Search

Significance Development of cortical areas begins in cortical stem cells through the action of morphogens controlling the graded expression of transcription factors (TFs). Here, we have systematically identified the TFs and gene regulatory elements (REs) that together control regional pattering of the cortical progenitor zone; these data have led us to propose a cortical regionalization TF network. To identify REs active in this network, we performed TF chromatin immunoprecipitation followed by sequencing (ChIP-seq) and chromatin-looping conformation experiments as well as assays for epigenomic marks and DNA accessibility in purified ventricular zone (VZ) progenitor cells in wild-type and patterning mutant mice. This integrated approach has laid the foundations to identify a TF network and cortical VZ REs involved in cortical regional patterning., We uncovered a transcription factor (TF) network that regulates cortical regional patterning in radial glial stem cells. Screening the expression of hundreds of TFs in the developing mouse cortex identified 38 TFs that are expressed in gradients in the ventricular zone (VZ). We tested whether their cortical expression was altered in mutant mice with known patterning defects (Emx2, Nr2f1, and Pax6), which enabled us to define a cortical regionalization TF network (CRTFN). To identify genomic programming underlying this network, we performed TF ChIP-seq and chromatin-looping conformation to identify enhancer–gene interactions. To map enhancers involved in regional patterning of cortical progenitors, we performed assays for epigenomic marks and DNA accessibility in VZ cells purified from wild-type and patterning mutant mice. This integrated approach has identified a CRTFN and VZ enhancers involved in cortical regional patterning in the mouse.

Acetylation of tau protein is dysregulated in Alzheimer's Disease (AD). It has been proposed that acetylation of specific sites in the KXGS motif of tau can regulate phosphorylation of nearby residues and reduce the propensity of tau to aggregate. Histone deacetylase 6 (HDAC6) is a cytoplasmic enzyme involved in deacetylation of multiple targets, including tau, and it has been suggested that inhibition of HDAC6 would increase tau acetylation at the KXGS motifs and thus may present a viable therapeutic approach to treat AD. To directly test the contribution of HDAC6 to tau pathology, we intracerebroventricularly injected an antisense oligonucleotide (ASO) directed against HDAC6 mRNA into brains of P301S tau mice (PS19 model), which resulted in a 70% knockdown of HDAC6 protein in the brain. Despite a robust decrease in levels of HDAC6, no increase in tau acetylation was observed. Additionally, no change of tau phosphorylation or tau aggregation was detected upon the knockdown of HDAC6. We conclude that HDAC6 does not impact tau pathology in PS19 mice.

SUMMARYWe uncovered a transcription factor (TF) network that regulates cortical regional patterning. Screening the expression of hundreds of TFs in the developing mouse cortex identified 38 TFs that are expressed in gradients in the ventricular zone (VZ). We tested whether their cortical expression was altered in mutant mice with known patterning defects (Emx2, Nr2f1andPax6), which enabled us to define a cortical regionalization TF network (CRTFN). To identify genomic programming underlying this network, we performed TF ChIP-seq and chromatin-looping conformation to identify enhancer-gene interactions. To map enhancers involved in regional patterning of cortical progenitors, we performed assays for epigenomic marks and DNA accessibility in VZ cells purified from wild-type and patterning mutant mice. This integrated approach has identified a CRTFN and VZ enhancers involved in cortical regional patterning.

Elucidating the transcriptional circuitry controlling forebrain development requires an understanding of enhancer activity and regulation. We generated stable transgenic mouse lines that express CreERT2 and GFP from ten different enhancer elements with activity in distinct domains within the embryonic basal ganglia. We used these unique tools to generate a comprehensive regional fate map of the mouse subpallium, including sources for specific subtypes of amygdala neurons. We then focused on deciphering transcriptional mechanisms that control enhancer activity. Using machine-learning computations, in vivo chromosomal occupancy of 13 transcription factors that regulate subpallial patterning and differentiation and analysis of enhancer activity in Dlx1/2 and Lhx6 mutants, we elucidated novel molecular mechanisms that regulate region-specific enhancer activity in the developing brain. Thus, these subpallial enhancer transgenic lines are data and tool resources to study transcriptional regulation of GABAergic cell fate.

