Relevant Human Model Research Articles

Antisense oligonucleotide therapy rescues aggresome formation in a novel spinocerebellar ataxia type 3 human embryonic stem cell line

Spinocerebellar ataxia type 3 (SCA3) is a fatal, late-onset neurodegenerative disorder characterized by selective neuropathology in the brainstem, cerebellum, spinal cord, and substantia nigra. Here we report the first NIH-approved human embryonic stem cell (hESC) line derived from an embryo harboring the SCA3 mutation. Referred to as SCA3-hESC, this line is heterozygous for the mutant polyglutamine-encoding CAG repeat expansion in the ATXN3 gene. We observed relevant molecular hallmarks of the human disease at all differentiation stages from stem cells to cortical neurons, including robust ATXN3 aggregation and altered expression of key components of the protein quality control machinery. In addition, SCA3-hESCs exhibit nuclear accumulation of mutant ATXN3 and form p62-positive aggresomes. Finally, antisense oligonucleotide-mediated reduction of ATXN3 markedly suppressed aggresome formation. The SCA3-hESC line offers a unique and highly relevant human disease model that holds strong potential to advance understanding of SCA3 disease mechanisms and facilitate the evaluation of candidate therapies for SCA3.

In vitro and in silico Models to Study Mosquito-Borne Flavivirus Neuropathogenesis, Prevention, and Treatment.

Mosquito-borne flaviviruses can cause disease in the nervous system, resulting in a significant burden of morbidity and mortality. Disease models are necessary to understand neuropathogenesis and identify potential therapeutics and vaccines. Non-human primates have been used extensively but present major challenges. Advances have also been made toward the development of humanized mouse models, but these models still do not fully represent human pathophysiology. Recent developments in stem cell technology and cell culture techniques have allowed the development of more physiologically relevant human cell-based models. In silico modeling has also allowed researchers to identify and predict transmission patterns and discover potential vaccine and therapeutic candidates. This review summarizes the research on in vitro and in silico models used to study three mosquito-borne flaviviruses that cause neurological disease in humans: West Nile, Dengue, and Zika. We also propose a roadmap for 21st century research on mosquito-borne flavivirus neuropathogenesis, prevention, and treatment.

Abstract 302: The combined inhibition of Plk1 and Notch1 results in a synergistic anti-proliferative response in human melanoma cells

Abstract Melanoma is the most aggressive form of skin cancer, and its incidence and mortality have been increasing over the past thirty years. Therefore, additional therapeutic strategies are needed for an effective management of this neoplasm. We previously demonstrated that polo-like kinase 1 (Plk1, a serine/threonine kinase involved in mitotic regulation) and Notch1 (a type I transmembrane protein deciding cell fate during development) are strongly correlated in melanoma (Cancer Res 2018; 78 (13 Suppl): Abstract nr 2530). In addition, a number of studies from our lab and elsewhere have suggested the important functions of Plk1- as well as Notch- signaling in melanoma progression. Based on these observations, in this study, we determined the potential efficacy of a combined inhibition of Plk1 and Notch1 against melanoma cells. Employing Plk1 inhibitor volasertib (BI6727) and Notch1 inhibitor MK-0752, we determined the effects of concomitant targeting of these pathways in A375 (wild type TP53) and SK-MEL-2 (mutant TP53) human melanoma cell lines. Employing RealTime-Glo and trypan blue exclusion assays, we found that volasertib (10 and 20 nM) and MK-0752 (50 and 100 μM) resulted in a significant decrease (60-80%) in viability and growth of human melanoma cells A375 and SK-MEL-2. The Combination Index (CI), as calculated using the Chou-Talalay theorem, was less than 1 when volasertib at 10 nM was given with MK-0752 at 50 and 100 μM, indicating a synergism between these two drugs. As a result of the synergism, the melanoma cells treated with combined drugs showed decreased colony formation ability compared to individual drugs. In addition, the combined treatment also resulted in enhanced apoptosis of melanoma cells. Interestingly, cell cycle analysis by flow cytometry showed that combined inhibition of Plk1 and Notch1 caused a G2-M phase arrest of melanoma cells. Furthermore, our data demonstrated that volasertib and MK-0752 combination caused a marked increase of cleaved PARP (poly ADP ribose polymerase) and cleaved caspase-3 in both melanoma cell lines, as well as increased expression of tumor suppressor protein TP53 and its target p21 (CDKN1A) in A375 melanoma cells. Taken together, our data demonstrated that a combined targeting of Plk1- and Notch1- signaling pathways imparts a synergistic anti-proliferative response against melanoma. However, additional experiments are needed i) to validate our in vitro data to in vivo situations in human relevant melanoma models, and ii) to define the interactive mechanism(s) of the observed synergistic response. Citation Format: Shengqin Su, Gagan Chhabra, Chandra K. Singh, Mary A. Ndiaye, Nihal Ahmad. The combined inhibition of Plk1 and Notch1 results in a synergistic anti-proliferative response in human melanoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 302.

