Monoacylglycerol acyltransferase 2 (MGAT2) is an important enzyme highly expressed in the human small intestine and liver for the regulation of triglyceride absorption and homeostasis. We report that treatment with BMS-963272, a potent and selective MGAT2 inhibitor, decreased inflammation and fibrosis in CDAHFD and STAM, two murine nonalcoholic steatohepatitis (NASH) models. In high-fat-diet-treated cynomolgus monkeys, in contrast to a selective diacylglycerol acyltransferase 1 (DGAT1) inhibitor, BMS-963272 did not cause diarrhea. In a Phase 1 multiple-dose trial of healthy human adults with obesity (NCT04116632), BMS-963272 was safe and well tolerated with no treatment discontinuations due to adverse events. Consistent with the findings in rodent models, BMS-963272 elevated plasma long-chain dicarboxylic acid, indicating robust pharmacodynamic biomarker modulation; increased gut hormones GLP-1 and PYY; and decreased body weight in human subjects. These data suggest MGAT2 inhibition is a promising therapeutic opportunity for NASH, a disease with high unmet medical needs.

Background The phase 3 CheckMate 214 trial demonstrated higher response rates and improved overall survival with nivolumab plus ipilimumab versus sunitinib in first-line therapy for advanced clear-cell renal cell carcinoma (RCC). An unmet need exists to identify patients with RCC who are most likely to benefit from treatment with nivolumab plus ipilimumab. Methods In exploratory analyses, pretreatment levels of programmed death ligand 1 were assessed by immunohistochemistry. Genomic and transcriptomic biomarkers (including tumor mutational burden and gene expression signatures) were also investigated. Results Biomarkers previously associated with benefit from immune checkpoint inhibitor-containing regimens in RCC were not predictive for survival in patients with RCC treated with nivolumab plus ipilimumab. Analysis of gene expression identified an association between an inflammatory response and progression-free survival with nivolumab plus ipilimumab. Conclusions The exploratory analyses reveal relationships between molecular biomarkers and provide supportive data on how the inflammation status of the tumor microenvironment may be important for identifying predictive biomarkers of response and survival with combination immunotherapy in patients with RCC. Further validation may help to provide biomarker-driven precision treatment for patients with RCC.

AbstractObjectiveSSc is a rheumatic autoimmune disease affecting roughly 20 000 people worldwide and characterized by excessive collagen accumulation in the skin and internal organs. Despite the high morbidity and mortality associated with SSc, there are no approved disease-modifying agents. Our objective in this study was to explore transcriptomic and model-based drug discovery approaches for SSc.MethodsIn this study, we explored the molecular basis for SSc pathogenesis in a well-studied mouse model of scleroderma. We profiled the skin and lung transcriptomes of mice at multiple timepoints, analysing the differential gene expression that underscores the development and resolution of bleomycin-induced fibrosis.ResultsWe observed shared expression signatures of upregulation and downregulation in fibrotic skin and lung tissue, and observed significant upregulation of key pro-fibrotic genes including GDF15, Saa3, Cxcl10, Spp1 and Timp1. To identify changes in gene expression in responses to anti-fibrotic therapy, we assessed the effect of TGF-β pathway inhibition via oral ALK5 (TGF-β receptor I) inhibitor SB525334 and observed a time-lagged response in the lung relative to skin. We also implemented a machine learning algorithm that showed promise at predicting lung function using transcriptome data from both skin and lung biopsies.ConclusionThis study provides the most comprehensive look at the gene expression dynamics of an animal model of SSc to date, provides a rich dataset for future comparative fibrotic disease research, and helps refine our understanding of pathways at work during SSc pathogenesis and intervention.