Deposition of tau aggregates in the brain is a pathological hallmark of several neurodegenerative diseases, termed tauopathies, such as Alzheimer's disease (AD), corticobasal degeneration, and progressive supranuclear palsy (PSP). As transcellular spread of pathological tau aggregates has been implicated in disease progression, immunotherapy is being considered as a treatment for tauopathies. Here we report a detailed biochemical and biophysical characterization of the tau-binding properties of gosuranemab, a humanized monoclonal antibody directed against N-terminal tau that is currently being investigated as a treatment for AD. Binding experiments showed that gosuranemab exhibited high affinity for tau monomer, tau fibrils, and insoluble tau from different tauopathies. Epitope mapping studies conducted using X-ray crystallography and mutagenesis showed that gosuranemab bound to human tau residues 15–22. Immunodepletion of pathological human brain homogenates and transgenic mouse interstitial fluid (ISF) with gosuranemab resulted in reduced tau aggregation in tau biosensor cells. Preincubation of seed-competent AD-tau with gosuranemab significantly inhibited tau aggregation in mouse primary cortical neurons. Gosuranemab also significantly reduced unbound N-terminal tau in cerebrospinal fluid (CSF) from individuals with PSP and AD, and in ISF and CSF of treated transgenic mice. These results are consistent with the >90% target engagement observed in the CSF of some clinical trial dosing cohorts and support the evaluation of gosuranemab as a potential treatment for AD.

© 2015 Elsevier Inc. We demonstrate using conditional mutagenesis that Pbx1, with and without Pbx2+/-sensitization, regulates regional identity and laminar patterning of the developing mouse neocortex in cortical progenitors (Emx1-Cre) and in newly generated neurons (Nex1-Cre). Pbx1/2 mutants have three salient molecular phenotypes of cortical regional and laminar organization: hypoplasia of the frontal cortex, ventral expansion of the dorsomedial cortex, and ventral expansion of Reelin expression in the cortical plate of the frontal cortex, concomitant with an inversion of cortical layering in the rostral cortex. Molecular analyses, including PBX ChIP-seq, provide evidence that PBX promotes frontal cortex identity by repressing genes that promote dorsocaudal fate.

Elucidating the genetic control of cerebral cortical (pallial) development is essential for understanding function, evolution, and disorders of the brain. Transcription factors (TFs) that embryonically regulate pallial regionalization are expressed in gradients, raising the question of how discrete domains are generated. We provide evidence that small enhancer elements active in protodomains integrate broad transcriptional information. CreERT2 and GFP expression from 14 different enhancer elements in stable transgenic mice allowed us to define the first comprehensive regional fate map of the pallium. We explored transcriptional mechanisms that control the activity of the enhancers using informatics, in vivo occupancy by TFs that regulate cortical patterning (CoupTFI, Pax6 and Pbx1), and analysis of enhancer activity in Pax6 mutants. Overall, the results provide novel insights into how broadly expressed patterning TFs regulate the activity of small enhancer elements that drive gene expression in pallial protodomains that fate map to distinct cortical regions.

SummaryThe mammalian telencephalon plays critical roles in cognition, motor function, and emotion. Though many of the genes required for its development have been identified, the distant-acting regulatory sequences orchestrating their in vivo expression are mostly unknown. Here, we describe a digital atlas of in vivo enhancers active in subregions of the developing telencephalon. We identified more than 4,600 candidate embryonic forebrain enhancers and studied the in vivo activity of 329 of these sequences in transgenic mouse embryos. We generated serial sets of histological brain sections for 145 reproducible forebrain enhancers, resulting in a publicly accessible web-based data collection comprising more than 32,000 sections. We also used epigenomic analysis of human and mouse cortex tissue to directly compare the genome-wide enhancer architecture in these species. These data provide a primary resource for investigating gene regulatory mechanisms of telencephalon development and enable studies of the role of distant-acting enhancers in neurodevelopmental disorders.

