A Therapeutically Targetable TAZ-TEAD2 Pathway Drives the Growth of Hepatocellular Carcinoma via ANLN and KIF23.

BACKGROUND & AIMS: Despite recent progress, long-term survival remains low for hepatocellular carcinoma (HCC). The most effective HCC therapies target the tumor immune microenvironment (TIME), and there are almost no therapies that directly target tumor cells. Here, we investigated the regulation and function of tumor cell-expressed Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ) in HCC. METHODS: HCC was induced in mice by Sleeping Beauty-mediated expression of MET, CTNNB1-S45Y, or TAZ-S89A, or by diethylnitrosamine plus CCl4. Hepatocellular TAZ and YAP were deleted in floxed mice via adeno-associated virus serotype 8-mediated expression of Cre. TAZ target genes were identified from RNA sequencing, confirmed by chromatin immunoprecipitation, and evaluated in a clustered regularly interspaced short palindromic repeats interference (CRISPRi) screen. TEA domain transcription factors (TEADs), anillin (ANLN), Kif23, and programmed cell death protein ligand 1 were knocked down by guide RNAs in dead clustered regularly interspaced short palindromic repeats-associated protein 9 (dCas9) knock-in mice. RESULTS: YAP and TAZ were up-regulated in murine and human HCC, but only deletion of TAZ consistently decreased HCC growth and mortality. Conversely, overexpression of activated TAZ was sufficient to trigger HCC. TAZ expression in HCC was regulated by cholesterol synthesis, as demonstrated by pharmacologic or genetic inhibition of 3-hydroxy-3-methylglutaryl- coenzyme A reductase (HMGCR), farnesyl pyrophosphate synthase, farnesyl-diphosphate farnesyltransferase 1 (FDFT1), or sterol regulatory element-binding protein 2 (SREBP2). TAZ- and MET/CTNNB1-S45Y-driven HCC required the expression of TEAD2 and, to a lesser extent, TEAD4. Accordingly, TEAD2 displayed the most profound effect on survival in patients with HCC. TAZ and TEAD2 promoted HCC via increased tumor cell proliferation, mediated by TAZ target genes ANLN and kinesin family member 23 (KIF23). Therapeutic targeting of HCC, using pan-TEAD inhibitors or the combination of a statin with sorafenib or anti-programmed cell death protein 1, decreased tumor growth. CONCLUSIONS: Our results suggest the cholesterol-TAZ-TEAD2-ANLN/KIF23 pathway as a mediator of HCC proliferation and tumor cell-intrinsic therapeutic target that could be synergistically combined with TIME-targeted therapies.

Emerging and potential use of CRISPR in human liver disease.

CRISPR is a gene editing tool adapted from naturally occurring defense systems from bacteria. It is a technology that is revolutionizing the interrogation of gene functions in driving liver disease, especially through genetic screens and by facilitating animal knockout and knockin models. It is being used in models of liver disease to identify which genes are critical for liver pathology, especially in genetic liver disease, hepatitis, and in cancer initiation and progression. It holds tremendous promise in treating human diseases directly by editing DNA. It could disable gene function in the case of expression of a maladaptive protein, such as blocking transthyretin as a therapy for amyloidosis, or to correct gene defects, such as restoring the normal functions of liver enzymes fumarylacetoacetate hydrolase or alpha-1 antitrypsin. It is also being studied for treatment of hepatitis B infection. CRISPR is an exciting, evolving technology that is facilitating gene characterization and discovery in liver disease and holds the potential to treat liver diseases safely and permanently.

Author Correction: Subepithelial telocytes are an important source of Wnts that supports intestinal crypts.

Change history: In this Letter, the surname of author Efi E. Massasa was misspelled 'Massassa'. This error has been corrected online.

Subepithelial telocytes are an important source of Wnts that supports intestinal crypts.

