The Experts below are selected from a list of 351 Experts worldwide ranked by ideXlab platform
Stuart L Schreiber - One of the best experts on this subject based on the ideXlab platform.
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small molecule inhibition of proteasome and aggresome function induces synergistic anti tumor activity in multiple myeloma
Blood, 2005Co-Authors: Teru Hideshima, Stuart L Schreiber, James E Bradner, Dharminder Chauhan, Paul G Richardson, Jason Wong, Kenneth C AndersonAbstract:Abstract Bortezomib (Velcade®) triggers significant anti-tumor activity in multiple myeloma (MM) both in preclinical models and in patients with relapsed refractory disease. However, 60–65% of patients did not respond to bortezomib, and mechanisms of resistance to bortezomib are not fully understood. Recent studies have revealed an alternative system to the proteasome for degradation of polyubiquitinated misfolded/unfolded proteins, termed the aggresome. Aggresome formation ultimately induces autophagic clearance, which terminates in lysosomal degradation. The aggresome pathway therefore likely provides a novel system for delivery of aggregated proteins from cytoplasm to lysosomes for degradation. In aggresomal protein degradation, histone deacetylase6 (HDAC6) has an essential role. We therefore hypothesized that inhibition of both mechanisms of protein catabolism could induce accumulation of ubiquitinated proteins, followed by significant cell stress and cytotoxicity in MM cells. We demonstrate that HDAC6 specific inhibitor Tubacin specifically triggers acetylation of α-tubulin, as a result of HDAC6 inhibition, in a dose- and time-dependent fashion. It induces cytotoxicity in MM cells, which is mediated via caspase-dependent apoptosis; no toxicity is observed in normal peripheral blood mononuclear cells. Tubacin inhibits the interaction of HDAC6 with dynein and induces marked accumulation of ubiquitinated proteins. It synergistically augments bortezomib-induced cytotoxicity via c-Jun NH2-terminal kinase (JNK)/caspase activation. Importantly, this combination also induces significant cytotoxicity in plasma cells isolated from MM patient bone marrow (BM). Adherence of MM cells to BM stromal cells confers growth and resistance to conventional treatments; in contrast, the combination of Tubacin and bortezomib triggers toxicity even in adherent MM cells (Hideshima et al. PNAS 102:8567–8572, 2005). In the present study, we further examined the biologic sequelae of inhibiting both proteasome and aggresome pathways in MM. Bortezomib induces aggresome formation, evidenced by immunohistochemical analyses using DAPI, ubiquitin and γ-tubulin Abs. Tubacin enhances 20S proteasome activity in a time-dependent fashion; conversely bortezomib significantly augments HDAC6 protein expression. Taken together, these results suggest that proteasome function could compensate for aggresome function and vise versa. Importantly, treatment with the combination of Tubacin and bortezomib induces caspase-12 cleavage and upregulation of Grp78 in MM cells suggesting endoplasmic reticulum (ER) stress, associated with increased apoptosis. Finally, heat shock protein (Hsp)90 is a chaperone for transport of ubiquitinated proteins to the proteasome. HDAC6 is also constitutively associated with Hsp90, and the HDAC6 inhibitor Tubacin augments Hsp90 acetylation. Ongoing studies will delineate the biologic role of HDAC6 in the chaperone function both in proteasome and aggresome protein degradation cascades. Our studies therefore demonstrate that Tubacin combined with bortezomib mediates significant anti-MM activity by blocking both the aggresome and proteasome, respectively, thereby providing the framework for clinical evaluation of combined therapy to improve patient outcome in MM.
