Metformin Promotes Antitumor Immunity via Endoplasmic-Reticulum-Associated Degradation of PD-L1.

Metformin has been reported to possess antitumor activity and maintain high cytotoxic T lymphocyte (CTL) immune surveillance. However, the functions and detailed mechanisms of metformin's role in cancer immunity are not fully understood. Here, we show that metformin increases CTL activity by reducing the stability and membrane localization of programmed death ligand-1 (PD-L1). Furthermore, we discover that AMP-activated protein kinase (AMPK) activated by metformin directly phosphorylates S195 of PD-L1. S195 phosphorylation induces abnormal PD-L1 glycosylation, resulting in its ER accumulation and ER-associated protein degradation (ERAD). Consistently, tumor tissues from metformin-treated breast cancer patients exhibit reduced PD-L1 levels with AMPK activation. Blocking the inhibitory signal of PD-L1 by metformin enhances CTL activity against cancer cells. Our findings identify a new regulatory mechanism of PD-L1 expression through the ERAD pathway and suggest that the metformin-CTLA4 blockade combination has the potential to increase the efficacy of immunotherapy.

The International Mouse Phenotyping Consortium: comprehensive knockout phenotyping underpinning the study of human disease.

The International Mouse Phenotyping Consortium (IMPC; https://www.mousephenotype.org/) web portal makes available curated, integrated and analysed knockout mouse phenotyping data generated by the IMPC project consisting of 85M data points and over 95,000 statistically significant phenotype hits mapped to human diseases. The IMPC portal delivers a substantial reference dataset that supports the enrichment of various domain-specific projects and databases, as well as the wider research and clinical community, where the IMPC genotype-phenotype knowledge contributes to the molecular diagnosis of patients affected by rare disorders. Data from 9,000 mouse lines and 750 000 images provides vital resources enabling the interpretation of the ignorome, and advancing our knowledge on mammalian gene function and the mechanisms underlying phenotypes associated with human diseases. The resource is widely integrated and the lines have been used in over 4,600 publications indicating the value of the data and the materials.

Patient-derived xenograft (PDX) models in basic and translational breast cancer research.

Patient-derived xenograft (PDX) models of a growing spectrum of cancers are rapidly supplanting long-established traditional cell lines as preferred models for conducting basic and translational preclinical research. In breast cancer, to complement the now curated collection of approximately 45 long-established human breast cancer cell lines, a newly formed consortium of academic laboratories, currently from Europe, Australia, and North America, herein summarizes data on over 500 stably transplantable PDX models representing all three clinical subtypes of breast cancer (ER+, HER2+, and "Triple-negative" (TNBC)). Many of these models are well-characterized with respect to genomic, transcriptomic, and proteomic features, metastatic behavior, and treatment response to a variety of standard-of-care and experimental therapeutics. These stably transplantable PDX lines are generally available for dissemination to laboratories conducting translational research, and contact information for each collection is provided. This review summarizes current experiences related to PDX generation across participating groups, efforts to develop data standards for annotation and dissemination of patient clinical information that does not compromise patient privacy, efforts to develop complementary data standards for annotation of PDX characteristics and biology, and progress toward "credentialing" of PDX models as surrogates to represent individual patients for use in preclinical and co-clinical translational research. In addition, this review highlights important unresolved questions, as well as current limitations, that have hampered more efficient generation of PDX lines and more rapid adoption of PDX use in translational breast cancer research.

Creation and Preliminary Characterization of Pregnane X Receptor and Constitutive Androstane Receptor Knockout Rats.

