PD-1 immune checkpoint inhibitors have produced encouraging results in patients with hepatocellular carcinoma (HCC). However, what determines resistance to anti-PD-1 therapies is unclear. We created a novel genetically engineered mouse model of HCC that enables interrogation of how different genetic alterations affect immune surveillance and response to immunotherapies. Expression of exogenous antigens in MYC;Trp53 −/− HCCs led to T cell-mediated immune surveillance, which was accompanied by decreased tumor formation and increased survival. Some antigen-expressing MYC;Trp53 −/− HCCs escaped the immune system by upregulating the β-catenin (CTNNB1) pathway. Accordingly, expression of exogenous antigens in MYC;CTNNB1 HCCs had no effect, demonstrating that β-catenin promoted immune escape, which involved defective recruitment of dendritic cells and consequently impaired T-cell activity. Expression of chemokine CCL5 in antigenexpressing MYC;CTNNB1 HCCs restored immune surveillance. Finally, β-catenin-driven tumors were resistant to anti-PD-1. In summary, β-catenin activation promotes immune escape and resistance to anti-PD-1 and could represent a novel biomarker for HCC patient exclusion. SIGNIFICANCE: Determinants of response to anti-PD-1 immunotherapies in HCC are poorly understood. Using a novel mouse model of HCC, we show that β-catenin activation promotes immune evasion and resistance to anti-PD-1 therapy and could potentially represent a novel biomarker for HCC patient exclusion.

See Covering the Cover synopsis on page 1997. BACKGROUND AND AIMS: The pattern of genetic alterations in cancer driver genes in patients with hepatocellular carcinoma (HCC) is highly diverse, which partially explains the low efficacy of available therapies. In spite of this, the existing mouse models only recapitulate a small portion of HCC intertumor heterogeneity, limiting the understanding of the disease and the nomination of personalized therapies. Here, we aimed at establishing a novel collection of HCC mouse models that captured human HCC diversity. METHODS: By performing hydrodynamic tail-vein injections, we tested the impact of altering a well-established HCC oncogene (either MYC or bcatenin) in combination with an additional alteration in one of eleven other genes frequently mutated in HCC. Of the 23 unique Gastroenterology 2020;159:2203-2220 BASIC AND TRANSLATIONAL LIVER pairs of genetic alterations that we interrogated, 9 were able to induce HCC. The established HCC mouse models were characterized at histopathological, immune, and transcriptomic level to identify the unique features of each model. Murine HCC cell lines were generated from each tumor model, characterized transcriptionally, and used to identify specific therapies that were validated in vivo. RESULTS: Cooperation between pairs of driver genes produced HCCs with diverse histopathology, immune microenvironments, transcriptomes, and drug responses. Interestingly, MYC expression levels strongly influenced b-catenin activity, indicating that inter-tumor heterogeneity emerges not only from specific combinations of genetic alterations but also from the acquisition of expression-dependent phenotypes. CONCLUSIONS: This novel collection of murine HCC models and corresponding cell lines establishes the role of driver genes in diverse contexts and enables mechanistic and translational studies.

Gastrointestinal symptoms are common in COVID-19 patients but the nature of the gut immune response to SARS-CoV-2 remains poorly characterized, partly due to the difficulty of obtaining biopsy specimens from infected individuals. In lieu of tissue samples, we measured cytokines, inflammatory markers, viral RNA, microbiome composition, and antibody responses in stool samples from a cohort of 44 hospitalized COVID-19 patients. SARS-CoV-2 RNA was detected in stool of 41% of patients and more frequently in patients with diarrhea. Patients who survived had lower fecal viral RNA than those who died. Strains isolated from stool and nasopharynx of an individual were the same. Compared to uninfected controls, COVID-19 patients had higher fecal levels of IL-8 and lower levels of fecal IL-10. Stool IL-23 was higher in patients with more severe COVID-19 disease, and we found evidence of intestinal virus-specific IgA responses associated with more severe disease. We provide evidence for an ongoing humeral immune response to SARS-CoV-2 in the gastrointestinal tract, but little evidence of overt inflammation.