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significance of hdac6 regulation via estrogen signaling for cell motility and prognosis in estrogen receptor positive breast cancer
Oncogene, 2005Co-Authors: Shigehira Saji, Stuart L Schreiber, Masayo Kawakami, Shinichi Hayashi, Nobuyuki Yoshida, Makiko Hirose, Shin Ichiro Horiguchi, Akihiro Itoh, Nobuaki Funata, Minoru YoshidaAbstract:Histone deacetylase (HDAC) 6 is a subtype of the HDAC family; it deacetylates alpha-tubulin and increases cell motility. Here, we investigate the impact of an alteration of HDAC6 expression in estrogen receptor alpha (ER)-positive breast cancer MCF-7 cells, as we identified that HDAC6 is a novel estrogen-regulated gene. MCF-7 treated with estradiol showed increased expression of HDAC6 mRNA and protein and a four-fold increase in cell motility in a migration assay. Cell motility was increased to the same degree by stably transfecting the HDAC6 expression vector into MCF-7 cells. In both cases, the cells changed in appearance from their original round shape to an axon-extended shape, like a neuronal cell. This HDAC6 accumulation caused the deacetylation of alpha-tubulin. Either the selective estrogen receptor modulator tamoxifen (TAM) or the pure antiestrogen ICI 182,780 prevented estradiol-induced HDAC6 accumulation and deacetylation of alpha-tubulin, leading to reduced cell motility. Tubacin, an inhibitory molecule that binds to the tubulin deacetylation domain of HDAC6, also prevented estradiol-stimulated cell migration. Finally, we evaluated HDAC6 protein expression in 139 consecutively archived human breast cancer tissues by immunohistochemical staining. The prognostic analyses for these patients revealed no significant differences based on HDAC6 expression. However, subset analysis of ER-positive patients who received adjuvant treatment with TAM (n = 67) showed a statistically significant difference in relapse-free survival and overall survival in favor of the HDAC6-positive group (P < 0.02 and P < 0.05, respectively). HDAC6 expression was an independent prognostic indicator by multivariate analysis (odds ratio = 2.82, P = 0.047). These results indicate the biological significance of HDAC6 regulation via estrogen signaling.
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small molecule inhibition of proteasome and aggresome function induces synergistic antitumor activity in multiple myeloma
Proceedings of the National Academy of Sciences of the United States of America, 2005Co-Authors: Teru Hideshima, Stuart L Schreiber, James E Bradner, Dharminder Chauhan, Paul G Richardson, Jason W H Wong, Kenneth C AndersonAbstract:We have shown that the proteasome inhibitor bortezomib (formerly known as PS-341) triggers significant antitumor activity in multiple myeloma (MM) in both preclinical models and patients with relapsed refractory disease. Recent studies have shown that unfolded and misfolded ubiquitinated proteins are degraded not only by proteasomes, but also by aggresomes, dependent on histone deacetylase 6 (HDAC6) activity. We therefore hypothesized that inhibition of both mechanisms of protein catabolism could induce accumulation of ubiquitinated proteins followed by significant cell stress and cytotoxicity in MM cells. To prove this hypothesis, we used bortezomib and Tubacin to inhibit the proteasome and HDAC6, respectively. Tubacin specifically triggers acetylation of α-tubulin as a result of HDAC6 inhibition in a dose- and time-dependent fashion. It induces cytotoxicity in MM cells at 72 h with an IC50 of 5–20 μM, which is mediated by caspase-dependent apoptosis; no toxicity is observed in normal peripheral blood mononuclear cells. Tubacin inhibits the interaction of HDAC6 with dynein and induces marked accumulation of ubiquitinated proteins. It synergistically augments bortezomib-induced cytotoxicity by c-Jun NH2-terminal kinase/caspase activation. Importantly, this combination also induces significant cytotoxicity in plasma cells isolated from MM patient bone marrow. Finally, adherence of MM cells to bone marrow stromal cells confers growth and resistance to conventional treatments; in contrast, the combination of Tubacin and bortezomib triggers toxicity even in adherent MM cells. Our studies therefore demonstrate that Tubacin combined with bortezomib mediates significant anti-MM activity, providing the framework for clinical evaluation of combined therapy to improve patient outcome in MM.
