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Haggarty, Stephen

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Haggarty

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Stephen

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Haggarty, Stephen
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    In Vivo Imaging of Histone Deacetylases (HDACs) in the Central Nervous System and Major Peripheral Organs
    (American Chemical Society, 2014) Wang, Changning; Schroeder, Frederick A.; Wey, Hsiao-Ying; Borra, Ronald; Wagner, Florence F.; Reis, Surya; Kim, Sung Won; Holson, Edward B.; Haggarty, Stephen; Hooker, Jacob
    Epigenetic enzymes are now targeted to treat the underlying gene expression dysregulation that contribute to disease pathogenesis. Histone deacetylases (HDACs) have shown broad potential in treatments against cancer and emerging data supports their targeting in the context of cardiovascular disease and central nervous system dysfunction. Development of a molecular agent for non-invasive imaging to elucidate the distribution and functional roles of HDACs in humans will accelerate medical research and drug discovery in this domain. Herein, we describe the synthesis and validation of an HDAC imaging agent, [11C]6. Our imaging results demonstrate that this probe has high specificity, good selectivity, and appropriate kinetics and distribution for imaging HDACs in the brain, heart, kidney, pancreas, and spleen. Our findings support the translational potential for [11C]6 for human epigenetic imaging.
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    Traditional and systems biology based drug discovery for the rare tumor syndrome neurofibromatosis type 2
    (Public Library of Science, 2018) Allaway, Robert; Angus, Steve P.; Beauchamp, Roberta L.; Blakeley, Jaishri O.; Bott, Marga; Burns, Sarah S.; Carlstedt, Annemarie; Chang, Long-Sheng; Chen, Xin; Clapp, D. Wade; Desouza, Patrick A.; Erdin, Serkan; Fernandez-Valle, Cristina; Guinney, Justin; Gusella, James; Haggarty, Stephen; Johnson, Gary L.; La Rosa, Salvatore; Morrison, Helen; Petrilli, Alejandra M.; Plotkin, Scott; Pratap, Abhishek; Ramesh, Vijaya; Sciaky, Noah; Stemmer-Rachamimov, Anat; Stuhlmiller, Tim J.; Talkowski, Michael; Welling, Duane; Yates, Charles W.; Zawistowski, Jon S.; Zhao, Wen-Ning
    Neurofibromatosis 2 (NF2) is a rare tumor suppressor syndrome that manifests with multiple schwannomas and meningiomas. There are no effective drug therapies for these benign tumors and conventional therapies have limited efficacy. Various model systems have been created and several drug targets have been implicated in NF2-driven tumorigenesis based on known effects of the absence of merlin, the product of the NF2 gene. We tested priority compounds based on known biology with traditional dose-concentration studies in meningioma and schwann cell systems. Concurrently, we studied functional kinome and gene expression in these cells pre- and post-treatment to determine merlin deficient molecular phenotypes. Cell viability results showed that three agents (GSK2126458, Panobinostat, CUDC-907) had the greatest activity across schwannoma and meningioma cell systems, but merlin status did not significantly influence response. In vivo, drug effect was tumor specific with meningioma, but not schwannoma, showing response to GSK2126458 and Panobinostat. In culture, changes in both the transcriptome and kinome in response to treatment clustered predominantly based on tumor type. However, there were differences in both gene expression and functional kinome at baseline between meningioma and schwannoma cell systems that may form the basis for future selective therapies. This work has created an openly accessible resource (www.synapse.org/SynodosNF2) of fully characterized isogenic schwannoma and meningioma cell systems as well as a rich data source of kinome and transcriptome data from these assay systems before and after treatment that enables single and combination drug discovery based on molecular phenotype.