Therapeutic intervention aimed at reactivation of fetal hemoglobin protein (HbF) is a promising approach for ameliorating sickle cell disease (SCD) and β-thalassemia. Previous studies showed genetic knockdown of histone deacetylase (HDAC) 1 or 2 is sufficient to induce HbF. Here we show that ACY-957, a selective chemical inhibitor of HDAC1 and 2 (HDAC1/2), elicits a dose and time dependent induction of γ-globin mRNA (HBG) and HbF in cultured primary cells derived from healthy individuals and sickle cell patients. Gene expression profiling of erythroid progenitors treated with ACY-957 identified global changes in gene expression that were significantly enriched in genes previously shown to be affected by HDAC1 or 2 knockdown. These genes included GATA2, which was induced greater than 3-fold. Lentiviral overexpression of GATA2 in primary erythroid progenitors increased HBG, and reduced adult β-globin mRNA (HBB). Furthermore, knockdown of GATA2 attenuated HBG induction by ACY-957. Chromatin immunoprecipitation and sequencing (ChIP-Seq) of primary erythroid progenitors demonstrated that HDAC1 and 2 occupancy was highly correlated throughout the GATA2 locus and that HDAC1/2 inhibition led to elevated histone acetylation at well-known GATA2 autoregulatory regions. The GATA2 protein itself also showed increased binding at these regions in response to ACY-957 treatment. These data show that chemical inhibition of HDAC1/2 induces HBG and suggest that this effect is mediated, at least in part, by histone acetylation-induced activation of the GATA2 gene.

Bcl11b is a transcription factor that, within the hematopoietic system, is expressed specifically in T cells. Although Bcl11b is required for T-cell differentiation in newborn Bcl11b-null mice, and for positive selection in the adult thymus of mice bearing a T-cell-targeted deletion, the gene network regulated by Bcl11b in T cells is unclear. We report herein that Bcl11b is a bifunctional transcriptional regulator, which is required for the correct expression of approximately 1000 genes in CD4(+)CD8(+)CD3(lo) double-positive (DP) thymocytes. Bcl11b-deficient DP cells displayed a gene expression program associated with mature CD4(+)CD8(-) and CD4(-)CD8(+) single-positive (SP) thymocytes, including upregulation of key transcriptional regulators, such as Zbtb7b and Runx3. Bcl11b interacted with regulatory regions of many dysregulated genes, suggesting a direct role in the transcriptional regulation of these genes. However, inappropriate expression of lineage-associated genes did not result in enhanced differentiation, as deletion of Bcl11b in DP cells prevented development of SP thymocytes, and that of canonical NKT cells. These data establish Bcl11b as a crucial transcriptional regulator in thymocytes, in which Bcl11b functions to prevent the premature expression of genes fundamental to the SP and NKT cell differentiation programs.

COUP-TF-interacting protein 2 (CTIP2), also known as Bcl11b, is a transcriptional regulatory protein that is highly expressed in and plays a critical role(s) during development of T lymphocytes and the central nervous system. We demonstrate herein that CTIP2 is also highly expressed in mouse skin during embryogenesis and in adulthood as revealed by immunohistochemical analyses. CTIP2 expression in the ectoderm was first detected at embryonic day 10.5 (E10.5), and became increasingly restricted to proliferating cells of the basal cell layer of the developing epidermis in later stages of fetal development and in adult skin. In addition, CTIP2 expression was also detected in some cells of the suprabasal layer of the developing epidermis, as well as in developing and mature hair follicles. Relatively fewer cells of the developing dermal component of skin were found to express CTIP2, and the adult dermis was devoid of CTIP2 expression. Some, but not all, of the cells present within hair follicle bulge were found to co-express CTIP2, keratin K15, but not CD34, indicating that a subset of K15(+) CD34(-) skin stem cells may express CTIP2. Considered together, these findings suggest that CTIP2 may play a role(s) in skin development and/or homeostasis.

Chemotherapy-induced peripheral neuropathy is one of the most common dose-limiting side effects of cancer treatment. Currently, there is no Food and Drug Administration-approved treatment available. Histone deacetylase 6 (HDAC6) is a microtubule-associated deacetylase whose function includes regulation of α-tubulin-dependent intracellular mitochondrial transport. Here, we examined the effect of HDAC6 inhibition on established cisplatin-induced peripheral neuropathy. We used a novel HDAC6 inhibitor ACY-1083, which shows 260-fold selectivity towards HDAC6 vs other HDACs. Our results show that HDAC6 inhibition prevented cisplatin-induced mechanical allodynia, and also completely reversed already existing cisplatin-induced mechanical allodynia, spontaneous pain, and numbness. These findings were confirmed using the established HDAC6 inhibitor ACY-1215 (Ricolinostat), which is currently in clinical trials for cancer treatment. Mechanistically, treatment with the HDAC6 inhibitor increased α-tubulin acetylation in the peripheral nerve. In addition, HDAC6 inhibition restored the cisplatin-induced reduction in mitochondrial bioenergetics and mitochondrial content in the tibial nerve, indicating increased mitochondrial transport. At a later time point, dorsal root ganglion mitochondrial bioenergetics also improved. HDAC6 inhibition restored the loss of intraepidermal nerve fiber density in cisplatin-treated mice. Our results demonstrate that pharmacological inhibition of HDAC6 completely reverses all the hallmarks of established cisplatin-induced peripheral neuropathy by normalization of mitochondrial function in dorsal root ganglia and nerve, and restoration of intraepidermal innervation. These results are especially promising because one of the HDAC6 inhibitors tested here is currently in clinical trials as an add-on cancer therapy, highlighting the potential for a fast clinical translation of our findings.

