En masse organoid phenotyping informs metabolic-associated genetic susceptibility to NASH.

Genotype-phenotype associations for common diseases are often compounded by pleiotropy and metabolic state. Here, we devised a pooled human organoid-panel of steatohepatitis to investigate the impact of metabolic status on genotype-phenotype association. En masse population-based phenotypic analysis under insulin insensitive conditions predicted key non-alcoholic steatohepatitis (NASH)-genetic factors including the glucokinase regulatory protein (GCKR)-rs1260326:C>T. Analysis of NASH clinical cohorts revealed that GCKR-rs1260326-T allele elevates disease severity only under diabetic state but protects from fibrosis under non-diabetic states. Transcriptomic, metabolomic, and pharmacological analyses indicate significant mitochondrial dysfunction incurred by GCKR-rs1260326, which was not reversed with metformin. Uncoupling oxidative mechanisms mitigated mitochondrial dysfunction and permitted adaptation to increased fatty acid supply while protecting against oxidant stress, forming a basis for future therapeutic approaches for diabetic NASH. Thus, "in-a-dish" genotype-phenotype association strategies disentangle the opposing roles of metabolic-associated gene variant functions and offer a rich mechanistic, diagnostic, and therapeutic inference toolbox toward precision hepatology. VIDEO ABSTRACT.

Mapping genotypes to chromatin accessibility profiles in single cells.

In somatic tissue differentiation, chromatin accessibility changes govern priming and precursor commitment towards cellular fates1-3. Therefore, somatic mutations are likely to alter chromatin accessibility patterns, as they disrupt differentiation topologies leading to abnormal clonal outgrowth. However, defining the impact of somatic mutations on the epigenome in human samples is challenging due to admixed mutated and wild-type cells. Here, to chart how somatic mutations disrupt epigenetic landscapes in human clonal outgrowths, we developed genotyping of targeted loci with single-cell chromatin accessibility (GoT-ChA). This high-throughput platform links genotypes to chromatin accessibility at single-cell resolution across thousands of cells within a single assay. We applied GoT-ChA to CD34+ cells from patients with myeloproliferative neoplasms with JAK2V617F-mutated haematopoiesis. Differential accessibility analysis between wild-type and JAK2V617F-mutant progenitors revealed both cell-intrinsic and cell-state-specific shifts within mutant haematopoietic precursors, including cell-intrinsic pro-inflammatory signatures in haematopoietic stem cells, and a distinct profibrotic inflammatory chromatin landscape in megakaryocytic progenitors. Integration of mitochondrial genome profiling and cell-surface protein expression measurement allowed expansion of genotyping onto DOGMA-seq through imputation, enabling single-cell capture of genotypes, chromatin accessibility, RNA expression and cell-surface protein expression. Collectively, we show that the JAK2V617F mutation leads to epigenetic rewiring in a cell-intrinsic and cell type-specific manner, influencing inflammation states and differentiation trajectories. We envision that GoT-ChA will empower broad future investigations of the critical link between somatic mutations and epigenetic alterations across clonal populations in malignant and non-malignant contexts.

Epigenetic dysregulation from chromosomal transit in micronuclei.

Chromosomal instability (CIN) and epigenetic alterations are characteristics of advanced and metastatic cancers1-4, but whether they are mechanistically linked is unknown. Here we show that missegregation of mitotic chromosomes, their sequestration in micronuclei5,6 and subsequent rupture of the micronuclear envelope7 profoundly disrupt normal histone post-translational modifications (PTMs), a phenomenon conserved across humans and mice, as well as in cancer and non-transformed cells. Some of the changes in histone PTMs occur because of the rupture of the micronuclear envelope, whereas others are inherited from mitotic abnormalities before the micronucleus is formed. Using orthogonal approaches, we demonstrate that micronuclei exhibit extensive differences in chromatin accessibility, with a strong positional bias between promoters and distal or intergenic regions, in line with observed redistributions of histone PTMs. Inducing CIN causes widespread epigenetic dysregulation, and chromosomes that transit in micronuclei experience heritable abnormalities in their accessibility long after they have been reincorporated into the primary nucleus. Thus, as well as altering genomic copy number, CIN promotes epigenetic reprogramming and heterogeneity in cancer.

Targeting histone acetylation dynamics and oncogenic transcription by catalytic P300/CBP inhibition.