APOE4 ACCELERATES NEURODEGENERATION AND EXACERBATES ALZHEIMER’S DISEASE RELATED PATHOLOGY IN CEREBRAL ORGANOIDS

APOE4 is the strongest genetic risk factor for late-onset Alzheimer's disease (AD) compared to the common APOE3 allele. The cerebral organoids derived from human induced pluripotent stem cells (iPSCs) have been shown to be a novel model system closely resembling the physiological condition of human brains. To understand how human apoE isoforms differentially impact neuron viability and AD-related pathologies, we developed iPSC lines with different APOE genotypes from individuals with either normal cognition or sporadic AD and further into cerebral organoids. We found that cerebral organoids from AD patients with APOE ε4/4 genotype showed accelerated differentiation pattern and had enhanced neuronal apoptosis in the ventricular zone-like area at day 90 when compared to those from normal controls or AD with the APOE ε3/3 genotype. Cerebral organoids from AD patients also had increased AD-related pathologies including the levels of Aβ40, Aβ42, and phosphorylation tau. More interestingly, we found that apoE4 exacerbates tau pathology in a disease status-independent manner. Transcriptomic analysis by RNA-sequencing revealed an enrichment of apoptosis pathway in cerebral organoids from AD patients with the APOE ε4/4 genotype. Together, our results using the cerebral organoids suggest that apoE4 accelerates neurodegeneration in addition to impacting AD-related pathology in a human-relevant model system. Both the cerebral organoid model system and the information on apoE4-relevant disease pathways should help the development of new therapeutics for AD.

Predicting Human Infection Risk: Do Rodent Host Resistance Models Add Value?

Use of genetically engineered rodents is often considered a valuable exercise to assess potential safety concerns associated with the inhibition of a target pathway. When there are potential immunomodulatory risks associated with the target, these genetically modified animals are often challenged with various pathogens in an acute setting to determine the risk to humans. However, the applicability of the results from infection models is seldom assessed when significant retrospective human data become available. Thus, the purpose of the current review is to compare the outcomes of infectious pathogen challenge in mice with genetic deficiencies in TNF-α, IL17, IL23, or Janus kinase pathways with infectious outcomes caused by inhibitors of these pathways in humans. In general, mouse infection challenge models had modest utility for hazard identification and were generally only able to predict overall trends in infection risk. These models did not demonstrate significant value in evaluating specific types of pathogens that are either prevalent (ie rhinoviruses) or of significant concern (ie herpes zoster). Similarly, outcomes in mouse models tended to overestimate the severity of infection risk in human patients. Thus, there is an emerging need for more human-relevant models that have better predictive value. Large meta-analyses of multiple clinical trials or post-marketing evaluations remains the gold-standard for characterizing the true infection risk to patients.

A human enteroid model to study the effect of short‐chain fatty acids on the intestinal epithelium