The nuclear receptors pregnane X receptor (PXR) and constitutive androstane receptor (CAR) are closely related transcription factors that regulate the expression of phase I (cytochrome P450s) and phase II metabolizing enzymes and transporter genes in response to stimulation from xenobiotics, including prescription drugs. PXR and CAR knockout and humanized mouse models have proven useful. However, the rat being bigger in size is a preferred model system for studying drug metabolism and pharmacokinetics. Here, we report the creation and preliminary characterization of PXR and CAR knockout rats and PXR/CAR double knockout rats. Whereas the expression of phase I and II enzymes and transporter genes were not upregulated by nuclear receptor-specific agonists pregnenlone-16α-carbonitrile and 1,4-bis-[2-(3,5-dichloropyridyloxy)] benzene in the knockout rats, confirming the disruption of respective nuclear receptor(s), our data demonstrate that PXR appears to suppress the basal expression levels of Cyp2b2, Cyp3a23/3a1, Cyp3a2, Cyp3a18, and Ugt2b1 genes, while CAR maintains Cyp2b2 and Ugt2b1 and suppresses Cyp3a9 basal expression levels. In wild-type rats, agonist binding of the nuclear receptors relieves the suppression, and target genes are expressed at levels comparable to knockout rats, with or without drug treatment. Overall, our findings are in good agreement with data obtained from human primary hepatocytes, nuclear receptor knockout cell lines, and mouse knockout models. We believe these models are a useful complement to their mouse counterparts for drug development and as importantly, for functional studies on metabolic pathways involving nuclear receptors.

Whole-rat conditional gene knockout via genome editing.

Animal models with genetic modifications under temporal and/or spatial control are invaluable to functional genomics and medical research. Here we report the generation of tissue-specific knockout rats via microinjection of zinc-finger nucleases (ZFNs) into fertilized eggs. We generated rats with loxP-flanked (floxed) alleles and a tyrosine hydroxylase promoter-driven cre allele and demonstrated Cre-dependent gene disruption in vivo. Pronuclear microinjection of ZFNs, shown by our data to be an efficient and rapid method for creating conditional knockout rats, should also be applicable in other species.

In silico and in vitro methods to identify ebola virus VP35-dsRNA inhibitors.

Ebola virus continues to be problematic as sporadic outbreaks in Africa continue to arise, and as terrorist organizations have considered the virus for bioterrorism use. Several proteins within the virus have been targeted for antiviral chemotherapy, including VP35, a dsRNA binding protein that promotes viral replication, protects dsRNA from degradation, and prevents detection of the viral genome by immune complexes. To augment the scope of our antiviral research, we have now employed molecular modeling techniques to enrich the population of compounds for further testing in vitro. In the initial docking of a static VP35 structure with an 80,000 compound library, 40 compounds were selected, of which four compounds inhibited VP35 with IC50 <200µM, with the best compounds having an IC50 of 20µM. By superimposing 26 VP35 structures, we determined four aspartic acid residues were highly flexible and the docking was repeated under flexible parameters. Of 14 compounds chosen for testing, five compounds inhibited VP35 with IC50 <200µM and one compound with an IC50 of 4µM. These studies demonstrate the value of docking in silico for enriching compounds for testing in vitro, and specifically using multiple structures as a guide for detecting flexibility and provide a foundation for further development of small molecule inhibitors directed towards VP35.

Phenotypic characterization of recessive gene knockout rat models of Parkinson's disease.

Recessively inherited loss-of-function mutations in the PTEN-induced putative kinase 1(Pink1), DJ-1 (Park7) and Parkin (Park2) genes are linked to familial cases of early-onset Parkinson's disease (PD). As part of its strategy to provide more tools for the research community, The Michael J. Fox Foundation for Parkinson's Research (MJFF) funded the generation of novel rat models with targeted disruption ofPink1, DJ-1 or Parkin genes and determined if the loss of these proteins would result in a progressive PD-like phenotype. Pathological, neurochemical and behavioral outcome measures were collected at 4, 6 and 8months of age in homozygous KO rats and compared to wild-type (WT) rats. Both Pink1 and DJ-1 KO rats showed progressive nigral neurodegeneration with about 50% dopaminergic cell loss observed at 8 months of age. ThePink1 KO and DJ-1 KO rats also showed a two to three fold increase in striatal dopamine and serotonin content at 8 months of age. Both Pink1 KO and DJ-1 KO rats exhibited significant motor deficits starting at 4months of age. However, Parkin KO rats displayed normal behaviors with no neurochemical or pathological changes. These results demonstrate that inactivation of the Pink1 or DJ-1 genes in the rat produces progressive neurodegeneration and early behavioral deficits, suggesting that these recessive genes may be essential for the survival of dopaminergic neurons in the substantia nigra (SN). These MJFF-generated novel rat models will assist the research community to elucidate the mechanisms by which these recessive genes produce PD pathology and potentially aid in therapeutic development.