To separate causal effects of histone acetylation on chromatin accessibility and transcriptional output, we used integrated epigenomic and transcriptomic analyses following acute inhibition of major cellular lysine acetyltransferases P300 and CBP in hematological malignancies. We found that catalytic P300/CBP inhibition dynamically perturbs steady-state acetylation kinetics and suppresses oncogenic transcriptional networks in the absence of changes to chromatin accessibility. CRISPR-Cas9 screening identified NCOR1 and HDAC3 transcriptional co-repressors as the principal antagonists of P300/CBP by counteracting acetylation turnover kinetics. Finally, deacetylation of H3K27 provides nucleation sites for reciprocal methylation switching, a feature that can be exploited therapeutically by concomitant KDM6A and P300/CBP inhibition. Overall, this study indicates that the steady-state histone acetylation-methylation equilibrium functions as a molecular rheostat governing cellular transcription that is amenable to therapeutic exploitation as an anti-cancer regimen.

Somatic mutations and cell identity linked by Genotyping of Transcriptomes.

Defining the transcriptomic identity of malignant cells is challenging in the absence of surface markers that distinguish cancer clones from one another, or from admixed non-neoplastic cells. To address this challenge, here we developed Genotyping of Transcriptomes (GoT), a method to integrate genotyping with high-throughput droplet-based single-cell RNA sequencing. We apply GoT to profile 38,290 CD34+ cells from patients with CALR-mutated myeloproliferative neoplasms to study how somatic mutations corrupt the complex process of human haematopoiesis. High-resolution mapping of malignant versus normal haematopoietic progenitors revealed an increasing fitness advantage with myeloid differentiation of cells with mutated CALR. We identified the unfolded protein response as a predominant outcome of CALR mutations, with a considerable dependency on cell identity, as well as upregulation of the NF-κB pathway specifically in uncommitted stem cells. We further extended the GoT toolkit to genotype multiple targets and loci that are distant from transcript ends. Together, these findings reveal that the transcriptional output of somatic mutations in myeloproliferative neoplasms is dependent on the native cell identity.

Development of an improved inhibitor of Lats kinases to promote regeneration of mammalian organs.

The Hippo signaling pathway acts as a brake on regeneration in many tissues. This cascade of kinases culminates in the phosphorylation of the transcriptional cofactors Yap and Taz, whose concentration in the nucleus consequently remains low. Various types of cellular signals can reduce phosphorylation, however, resulting in the accumulation of Yap and Taz in the nucleus and subsequently in mitosis. We earlier identified a small molecule, TRULI, that blocks the final kinases in the pathway, Lats1 and Lats2, and thus elicits proliferation of several cell types that are ordinarily postmitotic and aids regeneration in mammals. In the present study, we present the results of chemical modification of the original compound and demonstrate that a derivative, TDI-011536, is an effective blocker of Lats kinases in vitro at nanomolar concentrations. The compound fosters extensive proliferation in retinal organoids derived from human induced pluripotent stem cells. Intraperitoneal administration of the substance to mice suppresses Yap phosphorylation for several hours and induces transcriptional activation of Yap target genes in the heart, liver, and skin. Moreover, the compound initiates the proliferation of cardiomyocytes in adult mice following cardiac cryolesions. After further chemical refinement, related compounds might prove useful in protective and regenerative therapies.

Using a Function-First "Scout Fragment"-Based Approach to Develop Allosteric Covalent Inhibitors of Conformationally Dynamic Helicase Mechanoenzymes.

Helicases, classified into six superfamilies, are mechanoenzymes that utilize energy derived from ATP hydrolysis to remodel DNA and RNA substrates. These enzymes have key roles in diverse cellular processes, such as translation, ribosome assembly, and genome maintenance. Helicases with essential functions in certain cancer cells have been identified, and helicases expressed by many viruses are required for their pathogenicity. Therefore, helicases are important targets for chemical probes and therapeutics. However, it has been very challenging to develop chemical inhibitors for helicases, enzymes with high conformational dynamics. We envisioned that electrophilic "scout fragments", which have been used in chemical proteomic studies, could be leveraged to develop covalent inhibitors of helicases. We adopted a function-first approach, combining enzymatic assays with enantiomeric probe pairs and mass spectrometry, to develop a covalent inhibitor that selectively targets an allosteric site in SARS-CoV-2 nsp13, a superfamily-1 helicase. Further, we demonstrate that scout fragments inhibit the activity of two human superfamily-2 helicases, BLM and WRN, involved in genome maintenance. Together, our findings suggest an approach to discover covalent inhibitor starting points and druggable allosteric sites in conformationally dynamic mechanoenzymes.