Short‐chainfatty acids (SCFA) are dietary fiber derived bacterial metabolites thought to be beneficial to thehost. Of high interest is butyrate (BUT) which is the main fuel source for absorptivecells in the colon; as well as acetate (ACET) and propionate (PROP). Effects of BUT on intestinal physiology have been studied in several in vitro models, however, data in primary cells, includingintestinal stem cells (ISCs) and their distinct progenies is still not fullycharacterized. Recent advances inenteroid models have allowed these mechanisms to be studied in a more physiologically relevant human model. Therefore, our objective was to examine effects of BUT, ACET, and PROP on biomarkers of ISC activity, differentiation, barrier function and epithelial defense in the intestine using human 2‐dimensional (2D) and 3‐dimensional (3D) enteroid models. Duodenal 2D and 3D enteroids were treated with 10 mM BUT, ACET, or PROP for 24 h. Gene expression of stem cell marker Lgr5 was increased more than2‐fold in 3D enteroids treated with ACET. Conversely, enteroids treated with BUT and PROP had a greater than 10‐fold decrease in Lgr5 compared to controls (CON). Enterocyte biomarker alkaline phosphatase (Alpi) was increased due to all SCFA treatments (P<0.05). Gene expression of goblet cell marker mucin‐2 (Muc2) was increased 2 and 5‐fold due to ACET and BUT treatments respectively, but not due to PROP treatment. Paneth cell marker lysozyme (Lyz) increased due to BUT treatment and enteroendocrine cell marker chromogranin‐A (Chga) was increased 170‐fold in BUT treated enteroidsalone. Tuft cell marker Dclk1 was elevated due to ACET (7‐fold) and BUT (14‐fold) treatment compared to CON (P<0.05). Anti‐microbial molecule gene expression of Reg3b and Reg3g were not increased due to ACET treatment, however, were increased due to BUT (2.5‐fold and3‐fold, respectively) treatment. Defb1 gene expression increased 66‐fold and 75‐fold respectively in BUT and PROP treated enteroids (P<0.05). Tight junction marker expression of Ocln was only elevated in ACET treated organoids (4‐fold; P<0.05). However, Cldn3 and Tjp1 were increased due to both ACET and BUT treatments (P<0.05). 2Denteroid monolayers treated with BUT had a 2.4‐fold increase in relative transepithelial electrical resistance (TEER) compared to CON (P<0.05). PROP treatment also increased TEER (P < 0.05), while ACET had no effect. Protein expression of Interleukin‐8 in the media was induced by exposure to BUT (1.8‐fold increase) and PROP (1.5‐fold increase), but not ACET(P<0.05). Similarly, Interleukin‐18expression was increased by BUT (1.7‐fold increase) and PROP(1.3‐fold increase), while ACET was not different from CON. Intestinal Trefoil factor 3 expression in the media was increased1.3‐fold due to BUT treatment only (P<0.05). In conclusion, we determined that human enteroids are valuable and physiologically relevant models to study the effects of bacterial metabolites based upon the responses we examined. This work will be useful for future microbiomeand nutritional work using enteroid models.Support or Funding InformationFunding was provided by the Department of Defense Combat Feeding Research and Engineering Program through project “Gut Microbiomeand Performance Nutrition (TB 15–19).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Effect of environmental and pharmaceutical exposures on fetal testis development and function: a systematic review of human experimental data.

BACKGROUNDOverall, the incidence of male reproductive disorders has increased in recent decades. Testicular development during fetal life is crucial for subsequent male reproductive function. Non-genomic factors such as environmental chemicals, pharmaceuticals and lifestyle have been proposed to impact on human fetal testicular development resulting in subsequent effects on male reproductive health. Whilst experimental studies using animal models have provided support for this hypothesis, more recently a number of experimental studies using human tissues and cells have begun to translate these findings to determine direct human relevance.OBJECTIVE AND RATIONALEThe objective of this systematic review was to provide a comprehensive description of the evidence for effects of prenatal exposure(s) on human fetal testis development and function. We present the effects of environmental, pharmaceutical and lifestyle factors in experimental systems involving exposure of human fetal testis tissues and cells. Comparison is made with existing epidemiological data primarily derived from a recent meta-analysis.SEARCH METHODSFor identification of experimental studies, PubMed and EMBASE were searched for articles published in English between 01/01/1966 and 13/07/2018 using search terms including ‘endocrine disruptor’, ‘human’, ‘fetal’, ‘testis’, ‘germ cells’, ‘testosterone’ and related search terms. s were screened for selection of full-text articles for further interrogation. Epidemiological studies involving exposure to the same agents were extracted from a recent systematic review and meta-analysis. Additional studies were identified through screening of bibliographies of full-texts of articles identified through the initial searches.OUTCOMESA total of 25 experimental studies and 44 epidemiological studies were included. Consistent effects of analgesic and phthalate exposure on human fetal germ cell development are demonstrated in experimental models, correlating with evidence from epidemiological studies and animal models. Furthermore, analgesic-induced reduction in fetal testosterone production, which predisposes to the development of male reproductive disorders, has been reported in studies involving human tissues, which also supports data from animal and epidemiological studies. However, whilst reduced testosterone production has been demonstrated in animal studies following exposure(s) to a variety of environmental chemicals including phthalates and bisphenol A, these effects are not reproduced in experimental approaches using human fetal testis tissues.WIDER IMPLICATIONSDirect experimental evidence for effects of prenatal exposure(s) on human fetal testis development and function exists. However, for many exposures the data is limited. The increasing use of human-relevant models systems in which to determine the effects of environmental exposure(s) (including mixed exposures) on development and function of human tissues should form an important part of the process for assessment of such exposures by regulatory bodies to take account of animal–human differences in susceptibility.

