Citations

Background Adolescent idiopathic scoliosis (AIS) is a genetically heterogeneous disease characterised by three-dimensional deformity of the spine in the absence of a congenital spinal anomaly or neurological musculoskeletal disorder. The clinical variability and incomplete penetrance of some genes linked with AIS indicate that this disease constitutes an oligogenic trait. Objective We aimed to explore the oligogenic nature of this disease and identify novel AIS genes. Methods We analysed rare damaging variants within AIS-associated genes by using exome sequencing in 40 AIS trios and 183 sporadic patients. Results Multiple variants within AIS-associated genes were identified in eight AIS trios, and five individuals harboured rare damaging variants in the FLNB gene. The patients showed more frequent oligogenicity than the controls. In the gene-based burden test, the top signal resided in FLNB . In functional studies, we found that the AIS-associated FLNB variants altered the protein’s conformation and subcellular localisation and its interaction with other proteins (TTC26 and OFD1) involved in AIS. The most compelling evidence of an oligogenic basis was that the number of rare damaging variants was recognised as an independent prognostic factor for curve progression in Cox regression analysis. Conclusion Our data indicate that AIS is an oligogenic disease and identify FLNB as a susceptibility gene for AIS.

Abstract Background Hypothyroidism is known to be associated with adverse clinical outcomes in heart failure. The association between hypothyroidism and cardiac resynchronization therapy outcomes in patients with severe heart failure is not clear. Methods The study included 1,316 patients who received cardiac resynchronization therapy between 2002 and 2015. Baseline demographics and cardiac resynchronization therapy outcomes, including left ventricular ejection fraction, New York Heart Association class, appropriate implantable cardioverter-defibrillator therapy, and all-cause mortality, were collected from the electronic health record. Results Of the study cohort, 349 patients (26.5%) were classified as the hypothyroidism group. The median duration of follow-up was 3.6 years (interquartile range, 1.7-6.2). Hypothyroidism was associated with a greater all-cause mortality than euthyroidism (hazard ratio, 1.19; 95% CI, 1.01-1.38; P=0.04). In this group, the risk of appropriate implantable cardioverter-defibrillator therapy significantly increased in association with increased baseline thyroid-stimulating hormone level (hazard ratio, 1.27 per 5 mIU/L increase, 95% CI, 1.00-1.53, P=0.04). Conclusions Cardiac resynchronization therapy improves cardiac function in hypothyroid patients. Hypothyroidism has adverse effects on cardiac resynchronization therapy outcomes with reduced survival and increased implantable cardioverter-defibrillator therapies for ventricular arrhythmic events.

Nonalcoholic steatohepatitis (NASH) is an advanced form of nonalcoholic fatty liver disease (NAFLD), resulting from the complex interplay of genetic and environmental risk factors, e.g. diet. The development of novel therapies has been hampered by the lack of a preclinical animal model with high physiologic fidelity to the human condition. A single mutation (I148M) in the patatin‐like phospholipase domain‐containing 3 ( PNPLA3 ) gene is significantly associated with liver fat content and has been identified as the strongest genetic risk factor for NAFLD/NASH, cirrhosis, and hepatocellular carcinoma. We hypothesized that editing PNPLA3 I148M mutation into a genome of Ossabaw, a miniature pig that already has a natural propensity to develop metabolic syndrome, will develop phenotypes that hallmark NAFLD/NASH, and improve preclinical modeling over existing rodent models. PNPLA3 I148M/I148M founders were developed using TALEN‐stimulated homology directed repair in fibroblasts, followed by somatic cell nuclear transfer. Mutant pigs with age/gender‐matched wildtype controls, reared on standard (SD) and specialized western diet (WD), were clinically and histopathologically characterized. No significant differences in circulating triglyceride and liver enzyme levels was observed in our initial cohort on SD of a female mutant pig relative to the wildtype control. However, this mutant female exhibited increased ALP level and a more atherogenic lipoprotein profile with LDL particle concentration than the levels of the respective control. Histopathologically, the mutant female fed on the WD for 3 months exhibited augmented interlobular inflammation, ballooning, as well lipid and collagen buildup in the liver relative to the wildtype counterparts on the same WD or SD. Longitudinal studies in second larger cohort of barrows over six‐months period revealed significantly elevated liver enzymes (ALP, ALT, GGT), bile acids, and lipid profiles (cholesterol, triglyceride, triglyceride‐rich lipoprotein and vLDL) among the mutant versus wildtypes on western diet, substantiating liver pathology and the associated comorbidity of dyslipidemia. Liver histopathology from these barrows on WD over 6‐months did not reveal as significant difference as in the females of the initial cohort, implying gender differences and variances in western diet formulation in susceptibility to NAFLD/NASH that warrant future large‐scale studies. Levels of circulating extracellular vesicles (EVs) were significantly elevated in the sera of mutant pigs relative to wildtypes on the WD, validating this as an important biomarker in NAFLD/NASH pathology. Additionally, the mutant pigs on the WD had significantly greater coronary artery plaque burden than the control groups, in line with the predicted higher comorbidity level in mutants and reinforces the relevance to human clinical medicine. Support or Funding Information NIH (1R43OD023257‐01A1 to TM) and internal funds from Recombinetics Inc., Drs. Chalasani, Sturek, and Malhi.

