Histone Deacetylase 11 Research Articles

HDAC11 suppresses the thermogenic program of adipose tissue via BRD2.

Little is known about the biological function of histone deacetylase 11 (HDAC11), which is the lone class IV HDAC. Here, we demonstrate that deletion of HDAC11 in mice stimulates brown adipose tissue (BAT) formation and beiging of white adipose tissue (WAT). Consequently, HDAC11-deficient mice exhibit enhanced thermogenic potential and, in response to high-fat feeding, attenuated obesity, improved insulin sensitivity, and reduced hepatic steatosis. Ex vivo and cell-based assays revealed that HDAC11 catalytic activity suppresses the BAT transcriptional program, in both the basal state and in response to β-adrenergic receptor signaling, through a mechanism that is dependent on physical association with BRD2, a bromodomain and extraterminal (BET) acetyl-histone-binding protein. These findings define an epigenetic pathway for the regulation of energy homeostasis and suggest the potential for HDAC11-selective inhibitors for the treatment of obesity and diabetes.

HDAC11 deletion reduces fructose-induced cardiac dyslipidemia, apoptosis and inflammation by attenuating oxidative stress injury

Diabetes mellitus (DM) is a risk factor for abnormal heart development, but the molecular mechanism remains obscure. Histone deacetylase 11 (HDAC11), the most recently identified histone deacetylase, is the sole member of class IV HDACs. However, its role in diabetic cardiac injury is still poorly understood. In the present study, we attempted to explore the effects of HDAC11 on fructose (Fru)-induced cardiac injury using the wild type (HDAC11+/+) and knockout (HDAC11−/−) mice. The results indicated that HDAC11 was significantly expressed in human and mouse diabetic heart failure (DHF) hearts. HDAC11−/− reduced the body weight, inguinal fat-pad mass, and elevated blood pressure in Fru-fed mice. Compared to HDAC11+/+/Fru group, cardiac function was significantly improved in HDAC11−/−/Fru mice. HDAC11−/−/Fru mice exhibited reduced cardiac triacylglycerol (TG), total cholesterol (TC) and free fatty acid (FFA) levels, along with decreased mRNA levels of lipid synthesis-, lipid storage- and lipid oxidation-associated genes. In addition, HDAC11−/− attenuated apoptosis, oxidative stress and inflammation in the heart of Fru-fed mice, as evidenced by the reduced cleavage of Caspase-3, nicotinamide adenine dinucleotide phosphate (NADPH), and xanthine oxidase (XOD) activity, enhanced superoxide dismutase (SOD) activity, as well as the decreased interleukin 1β (IL-1β) and tumor necrosis factor-α (TNF-α) levels, which was accompanied with down-regulated p-NF-κB. The results above were verified in Fru-treated primary cardiomyocytes isolated from HDAC11+/+ or HDAC11−/− mice. Intriguingly, suppressing the expressions of anti-oxidants using zinc protoporphyrin (ZnPP) or siNrf-2 siRNA markedly abolished the results that HDAC11 suppression-induced reduction of apoptosis, reactive oxygen species (ROS) production, inflammation, as well as the improvement of dyslipidemia in Fru-incubated primary cardiomyocytes. Thus, ROS production was responsible for HDAC11-modulated diabetic heart injury. These findings suggested that suppressing HDAC11 has therapeutic potential for treating diabetes mellitus-associated cardiac injury.

Histone Deacetylase 11 Is a Fatty-Acid Deacylase

Histone deacetylase 11 (HDAC11) is a sole member of the class IV HDAC subfamily with negligible intrinsic deacetylation activity. Here, we report in vitro profiling of HDAC11 deacylase activities, and our data unequivocally show that the enzyme efficiently removes acyl moieties spanning 8-18 carbons from the side chain nitrogen of the lysine residue of a peptidic substrate. Additionally, N-linked lipoic acid and biotin are removed by the enzyme, although with lower efficacy. Catalytic efficiencies toward dodecanoylated and myristoylated peptides were 77 700 and 149 000 M-1 s-1, respectively, making HDAC11 the most proficient fatty-acid deacylase of the HDAC family. Interestingly, HDAC11 is strongly inhibited by free myristic, palmitic, and stearic acids with inhibition constants of 6.5, 0.9, and 1.6 μM, respectively. At the same time, its deacylase activity is stimulated more than 2.5-fold by both palmitoyl-coenzyme A and myristoyl-coenzyme A, pointing toward metabolic control of the enzymatic activity by fatty-acid metabolites. Our data reveal novel enzymatic activity of HDAC11 that can, in turn, facilitate the uncovering of additional biological functions of the enzyme as well as the design of isoform-specific HDAC inhibitors.

Disruption to schizophrenia-associated gene Fez1 in the hippocampus of HDAC11 knockout mice

Histone Deacetylase 11 (HDAC11) is highly expressed in the central nervous system where it has been reported to have roles in neural differentiation. In contrast with previous studies showing nuclear and cytoplasmic localisation, we observed synaptic enrichment of HDAC11. Knockout mouse models for HDACs 1–9 have been important for guiding the development of isoform specific HDAC inhibitors as effective therapeutics. Given the close relationship between HDAC11 and neural cells in vitro, we examined neural tissue in a previously uncharacterised Hdac11 knockout mouse (Hdac11KO/KO). Loss of HDAC11 had no obvious impact on brain morphology and neural stem/precursor cells isolated from Hdac11KO/KO mice had comparable proliferation and differentiation characteristics. However, in differentiating neural cells we observed decreased expression of schizophrenia-associated gene Fez1 (fasciculation and elongation protein zeta 1), a gene previously reported to be regulated by HDAC11 activity. FEZ1 has been associated with the dendritic growth of neurons and risk of schizophrenia via its interaction with DISC1 (disrupted in schizophrenia 1). Examination of cortical, cerebellar and hippocampal tissue reveal decreased Fez1 expression specifically in the hippocampus of adult mice. The results of this study demonstrate that loss of HDAC11 has age dependent and brain-region specific consequences.