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multidimensional chemical genetic analysis of diversity oriented synthesis derived deacetylase inhibitors using cell based assays
Chemistry & Biology, 2003Co-Authors: Stephen J Haggarty, Kathryn M Koeller, Jason C Wong, Rebecca A Butcher, Stuart L SchreiberAbstract:Abstract Systematic chemical genetics aims to explore the space representing interactions between small molecules and biological systems. Beyond measuring binding interactions and enzyme inhibition, measuring changes in the activity of proteins in intact signaling networks is necessary. Toward this end, we are partitioning chemical space into regions with different biological activities using a panel of cell-based assays and small molecule "chemical genetic modifiers." Herein, we report on the use of this methodology for the discovery of 617 small molecule inhibitors of histone deacetylases from a multidimensional screen of an encoded, diversity-oriented synthesis library. Following decoding of chemical tags and resynthesis, we demonstrate the selectivity of one inhibitory molecule (Tubacin) toward α-tubulin deacetylation and another (histacin) toward histone deacetylation. These small molecules will facilitate dissecting the role of acetylation in a variety of cell biological processes.
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domain selective small molecule inhibitor of histone deacetylase 6 hdac6 mediated tubulin deacetylation
Proceedings of the National Academy of Sciences of the United States of America, 2003Co-Authors: Stephen J Haggarty, Kathryn M Koeller, Jason C Wong, Christina M Grozinger, Stuart L SchreiberAbstract:Protein acetylation, especially histone acetylation, is the subject of both research and clinical investigation. At least four small-molecule histone deacetylase inhibitors are currently in clinical trials for the treatment of cancer. These and other inhibitors also affect microtubule acetylation. A multidimensional, chemical genetic screen of 7,392 small molecules was used to discover “Tubacin,” which inhibits α-tubulin deacetylation in mammalian cells. Tubacin does not affect the level of histone acetylation, gene-expression patterns, or cell-cycle progression. We provide evidence that class II histone deacetylase 6 (HDAC6) is the intracellular target of Tubacin. Only one of the two catalytic domains of HDAC6 possesses tubulin deacetylase activity, and only this domain is bound by Tubacin. Tubacin treatment did not affect the stability of microtubules but did decrease cell motility. HDAC6 overexpression disrupted the localization of p58, a protein that mediates binding of Golgi elements to microtubules. Our results highlight the role of α-tubulin acetylation in mediating the localization of microtubule-associated proteins. They also suggest that small molecules that selectively inhibit HDAC6-mediated α-tubulin deacetylation, a first example of which is Tubacin, might have therapeutic applications as antimetastatic and antiangiogenic agents.
Ralph Mazitschek - One of the best experts on this subject based on the ideXlab platform.
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discovery of selective small molecule hdac6 inhibitor for overcoming proteasome inhibitor resistance in multiple myeloma
Proceedings of the National Academy of Sciences of the United States of America, 2016Co-Authors: Teru Hideshima, Ralph Mazitschek, Ronald M Paranal, Weiping Tang, Edward Greenberg, Nathan West, Meaghan E Colling, Guillermina Estiu, Jennifer A PerryAbstract:Multiple myeloma (MM) has proven clinically susceptible to modulation of pathways of protein homeostasis. Blockade of proteasomal degradation of polyubiquitinated misfolded proteins by the proteasome inhibitor bortezomib (BTZ) achieves responses and prolongs survival in MM, but long-term treatment with BTZ leads to drug-resistant relapse in most patients. In a proof-of-concept study, we previously demonstrated that blocking aggresomal breakdown of polyubiquitinated misfolded proteins with the histone deacetylase 6 (HDAC6) inhibitor Tubacin enhances BTZ-induced cytotoxicity in MM cells in vitro. However, these foundational studies were limited by the pharmacologic liabilities of Tubacin as a chemical probe with only in vitro utility. Emerging from a focused library synthesis, a potent, selective, and bioavailable HDAC6 inhibitor, WT161, was created to study the mechanism of action of HDAC6 inhibition in MM alone and in combination with BTZ. WT161 in combination with BTZ triggers significant accumulation of polyubiquitinated proteins and cell stress, followed by caspase activation and apoptosis. More importantly, this combination treatment was effective in BTZ-resistant cells and in the presence of bone marrow stromal cells, which have been shown to mediate MM cell drug resistance. The activity of WT161 was confirmed in our human MM cell xenograft mouse model and established the framework for clinical trials of the combination treatment to improve patient outcomes in MM.