Tissues that undergo rapid cellular turnover, such as the mammalian haematopoietic system or the intestinal epithelium, are dependent on stem and progenitor cells that proliferate to provide differentiated cells to maintain organismal health. Stem and progenitor cells, in turn, are thought to rely on signals and growth factors provided by local niche cells to support their function and self-renewal. Several cell types have been hypothesized to provide the signals required for the proliferation and differentiation of the intestinal stem cells in intestinal crypts1-6. Here we identify subepithelial telocytes as an important source of Wnt proteins, without which intestinal stem cells cannot proliferate and support epithelial renewal. Telocytes are large but rare mesenchymal cells that are marked by expression of FOXL1 and form a subepithelial plexus that extends from the stomach to the colon. While supporting the entire epithelium, FOXL1+ telocytes compartmentalize the production of Wnt ligands and inhibitors to enable localized pathway activation. Conditional genetic ablation of porcupine (Porcn), which is required for functional maturation of all Wnt proteins, in mouse FOXL1+ telocytes causes rapid cessation of Wnt signalling to intestinal crypts, followed by loss of proliferation of stem and transit amplifying cells and impaired epithelial renewal. Thus, FOXL1+ telocytes are an important source of niche signals to intestinal stem cells.

Cytoplasmic chromatin triggers inflammation in senescence and cancer.

Chromatin is traditionally viewed as a nuclear entity that regulates gene expression and silencing. However, we recently discovered the presence of cytoplasmic chromatin fragments that pinch off from intact nuclei of primary cells during senescence, a form of terminal cell-cycle arrest associated with pro-inflammatory responses. The functional significance of chromatin in the cytoplasm is unclear. Here we show that cytoplasmic chromatin activates the innate immunity cytosolic DNA-sensing cGAS-STING (cyclic GMP-AMP synthase linked to stimulator of interferon genes) pathway, leading both to short-term inflammation to restrain activated oncogenes and to chronic inflammation that associates with tissue destruction and cancer. The cytoplasmic chromatin-cGAS-STING pathway promotes the senescence-associated secretory phenotype in primary human cells and in mice. Mice deficient in STING show impaired immuno-surveillance of oncogenic RAS and reduced tissue inflammation upon ionizing radiation. Furthermore, this pathway is activated in cancer cells, and correlates with pro-inflammatory gene expression in human cancers. Overall, our findings indicate that genomic DNA serves as a reservoir to initiate a pro-inflammatory pathway in the cytoplasm in senescence and cancer. Targeting the cytoplasmic chromatin-mediated pathway may hold promise in treating inflammation-related disorders.

Comparing telemedicine and in-person gastrointestinal cancer genetic appointment outcomes during the COVID-19 pandemic.

BACKGROUND: The study purpose is to compare outcomes associated with completion of genetic testing between telemedicine and in-person gastrointestinal cancer risk assessment appointments during the COVID-19 pandemic. METHODS: Data was collected on patients with scheduled appointments between July 2020 and June 2021 in a gastrointestinal cancer risk evaluation program (GI-CREP) that utilized both telemedicine and in-person visits throughout the COVID-19 pandemic, and a survey was administered. RESULTS: A total of 293 patients had a GI-CREP appointment scheduled and completion rates of in-person versus telemedicine appointments were similar. Individuals diagnosed with cancer and those with Medicaid insurance had lower rates of appointment completion. Although telehealth was the preferred visit modality, there were no differences in recommending genetic testing nor in the consent rate for genetic testing between in-person and telemedicine visits. However, of patients who consented for genetic testing, more than three times more patients seen via telemedicine did not complete genetic testing compared to those seen in-person (18.3% versus 5.2%, p = 0.008). Furthermore, telemedicine visits had a longer turnaround time for genetic test reporting (32 days versus 13 days, p < 0.001). CONCLUSIONS: Compared to in-person GI-CREP appointments, telemedicine was associated with lower rates of genetic testing completion, and longer turnaround time for results.

A genetic screen reveals Foxa3 and TNFR1 as key regulators of liver repopulation.

The fundamental question of which genes are most important in controlling liver regeneration remains unanswered. We employed a parallel screen to test the impact of 43 selected genes on liver repopulation in the Fah(-/-) mouse model of hereditary tyrosinemia. We discovered that the transcription factor Foxa3 was a strong promoter of liver regeneration, while tumor necrosis factor receptor 1 (TNFR1) was the most significant suppressor of repopulation among all of the genes tested. Our approach enabled the identification of these factors as important regulators of liver repopulation and potential drug targets for the promotion of liver repopulation.

A High-Content Screen Identifies MicroRNAs That Regulate Liver Repopulation After Injury in Mice.