MAP4K4 Inhibition Promotes Survival of Human Stem Cell-Derived Cardiomyocytes and Reduces Infarct Size InVivo.

SummaryHeart disease is a paramount cause of global death and disability. Although cardiomyocyte death plays a causal role and its suppression would be logical, no clinical counter-measures target the responsible intracellular pathways. Therapeutic progress has been hampered by lack of preclinical human validation. Mitogen-activated protein kinase kinase kinase kinase-4 (MAP4K4) is activated in failing human hearts and relevant rodent models. Using human induced-pluripotent-stem-cell-derived cardiomyocytes (hiPSC-CMs) and MAP4K4 gene silencing, we demonstrate that death induced by oxidative stress requires MAP4K4. Consequently, we devised a small-molecule inhibitor, DMX-5804, that rescues cell survival, mitochondrial function, and calcium cycling in hiPSC-CMs. As proof of principle that drug discovery in hiPSC-CMs may predict efficacy in vivo, DMX-5804 reduces ischemia-reperfusion injury in mice by more than 50%. We implicate MAP4K4 as a well-posed target toward suppressing human cardiac cell death and highlight the utility of hiPSC-CMs in drug discovery to enhance cardiomyocyte survival.

3213 Unraveling the role of Phospholamban (PLN) in humans via the characterization of Induced Pluripotent Stem Cell (iPSC) Cardiomyocytes (CM) derived from carriers of a lethal PLN mutation

OBJECTIVES/SPECIFIC AIMS: To study the biology of Phosholamban (PLN) in a human relevant model. METHODS/STUDY POPULATION: State of the art stem-cell technologies using iPSC-CMs derived from carriers of a lethal PLN mutation. RESULTS/ANTICIPATED RESULTS: Our preliminary data demonstrate that this particular PLN mutation (L39) results in reduced expression and mis-localization of PLN as well as increased incidence of early after depolarization in isolated iPSC-CMs. DISCUSSION/SIGNIFICANCE OF IMPACT: Phospholamban (PLN) is a critical regulator of Ca++ homeostasis yet many uncertainties still remain regarding its role in humans. Our study will provide unique insights into the pathophysiology of this protein in HF.

Long-term impact of maternal high-fat diet on offspring cardiac health: role of micro-RNA biogenesis

Heart failure is a worldwide leading cause of death. Diet and obesity are particularly of high concern in heart disease etiology. Gravely, altered nutrition during developmental windows of vulnerability can have long-term impact on heart health; however, the underlying mechanisms are poorly understood. In the understanding of the initiation of chronic diseases related to developmental exposure to environmental challenges, deregulations in epigenetic mechanisms including micro-RNAs have been proposed as key events. In this context, we aimed at delineating the role of micro-RNAs in the programming of cardiac alterations induced by early developmental exposure to nutritional imbalance. To reach our aim, we developed a human relevant model of developmental exposure to nutritional imbalance by maternally exposing rat to high-fat diet during gestation and lactation. In this model, offspring exposed to maternal high-fat diet developed cardiac hypertrophy and increased extracellular matrix depot compared to those exposed to chow diet. Microarray approach performed on cardiac tissue allowed the identification of a micro-RNA subset which was down-regulated in high-fat diet-exposed animals and which were predicted to regulate transforming growth factor-beta (TGFβ)-mediated remodeling. As indicated by in vitro approaches and gene expression measurement in the heart of our animals, decrease in DiGeorge critical region 8 (DGCR8) expression, involved in micro-RNA biogenesis, seems to be a critical point in the alterations of the micro-RNA profile and the TGFβ-mediated remodeling induced by maternal exposure to high-fat diet. Finally, increasing DGCR8 activity and/or expression through hemin treatment in vitro revealed its potential in the rescue of the pro-fibrotic phenotype in cardiomyocytes driven by DGCR8 decrease. These findings suggest that cardiac alterations induced by maternal exposure to high-fat diet is related to abnormalities in TGFβ pathway and associated with down-regulated micro-RNA processing. Our study highlighted DGCR8 as a potential therapeutic target for heart diseases related to early exposure to dietary challenge.