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abstract a11 regulation of cd133 by the tubulin deacetylase hdac6 can alter cancer cell state
Clinical Cancer Research, 2010Co-Authors: Saranya Kittanakom, Ginny I Chen, Ralph Mazitschek, Anneclaude Gingras, Igor Stagljar, Jason MoffatAbstract:The CD133 antigen AC133/1 is a putative marker for enriching certain stem cells and cancer progenitor cells. Despite its wide application, the function and regulation of its expression remains unclear. Given the presence of its transcript in more differentiated cells, epigenetic regulation of CD133 transcripts alone cannot vouch for its utility as a marker. Therefore, understanding the regulation of CD133 protein expression is important for further defining the cellular state of tumor-initiating cells. We used the MAPLE system (Mak et al, Mol Cell Proteomics, 2010) to identify bona fide protein interaction partners of CD133. We present the microtubule-associated histone deacetylase 6 (HDAC6) as a physical interaction partner of CD133. This interaction was validated by co-immunoprecipitation/Western Blot analyses and by a membrane yeast two-hybrid (MYTH) assay. In order to understand the functional consequence of the HDAC6 and CD133 interaction, we used lentiviral short-hairpin RNAs to knockdown HDAC6 in multiple cell types, including the human epithelial colorectal adenocarcinoma lines Caco- 2 and HT-29 and in the retinoblastoma cell line Weri-Rb-1. Interestingly, we observed a significant reduction of CD133 protein expression. This reduction was shown to be dependent on HDAC6 deacetylase activity using a deacetlyase deficient HDAC6 mutant as well as when using the small molecule inhibitor of HDAC6, Tubacin. HDAC6 promotes de-acetylation of alpha-tubulin to influence re-cycling of cell surface proteins. Furthermore, when we performed a CD133 MYTH screen against a cDNA library isolated from an adult human brain, we identified the late endosomal- and lysosomal-associated marker CD63 as an interaction partner of CD133. In fact, treatment of Caco-2 cells with Tubacin resulted in trafficking of CD133 into endosomes for lysosomal degradation as determined by increased co-localization with CD63. In an attempt to study the affect of HDAC6 knockdown on cell viability, we noticed that CD133 knockdown significantly impacted cell proliferation and clonogenicity of Caco-2 cells. We determined that the cause of this is likely due to increased differentiation as determined by the level of alkaline phosphatase activity and by quantitative realtime PCR for markers of differentiated colon cells. To assess the tumorigenic potential of Caco-2 cells with reduced CD133 expression in vitro we performed soft agar colony-forming assay, which resulted in the loss of anchorage independent growth, a well-known hallmark of cancer. Together, these results suggest that CD133 can exercise a function in establishing and maintaining a primitive cancer cell state and loss or reduction of its expression marks more differentiated cancer cells that may be nontumorigenic. Citation Information: Clin Cancer Res 2010;16(14 Suppl):A11.
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Regulation of microtubule dynamics by inhibition of the tubulin deacetylase HDAC6.