Abstract Introduction: CAYA with R/R cHL without CMR have suboptimal outcomes. Biomarkers associated with poor outcomes in cHL include PD-L1 overexpression and presence of tumor-associated macrophages (TAMs). In the phase 2 CheckMate 744 study (NCT02927769), CAYA with R/R cHL treated with a risk-adapted approach using nivo + BV induction showed a CMR rate per BICR of 59% (26/44) in the standard risk cohort; 41% of patients (pts) did not achieve CMR (non-CMR). Here we compared biomarker data from pts achieving CMR vs non-CMR. Methods: Pts aged 5-30 y with R/R cHL received 4 cycles of nivo + BV induction. Tumor biopsy and peripheral blood samples were collected at baseline (BL) and 4 prespecified timepoints during induction. CD163+ CD68+ TAMs were quantified using multiplex immunohistochemistry. Cytokine expression in pts with CMR vs non-CMR was assessed by the limma workflow and a linear model based on changes from BL. Immune cells were analyzed by flow cytometry. Statistical testing was adjusted for repeated measures across visits; values are reported from 39/39 cytokines and 13/16 cell subsets tested. Cytokine ratios to BL were compared across all timepoints and between response groups by ANOVA. Results: Pts with CMR (n = 24/26) after nivo + BV induction had more CD163+ CD68+ TAMs than pts with non-CMR (n = 17/18) in the BL tumor microenvironment (P = 0.018). Hierarchical clustering of cytokine fold changes from BL to the indicated timepoint, between pts with CMR vs non-CMR, showed reduced levels of haptoglobin, RANTES, CRP, PARC, EN-RAGE, and TARC/CCL17, and increased levels of MCP-2, IFN-γ, IP-10, and MIG. In all pts, median concentrations of TARC, IL-2Rα, CRP, and ICAM-1 decreased significantly (all P < 0.05); a greater reduction in TARC and CRP was observed in early cycles in pts with CMR vs non-CMR. In contrast, IP-10 levels increased from BL in all pts (P = 0.0012). Four cytokines showed higher concentrations in pts with CMR vs non-CMR: TNFR2, MCP-2, vWF, and TIMP-1 (all P < 0.05). Increased numbers of lymphocytes and monocytes and decreased numbers of granulocytes were observed after induction in all pts (all P < 0.01). Pts with non-CMR had more naive (CCR7+ CD45RA+) CD4, and naive CD8 T cells than pts with CMR at BL, and more overall CD4 (CD3+ CD4+) T cells at the end of cycle 4 (all P < 0.05). Conclusions: This biomarker analysis from CheckMate 744 suggests that different immunological parameters are associated with CMR vs non-CMR to nivo + BV induction in CAYA with R/R cHL. These include increased TAMs at BL in pts with CMR, changes in cytokine levels associated with macrophage biology, and greater numbers of naive lymphocytes and CD4 T cells in pts with non-CMR. TAMs have been reported to express high PD-L1 levels; thus, increased TAM numbers may sensitize pts to nivo + BV. Citation Format: Charlie Garnett-Benson, Ron Ammar, Russell Crowe, Stephen Francis, Sahar Ansari, Alev Akyol. Biomarkers associated with complete metabolic response (CMR) to nivolumab and brentuximab vedotin (nivo + BV) for the treatment of children, adolescents, and young adults (CAYA) with relapsed or refractory (R/R) classical Hodgkin lymphoma (cHL) in the CheckMate 744 study [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 5222.

Background Idiopathic pulmonary fibrosis (IPF) is a fatal lung disease with a significant unmet medical need. Development of transformational therapies for IPF is challenging in part to due to lack of robust predictive biomarkers of prognosis and treatment response. Importantly, circulating biomarkers of IPF are limited and none are in clinical use. Methods We previously reported dysregulated pathways and new disease biomarkers in advanced IPF through RNA sequencing of lung tissues from a cohort of transplant-stage IPF patients (n = 36) in comparison to normal healthy donors (n = 19) and patients with acute lung injury (n = 11). Here we performed proteomic profiling of matching plasma samples from these cohorts through the Somascan-1300 SomaLogics platform. Results Comparative analyses of lung transcriptomic and plasma proteomic signatures identified a set of 34 differentially expressed analytes (fold change (FC) ≥ ± 1.5, false discovery ratio (FDR) ≤ 0.1) in IPF samples compared to healthy controls. IPF samples showed strong enrichment of chemotaxis, tumor infiltration and mast cell migration pathways and downregulated extracellular matrix (ECM) degradation. Mucosal (CCL25 and CCL28) and Th2 (CCL17 and CCL22) chemokines were markedly upregulated in IPF and highly correlated within the subjects. The mast cell maturation chemokine, CXCL12, was also upregulated in IPF plasma (fold change 1.92, FDR 0.006) and significantly correlated (Pearson r = − 0.38, p = 0.022) to lung function (%predicted FVC), with a concomitant increase in the mast cell Tryptase, TPSB2. Markers of collagen III and VI degradation (C3M and C6M) were significantly downregulated (C3M p < 0.001 and C6M p < 0.0001 IPF vs control) and correlated, Pearson r = 0.77) in advanced IPF consistent with altered ECM homeostasis. Conclusions Our study identifies a panel of tissue and circulating biomarkers with clinical utility in IPF that can be validated in future studies across larger cohorts.

