same patient to perform a drug screen using twelve candidate compounds. One drug, JQ1, demonstrated increased destruction of liver organoids from hepatoblastoma tumor tissue relative to organoids from the adjacent non-tumor liver. Our findings suggest that hepatoblastoma organoids could be used for a variety of applications and have the potential to improve treatment options for the subset of hepatoblastoma patients who do not respond to existing treatments.

Fibrolamellar Hepatocellular Carcinoma (FLC) is a usually lethal liver cancer affecting adolescents and young adults without previous liver disease. FLC is characterized by a ~400kb nucleotide deletion resulting in a fusion of a heatshock protein cofactor, DNAJB1, with the catalytic subunit of protein kinase A (PRKACA). One limitation to the study of this disease is the lack of in vitro models. Organoids have shown utility as a model system for studying many diseases, including cancer. This study describes the development and characterization of FLC tumor organoids. With IRB approval, normal liver and FLC tumor tissue was obtained from patients undergoing surgical resection. Liver cells were isolated from the tissue samples and placed into a 3D basement membrane matrix and the media supplemented with growth factors specific to promote liver organoid expansion. Normal and tumor organoids were analyzed by microscopy for morphology, by PCR for presence of the DNAJB1-PRKACA chimeric transcript, by Western blot for chimeric protein expression, and by RNA sequencing for transcriptomic changes. Eight organoid lines have been developed from normal liver and eight from FLC tumor tissue, including multiple independent organoids developed from different metastases from the same patient. A distinct morphological difference between normal liver organoids and FLC tumor organoids can be seen with either brightfield microscopy or with H&E. The DNAJB1-PRKACA chimeric transcript was detected in all of the tumor tissue and tumor organoids by RT-PCR and not detected in any of the normal tissue or organoids. The presence of the DNAJB1-PRKACA fusion protein was verified by Western blot in tumor organoids and tissue but not detectable in the normal tissue. The RNA sequencing analysis shows clustering of tumor organoids with tumor tissue. Tumor organoids injected subcutaneously in mice grew as tumors. Now that the normal and tumor organoids have been developed and validated, they are being used for screening for potential therapeutics. Citation Format: Nicole J. Croteau, David Requena, Gadi Lalazar, Denise Ng, Solomon Levin, Bassem Shebl, Ruisi Wang, William J. Hammond, James A. Saltsman, Helmuth Gehart, Hans Clevers, Michael P. LaQuaglia, Sanford Simon. Human liver organoids for disease modeling of fibrolamellar hepatocellular carcinoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3905.

Introduction: Fibrolamellar hepatocellular carcinoma (FLC) is a liver cancer affecting adolescents and young adults without underlying liver disease. Patients with FLC are often diagnosed with advanced-stage disease and have a poor 5-year survival. Recently we have made advances in both understanding the etiology of FLC as well as developing therapeutics. We have found that the bulk of the genome lacks recurrent single-nucleotide or structural variants (inversions, amplifications) with the exception of a deletion in one copy of chromosome 19. This results in a fusion of the first exon of the heat shock protein, DNAJB1, with the 2nd through 10th exons of PRKACA, the catalytic subunit of protein kinase A. Expression of this fusion in the livers of mice, either by CRISPR or transposon, is sufficient to phenocopy the disease. The kinase activity of the fusion protein is essential for transformation, but equivalent expression of the native kinase is not sufficient. Results: Etiology: We have tested the ability of the native kinase and fusion oncokinase to phosphorylate potential substrates in human liver. We have found a discrete set of substrates that are phosphorylated in human liver for which we are now testing, which are critical for the pathogenesis of this cancer. Therapeutics: We have purified the fusion oncokinase without a tag and used a high-throughput screen to find inhibitors. We have found inhibitors in the nanomolar range for which we are using structural analysis to inform our medicinal chemistry. Additionally, we have developed several “never-in-plastic” patient-derived xenograft (PDX) models that retain expression of the driver, histology, transcriptome, and proteome of the original human tumor. We have also screened for compounds that induce tumor cell death and tumor regression in FLC PDXs. Conclusion: Subtle changes in the kinase substrate profile of the fusion kinase provide new leads for therapeutics for FLC. The ability to find inhibitors of the kinase in the nanomolar range, as well as the ability to screen for inhibitors of the PDX, offers the first hope for treatment against this lethal tumor. Citation Format: Gadi Lalazar, Melissa Jarmel, Ruisi Wang, Michael Tomasini, David Requena, Denise Ng, Sanford M. Simon. Advances in the etiology and therapeutics of a lethal childhood cancer, fibrolamellar hepatocellular carcinoma [abstract]. In: Proceedings of the AACR Special Conference on the Advances in Pediatric Cancer Research; 2019 Sep 17-20; Montreal, QC, Canada. Philadelphia (PA): AACR; Cancer Res 2020;80(14 Suppl):Abstract nr A51.