Journal of cell science, 2009Co-Authors: Yuliya Zilberman, Ralph Mazitschek, Christoph Ballestrem, Letizia Carramusa, Saadi Khochbin, Alexander BershadskyAbstract:We studied the role of a class II histone deacetylase, HDAC6, known to function as a potent alpha-tubulin deacetylase, in the regulation of microtubule dynamics. Treatment of cells with the class I and II histone deacetylase inhibitor TSA, as well as the selective HDAC6 inhibitor Tubacin, increased microtubule acetylation and significantly reduced velocities of microtubule growth and shrinkage. siRNA-mediated knockdown of HDAC6 also increased microtubule acetylation but, surprisingly, had no effect on microtubule growth velocity. At the same time, HDAC6 knockdown abolished the effect of Tubacin on microtubule growth, demonstrating that Tubacin influences microtubule dynamics via specific inhibition of HDAC6. Thus, the physical presence of HDAC6 with impaired catalytic activity, rather than tubulin acetylation per se, is the factor responsible for the alteration of microtubule growth velocity in HDAC6 inhibitor-treated cells. In support of this notion, HDAC6 mutants bearing inactivating point mutations in either of the two catalytic domains mimicked the effect of HDAC6 inhibitors on microtubule growth velocity. In addition, HDAC6 was found to be physically associated with the microtubule end-tracking protein EB1 and a dynactin core component, Arp1, both of which accumulate at the tips of growing microtubules. We hypothesize that inhibition of HDAC6 catalytic activity may affect microtubule dynamics by promoting the interaction of HDAC6 with tubulin and/or with other microtubule regulatory proteins.
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targeting histone deacetylase 6 and the aggresome pathway in acute lymphoblastic leukemia cells
Blood, 2007Co-Authors: Augustin Rodriguezgonzalez, Ralph Mazitschek, James Chng, Alan K Ikeda, Tara L Lin, Bita Bahrami, James E Bradner, Kathleen M SakamotoAbstract:Acute lymphoblastic leukemia (ALL) is the most common form of childhood cancer. Despite effective chemotherapy, 20% of patients will relapse. Therefore, it is critical that we identify novel therapies to treat ALL. We are studying a new small molecule compound known as Tubacin (tubulin acetylation inducer) that selectively inhibits histone deacetylase 6 (HDAC6). HDAC6 binds to polyubiquitinated misfolded proteins and to dynein motor proteins, including alpha-tubulin, thereby recruiting misfolded or unwanted proteins to aggresomes and subsequent degradation by the lysosome. Tubacin was discovered through a chemical genetic screen of a 7,392 small molecule library and was found to induce acetylation of alpha-tubulin by inhibiting one of the two catalytic domains of HDAC6. This inhibition disrupts the interaction of HDAC6 with dynein resulting in marked accumulation of ubiquitinated proteins. Previous work demonstrated that treatment of multiple myeloma cells with Tubacin inhibited growth at low micromolar concentrations. Tubacin does not appear to affect global histone acetylation, gene expression, or cell cycle regulation. To determine the effects of Tubacin in human ALL cells, we treated both B- and T-cell ALL cell lines with varying concentrations of the drug and performed MTT assays. In T-ALL cells (Jurkat, Loucy), the IC50 of Tubacin was found to be 1 to 3 uM, while in B-cell ALL cells (REH, Nalm-6), the IC50 was 2 to 5uM. Tubacin induced apoptosis of Jurkat and Loucy cells stained with Annexin V and propidium iodide. Within 12 hours, we observed increased protein polyubiquitination and PARP cleavage, but no difference in Rb phosphorylation in cells treated with Tubacin. Furthermore, Tubacin treatment increased acetylation of alpha-tubulin in Loucy and Jurkat cells within 3 hours. To study whether Tubacin was toxic to normal hematopoietic cells, we treated human bone marrow cells cultured in methylcellulose containing IL-3, IL-6, and Stem Cell Factor with varying concentrations of Tubacin. The IC50 of 20uM was determined from numbers of colonies plated in triplicate. Similarly, treatment of normal human lymphocytes cultured in IL-2, demonstrated an IC50 of 16uM. Finally, we examined the effects of Tubacin on growth of primary ALL cells. Bone marrow cells from three patients with B-cell ALL at diagnosis were cultured in Tubacin at varying concentrations and MTT assays were performed. In two of the three ALL samples, the IC50 was less than 5uM. Experiments to study the effects of Tubacin in mouse models of leukemia are in progress. Our results suggest that inhibition of HDAC6 and the aggresome pathway provides a novel approach to treat ALL.
