Genome-first evaluation with exome sequence and clinical data uncovers underdiagnosed genetic disorders in a large healthcare system.

Population-based genomic screening may help diagnose individuals with disease-risk variants. Here, we perform a genome-first evaluation for nine disorders in 29,039 participants with linked exome sequences and electronic health records (EHRs). We identify 614 individuals with 303 pathogenic/likely pathogenic or predicted loss-of-function (P/LP/LoF) variants, yielding 644 observations; 487 observations (76%) lack a corresponding clinical diagnosis in the EHR. Upon further investigation, 75 clinically undiagnosed observations (15%) have evidence of symptomatic untreated disease, including familial hypercholesterolemia (3 of 6 [50%] undiagnosed observations with disease evidence) and breast cancer (23 of 106 [22%]). These genetic findings enable targeted phenotyping that reveals new diagnoses in previously undiagnosed individuals. Disease yield is greater with variants in penetrant genes for which disease is observed in carriers in an independent cohort. The prevalence of P/LP/LoF variants exceeds that of clinical diagnoses, and some clinically undiagnosed carriers are discovered to have disease. These results highlight the potential of population-based genomic screening.

An Integrative Multiomics Framework for Identification of Therapeutic Targets in Pulmonary Fibrosis.

Pulmonary fibrosis (PF) is a heterogeneous disease with a poor prognosis. Therefore, identifying additional therapeutic modalities is required to improve outcome. However, the lack of biomarkers of disease progression hampers the preclinical to clinical translational process. Here, this work assesses and identifies progressive alterations in pulmonary function, transcriptomics, and metabolomics in the mouse lung at 7, 14, 21, and 28 days after a single dose of oropharyngeal bleomycin. By integrating multi-omics data, this work identifies two central gene subnetworks associated with multiple critical pathological changes in transcriptomics and metabolomics as well as pulmonary function. This work presents a multi-omics-based framework to establish a translational link between the bleomycin-induced PF model in mice and human idiopathic pulmonary fibrosis to identify druggable targets and test therapeutic candidates. This work also indicates peripheral cannabinoid receptor 1 (CB1 R) antagonism as a rational therapeutic target for clinical translation in PF. Mouse Lung Fibrosis Atlas can be accessed freely at https://niaaa.nih.gov/mouselungfibrosisatlas.

Phenome-wide Mendelian randomization study of plasma triglyceride levels and 2600 disease traits.

Background: Causality between plasma triglyceride (TG) levels and atherosclerotic cardiovascular disease (ASCVD) risk remains controversial despite more than four decades of study and two recent landmark trials, STRENGTH, and REDUCE-IT. Further unclear is the association between TG levels and non-atherosclerotic diseases across organ systems. Methods: Here, we conducted a phenome-wide, two-sample Mendelian randomization (MR) analysis using inverse-variance weighted (IVW) regression to systematically infer the causal effects of plasma TG levels on 2600 disease traits in the European ancestry population of UK Biobank. For replication, we externally tested 221 nominally significant associations (p<0.05) in an independent cohort from FinnGen. To account for potential horizontal pleiotropy and the influence of invalid instrumental variables, we performed sensitivity analyses using MR-Egger regression, weighted median estimator, and MR-PRESSO. Finally, we used multivariable MR (MVMR) controlling for correlated lipid fractions to distinguish the independent effect of plasma TG levels. Results: Our results identified seven disease traits reaching Bonferroni-corrected significance in both the discovery (p<1.92 × 10-5) and replication analyses (p<2.26 × 10-4), suggesting a causal relationship between plasma TG levels and ASCVDs, including coronary artery disease (OR 1.33, 95% CI 1.24-1.43, p=2.47 × 10-13). We also identified 12 disease traits that were Bonferroni-significant in the discovery or replication analysis and at least nominally significant in the other analysis (p<0.05), identifying plasma TG levels as a novel potential risk factor for nine non-ASCVD diseases, including uterine leiomyoma (OR 1.19, 95% CI 1.10-1.29, p=1.17 × 10-5). Conclusions: Taking a phenome-wide, two-sample MR approach, we identified causal associations between plasma TG levels and 19 disease traits across organ systems. Our findings suggest unrealized drug repurposing opportunities or adverse effects related to approved and emerging TG-lowering agents, as well as mechanistic insights for future studies. Funding: RD is supported by the National Institute of General Medical Sciences of the National Institutes of Health (NIH) (R35-GM124836) and the National Heart, Lung, and Blood Institute of the NIH (R01-HL139865 and R01-HL155915).