Protein degradation by the ubiquitin–proteasome system (UPS) is central to protein homeostasis and cell survival. The active 26S proteasome is a large protease complex consisting of a catalytic 20S subunit and 19S regulatory particles. Cancer cells are exposed to considerable protein overload due to high metabolic rates, reprogrammed energy metabolism, and aneuploidy. Here we report a mechanism that facilitates the assembly of active 26S proteasomes in malignant cells. Upon tumorigenic transformation of the gut epithelium, 26S proteasome assembly was significantly enhanced, but levels of individual subunits were not changed. This enhanced assembly of 26S proteasomes increased further with tumor progression and was observed specifically in transformed cells, but not in other rapidly dividing cells. Moreover, expression of PSMD5, an inhibitor of proteasome assembly, was reduced in intestinal tumors and silenced with tumor progression. Reexpression of PSMD5 in tumor cells caused decreased 26S assembly and accumulation of polyubiquitinated proteins. These results suggest that inhibition of cancer-associated proteasome assembly may provide a novel therapeutic strategy to selectively kill cancer cells. Significance: Enhanced cancer-associated proteasome assembly is a major stress response that allows tumors to adapt to and to withstand protein overload. Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/78/13/3458/F1.large.jpg. Cancer Res; 78(13); 3458–68. ©2018 AACR.

β-glucosylceramide (GC) is a naturally occurring glycosphingolipid that was shown to improve hepatic steatosis, steatohepatitis, and insulin resistance in animal models of nonalcoholic fatty liver disease. In this study, we evaluated the safety and efficacy of oral administration of GC in subjects with nonalcoholic steatohepatitis (NASH). Twenty-three patients with biopsy proven NASH were enrolled in a double-blind, placebo-controlled trial. Patients were orally administered daily with 7.5 mg of GC. Patients were followed for safety, liver enzymes, HbA1c, insulin sensitivity, lipid profile, hepatic fat content as measured by magnetic resonance imaging (MRI), and NASH score on liver biopsy. No treatment-related adverse events were observed during treatment. In a per protocol analysis of data, oral administration of GC decreased the hepatic fat content as measured by MRI in GC-treated compared with placebo. HbA1C decreased in patients treated with GC. GC treatment was associated with a milder decrease in the high-density lipoprotein serum levels. The beneficial effects were associated with a decrease in CD4 and NKT cell subsets of lymphocytes. Due to the small number of subjects enrolled, differences did reach statistical significance. Oral administration of GC is safe and biologically active in patients with NASH and insulin resistance.

A previously healthy, 25-year-old man was admitted to the hospital because of abdominal pain, nausea, vomiting, and weight loss. Two weeks before his admission, fever (temperature up to 40°C), chills, and weakness developed. To control his fever, the patient ingested ibuprofen at a dose of 400 mg four times a day for more than a week. Subsequently, abdominal discomfort and nausea developed, and he presented to the emergency department owing to worsening of epigastric pain and vomiting. Since his gastrointestinal symptoms had started, he had lost about 4 kg of body weight. He reported no hematemesis, hematochezia, or melena. His medical history was remarkable only for a left inguinal hernioplasty 3 years earlier. He did not smoke, ingest alcohol, or use illicit drugs. This patient’s symptoms of fever, chills, and malaise are nonspecific and may occur with viral or bacterial infection or noninfectious inflammation. Protracted vomiting or nausea may lead to Mallory–Weiss tears at the gastroesophageal junction, causing epigastric pain and possibly upper gastrointestinal bleeding. Pancreatitis may also explain this constellation of symptoms, although the patient reports no alcohol ingestion and has no known history of gallstones. Intestinal obstruction is another, more remote possibility. The patient’s use of ibuprofen puts him at risk for nonsteroidal antiinflammatory drug (NSAID)–induced gastritis; prepyloric edema could lead to partial gastric obstruction and vomiting. Treatment with a proton-pump inhibitor may alleviate his symptoms pending further evaluation. On physical examination, the patient was pale and weak but did not appear to be in distress. His blood pressure was 125/75 mm Hg in a supine position and 105/70 mm Hg while he was standing. The pulse rate was 96 beats per minute, and the temperature was 36°C. His tongue appeared dry. His tonsils were not enlarged, and there was no exudate. There was no cervical or axillary adenopathy. He had marked epigastric tenderness without rebound or abdominal rigidity. The spleen and liver were mildly enlarged. There was no rash. The patient’s physical examination is consistent with dehydration, but there is no evidence of peritonitis, which would suggest perforation. The mild hepatic and splenic enlargement may be consistent with a viral illness, which could also explain his recent fever and its absence on admission. Alternatively, the patient’s fever may be intermittent, as in Hodgkin’s disease (Pel–Ebstein fever); the abdominal pain, weight loss, and hepatic and splenic enlargement could also be consistent with this diagnosis.

