Keith Q. Tanis

Transcriptional responses to loss or gain of function of the leucine-rich repeat kinase 2 (LRRK2) gene uncover biological processes modulated by LRRK2 activity

Mutations in the leucine-rich repeat kinase 2 gene (LRRK2) are the most common genetic cause of Parkinsons disease (PD) and cause both autosomal dominant familial and sporadic PD. Currently, the physiological and pathogenic activities of LRRK2 are poorly understood. To decipher the biological functions of LRRK2, including the genes and pathways modulated by LRRK2 kinase activity in vivo, we assayed genome-wide mRNA expression in the brain and peripheral tissues from LRRK2 knockout (KO) and kinase hyperactive G2019S (G2019S) transgenic mice. Subtle but significant differences in mRNA expression were observed relative to wild-type (WT) controls in the cortex, striatum and kidney of KO animals, but only in the striatum in the G2019S model. In contrast, robust, consistent and highly significant differences were identified by the direct comparison of KO and G2019S profiles in the cortex, striatum, kidney and muscle, indicating opposite effects on mRNA expression by the two models relative to WT. Ribosomal and glycolytic biological functions were consistently and significantly up-regulated in LRRK2 G2019S compared with LRRK2 KO tissues. Genes involved in membrane-bound organelles, oxidative phosphorylation, mRNA processing and the endoplasmic reticulum were down-regulated in LRRK2 G2019S mice compared with KO. We confirmed the expression patterns of 35 LRRK2-regulated genes using quantitative reverse transcription polymerase chain reaction. These findings provide the first description of the transcriptional responses to genetically modified LRRK2 activity and provide preclinical target engagement and/or pharmacodynamic biomarker strategies for LRRK2 and may inform future therapeutic strategies for LRRK2-associated PD.

Abstract 26: Notch pathway inhibition with MK-0752 leads to dose- and time-dependent transcriptional alterations in proliferation, PI3K, and Wnt pathway genes in plucked human hair follicles

Introduction: Gamma-secretase inhibitors (GSIs) inhibit Notch signaling and have potential as cancer therapeutics. A clinical trial of the oral GSI MK-0752 was conducted in healthy subjects using transcriptional profiling of human plucked hair follicles (PHFs). Data from this study were analyzed using pre-specified gene signatures reflecting Notch, proliferation, and PI3K pathway activity. De novo signature analysis was performed to identify late patterns of transcriptional response. Procedures: A randomized, placebo-controlled (PBO) trial evaluated the effects of a single 350mg or 1000mg dose of MK-0752 on PHF gene expression in healthy males. Plasma and pooled PHFs were collected for PK and PD (mRNA profiling) analyses. Results: In addition to a significant decrease in a Notch signature score (NSS, primary endpoint) which was maximal at 8.5h and significant up to 96h following a single 1000mg dose of MK-0752 compared to PBO, in a post hoc analysis we saw a significant decrease in a 101-gene Growth Factor Signature (GFS) score associated with downregulation of PI3K pathway signaling (effect size = −1.02, p Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 26.

Abstract B41: Identification of a time‐ and dose‐responsive transcriptional signature of Notch pathway inhibition in plucked human hair follicles following exposure to the gamma‐secretase inhibitor MK‐0752

Introduction: Gamma‐secretase inhibitors (GSIs) inhibit Notch pathway signaling and have potential as cancer therapeutics. Clinical development of GSIs has been complicated by mechanism‐related goblet cell hyperplasia and dose‐limiting diarrhea. A non‐invasive biomarker of Notch pathway inhibition would facilitate the identification of a well‐tolerated dose/schedule with maximal biological effect. In this study, a transcriptional biomarker of Notch pathway inhibition was developed from whole genome profiling of human plucked hair follicles (PHFs) for use in demonstrating target engagement and defining a PK/PD relationship. Procedures: This randomized, placebo‐controlled, 3‐period crossover study evaluated the effects of 350mg or 1000mg of the oral GSI MK‐0752 on PHF gene expression in 30 healthy male subjects. Pooled PHFs, single anagen hairs, and whole blood samples were collected at various timepoints for pharmacokinetic and pharmacodynamic (mRNA profiling) analysis. Results: There was a significant decrease in a pre‐specified 15‐gene Notch signature score (NSS) at 8.5, 28.5, 48 and 96 hours in response to either 350mg or 1000mg MK‐0752, compared to placebo, at the nominal 1‐sided 0.05 level. The maximum response in pooled PHFs was seen following administration 1000mg MK‐0752, which resulted in a significant (p Conclusions: The results of this study demonstrate the development of a compound‐specific biomarker in a novel surrogate tissue. This is the first known report of whole genome mRNA profiling of human hair follicles in response to a GSI, and demonstrates that PHFs contain cells with intact Notch signaling pathways responsive to MK‐0752. A 1000mg dose of MK‐0752, which is smaller than the clinically‐tolerated dose of 1800mg/week, decreases expression of canonical Notch pathway genes for up to 96 hours. In addition, treatment with a GSI results in various biological changes in PHFs, including early downregulation of PI3K pathway and proliferation genes, and a late (48h) upregulation of other signaling pathways that are potentially relevant to cancer cell proliferation. This novel plucked hair follicle biomarker provides important insights into the effect of GSIs on cell signaling, and also facilitates the development of gamma secretase targeted drugs for oncology indications. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B41.