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HDAC6 deacetylation of tubulin modulates dynamics of cellular adhesions.
Journal of cell science, 2007Co-Authors: Andy Dong-anh Tran, Ralph Mazitschek, Timothy P Marmo, Ambar A Salam, Sally Che, Erik Finkelstein, Rafi Kabarriti, Harry S Xenias, Charlotte Hubbert, Yoshiharu KawaguchiAbstract:Genetic or pharmacological alteration of the activity of the histone deacetylase 6 (HDAC6) induces a parallel alteration in cell migration. Using Tubacin to block deacetylation of alpha-tubulin, and not other HDAC6 substrates, yielded a motility reduction equivalent to agents that block all NAD-independent HDACs. Accordingly, we investigated how the failure to deacetylate tubulin contributes to decreased motility in HDAC6-inhibited cells. Testing the hypothesis that motility is reduced because cellular adhesion is altered, we found that inhibiting HDAC6 activity towards tubulin rapidly increased total adhesion area. Next, we investigated the mechanism of the adhesion area increase. Formation of adhesions proceeded normally and cell spreading was more rapid in the absence of active HDAC6; however, photobleaching assays and adhesion breakdown showed that adhesion turnover was slower. To test the role of hyperacetylated tubulin in altering adhesion turnover, we measured microtubule dynamics in HDAC6-inhibited cells because dynamic microtubules are required to target adhesions for turnover. HDAC6 inhibition yielded a decrease in microtubule dynamics that was sufficient to decrease focal adhesion turnover. Thus, our results suggest a scenario in which the decreased dynamics of hyperacetylated microtubules in HDAC6-inhibited cells compromises their capacity to mediate the focal adhesion dynamics required for rapid cell migration.
M Crudden - One of the best experts on this subject based on the ideXlab platform.
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identification of an acetylation dependant ku70 flip complex that regulates flip expression and hdac inhibitor induced apoptosis
Cell Death & Differentiation, 2012Co-Authors: Emma M Kerr, Caitriona Holohan, Kirsty M Mclaughlin, Joanna Majkut, Sharron Dolan, Keara Redmond, Joel S Riley, Kirsty Mclaughlin, Izabela Stasik, M CruddenAbstract:FLIP is a potential anti-cancer therapeutic target that inhibits apoptosis by blocking caspase 8 activation by death receptors. We report a novel interaction between FLIP and the DNA repair protein Ku70 that regulates FLIP protein stability by inhibiting its polyubiquitination. Furthermore, we found that the histone deacetylase (HDAC) inhibitor Vorinostat (SAHA) enhances the acetylation of Ku70, thereby disrupting the FLIP/Ku70 complex and triggering FLIP polyubiquitination and degradation by the proteasome. Using in vitro and in vivo colorectal cancer models, we further demonstrated that SAHA-induced apoptosis is dependant on FLIP downregulation and caspase 8 activation. In addition, an HDAC6-specific inhibitor Tubacin recapitulated the effects of SAHA, suggesting that HDAC6 is a key regulator of Ku70 acetylation and FLIP protein stability. Thus, HDAC inhibitors with anti-HDAC6 activity act as efficient post-transcriptional suppressors of FLIP expression and may, therefore, effectively act as ‘FLIP inhibitors'.
James E Bradner - One of the best experts on this subject based on the ideXlab platform.