Background Nivolumab is an immune checkpoint inhibitor targeting the programmed death-1 receptor that improves survival in a subset of patients with clear cell renal cell carcinoma (ccRCC). In contrast to other tumor types that respond to immunotherapy, factors such as programmed death ligand-1 (PD-L1) status and tumor mutational burden show limited predictive utility in ccRCC. To address this gap, we report here the first molecular characterization of nivolumab response using paired index lesions, before and during treatment of metastatic ccRCC. Methods We analyzed gene expression and T-cell receptor (TCR) clonality using lesion-paired biopsies provided in the CheckMate 009 trial and integrated the results with their PD-L1/CD4/CD8 status, genomic mutation status and serum cytokine assays. Statistical tests included linear mixed models, logistic regression models, Fisher’s exact test, and Kruskal-Wallis rank-sum test. Results We identified transcripts related to response, both at baseline and on therapy, including several that are amenable to peripheral bioassays or to therapeutic intervention. At both timepoints, response was positively associated with T-cell infiltration but not associated with TCR clonality, and some non-Responders were highly infiltrated. Lower baseline T-cell infiltration correlated with elevated transcription of Wnt/β-catenin signaling components and hypoxia-regulated genes, including the Treg chemoattractant CCL28. On treatment, analysis of the non-responding patients whose tumors were highly T-cell infiltrated suggests association of the RIG-I-MDA5 pathway in their nivolumab resistance. We also analyzed our data using previous transcriptional classifications of ccRCC and found they concordantly identified a molecular subtype that has enhanced nivolumab response but is sunitinib-resistant. Conclusion Our study describes molecular characteristics of response and resistance to nivolumab in patients with metastatic ccRCC, potentially impacting patient selection and first-line treatment decisions. Trial registration number NCT01358721.

5009 Background: In CheckMate 214 (NCT02231749), N+I demonstrated superior clinical outcomes vs S in patients (pts) with aRCC. Here, we report exploratory biomarker analyses of pretreatment tumor samples relative to outcomes. Methods: Formalin-fixed, paraffin-embedded aRCC samples were characterized by immunohistochemistry (tumor programmed death ligand 1 [PD-L1], n = 992; PD-L1 combined positive score [CPS], n = 980), whole exome sequencing (WES; tumor mutational burden, indel burden, HLA class I zygosity, and PBRM1 mutation status; n = 481), and RNAseq (n = 213). Gene signature scores were calculated as the median value of Z-scored expression for transcripts. Association with outcome was tested using Fisher’s exact test and Cox proportional hazards model. With ≥ 42 mo follow-up, prolonged progression-free survival (PFS) with N+I ( < or ≥ 18 mo; n = 82 vs 27, respectively) was analyzed by limma and gene set enrichment analysis of Hallmark gene sets (MSigDB). Results: PD-L1 CPS did not have improved predictive power over tumor PD-L1. The WES-derived biomarkers were not associated with outcomes in pts treated with N+I or S. For the RNAseq cohort, objective response rates (ORR) for pts with ≥ median scores, and hazard ratios (HR) relative to < median are shown (Table). A higher angiogenesis (Angio) gene signature score was associated with higher ORR and PFS for S; lower Angio score was associated with higher ORR in N+I. Immune signature scores were not predictive for ORR in N+I. Prolonged PFS with N+I (≥ 18 mo, n = 27) was associated with higher expression of Hallmark inflammatory response and Hallmark epithelial mesenchymal transition (EMT) gene sets (both adjusted P = 0.002). Conclusions: The Angio gene signature was associated with ORR for S (high score) and N+I (low score). Prolonged PFS in 27 pts receiving N+I was linked with inflammation (consistent with findings from PD-1 blockade monotherapy), but associated with EMT-related transcripts (in contrast with findings in other tumor types). Further validation of these exploratory analyses will be required. Clinical trial information: NCT02231749 . [Table: see text]