Objective To investigate the effect of β-glucosylceramide (GC), a natural glycolipid, on hepatic fat accumulation and regenerative response after partial hepatectomy (PH). Methods Male C57Bl/6 mice were assigned to either 70% PH or sham surgery after receiving daily intraperitoneal injection of GC or vehicle for 3 days. Hepatic fat accumulation, cytokines, cell cycle proteins and adipogenic genes expression were assessed at various time points after PH. Results The administration of GC delayed hepatic triglyceride accumulation during hepatic regeneration. This observation was closely correlated with alterations in the expression of four major adipogenic genes during the course of liver regeneration, with reduced expression 3 h after PH and increased expression 48 h post-surgery. GC significantly reduced hepatocellular proliferation 48 h after PH. In GC-treated mice, both tumor necrosis factor-α and interleukin-6 levels were lower 3, 48 and 72 h after PH compared with the control group. Conclusions Administration of GC delayed hepatic triglyceride accumulation and suppressed early adipogenic gene expression during the hepatic regenerative response. These changes are closely associated with early inhibition of liver regeneration and temporal alteration of cytokine secretion.

BACKGROUND AND AIM Distinguishing biliary atresia (BA) from other causes of neonatal cholestasis (NC) is challenging. Continuous BreathID C-methacetin breath test (MBT) is a novel method that determines liver function. Methacetin is metabolized uniquely by the liver and CO2 is measured passively, through a nasal cannula in the exhaled breath. The aim of this study was to assess the ability of MBT to differentiate BA from other causes of NC. METHODS MBT was performed in infants with NC before any invasive procedure. Percent dose recovered (PDR) peak and time to peak (TTPP) of C recovered were correlated with blood test results and degree of fibrosis on liver biopsy. RESULTS Fifteen infants were enrolled in the study. Eight were eventually diagnosed as having BA. MBT showed that infants with NC from various causes reached the PDR peak after 44.5 ± 6.7 minutes, whereas infants with BA reached the PDR peak value after 54.7 ± 4.3 minutes (P < 0.005). This suggested low cytochrome P450 1A2 activity in the BA group. The area under the curve (AUC) was 0.95 (95% confidence interval [CI] 0.83-1), sensitivity of 88%, and specificity of 100%. CONCLUSIONS This pilot study shows that MBT can differentiate between BA and other causes of NC by time to peak of methacetin metabolism. The results suggest that MBT may be used as part of the diagnostic algorithm in infants with liver disease. Larger-scale studies should be conducted to confirm these initial observations.

Stressed-out dads are clearly a torment to their children, but new research suggests that even unborn offspring aren’t protected from frazzled fathers. Genetic and environmental risk factors have historically taken center stage in the understanding of metabolic diseases such as type II diabetes. However, recent studies of nongenetic inheritance mechanisms suggest that parents’ behavioral patterns—even prior to conception—can have far reaching effects on the metabolic programming of their offspring. Stress is a common affliction of modern society and increases the risk of type II diabetes, but whether paternal stress could increase this risk in offspring was unknown. Now, Wu et al . show that in mice, paternal stress leads to hyperglycemia in offspring through up-regulation of the enzyme phosphoenolpyruvate carboxykinase (PEPCK). Mice (the F0 generation) subjected to a daily restraint stress model for 14 days exhibited hyperglycemia, as did their offspring (F1). Abnormal pyruvate tolerance in the F1 mice, in the presence of normal insulin sensitivity, implicated hyperactive hepatic gluconeogenesis, an effect that was most prominent in F1 males. Examination of the key enzymes that regulate gluconeogenesis revealed control amounts of PEPCK mRNA but significantly increased protein levels. To investigate posttranslational regulation of gene expression, the authors conducted a hepatic microRNA (miR) microarray analysis; it revealed down-regulation of miR-466b-3p in F1 mice, which targets the PEPCK 3ʹ untranslated region. Using multiple approaches, the authors showed that miR-466b-3p represses the translation of PEPCK, preventing excess gluconeogenesis. miR-466b-3p is encoded within the Sfmbt2, a polycomb gene, and the transcription of miR-466b-3p was shown to be negatively regulated by methylation of the Sfmbt2 promoter. Indeed, increased methylation of this promoter was demonstrated in both the sperm of stressed mice and specifically in the livers of their male offspring. This pattern of methylation was replicated by injecting exogenous glucocorticoids into nonstressed male F0 mice and blocked by injecting a glucocorticoid inhibitor into stressed F0 male mice. These observations implicate stress-induced glucocorticoid release as the mechanism of inducing posttranslational regulation of PEPCK. Although genetic and environmental risk factors are clearly important to the pathogenesis of type II diabetes, behavior patterns—including paternal stress—appear to have a deleterious effect on male offspring’s health, through nongenetic inheritance. If these findings prove true in humans, avoiding stress might be prudent not only for an individual’s health, but also for that of their children. L. Wu et al ., Paternal psychological stress reprograms hepatic gluconeogenesis in offspring. Cell Metab. 10.1016/j.cmet.2016.01.014 (2016). [[Full Text]][1] [1]: http://www.cell.com/cell-metabolism/pdfExtended/S1550-4131(16)30006-7