Predictors of success in establishing orthotopic patient-derived xenograft models of triple negative breast cancer.

Patient-derived xenograft (PDX) models of breast cancer are an effective discovery platform and tool for preclinical pharmacologic testing and biomarker identification. We established orthotopic PDX models of triple negative breast cancer (TNBC) from the primary breast tumors of patients prior to and following neoadjuvant chemotherapy (NACT) while they were enrolled in the ARTEMIS trial (NCT02276443). Serial biopsies were obtained from patients prior to treatment (pre-NACT), from poorly responsive disease after four cycles of Adriamycin and cyclophosphamide (AC, mid-NACT), and in cases of AC-resistance, after a 3-month course of different experimental therapies and/or additional chemotherapy (post-NACT). Our study cohort includes a total of 269 fine needle aspirates (FNAs) from 217 women, generating a total of 62 PDX models (overall success-rate = 23%). Success of PDX engraftment was generally higher from those cancers that proved to be treatment-resistant, whether poorly responsive to AC as determined by ultrasound measurements mid-NACT (p = 0.063), RCB II/III status after NACT (p = 0.046), or metastatic relapse within 2 years of surgery (p = 0.008). TNBC molecular subtype determined from gene expression microarrays of pre-NACT tumors revealed no significant association with PDX engraftment rate (p = 0.877). Finally, we developed a statistical model predictive of PDX engraftment using percent Ki67 positive cells in the patient's diagnostic biopsy, positive lymph node status at diagnosis, and low volumetric reduction of the patient's tumor following AC treatment. This novel bank of 62 PDX models of TNBC provides a valuable resource for biomarker discovery and preclinical therapeutic trials aimed at improving neoadjuvant response rates for patients with TNBC.

Encapsulation and release of Amphotericin B from an ABC triblock fluorous copolymer.

PURPOSE: PEG-phospholipid-based micelles have been successfully used for the solubilization of several hydrophobic drugs but generally lack sustained stability in blood. Our novel PEG-Fluorocarbon-DSPE polymers were designed to increase stability and improve time-release properties of drug-loaded micelles. METHODS: Novel ABC fluorous copolymers were synthesized, characterized, and used for encapsulation release of amphotericin B. FRET studies were used to study micelle stability. RESULTS: The micelles formed by the new polymers showed lower critical micelle concentrations and higher viscosity cores than those formed by the polymers lacking the fluorous block. FRET studies indicated that fluorocarbon-containing micelles had increased stability in presence of human serum. Physicochemical properties and in vitro release profile of micelles loaded with Amphotericin B (AmB) were studied. CONCLUSIONS: The effect of PEG length and fluorocarbon incorporation were investigated. The shorter hydrophilic PEG2K induced greater stability than PEG5K by decreasing the proportion of hydrophilic block of the polymer. The fluorocarbon placed between hydrophilic and hydrophobic block formed a fluorous shell contributing to the enhanced thermodynamic stability of micelles and to the drug sustained release. Polymer mPEG2K-F(10)-DSPE, bearing both a fluorocarbon block and a shorter mPEG, showed the greatest stability and the longest half-life for AmB release.

Application of a convolutional neural network to the quality control of MRI defacing.