T cells lacking HDAC11 have increased effector functions and mediate enhanced alloreactivity in a murine model

AbstractHistone acetylation and the families of enzymes responsible for controlling these epigenetic marks have been implicated in regulating T-cell maturation and phenotype. Here, we demonstrate a previously undefined role of histone deacetylase 11 (HDAC11) in regulating T-cell effector functions. Using EGFP-HDAC11 transgenic reporter mice, we found that HDAC11 expression was lower in effector relative to naive and central memory T-cell populations, and activation of resting T cells resulted in its decreased expression. Experiments using HDAC11 knockout (KO) mice revealed that T cells from these mice displayed enhanced proliferation, proinflammatory cytokine production, and effector molecule expression. In addition, HDAC11KO T cells had increased expression of Eomesodermin (Eomes) and TBX21 (Tbet), transcription factors previously shown to regulate inflammatory cytokine and effector molecule production. Conversely, overexpression of HDAC11 resulted in decreased expression of these genes. Chromatin immunoprecipitation showed the presence of HDAC11 at the Eomes and Tbet gene promoters in resting T cells, where it rapidly disassociated following T-cell activation. In vivo, HDAC11KO T cells were refractory to tolerance induction. HDAC11KO T cells also mediated accelerated onset of acute graft-versus-host disease (GVHD) in a murine model, characterized by increased proliferation of T cells and expression of interferon-γ, tumor necrosis factor, and EOMES. In addition, adoptive transfer of HDAC11KO T cells resulted in significantly reduced tumor burden in a murine B-cell lymphoma model. Taken together, these data demonstrate a previously unknown role of HDAC11 as a negative epigenetic regulator of T-cell effector phenotype and function.

Essential role for histone deacetylase 11 (HDAC11) in neutrophil biology

Epigenetic changes in chromatin structure have been recently associated with the deregulated expression of critical genes in normal and malignant processes. HDAC11, the newest member of the HDAC family of enzymes, functions as a negative regulator of IL-10 expression in APCs, as previously described by our lab. However, at the present time, its role in other hematopoietic cells, specifically in neutrophils, has not been fully explored. In this report, for the first time, we present a novel physiologic role for HDAC11 as a multifaceted regulator of neutrophils. Thus far, we have been able to demonstrate a lineage-restricted overexpression of HDAC11 in neutrophils and committed neutrophil precursors (promyelocytes). Additionally, we show that HDAC11 appears to associate with the transcription machinery, possibly regulating the expression of inflammatory and migratory genes in neutrophils. Given the prevalence of neutrophils in the peripheral circulation and their central role in the first line of defense, our results highlight a unique and novel role for HDAC11. With the consideration of the emergence of new, selective HDAC11 inhibitors, we believe that our findings will have significant implications in a wide range of diseases spanning malignancies, autoimmunity, and inflammation.

Vitamin D3 induces vitamin D receptor and HDAC11 binding to relieve the promoter of the tight junction proteins.

Intestinal epithelial barrier dysfunction and vitamin D (VitD)-deficiency play a critical role in a large number of diseases. The histone deacetylases (HDAC) are associated with a large number of immune diseases. This study tests a hypothesis that the interaction between VitD and HDAC is associated with the regulation of epithelial barrier functions. In this study, human intestinal epithelial cell line, T84 cells, was cultured into monolayers to be used as a model to test the epithelial barrier functions. We observed that in a VitD-deficient environment, the T84 monolayer barrier function was compromised. Exposure to calcitriol (the active form of VitD3) in the culture increased the expression of VitD receptor (VDR) in T84 cells. In a VitD-sufficient environment, VDR formed a complex with histone deacetylase-11 (HDAC11); the complex was markedly decreased in a VitD-deficient environment. We also observed that significantly more binding of HDAC11 to the promoter of the tight junction proteins inhibit the gene transcription activities of these loci in the VitD-deficient environment, which were abolished by the presence of calcitriol in the culture. In conclusion, the interaction between VDR and HDAC11 plays a crucial role in the maintenance of the epithelial barrier integrity.

Histone Deacetylase 11 (HDAC11) Interaction with Ikaros Represent a Novel Mechanism of Regulation of Essential Transcriptional Factors in CD4+ T Cells