A link between altered levels of various gangliosides and the development of insulin resistance was described in transgenic mice. Naturally occurring glycosphingolipids were shown to exert immunomodulatory effects in a natural killer T (NKT) cell-dependent manner. This study examined whether glycosphingolipid-induced modulation of the immune system may reduce pancreatic and liver steatosis and stimulate insulin secretion in the Cohen diabetes-sensitive (CDS) rat, a lean model of non-insulin-resistant, nutritionally induced diabetes. Four groups of CDS rats fed a diabetogenic diet were treated with daily intraperitoneal injections of glycosphingolipids β-glucosylceramide, β-lactosylceramide, a combination of both (IGL), or vehicle (PBS) for up to 45 days. Immune modulation was assessed by fluorescence-activated cell sorting analysis of intrahepatic and intrasplenic lymphocytes. Steatosis was assessed by MRI imaging and histological examination of liver and pancreas, Blood glucose and plasma insulin concentrations were assessed during an oral glucose tolerance test. Administration of glycosphingolipids, particularly IGL, increased intrahepatic trapping of CD8 T and NKT lymphocytes. Pancreatic and liver histology were markedly improved and steatosis was reduced in all treated groups compared with vehicle-treated rats. Insulin secretion was restored after glycosphingolipid treatment, resulting in improved glucose tolerance. The immunomodulatory effect of β-glycosphingolipids improved the β-cell function of the hyperglycemic CDS rat. Thus our results suggest a role for the immune system in the pathogenesis of diabetes in this model.

Summary. Up to 30% of patients with hepatitis C virus (HCV) infection and normal serum alanine aminotransferase (NALT) have significant liver disease. Currently, many of these patients undergo a liver biopsy to guide therapeutic decisions. The BreathID® continuous online 13C-methacetin breath test (MBT) reflects hepatic microsomal function and correlates with hepatic fibrosis. To assess its role in identifying intrahepatic inflammation and fibrosis in NALT patients, we tested 100 patients with untreated chronic HCV infection, and 100 age- and sex-matched healthy volunteers using 13C MBT following ingestion of 75 mg methacetin. All HCV patients had undergone a liver biopsy within 12 months of performing the MBT. Patients with a necroinflammatory grade ≤4 or >4, based on Ishak (modified HAI) score, HAIa + HAIb + HAIc + HAId, were defined as having low or high inflammation, respectively. Patients with a histological activity fibrosis stage ≤2 or >2, were defined as having nonsignificant or significant fibrosis, respectively. A proprietary algorithm to differentiate intrahepatic inflammation within chronic HCV patients with NALT achieved an area under the curve (AUC) of 0.90. Setting a threshold on the point of best agreement (at 83%) results in 82% sensitivity and 84% specificity. With application of another proprietary algorithm to differentiate patients with nonsignificant or significant fibrosis, 67% of liver biopsies performed in the patient group could have been avoided. This algorithm achieved an AUC of 0.92, with a sensitivity of 91% and a specificity of 88%. There was no correlation between body mass index (BMI) and MBT scores for patients with the same histological score. The continuous BreathID®13C MBT is an accurate tool for measuring the degree of inflammation and fibrosis in patients with chronic HCV infection and NALT. As such, it may prove to be a powerful, noninvasive alternative to liver biopsy in the management of this patient population.

The integrity of lipid rafts in cell membranes is important for signal transduction. Aim To determine the distinct effects of β-glycolipids on the composition of lipid rafts in natural killer T (NKT) cells and on the level of expression of flotillin-2, leukocyte-specific protein tyrosine kinase (Lck), and STAT1-associated pathways. Methods The effects of glycolipids were determined by composition analysis of the raft domains, FACS analysis of the distribution patterns for the raft ganglioside, GM1, and fluorescence microscopy of raft patching. To evaluate the effects of the immune environment on glycolipid-associated alteration of lipid rafts, hepatitis was induced by an intravenous injection of concanavalin A (ConA) in mice treated with various glycolipids. Results The administration of β-glucosylceramide, β-lactosylceramide, and a combination of both significantly altered GM1 content in lymphocyte membranes in an environment-dependent manner. These effects were associated with altered expression levels of flotillin-2, Lck, and STAT1, and with a significant decrease in intrahepatic CD8+ lymphocyte trapping and the alleviation of ConA-induced hepatitis. The administration of α-glycolipids failed to induce similar effects. Conclusions The alteration in the expression levels of flotillin-2, Lck, and STAT1 that occurs concomitantly with changes in lipid raft composition and structure following the administration of β-glycolipids in ConA-induced hepatitis is microenvironment-dependent and is associated with decreased intrahepatic CD8+ T lymphocyte trapping and amelioration of immune-mediated hepatitis.