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hdac6 inhibition enhances 17 aag mediated abrogation of hsp90 chaperone function in human leukemia cells
Blood, 2008Co-Authors: Rekha Rao, James E Bradner, Warren Fiskus, Yonghua Yang, Pearl Lee, Rajeshree Joshi, Pravina Fernandez, Aditya Mandawat, Peter Atadja, Kapil N BhallaAbstract:Histone deacetylase 6 (HDAC6) is a heat shock protein 90 (hsp90) deacetylase. Treatment with pan-HDAC inhibitors or depletion of HDAC6 by siRNA induces hyperacetylation and inhibits ATP binding and chaperone function of hsp90. Treatment with 17-allylamino-demothoxy geldanamycin (17-AAG) also inhibits ATP binding and chaperone function of hsp90, resulting in polyubiquitylation and proteasomal degradation of hsp90 client proteins. In this study, we determined the effect of hsp90 hyperacetylation on the anti-hsp90 and antileukemia activity of 17-AAG. Hyperacetylation of hsp90 increased its binding to 17-AAG, as well as enhanced 17-AAG-mediated attenuation of ATP and the cochaperone p23 binding to hsp90. Notably, treatment with 17-AAG alone also reduced HDAC6 binding to hsp90 and induced hyperacetylation of hsp90. This promoted the proteasomal degradation of HDAC6. Cotreatment with 17-AAG and siRNA to HDAC6 induced more inhibition of hsp90 chaperone function and depletion of BCR-ABL and c-Raf than treatment with either agent alone. In addition, cotreatment with 17-AAG and Tubacin augmented the loss of survival of K562 cells and viability of primary acute myeloid leukemia (AML) and chronic myeloid leukemia (CML) samples. These findings demonstrate that HDAC6 is an hsp90 client protein and hyperacetylation of hsp90 augments the anti-hsp90 and antileukemia effects of 17-AAG.
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targeting histone deacetylase 6 and the aggresome pathway in acute lymphoblastic leukemia cells
Blood, 2007Co-Authors: Augustin Rodriguezgonzalez, Ralph Mazitschek, James Chng, Alan K Ikeda, Tara L Lin, Bita Bahrami, James E Bradner, Kathleen M SakamotoAbstract:Acute lymphoblastic leukemia (ALL) is the most common form of childhood cancer. Despite effective chemotherapy, 20% of patients will relapse. Therefore, it is critical that we identify novel therapies to treat ALL. We are studying a new small molecule compound known as Tubacin (tubulin acetylation inducer) that selectively inhibits histone deacetylase 6 (HDAC6). HDAC6 binds to polyubiquitinated misfolded proteins and to dynein motor proteins, including alpha-tubulin, thereby recruiting misfolded or unwanted proteins to aggresomes and subsequent degradation by the lysosome. Tubacin was discovered through a chemical genetic screen of a 7,392 small molecule library and was found to induce acetylation of alpha-tubulin by inhibiting one of the two catalytic domains of HDAC6. This inhibition disrupts the interaction of HDAC6 with dynein resulting in marked accumulation of ubiquitinated proteins. Previous work demonstrated that treatment of multiple myeloma cells with Tubacin inhibited growth at low micromolar concentrations. Tubacin does not appear to affect global histone acetylation, gene expression, or cell cycle regulation. To determine the effects of Tubacin in human ALL cells, we treated both B- and T-cell ALL cell lines with varying concentrations of the drug and performed MTT assays. In T-ALL cells (Jurkat, Loucy), the IC50 of Tubacin was found to be 1 to 3 uM, while in B-cell ALL cells (REH, Nalm-6), the IC50 was 2 to 5uM. Tubacin induced apoptosis of Jurkat and Loucy cells stained with Annexin V and propidium iodide. Within 12 hours, we observed increased protein polyubiquitination and PARP cleavage, but no difference in Rb phosphorylation in cells treated with Tubacin. Furthermore, Tubacin treatment increased acetylation of alpha-tubulin in Loucy and Jurkat cells within 3 hours. To study whether Tubacin was toxic to normal hematopoietic cells, we treated human bone marrow cells cultured in methylcellulose containing IL-3, IL-6, and Stem Cell Factor with varying concentrations of Tubacin. The IC50 of 20uM was determined from numbers of colonies plated in triplicate. Similarly, treatment of normal human lymphocytes cultured in IL-2, demonstrated an IC50 of 16uM. Finally, we examined the effects of Tubacin on growth of primary ALL cells. Bone marrow cells from three patients with B-cell ALL at diagnosis were cultured in Tubacin at varying concentrations and MTT assays were performed. In two of the three ALL samples, the IC50 was less than 5uM. Experiments to study the effects of Tubacin in mouse models of leukemia are in progress. Our results suggest that inhibition of HDAC6 and the aggresome pathway provides a novel approach to treat ALL.