Histone deacetylase 11 (HDAC11), the most recently identified histone deacetylase, is the sole member of class IV HDACs [1]. Since its discovery, no biological function was assigned to this HDAC until we demonstrated its central role in negatively regulating IL-10 production in antigen presenting cells (APCs) [2]. More recently, we have found that disruption of HDAC11 in T cells is associated with an enhanced pro-inflammatory cytokine profile and effector molecule production. Furthermore, T-cells lacking HDAC11 were less susceptible to regulatory T-cell (Treg) suppression in vitro, were refractory to tolerance induction in vivo and displayed enhanced allo-reactivity and anti-tumor responses in murine models. Of note, T-cells lacking HDAC11 expressed higher levels of the transcription factors Eomes and Tbet. Conversely, overexpression of HDAC11 in T-cells decreased the expression of both transcription factors. The molecular mechanism(s) by which HDAC11 regulates the expression of these transcription factors have remained unknown.By using chromatin immunoprecipitation (ChIP) assay we found that in resting T-cells HDAC11 is present at the Eomes and Tbet gene promoters where it maintains histone deacetylation, a compacted chromatin and gene repression. Following T-cell stimulation, HDAC11 was largely absent from both promoters, which resulted in increased histone 3 (H3) acetylation and gene transcriptional activity. These findings were confirmed in T-cells isolated from HDAC11 knock out (KO) mice which also displayed an increase in H3 acetylation at the Tbet and Eomes gene promoter regions. Conversely, H3 acetylation was decreased in both gene promoters in T-cells overexpressing HDAC11 as compared to empty-vector transfected cells. Given that HDACs do not bind to DNA, we asked next which transcription factor(s) HDAC11 might be associated with, in order to regulate Tbet and Eomes gene transcriptional activity.In prior studies we have found that HDAC11 form a molecular complex with another member of the HDAC family, HDAC6, which physically interacts with the transcription factor, STAT3 in both the cytoplasmic and nuclear compartments. However, in T-cells no direct interaction of HDAC11 with STAT3 was detected in either compartment. In contrast, we found for the first time that HDAC11 physically associates with Ikaros (Ikzf1), a member of the Ikaros zinc finger transcription factor family that has been previously implicated in the regulation of T-bet gene expression and IFN-g production in T-cells [3-5]. The protein complex HDAC11-Ikaros was mainly detected in the nuclear compartment and both proteins were present at the T-bet gene promoter. Collectively, these results point to the HDAC11-Ikaros complex as a novel epigenetic mechanism of regulation of Tbet and Eomes, transcription factors that are essential for T cell development and function. DisclosuresWoods:BMS: Other: Stock; HDAC11: Patents & Royalties: Patent for targeting HDAC11; Lion Biotech: Other: Stock.

Effects of Sodium Butyrate Treatment on Histone Modifications and the Expression of Genes Related to Epigenetic Regulatory Mechanisms and Immune Response in European Sea Bass (Dicentrarchus Labrax) Fed a Plant-Based Diet.

Bacteria that inhabit the epithelium of the animals’ digestive tract provide the essential biochemical pathways for fermenting otherwise indigestible dietary fibers, leading to the production of short-chain fatty acids (SCFAs). Of the major SCFAs, butyrate has received particular attention due to its numerous positive effects on the health of the intestinal tract and peripheral tissues. The mechanisms of action of this four-carbon chain organic acid are different; many of these are related to its potent regulatory effect on gene expression since butyrate is a histone deacetylase inhibitor that play a predominant role in the epigenetic regulation of gene expression and cell function. In the present work, we investigated in the European sea bass (Dicentrarchus labrax) the effects of butyrate used as a feed additive on fish epigenetics as well as its regulatory role in mucosal protection and immune homeostasis through impact on gene expression. Seven target genes related to inflammatory response and reinforcement of the epithelial defense barrier [tnfα (tumor necrosis factor alpha) il1β, (interleukin 1beta), il-6, il-8, il-10, and muc2 (mucin 2)] and five target genes related to epigenetic modifications [dicer1(double-stranded RNA-specific endoribonuclease), ehmt2 (euchromatic histone-lysine-N-methyltransferase 2), pcgf2 (polycomb group ring finger 2), hdac11 (histone deacetylase-11), and jarid2a (jumonji)] were analyzed in fish intestine and liver. We also investigated the effect of dietary butyrate supplementation on histone acetylation, by performing an immunoblotting analysis on liver core histone extracts. Results of the eight-week-long feeding trial showed no significant differences in weight gain or SGR (specific growth rate) of sea bass that received 0.2% sodium butyrate supplementation in the diet in comparison to control fish that received a diet without Na-butyrate. Dietary butyrate led to a twofold increase in the acetylation level of histone H4 at lysine 8, but showed no effect on the histone H3 at Lys9. Moreover, two different isoforms of histone H3 that might correspond to the H3.1 and H3.2 isoforms previously found in terrestrial animals were separated on the immunoblots. The expression of four (il1 β, il8, irf1, and tnfα) out of seven analyzed genes related to mucosal protection and inflammatory response was significantly different between the two analyzed tissues but only il10 showed differences in expression due to the interaction between tissue and butyrate treatment. In addition, butyrate caused significant changes in vivo in the expression of genes related to epigenetic regulatory mechanisms such as hdac11, ehmt2, and dicer1. Statistical analysis by two-way ANOVA for these genes showed not only significant differences due to the butyrate treatment, but also due to the interaction between tissue and treatment.

Functional Analysis of Histone Deacetylase 11 (HDAC11).

The physiological role of histone deacetylase 11 (HDAC11), the newest member of the HDAC family, remained largely unknown until the discovery of its regulatory function in immune cells. Among them, the regulation of cytokine production by antigen-presenting cells and the modulation of the suppressive ability of myeloid-derived suppressor cells (MDSCs) (Sahakian et al. Mol Immunol 63: 579-585, 2015; Wang et al. J Immunol 186: 3986-3996, 2011; Villagra et al. Nat Immunol 10: 92-100, 2009). Our earlier data has demonstrated that HDAC11, by interacting at the chromatin level with the IL-10 promoter, downregulates il-10 transcription in both murine and human APCs in vitro and ex vivo models (Villagra et al. Nat Immunol 10: 92-100, 2009). However the role of HDAC11 in other cell types still remains unknown. Here we present several methods that can potentially be used to identify the functional role of HDAC11, assigning special attention to the evaluation of immunological parameters.