Carbohydrates are an important source of energy for many animal species, but excessive consumption can cause obesity, liver toxicity, and metabolic syndrome. Fibroblast growth factor 21 (FGF21) is a pleotropic liver-derived hormone that regulates diverse aspects of energy homeostasis, circadian behavior, growth, and female reproduction. Now, Talukdar et al . show that FGF21 suppresses sweet and alcohol preferences in mice and sweet preference in monkeys by decreasing dopamine, a key neurotransmitter in reward pathways in the brain. FGF21 levels increase in response to carbohydrate consumption in rodents and humans, and certain SNPs in the FGF21 gene are associated with increased carbohydrate consumption in humans. FGF21 is known to elicit its effects in the brain through a cell surface receptor that is composed of FGF receptor 1 and the β-Klotho receptor, a β-glucuronidase implicated in insulin sensitivity and aging. Transgenic Fgf21 mice that express supraphysiological levels of FGF21 or wild-type mice that received exogenous FGF21 showed significant decreases in both sucrose and saccharin preferences. These decreases were abrogated in mice that specifically lacked the β-Klotho receptor in the brain. Administration of FGF21 similarly decreased ethanol preference in mice but had no effect on responses to fatty acids and bitter taste. The effect of FGF21 on carbohydrate preference was also observed in nonhuman primates (obese cynomolgus monkeys). FGF21 receptor expression was localized specifically in the brain’s mesolimbic pathway [ventral tegmental area (VTA) and nucleus accumbens (NAc)], which regulates reward behavior primarily through dopamine signaling. Indeed, FGF21 administration decreased dopamine and its metabolites in the NAc. Long-acting FGF21 analogues are currently in clinical trials for the treatment of obesity and type 2 diabetes. Although the mechanism by which FGF21 reduces dopamine concentrations remains unknown, these findings warrant additional studies to evaluate FGF21’s effects on carbohydrate preference and other reward behavior in humans. S. Talkudar et al ., FGF21 regulates sweet and alcohol preference. Cell Metab. 10.1016/j.cmet.2015.12.008 (2015). [[Abstract]][1] [1]: http://dx.doi.org/10.1016/j.cmet.2015.12.008

Natural killer T (NKT) cells are a subset of immune regulatory cells. Several glycolipids and phospholipids derived from mammalian, bacterial, protozoan and plant species have recently been identified as possible natural ligands for NKT cells. One such compound, sulfatide, which is a major glycolipid of myelin, has been shown to modulate NKT cell activity. This patent application claims to ameliorate or prevent symptoms of immune related disorders by application of sulfatides. Although preclinical studies have effectively targeted NKT cells for immunotherapy, little is known regarding the early in vivo response of these cells to antigenic stimulation. The data in support of the present patent may suggest that the naturally β-anomeric glycosphingolipids (sulfatides) may be ligands for NKT lymphocytes.

Primary biliary cirrhosis (PBC) and systemic sarcoidosis are granulomatous diseases of unknown etiology whose hepatic manifestations may infrequently be imitative of one another. Described herein is the first reported case in the medical literature of systemic sarcoidosis developing after liver transplantation for PBC. The presented patient, who suffered from typical clinical, laboratory, and pathologic manifestations of PBC, developed decompensated liver cirrhosis within a course of 8 years, necessitating orthotopic liver transplantation. A year and a half after transplantation, the patient developed diffuse, biopsy-proven, dermatologic and pulmonary manifestations of systemic sarcoidosis, which promptly responded to corticosteroid treatment. In retrospect, the patient's longstanding liver disease was probably caused by an unrecognizable, isolated hepatic form of sarcoidosis or an overlap between PBC and sarcoidosis. This patient illustrates the complexity that may be rarely encountered in differentiating between PBC and hepatic sarcoidosis. Discussed are the clinical, laboratory, and pathologic overlaps between hepatic sarcoidosis and PBC, and clues that may aid in the diagnosis and differentiation between the 2 disorders. Hepatologists and liver transplantation specialists should be aware of the rare possibility of hepatic sarcoidosis imitating PBC, and exacerbating systemically after liver transplantation.