Human-relevant preclinical in vitro models for studying hepatobiliary development and liver diseases using induced pluripotent stem cells.

In this review, we address the potential of human-induced pluripotent stem cell-based hepatobiliary differentiation technology as a means to study human liver development and cell fate determination, and to model liver diseases in an effort to develop a new human-relevant preclinical platform for drug development.

Optimized protocol for induction of cytochrome P450 enzymes 1A2 and 3A4 in human primary-like hepatocyte cell strain HepaFH3 to study in vitro toxicology

Drug induced liver injury (DILI) is the most frequent cause for failure of new drugs in clinical studies. Thus, toxicity studies are indispensable during drug development. The proliferative human liver cell strain HepaFH3 with promising primary-like cellular properties might be a suitable liver model for such studies, but its cytochrome-P450 (CYP) expression is still in low ranges compared to freshly isolated primary human hepatocytes. We aimed to optimize the induction protocol for CYP1A2 and CYP3A4 in HepaFH3 to obtain a physiologically relevant in vitro liver model. CYP1A2 and CYP3A4 were induced by omeprazole and rifampicin, respectively. Induction of the two CYPs was measured by qRT-PCR, immunofluorescence and by P450 Glo enzyme activity assays. The optimized protocol made the experimental design six days shorter than the original procedure. CYP1A2 mRNA levels were induced 118-fold, CYP3A4 levels 36-fold. This result was also reflected at protein level. Enzymatic activity of CYP1A2 increased 3.7-fold and CYP3A4 activity increased 9.8-fold after induction. We succeeded in optimizing the induction protocol for HepaFH3 to such an extent that CYP1A2 and CYP3A4 are expressed in sufficient amounts that the cell strain can be used as a physiological relevant human liver model for in vitro toxicology studies.

Patient-Derived Xenografts Can Be Reliably Generated from Patient Clinical Biopsy Specimens

BackgroundPatient-derived xenografts (PDX) are clinically relevant human cancer models that can be used to guide individualized medicine. We aimed to generate PDX models from clinically obtained biopsy specimens (surgical or image-guided) hypothesizing that low volume biopsy specimens could provide sufficient viable tissue to successfully engraft PDX models from patients with unresectable or metastatic disease. Materials and MethodsWe maintain a prospective high volume gastrointestinal malignancy PDX program. With informed consent and institutional approval, biopsy specimens (surgical or image-guided) were obtained from patients with unresectable or metastatic tumors: pancreatic adenocarcinoma (PDAC), cholangiocarcinoma, gastric and gallbladder carcinoma. Biopsies were implanted into immunodeficient mice. Tumor growth was monitored, viable tumor was passed into subsequent generations, and histopathology was confirmed. ResultsIn this study, biopsy specimens from 29 patients were used for PDX engraftment. Successful PDX engraftment was variable with highest engraftment rates in gastric and gallbladder carcinoma specimens (100%) compared to engraftment rates of 33% and 29% in PDAC and cholangiocarcinoma respectively. PDX models created from metastasis biopsies compared to unresectable primary tumor tissue demonstrated higher engraftment rates (69% versus 15.4%, p = 0.001). PDX models demonstrated higher engraftment rates when biopsies were obtained during surgical operations (n = 15) compared to image-guided (n = 14) (73% versus 14%, p = 0.003). Patient age, pretreatment status, or ischemic time was not different between biopsy methods. ConclusionsPDX models can be successfully created from clinical biopsy specimens in patients with metastatic or unresectable GI cancers. The use of clinical biopsy specimens for PDX engraftment can expand the repertoire of stage-specific PDX models for downstream basic/translational research.

Next-Generation Compound Delivery Platforms to Support Miniaturized Biology.