Histone deacetylase 11 (HDAC11) is critical for plasma cell development and multiple myeloma survival

Histone deacetylase 11 (HDAC11) is critical for plasma cell development and multiple myeloma survival

A Novel Role for Histone Deacetylase 11 (HDAC11) in B Cell Lymphopoiesis and Plasma Cell Survival in Multiple Myeloma

While multiple myeloma (MM) remains incurable presently, expanded therapeutic options over the past decade have improved patient survival markedly. Proteasome inhibitors have redefined the treatment paradigm for myeloma, often serving as the backbone of front-line treatment. Histone deacetylase (HDAC) inhibitors (HDI), although only marginally active as single agent therapy in hematological malignancies, have demonstrated an ability to salvage bortezomib responsiveness in refractory patients, prompting heightened interest in this class of targeted therapeutics in myeloma. HDAC’s represent a family of enzymes, currently with 11 known members in the classical HDAC family, and subdivided into 4 sub-classes. HDAC11 is currently the only member of the sub-class IV and, as the newest member of the HDAC family, its impact on B cell lymphopoiesis and myeloma development is only starting to be unveiled. Intriguingly, we show that mice with germ-line silencing of HDAC11 (HDAC11KO mice) exhibit a 50% decrease in plasma cells in both the bone marrow and peripheral blood plasma cell compartments relative to wild-type mice. Consistent with this, Tg-HDAC11-eGFP mice, a transgenic strain engineered to express GFP under control of the HDAC11 promoter (Heinz, N Nat. Rev. Neuroscience 2001) reveals that HDAC11 expression is increased in the plasma cell population and to a lesser extent B1 B cells, as compared to earlier lineage stages. Similar observations based on measurements of HDAC11 mRNA were seen in normal human plasma cells. Significant increases in HDAC11 mRNA expression were observed in 7 of 11 primary human multiple myeloma samples and 11 of 12 human myeloma cell lines as compared to normal plasma cells, further emphasizing the potential relevance of HDAC11 to the underlying pathologic processes driving myeloma development and/or survival. Targeted silencing of HDAC11 in RPMI-8226 cells lines using siRNA results in a modest decrease in cell viability as measured by Annexin/PI staining and detection of activated caspase-3. Quisinostat, a second generation pan-HDI, has previously demonstrated activity against human myeloma cell lines in vitro (Stuhmer, Brit J Haematol, 2010), and suppressed bone destruction in an in vivo murine myeloma model (Deleu, Cancer Res, 2009). We similarly observe dose-dependent survival impairment in 10 human myeloma cell lines when cultured in the presence of quisinostat, with EC50’s consistently in the 1-10nM range. Importantly, quisinostat acts synergistically with proteasome inhibitiors (bortezomib and carfilzomib) in RPMI-8226 cells; more importantly, the degree of synergism is amplified in the RPMI-6226-B25 bortezomib-resistant cell line. Although a clear mechanism of action remains to be elucidated, preliminary data suggests that RPMI-8226 cells exposed to quisinostat appear to exhibit a decrease nuclear, but not cytosolic HDAC11. Collectively, these data illustrate a previously unknown role for HDAC11 in plasma cell differentiation and survival. Increased HDAC11 expression seen in myeloma patient specimens and primary myeloma cell lines highlights the potential of HDAC11 as a therapeutic target. Furthermore, we show that quisinostat, a pan-HDI with selectivity towards HDAC11 at lower dosing, acts synergistically with proteasome inhibitors in vitro in proteasome inhibitor sensitive and resistant cell lines. Future work will focus on further elucidating the role of HDAC11 in myeloma survival and drug response, with particular emphasis on proteasome inhibitors. DisclosuresNo relevant conflicts of interest to declare.

Histone Deacetylase 11 (HDAC11) As a Novel Transcriptional Regulator of C/EBP-β, in Immature Myeloid Cell to Myeloid Derived Suppressor Cell Transition