Peripheral Hybrid CB1R and iNOS Antagonist MRI-1867 Displays Anti-Fibrotic Efficacy in Bleomycin-Induced Skin Fibrosis.

Scleroderma, or systemic sclerosis, is a multi-organ connective tissue disease resulting in fibrosis of the skin, heart, and lungs with no effective treatment. Endocannabinoids acting via cannabinoid-1 receptors (CB1R) and increased activity of inducible NO synthase (iNOS) promote tissue fibrosis including skin fibrosis, and joint targeting of these pathways may improve therapeutic efficacy. Recently, we showed that in mouse models of liver, lung and kidney fibrosis, treatment with a peripherally restricted hybrid CB1R/iNOS inhibitor (MRI-1867) yields greater anti-fibrotic efficacy than inhibiting either target alone. Here, we evaluated the therapeutic efficacy of MRI-1867 in bleomycin-induced skin fibrosis. Skin fibrosis was induced in C57BL/6J (B6) and Mdr1a/b-Bcrp triple knock-out (KO) mice by daily subcutaneous injections of bleomycin (2 IU/100 µL) for 28 days. Starting on day 15, mice were treated for 2 weeks with daily oral gavage of vehicle or MRI-1867. Skin levels of MRI-1867 and endocannabinoids were measured by mass spectrometry to assess target exposure and engagement by MRI-1867. Fibrosis was characterized histologically by dermal thickening and biochemically by hydroxyproline content. We also evaluated the potential increase of drug-efflux associated ABC transporters by bleomycin in skin fibrosis, which could affect target exposure to test compounds, as reported in bleomycin-induced lung fibrosis. Bleomycin-induced skin fibrosis was comparable in B6 and Mdr1a/b-Bcrp KO mice. However, the skin level of MRI-1867, an MDR1 substrate, was dramatically lower in B6 mice (0.023 µM) than in Mdr1a/b-Bcrp KO mice (8.8 µM) due to a bleomycin-induced increase in efflux activity of MDR1 in fibrotic skin. Furthermore, the endocannabinoids anandamide and 2-arachidonylglycerol were elevated 2-4-fold in the fibrotic vs. control skin in both mouse strains. MRI-1867 treatment attenuated bleomycin-induced established skin fibrosis and the associated increase in endocannabinoids in Mdr1a/b-Bcrp KO mice but not in B6 mice. We conclude that combined inhibition of CB1R and iNOS is an effective anti-fibrotic strategy for scleroderma. As bleomycin induces an artifact in testing antifibrotic drug candidates that are substrates of drug-efflux transporters, using Mdr1a/b-Bcrp KO mice for preclinical testing of such compounds avoids this pitfall.

CB1 R and iNOS are distinct players promoting pulmonary fibrosis in Hermansky-Pudlak syndrome.