IL-31 levels correlate with pruritus in patients with cholestatic and metabolic liver diseases and is farnesoid X receptor responsive in NASH.

BACKGROUND AND AIMS: Pruritus is associated with multiple liver diseases, particularly those with cholestasis, but the mechanism remains incompletely understood. Our aim was to evaluate serum IL-31 as a putative biomarker of pruritus in clinical trials of an farnesoid X receptor (FXR) agonist, cilofexor, in patients with NASH, primary sclerosing cholangitis (PSC), and primary biliary cholangitis (PBC). APPROACH AND RESULTS: Serum IL-31 was measured in clinical studies of cilofexor in NASH, PSC, and PBC. In patients with PSC or PBC, baseline IL-31 was elevated compared to patients with NASH and healthy volunteers (HVs). IL-31 correlated with serum bile acids among patients with NASH, PBC, and PSC. Baseline IL-31 levels in PSC and PBC were positively correlated with Visual Analog Scale for pruritus and 5-D itch scores. In patients with NASH, cilofexor dose-dependently increased IL-31 from Week (W)1 to W24. In patients with NASH receiving cilofexor 100 mg, IL-31 was higher in those with Grade 2-3 pruritus adverse events (AEs) than those with Grade 0-1 pruritus AEs. IL-31 weakly correlated with C4 at baseline in patients with NASH, and among those receiving cilofexor 100 mg, changes in IL-31 and C4 from baseline to W24 were negatively correlated. IL-31 messenger RNA (mRNA) was elevated in hepatocytes from patients with PSC and NASH compared to HVs. In a humanized liver murine model, obeticholic acid increased IL-31 mRNA expression in human hepatocytes and serum levels of human IL-31. CONCLUSIONS: IL-31 levels correlate with pruritus in patients with cholestatic disease and NASH, with FXR agonist therapy resulting in higher serum levels in the latter group. IL-31 appears to derive in part from increased hepatocyte expression. These findings have therapeutic implications for patients with liver disease and pruritus.

Liver stiffness thresholds to predict disease progression and clinical outcomes in bridging fibrosis and cirrhosis.

OBJECTIVE: In retrospective studies, liver stiffness (LS) by vibration-controlled transient elastography (VCTE) is associated with the risk of liver decompensation in patients with non-alcoholic steatohepatitis (NASH), but prospective data in biopsy-confirmed cohorts with advanced fibrosis are limited. We aimed to establish thresholds for LS by VCTE that predict progression to cirrhosis among patients with bridging fibrosis and hepatic decompensation among patients with cirrhosis due to NASH. DESIGN: We used data from four randomised placebo-controlled trials of selonsertib and simtuzumab in participants with advanced fibrosis (F3-F4). The trials were discontinued due to lack of efficacy. Liver fibrosis was staged centrally at baseline and week 48 (selonsertib study) or week 96 (simtuzumab study). Associations between LS by VCTE with disease progression were determined using Cox proportional hazards regression analysis. RESULTS: Progression to cirrhosis occurred in 16% (103/664) of participants with bridging fibrosis and adjudicated liver-related events occurred in 4% (27/734) of participants with baseline cirrhosis. The optimal baseline LS thresholds were ≥16.6 kPa for predicting progression to cirrhosis, and ≥30.7 kPa for predicting liver-related events. Baseline LS ≥16.6 kPa (adjusted HR 3.99; 95% CI 2.66 to 5.98, p<0.0001) and a ≥5 kPa (and ≥20%) increase (adjusted HR 1.98; 95% CI 1.20 to 3.26, p=0.008) were independent predictors of progression to cirrhosis in participants with bridging fibrosis, while baseline LS ≥30.7 kPa (adjusted HR 10.13, 95% CI 4.38 to 23.41, p<0.0001) predicted liver-related events in participants with cirrhosis. CONCLUSION: The LS thresholds identified in this study may be useful for risk stratification of NASH patients with advanced fibrosis.

Acetyl-CoA carboxylase inhibitor increases LDL-apoB production rate in NASH with cirrhosis: prevention by fenofibrate.