Obesity is linked to increased mortality. However, BMI (body mass index), the usual definition of obesity, may not actually be the best measure. Other metrics like WHR (waist-to-hip ratio), which measures central obesity (adipose tissue carried in the abdomen), may be better predictors of morbidity and mortality. Now Sahakyan et al . present data showing that normal-weight U.S. adults with central obesity have poorer long-term survival than those who have normal fat distribution, regardless of BMI. These authors surveyed a nationally representative sample of 30,818 individuals. About half of these had a BMI greater than 18.5 and available WHR information. During a mean follow-up of 14.3 years, there were 3222 deaths; of these, 1404 were due to cardiovascular disease. A multivariable Cox proportional hazards analysis showed that WHR—but not BMI—was associated with a higher risk of mortality. Men of normal weight but with central obesity had a higher risk of death than men with a similar BMI and no central obesity, or men with any other combination of BMI and WHR (HR range 1.22 to 2.42), even when compared with obese men with central obesity. A similar but less pronounced pattern was seen in women, and mortality risk further increased with age in both sexes. Possible mechanisms linking WHR with increased mortality risk include visceral fat accumulation, decreased muscle mass, and decreased leg and buttock adiposity. The risk of central obesity was also apparent when only mortality from cardiovascular causes was considered (even beyond the influence of obesity-related cardiovascular risk factors). Some caution should be exercised regarding these results because comorbidities were self-reported by the participants, and WHR was not measured with the commonly recommended technique (classification according to baseline parameters). Nonetheless, this study identifies people with normal-weight central obesity as a previously underappreciated target population for preventative strategies. K. R. Sahakyan et al ., Normal-weight central obesity: Implications for total and cardiovascular mortality. Ann. Intern. Med. 10.7326/M14-2525 (2015) [[Abstract]][1] [1]: http://annals.org/article.aspx?articleid=2468805

Fibrolamellar hepatocellular carcinoma (FLHCC) tumors all carry a deletion of ∼ 400 kb in chromosome 19, resulting in a fusion of the genes for the heat shock protein, DNAJ (Hsp40) homolog, subfamily B, member 1, DNAJB1, and the catalytic subunit of protein kinase A, PRKACA. The resulting chimeric transcript produces a fusion protein that retains kinase activity. No other recurrent genomic alterations have been identified. Here we characterize the molecular pathogenesis of FLHCC with transcriptome sequencing (RNA sequencing). Differential expression (tumor vs. adjacent normal tissue) was detected for more than 3,500 genes (log2 fold change ≥ 1, false discovery rate ≤ 0.01), many of which were distinct from those found in hepatocellular carcinoma. Expression of several known oncogenes, such as ErbB2 and Aurora Kinase A, was increased in tumor samples. These and other dysregulated genes may serve as potential targets for therapeutic intervention.

Four decades ago, Richard Peto, a statistical epidemiologist from Oxford, made the observation that at the species level, the incidence of cancer does not appear to correlate with the number of cells or the life span of an organism. This became known as “Peto’s paradox.” However, the mechanism

Human tumors display a surprising paucity of mutations when compared with an equivalent mass of normal cells. Remarkably, even within large tumors, the majority of mutations, especially driver mutations, are contained within most neoplastic cells, and genetic heterogeneity is limited. This

Modern lifestyle is associated with longer waking hours and a shift to a more nocturnal schedule. Disruption of the circadian rhythm has been shown to promote obesity and metabolic complications. Recent research has shown that circadian disruption can abolish the normal diurnal variation in serum leptin, independently of changes in external food cues or physical activity. However, the underlying mechanism for this phenomenon and how it leads to weight gain were unknown. Now, Kettner et al . show that the peripheral circadian clock directly regulates leptin transcription in adipose tissue, and that the central circadian clock plays a dominant role in response to leptin signaling. Furthermore, the authors show that circadian disruption induces leptin resistance independent of other risk factors for obesity. The authors used a mouse jet-lag model to simulate circadian dysfunction and found that chronic jet-lag resulted in high, arrhythmic serum concentrations of leptin, which correlated with weight gain and increased fat mass, but no change in the diurnal profiles of food intake, physical activity, or energy expenditure. At the level of the adipose tissue, jet-lagged mice showed a disruption of circadian leptin expression. The key endogenous clock heterodimer BMAL1/CLOCK was shown to regulate leptin transcription through C/EBPα, and the circadian rhythm of this regulation was achieved through a differential effect of low (stimulating) versus high (inhibiting) concentrations of BMAL1/CLOCK, which correlate with different stages of sleep. Surprisingly, knock-out (KO) mice lacking key circadian genes (BMAL1, Cry, or Per), which were expected to have similar circadian dysfunction, exhibited distinct metabolic phenotypes. Although KO and chronic jet-lagged mice showed similar adipose tissue leptin dysregulation, striking differences were found in central leptin-mediated melanocortin signaling. Importantly, chronic jet-lagged mice had arrhythmic dampened pSTAT3 and POMC expression in arcuate nucleus neurons, despite high serum leptin concentrations. This was not rescued by exogenous leptin, indicating central leptin resistance. The mechanism for the differential central effects of different circadian gene KOs has yet to be established. Nevertheless, the demonstration that chronic jet-lag in WT mice was sufficient to induce leptin resistance and a progressive gain in body fat and weight, independent of all previously identified obesity risk factors, implicates chronic circadian dysfunction as a contributing factor to the obesity epidemic. N. M. Kettner et al ., Circadian dysfunction induces leptin resistance in mice. Cell Metab. 10.1016/j.cmet.2015.06.005 (2015). [[Abstract]][1] [1]: http://www.sciencedirect.com/science/article/pii/S1550413115002727