ACE2 (angiotensin-converting enzyme 2) is a key component of the renin-angiotensin-aldosterone system. Yet, little is known about the clinical and biologic correlates of circulating ACE2 levels in humans. We assessed the clinical and proteomic correlates of plasma (soluble) ACE2 protein levels in human heart failure. We measured plasma ACE2 using a modified aptamer assay among PHFS (Penn Heart Failure Study) participants (n=2248). We performed an association study of ACE2 against ≈5000 other plasma proteins measured with the SomaScan platform. Plasma ACE2 was not associated with ACE inhibitor and angiotensin-receptor blocker use. Plasma ACE2 was associated with older age, male sex, diabetes mellitus, a lower estimated glomerular filtration rate, worse New York Heart Association class, a history of coronary artery bypass surgery, and higher pro-BNP (pro-B-type natriuretic peptide) levels. Plasma ACE2 exhibited associations with 1011 other plasma proteins. In pathway overrepresentation analyses, top canonical pathways associated with plasma ACE2 included clathrin-mediated endocytosis signaling, actin cytoskeleton signaling, mechanisms of viral exit from host cells, EIF2 (eukaryotic initiation factor 2) signaling, and the protein ubiquitination pathway. In conclusion, in humans with heart failure, plasma ACE2 is associated with various clinical factors known to be associated with severe coronavirus disease 2019 (COVID-19), including older age, male sex, and diabetes mellitus, but is not associated with ACE inhibitor and angiotensin-receptor blocker use. Plasma ACE2 protein levels are prominently associated with multiple cellular pathways involved in cellular endocytosis, exocytosis, and intracellular protein trafficking. Whether these have a causal relationship with ACE2 or are relevant to novel coronavirus-2 infection remains to be assessed in future studies.

Hundreds of dominant-negative myosin mutations have been identified that lead to hypertrophic cardiomyopathy, and the biomechanical link between mutation and disease is heterogeneous across this patient population. To increase the therapeutic feasibility of treating this diverse genetic population, we investigated the ability of locked nucleic acid (LNA)-modified antisense oligonucleotides (ASOs) to selectively knock down mutant myosin transcripts by targeting single-nucleotide polymorphisms (SNPs) that were found to be common in the myosin heavy chain 7 (MYH7) gene. We identified three SNPs in MYH7 and designed ASO libraries to selectively target either the reference or alternate MYH7 sequence. We identified ASOs that selectively knocked down either the reference or alternate allele at all three SNP regions. We also show allele-selective knockdown in a mouse model that was humanized on one allele. These results suggest that SNP-targeting ASOs are a promising therapeutic modality for treating cardiac pathology.

Idiopathic pulmonary fibrosis (IPF) is a chronic and progressive lung disease affecting ~5 million people globally. We have constructed an accurate model of IPF disease status using elastic net regularized regression on clinical gene expression data. Leveraging whole transcriptome microarray data from 230 IPF and 89 control samples from Yang et al. (2013), sourced from the Lung Tissue Research Consortium (LTRC) and National Jewish Health (NJH) cohorts, we identify an IPF gene expression signature. We performed optimal feature selection to reduce the number of transcripts required by our model to a parsimonious set of 15. This signature enables our model to accurately separate IPF patients from controls. Our model outperforms existing published models when tested with multiple independent clinical cohorts. Our study underscores the utility of elastic nets for gene signature/panel selection which can be used for the construction of a multianalyte biomarker of disease. We also filter the gene sets used for model input to construct a model reliant on secreted proteins. Using this approach, we identify the preclinical bleomycin rat model that is most congruent with human disease at day 21 post-bleomycin administration, contrasting with earlier timepoints suggested by other studies.