INTRODUCTION: Neuroblastoma (NB) is an extra-cranial solid cancer and is among the most common cancers in infants less than 1 year of age, with 650 new cases each year in the United States. Half of the children with NB have high risk disease and 20-50% of those will fail to respond adequately to current therapies, illustrating an urgent unmet medical need. Current treatment for high-risk disease is aggressive, including chemotherapy, surgery, radiation with stem cell transplant, anti-GD2/cytokine immunotherapy and retinoic acid (RA) treatment. RA is a pro-differentiation agent that slows growth and promotes cell death. A gene expression pattern associated with RA-induced NB differentiation was identified, and chemical inhibition of HDAC1/2 was shown to induce a similar expression pattern. METHODS: In this work, we examine the activity of an orally bioavailable HDAC1/2 inhibitor (HDAC1/2i) on NB cell differentiation, proliferation and apoptosis. RA combined with HDAC1/2i enhances gene expression patterns associated with differentiation, slows cellular proliferation and more rapidly induces dendrite formation than RA can achieve alone. The mechanisms leading to the differentiated phenotype were examined by RT-PCR, gene expression microarray and retinoic acid receptor (RAR) chromatin immunoprecipitation followed by high-througput sequening (ChIP-Seq). RESULTS: HDAC1/2i and RA together caused increased localization of the RAR to its own RARα and RARβ promoter regions, and increased in RAR mRNA and protein relative to the RA treatment condition alone. Additionally, expression of Cyp26, an enzyme responsible for clearing intercellular RA, was reduced in the combination setting. Gene set enrichment analysis of the microarray data comparing the combination setting against RA as a single agent suggested that the addition of HDAC1/2i enhanced apoptotic pathways and decreased E2F driven cell cycle signaling. We confirmed enhanced apoptosis in the combination setting by measuring caspase 3 and PARP cleavage. Consistent with this finding, we observed reduced proliferation, increased sub-G1 cell frequency in cell cycle assays and ablation of emergent RA-resistant NB colonies after combination treatment. Further, combination treatment reduced the E2F-activators CDK4 and CDK6 at the protein level while the CDK inhibitor, p21, was dramatically increased. Hypo-phosphorylation of retinoblastoma protein, directly linked to E2F complex inactivation, was also observed and consistent with reduced proliferation and the decreased frequency of S-phase cells observed in EDU incorporation assays. Xenograft models of NB with RA and HDAC1/2i are in progress, as are HDAC1, HDAC2, acetylated histone H3K9 ChIP-seq experiments, and will be discussed. Taken together, these findings support a role for selective HDAC1/2i in combination with RA for the treatment of patients with high risk NB. Citation Format: David L. Tamang, Pengyu Huang, Olga Golonzhka, Jeffrey R. Shearstone, Steven N. Quayle, Simon S. Jones, Min Yang. An orally bioavailable and selective histone deacetylase (HDAC) 1 & 2 inhibitor enhances retinoic acid mediated differentiation of neuroblastoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2477.

The transcription factor Ctip2/Bcl11b plays essential roles in developmental processes of the immune and central nervous systems and skin. Here we show that Ctip2 also plays a key role in tooth development. Ctip2 is highly expressed in the ectodermal components of the developing tooth, including inner and outer enamel epithelia, stellate reticulum, stratum intermedium, and the ameloblast cell lineage. In Ctip2 −/− mice, tooth morphogenesis appeared to proceed normally through the cap stage but developed multiple defects at the bell stage. Mutant incisors and molars were reduced in size and exhibited hypoplasticity of the stellate reticulum. An ameloblast-like cell population developed ectopically on the lingual aspect of mutant lower incisors, and the morphology, polarization, and adhesion properties of ameloblasts on the labial side of these teeth were severely disrupted. Perturbations of gene expression were also observed in the mandible of Ctip2 −/− mice: expression of the ameloblast markers amelogenin , ameloblastin , and enamelin was down-regulated, as was expression of Msx2 and epiprofin , transcription factors implicated in the tooth development and ameloblast differentiation. These results suggest that Ctip2 functions as a critical regulator of epithelial cell fate and differentiation during tooth morphogenesis.