In normal myelopoiesis, immature myeloid cells (IMCs) differentiate into macrophages, dendritic cells, and neutrophils. However, in pathological conditions such as in cancer, these immature cells differentiate into myeloid derived suppressor cells (MDSCs). Transcription factor CCAAT-enhancer-binding protein beta (C/EBP-β) plays a crucial role in the accumulation of MDSCs in several pathological conditions. Of note, mice lacking C/EBP-β in the bone marrow compartment lose the ability to differentiate IMCs into pathologically active MDSCs2.In contrast, up-regulation of C/EBP-β can partly induce MDSC expansion through a mechanism involving the STAT3 pathway3.Epigenetic changes, such as histone acetylation status, have been recently shown to modulate the regulatory function of MDSCs4. However, the epigenetic mechanism(s) involved in regulation of C/EBP-β gene expression in MDSCs are poorly understood. Here we show for the first time that among all the HDACs known, HDAC11, the newest member of this family of enzymes, is recruited to the C/EBP-β gene promoter region of primary myeloid cells isolated from C57BL/6 mice. Such a finding led us to explore the functional consequences of disrupting HDAC11 in myeloid cells. First, we observed a markedly higher expression of C/EBP-β mRNA (194 fold difference) in the CD11b+/Ly6G+ granulocytic compartment and a 6.7 fold difference in the CD11b+/Ly6C+monocytic compartments of HDAC11KO mice relative to control wild type mice. Second, MDSCs from HDAC11 KO mice display a higher suppressive phenotype and mechanistically we have found that they have increased expression of the immunosuppressive STAT3/IL10 axis. Third, inoculation of EL4 tumor cells into HDAC11KO mice and controls resulted in a higher expansion capacity of splenic MDSCs in mice devoid of HDAC11. Such an effect was associated with a more rapid tumor growth in HDAC11KO mice as compared to WT mice. Similar aggressive tumor growth was also observed in HDAC11 myeloid conditional KO mice (LyZ-Cre-HDAC11KO).Taken together, we have uncovered a previously unknown role for HDAC11 as a transcriptional regulator of C/EBP-β in MDSCs. Indeed, in the absence of this epigenetic checkpoint of C/EBP-β gene expression, the fully suppressive potential of MDSCs was fully unleashed. A better understanding of this novel role of HDAC11 in myeloid biology will ultimately lead to targeted epigenetic therapies to manipulate the suppressive abilities of these immunoregulatory cells.1. Hirai H, Zhang P, Dayaram T, et al. C/EBPbeta is required for 'emergency’ granulopoiesis. Nat Immunol 2006; 7(7): 732-9.2. Marigo I, Bosio E, Solito S, et al. Tumor-induced tolerance and immune suppression depend on the C/EBPbeta transcription factor. Immunity 2010; 32(6): 790-802.3. Condamine T, Gabrilovich DI. Molecular mechanisms regulating myeloid-derived suppressor cell differentiation and function. Trends Immunol 2011; 32(1): 19-25.4. Youn JI, Kumar V, Collazo M, et al. Epigenetic silencing of retinoblastoma gene regulates pathologic differentiation of myeloid cells in cancer. Nat Immunol 2013; 14(3): 211-20. DisclosuresNo relevant conflicts of interest to declare.

Abstract 5537: Histone deacetylase 11 (HDAC11) regulates B cell lymphopoiesis and potentiates plasma cell survival in multiple myeloma

Abstract Initially studied mainly for its role as a regulator of neural cell differentiation and development, expression of HDAC11 was once thought to be restricted exclusively to brain, kidney and testes. Hence, our recent discovery that HDAC11 acts as an important modulator of antigen presentation and T cell activation, downregulating IL-10 transcription via interactions with the IL-10 promoter at the chromatin level, exposes a previously unknown capacity and tissue specificity for this enzyme. Transgenic mice harboring an eGFP reporter construct driven by the HDAC11 promoter (Tg-HDAC11-eGFP) (Heinz, N Nat. Rev. Neuroscience 2001) clearly illustrate the dynamic changes in HDAC11 gene expression in hematopoietic cell lineages, additionally unveiling an important role for HDAC11 in B cell lymphopoiesis and plasma cell biology. While common lymphoid progenitors appear to be devoid of HDAC11 transcriptional activation as indicated by minimal detectable eGFP expression, eGFP intensity markedly increases in the B-1 stage of differentiation in the periphery. Interestingly, examination of both the bone marrow (BM) and peripheral blood (PB) plasma cell compartment demonstrates an increase in expression of eGFP/HDAC11 mRNA at the steady-state, and these results are consistent with HDAC11 expression measured in PB from healthy human subjects. Furthermore, mice globally deficient in HDAC11 expression (HDAC11KO mice) exhibit a 50% decrease in plasma cells in both the bone marrow and peripheral blood plasma cell compartments relative to wild-type mice. The concordance of HDAC11 expression and plasma cell differentiation leads us to hypothesize that HDAC11 may also be critical to malignant plasma cell survival. A comparison of normal bone marrow and malignant plasma cells isolated from multiple myeloma patient samples reveals a significantly higher level of HDAC11 expression associated with malignancy. Similar results are observed in 8 of 12 myeloma cell lines suggesting that HDAC11 expression may provide a distinct survival advantage to malignant plasma cells. Further stratification of patients into “newly diagnosed” and “proteasome inhibitor resistant” categories defines a positive correlation between HDAC11 expression and refractory disease. Treatment of the myeloma cell lines with Quisinostat, a second-generation HDAC inhibitor with enhanced selectivity for HDAC 1, 2, 4, 10 and 11 induces growth retardation at low nanomolar concentrations. Future studies will entail direct targeting of HDAC11 in myeloma cell lines and patient specimens to determine the contribution of HDAC11 to Quinsinostat activity. Taken together, we have unveiled a previously unknown role for HDAC11 in plasma cell differentiation and survival. The demonstration of HDAC11 overexpression in primary human myeloma cells provides a framework for therapeutics targeting this HDAC in multiple myeloma. Citation Format: Eva Sahakian, Jason Brayer, John Powers, Mark Meads, Susan Deng, Allison Distler, Melissa Alsina, Taiga Nishihori, Rachid Baz, Alejandro Villagra, Javier Pinilla-Ibarz, Eduardo Sotomayor, Kenneth Shain. Histone deacetylase 11 (HDAC11) regulates B cell lymphopoiesis and potentiates plasma cell survival in multiple myeloma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5537. doi:10.1158/1538-7445.AM2014-5537

Divergent roles of histone deacetylase 6 (HDAC6) and histone deacetylase 11 (HDAC11) on the transcriptional regulation of IL10 in antigen presenting cells