Recent advancements in science and engineering are revolutionizing our understanding of an individual's disease, and with this knowledge we are gaining an increasingly sophisticated understanding of how discovery can be transformed to deliver personalized medicines. To reach this future state, we must reengineer our approach to enable the use of more relevant human cellular models earlier in the drug discovery process. Stem cells and primary human cells represent more disease-relevant models than immortalized cell lines; however, due to both availability and cost, their use is limited in lead generation activities. Miniaturization of cellular assays below microtiter plate volumes will enable the use of more relevant cells in screening, but this would require a change in how test molecules are introduced to the biology. With these shifting paradigms, Discovery Supply teams at GlaxoSmithKline (GSK) are modernizing our sample handling approaches. Various emerging technologies such as microarrays, nanowells, and microfluidic devices could bring fundamental changes in conventional sample handling support as we transition from microtiter plates to well-less platforms. The discussion here is exploratory in nature and reviews ongoing proof-of-concept experiments. Our ultimate goal is to industrialize the sample management platforms to support future miniaturized biological assay systems.

A Novel Human Neuronal Cell Model to Study Iron Accumulation in Parkinson’s Disease

Objectives: With an estimated seven to ten million sufferers worldwide, Parkinson’s disease (PD) is the second most common age-related neurodegenerative disorder. Progress in elucidating its causes has been slow, partly due to the lack of human-relevant models. Similarly, while the contribution of iron is increasingly advocated, identifying its role in disease progression remains challenging mainly due to the lack of valid model. In this study, we created Parkinson-like conditions in a human neuron model and conducted preliminary studies on iron-related parameters to assess whether these cells replicated iron accumulation observed in Parkinsonism. Methods: ReNcell VM (human neural progenitor) were differentiated into dopaminergic neurons (dDCNs) and treated with neurotoxin 6-hydroxy dopamine (100 μM) to mimic Parkinsonism. Total intracellular, mitochondrial and cytoplasmic iron was measured by ferrozine assay. Expression of iron-related genes TFRC, SLC40A1, HAMP and SLC25A37 were assessed through real-time PCR. Results: Data showed that the treated dDCNs accumulated iron over time and exceeded levels measured in untreated dDCNs by 2.5-fold at 48 h (p<0.02). Following the treatment, the treated cells showed lower expression of TFRC (p<0.05), but substantially higher mRNA expressions of SLC40A1 (9-fold; p<0.02) and HAMP (5.7-fold; p<0.05), along with higher intracellular iron (p<0.05). Higher iron accumulation in the mitochondria than cytosol (p<0.05), was also observed with increased expression of the mitochondrial iron-importer SLC25A37 (p=0.08). Conclusion: Our Parkinsonian model demonstrates iron accumulation and elevated HAMP expression as previously described in PD phenotype. The observed mitochondrial iron shuttling, which is proposed to be one of the primary contributors of oxidative stress in PD, calls for further investigation. The differences observed in distribution of iron in our human model and with the expression of major iron-related proteins, indicate that our model reproduces the disease state successfully, and suggests that further study could help in advancing our understanding of PD.

Preparation and Utilization of a 3D Human Liver Microtissue Model for Nanotoxicological Assessment.

The liver is the principal detoxification center of the body, removing xenobiotics and waste products which could potentially include some nanomaterials (NM). With the ever increasing public and occupational exposure associated with accumulative production of nanomaterials, there is an urgent need to consider the possibility of harmful health consequences of engineered NM exposure. It is understood that following exposure via inhalation, ingestion, or direct intravenous injection a fraction of NMs reach the liver. Traditional in vitro or ex vivo hepatic nanotoxicology models are often limiting and/or troublesome (i.e., reduced metabolism enzymes, lacking important cell populations, unstable with very high variability, etc.). This chapter highlights a methodology for the preparation of a physiologically relevant 3D human liver microtissue model which addresses most of the negative issues associated with the models used in traditional in vitro hepatic toxicological investigations. The spheroids are a very promising model for the assessment of the toxicological effects associated with engineered NM exposure.