Large-scale neuroimaging datasets present unique challenges for automated processing pipelines. Motivated by a large clinical trials dataset with over 235,000 MRI scans, we consider the challenge of defacing - anonymisation to remove identifying facial features. The defacing process must undergo quality control (QC) checks to ensure that the facial features have been removed and that the brain tissue is left intact. Visual QC checks are time-consuming and can cause delays in preparing data. We have developed a convolutional neural network (CNN) that can assist with the QC of the application of MRI defacing; our CNN is able to distinguish between scans that are correctly defaced and can classify defacing failures into three sub-types to facilitate parameter tuning during remedial re-defacing. Since integrating the CNN into our anonymisation pipeline, over 75,000 scans have been processed. Strict thresholds have been applied so that ambiguous classifications are referred for visual QC checks, however all scans still undergo an efficient verification check before being marked as passed. After applying the thresholds, our network is 92% accurate and can classify nearly half of the scans without the need for protracted manual checks. Our model can generalise across MRI modalities and has comparable performance when tested on an independent dataset. Even with the introduction of the verification checks, incorporation of the CNN has reduced the time spent undertaking QC checks by 42% during initial defacing, and by 35% overall. With the help of the CNN, we have been able to successfully deface 96% of the scans in the project whilst maintaining high QC standards. In a similarly sized new project, we would expect the model to reduce the time spent on manual QC checks by 125 h. Our approach is applicable to other projects with the potential to greatly improve the efficiency of imaging anonymisation pipelines.

Resistance to neoadjuvant chemotherapy in triple-negative breast cancer mediated by a reversible drug-tolerant state.

Eradicating triple-negative breast cancer (TNBC) resistant to neoadjuvant chemotherapy (NACT) is a critical unmet clinical need. In this study, patient-derived xenograft (PDX) models of treatment-naïve TNBC and serial biopsies from TNBC patients undergoing NACT were used to elucidate mechanisms of chemoresistance in the neoadjuvant setting. Barcode-mediated clonal tracking and genomic sequencing of PDX tumors revealed that residual tumors remaining after treatment with standard frontline chemotherapies, doxorubicin (Adriamycin) combined with cyclophosphamide (AC), maintained the subclonal architecture of untreated tumors, yet their transcriptomes, proteomes, and histologic features were distinct from those of untreated tumors. Once treatment was halted, residual tumors gave rise to AC-sensitive tumors with similar transcriptomes, proteomes, and histological features to those of untreated tumors. Together, these results demonstrated that tumors can adopt a reversible drug-tolerant state that does not involve clonal selection as an AC resistance mechanism. Serial biopsies obtained from patients with TNBC undergoing NACT revealed similar histologic changes and maintenance of stable subclonal architecture, demonstrating that AC-treated PDXs capture molecular features characteristic of human TNBC chemoresistance. Last, pharmacologic inhibition of oxidative phosphorylation using an inhibitor currently in phase 1 clinical development delayed residual tumor regrowth. Thus, AC resistance in treatment-naïve TNBC can be mediated by nonselective mechanisms that confer a reversible chemotherapy-tolerant state with targetable vulnerabilities.

Effect of intermittent hypoxia on arcuate nucleus in the leptin-deficient rat.

Intermittent hypoxia (IH) is a major pathophysiological consequence of obstructive sleep apnea. Recently, it has been shown that IH results in changes in body energy balance, leptin secretion and concomitant alterations in arcuate nucleus (ARC). In this study, the role of leptin on these changes was investigated in leptin-deficient rats exposed to IH or normoxic control conditions. Body weights, consumatory and locomotor behaviours, and protein signaling in ARC were assessed immediately after IH exposure. Compared to normoxia, IH altered body weight, food intake, locomotor pattern, and the plasma concentration of leptin and angiotensin II in the wild-type rat. However, these changes were not observed in the leptin-deficient rat. Within ARC of wild-type animals, IH increased phosphorylated signal transducer and activator of transcription 3 and pro-opiomelanocortin protein expression, but not in the leptin-deficient rat. The long-form leptin receptor protein expression was not altered following IH in either rat strain. These data suggest that leptin is involved in mediating the alterations to body energy balance and ARC activity following IH.

