Aberrant EVI1 splicing contributes to EVI1-rearranged leukemia.

Detailed genomic and epigenomic analyses of MECOM (the MDS1 and EVI1 complex locus) have revealed that inversion or translocation of chromosome 3 drives inv(3)/t(3;3) myeloid leukemias via structural rearrangement of an enhancer that upregulates transcription of EVI1. Here, we identify a novel, previously unannotated oncogenic RNA-splicing derived isoform of EVI1 that is frequently present in inv(3)/t(3;3) acute myeloid leukemia (AML) and directly contributes to leukemic transformation. This EVI1 isoform is generated by oncogenic mutations in the core RNA splicing factor SF3B1, which is mutated in >30% of inv(3)/t(3;3) myeloid neoplasm patients and thereby represents the single most commonly cooccurring genomic alteration in inv(3)/t(3;3) patients. SF3B1 mutations are statistically uniquely enriched in inv(3)/t(3;3) myeloid neoplasm patients and patient-derived cell lines compared with other forms of AML and promote mis-splicing of EVI1 generating an in-frame insertion of 6 amino acids at the 3' end of the second zinc finger domain of EVI1. Expression of this EVI1 splice variant enhanced the self-renewal of hematopoietic stem cells, and introduction of mutant SF3B1 in mice bearing the humanized inv(3)(q21q26) allele resulted in generation of this novel EVI1 isoform in mice and hastened leukemogenesis in vivo. The mutant SF3B1 spliceosome depends upon an exonic splicing enhancer within EVI1 exon 13 to promote usage of a cryptic branch point and aberrant 3' splice site within intron 12 resulting in the generation of this isoform. These data provide a mechanistic basis for the frequent cooccurrence of SF3B1 mutations as well as new insights into the pathogenesis of myeloid leukemias harboring inv(3)/t(3;3).

Lipid Profiling of Serum and Lipoprotein Fractions in Response to Pitavastatin Using an Animal Model of Familial Hypercholesterolemia.

Statins are widely used for the treatment of atherosclerotic cardiovascular diseases. They inhibit cholesterol biosynthesis in the liver and cause pleiotropic effects, including anti-inflammatory and antioxidant effects. To develop novel therapeutic drugs, the effect of blood-borne lipid molecules on the pleiotropic effects of statins must be elucidated. Myocardial infarction-prone Watanabe heritable hyperlipidemic (WHHLMI) rabbits, an animal model for hypercholesterolemia, are suitable for the determination of lipid molecules in the blood in response to statins because their lipoprotein metabolism is similar to that of humans. Herein, lipid molecules were investigated by lipidome analysis in response to pitavastatin using WHHLMI rabbits. Various lipid molecules in the blood were measured using a supercritical fluid chromatography triple quadrupole mass spectrometry. Cholesterol and cholesterol ester blood concentrations decreased by reducing the secretion of very low density lipoproteins from the liver. Independent of the inhibition effects of cholesterol biosynthesis, the concentrations of some lipids with anti-inflammation and antioxidant effects (phospholipid molecules with n-6 fatty acid side chains, lysophosphatidylcholines, phosphatidylethanolamine plasmalogens, and ceramide molecules) were significantly altered. These findings may lead to further investigation of the mechanism of statin action.

Deficiency of Cholesteryl Ester Transfer Protein Protects Against Atherosclerosis in Rabbits.

OBJECTIVE: CETP (cholesteryl ester transfer protein) plays an important role in lipoprotein metabolism; however, whether inhibition of CETP activity can prevent cardiovascular disease remains controversial. APPROACH AND RESULTS: We generated CETP knockout (KO) rabbits by zinc finger nuclease gene editing and compared their susceptibility to cholesterol diet-induced atherosclerosis to that of wild-type (WT) rabbits. On a chow diet, KO rabbits showed higher plasma levels of high-density lipoprotein (HDL) cholesterol than WT controls, and HDL particles of KO rabbits were essentially rich in apolipoprotein AI and apolipoprotein E contents. When challenged with a cholesterol-rich diet for 18 weeks, KO rabbits not only had higher HDL cholesterol levels but also lower total cholesterol levels than WT rabbits. Analysis of plasma lipoproteins revealed that reduced plasma total cholesterol in KO rabbits was attributable to decreased apolipoprotein B-containing particles, while HDLs remained higher than that in WT rabbits. Both aortic and coronary atherosclerosis was significantly reduced in KO rabbits compared with WT rabbits. Apolipoprotein B-depleted plasma isolated from CETP KO rabbits showed significantly higher capacity for cholesterol efflux from macrophages than that from WT rabbits. Furthermore, HDLs isolated from CETP KO rabbits suppressed tumor necrosis factor-α-induced vascular cell adhesion molecule 1 and E-selectin expression in cultured endothelial cells. CONCLUSIONS: These results provide evidence that genetic ablation of CETP activity protects against cholesterol diet-induced atherosclerosis in rabbits.