Treatment with acetyl-CoA carboxylase inhibitors (ACCi) in nonalcoholic steatohepatitis (NASH) may increase plasma triglycerides (TGs), with variable changes in apoB concentrations. ACC is rate limiting in de novo lipogenesis and regulates fatty acid oxidation, making it an attractive therapeutic target in NASH. Our objectives were to determine the effects of the ACCi, firsocostat, on production rates of plasma LDL-apoB in NASH and the effects of combined therapy with fenofibrate. Metabolic labeling with heavy water and tandem mass spectrometric analysis of LDL-apoB enrichments was performed in 16 NASH patients treated with firsocostat for 12 weeks and in 29 NASH subjects treated with firsocostat and fenofibrate for 12 weeks. In NASH on firsocostat, plasma TG increased significantly by 17% from baseline to week 12 (P = 0.0056). Significant increases were also observed in LDL-apoB fractional replacement rate (baseline to week 12: 31 ± 20.2 to 46 ± 22.6%/day, P = 0.03) and absolute synthesis rate (ASR) (30.4-45.2 mg/dl/day, P = 0.016) but not plasma apoB concentrations. The effect of firsocostat on LDL-apoB ASR was restricted to patients with cirrhosis (21.0 ± 9.6 at baseline and 44.2 ± 17 mg/dl/day at week 12, P = 0.002, N = 8); noncirrhotic patients did not change (39.8 ± 20.8 and 46.3 ± 14.8 mg/dl/day, respectively, P = 0.51, N = 8). Combination treatment with fenofibrate and firsocostat prevented increases in plasma TG, LDL-apoB fractional replacement rate, and ASR. In summary, in NASH with cirrhosis, ACCi treatment increases LDL-apoB100 production rate and this effect can be prevented by concurrent fenofibrate therapy.

Enhanced diagnosis of advanced fibrosis and cirrhosis in individuals with NAFLD using FibroScan-based Agile scores.

BACKGROUND & AIMS: Currently available non-invasive tests, including fibrosis-4 index (FIB-4) and liver stiffness measurement (LSM by VCTE), are highly effective at excluding advanced fibrosis (AF) (F ≥3) or cirrhosis in people with non-alcoholic fatty liver disease (NAFLD), but only have moderate ability to rule-in these conditions. Our objective was to develop and validate two new scores (Agile 4 and Agile 3+) to identify cirrhosis or AF, respectively, with optimized positive predictive value and fewer indeterminate results, in individuals with NAFLD attending liver clinics. METHODS: This international study included seven adult cohorts with suspected NAFLD who underwent liver biopsy, LSM and blood sampling during routine clinical practice or screening for trials. The population was randomly divided into a training set and an internal validation set, on which the best-fitting logistic regression model was built, and performance and goodness of fit were assessed, respectively. Furthermore, both scores were externally validated on two large cohorts. Cut-offs for high sensitivity and specificity were derived in the training set to rule-out and rule-in cirrhosis or AF and then tested in the validation set and compared to FIB-4 and LSM. RESULTS: Each score combined LSM, AST/ALT ratio, platelets, sex and diabetes status, as well as age for Agile 3+. Calibration plots for Agile 4 and Agile 3+ indicated satisfactory to excellent goodness of fit. Agile 4 and Agile 3+ outperformed FIB-4 and LSM in terms of AUROC, percentage of patients with indeterminate results and positive predictive value to rule-in cirrhosis or AF. CONCLUSIONS: The two novel non-invasive scores improve identification of cirrhosis or AF among individuals with NAFLD attending liver clinics and reduce the need for liver biopsy in this population. IMPACT AND IMPLICATIONS: Non-invasive tests currently used to identify patients with advanced fibrosis or cirrhosis, such as fibrosis-4 index and liver stiffness measurement by vibration-controlled transient elastography, have high negative predictive values but high false positive rates, while results are indeterminate for a large number of cases. This study provides scores that will help the clinician diagnose advanced fibrosis or cirrhosis. These new easy-to-implement scores will help liver specialists to better identify (1) patients who need more intensive follow-up, (2) patients who should be referred for inclusion in therapeutic trials, and (3) which patients should be treated with pharmacological agents when effective therapies are approved.

The Potential Role of Fatigue in Identifying Patients With NASH and Advanced Fibrosis Who Experience Disease Progression.