The anti-inflammatory cytokine IL-10 is a key modulator of immune responses. A better understanding of the regulation of this cytokine offers the possibility of tipping the balance of the immune response toward either tolerance, or enhanced immune responses. Histone deacetylases (HDACs) have been widely described as negative regulators of transcriptional regulation, and in this context, the primarily nuclear protein HDAC11 was shown to repress il-10 gene transcriptional activity in antigen-presenting cells (APCs). Here we report that another HDAC, HDAC6, primarily a cytoplasmic protein, associates with HDAC11 and modulates the expression of IL-10 as a transcriptional activator. To our knowledge, this is the first demonstration of two different HDACs being recruited to the same gene promoter to dictate divergent transcriptional responses. This dynamic interaction results in dynamic changes in the expression of IL-10 and might help to explain the intrinsic plasticity of the APC to determine T-cell activation versus T-cell tolerance.

A Novel Role For Histone Deacetylase 11 (HDAC11) In Plasma Cell Differentation and Survival

The role of HDACs in cellular biology, initially limited to their effects upon histones, is now appreciated to encompass more complex regulatory functions that are dependent on their tissue expression, cellular compartment distribution, and the stage of cellular differentiation. Recently, our group has demonstrated that the newest member of the HDAC family of enzymes, HDAC11, is an important regulator of IL-10 gene expression in myeloid cells (Villagra A Nat Immunol. 2009). The role of this specific HDAC in B-cell development and differentiation is however unknown. To answer this question, we have utilized a HDAC11 promoter-driven eGFP reporter transgenic mice (TgHDAC11-eGFP) which allows the monitoring of the dynamic changes in HDAC11 gene expression/promoter activity in B-cells at different maturation stages (Heinz, N Nat. Rev. Neuroscience 2001). First, common lymphoid progenitors are devoid of HDAC11 transcriptional activation as indicated by eGFP expression. In the bone marrow, expression of eGFP moderately increases in Pro-B-cells and transitions to the Pre- and Immature B-cells respectively. Expression of eGFP doubles in the B-1 stage of differentiation in the periphery. Of note, examination of both the bone marrow and peripheral blood plasma cell compartment demonstrated increased expression of eGFP/HDAC11 mRNA at the steady-state. These results were confirmed in plasma cells isolated from normal human subjects in which HDAC11 mRNA expression was demonstrated. Strikingly, analysis of primary human multiple myeloma cells demonstrated a significantly higher HDAC11 mRNA expression in malignant cells as compared to normal plasma cells. Similar results were observed in 4/5 myeloma cell lines suggesting that perhaps HDAC11 expression might provide survival advantage to malignant plasma cells. Support to this hypothesis was further provided by studies in HDAC11KO mice in which we observed a 50% decrease in plasma cells in both the bone marrow and peripheral blood plasma cell compartments relative to wild-type mice. Taken together, we have unveiled a previously unknown role for HDAC11 in plasma cell differentiation and survival. The additional demonstration that HDAC11 is overexpressed in primary human myeloma cells provide the framework for specifically targeting this HDAC in multiple myeloma. Disclosures:Alsina:Millennium: Membership on an entity's Board of Directors or advisory committees, Research Funding. Baz:Celgene Corporation: Research Funding; Millenium: Research Funding; Bristol Myers Squibb: Research Funding; Novartis: Research Funding; Karyopharm: Research Funding; Sanofi: Research Funding.

A Novel Role For Histone Deacetylase 11 (HDAC11) As a Regulator Of Neutrophil Function and Differentiation In Normal and Malignant Hematopoesis

Histone Deacetylase 11 (HDAC11) is the newest member of the HDAC family of enzymes, which we have previously reported to function as a negative regulator of IL-10 expression in macrophages and dendritic cells. Thus far, its role in other hematopoietic cells has not been completely elucidated. We hereby report for the first time a lineage-restricted over-expression of HDAC11 in neutrophils, committed neutrophil precursors and myeloid leukemias exhibiting neutrophilic differentiation demonstrating a novel physiological role of HDAC11 as a negative regulator of neutrophil cytokine production.Leukocyte subpopulations from murine bone marrow and spleen were flow-sorted and analyzed by qRT-PCR for HDAC11 mRNA, revealing a higher level of mRNA expression on neutrophils and promyelocytes, as compared to monocytes and lymphoid subsets. Similarly, sorted human peripheral blood leukocytes from normal donors, showed higher levels of HDAC11 mRNA in neutrophils, as compared to monocytes. To further investigate the transcriptional activity of HDAC11 in myeloid and lymphoid cells, we utilized a HDAC11 promoter-driven eGFP reporter mice, where eGFP expression indicates HDAC11 transcription (Heintz, N Nat. Rev. Neuroscience 2001). Using multiparametric flow cytometry with lineage-specific markers on this mouse model, we confirmed a marked over-expression of HDAC11 on neutrophils, compared to other subpopulations including monocytes, B-cell, T-cells, NK cells and plasma cells. Furthermore, analysis of bone marrow hematopoietic cells revealed a swift over-expression of HDAC11 at the promyelocyte stage of neutrophil differentiation, with low to undetectable expression in upstream uncommitted common myeloid progenitors and lineage-unrelated monocytic precursors. To study whether this lineage-specific overexpression applies to malignant processes, we studied human cell lines and found overt overexpression of HDAC11 in the acute promyelocytic leukemia cell line NB4, as compared to the myeloblastic cell line Kasumi and two monocyte/macrophage cell lines U937 and THP1. Moreover, in-vitro maturation of the differentiation-inducible myeloid cell line HL60 demonstrated a marked increase in HDAC11 mRNA, paralleling the acquisition of nuclear segmentation characteristic of neutrophil maturation. In order to investigate the physiologic role of HDAC11 overexpression on neutrophils, we utilized a model of germline-HDAC11KO mice. Surprisingly, highly purified neutrophils lacking HDAC11 showed an overt overproduction of TNF-alpha and IL-6 upon stimulation with LPS, as compared to their wild type counterparts.We hereby report a previously un-described lineage-specific over-expression of HDAC11 in neutrophils and its precursors, which actively functions as a physiological repressor of cytokine production and possibly involved in their regulation. Given the predominance of neutrophils which account for 70% of leukocytes in the peripheral blood, and their pivotal role in the first line of defense, results highlight a novel mechanism for HDAC11, as a key regulator and modulator of neutrophil cytokine production with potential implications for autoimmunity, inflammation, and infection. Disclosures:No relevant conflicts of interest to declare.