Cystic fibrosis rabbits develop spontaneous hepatobiliary lesions and CF-associated liver disease (CFLD)-like phenotypes.

Cystic fibrosis (CF) is an autosomal recessive genetic disease affecting multiple organs. Approximately 30% CF patients develop CF-related liver disease (CFLD), which is the third most common cause of morbidity and mortality of CF. CFLD is progressive, and many of the severe forms eventually need liver transplantation. The mechanistic studies and therapeutic interventions to CFLD are unfortunately very limited. Utilizing the CRISPR/Cas9 technology, we recently generated CF rabbits by introducing mutations to the rabbit CF transmembrane conductance regulator (CFTR) gene. Here we report the liver phenotypes and mechanistic insights into the liver pathogenesis in these animals. CF rabbits develop spontaneous hepatobiliary lesions and abnormal biliary secretion accompanied with altered bile acid profiles. They exhibit nonalcoholic steatohepatitis (NASH)-like phenotypes, characterized by hepatic inflammation, steatosis, and fibrosis, as well as altered lipid profiles and diminished glycogen storage. Mechanistically, our data reveal that multiple stress-induced metabolic regulators involved in hepatic lipid homeostasis were up-regulated in the livers of CF-rabbits, and that endoplasmic reticulum (ER) stress response mediated through IRE1α-XBP1 axis as well as NF-κB- and JNK-mediated inflammatory responses prevail in CF rabbit livers. These findings show that CF rabbits manifest many CFLD-like phenotypes and suggest targeting hepatic ER stress and inflammatory pathways for potential CFLD treatment.

SETBP1 is dispensable for normal and malignant hematopoiesis.

SETBP1 is a potential epigenetic regulator whose hotspot mutations preventing proteasomal degradation are recurrently detected in myeloid malignancies with poor prognosis. It is believed that the mutant SETBP1 exerts amplified effects of wild-type SETBP1 rather than neomorphic functions. This indicates that dysregulated quantitative control of SETBP1 would result in the transformation of hematopoietic cells. However, little is known about the roles of endogenous SETBP1 in malignant and normal hematopoiesis. Thus, we integrated the analyses of primary AML and healthy samples, cancer cell lines, and a newly generated murine model, Vav1-iCre;Setbp1fl/fl. Despite the expression in long-term hematopoietic stem cells, SETBP1 depletion in normal hematopoiesis minimally alters self-renewal, differentiation, or reconstitution in vivo. Indeed, its loss does not profoundly alter transcription or chromatin accessibilities. Furthermore, although AML with high SETBP1 mRNA is associated with genetic and clinical characteristics for dismal outcomes, SETBP1 is dispensable for the development or maintenance of AML. Contrary to the evidence that SETBP1 mutations are restricted to myeloid malignancies, dependency on SETBP1 mRNA expression is not observed in AML. These unexpected results shed light on the unrecognized idea that a physiologically nonessential gene can act as an oncogene when the machinery of protein degradation is damaged.

BRD9 determines the cell fate of hematopoietic stem cells by regulating chromatin state.

ATP-dependent chromatin remodeling SWI/SNF complexes exist in three subcomplexes: canonical BAF (cBAF), polybromo BAF (PBAF), and a newly described non-canonical BAF (ncBAF). While cBAF and PBAF regulate fates of multiple cell types, roles for ncBAF in hematopoietic stem cells (HSCs) have not been investigated. Motivated by recent discovery of disrupted expression of BRD9, an essential component of ncBAF, in multiple cancers, including clonal hematopoietic disorders, we evaluate here the role of BRD9 in normal and malignant HSCs. BRD9 loss enhances chromatin accessibility, promoting myeloid lineage skewing while impairing B cell development. BRD9 significantly colocalizes with CTCF, whose chromatin recruitment is augmented by BRD9 loss, leading to altered chromatin state and expression of myeloid-related genes within intact topologically associating domains. These data uncover ncBAF as critical for cell fate specification in HSCs via three-dimensional regulation of gene expression and illuminate roles for ncBAF in normal and malignant hematopoiesis.

HDL quality features revealed by proteome‒lipidome connectivity are associated with atherosclerotic disease.