The majority of patients with cancer die of metastatic disease, and there is a major effort to understand and formulate effective ways to treat cancer metastasis. Pancreatic ductal adenocarcinoma (PDAC) is a particularly deadly cancer, with a high metastatic potential and a median survival of only 6 months. Exosomes are secreted membrane vesicles that mediate intercellular communication between cancer cells and their microenvironment. Although exosomes have previously been implicated in the formation of premetastatic niches in target organs, the mechanism for this phenomenon was unclear. Now, Costa-Silva et al . present evidence that PDAC-derived exosomal macrophage migration inhibitory factor (MIF) promotes the formation of premetastatic niches in the liver through induction of transforming growth factor–β (TGF-β) release by Kupffer cells (KCs). When PDAC-derived exosomes were administered to mice prior to tumor cell injection, liver metastatic burden increased. Fluorescently labeled tumor exosomes were preferentially taken up by KCs, and in vitro “education” of KCs by PDAC-derived exosomes up-regulated soluble factors associated with liver fibrosis, including TGF-β. Examination of exosome-educated livers showed a marked increase in extracellular fibronectin deposited by hepatic stellate cells (hStCs), followed by an influx of bone marrow–derived macrophages. Moreover, injected PDAC cells were shown to localize adjacent to KCs. Treatment with a TGF-β type I receptor inhibitor (A83-01) during exosome education decreased hStCs, fibronectin deposition, and macrophage migration to the liver. Furthermore, knocking out fibronectin or depleting recruited macrophages caused a reversion of the prometastatic phenotype. Mass spectrometry of PDAC-derived exosomes revealed high expression of MIF. Knockdown of MIF in exosomes resulted in pronounced reduction in TGF-β, fibronectin deposition, macrophage recruitment, and liver metastatic burden, without affecting exosome binding to KCs. Baseline exosomal MIF concentrations were markedly higher in patients with subsequent progressive disease compared with patients without progression, confirming the importance of this process in human disease. Last, in a genetic model of PDAC, an increase in exosomal MIF and TGF-β expression by liver KCs was observed in the early pretumoral stage and increased in correlation with disease progression. This study demonstrates the importance of PDAC-derived exosomal MIF in premetastatic niche formation and efficient liver metastasis, which correlates with patient outcomes. These findings suggest that exosomal MIF can potentially serve as both a therapeutic target and a prognostic marker for PDAC. B. Costa-Silva et al ., Pancreatic cancer exosomes initiate pre-metastatic niche formation in the liver. Nat. Cell Biol . 17 , 816–826 (2015). [[Abstract]][1] [1]: http://www.nature.com/ncb/journal/v17/n6/pdf/ncb3169.pdf

To evaluate the safety and efficacy of oral administration of Alequel™, an autologous protein-containing colon extract.A total of 43 patients were enrolled in a randomized, placebo-controlled, double-blind trial. Patients were orally administered with autologous protein-containing colon extract three doses of autologous study drug per week for 15 wk, for a total of 45 doses. Patients were followed for safety parameters. Remission was defined as a Crohn's disease activity index (CDAI) score of less than or equal to 150. All patients were followed for changes in subsets of T cells by fluorescence-activated cell sorting analysis.Analysis was performed on a total number of evaluable patients of 14 in the study drug group and 15 in the placebo group. Treatment was well tolerated by all patients. No major treatment-related adverse events were reported or observed in any of the treated patients during the feeding or follow-up periods. Between weeks 6 and 9 of the study, six of the 14 (43%) evaluable subjects who received the study drug achieved a CDAI of 150 or lower. In contrast, five of the 15 (33%) evaluable subjects in the placebo group achieved remission. Between weeks 9 and 12, the remission rates were 50% and 33% for the drug group and placebo group, respectively. Among the drug-treated subjects who achieved remission, the effect of the drug was judged as stable in eight of the 14 subjects as measured by at least two CDAI scores indicating remission in the 15-wk treatment period. A decreased percentage of peripheral natural killer T regulatory cells (a decrease of 28% vs an increase of 16%) and an increased ratio of CD4(+)/CD8(+) T lymphocytes (an increase of 11% vs a decrease of 9%) were noted in subjects with a significant clinical response.Oral administration of the autologous colonic extract could be a safe and effective for the treatment of patients with moderate to severe Crohn's disease.