Abstract From a high throughput RNAi screen of the human druggable genome targeting the KMS11 cell line, we identified the suppression of the vacuolar H+- ATPase (V-ATPase) family as cytotoxic to myeloma cells. In the screen, two oligos against each gene for the V-ATPase subunits ATP6V1A and ATP6V1B1 resulted in suppression of cell growth (50% and 60% inhibition of cell viability respectively). We further confirmed this result using both lentiviral shRNA knockdown and two small molecule inhibitors specific for V-ATPase. Silencing of ATP6V1A by lentiviral shRNA knock-down in KMS11 and in OPM1 myeloma cell lines caused 75–80% reduction of cell viability at 5 days post infection (measured by MTT assay). Consistent with this result, the V-ATPase specific inhibitors, bafilomycin A1 and REATA 203, both inhibited the growth of a genetically heterogeneous and standardized panel of 14 human myeloma cell lines in vitro with an IC50 ranging from 2.2 – 8.9 nM (mean 5.25 nM) for bafilomycin A1 and 46–1594 nM (mean 542.5 nM)) for REATA 203. We further demonstrated that patient samples (n=10) were sensitive to 20nM bafilomycin A1 which induced a mean of 58% of MM cells to undergo apoptosis (range 10% to 93%) after 24 hours of treatment. Similar to bafilomycin A1, treatment of primary patient-derived MM cells with 500 nM REATA 203 for 72 hours resulted in a mean 69% apoptosis (range 24% to 97%). In contrast, non-myeloma cells (the CD138- fractions of the bone marrow samples) were less sensitive - mean 9% apoptosis (range from 0% to 34%) under the same treatment conditions. Of high interest, however, unlike most drugs we have studied in pre-clinical myeloma models, the cytotoxicity induced by bafilomycin A1 in MM cell lines is abrogated by co-culture with patient bone marrow stromal cells but is not affected by IL-6 or IGF-1 treatment. Dexamethasone- or melphalan-resistant MM cell lines were also highly sensitive to both bafilomycin A1 and REATA 203. In a xenographic JJN3 mouse model, bafilomycin A1 suppresses and delays growth of tumor in a dose-dependent fashion. Gene expression analysis of normal-donor bone marrow plasma cells (n=19), primary tumor samples from MM patients (n=107) and normal somatic tissues demonstrates ubiquitous expression of most subunits of V-ATPase, however, some subunits are preferentially expressed in myeloma cells compared with normal plasma cells, including ATP6V1F (84% vs. 11%), ATP6V1E1 (29% vs. 5%), ATP6V1G2 (17% vs. 0%) and ATP6V0E 2 (36% vs. 16%). In conclusion, our data indicate that vacuolar H+-ATPase inhibitors are of interest as potential therapeutics for MM.