BACKGROUND & AIMS: Liver regeneration is impaired in mice with hepatocyte-specific deficiencies in microRNA (miRNA) processing, but it is not clear which miRNAs regulate this process. We developed a high-throughput screen to identify miRNAs that regulate hepatocyte repopulation after toxic liver injury using fumarylacetoacetate hydrolase-deficient mice. METHODS: We constructed plasmid pools encoding more than 30,000 tough decoy miRNA inhibitors (hairpin nucleic acids designed to specifically inhibit interactions between miRNAs and their targets) to target hepatocyte miRNAs in a pairwise manner. The plasmid libraries were delivered to hepatocytes in fumarylacetoacetate hydrolase-deficient mice at the time of liver injury via hydrodynamic tail-vein injection. Integrated transgene-containing transposons were quantified after liver repopulation via high-throughput sequencing. Changes in polysome-bound transcripts after miRNA inhibition were determined using translating ribosome affinity purification followed by high-throughput sequencing. RESULTS: Analyses of tough decoy abundance in hepatocyte genomic DNA and input plasmid pools identified several thousand miRNA inhibitors that were significantly depleted or increased after repopulation. We classified a subset of miRNA binding sites as those that have strong effects on liver repopulation, implicating the targeted hepatocyte miRNAs as regulators of this process. We then generated a high-content map of pairwise interactions between 171 miRNA-binding sites and identified synergistic and redundant effects. CONCLUSIONS: We developed a screen to identify miRNAs that regulate liver repopulation after injury in live mice.

Liver Cancer Gene Discovery Using Gene Targeting, Sleeping Beauty, and CRISPR/Cas9.

Hepatocellular carcinoma (HCC) is a devastating and prevalent cancer with limited treatment options. Technological advances have enabled genetic screens to be employed in HCC model systems to characterize genes regulating tumor initiation and growth. Relative to traditional methods for studying cancer biology, such as candidate gene approaches or expression analysis, genetic screens have several advantages: they are unbiased, with no a priori selection; can directly annotate gene function; and can uncover gene-gene interactions. In HCC, three main types of screens have been conducted and are reviewed here: (1) transposon-based mutagenesis screens, (2) knockdown screens using RNA interference (RNAi) or the CRISPR/Cas9 system, and (3) overexpression screens using CRISPR activation (CRISPRa) or cDNAs. These methods will be valuable in future genetic screens to delineate the mechanisms underlying drug resistance and to identify new treatments for HCC.

Combinatorial genetics in liver repopulation and carcinogenesis with a in vivo CRISPR activation platform.

Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 activation (CRISPRa) systems have enabled genetic screens in cultured cell lines to discover and characterize drivers and inhibitors of cancer cell growth. We adapted this system for use in vivo to assess whether modulating endogenous gene expression levels can result in functional outcomes in the native environment of the liver. We engineered the catalytically dead CRISPR-associated 9 (dCas9)-positive mouse, cyclization recombination-inducible (Cre) CRISPRa system for cell type-specific gene activation in vivo. We tested the capacity for genetic screening in live animals by applying CRISPRa in a clinically relevant model of liver injury and repopulation. We targeted promoters of interest in regenerating hepatocytes using multiple single guide RNAs (gRNAs), and employed high-throughput sequencing to assess enrichment of gRNA sequences during liver repopulation and to link specific gRNAs to the initiation of carcinogenesis. All components of the CRISPRa system were expressed in a cell type-specific manner and activated endogenous gene expression in vivo. Multiple gRNA cassettes targeting a proto-oncogene were significantly enriched following liver repopulation, indicative of enhanced division of cells expressing the proto-oncogene. Furthermore, hepatocellular carcinomas developed containing gRNAs that activated this oncogene, indicative of cancer initiation events. Also, we employed our system for combinatorial cancer genetics in vivo as we found that while clonal hepatocellular carcinomas were dependent on the presence of the oncogene-inducing gRNAs, they were depleted for multiple gRNAs activating tumor suppressors. CONCLUSION: The in vivo CRISPRa platform developed here allows for parallel and combinatorial genetic screens in live animals; this approach enables screening for drivers and suppressors of cell replication and tumor initiation. (Hepatology 2017).

Genetic lineage tracing analysis of the cell of origin of hepatotoxin-induced liver tumors in mice.