Leptin dependent changes in the expression of tropomyosin receptor kinase B protein in nucleus of the solitary tract to acute intermittent hypoxia.

To investigate the possibility that leptin exerts an effect in NTS by inducing changes in the expression of pre- and/or post-synaptic proteins, experiments were done in Sprague-Dawley wild-type rats (WT) rats and leptin-deficient rats (Lep(Δ151/Δ151); KILO rat) exposed to 8h of continuous intermittent hypoxia (IH) or normoxia. Protein was extracted from the caudal medial NTS and analyzed by western blot for the expression of brain-derived neurotrophic factor (BDNF), tropomyosin receptor kinase B (TrkB), synaptophysin, synaptopodin and growth-associated protein-43 (GAP-43). In WT rats, BDNF and GAP 43 protein expression levels were not altered after IH or normoxia, although there was a trend towards an increase in BDNF expression. On the other hand, after IH, protein expression of both isoforms of the BDNF receptor TrkB (gp95 and gp145) was higher. Furthermore, synaptophysin protein expression was lower compared to normoxic WT rats. In the KILO rat, no changes were observed in the protein expression of BDNF, TrkB, or GAP 43 after IH when compared to KILO normoxic controls. However, synaptophysin was lower in the IH exposed KILO rat compared to normoxic controls, as found in the WT rat. Expression of synaptopodin was not detected in NTS in either IH or normoxic animals of all groups. These results suggest that leptin released during IH may contribute to neurotrophic changes occurring within NTS and that these changes may be associated with altered chemoreceptor reflex function.

Fmr1 and Nlgn3 knockout rats: novel tools for investigating autism spectrum disorders.

Animal models are critical for gaining insights into autism spectrum disorder (ASD). Despite their apparent advantages to mice for neural studies, rats have not been widely used for disorders of the human CNS, such as ASD, for the lack of convenient genome manipulation tools. Here we describe two of the first transgenic rat models for ASD, developed using zinc-finger nuclease (ZFN) methodologies, and their initial behavioral assessment using a rapid juvenile test battery. A syndromic and nonsyndromic rat model for ASD were created as two separate knockout rat lines with heritable disruptions in the genes encoding Fragile X mental retardation protein (FMRP) and Neuroligin3 (NLGN3). FMRP, a protein with numerous proposed functions including regulation of mRNA and synaptic protein synthesis, and NLGN3, a member of the neuroligin synaptic cell-adhesion protein family, have been implicated in human ASD. Juvenile subjects from both knockout rat lines exhibited abnormalities in ASD-relevant phenotypes including juvenile play, perseverative behaviors, and sensorimotor gating. These data provide important first evidence regarding the utility of rats as genetic models for investigating ASD-relevant genes.

Creation and preliminary characterization of a Tp53 knockout rat.

The tumor suppressor TP53 plays a crucial role in cancer biology, and the TP53 gene is the most mutated gene in human cancer. Trp53 knockout mouse models have been widely used in cancer etiology studies and in search for a cure of cancer with some limitations that other model organisms might help overcome. Via pronuclear microinjection of zinc finger nucleases (ZFNs), we created a Tp53 knockout rat that contains an 11-bp deletion in exon 3, resulting in a frameshift and premature terminations in the open reading frame. In cohorts of 25 homozygous (Tp53(Δ11/Δ11)), 37 heterozygous (Tp53(Δ11/+)) and 30 wild-type rats, the Tp53(Δ11/Δ11) rats lived an average of 126 days before death or removal from study because of clinical signs of abnormality or formation of tumors. Half of Tp53(Δ11/+) were removed from study by 1 year of age because of tumor formation. Both Tp53(Δ11/+) and Tp53(Δ11/Δ11) rats developed a wide spectrum of tumors, most commonly sarcomas. Interestingly, there was a strikingly high incidence of brain lesions, especially in Tp53(Δ11/Δ11) animals. We believe that this mutant rat line will be useful in studying cancer types rarely observed in mice and in carcinogenicity assays for drug development.