Abstract Cyclin Ds are key regulators of the cell cycle that are frequently over-expressed in multiple myeloma and leukemia and act to promote the phosphorylation of Rb, thereby facilitating the transition from G1 to S phase. Over-expression of cyclin Ds increases cell proliferation and chemoresistance. In contrast, reducing cyclin Ds levels directly or indirectly through G1 arrest can decrease cellular proliferation and induce apoptosis. To identify novel pharmaceutical inhibitors of cyclin D transactivation, we screened the LOPAC and Prestwick libraries of drugs and natural compounds (n = 2400) using NIH 3T3 cells stably expressing the Cyclin D2 promoter-driving a luciferase reporter gene. From this library screen and subsequent validation experiments we identified Cyproheptadine as a novel inhibitor of Cyclin D2 transactivation. Cyproheptadine has been used previously for the treatment of atopic dermatitis and anorexia, but its ability to inhibit cyclin D expression has not previously been reported. By immunoblotting, Cyproheptadine decreased expression of cyclin D1, D2 and D3 proteins in human myeloma and leukemia cell lines at low micromolar concentrations. Consistent with effects on the cyclin Ds, Cyproheptadine arrested cells in the G1 phase at concentrations associated with reduction in cyclin D expression. Decreased cyclin D expression and G1 arrest can induce apoptosis, so we tested the effects of Cyproheptadine on cell viability. Myeloma and leukemia cell lines were treated with increasing concentrations of Cyproheptadine and viability was measured by the MTS assay. Cyproheptadine reduced the viability of 7/10 myeloma and 7/8 AML cells lines with an IC50 ranging from 10–25μM. In contrast, it was less toxic to HeLa or NIH3T3 cells with IC20 > 50 μM. Cyproheptadine also reduced the viability of primary myeloma (8/8) and AML patient samples (7/9) with an IC50 <25 μM, but was less toxic to normal hematopoietic cells (IC20 > 50 μM). In a MDAY-D2 mouse model of leukemia, treatment with Cyproheptadine (50mg/kg/d) abolished formation of malignant leukemic ascites without untoward toxicity. Cyproheptadine-induced cell death was associated with reductions in mitochondrial membrane potential. Furthermore, reductions of pro-caspases -3 and -9 were observed prior to the reduction in pro-caspase-8, indicating that Cyproheptadine activates the mitochondrial pathway of caspase activation. Cyproheptadine is a known H1 histamine and serotonin receptor inhibitor, but pre-incubation with histamine, serotonin, or a combination of histamine and serotonin did not abrogate Cyproheptadine-induced cell death. Moreover, the structurally related H1 receptor inhibitor loratadine did not decrease cyclin D expression or reduce cell viability. Therefore, the pro-apoptotic activity of Cyproheptadine is not due to a competitive inhibition of the H1 and/or serotonin receptors, suggesting that Cyproheptadine has additional targets. In summary, Cyproheptadine arrests cells in G1, reduces cyclin D expression, and induces apoptosis via the mitochondrial pathway of caspase activation. Given the prior safety and toxicity record of Cyproheptadine, this drug could be rapidly advanced into clinical trial for the treatment of hematologic malignancies.