BACKGROUND AND AIMS: Fatigue is common in patients with advanced liver disease. We investigated fatigue and clinical outcomes among patients with advanced nonalcoholic steatohepatitis (NASH). METHODS: In this study, patients with biopsy confirmed NASH and bridging fibrosis (F3) or compensated cirrhosis (F4) were followed for up to 2 years. The Chronic Liver Disease Questionnaire for Nonalcoholic Steatohepatitis (CLDQ-NASH) fatigue domain at baseline (range, 1-7; lower score indicating worse fatigue) quantified fatigue. The Cox proportional hazards model was used to study time to liver-related clinical events (progression to histologic cirrhosis or hepatic decompensation in F3, hepatic decompensation in F4). RESULTS: Of the 1679 NASH patients with fibrosis, 802 had F3 and 877 had F4 (58 ± 9 years of age, 40% male, 74% type 2 diabetes). During median follow-up of 16 months (interquartile range, 14-18), 15% (n = 123) of NASH F3 patients experienced liver-related events and 3.5% (n = 31) of NASH F4 patients experienced hepatic decompensation. Mean baseline CLDQ-NASH fatigue score in F3 patients was 4.77 ± 1.36; NASH F3 patients who experienced liver-related events had lower baseline scores: 4.47 ± 1.36 vs 4.83 ± 1.35 (P = .0091). The mean fatigue score in F4 was 4.56 ± 1.44; these scores were lower in patients who decompensated in follow-up: 3.74 ± 1.31 vs 4.59 ± 1.43 (P = .0011). The association of lower fatigue scores and risk of liver-related or decompensation events was significant after adjustment for confounders (adjusted hazard ratio per 1 point in fatigue score in F3, 0.85; 95% confidence interval, 0.74-0.97; P = .02; adjusted hazard ratio in F4, 0.62; 95% confidence interval, 0.48-0.81; P = .0004). CONCLUSION: Worse fatigue at baseline is associated with a higher risk of adverse clinical events in patients with NASH-related advanced fibrosis and cirrhosis.

Fenofibrate Mitigates Hypertriglyceridemia in Nonalcoholic Steatohepatitis Patients Treated With Cilofexor/Firsocostat.

BACKGROUND & AIMS: Patients with advanced fibrosis due to nonalcoholic steatohepatitis (NASH) are at high risk of morbidity and mortality. We previously found that a combination of the farnesoid X receptor agonist cilofexor (CILO) and the acetyl-CoA carboxylase inhibitor firsocostat (FIR) improved liver histology and biomarkers in NASH with advanced fibrosis but was associated with hypertriglyceridemia. We evaluated the safety and efficacy of icosapent ethyl (Vascepa) and fenofibrate to mitigate triglyceride elevations in patients with NASH treated with CILO and FIR. METHODS: Patients with NASH with elevated triglycerides (≥150 and <500 mg/dL) were randomized to Vascepa 2 g twice daily (n = 33) or fenofibrate 145 mg daily (n = 33) for 2 weeks, followed by the addition of CILO 30 mg and FIR 20 mg daily for 6 weeks. Safety, lipids, and liver biochemistry were monitored. RESULTS: All treatments were well-tolerated; most treatment-emergent adverse events were Grade 1 to 2 severity, and there were no discontinuations due to adverse events. At baseline, median (interquartile range [IQR]) triglycerides were similar in the Vascepa and fenofibrate groups (median, 177 [IQR, 154-205] vs 190 [IQR, 144-258] mg/dL, respectively). Median changes from baseline in triglycerides for Vascepa vs fenofibrate after 2 weeks of pretreatment were -12 mg/dL (IQR, -33 to 7 mg/dL; P = .09) vs -32 mg/dL (IQR, -76 to 6 mg/dL; P = .012) and at 6 weeks were +41 mg/dL (IQR, 16-103 mg/dL; P < .001) vs -2 mg/dL (IQR, -42 to 54 mg/dL; P = .92). In patients with baseline triglycerides <250 mg/dL, fenofibrate was more effective vs Vascepa in mitigating triglyceride increases after 6 weeks of combination treatment (+6 vs +39 mg/dL); similar trends were observed in patients with baseline triglycerides ≥250 mg/d (-61 vs +99 mg/dL). CONCLUSIONS: In patients with NASH with hypertriglyceridemia treated with CILO and FIR, fenofibrate was safe and effectively mitigated increases in triglycerides associated with acetyl-CoA carboxylase inhibition. CLINICALTRIALS: gov, Number: NCT02781584.

Performance of Noninvasive Tests of Fibrosis Among Asians, Hispanic, and non-Hispanic Whites in the STELLAR Trials.