Lipoprotein, especially high-density lipoprotein (HDL), particles are composed of multiple heterogeneous subgroups containing various proteins and lipids. The molecular distribution among these subgroups is closely related to cardiovascular disease (CVD). Here, we established high-resolution proteomics and lipidomics (HiPL) methods to depict the molecular profiles across lipoprotein (Lipo-HiPL) and HDL (HDL-HiPL) subgroups by optimizing the resolution of anion-exchange chromatography and comprehensive quantification of proteins and lipids on the omics level. Furthermore, based on the Pearson correlation coefficient analysis of molecular profiles across high-resolution subgroups, we achieved the relationship of proteome‒lipidome connectivity (PLC) for lipoprotein and HDL particles. By application of these methods to high-fat, high-cholesterol diet-fed rabbits and acute coronary syndrome (ACS) patients, we uncovered the delicate dynamics of the molecular profile and reconstruction of lipoprotein and HDL particles. Of note, the PLC features revealed by the HDL-HiPL method discriminated ACS from healthy individuals better than direct proteome and lipidome quantification or PLC features revealed by the Lipo-HiPL method, suggesting their potential in ACS diagnosis. Together, we established HiPL methods to trace the dynamics of the molecular profile and PLC of lipoprotein and even HDL during the development of CVD.

Development of the Nude Rabbit Model.

Loss-of-function mutations in the forkhead box N1 (FOXN1) gene lead to nude severe combined immunodeficiency, a rare inherited syndrome characterized by athymia, severe T cell immunodeficiency, congenital alopecia, and nail dystrophy. We recently produced FOXN1 mutant nude rabbits (NuRabbits) by using CRISPR-Cas9. Here we report the establishment and maintenance of the NuRabbit colony. NuRabbits, like nude mice, are hairless, lack thymic development, and are immunodeficient. To demonstrate the functional applications of NuRabbits in biomedical research, we show that they can successfully serve as the recipient animals in xenotransplantation experiments using human induced pluripotent stem cells or tissue-engineered blood vessels. Our work presents the NuRabbit as a new member of the immunodeficient animal model family. The relatively large size and long lifespan of NuRabbits offer unique applications in regenerative medicine, cancer research, and the study of a variety of other human conditions, including immunodeficiency.

Human apolipoprotein A-II reduces atherosclerosis in knock-in rabbits.

BACKGROUND AND AIMS: Apolipoprotein A-II (apoAII) is the second major apolipoprotein of the high-density lipoprotein (HDL) particle, after apoAI. Unlike apoAI, the biological and physiological functions of apoAII are unclear. We aimed to gain insight into the specific roles of apoAII in lipoprotein metabolism and atherosclerosis using a novel rabbit model. METHODS: Wild-type (WT) rabbits are naturally deficient in apoAII, thus their HDL contains only apoAI. Using TALEN technology, we replaced the endogenous apoAI in rabbits through knock-in (KI) of human apoAII. The newly generated apoAII KI rabbits were used to study the specific function of apoAII, independent of apoAI. RESULTS: ApoAII KI rabbits expressed exclusively apoAII without apoAI, as confirmed by RT-PCR and Western blotting. On a standard diet, the KI rabbits exhibited lower plasma triglycerides (TG, 52%, p < 0.01) due to accelerated clearance of TG-rich particles and higher lipoprotein lipase activity than the WT littermates. ApoAII KI rabbits also had higher plasma HDL-C (28%, p < 0.05) and their HDL was rich in apoE, apoAIV, and apoAV. When fed a cholesterol-rich diet for 16 weeks, apoAII KI rabbits were resistant to diet-induced hypertriglyceridemia and developed significantly less aortic atherosclerosis compared to WT rabbits. HDL isolated from rabbits with apoAII KI had similar cholesterol efflux capacity and anti-inflammatory effects as HDL isolated from the WT rabbits. CONCLUSIONS: ApoAII KI rabbits developed less atherosclerosis than WT rabbits, possibly through increased plasma HDL-C, reduced TG and atherogenic lipoproteins. These results suggest that apoAII may serve as a potential target for the treatment of atherosclerosis.

Cl-Amidine Improves Survival and Attenuates Kidney Injury in a Rabbit Model of Endotoxic Shock.