International audience; COUP-TF-interacting protein 2 (CTIP2; also known as Bcl11b) is a transcription factor that plays key roles in the development of the central nervous and immune systems. CTIP2 is also highly expressed in the developing epidermis, and at lower levels in the dermis and in adult skin. Analyses of mice harboring a germline deletion of CTIP2 revealed that the protein plays critical roles in skin during development, particularly in keratinocyte proliferation and late differentiation events, as well as in the development of the epidermal permeability barrier. At the core of all of these actions is a relatively large network of genes, described herein, that is regulated directly or indirectly by CTIP2. The analysis of conditionally null mice, in which expression of CTIP2 was ablated specifically in epidermal keratinocytes, suggests that CTIP2 functions in both cell and non-cell autonomous contexts to exert regulatory influence over multiple phases of skin development, including barrier establishment. Considered together, our results suggest that CTIP2 functions as a top-level regulator of skin morphogenesis.

Neuroblastoma (NB) is an extra-cranial solid cancer arising from the neural crest and is among the most common cancers in infants less than 1 year of age (Park, JR et al., 2008). Approximately one child per 100,000 is diagnosed with NB, resulting in 650 new cases each year in the United States. Half of the children with NB have high risk disease and 20-50% of those will fail to respond adequately to current therapies, illustrating a clear unmet medical need. Current treatment for high-risk disease is aggressive, including chemotherapy, surgery, radiation with stem cell transplant, anti-GD2/cytokine immunotherapy and retinoic acid (RA) (Yang, RK et. al., 2010; Cheung, NK et. al., 2012). RA is a pro-differentiation agent that slows growth and promotes cell death. A gene expression pattern associated with RA-induced NB differentiation was identified (Hahn, CK et. al., 2008; Frumm, SM et. al., 2013), and inhibition of HDAC1/2 was shown to induce a similar expression pattern. In this work, we examine the activity of an orally bioavailable HDAC1/2 inhibitor (HDAC1/2i) on NB cell differentiation, proliferation and apoptosis. RA combined with HDAC1/2i enhances gene expression patterns associated with differentiation, slows cellular proliferation and more rapidly induces dendrite formation than RA can achieve alone. The mechanisms leading to the differentiated phenotype were examined by microarray and retinoic acid receptor (RAR) ChIP-seq. HDAC1/2i and RA together caused increased localization of the RAR to its own RARa and RARß promoter regions, and an increase in mRNA and protein relative to the RA treatment condition alone. Additionally, expression of Cyp26a1/b1, enzymes responsible for clearing intercellular RA, were reduced in the combination setting. Gene set enrichment analysis of the microarray data comparing the combination setting against RA as a single agent suggested that the addition of HDAC1/2i was enhancing apoptotic pathways and decreasing E2F driven cell cycle signaling. In further experiments, we confirmed enhanced apoptosis in the combination setting by measuring caspase 3 and PARP cleavage, which is consistent with reduced proliferation, increased sub-G1 cell frequency in cell cycle assays and ablation of emergent RA-resistant NB colonies. Further, the E2F-activators, CDK4 and CDK6, were reduced at the protein level in the combination setting while the CDK inhibitor, p21, was dramatically increased. Hypo-phosphorylation of retinoblastoma protein, directly linked to E2F complex inactivation, was also observed and consistent with reduced proliferation and the decreased frequency of S-phase cells observed in EDU incorporation assays. Xenograft models of NB with RA and HDAC1/2i are in progress and will be discussed. Taken together, these findings support a role for selective HDAC1/2i in combination with RA for the treatment of patients with high risk NB. Citation Format: David L. Tamang, Pengyu Haung, Olga Golonzhka, Jeff Shearstone, Steven N. Quayle, Simon S. Jones, Min Yang. A novel, orally bioavailable and selective histone deacetylase (HDAC) 1 & 2 inhibitor enhances retinoic acid mediated differentiation of neuroblastoma. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A136.