Hermansky-Pudlak syndrome (HPS) is a rare genetic disorder which, in its most common and severe form, HPS-1, leads to fatal adult-onset pulmonary fibrosis (PF) with no effective treatment. We evaluated the role of the endocannabinoid/CB1 R system and inducible nitric oxide synthase (iNOS) for dual-target therapeutic strategy using human bronchoalveolar lavage fluid (BALF), lung samples from patients with HPS and controls, HPS-PF patient-derived lung fibroblasts, and bleomycin-induced PF in pale ear mice (HPS1ep/ep ). We found overexpression of CB1 R and iNOS in fibrotic lungs of HPSPF patients and bleomycin-infused pale ear mice. The endocannabinoid anandamide was elevated in BALF and negatively correlated with pulmonary function parameters in HPSPF patients and pale ear mice with bleomycin-induced PF. Simultaneous targeting of CB1 R and iNOS by MRI-1867 yielded greater antifibrotic efficacy than inhibiting either target alone by attenuating critical pathologic pathways. Moreover, MRI-1867 treatment abrogated bleomycin-induced increases in lung levels of the profibrotic interleukin-11 via iNOS inhibition and reversed mitochondrial dysfunction via CB1 R inhibition. Dual inhibition of CB1 R and iNOS is an effective antifibrotic strategy for HPSPF.

The Heterogeneity of Lipid Metabolism in Cancer.

The study of cancer cell metabolism has traditionally focused on glycolysis and glutaminolysis. However, lipidomic technologies have matured considerably over the last decade and broadened our understanding of how lipid metabolism is relevant to cancer biology [1-3]. Studies now suggest that the reprogramming of cellular lipid metabolism contributes directly to malignant transformation and progression [4, 5]. For example, de novo lipid synthesis can supply proliferating tumor cells with phospholipid components that comprise the plasma and organelle membranes of new daughter cells [6, 7]. Moreover, the upregulation of mitochondrial ß-oxidation can support tumor cell energetics and redox homeostasis [8], while lipid-derived messengers can regulate major signaling pathways or coordinate immunosuppressive mechanisms [9-11]. Lipid metabolism has, therefore, become implicated in a variety of oncogenic processes, including metastatic colonization, drug resistance, and cell differentiation [10, 12-16]. However, whether we can safely and effectively modulate the underlying mechanisms of lipid metabolism for cancer therapy is still an open question.

Discovery of a NAPE-PLD inhibitor that modulates emotional behavior in mice.

N-acylethanolamines (NAEs), which include the endocannabinoid anandamide, represent an important family of signaling lipids in the brain. The lack of chemical probes that modulate NAE biosynthesis in living systems hamper the understanding of the biological role of these lipids. Using a high-throughput screen, chemical proteomics and targeted lipidomics, we report here the discovery and characterization of LEI-401 as a CNS-active N-acylphosphatidylethanolamine phospholipase D (NAPE-PLD) inhibitor. LEI-401 reduced NAE levels in neuroblastoma cells and in the brain of freely moving mice, but not in NAPE-PLD KO cells and mice, respectively. LEI-401 activated the hypothalamus-pituitary-adrenal axis and impaired fear extinction, thereby emulating the effect of a cannabinoid CB1 receptor antagonist, which could be reversed by a fatty acid amide hydrolase inhibitor. Our findings highlight the distinctive role of NAPE-PLD in NAE biosynthesis in the brain and suggest the presence of an endogenous NAE tone controlling emotional behavior.

Bleomycin Induces Drug Efflux in Lungs. A Pitfall for Pharmacological Studies of Pulmonary Fibrosis.