UNLABELLED: The expression of biliary/progenitor markers by hepatocellular carcinoma (HCC) is often associated with poor prognosis and stem cell-like behaviors of tumor cells. Hepatocellular adenomas (HCAs) also often express biliary/progenitor markers and frequently act as precursor lesions for HCC. However, the cell of origin of HCA and HCC that expresses these markers remains unclear. Therefore, to evaluate if mature hepatocytes give rise to HCA and HCC tumors and to understand the molecular pathways involved in tumorigenesis, we lineage-labeled hepatocytes by injecting adeno-associated virus containing thyroxine-binding globulin promoter-driven causes recombination (AAV-TBG-Cre) into Rosa(YFP) mice. Yellow fluorescent protein (YFP) was present in >96% of hepatocytes before exposure to carcinogens. We treated AAV-TBG-Cre; Rosa(YFP) mice with diethylnitrosamine (DEN), followed by multiple injections of carbon tetrachloride to induce carcinogenesis and fibrosis and found that HCA and HCC nodules were YFP(+) lineage-labeled; positive for osteopontin, SRY (sex determining region Y)-box 9, and epithelial cell adhesion molecule; and enriched for transcripts of biliary/progenitor markers such as prominin 1, Cd44, and delta-like 1 homolog. Next, we performed the converse experiment and lineage-labeled forkhead box protein L1(Foxl1)-positive hepatic progenitor cells simultaneously with exposure to carcinogens. None of the tumor nodules expressed YFP, indicating that Foxl1-expressing cells are not the origin for hepatotoxin-induced liver tumors. We confirmed that HCA and HCC cells are derived from mature hepatocytes and not from Foxl1-Cre-marked cells in a second model of toxin-induced hepatic neoplasia, using DEN and 3,3',5,5'-tetrachloro-1,4-bis(pyridyloxy)benzene (TCPOBOP). CONCLUSION: Hepatocytes are the cell of origin of HCA and HCC in DEN/carbon tetrachloride and DEN/TCPOBOP induced liver tumors. (Hepatology 2016;64:1163-1177).

Natural History of Patients Presenting with Autoimmune Hepatitis and Coincident Nonalcoholic Fatty Liver Disease.

BACKGROUND: Given the increase of nonalcoholic fatty liver disease (NAFLD) in the general population, a similar rise might be expected in autoimmune hepatitis (AIH) patients. AIMS: We sought to determine the clinical outcome of patients with coincident AIH and NAFLD. METHODS: We identified all intradepartmental AIH cases, and those meeting study criteria were placed into one of three cohorts: AIH only, AIH and simple steatosis (SS), and AIH and nonalcoholic steatohepatitis (NASH). The following outcome and clinical data were analyzed: incidence of all-cause mortality, incidence of liver-related mortality, incidence of liver-related adverse outcomes, and prevalence of cirrhosis at index biopsy. RESULTS: Out of a total 73 study patients, 14 % classified as AIH with SS and 16 % as AIH and NASH. Fifty percent of AIH and NASH patients had cirrhosis at index biopsy as compared to 18 % of AIH-only patients (p = 0.032). Patients with AIH and NASH had a relative risk of 7.65 (95 % CI 1.43-40.8) for liver-related mortality and 2.55 (95 % CI 0.92-7.09) for liver-related adverse outcomes, as compared to the AIH-only cohort. No significant difference in outcome measures existed in comparing (AIH only) with (AIH and SS) cohorts. DISCUSSION: Patients with coincident AIH and NASH were more likely to present with cirrhosis and more likely to develop adverse clinical outcome with decreased survival as compared to AIH-only patients. These findings suggest that simultaneous exposure confers a clinically significant increased risk, which may warrant closer follow-up and surveillance.

Reversal of hepatocyte senescence after continuous in vivo cell proliferation.