Neurotoxic side-effects of chemotherapy, including pain and numbness in hands and feet, frequently leads to dose reduction. This chemotherapy-induced peripheral neuropathy (CIPN) often persists long into survivorship and negatively affects quality of life. No drugs are available to prevent or treat CIPN and underlying mechanisms are only partially understood. Animal models for investigating chemotherapy-induced numbness are lacking. We have developed a tool for measuring numbness in mice; the adhesive removal task (ART). This task measures the time it takes an animal to show a behavioral response to an adhesive patch on its paw. We show that cisplatin induces mechanical hyperalgesia and impairs performance in the ART without altering general locomotor activity or motor coordination. Here we investigated the capacity of a specific histone deacetylase 6 (HDAC6) inhibitor to treat cisplatin-induced numbness and pain in mice. We show that the HDAC6 inhibitor, ACY-1083, effectively treats cisplatin-induced numbness. ACY-1083 also reverses existing cisplatin-induced mechanical hyperalgesia. ACY-1083 alone did not affect sensitivity to mechanical stimulation or performance in the ART. Analysis of cultured dorsal root ganglia revealed that cisplatin treatment decreased mitochondrial axonal transport, while co-culture with ACY-1083 attenuated this decrease. This is important because HDAC6 acts by deacetylating alpha-tubulin, which is thought to promote axonal transport. To our knowledge this is the first identified drug capable of treating already existing chemotherapy-induced numbness and pain.

Neuroblastoma is an extra-cranial solid cancer arising from the neural crest and is among the most common cancers in infants less than 1 year of age (Park, JR et al., 2008). Approximately one child per 100,000 is diagnosed with neuroblastoma, resulting in 650 new cases each year in the United States. Half of the children with neuroblastoma have high risk disease and 20% - 50% of those children will fail to respond adequately to current therapies, illustrating a clear unmet medical need. Current treatment for high-risk disease is aggressive, including chemotherapy, surgery, radiation with stem cell transplant, anti-GD2/cytokine immunotherapy and retinoic acid (Yang, RK et. al., 2010; Cheung, NK et. al., 2012). Retinoic acid is a pro-differentiation agent that acts on neuroblastoma cells to slow growth and promote cell death. A gene expression pattern associated with retinoic acid induced neuroblastoma differentiation was recently identified (Hahn, CK et. al., 2008; Frumm, SM et. al., 2013), and it was further shown that inhibition of HDAC1/2 was able to induce a similar expression pattern. In this work, we demonstrate that next generation selective and orally bioavailable HDAC1/2 inhibitors can induce gene expression changes in neuroblastoma cells consistent with differentiation. The action of HDAC1/2 inhibitors potently enhances the retinoic acid differentiation effect at sub-optimal concentrations of retinoic acid or HDAC inhibitor, as well as with intermittent (pulse) HDAC1/2 inhibition. Retinoic acid alone and in combination with HDAC1/2 inhibitors is able to slow cell proliferation in long term growth assays and alter morphology in a manner consistent with differentiation. The observed enhancement of differentiation by selective HDAC1/2 inhibitors occurs at concentrations below that required for cell death as evidenced by viability assays and caspase 3/7 activation. Acute toxicity is induced by elevated concentrations of HDAC1/2 inhibitors, and synergy is observed in combination with retinoic acid. Ongoing studies exploring global gene expression changes, ChIP-seq examining retinoic acid receptor and HDAC1/2 chromatin binding, and activity of the selective HDAC1/2 inhibitor in combination with retinoic acid in animal models of neuroblastoma will be discussed. Taken together, these findings support a role for selective HDAC1/2 inhibitors in combination with retinoic acid for the treatment of patients with high risk neuroblastoma. Citation Format: David L. Tamang, Emily Lurier, Olga Golonzhka, Steven N. Quayle, Simon S. Jones, Min Yang. Novel and selective histone deacetylase (HDAC) 1 & 2 inhibitors enhance differentiation of neuroblastoma cells in combination with retinoic acid. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2196. doi:10.1158/1538-7445.AM2015-2196