Toxicogenomics in drug development: a match made in heaven?

Compound toxicity accounts for approximately half of all drug failures during development. Currently accepted preclinical studies for drug safety evaluation are time, resource, and animal intensive with often limited clinical predictivity. It is thus highly desirable to develop more efficient and predictive tools for early detection and assessment of potential compound liabilities. The emergence of genomics technologies over the last two decades promised to provide a solution. The premise of toxicogenomics (TGx) is straight forward: compounds with similar toxicity mechanisms and outcomes should perturb the transcriptome similarly and these perturbations could be used as more efficient and/or more predictive biomarkers of downstream toxicity outcome. This concept was reinforced by a number of pioneering studies demonstrating, for example, strong correlations between histopathology, clinical chemistry, and gene expression when different hepatocellular injuries were induced by chemical agents as reviewed in.[1,2] With such early advances, TGx was poised for earlier detection of a vast variety of drug-related outcomes, covering histopathologies across various organs, carcinogenicity, reproductive toxicity, etc., while deciphering mechanisms of action to create a more predictive and resource-sparing battery of tests for hazard identification, risk assessment, toxicity monitoring, and problem-solving across the drug development pipeline. This paradigm shift was anticipated to liberate the pharmaceutical and chemical industries from the current burden of toxicity liabilities, by enabling faster development of clinically safer compounds while reducing cost, infrastructure, and animal requirements.[1–3] TGx and drug discovery/development was expected to be a match made in heaven.

Aging African green monkeys manifest transcriptional, pathological, and cognitive hallmarks of human Alzheimer’s disease

While many preclinical models of Alzheimers disease (AD) have been reported, none fully recapitulate the disease. In an effort to identify an appropriate preclinical disease model, we characterized age-related changes in 2 higher order species, the African green monkey (AGM) and the rhesus macaque. Gene expression profiles in the dorsolateral prefrontal cortex and the visual cortex showed age-related changes in AGMs that are strikingly reminiscent of AD, whereas aged rhesus were most similar to healthy elderly humans. Biochemically, age-related changes in AGM cerebrospinal fluid levels of tau, phospho-tau, and amyloid beta were consistent with AD. Histologically, aged AGMs displayed pathological hallmarks of the disease, plaques, and 2 AGMs showed evidence of neurofibrillary tangle-like structures. We hypothesized and confirmed that AGMs have age-related cognitive deficits via a prefrontal cortex-dependent cognition test, and that symptomatic treatments that improve cognition in AD patients show efficacy in AGMs. These data suggest that the AGM could represent a novel and improved translational model to assist in the development of therapeutics for AD.

Transcriptional Profiling of Cholinergic Neurons From Basal Forebrain Identifies Changes in Expression of Genes Between Sleep and Wake

Study objective To assess differences in gene expression in cholinergic basal forebrain cells between sleeping and sleep-deprived mice sacrificed at the same time of day. Methods Tg(ChAT-eGFP)86Gsat mice expressing enhanced green fluorescent protein (eGFP) under control of the choline acetyltransferase (Chat) promoter were utilized to guide laser capture of cholinergic cells in basal forebrain. Messenger RNA expression levels in these cells were profiled using microarrays. Gene expression in eGFP(+) neurons was compared (1) to that in eGFP(-) neurons and to adjacent white matter, (2) between 7:00 am (lights on) and 7:00 pm (lights off), (3) between sleep-deprived and sleeping animals at 0, 3, 6, and 9 hours from lights on. Results There was a marked enrichment of ChAT and other markers of cholinergic neurons in eGFP(+) cells. Comparison of gene expression in these eGFP(+) neurons between 7:00 am and 7:00 pm revealed expected differences in the expression of clock genes (Arntl2, Per1, Per2, Dbp, Nr1d1) as well as mGluR3. Comparison of expression between spontaneous sleep and sleep-deprived groups sacrificed at the same time of day revealed a number of transcripts (n = 55) that had higher expression in sleep deprivation compared to sleep. Genes upregulated in sleep deprivation predominantly were from the protein folding pathway (25 transcripts, including chaperones). Among 42 transcripts upregulated in sleep was the cold-inducible RNA-binding protein. Conclusions Cholinergic cell signatures were characterized. Whether the identified genes are changing as a consequence of differences in behavioral state or as part of the molecular regulatory mechanism remains to be determined.