UNLABELLED: A better understanding of hepatocyte senescence could be used to treat age-dependent disease processes of the liver. Whether continuously proliferating hepatocytes could avoid or reverse senescence has not yet been fully elucidated. We confirmed that the livers of aged mice accumulated senescent and polyploid hepatocytes, which is associated with accumulation of DNA damage and activation of p53-p21 and p16(ink4a)-pRB pathways. Induction of multiple rounds continuous cell division is hard to apply in any animal model. Taking advantage of serial hepatocyte transplantation assays in the fumarylacetoacetate hydrolase-deficient (Fah(-/-)) mouse, we studied the senescence of hepatocytes that had undergone continuous cell proliferation over a long time period, up to 12 rounds of serial transplantations. We demonstrated that the continuously proliferating hepatocytes avoided senescence and always maintained a youthful state. The reactivation of telomerase in hepatocytes after serial transplantation correlated with reversal of senescence. Moreover, senescent hepatocytes harvested from aged mice became rejuvenated upon serial transplantation, with full restoration of proliferative capacity. The same findings were also true for human hepatocytes. After serial transplantation, the high initial proportion of octoploid hepatocytes decreased to match the low level of youthful liver. CONCLUSION: These findings suggest that the hepatocyte "ploidy conveyer" is regulated differently during aging and regeneration. The findings of reversal of hepatocyte senescence could enable future studies on liver aging and cell therapy.

Idiopathic Myointimal Hyperplasia of the Mesenteric Veins Diagnosed Preoperatively.

Idiopathic myointimal hyperplasia of the mesenteric veins (IMHMV) of the colon is a very rare disease that in previously reported cases was often mistaken for inflammatory bowel disease because of similar clinical characteristics. In our review of the literature, we found a total of 15 cases described, generally featuring sigmoid and rectal colitis and symptoms of abdominal pain, diarrhea, and hematochezia refractory to treatment with immunosuppressants. In all previously reported cases, the diagnosis was achieved only after surgical resection of the affected area. Herein, we report a case of IMHMV that was diagnosed preoperatively based on clinical information and endoscopy with biopsies. This led to the withdrawal of immunosuppression before a carefully planned surgical resection, with confirmation of the diagnosis in the resected tissue. To our knowledge, our case of IMHMV is the first to be diagnosed preoperatively.

Germline Genetic Associations for Hepatobiliary Cancers.

Hepatobiliary cancers (HBCs) include hepatocellular carcinoma, cholangiocarcinoma, and gallbladder carcinoma, which originate from the liver, bile ducts, and gallbladder, respectively. They are responsible for a substantial burden of cancer-related deaths worldwide. Despite knowledge of risk factors and advancements in therapeutics and surgical interventions, the prognosis for most patients with HBC remains bleak. There is evidence from familial aggregation and case-control studies to suggest a familial risk component in HBC susceptibility. Recent progress in genomics research has led to the identification of germline variants including single nucleotide polymorphisms (SNPs) and pathogenic or likely pathogenic (P/LP) variants in cancer-associated genes associated with HBC risk. These findings emerged from genome-wide association studies and next-generation sequencing techniques such as whole-exome sequencing. Patients with other cancer types, including breast, colon, ovarian, prostate, and pancreatic cancer, are recommended by guidelines to undergo germline genetic testing, but similar recommendations are lagging in HBC. This prompts the question of whether multi-gene panel testing should be integrated into clinical guidelines for HBC management. Here, we review the hereditary genetics of HBC, explore studies investigating SNPs and P/LP variants in HBC patients, discuss the clinical implications and potential for personalized treatments and impact on patient's family members, and conclude that additional studies are needed to examine how genetic testing can be applied clinically.

Lipid Nanoparticle Delivery of Small Proteins for Potent In Vivo RAS Inhibition.

Mutated RAS proteins are potent oncogenic drivers and have long been considered "undruggable". While RAS-targeting therapies have recently shown promise, there remains a clinical need for RAS inhibitors with more diverse targets. Small proteins represent a potential new therapeutic option, including K27, a designed ankyrin repeat protein (DARPin) engineered to inhibit RAS. However, K27 functions intracellularly and is incapable of entering the cytosol on its own, currently limiting its utility. To overcome this barrier, we have engineered a lipid nanoparticle (LNP) platform for potent delivery of functional K27-D30─a charge-modified version of the protein─intracellularly in vitro and in vivo. This system efficiently encapsulates charge-modified proteins, facilitates delivery in up to 90% of cells in vitro, and maintains potency after at least 45 days of storage. In vivo, these LNPs deliver K27-D30 to the cytosol of cancerous cells in the liver, inhibiting RAS-driven growth and ultimately reducing tumor load in an HTVI-induced mouse model of hepatocellular carcinoma. This work shows that K27 holds promise as a new cancer therapeutic when delivered using this LNP platform. Furthermore, this technology has the potential to broaden the use of LNPs to include new cargo types─beyond RNA─for diverse therapeutic applications.