BACKGROUND & AIMS: The effect of race on routinely available noninvasive tests of fibrosis is incompletely understood. This study evaluated the performance of noninvasive tests among white and Asian patients in the STELLAR trials (NCT03053050 and NCT03053063), which evaluated selonsertib in patients with advanced (F3-F4) fibrosis due to nonalcoholic steatohepatitis (NASH). METHODS: Baseline liver biopsies were centrally read using the NASH Clinical Research Network system, and 4 noninvasive tests (Nonalcoholic fatty liver disease fibrosis score [NFS], Fibrosis-4 index [FIB-4], Enhanced Liver Fibrosis test [ELF], and liver stiffness by vibration-controlled transient elastography) were measured. The performance of these tests to discriminate advanced fibrosis was evaluated using areas under the receiver operating characteristics curves with 5-fold cross-validation repeated 100 times. RESULTS: Among 3207 patients screened with evaluable liver histology, 2281 were whites and 762 were Asians. Seventy-two percent of whites and 67% of Asians had advanced fibrosis. The areas under the receiver operating characteristics curves of the noninvasive tests for advanced fibrosis were similar in whites and Asians: 0.73 and 0.75 for NFS, 0.78 and 0.80 for FIB-4, 0.79 and 0.81 for ELF, and 0.80 and 0.83 for liver stiffness, respectively. At the published cutoffs, the tests had similar sensitivities and specificities in the 2 groups. However, the sensitivities of NFS, FIB-4, and ELF were low in both white and Asian patients younger than 40 years. CONCLUSIONS: In the global phase III STELLAR trials, the diagnostic performance of routinely available noninvasive tests for the detection of advanced fibrosis due to NASH was acceptable and similar between white and Asian patients.

Safety and Sustained Efficacy of the Farnesoid X Receptor (FXR) Agonist Cilofexor Over a 96-Week Open-label Extension in Patients With PSC.

BACKGROUND & AIMS: Primary sclerosing cholangitis (PSC) is a major unmet medical need in clinical hepatology. Cilofexor is a nonsteroidal farnesoid X receptor agonist being evaluated for the treatment of PSC. Here, we describe the safety and preliminary efficacy of cilofexor in a 96-week, open-label extension (OLE) of a phase II trial. METHODS: Noncirrhotic subjects with large-duct PSC who completed the 12-week, blinded phase of a phase II study (NCT02943460) were eligible, after a 4-week washout period, for a 96-week OLE with cilofexor 100 mg daily. Safety, liver biochemistry, and serum markers of fibrosis, cellular injury, and pharmacodynamic effects of cilofexor (fibroblast growth factor 19, C4, and bile acids [BAs]) were evaluated. RESULTS: Among 52 subjects enrolled in the phase II study, 47 (90%) continued in the OLE phase (median age, 44 years; 60% male patients, 60% with inflammatory bowel disease, and 45% on ursodeoxycholic acid [UDCA]). At OLE baseline (BL), the median serum alkaline phosphatase (ALP) and gamma-glutamyl transferase (GGT) were 368 U/L (interquartile range [IQR], 277-468 U/L) and 417 U/L (IQR, 196-801 U/L), respectively. Of the 47 subjects enrolled, 15 (32%) discontinued treatment prematurely (pruritus [n = 5], other adverse events [n = 5], subject decision/investigator discretion [n = 5]). At week 96, reductions in liver biochemistry parameters occurred, including serum ALP (median, -8.3% [IQR, -25.9% to 11.0%]; P = .066), GGT (-29.8% [IQR, -42.3% to -13.9%]; P < .001), alanine aminotransaminase (ALT) (-29.8% [IQR, -43.7% to -6.6%]; P = .002), and aspartate aminotransaminase (AST) (-16.7% [IQR, -35.3% to 1.0%]; P = .010), and rebounded after 4 weeks of untreated follow-up. ALP response (≥20% reduction from BL to week 96) was similar in the presence or absence of UDCA therapy (29% vs 39%; P = .71). At week 96, cilofexor treatment was associated with a significant reduction in serum 7α-hydroxy-4-cholesten-3-one (C4) (-29.8% [IQR, -64.3% to -8.5%]; P = .001). In subjects with detectable serum BAs at BL (n = 40), BAs decreased -23.9% (IQR, -44.4% to -0.6%; P = .006) at week 48 (n = 28) and -25.7% (IQR, -35.9% to 53.7%; P = .91) at week 96 (n = 26). Serum cytokeratin 18 (CK18) M30 and M65 were reduced throughout the OLE; significant reductions were observed at week 72 (CK18 M30, -17.3% [IQR, -39.3% to 8.8%]; P = .018; CK18 M65, -43.5% [IQR, -54.9% to 15.3%]; P = .096). At week 96, a small, but statistically significant absolute increase of 0.15 units in Enhanced Liver Fibrosis score was observed compared with BL (median, 9.34 vs 9.53; P = .028). CONCLUSIONS: In this 96-week OLE of a phase II study of PSC, cilofexor was safe and improved liver biochemistry and biomarkers of cholestasis and cellular injury. CLINICALTRIALS: gov identifier: NCT02943460.