AS2762900-00, a potent anti-human IL-23 receptor monoclonal antibody, prevents epidermal hyperplasia in a psoriatic human skin xenograft model

Interleukin (IL)-23 is thought to be critical in the pathogenesis of psoriasis, and anti-IL-23 monoclonal antibodies (mAbs) have been approved for the treatment of psoriasis. We speculated that an anti-IL-23 receptor mAb might have greater efficacy than an anti-IL-23 mAb in the treatment of local inflamed lesions with high IL-23 levels. We previously generated an anti-human IL-23 receptor mAb, AS2762900-00, which potently blocked IL-23-induced cell proliferation, regardless of the concentration of IL-23. Here, we evaluated the therapeutic potential of AS2762900-00 in the treatment of psoriasis. Compared with untreated control, AS2762900-00 significantly reduced the epidermal thickness of lesions in a clinically relevant psoriatic human skin xenograft model. The expression of inflammatory genes including genes downstream of IL-23 signaling in the lesion tended to be lower in the AS2762900-00 group than the untreated group, suggesting that the inhibitory effects of AS2762900-00 in the psoriatic human skin xenograft model might occur via blockade of IL-23 signaling pathways. Further, AS2762900-00 showed an inhibitory effect on signal transducer and activator of transcription 3 (STAT3) phosphorylation as a downstream signal of IL-23 receptor activation in whole blood from patients with psoriasis. We also confirmed that AS2762900-00 inhibited IL-23-induced STAT3 phosphorylation in a concentration-dependent manner using whole blood from healthy donors. These data suggest that AS2762900-00 is a promising drug candidate for the treatment of psoriasis. In addition, STAT3 phosphorylation in whole blood may be a useful biomarker for the evaluation of the pharmacodynamic effects of AS2762900-00 in healthy volunteers in clinical development.

MicroRNA Alterations in the Brain and Body Fluids of Humans and Animal Prion Disease Models: Current Status and Perspectives.

Prion diseases are transmissible progressive neurodegenerative conditions characterized by rapid neuronal loss accompanied by a heterogeneous neuropathology, including spongiform degeneration, gliosis and protein aggregation. The pathogenic mechanisms and the origins of prion diseases remain unclear on the molecular level. Even though neurodegenerative diseases, including prion diseases, represent distinct entities, their pathogenesis shares a number of features including disturbed protein homeostasis, an overload of protein clearance pathways, the aggregation of pathological altered proteins, and the dysfunction and/or loss of specific neuronal populations. Recently, direct links have been established between neurodegenerative diseases and miRNA dysregulated patterns. miRNAs are a class of small non-coding RNAs involved in the fundamental post-transcriptional regulation of gene expression. Studies of miRNA alterations in the brain and body fluids in human prion diseases provide important insights into potential miRNA-associated disease mechanisms and biomarker candidates. miRNA alterations in prion disease models represent a unique tool to investigate the cause-consequence relationships of miRNA dysregulation in prion disease pathology, and to evaluate the use of miRNAs in diagnosis as biomarkers. Here, we provide an overview of studies on miRNA alterations in human prion diseases and relevant disease models, in relation to pertinent studies on other neurodegenerative diseases.

Abstract 5034: Time-lapse analysis of drug response and invasive capacity using patient-derived CRC microfluidic 3D cancer models under oxygen-controlled conditions

Abstract The development of relevant, reliable, human-derived, and cost-effective cellular models is a present need to bring precision medicine to fruition and to accelerate drug discovery. 3D cultures show robust genomic stability and tissue-specific gene expression with high fidelity to their tissue of origin, making them ideal for preserving healthy and cancer tissue molecular and morphological traits. Our objective is to expand the number of relevant human cancer models available and implement methods that allow cellular response quantification of 3D cultures. We sought to establish and implement 3D cell-based assays suitable for detection of known molecular biomarkers in cancer using spherical cancer models (multicellular tumor spheroids and tissue-derived tumor spheres). We validate our procedures by detecting colorectal cancer (CRC) biomarkers both in patient-derived cultures and cell-lines (HCT116, HT29, LS174T). We expanded the study's scope to the analysis of specific CRC transcriptional and proteomic signatures in 3D. To this end, we employ primary cultures derived from CRC patients, and cell lines. Our methodology takes advantage of multiplex antibody microarrays to detect proteomic levels of 10 metalloproteinases of which MMP-13 and MMP- 9, as well as of surface proteins known to be involved in colorectal cancer invasion. Alterations in oxygen tension are milestones in tumor progression, and low oxygen tension (hypoxia) has been demonstrated in CRC. The Hypoxia Inducible Factor (HIF) transcription factor family is central to the response to hypoxic stress and is correlated with CRC carcinogenesis, invasion, VEGF expression, and poorer diagnosis. To increase the relevance of our model we use low oxygen conditions (3-5% physioxia conditions), and atmospheric oxygen levels (normoxia conditions) finding relevant differences at the proteomic, transcriptional and miRNA levels (also verified by qRT-PCR). We test the spherical cancer models on microfluidics chip and assess their vessel invasion capacity in normoxia and hypoxia, finding a correlation between hypoxia stress response and augmented invasiveness. Additionally, we test cellular responses to standard drugs and vasculature invasive capacity by time-lapse microscopy on-chip. We design, develop and validate cellular assays for human cancer 3D culture generation, long-term maintenance and analysis. We analyze and quantify cellular responses to cytotoxic drugs, image and quantify invasion in fluidic chambers, determine levels of apoptosis, cell proliferation. We demonstrate that a combination of controllable assays can successfully be used for identification of biomarker expression and compound screening in preclinical studies. 3D models are, therefore, a cost-effective in vitro model capable of recapitulating reliably physiological responses. Citation Format: Dora Sabino, Isabelle Fixe, Alexandra Foucher, Eric Mennesson, Nadia Normand. Time-lapse analysis of drug response and invasive capacity using patient-derived CRC microfluidic 3D cancer models under oxygen-controlled conditions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5034.