A novel link between clusterin and tau pathology in Alzheimer's disease

Ikuo Hayashi, Jacky Wong, Kate Tugusheva, Jacob Marcus, Xiaohai Wang, Keith Tanis, John Renger, Celina Zerbinatti, Merck Research Laboratories, West Point, Pennsylvania, United States; Merck & Co., Inc., West Point, Pennsylvania, United States; Merck & Co., Inc., West Point, Pennsylvania, United States; Merck and Company, West Point, Pennsylvania, United States. Contact e-mail: ikuo_hayashi@merck.com

Comparative transcriptomics of choroid plexus in Alzheimer’s disease, frontotemporal dementia and Huntington’s disease: implications for CSF homeostasis

BackgroundIn Alzheimer’s disease, there are striking changes in CSF composition that relate to altered choroid plexus (CP) function. Studying CP tissue gene expression at the blood–cerebrospinal fluid barrier could provide further insight into the epithelial and stromal responses to neurodegenerative disease states.MethodsTranscriptome-wide Affymetrix microarrays were used to determine disease-related changes in gene expression in human CP. RNA from post-mortem samples of the entire lateral ventricular choroid plexus was extracted from 6 healthy controls (Ctrl), 7 patients with advanced (Braak and Braak stage III–VI) Alzheimer’s disease (AD), 4 with frontotemporal dementia (FTD) and 3 with Huntington’s disease (HuD). Statistics and agglomerative clustering were accomplished with MathWorks, MatLab; and gene set annotations by comparing input sets to GeneGo (http://www.genego.com) and Ingenuity (http://www.ingenuity.com) pathway sets. Bonferroni-corrected hypergeometric p-values of < 0.1 were considered a significant overlap between sets.ResultsPronounced differences in gene expression occurred in CP of advanced AD patients vs. Ctrls. Metabolic and immune-related pathways including acute phase response, cytokine, cell adhesion, interferons, and JAK-STAT as well as mTOR were significantly enriched among the genes upregulated. Methionine degradation, claudin-5 and protein translation genes were downregulated. Many gene expression changes in AD patients were observed in FTD and HuD (e.g., claudin-5, tight junction downregulation), but there were significant differences between the disease groups. In AD and HuD (but not FTD), several neuroimmune-modulating interferons were significantly enriched (e.g., in AD: IFI-TM1, IFN-AR1, IFN-AR2, and IFN-GR2). AD-associated expression changes, but not those in HuD and FTD, were enriched for upregulation of VEGF signaling and immune response proteins, e.g., interleukins. HuD and FTD patients distinctively displayed upregulated cadherin-mediated adhesion.ConclusionsOur transcript data for human CP tissue provides genomic and mechanistic insight for differential expression in AD vs. FTD vs. HuD for stromal as well as epithelial components. These choroidal transcriptome characterizations elucidate immune activation, tissue functional resiliency, and CSF metabolic homeostasis. The BCSFB undergoes harmful, but also important functional and adaptive changes in neurodegenerative diseases; accordingly, the enriched JAK-STAT and mTOR pathways, respectively, likely help the CP in adaptive transcription and epithelial repair and/or replacement when harmed by neurodegeneration pathophysiology. We anticipate that these precise CP translational data will facilitate pharmacologic/transgenic therapies to alleviate dementia.

The Transcriptomic Signature Of Disease Development And Progression Of Nonalcoholic Fatty Liver Disease

A longitudinal molecular model of the development and progression of nonalcoholic fatty liver disease (NAFLD) over time is lacking. We have recently validated a high fat/sugar water-induced animal (an isogenic strain of C57BL/6 J:129S1/SvImJ mice) model of NAFLD that closely mimics most aspects of human disease. The hepatic transcriptome of such mice with fatty liver (8 weeks), steatohepatitis with early fibrosis (16–24 weeks) and advanced fibrosis (52 weeks) after initiation of the diet was evaluated and compared to mice on chow diet. Fatty liver development was associated with transcriptional activation of lipogenesis, FXR-RXR, PPAR-α mediated lipid oxidation and oxidative stress pathways. With progression to steatohepatitis, metabolic pathway activation persisted with additional activation of IL-1/inhibition of RXR, granulocyte diapedesis/adhesion, Fc macrophage activation, prothrombin activation and hepatic stellate cell activation. Progression to advanced fibrosis was associated with dampening of metabolic, oxidative stress and cell stress related pathway activation but with further Fc macrophage activation, cell death and turnover and activation of cancer-related networks. The molecular progression of NAFLD involves a metabolic perturbation which triggers subsequent cell stress and inflammation driving cell death and turnover. Over time, inflammation and fibrogenic pathways become dominant while in advanced disease an inflammatory-oncogenic profile dominates.

An accessible pharmacodynamic transcriptional biomarker for notch target engagement

γ‐Secretase mediates amyloid production in Alzheimers disease (AD) and oncogenic activity of Notch. γ‐Secretase inhibitors (GSIs) are thus of interest for AD and oncology. A peripheral biomarker of Notch activity would aid determination of the therapeutic window and dosing regimen for GSIs, given toxicities associated with chronic Notch inhibition. This study examined the effects of GSI MK‐0752 on blood and hair follicle transcriptomes in healthy volunteers. The effects of a structurally diverse GSI on rhesus blood and hair follicles were also compared. Significant dose‐related effects of MK‐0752 on transcription were observed in hair follicles, but not blood. The GSI biomarker identified in follicles exhibited 100% accuracy in a clinical test cohort, and was regulated in rhesus by a structurally diverse GSI. This study identified a translatable, accessible pharmacodynamic biomarker of GSI target engagement and provides proof of concept of hair follicle RNA as a translatable biomarker source.