Beware the low HDAC11: males at risk for ischemic kidney injury

plasminogen activator inhibitor type 1 (PAI-1) is a member of the serine protease inhibitor (serpin) family. As such, it is well known as a physiological inhibitor of fibrinolysis, but PAI-1 also plays a key role in inflammation and production of extracellular matrix proteins (reviewed in Ref. 1). PAI-1 is implicated in the pathology of fibrosis in the heart, lung, kidney, liver, and skin. In pathologic states, excessive PAI-1 contributes to accumulation of collagen and other extracellular matrix proteins, while lack of PAI-1 protects from fibrosis in response to injury-related profibrotic signals. Involvement of PAI-1 has been implicated in disease states that include insulin resistance, diabetes, cardiovascular disease, kidney fibrosis in unilateral ureteral obstruction, and aldosterone-induced glomerular injury. Regulation of PAI-1 levels is complicated and occurs through transcriptional, posttranscriptional, and posttranslational mechanisms (reviewed in Ref. 1). Multiple cytokines and growth factors, including transforming growth factor (TGF)-β, epidermal growth factor, and insulin, and endotoxin and oxidative stress impact local PAI-1 production through these mechanisms. Thus PAI-1 may have a role in outcomes in chronic kidney disease and especially acute kidney injury. In an issue of the American Journal of Physiology-Renal Physiol, Kim et al. (4) examined the effect of both PAI-1 expression and gender in outcome of kidney ischemia/reperfusion (I/R)-induced injury in mice. Male gender and male hormone testosterone increase susceptibility to renal I/R injury (5); mechanisms that underlie these effects were elucidated by Kim et al. (4). They noticed an unimpressive increase in renal PAI-1-encoding gene (Serpine1) expression in females with or without ovariectomy 24 h after a 30-min I/R injury. In contrast, PAI-1-encoding gene expression in orchiectomized males was also low, but administration of dihydrotestosterone (DHT) triggered an increase to levels comparable to those seen in males without orchiectomy. Because I/R injury represents a well-established model of gender dimorphism in acute kidney injury (AKI) outcomes and since pharmacological PAI-1 inhibition attenuated adverse renal I/R effects, the authors then examined the gender-specific mechanisms regulating PAI-1 expression in this model. They observed that renal expression of several histone deacetylase (HDAC)-encoding genes (i.e., Hdac1, 5, 8, 9, and 11) decreased following 30 min of renal I/R injury. However, decreased expression of Hdac9 and Hdac11 did not occur in orchiectomized males exposed to kidney I/R injury. In addition, among the five studied HDAC isotypes, only HDAC11 inhibited PAI-1 expression (using an activated monocyte/macrophage cell line and treatment with HDAC11 small interfering RNA). Based on chromatin immunoprecipitation assay, this inhibition was mediated by direct HDAC11 interaction with the PAI-1 gene (Serpine1) promoter. The I/R-induced release of HDAC11 from PAI-1 promoter then led to increased histone H3 acetylation. This effect was observed only in males without orchiectomy but not in orchiectomized males or intact or ovariectomized females. This observation, coupled with the fact that DHT reversed the effect of orchiectomy on I/R-induced decreased HDAC11 expression, pointed to a testosterone-HDAC11-PAI-1 axis as a likely pathway regulating gender-induced differences in I/R-induced renal injury (Fig. 1). Fig. 1. Schema of the effects of testosterone on plasminogen activator inhibitor type 1 (PAI-1)-enhanced renal injury. Testosterone increases severity of kidney ischemia/reperfusion (I/R)-induced renal injury in mice by inhibiting histone deacetylase 11 (HDAC11). ... Gender is an important regulator of susceptibility to AKI in a wide range of renal insults (reviewed in Ref. 3). These gender-induced differences have been attributed to biological effects of male and female sex hormones. While some of these differences may be explained by testosterone-enhanced susceptibility to endoplasmic reticulum stress (2), additional specific mechanisms underlying the sex hormone effects in AKI have remained elusive. The current study by Kim et al. (4) points to testosterone-induced HDAC11-regulated histone H3 acetylation as a key mechanism responsible for gender-related susceptibility to AKI. This new concept offers a novel avenue for development of gender-based therapeutic strategies, an important component of emerging personalized medical care (7). While such strategies hold potential for reducing adverse outcomes in clinical care (e.g., in males with high risk of AKI), first it is necessary to confirm that the above concept is not murine specific but can be also applied to humans and other mammals (e.g., unlike the outcome of the current study on mice, testosterone protected kidneys from I/R-induced AKI in rats; Ref. 6). In addition, the findings in the paper also raise questions of potentially unpredictable adverse effect of non-selective HDAC inhibitors in AKI. Unfortunately, specific HDAC11 activity-enhancing therapeutics have not been described; their development may hasten potential translation of this discovery to clinical care.