Abstract 2568: CAR T cells secreting an immune checkpoint blockade scFv have enhanced anti-tumor efficacy

Abstract T cell therapies have had valuable clinical responses in patients with cancer. Chimeric antigen receptor (CAR) T cells are genetically engineered to recognize tumor cells and CAR T cell therapy has had impressive results in the setting of B cell acute lymphoblastic leukemia but has been less effective in treating other types of hematologic and solid tumors. The inhibitory tumor microenvironment (TME), including expression of ligands that bind inhibitory receptors on T cells, e.g. programmed death receptor 1 (PD-1), can dampen CAR T cell responses. To prevent PD-1-mediated dampening of CAR T cell function, we have co-modified CAR T cells to secrete PD-1 blocking single chain variable fragments (scFv). We first designed mouse constructs with which we could investigate the scFv-secreting CAR T cells in the context of a syngeneic immune-competent intact TME. CAR constructs were engineered directed against either human CD19 or MUC-16 (ecto) and an anti-mouse PD-1 scFv. Mouse T cells transduced with these constructs expressed the CAR on the surface and secreted detectable amounts of scFv that bound to mouse PD-1. The scFv-secreting CAR T cells were cytotoxic and produced IFN- γ when co-cultured with PD-L1 expressing tumors in vitro. We utilized a syngeneic mouse model to study scFv secreting CAR T cells in a model with an intact TME. In tumor-bearing mice treated with CAR T cells, scFv-secreting CAR T cells enhanced survival as compared to second generation CAR T cells. The survival benefit achieved with scFv-secreting CAR T cells was comparable to that achieved with systemic infusion of PD-1 blocking antibody, but with localized delivery of PD-1 blockade. Mice treated with scFv-secreting CAR T cells had detectable scFv in vivo in the TME. Lastly, long term surviving mice had detectable CAR T cells in the bone marrow by PCR, demonstrating persistence and suggesting an immunological memory. We next aimed to translate PD-1 blocking scFv CAR T cells to a clinically relevant human model utilizing a novel anti-human PD-1 blocking scFv. Human T cells modified with the CAR constructs express the CAR on the surface and secrete detectable amounts of PD-1 blocking scFv. The scFv binds to human PD-1 and scFv-secreting CAR T cells are cytotoxic to PD-L1 expressing tumors. Expression of PD-1-blocking scFv enhances CAR T cell function against PD- L1 expressing tumors in xenograft models of hematological and solid tumors by enhancing survival in tumor-bearing mice as compared to second generation CAR T cells. Furthermore, scFv-secreting CAR T cells exhibit in vivo bystander T cell enhancement of function, suggesting scFv-secreting CAR T cells can reactivate endogenous TILs in the TME. These data support the novel concept that localized delivery of scFv by CAR T cells can successfully block PD-1 binding to PD-L1 to enhance the overall anti-tumor efficacy of CAR T cell therapy. Citation Format: Sarwish Rafiq, Oladapo Yeku, Hollie Jackson, Terence purdon, Dayenne van Leeuwen, Su Yan, Pei Wang, Jingyi Xiang, Cheng Liu, Venkatraman Seshan, Renier Brentjens. CAR T cells secreting an immune checkpoint blockade scFv have enhanced anti-tumor efficacy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2568.