Metabolic reprogramming of the intestinal microbiome with functional bile acid changes underlie the development of NAFLD.

BACKGROUND AND AIMS: Bile acids are hepatic metabolites and have many properties considered to be relevant to the pathophysiology of NAFLD. Circulating levels of the intestinal microbiome-modified bile acid deoxycholate are increased in cirrhosis. APPROACH AND RESULTS: To further elucidate the role of bile acids and intestinal microbiota linked to bile acids in progressively severe NAFLD, a multiomic study of feces including 16S rRNA sequencing, microbial transcriptomics and metabolomics was performed in a cohort with varying phenotypes of NAFLD. Several bile acids of microbial origin derived from deoxycholic acid (DCA) (glycodeoxycholate, 7-ketodeoxycholic acid, dehydrocholic acid) increased with disease activity and fibrosis stage. These were linked to increased expression of microbial bile salt hydrolase, bile acid operon (BaiCD) and hydroxysteroid dehydrogenases (hdhA) required for DCA and downstream metabolite synthesis providing a mechanistic basis for altered bile acid profiles with disease progression. Bacteroidetes and several genera of Lachnospiraceae family containing DCA generating genes increased with increasing disease severity, whereas several potentially beneficial microbes sensitive to antibacterial effects of DCA e.g., Ruminococcaceae were decreased. The clinical relevance of these data was confirmed in an independent cohort enrolled in a clinical trial for NASH where at entry DCA and its conjugates were associated with advanced fibrosis. In patients treated with placebo, DCA declined in those with fibrosis regression and increased in those with fibrosis progression. DCA rose further in those with compensated cirrhosis when they experienced decompensation. CONCLUSIONS: These findings demonstrate a role for bile acids and the bile acid dependent microbiome in the development and progression of NAFLD and set the stage to leverage these findings for NASH biomarker development and for therapeutics.

Cirrhosis regression is associated with improved clinical outcomes in patients with nonalcoholic steatohepatitis.

BACKGROUND AND AIMS: Surrogate endpoints that predict complications are necessary for assessment and approval of NASH therapies. We assessed associations between histologic and noninvasive tests (NITs) of fibrosis with liver-related complications in patients with NASH cirrhosis. APPROACH AND RESULTS: Patients with compensated cirrhosis due to NASH were enrolled in two placebo-controlled trials of simtuzumab and selonsertib. Liver fibrosis at baseline and week 48 (W48) was staged by NASH Clinical Research Network (CRN) and Ishak classifications and a machine learning (ML) approach, hepatic collagen and alpha-smooth muscle actin (α-SMA) expression were quantified by morphometry, liver stiffness (LS) was measured by transient elastography, and serum NITs (enhanced liver fibrosis [ELF], NAFLD fibrosis score [NFS], and Fibrosis-4 index [FIB-4]) were calculated. Cox regression determined associations between these parameters at baseline and their changes over time with adjudicated liver-related clinical events. Among 1,135 patients, 709 (62%) had Ishak stage 6 fibrosis, and median ELF and LS were 10.66 and 21.1 kPa, respectively. During a median follow-up of 16.6 months, 71 (6.3%) had a liver-related event; associated baseline factors included Ishak stage 6 fibrosis, and higher hepatic collagen, α-SMA expression, ML-based fibrosis parameters, LS, ELF, NFS, and FIB-4. Cirrhosis regression observed in 16% (176/1,135) between BL and W48 was associated with a lower risk of events versus nonregression (1.1% [2/176] vs. 7.2% [69/957]; HR, 0.16; 95% CI, 0.04, 0.65 [p = 0.0104]). Conversely, after adjustment for baseline values, increases in hepatic collagen, α-SMA, ML-based fibrosis parameters, NFS, and LS were associated with an increased risk of events. CONCLUSIONS: In patients with compensated cirrhosis due to NASH, regression of fibrosis is associated with a reduction in liver-related complications. These data support the utility of histologic fibrosis regression and NITs as clinical trial endpoints for NASH cirrhosis.

Limb salvage in octogenarians with critical limb ischemia after lower extremity bypass surgery.