Hepatitis B virus (HBV) lurks in the liver, causing a chronic liver infection that, although asymptomic in many individuals, can eventually lead to cirrhosis and liver cancer. Immune cells attempt to seek out these hidden viruses through a process called immune surveillance; however, exactly how immune cells probe the liver has remained unclear. Effector CD8+ T lymphocytes (CD8 TE) have been believed to recognize pathogen-infected liver parenchymal cells and then kill the infected cells after extravasation from the post-capillary venules through the process of leukocyte diapedesis. However, liver sinusoids are relatively porous, and sinusoidal leukocyte adhesion often occurs independent of rolling. This led the authors to hypothesize that CD8 TE migration and function in the liver may differ from the classic paradigm. In their elegant study, Guidotti et al. show that hepatic CD8 TE homing is independent of adhesion molecules, chemokines, or antigen recognition, and instead depends on platelet “docking,” which allows CD8 TEs to perform immune surveillance and effector functions while still in the intravascular space. When HBV-specific CD8 TE cells were transferred into HBV replication-competent mice, they adhered to hepatic sinusoids (rather than post-capillary venules), regardless of the location of HBV-antigen–producing hepatocytes. Platelet depletion, but not inhibition of adhesion molecules, chemokines, or the degree of liver inflammation, reduced CD8 TE accumulation. CD8 TEs docked preferentially to sites of transient platelet micro-aggregates on liver sinusoidal epithelial cells (LSEC), and this required platelet-expressed CD44 interacting with sinusoidal hyaluronan. Once “docked,” CD8 TEs crawled along liver sinusoids, and after recognizing hepatocellular antigens, expressed IFN-γ. Indeed, apoptotic hepatocytes were preferentially detected juxtaposed to these cells. Using a combination of three-dimensional (3D) confocal fluorescence and transmission electron microscopy, the authors demonstrated that CD8 TEs extended small protrusions through LSEC fenestrations to probe underlying hepatocytes. Chronic treatment with arsenite, which reduces LSEC fenestrations, or CCl4, which leads to deposition of extracellular matrix in the space of Disse, reduced in vivo CD8 TE antigen recognition and liver damage, but not hepatic platelet-dependent homing. Although the authors did not demonstrate a direct link between impaired intravascular CD8 TE immune surveillance and hepatic carcinogenesis, their findings offer a model by which liver fibrosis creates a physical barrier to T cell immune surveillance and an environment that favors the development of hepatocellular carcinoma. L. G. Guidotti et al ., Immunosurveillance of the liver by intravascular effector CD8+ T cells. Cell 161 , 486–500 (2015). [[Abstract]][1] [1]: http://www.cell.com/cell/abstract/S0092-8674%2815%2900258-5

Recent evidence that excessive lipid accumulation can decrease cellular levels of autophagy and that autophagy regulates immune responsiveness suggested that impaired macrophage autophagy may promote the increased innate immune activation that underlies obesity. Primary bone marrow-derived macrophages (BMDM) and peritoneal macrophages from high-fat diet (HFD)-fed mice had decreased levels of autophagic flux indicating a generalized impairment of macrophage autophagy in obese mice. To assess the effects of decreased macrophage autophagy on inflammation, mice with a Lyz2-Cre-mediated knockout of Atg5 in macrophages were fed a HFD and treated with low-dose lipopolysaccharide (LPS). Knockout mice developed systemic and hepatic inflammation with HFD feeding and LPS. This effect was liver specific as knockout mice did not have increased adipose tissue inflammation. The mechanism by which the loss of autophagy promoted inflammation was through the regulation of macrophage polarization. BMDM and Kupffer cells from knockout mice exhibited abnormalities in polarization with both increased proinflammatory M1 and decreased anti-inflammatory M2 polarization as determined by measures of genes and proteins. The heightened hepatic inflammatory response in HFD-fed, LPS-treated knockout mice led to liver injury without affecting steatosis. These findings demonstrate that autophagy has a critical regulatory function in macrophage polarization that downregulates inflammation. Defects in macrophage autophagy may underlie inflammatory disease states such as the decrease in macrophage autophagy with obesity that leads to hepatic inflammation and the progression to liver injury.

Fibrolamellar hepatocellular carcinoma (FLHCC) is a rare liver malignancy in adolescents and young adults. Surgery is the mainstay of therapy for primary and metastatic disease. Most patients relapse, with development of both local and distant metastases. Brain metastases from solid tumors are rare in the pediatric and young adult population. Here, we document three patients with brain metastases from FLHCC, confirmed by histology and molecular characterization of the chimeric fusion DNAJB1-PRKACA, each necessitating neurosurgical intervention. These observations highlight the ability of FLHCC to metastasize to the brain and suggest the need for surveillance neuroimaging for patients with advanced-stage disease.