ATP-binding cassette (ABC) transporters are evolutionarily conserved membrane proteins that pump a variety of endogenous substrates across cell membranes. Certain subfamilies are known to interact with pharmaceutical compounds, potentially influencing drug delivery and treatment efficacy. However, the role of drug resistance-associated ABC transporters has not been examined in idiopathic pulmonary fibrosis (IPF) or its animal model: the bleomycin (BLM)-induced murine model. Here, we investigate the expression of two ABC transporters, P-gp (permeability glycoprotein) and BCRP (breast cancer resistance protein), in human IPF lung tissue and two different BLM-induced mouse models of pulmonary fibrosis. We obtained human IPF specimens from patients during lung transplantation and administered BLM to male C57BL/6J mice either by oropharyngeal aspiration (1 U/kg) or subcutaneous osmotic infusion (100 U/kg over 7 d). We report that P-gp and BCRP expression in lungs of patients with IPF was comparable to controls. However, murine lungs expressed increased levels of P-gp and BCRP after oropharyngeal and subcutaneous BLM administration. We localized this upregulation to multiple pulmonary cell types, including alveolar fibroblasts, endothelial cells, and type 2 epithelial cells. Functionally, this effect reduced murine lung exposure to nintedanib, a U.S. Food and Drug Administration-approved IPF therapy known to be a P-gp substrate. The study reveals a discrepancy between IPF pathophysiology and the common animal model of lung fibrosis. BLM-induced drug efflux in the murine lungs may present an uncontrolled confounding variable in the preclinical study of IPF drug candidates, and these findings will facilitate disease model validation and enhance new drug discoveries that will ultimately improve patient outcomes.

The Heterogeneity of Lipid Metabolism in Cancer.

KEY POINTS: The study of cancer cell metabolism has traditionally focused on glycolysis and glutaminolysis. However, lipidomic technologies have matured considerably over the last decade and broadened our understanding of how lipid metabolism is relevant to cancer biology [1­3]. Studies now suggest that the reprogramming of cellular lipid metabolism contributes directly to malignant transformation and progression [4, 5]. For example, de novo lipid synthesis can supply proliferating tumor cells with phospholipid components that comprise the plasma and organelle membranes of new daughter cells [6, 7]. Moreover, the upregulation of mitochondrial ß-oxidation can support tumor cell energetics and redox homeostasis [8], while lipid-derived messengers can regulate major signaling pathways or coordinate immunosuppressive mechanisms [9­11]. Lipid metabolism has therefore become implicated in a variety of oncogenic processes, including metastatic colonization, drug resistance, and cell differentiation [10, 12­16]. However, whether we can safely and effectively modulate the underlying mechanisms for cancer therapy is still an open question.

Network analyses identify liver-specific targets for treating liver diseases.

We performed integrative network analyses to identify targets that can be used for effectively treating liver diseases with minimal side effects. We first generated co-expression networks (CNs) for 46 human tissues and liver cancer to explore the functional relationships between genes and examined the overlap between functional and physical interactions. Since increased de novo lipogenesis is a characteristic of nonalcoholic fatty liver disease (NAFLD) and hepatocellular carcinoma (HCC), we investigated the liver-specific genes co-expressed with fatty acid synthase (FASN). CN analyses predicted that inhibition of these liver-specific genes decreases FASN expression. Experiments in human cancer cell lines, mouse liver samples, and primary human hepatocytes validated our predictions by demonstrating functional relationships between these liver genes, and showing that their inhibition decreases cell growth and liver fat content. In conclusion, we identified liver-specific genes linked to NAFLD pathogenesis, such as pyruvate kinase liver and red blood cell (PKLR), or to HCC pathogenesis, such as PKLR, patatin-like phospholipase domain containing 3 (PNPLA3), and proprotein convertase subtilisin/kexin type 9 (PCSK9), all of which are potential targets for drug development.

Developmental Role of Macrophage Cannabinoid-1 Receptor Signaling in Type 2 Diabetes.