OBJECTIVES: Peripheral artery disease is a worldwide epidemic that affects millions of patients, especially the elderly. It has a prevalence of 20% in individuals >80 years old. Although peripheral artery disease affects >20% of octogenarians, information about limb salvage rates in this patient population is limited. Therefore, this study aims to understand the impact of bypass surgery on limb salvage in patients aged >80 years with critical limb ischemia. METHODS: We conducted a retrospective analysis by querying the electronic medical records at a single institution from 2016 through 2022 to identify the population of interest and analyzed their outcomes after lower extremity bypass. The primary outcomes were limb salvage and primary patency, with hospital length of stay and 1-year mortality as secondary outcomes. RESULTS: We identified 137 patients who met the inclusion criteria. The lower extremity bypass population was divided into two cohorts: <80 years old (n = 111) with a mean age of 66 or ≥80 years old (n = 26) with a mean age of 84 years. The gender distribution was similar (P = .163). No significant difference was found in the two cohorts when it came to coronary artery disease, chronic kidney disease, or diabetes mellitus. However, when current and former smokers were grouped together, they were significantly more common in the younger cohort when compared with nonsmokers (P = .028). The primary end point of limb salvage was not significantly different between the two cohorts. Hospital length of stay was not significantly different between the two cohorts with 4.13 days vs 4.17 days in the younger vs octogenarian cohorts, respectively (P = .95). The 30-day all-cause readmissions were also not found to be significantly different between the two groups. The primary patency at 1 year was 75% and 77% (P = .16) for the <80-year-old and ≥80-year-old cohorts, respectively. Mortality was low in both cohorts, with two and three for the younger and octogenarian populations, respectively; thus, no analysis was performed. CONCLUSIONS: Our study shows that octogenarians who undergo the same preoperative risk assessment as younger populations have similar outcomes when it comes to primary patency, hospital length of stay, and limb salvage when comorbidities were considered. Further studies need to be done to determine the statistical impact on mortality in this population with a larger cohort.

Scaffold Hopping and Optimization of Small Molecule Soluble Adenyl Cyclase Inhibitors Led by Free Energy Perturbation.

Free energy perturbation is a computational technique that can be used to predict how small changes to an inhibitor structure will affect the binding free energy to its target. In this paper, we describe the utility of free energy perturbation with FEP+ in the hit-to-lead stage of a drug discovery project targeting soluble adenyl cyclase. The project was structurally enabled by X-ray crystallography throughout. We employed free energy perturbation to first scaffold hop to a preferable chemotype and then optimize the binding affinity to sub-nanomolar levels while retaining druglike properties. The results illustrate that effective use of free energy perturbation can enable a drug discovery campaign to progress rapidly from hit to lead, facilitating proof-of-concept studies that enable target validation.

PRIMIS: design of a pivotal, randomized, phase 3 study evaluating the safety and efficacy of the nonsteroidal farnesoid X receptor agonist cilofexor in noncirrhotic patients with primary sclerosing cholangitis.

BACKGROUND: Primary sclerosing cholangitis (PSC) is a chronic progressive liver disease leading to biliary fibrosis and cirrhosis. Cilofexor is a nonsteroidal farnesoid X receptor agonist that demonstrated significant improvements in liver biochemistry and markers of cholestasis in patients with PSC in a phase 2 study. We describe here the rationale, design, and implementation of the phase 3 PRIMIS trial, the largest placebo-controlled trial in PSC. METHODS: Adults with large-duct PSC without cirrhosis are randomized 2:1 to receive oral cilofexor 100 mg once daily or placebo for up to 96 weeks during the blinded phase. Patients completing the blinded phase are eligible to receive open-label cilofexor 100 mg daily for up to 96 weeks. The primary objective is to evaluate whether cilofexor reduces the risk of fibrosis progression compared with placebo. Liver biopsy is performed at screening and Week 96 of the blinded phase for histologic assessment of fibrosis. The primary endpoint-chosen in conjunction with guidance from the U.S. Food and Drug Administration-is the proportion of patients with ≥ 1-stage increase in fibrosis according to Ludwig histologic classification at week 96. Secondary objectives include evaluation of changes in liver biochemistry, serum bile acids, liver fibrosis assessed by noninvasive methods, health-related quality of life, and safety of cilofexor. CONCLUSION: The phase 3 PRIMIS study is the largest randomized, double-blind, placebo-controlled trial in PSC to date and will allow for robust evaluation of the efficacy and safety of cilofexor in noncirrhotic patients with large-duct PSC. TRIAL REGISTRATION: ClinicalTrials.gov NCT03890120; registered 26/03/2019.