Association of Statin Use With Risk of Liver Disease, Hepatocellular Carcinoma, and Liver-Related Mortality.

Importance: Given the burden of chronic liver disease on the health care system, more information on the hepatoprotective association of statins in the general population is needed. Objective: To examine whether regular statin use is associated with a reduction in liver disease, particularly hepatocellular carcinoma (HCC) and liver-related deaths, in the general population. Design, Setting, and Participants: This cohort study used data from the UK Biobank (UKB) (individuals aged 37-73 years) collected from baseline (2006-2010) to the end of follow-up in May 2021, from the TriNetX cohort (individuals aged 18-90 years) enrolled from baseline (2011-2020) until end of follow-up in September 2022, and from the Penn Medicine Biobank (PMBB) (individuals aged 18-102 years) with ongoing enrollment starting in 2013 to the end of follow-up in December 2020. Individuals were matched using propensity score matching according to the following criteria: age, sex, body mass index, ethnicity, diabetes with or without insulin or biguanide use, hypertension, ischemic heart disease, dyslipidemia, aspirin use, and number of medications taken (UKB only). Data analysis was performed from April 2021 to April 2023. Exposure: Regular statin use. Main Outcomes and Measures: Primary outcomes were liver disease and HCC development as well as liver-associated death. Results: A total of 1 785 491 individuals were evaluated after matching (aged 55 to 61 years on average, up to 56% men, and up to 49% women). A total of 581 cases of liver-associated death, 472 cases of incident HCC, and 98 497 new liver diseases were registered during the follow-up period. Individuals were aged 55-61 years on average, with a slightly higher proportion of men (up to 56%). In UKB individuals (n = 205 057) without previously diagnosed liver disease, statin users (n = 56 109) had a 15% lower hazard ratio (HR) for the association of developing a new liver disease (HR, 0.85; 95% CI, 0.78-0.92; P < .001). In addition, statin users demonstrated a 28% lower HR for the association with liver-related death (HR, 0.72; 95% CI, 0.59-0.88; P = .001) and a 42% lower HR for the development of HCC (HR, 0.58; 95% CI, 0.35-0.96; P = .04). In TriNetX individuals (n = 1 568 794), the HR for the association of HCC was reduced even further for statin users (HR, 0.26; 95% CI, 0.22-0.31; P = .003). The hepatoprotective association of statins was time and dose dependent, with a significant association in PMBB individuals (n = 11 640) for incident liver diseases after 1 year of statin use (HR, 0.76; 95% CI, 0.59-0.98; P = .03). Taking statins was particularly beneficial in men, individuals with diabetes, and individuals with a high Fibrosis-4 index at baseline. Carriers of the heterozygous minor allele of PNPLA3 rs738409 benefited from statin use and had a 69% lower HR for the association with HCC (UKB HR, 0.31; 95% CI, 0.11-0.85; P = .02). Conclusions and Relevance: This cohort study indicates substantial preventive associations of statins against liver disease, with an association with duration and dose of intake.

Cellular origins of regenerating liver and hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is the predominant primary cancer arising from the liver and is one of the major causes of cancer-related mortality worldwide. The cellular origin of HCC has been a topic of great interest due to conflicting findings regarding whether it originates in hepatocytes, biliary cells, or facultative stem cells. These cell types all undergo changes during liver injury, and there is controversy about their contribution to regenerative responses in the liver. Most HCCs emerge in the setting of chronic liver injury from viral hepatitis, fatty liver disease, alcohol, and environmental exposures. The injuries are marked by liver parenchymal changes such as hepatocyte regenerative nodules, biliary duct cellular changes, expansion of myofibroblasts that cause fibrosis and cirrhosis, and inflammatory cell infiltration, all of which may contribute to carcinogenesis. Addressing the cellular origin of HCC is the key to identifying the earliest events that trigger it. Herein, we review data on the cells of origin in regenerating liver and HCC and the implications of these findings for prevention and treatment. We also review the origins of childhood liver cancer and other rare cancers of the liver.