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    MeCP2-regulated miRNAs control early human neurogenesis through differential effects on ERK and AKT signaling
    (2018) Mellios, Nikolaos; Feldman, Danielle A.; Sheridan, Steven D.; Ip, Jacque P.K.; Kwok, Showming; Amoah, Stephen K.; Rosen, Bess; Rodriguez, Brian A.; Crawford, Benjamin; Swaminathan, Radha; Chou, Stephanie; Li, Yun; Ziats, Mark; Ernst, Carl; Jaenisch, Rudolf; Haggarty, Stephen; Sur, Mriganka
    Rett Syndrome (RTT) is an X-linked, neurodevelopmental disorder caused primarily by mutations in the Methyl-CpG-binding protein 2 (MECP2) gene, which encodes a multifunctional epigenetic regulator with known links to a wide spectrum of neuropsychiatric disorders. While postnatal functions of MeCP2 have been thoroughly investigated, its role in prenatal brain development remains poorly understood. Given the well-established importance of miRNAs in neurogenesis, we employed isogenic human RTT patient-derived induced pluripotent stem cell (iPSC) and MeCP2 shRNA knockdown approaches to identify novel MeCP2-regulated miRNAs enriched during early human neuronal development. Focusing on the most dysregulated miRNAs, we found miR-199 and miR-214 to be increased during early brain development and to differentially regulate extracellular signal-regulated kinase (ERK/MAPK) and protein kinase B (PKB/AKT) signaling. In parallel, we characterized the effects on human neurogenesis and neuronal differentiation brought about by MeCP2 deficiency using both monolayer and 3D (cerebral organoid) patient-derived and MeCP2-deficient neuronal culture models. Inhibiting miR-199 or miR-214 expression in iPSC-derived neural progenitors (NPs) deficient in MeCP2 restored AKT and ERK activation, respectively, and ameliorated the observed alterations in neuronal differentiation. Moreover, overexpression of miR-199 or miR-214 in WT mouse embryonic brains was sufficient to disturb neurogenesis and neuronal migration in a similar manner to Mecp2 knockdown. Taken together, our data support a novel miRNA-mediated pathway downstream of MeCP2 that influences neurogenesis via interactions with central molecular hubs linked to autism spectrum disorders.
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    Combining NGN2 Programming with Developmental Patterning Generates Human Excitatory Neurons with NMDAR-Mediated Synaptic Transmission
    (2018) Nehme, Ralda; Zuccaro, Emanuela; Dia Ghosh, Sulagna; Li, Chenchen; Sherwood, John; Pietilainen, Olli; Barrett, Lindy; Limone, Francesco; Worringer, Kathleen A.; Kommineni, Sravya; Zang, Ying; Cacchiarelli, Davide; Meissner, Alex; Adolfsson, Rolf; Haggarty, Stephen; Madison, Jon; Muller, Matthias; Arlotta, Paola; Fu, Zhanyan; Feng, Guoping; Eggan, Kevin
    SUMMARY Transcription factor programming of pluripotent stem cells (PSCs) has emerged as an approach to generate human neurons for disease modeling. However, programming schemes produce a variety of cell types, and those neurons that are made often retain an immature phenotype, which limits their utility in modeling neuronal processes, including synaptic transmission. We report that combining NGN2 programming with SMAD and WNT inhibition generates human patterned induced neurons (hpiNs). Single-cell analyses showed that hpiN cultures contained cells along a developmental continuum, ranging from poorly differentiated neuronal progenitors to well-differentiated, excitatory glutamatergic neurons. The most differentiated neurons could be identified using a CAMK2A::GFP reporter gene and exhibited greater functionality, including NMDAR-mediated synaptic transmission. We conclude that utilizing single-cell and reporter gene approaches for selecting successfully programmed cells for study will greatly enhance the utility of hpiNs and other programmed neuronal populations in the modeling of nervous system disorders.
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    Class I HDAC Inhibition Blocks Cocaine-Induced Plasticity Through Targeted Changes in Histone Methylation
    (2013) Kennedy, Pamela J.; Feng, Jian; Robison, A.J.; Maze, Ian; Badimon, Ana; Mouzon, Ezekiell; Chaudhury, Dipesh; Damez-Werno, Diane M.; Haggarty, Stephen; Han, Ming-Hu; Bassel-Duby, Rhonda; Olson, Eric N.; Nestler, Eric J.