Islet inflammation promotes ß-cell loss and type 2 diabetes (T2D), a process replicated in Zucker Diabetic Fatty (ZDF) rats in which ß-cell loss has been linked to cannabinoid-1 receptor (CB1R)-induced proinflammatory signaling in macrophages infiltrating pancreatic islets. Here, we analyzed CB1R signaling in macrophages and its developmental role in T2D. ZDF rats with global deletion of CB1R are protected from ß-cell loss, hyperglycemia, and nephropathy that are present in ZDF littermates. Adoptive transfer of CB1R-/- bone marrow to ZDF rats also prevents ß-cell loss and hyperglycemia but not nephropathy. ZDF islets contain elevated levels of CB1R, interleukin-1ß, tumor necrosis factor-α, the chemokine CCL2, and interferon regulatory factor-5 (IRF5), a marker of inflammatory macrophage polarization. In primary cultured rodent and human macrophages, CB1R activation increased Irf5 expression, whereas knockdown of Irf5 blunted CB1R-induced secretion of inflammatory cytokines without affecting CCL2 expression, which was p38MAPKα dependent. Macrophage-specific in vivo knockdown of Irf5 protected ZDF rats from ß-cell loss and hyperglycemia. Thus, IRF5 is a crucial downstream mediator of diabetogenic CB1R signaling in macrophages and a potential therapeutic target.

Combination therapy with BPTES nanoparticles and metformin targets the metabolic heterogeneity of pancreatic cancer.

Targeting glutamine metabolism via pharmacological inhibition of glutaminase has been translated into clinical trials as a novel cancer therapy, but available drugs lack optimal safety and efficacy. In this study, we used a proprietary emulsification process to encapsulate bis-2-(5-phenylacetamido-1,2,4-thiadiazol-2-yl)ethyl sulfide (BPTES), a selective but relatively insoluble glutaminase inhibitor, in nanoparticles. BPTES nanoparticles demonstrated improved pharmacokinetics and efficacy compared with unencapsulated BPTES. In addition, BPTES nanoparticles had no effect on the plasma levels of liver enzymes in contrast to CB-839, a glutaminase inhibitor that is currently in clinical trials. In a mouse model using orthotopic transplantation of patient-derived pancreatic tumor tissue, BPTES nanoparticle monotherapy led to modest antitumor effects. Using the HypoxCR reporter in vivo, we found that glutaminase inhibition reduced tumor growth by specifically targeting proliferating cancer cells but did not affect hypoxic, noncycling cells. Metabolomics analyses revealed that surviving tumor cells following glutaminase inhibition were reliant on glycolysis and glycogen synthesis. Based on these findings, metformin was selected for combination therapy with BPTES nanoparticles, which resulted in significantly greater pancreatic tumor reduction than either treatment alone. Thus, targeting of multiple metabolic pathways, including effective inhibition of glutaminase by nanoparticle drug delivery, holds promise as a novel therapy for pancreatic cancer.

Hybrid inhibitor of peripheral cannabinoid-1 receptors and inducible nitric oxide synthase mitigates liver fibrosis.

Liver fibrosis, a consequence of chronic liver injury and a way station to cirrhosis and hepatocellular carcinoma, lacks effective treatment. Endocannabinoids acting via cannabinoid-1 receptors (CB1R) induce profibrotic gene expression and promote pathologies that predispose to liver fibrosis. CB1R antagonists produce opposite effects, but their therapeutic development was halted due to neuropsychiatric side effects. Inducible nitric oxide synthase (iNOS) also promotes liver fibrosis and its underlying pathologies, but iNOS inhibitors tested to date showed limited therapeutic efficacy in inflammatory diseases. Here, we introduce a peripherally restricted, orally bioavailable CB1R antagonist, which accumulates in liver to release an iNOS inhibitory leaving group. In mouse models of fibrosis induced by CCl4 or bile duct ligation, the hybrid CB1R/iNOS antagonist surpassed the antifibrotic efficacy of the CB1R antagonist rimonabant or the iNOS inhibitor 1400W, without inducing anxiety-like behaviors or CB1R occupancy in the CNS. The hybrid inhibitor also targeted CB1R-independent, iNOS-mediated profibrotic pathways, including increased PDGF, Nlrp3/Asc3, and integrin αvß6 signaling, as judged by its ability to inhibit these pathways in cnr1-/- but not in nos2-/- mice. Additionally, it was able to slow fibrosis progression and to attenuate established fibrosis. Thus, dual-target peripheral CB1R/iNOS antagonists have therapeutic potential in liver fibrosis.