AbstractBackground: Retinoblastoma (RB) is the most frequent pediatric retinal tumor. In the present study, to elucidate chemoresistance mechanisms and identify potential biomarkers in RB, we utilized RNA sequencing (RNAseq) technological platforms to reveal transcriptome profiles and identify any differentially expressed genes (DEGs) between an etoposide drug-resistant subline (Y79/EDR) and parental Y79 cells. Methods: To test whether Y79/EDR cells showed resistance to antineoplastic agents for RB, we treated the cells with etoposide, carboplatin and vincristine and analyzed them with a Cell Counting Kit-8 (CCK-8). Y79/EDR and parental Y79 cells were used for RNAseq and bioinformatics analysis to enable a genome-wide review of DEGs between the two lines using the DESeq R package (1.10.1). Then, DEG enrichment in Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways was analyzed with KOBAS software. Next, real-time quantitative reverse transcription polymerase chain reaction (real time QRT-PCR) and cytotoxicity assays were performed to experimentally and functionally validate the identified candidate biomarkers. Results: Y79/EDR cells showed resistance to etoposide, carboplatin and vincristine at different concentrations. In total, 524 transcripts were differentially expressed in Y79/EDR cells based on analysis of fragments per kilobase of transcript per million fragments mapped (FPKM); among these, 57 genes were downregulated and 467 genes were upregulated in Y79/EDR cells compared to parental Y79 cells. We selected candidate DEGs, including ARHGAP9 , HIST1H4H , RELN , DDIT4 , HK2 , STC1 and PFKFB4, for mRNA expression validation with real time QRT-PCR assays and found that the expression levels determined by real time QRT-PCR were consistent with the RNAseq data. Further studies involving downregulation of ARHGAP9 with a specific siRNA showed that ARHGAP9 altered the cellular sensitivity of Y79 cells to etoposide and carboplatin.Conclusion: Our initial findings provided a genomic view of the transcription profiles of etoposide-induced acquired resistance in RB. Follow-up studies indicated that ARHGAP9 might be a chemoresistance biomarker in RB, providing insight into potential therapeutic targets for overcoming acquired chemoresistance in RB. These findings can aid in understanding and overcoming chemoresistance during treatment of RB in the clinic.

OBJECTIVE Convection-enhanced delivery (CED) and osmotic pump delivery both have been promoted as promising techniques to deliver drugs to pediatric diffuse intrinsic pontine gliomas (DIPGs). Correspondingly, the aim of this study was to understand how infusate molecular weight (MW), duration of delivery, and mechanism of delivery (CED or osmotic pump) affect volume of distribution (V d ) in the brainstem, to better inform drug selection and delivery in future DIPG investigations. METHODS A series of in vivo experiments were conducted using rat models. CED and osmotic pump delivery systems were surgically implanted in the brainstem, and different MW fluorescent dextran beads were infused either once (acute) or daily for 5 days (chronic) in a volume infused (V i ). Brainstems were harvested after the last infusion, and V d was quantified using serial sectioning and fluorescence imaging. RESULTS Fluorescence imaging showed infusate uptake within the brainstem for both systems without complication. A significant inverse relationship was observed between infusate MW and V d in all settings, which was distinctly exponential in nature in the setting of acute delivery across the 570-Da to 150-kDa range. Chronic duration and CED technique resulted in significantly greater V d compared to acute duration or osmotic pump delivery, respectively. When accounting for V i , acute infusion yielded significantly greater V d /V i than chronic infusion. The distribution in CED versus osmotic pump delivery was significantly affected by infusate MW at higher weights. CONCLUSIONS Here the authors demonstrate that infusate MW, duration of infusion, and infusion mechanism all impact the V d of an infused agent and should be considered when selecting drugs and infusion parameters for novel investigations to treat DIPGs.

We clarify that DNA damage triggers autophagy by CHK2-FOXK axis, and misregulation of this pathway leads to chemoresistance.

Drug safety is a severe clinical pharmacology and toxicology problem that has caused immense medical and social burdens every year. Regretfully, a reproducible method to assess drug safety systematically and quantitatively is still missing. In this study, we developed an advanced machine learning model for de novo drug safety assessment by solving the multilayer drug‐gene‐adverse drug reaction (ADR) interaction network. For the first time, the drug safety was assessed in a broad landscape of 1,156 distinct ADRs. We also designed a parameter ToxicityScore to quantify the overall drug safety. Moreover, we determined association strength for every 3,807,631 gene‐ADR interactions, which clues mechanistic exploration of ADRs. For convenience, we deployed the model as a web service ADRAlert‐gene at http://www.bio-add.org/ADRAlert/. In summary, this study offers insights into prioritizing safe drug therapy. It helps reduce the attrition rate of new drug discovery by providing a reliable ADR profile in the early preclinical stage.