Creation and preliminary characterization of a leptin knockout rat.

Leptin, a cytokine-like hormone secreted mainly by adipocytes, regulates various pathways centered on food intake and energy expenditure, including insulin sensitivity, fertility, immune system, and bone metabolism. Here, using zinc finger nuclease technology, we created the first leptin knockout rat. Homozygous leptin null rats are obese with significantly higher serum cholesterol, triglyceride, and insulin levels than wild-type controls. Neither gender produced offspring despite of repeated attempts. The leptin knockout rats also have depressed immune system. In addition, examination by microcomputed tomography of the femurs of the leptin null rats shows a significant increase in both trabecular bone mineral density and bone volume of the femur compared with wild-type littermates. Our model should be useful for many different fields of studies, such as obesity, diabetes, and bone metabolism-related illnesses.

Efficient catalytic cycloalkane oxidation employing a "helmet" phthalocyaninato iron(III) complex.

We have examined the catalytic activity of an iron(III) complex bearing the 14,28-[1,3-diiminoisoindolinato]phthalocyaninato (diiPc) ligand in oxidation reactions with three substrates (cyclohexane, cyclooctane, and indan). This modified metallophthalocyaninato complex serves as an efficient and selective catalyst for the oxidation of cyclohexane and cyclooctane, and to a far lesser extent indan. In the oxidations of cyclohexane and cyclooctane, in which hydrogen peroxide is employed as the oxidant under inert atmosphere, we have observed turnover numbers of 100.9 and 122.2 for cyclohexanol and cyclooctanol, respectively. The catalyst shows strong selectivity for alcohol (vs. ketone) formation, with alcohol to ketone (A/K) ratios of 6.7 and 21.0 for the cyclohexane and cyclooctane oxidations, respectively. Overall yields (alcohol + ketone) were 73% for cyclohexane and 92% for cyclooctane, based upon the total hydrogen peroxide added. In the catalytic oxidation of indan under similar conditions, the TON for 1-indanol was 10.1, with a yield of 12% based upon hydrogen peroxide. No 1-indanone was observed in the product mixture.

Resolution of enantiomers of a series of chiral alkoxy-modified phthalocyaninato nickel(II) complexes by enantioselective HPLC.

We have resolved the enantiomers of a series of chiral modified metallophthalocyaninato complexes of nickel bearing alkoxy groups at the 14 and 28 positions on what would otherwise be a normal phthalocyaninato ligand and conforming to the general formula [14,28-(RO)(2)Pc]Ni(ii), where R = Me, Et, or n-Pr. The complex for which R = n-Pr is reported here for the first time. Resolution of the enantiomers of these complexes was accomplished via HPLC utilizing an immobilized carbohydrate-based stationary phase, resulting in baseline resolution of peaks corresponding to enantiomers of the complexes, with R(s) values in excess of five. Isolation of milligram quantities of the complexes bearing methoxy and n-propoxy groups in high enantiomeric excess has been achieved via semi-preparative-scale HPLC on the same stationary phase. Resolved samples of these compounds do not appear to racemize at an appreciable rate, nor do they readily exchange alkoxy groups with alcohols while stirring in alcoholic solution. The spectroscopic details and the crystallographically-determined solid-state structure for the complex where R = n-Pr are reported, and are highly similar to those that have been observed for the previously reported analogues. It has been shown by NMR that the chirality and C(2) molecular symmetry of the complex bearing n-propoxy groups is maintained in solution.