Idiopathic pulmonary fibrosis (IPF), the scarring of lung parenchyma resulting in the loss of lung function, remains a fatal disease with a significant unmet medical need. Patients with severe IPF often develop acute exacerbations resulting in the rapid deterioration of lung function, requiring transplantation. Understanding the pathophysiological mechanisms contributing to IPF is key to develop novel therapeutic approaches for end-stage disease. We report here RNA-sequencing analyses of lung tissues from a cohort of patients with transplant-stage IPF (n=36), compared with acute lung injury (ALI) (n=11) and nondisease controls (n=19), that reveal a robust gene expression signature unique to end-stage IPF. In addition to extracellular matrix remodelling pathways, we identified pathways associated with T-cell infiltration/activation, tumour development, and cholesterol homeostasis, as well as novel alternatively spliced transcripts that are differentially regulated in the advanced IPF lung versus ALI or nondisease controls. Additionally, we show a subset of genes that are correlated with percent predicted forced vital capacity and could reflect disease severity. Our results establish a robust transcriptomic fingerprint of an advanced IPF lung that is distinct from previously reported microarray signatures of moderate, stable or progressive IPF and identifies hitherto unknown candidate targets and pathways for therapeutic intervention in late-stage IPF as well as biomarkers to characterise disease progression and enable patient stratification.

Precision-cut liver tissue slice (PCLS) contains all major cell types of the liver parenchyma and preserves the original cell-cell and cell-matrix contacts. It represents a promising ex vivo model to study liver fibrosis and test the antifibrotic effect of experimental compounds in a physiological environment. In this study using RNA sequencing, we demonstrated that various pathways functionally related to fibrotic mechanisms were dysregulated in PCLSs derived from rats subjected to bile duct ligation. The activin receptor-like kinase-5 (Alk5) inhibitor SB525334, nintedanib, and sorafenib each reversed a subset of genes dysregulated in fibrotic PCLSs, and of those genes we identified 608 genes whose expression was reversed by all three compounds. These genes define a molecular signature characterizing many aspects of liver fibrosis pathology and its attenuation in the model. A panel of 12 genes and 4 secreted biomarkers including procollagen I, hyaluronic acid (HA), insulin-like growth factor binding protein 5 (IGFBP5), and WNT1-inducible signaling pathway protein 1 (WISP1) were further validated as efficacy end points for the evaluation of antifibrotic activity of experimental compounds. Finally, we showed that blockade of αV-integrins with a small molecule inhibitor attenuated the fibrotic phenotype in the model. Overall, our results suggest that the rat fibrotic PCLS model may represent a valuable system for target validation and determining the efficacy of experimental compounds. NEW & NOTEWORTHY We investigated the antifibrotic activity of three compounds, the activin receptor-like kinase-5 (Alk5) inhibitor SB525334, nintedanib, and sorafenib, in a rat fibrotic precision-cut liver tissue slice model using RNA sequencing analysis. A panel of 12 genes and 4 secreted biomarkers including procollagen I, hyaluronic acid (HA), insulin-like growth factor binding protein 5 (IGFBP5), and WNT1-inducible signaling pathway protein 1 (WISP1) were then established as efficacy end points to validate the antifibrotic activity of the αV-integrin inhibitor CWHM12. This study demonstrated the value of the rat fibrotic PCLS model for the evaluation of antifibrotic drugs.

The translation of novel pulmonary fibrosis therapies from preclinical models into the clinic represents a major challenge demonstrated by the high attrition rate of compounds that showed efficacy in preclinical models but demonstrated no significant beneficial effects in clinical trials. A precision-cut lung tissue slice (PCLS) contains all major cell types of the lung and preserves the original cell-cell and cell-matrix contacts. It represents a promising ex vivo model to study pulmonary fibrosis. In this study, using RNA sequencing, we demonstrated that transforming growth factor-β1 (TGFβ1) induced robust fibrotic responses in the rat PCLS model, as it changed the expression of genes functionally related to extracellular matrix remodeling, cell adhesion, epithelial-to-mesenchymal transition, and various immune responses. Nintedanib, pirfenidone, and sorafenib each reversed a subset of genes modulated by TGFβ1, and of those genes we identified 229 whose expression was reversed by all three drugs. These genes define a molecular signature characterizing many aspects of pulmonary fibrosis pathology and its attenuation in the rat PCLS fibrosis model. A panel of 12 genes and three secreted biomarkers, including procollagen I, hyaluronic acid, and WNT1-inducible signaling pathway protein 1 were validated as efficacy end points for the evaluation of antifibrotic activity of experimental compounds. Finally, we showed that blockade of αV-integrins suppressed TGFβ1-induced fibrotic responses in the rat PCLS fibrosis model. Overall, our results suggest that the TGFβ1-induced rat PCLS fibrosis model may represent a valuable system for target validation and to determine the efficacy of experimental compounds.