Abstract 1742: Potential roles of histone deacetylase 11 in cell cycle regulation and cancer.

Abstract Histone deacetylases (HDACs) are enzymes that deacetylate lysine residues on histone and non-histone proteins. They regulate various cellular functions including transcriptional regulation, cell proliferation, apoptosis, and cellular metabolism. Broad spectrum HDAC inhibitors, including Vorinostat and Romidepsin, have been approved for cancer therapy, but their use is limited due to toxicity and side effects. A deeper understanding of the function of individual histone deacetylases could lead to the development of more specific and potentially more effective anti-cancer agents. In this context, Histone Deacetylase 11 (HDAC11) could be a potential target for anticancer therapeutics. HDAC11 is the most recently discovered Histone Deacetylase and, therefore, the least characterized among the 18 HDACs. Its expression is altered in various cancers and recent studies have suggested that HDAC11 might inhibit the activity and function of the DNA replication licensing factor Cdt1. To examine whether HDAC11 controls Cdt1-mediated cell cycle regulation, HDAC11 was overexpressed in human U87 glioblastoma cells. We found that overexpression of HDAC11 represses Cdt1 expression levels. Most importantly, we observed that HDAC11 overexpression promotes earlier cell entry into the G2/M phase of the cell cycle than non-HDAC11-overexpressed cells. Finally, we discovered that HDAC11 levels change throughout the cell cycle in a manner that is contrary to the Cdt1 expression levels. These results indicate that HDAC11 negatively regulates Cdt1, and uncover an important and previously unknown role of this enzyme in cell cycle regulation. Together, our findings suggest that development of drugs that target HDAC11 might be a viable strategy for overcoming cancers where this protein is overexpressed. Citation Format: Thejal Srikumar, Elphine Telles, Edward Seto. Potential roles of histone deacetylase 11 in cell cycle regulation and cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1742. doi:10.1158/1538-7445.AM2013-1742

Novel Role of Histone Deacetylase 11 (HDAC11) in Hematopoiesis

AbstractAbstract 4728HDAC11 is the newest member of the HDAC family. The physiological role of this HDAC was mainly unknown until the discovery by our group that HDAC11 regulates IL-10 gene expression in immune cells in-vitro1. To better elucidate the role of HDAC11 in lineage differentiation and hematopoiesis, we have utilized an HDAC11 promoter-driven eGFP reporter transgenic mice (TgHDAC11-eGFP) which allow us to “visualize” dynamic changes in HDAC11 gene expression/transcriptional activity in immune cell compartments in vivo. Thus far, our data indicates that in hematopoietic stem cells (CD34+/Lin−), transcriptional activation of HDAC11, indicated by eGFP expression appears to be absent. Also, no eGFP expression is seen in the common lymphoid progenitors (CLP-CD34+/CD127+/CD117low/Lin−) and/or the common myeloid progenitors (CMP-CD34+/CD127−/CD117high/Lin−). In the T-cell compartment, transcriptional activation of HDAC11 increases from CD4−/CD8− T-cells to CD4+/CD8+ T-cells to single positive CD4+ and CD8+ T-cells. The expression of eGFP then decreases from naive to effector memory, but then increases again at terminal effector memory. Expression of eGFP, in the bone marrow moderately increase transitioning from Pro-B-cells (CD3−/CD200+/CD19low/CD43high), Pre-B-cells (CD3−/CD200+/CD19int/CD43int), and Immature (CD3−/CD200+/CD19high/CD43low) respectively. Interestingly eGFP expression doubles in the B-1 (CD3−/CD19+/CD200low/−) stage of differentiation in the periphery. Remarkably, eGFP expression appears to be at its highest in the plasma cell compartment of the bone marrow. A second murine model also available to us, HDAC11 knockout mice (HDAC11KO) were also utilize to confirm these findings. When compared to wild-type mice, HDAC11KO mice have increased B-1 B-cells and decreased plasma cells. In the myeloid compartment, using TgHDAC11-eGFP mice, expression of HDAC11 transcript in myeloblasts (CD34+/CD45dim/CD117+/Lin-) appears to be absent. However the expression increases to 50% in the promyelocytes (Side Scatter high/CD45dim/+/CD34−/CD117+) and to 98% in the granulocytes specifically Neutrophils (Side scatter high/CD45dim+/CD34−/CD117−/CD14−/Ly6Gbright+). Strikingly, monocytes (dendritic cells and macrophages) showed no expression of eGFP.Taken together, HDAC11 appears to be essential for proper B-cells and T-cell differentiation. It also seems to play a critical role in differentiation of granulocytes and monocytes. Therefore it is plausible that HDAC11 might function as a regulator of hematopoietic differentiation and expansion in vivo. A better understanding of this previously unknown role of HDAC11 in hematopoiesis might lead to targeted epigenetic therapies in hematological malignancies to influence the appropriate differentiation of these cells, and possibly augmenting the efficacy of immunotherapeutic approaches against malignancies. Disclosures:No relevant conflicts of interest to declare.