The progression of genetically inherited cardiomyopathies from an altered protein structure to clinical presentation of disease is not well understood. One of the main roadblocks to mechanistic insight remains a lack of high-resolution structural information about multiprotein complexes within the cardiac sarcomere. One example is the tropomyosin (Tm) overlap region of the thin filament that is crucial for the function of the cardiac sarcomere. To address this central question, we devised coupled experimental and computational modalities to characterize the baseline function and structure of the Tm overlap, as well as the effects of mutations causing divergent patterns of ventricular remodeling on both structure and function. Because the Tm overlap contributes to the cooperativity of myofilament activation, we hypothesized that mutations that enhance the interactions between overlap proteins result in more cooperativity, and conversely, those that weaken interaction between these elements lower cooperativity. Our results suggest that the Tm overlap region is affected differentially by dilated cardiomyopathy-associated Tm D230N and hypertrophic cardiomyopathy-associated human cardiac troponin T (cTnT) R92L. The Tm D230N mutation compacts the Tm overlap region, increasing the cooperativity of the Tm filament, contributing to a dilated cardiomyopathy phenotype. The cTnT R92L mutation causes weakened interactions closer to the N-terminal end of the overlap, resulting in decreased cooperativity. These studies demonstrate that mutations with differential phenotypes exert opposite effects on the Tm–Tn overlap, and that these effects can be directly correlated to a molecular level understanding of the structure and dynamics of the component proteins.

Background and aims: Immune dysregulation caused by SARS-CoV-2 infection is thought to play a pathogenic role in COVID-19. SARS-CoV-2 can infect a variety of host cells, including intestinal epithelial cells. We sought to characterize the role of the gastrointestinal immune system in the pathogenesis of the inflammatory response associated with COVID-19. Methods: We measured cytokines, inflammatory markers, viral RNA, microbiome composition and antibody responses in stool and serum samples from a prospectively enrolled cohort of 44 hospitalized COVID-19 patients. Results: SARS-CoV-2 RNA was detected in stool of 41% of patients and was found more frequently in patients with diarrhea than those without (16[44%] vs 5[19%], p=0.06). Patients who survived had lower median viral genome copies than those who did not (p=0.021). Compared to uninfected controls, COVID-19 patients had higher median fecal levels of IL-8 (166.5 vs 286.5 pg/mg; p=0.05) and lower levels of fecal IL-10 (678 vs 194 pg/mg; p<0.001) compared to uninfected controls. Stool IL-23 was higher in patients with more severe COVID-19 disease (223.8 vs 86.6 pg/mg; p=0.03) and we find evidence of intestinal virus-specific IgA responses, which was associated with more severe disease. Fecal cytokines and calprotectin levels were not correlated with gastrointestinal symptoms or with the level of virus detected. Conclusions: Although SARS-CoV-2 RNA was detectable in the stools of COVID-19 patients and select individuals had evidence for a specific mucosal IgA response, intestinal inflammation was limited, even in patients presenting with gastrointestinal symptoms.

To the Editor-Clostridioides difficile infection (CDI) is the most common healthcare-associated infection in the United States. 1 CDI affects 13 in every 1,000 patients, and~75% of cases are classified as hospital onset. Antimicrobial stewardship and compliance with hand hygiene and personal protective equipment (PPE) protocols are paramount in efforts to reduce horizontal CDI transmission. 2 As an epicenter of the coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), New York City hospitals saw a dramatic increase in admissions and ICU utilization. 3 To understand the impact of COVID-19 on hospital-onset CDI, we examined antibiotic prescribing patterns, standardized infection ratios (SIRs), and baseline variables in hospitalized adult patients prior to and during the COVID-19 pandemic. We hypothesized that increased antibiotics exposure during the COVID-19 pandemic would lead to a higher incidence of CDI in hospitalized patients. We conducted a retrospective cohort analysis at a high-volume tertiary-care center comparing a pre-COVID-19 cohort (February-June 2019) of all adult patients who were diagnosed with CDI on admission or during their hospitalization with a cohort during the COVID-19 pandemic (February-June 2020). Baseline categorical variables were compared using χ 2 tests and continuous variables were compared using the Student t test and Mann-Whitney-Wilcoxon test. All analysis was performed in SAS version 9.4 software (SAS Institute, Cary, NC). Primary outcomes of interest included rates of hospital-onset CDI (HO-CDI, defined as a positive C. difficile test over 3 days after admission), 4 antibiotic prescribing and length of stay. HO-CDI incidence was described by the standardized infection ratios (SIR), which adjusts for facility and patient-level factors that contribute to hospitalonset infection risk within each facility. Antibiotic prescriptions were measured by antibiotic days per 1,000 days present. The study was approved by the Institutional Review Board of the Icahn School of Medicine at Mount Sinai. Overall, HO-CDI SIR 5 was not statistically different during the COVID-19 period than during the 2019 period (P = 0.69)