Induction of HbF is an established therapeutic strategy for the treatment of sickle cell disease, and could also be effective in treating beta-thalassemia. Genetic ablation of HDAC1 or HDAC2, but not HDAC3, results in the induction of the fetal beta-like globin gene (HbG) transcript (Bradner JE, Proc Natl Acad Sci, 2010). We have previously shown that selective chemical inhibitors of HDAC1/2 elicit a dose and time dependent induction of HbG mRNA and HbF protein in cultured human CD34+ bone marrow cells undergoing erythroid differentiation (Shearstone JS, ASH Annual Meeting Abstracts, 2012). In this work, we have utilized our proof of concept molecule ACY-957, a selective inhibitor of HDAC1/2, to discover a novel role for Gata2 in the activation of HbG. To identify genes affected by HDAC1/2 inhibition, CD34+ bone marrow cells undergoing erythroid differentiation were treated with ACY-957 or vehicle, followed by mRNA expression profiling. Among the genes differentially regulated by both pharmacological inhibition and genetic ablation of HDAC1/2 were Bcl11a and Sox6, known HbG repressors, and Gata2, a potential HbG activator. Quantitative real time PCR (QRT-PCR) time course experiments confirmed that ACY-957 treatment leads to a 2-fold and 10-fold decrease in Bcl11A and Sox6, respectively, and an 8-fold increase in Gata2 mRNA. Unlike Bcl11a and Sox6, Gata2 induction by ACY-957 was highly correlated with HbG induction, suggesting a possible role for this transcription factor in the direct activation of HbG. To investigate this possibility, lentiviral infection was utilized to overexpress full length Gata2 transcript in differentiating primary erythroblasts. After 5 days of differentiation, Gata2 overexpression resulted in a 2.5-fold increase in HbG mRNA, while the level of the major adult beta-like globin chain (HbB) mRNA was unaffected. HbG mRNA remained elevated by Gata2 overexpression at day 7 of differentiation, while HbB was reduced by 1.6-fold. Gata2 overexpression appeared to have minimal effect on cell differentiation, as determined by the cell surface markers CD71 and GlycophorinA, a finding consistent with observations in ACY-957 treated cells with elevated Gata2. Furthermore, lentiviral delivery of short hairpin RNA (shRNA) targeting Gata2, attenuated HbG induction by ACY-957. These data suggest that elevated levels of Gata2 resulting from HDAC1/2 inhibition is sufficient to induce HbG at early stages of erythroid cell differentiation. To understand how HDAC1/2 inhibition drives Gata2 activation, chromatin immunoprecipitation coupled with either next generation sequencing (ChIP-seq) or QRT-PCR was performed in ACY-957 and vehicle treated cells. HDAC1 and HDAC2 were present throughout the Gata2 gene body and promoter regions, and HDAC1/2 binding levels were highly correlated, suggesting co-occupancy of these enzymes at this locus. ACY-957 treatment led to elevated histone acetylation at previously described Gata2 gene regulatory regions (Bresnick et. al. 2010, J Biol Chem). Specifically, the -1.8 kb and -2.8 kb regulatory regions showed a 6-fold increase in histone H3K9 and H2BK5 acetylation, while the +9.5 kb and -3.9 kb regions showed a 3-fold increase. The Gata2 protein showed increased binding at these regulatory regions in response to ACY-957 treatment, with a maximum increase of 3-fold at the -1.8 kb region. This finding is consistent with the known positive autoregulation of the Gata2 gene. Taken together, these data suggest that selective inhibition of HDAC1/2 leads to elevated Gata2 through acetylation-induced activation of a positive autoregulatory loop. The tight temporal correlation between Gata2 and HbG activation following HDAC1/2 inhibition argues that Gata2 may affect the beta-globin locus directly. ChIP-seq data across the 70-kb beta-globin locus demonstrated that ACY-957 treatment altered Gata2 binding only at a single region, lying within the promoter for delta globin. This region is suspected in playing a role in switching from fetal to adult globin during development, as naturally occurring deletions of this region are associated with elevated fetal hemoglobin in adults (Sankaran et. al. 2011, NEJM). Whether the change in GATA2 binding to this region is responsible for the increased expression of HbG in cells treated with HDAC1/2-selective inhibitors is under investigation. Disclosures Shearstone: Acetylon Pharmaceuticals, Inc.: Employment, Equity Ownership. Golonzhka:Acetylon Pharmaceuticals, Inc.: Employment, Equity Ownership. Chonkar:Acetylon Pharmaceuticals, Inc.: Employment, Equity Ownership. Jarpe:Acetylon Pharmaceuticals, Inc.: Employment, Equity Ownership.