Objective: Sepsis causes millions of deaths on a global scale annually. Activation of peptidylarginine deiminase (PAD) enzymes in sepsis causes citrullination of histones, which results in neutrophil extracellular trap formation and sepsis progression. This study evaluates pan-PAD inhibitor, Cl-amidine, in a model of lipopolysaccharide (LPS)-induced endotoxic shock in rabbits. We hypothesized that Cl-amidine would improve survival and attenuate kidney injury. Methods: In the survival model, rabbits were injected injected intravenously with 1 mg/kg of LPS, and then randomly assigned either to receive dimethyl sulfoxide (DMSO; 1 mcL/g) or Cl-amidine (10 mg/kg diluted in 1 mcL/g DMSO). They were then monitored for 14 days to evaluate survival. In the non-survival experiment, the same insult and treatment were administered, however; the animals were euthanized 12 hours after LPS injection for kidney harvest. Acute kidney injury (AKI) scoring was performed by a histopathologist who was blinded to the group assignment. Serial blood samples were also collected and compared. Results: Rabbits that received Cl-amidine had a higher survival (72%) compared with the rabbits that received DMSO (14%; p < 0.05). Cl-amidine-treated rabbits had lower (p < 0.05) histopathologic AKI scores, as well as plasma creatinine and blood urea nitrogen (BUN) levels 12 hours after insult. Conclusions: Pan-PAD inhibitor Cl-amidine improves survival and attenuates kidney injury in LPS-induced endotoxic shock in rabbits.

Glycine-based treatment ameliorates NAFLD by modulating fatty acid oxidation, glutathione synthesis, and the gut microbiome.

Nonalcoholic fatty liver disease (NAFLD) including nonalcoholic steatohepatitis (NASH) has reached epidemic proportions with no pharmacological therapy approved. Lower circulating glycine is consistently reported in patients with NAFLD, but the causes for reduced glycine, its role as a causative factor, and its therapeutic potential remain unclear. We performed transcriptomics in livers from humans and mice with NAFLD and found suppression of glycine biosynthetic genes, primarily alanine-glyoxylate aminotransferase 1 (AGXT1). Genetic (Agxt1 -/- mice) and dietary approaches to limit glycine availability resulted in exacerbated diet-induced hyperlipidemia and steatohepatitis, with suppressed mitochondrial/peroxisomal fatty acid ß-oxidation (FAO) and enhanced inflammation as the underlying pathways. We explored glycine-based compounds with dual lipid/glucose-lowering properties as potential therapies for NAFLD and identified a tripeptide (Gly-Gly-L-Leu, DT-109) that improved body composition and lowered circulating glucose, lipids, transaminases, proinflammatory cytokines, and steatohepatitis in mice with established NASH induced by a high-fat, cholesterol, and fructose diet. We applied metagenomics, transcriptomics, and metabolomics to explore the underlying mechanisms. The bacterial genus Clostridium sensu stricto was markedly increased in mice with NASH and decreased after DT-109 treatment. DT-109 induced hepatic FAO pathways, lowered lipotoxicity, and stimulated de novo glutathione synthesis. In turn, inflammatory infiltration and hepatic fibrosis were attenuated via suppression of NF-κB target genes and TGFß/SMAD signaling. Unlike its effects on the gut microbiome, DT-109 stimulated FAO and glutathione synthesis independent of NASH. In conclusion, impaired glycine metabolism may play a causative role in NAFLD. Glycine-based treatment attenuates experimental NAFLD by stimulating hepatic FAO and glutathione synthesis, thus warranting clinical evaluation.

Ameliorating effects of D-47, a newly developed compound, on lipid metabolism in an animal model of familial hypercholesterolemia (WHHLMI rabbits).

Improvements induced in lipid metabolism in the liver by D-47, a newly developed compound, were examined herein. WHHLMI rabbits, an animal model of hypercholesterolemia and coronary atherosclerosis, was fed D-47-supplemented chow for 5 weeks at a dose of 30mg/kg. Lipid concentration were assayed using enzymatic methods. Plasma lipoproteins were fractionated with an ultracentrifuge. mRNA expression was analyzed with real-time PCR. Lipidome analyses of lipoproteins were performed using supercritical fluid chromatography mass spectrometry. In the D-47-treated group, serum lipid levels decreased by 23% for total cholesterol and by 40% for triglycerides. These reductions were mainly attributed to decreases in the VLDL fraction. Compared with the control, in the D-47 group, lipid contents in the liver were decreased by 22% in cholesterol and by 69% in triglycerides, and fat accumulation was decreased by 57% in pericardial fat and by 17% in mesenteric fat. In lipidome analyses of VLDL fraction, lysophosphatidylcholine, phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylethanolamine plasmalogen, sphingomyelin, and ceramide were decreased by the D-47 treatment. mRNA expression in the liver was 51% lower for FAS and 24% lower for MTP, but 5.9- and 5.1-fold higher for CYP7A1 and CPT-1, respectively, in the D-47 group than in the control. mRNA expression was 72%, 64%, and 36% higher for LPL, CTP-1, and PPARγ, respectively, in mesenteric fat in the D-47 group. D-47 is a potent lipid-lowering compound that uses a different mechanism of action from that of statins. It has potential as a compound in the treatment of steatohepatitis and metabolic syndrome.