Hypertrophic cardiomyopathy (HCM) is a primary disease of cardiac muscle, and one of the most common causes of sudden cardiac death (SCD) in young people. Many mutations in cardiac troponin T (cTnT) lead to a complex form of HCM with varying degrees of ventricular hypertrophy and ~65% of all cTnT mutations occur within or flanking the elongated N-terminal TNT1 domain. Biophysical studies have predicted that distal TNT1 mutations, including Δ160E, cause disease by a novel, yet unknown mechanism as compared to N-terminal mutations. To begin to address the specific effects of this commonly observed cTnT mutation we generated two independent transgenic mouse lines carrying variant doses of the mutant transgene. Hearts from the 30% and 70% cTnT Δ160E lines demonstrated a highly unique, dose-dependent disruption in cellular and sarcomeric architecture and a highly progressive pattern of ventricular remodeling. While adult ventricular myocytes isolated from Δ160E transgenic mice exhibited dosage-independent mechanical impairments, decreased sarcoplasmic reticulum calcium load and SERCA2a calcium uptake activity, the observed decreases in calcium transients were dosage-dependent. The latter findings were concordant with measures of calcium regulatory proteins abundance and phosphorylation state. Finally, studies of whole heart physiology in the isovolumic mode demonstrated dose-dependent differences in the degree of cardiac dysfuction. We conclude that the observed clinical severity of the cTnT Δ160E mutation is caused by a combination of direct sarcomeric disruption coupled to a profound disregulation of Ca2+ homeostasis at the cellular level that results in a unique and highly progressive pattern of ventricular remodeling.

Recently, linkage analysis of two large unrelated multigenerational families identified a novel dilated cardiomyopathy (DCM)-linked mutation in the gene coding for alpha-tropomyosin (TPM1) resulting in the substitution of an aspartic acid for an asparagine (at residue 230). To determine how a single amino acid mutation in α-tropomyosin (Tm) can lead to a highly penetrant DCM we generated a novel transgenic mouse model carrying the D230N mutation. The resultant mouse model strongly phenocopied the early onset of cardiomyopathic remodeling observed in patients as significant systolic dysfunction was observed by 2 months of age. To determine the precise cellular mechanism(s) leading to the observed cardiac pathology we examined the effect of the mutation on Ca2+ handling in isolated myocytes and myofilament activation in vitro. D230N-Tm filaments exhibited a reduced Ca2+ sensitivity of sliding velocity. This decrease in sensitivity was coupled to increase in the peak amplitude of Ca2+ transients. While significant, and consistent with other DCMs, these measurements are comprised of complex inputs and did not provide sufficient experimental resolution. We then assessed the primary structural effects of D230N-Tm. Measurements of the thermal unfolding of D230N-Tm vs WT-Tm revealed an increase in stability primarily affecting the C-terminus of the Tm coiled-coil. We conclude that the D230N-Tm mutation induces a decrease in flexibility of the C-terminus via propagation through the helical structure of the protein, thus decreasing the flexibility of the Tm overlap and impairing its ability to regulate contraction. Understanding this unique structural mechanism could provide novel targets for eventual therapeutic interventions in patients with Tm-linked cardiomyopathies.