    Induction of histone acetylation in the nucleus accumbens (NAc), a key brain reward region, promotes cocaine-induced alterations in gene expression. Histone deacetylases (HDACs) tightly regulate the acetylation of histone tails, but little is known about the functional specificity of different HDAC isoforms in the development and maintenance of cocaine-induced plasticity, and prior studies of HDAC inhibitors report conflicting effects on cocaine-elicited behavioral adaptations. Here, we demonstrate that specific and prolonged blockade of HDAC1 in NAc of mice increased global levels of histone acetylation, but also induced repressive histone methylation and antagonized cocaine-induced changes in behavior, an effect mediated in part via a chromatin-mediated suppression of GABAA receptor subunit expression and inhibitory tone on NAc neurons. Our findings suggest a novel mechanism by which prolonged and selective HDAC inhibition can alter behavioral and molecular adaptations to cocaine and inform the development of novel therapeutics for cocaine addiction.
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    Cis-Acting Regulation of Brain-Specific ANK3 Gene Expression by a Genetic Variant Associated with Bipolar Disorder
    (2013) Rueckert, Erroll H.; Barker, Douglas; Ruderfer, Douglas; Bergen, Sarah E.; O’Dushlaine, Colm; Luce, Catherine J.; Sheridan, Steven D.; Theriault, Kraig M.; Chambert, Kimberly; Moran, Jennifer; Purcell, Shaun M.; Madison, Jon Morrow; Haggarty, Stephen; Sklar, Pamela
    Several genome-wide association studies (GWAS) for bipolar disorder (BD) have found a strong association of the Ankyrin3 (ANK3) gene. This association spans numerous linked single nucleotide polymorphisms (SNPs) in a ~250 kb genomic region overlapping ANK3. The associated region encompasses predicted regulatory elements as well as two of six validated alternative first exons, which encode distinct protein domains at the N-terminus of the protein also known as ankyrin-G (AnkG). Using RNA Ligase-Mediated Rapid Amplification of cDNA Ends (RLM-RACE) to identify novel transcripts in conjunction with a highly sensitive, exon-specific multiplexed mRNA expression assay, we detected differential regulation of distinct ANK3 transcription start sites (TSSs) and coupling of specific 5’ ends with 3’ mRNA splicing events in post-mortem human brain and human stem cell-derived neural progenitors and neurons. Furthermore, allelic variation at the BD–associated SNP rs1938526 correlated with a significant difference in cerebellar expression of a brain-specific ANK3 transcript. These findings suggest a brain-specific cis-regulatory transcriptional effect of ANK3 may be relevant to BD pathophysiology.
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    A Selective HDAC 1/2 Inhibitor Modulates Chromatin and Gene Expression in Brain and Alters Mouse Behavior in Two Mood-Related Tests
    (Public Library of Science, 2013) Schroeder, Frederick A; Lewis, Michael C.; Fass, Daniel M.; Wagner, Florence F.; Zhang, Yan-Ling; Hennig, Krista M.; Gale, Jennifer; Zhao, Wen-Ning; Reis, Surya; Barker, Douglas D.; Berry-Scott, Erin; Kim, Sung Won; Clore, Elizabeth L.; Hooker, Jacob; Holson, Edward B.; Haggarty, Stephen; Petryshen, Tracey L.
    Psychiatric diseases, including schizophrenia, bipolar disorder and major depression, are projected to lead global disease burden within the next decade. Pharmacotherapy, the primary – albeit often ineffective – treatment method, has remained largely unchanged over the past 50 years, highlighting the need for novel target discovery and improved mechanism-based treatments. Here, we examined in wild type mice the impact of chronic, systemic treatment with Compound 60 (Cpd-60), a slow-binding, benzamide-based inhibitor of the class I histone deacetylase (HDAC) family members, HDAC1 and HDAC2, in mood-related behavioral assays responsive to clinically effective drugs. Cpd-60 treatment for one week was associated with attenuated locomotor activity following acute amphetamine challenge. Further, treated mice demonstrated decreased immobility in the forced swim test. These changes are consistent with established effects of clinical mood stabilizers and antidepressants, respectively. Whole-genome expression profiling of specific brain regions (prefrontal cortex, nucleus accumbens, hippocampus) from mice treated with Cpd-60 identified gene expression changes, including a small subset of transcripts that significantly overlapped those previously reported in lithium-treated mice. HDAC inhibition in brain was confirmed by increased histone acetylation both globally and, using chromatin immunoprecipitation, at the promoter regions of upregulated transcripts, a finding consistent with in vivo engagement of HDAC targets. In contrast, treatment with suberoylanilide hydroxamic acid (SAHA), a non-selective fast-binding, hydroxamic acid HDAC 1/2/3/6 inhibitor, was sufficient to increase histone acetylation in brain, but did not alter mood-related behaviors and had dissimilar transcriptional regulatory effects compared to Cpd-60. These results provide evidence that selective inhibition of HDAC1 and HDAC2 in brain may provide an epigenetic-based target for developing improved treatments for mood disorders and other brain disorders with altered chromatin-mediated neuroplasticity.
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    Class I HDAC imaging using [3H]CI-994 autoradiography
    (Landes Bioscience, 2013) Wang, Yajie; Zhang, Yan-Ling; Hennig, Krista; Gale, Jennifer P.; Hong, Yijia; Cha, Anna; Riley, Misha; Wagner, Florence; Haggarty, Stephen; Holson, Edward; Hooker, Jacob
    [3H]CI-994, a radioactive isotopologue of the benzamide CI-994, a class I histone deacetylase inhibitor (HDACi), was evaluated as an autoradiography probe for ex vivo labeling and localizing of class I HDAC (isoforms 1–3) in the rodent brain. After protocol optimization, up to 80% of total binding was attributed to specific binding. Notably, like other benzamide HDACi, [3H]CI-994 exhibits slow binding kinetics when measured in vitro with isolated enzymes and ex vivo when used for autoradiographic mapping of HDAC1–3 density. The regional distribution and density of HDAC1–3 was determined through a series of saturation and kinetics experiments. The binding properties of [3H]CI-994 to HDAC1–3 were characterized and the data were used to determine the regional Bmax of the target proteins. Kd values, determined from slice autoradiography, were between 9.17 and 15.6 nM. The HDAC1–3 density (Bmax), averaged over whole brain sections, was of 12.9 picomol · mg−1 protein. The highest HDAC1–3 density was found in the cerebellum, followed by hippocampus and cortex. Moderate to low receptor density was found in striatum, hypothalamus and thalamus. These data were correlated with semi-quantitative measures of each HDAC isoform using western blot analysis and it was determined that autoradiographic images most likely represent the sum of HDAC1, HDAC2, and HDAC3 protein density. In competition experiments, [3H]CI-994 binding can be dose-dependently blocked with other HDAC inhibitors, including suberoylanilide hydroxamic acid (SAHA). In summary, we have developed the first known autoradiography tool for imaging class I HDAC enzymes. Although validated in the CNS, [3H]CI-994 will be applicable and beneficial to other target tissues and can be used to evaluate HDAC inhibition in tissues for novel therapies being developed. [3H]CI-994 is now an enabling imaging tool to study the relationship between diseases and epigenetic regulation.
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    Design, Synthesis, and Evaluation of Hydroxamic Acid-Based Molecular Probes for In Vivo Imaging of Histone Deacetylase (HDAC) in Brain
    (e-Century Publishing, 2014) Hooker, Jacob; Wang, Changning; Eessalu, Thomas E.; Barth, Vanessa N.; Mitch, Charles H.; Wagner, Florence F.; Hong, Yijia; Neelamegam, Ramesh; Schroeder, Frederick A; Holson, Edward B.; Haggarty, Stephen
    Hydroxamic acid-based histone deacetylase inhibitors (HDACis) are a class of molecules with therapeutic potential currently reflected in the use of suberoylanilide hydroxamic acid (SAHA; Vorinostat) to treat cutaneous T-cell lymphomas (CTCL). HDACis may have utility beyond cancer therapy, as preclinical studies have ascribed HDAC inhibition as beneficial in areas such as heart disease, diabetes, depression, neurodegeneration, and other disorders of the central nervous system (CNS). However, little is known about the pharmacokinetics (PK) of hydroxamates, particularly with respect to CNS-penetration, distribution, and retention. To explore the rodent and non-human primate (NHP) brain permeability of hydroxamic acid-based HDAC inhibitors using positron emission tomography (PET), we modified the structures of belinostat (PXD101) and panobinostat (LBH-589) to incorporate carbon-11. We also labeled PCI 34051 through carbon isotope substitution. After characterizing the in vitro affinity and efficacy of these compounds across nine recombinant HDAC isoforms spanning Class I and Class II family members, we determined the brain uptake of each inhibitor. Each labeled compound has low uptake in brain tissue when administered intravenously to rodents and NHPs. In rodent studies, we observed that brain accumulation of the radiotracers were unaffected by the pre-administration of unlabeled inhibitors. Knowing that CNS-penetration may be desirable for both imaging applications and therapy, we explored whether a liquid chromatography, tandem mass spectrometry (LC-MS-MS) method to predict brain penetrance would be an appropriate method to pre-screen compounds (hydroxamic acid-based HDACi) prior to PET radiolabeling. LC-MS-MS data were indeed useful in identifying additional lead molecules to explore as PET imaging agents to visualize HDAC enzymes in vivo. However, HDACi brain penetrance predicted by LC-MS-MS did not strongly correlate with PET imaging results. This underscores the importance of in vivo PET imaging tools in characterizing putative CNS drug lead compounds and the continued need to discover effect PET tracers for neuroepigenetic imaging.
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    FDG-PET Imaging Reveals Local Brain Glucose Utilization Is Altered by Class I Histone Deacetylase Inhibitors
    (Elsevier BV, 2013) Schroeder, Frederick A; Chonde, Daniel B.; Riley, Misha M.; Moseley, Christian K.; Granda, Michael L.; Wilson, Colin M.; Wagner, Florence F.; Zhang, Yan-Ling; Gale, Jennifer; Holson, Edward B.; Haggarty, Stephen; Hooker, Jacob
    The purpose of this work – the first of its kind – was to evaluate the impact of chronic selective histone deacetylase (HDAC) inhibitor treatment on brain activity using uptake of the radioligand 18F-fluorodeoxyglucose and positron emission tomography (18FDG-PET). HDAC dysfunction and other epigenetic mechanisms are implicated in diverse CNS disorders and animal research suggests HDAC inhibition may provide a lead toward developing improved treatment. To begin to better understand the role of the class I HDAC subtypes HDAC 1, 2 and 3 in modulating brain activity, we utilized two benzamide inhibitors from the literature, compound 60 (Cpd-60) and CI-994 which selectively inhibit HDAC 1 and 2 or HDACs 1, 2 and 3, respectively. One day after the seventh treatment with Cpd-60 (22.5 mg/kg) or CI-994 (5 mg/kg), 18FDG-PET experiments (n = 11–12 rats per treatment group) revealed significant, local changes in brain glucose utilization. These 2–17% changes were represented by increases and decreases in glucose uptake. The pattern of changes was similar but distinct between Cpd-60 and CI-994, supporting that 18FDG-PET is a useful tool to examine the relationship between HDAC subtype activity and brain activity. Further work using additional selective HDAC inhibitors will be needed to clarify these effects as well as to understand how brain activity changes influence behavioral response.