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histone deacetylase 6 hdac6 modulates akt and stat3 activity via heat shock protein hsp 90 in human multiple myeloma mm cells
Blood, 2006Co-Authors: Teru Hideshima, James E Bradner, Hiroshi Yasui, Noopur Raje, Dharminder Chauhan, Klaus Podar, Yutzu Tai, Constantine S Mitsiades, Paul G RichardsonAbstract:Histone deacetylase 6 (HDAC6) has an essential role to recruit ubiquitinated proteins to transport to aggresomes, which ultimately induces lysosomal protein degradation. We have shown that inhibition of proteasomes with bortezomib and of aggresomes with HDAC6 inhibitor Tubacin demonstrated significant cytotoxicity in MM cell lines and MM patient tumor cells in vitro (Hideshima T et al., PNAS2005, 102: 8597–8572). In this study, we further examined the biologic significance of HDAC6 inhibition by Tubacin in MM cells. We found that HDAC6 is constitutively associated with heat shock protein (Hsp) 90 in MM cell lines which is enhanced by Tubacin, as evidenced by co-immunoprecipitation. Since Akt and STAT3 have been shown to play important role in proliferation, anti-apoptosis, and drug resistance in MM cells; and all are client proteins of Hsp90, we next further examined whether inhibition of HDAC6 could modulate activities of these proteins via Hsp90. Importantly, Tubacin enhanced phosphorylation of Akt, associated with augmentation of Hsp90 acetylation. Hsp90 inhibitor 17-AAG downregulated Akt phosphorylation associated with enhanced interaction of Hsp90 with Akt, which was partially blocked by Tubacin. On the other hand, 17-AAG did not enhance acetylation of α-tubulin or ubiquitination of proteins, suggesting that Hsp90 does not affect HDAC6 function. Furthermore, we found that STAT3 is also constitutively associated with Hsp90. Importantly, both Tubacin and 17-AAG inhibit phosphorylation of STAT3 in a dose- and time-dependent fashion in MM cells. Taken together, our data indicate that HDAC6 has an important role not only in aggresomal protein degradation, but also in MM cell pathogenesis by modulating Akt and STAT3 signaling cascades via Hsp90 acetylation in MM cells.
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lymphocyte chemotaxis is regulated by histone deacetylase 6 independently of its deacetylase activity
Molecular Biology of the Cell, 2006Co-Authors: Roman J Cabrero, Ralph Mazitschek, Juan M Serrador, Olga Barreiro, Maria Mittelbrunn, Salvador Naranjosuarez, Noa B Martincofreces, Miguel Vicentemanzanares, James E BradnerAbstract:In this work, the role of HDAC6, a type II histone deacetylase with tubulin deacetylase activity, in lymphocyte polarity, motility, and transmigration was explored. HDAC6 was localized at dynamic subcellular structures as leading lamellipodia and the uropod in migrating T-cells. However, HDAC6 activity did not appear to be involved in the polarity of migrating lymphocytes. Overexpression of HDAC6 in freshly isolated lymphocytes and T-cell lines increased the lymphocyte migration mediated by chemokines and their transendothelial migration under shear flow. Accordingly, the knockdown of HDAC6 expression in T-cells diminished their chemotactic capability. Additional experiments with HDAC6 inhibitors (trichostatin, Tubacin), other structural related molecules (nilTubacin, MAZ-1391), and HDAC6 dead mutants showed that the deacetylase activity of HDAC6 was not involved in the modulatory effect of this molecule on cell migration. Our results indicate that HDAC6 has an important role in the chemotaxis of T-lymphocytes, which is independent of its tubulin deacetylase activity.
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Lymphocyte Chemotaxis Is Regulated by Histone Deacetylase 6, Independently of Its Deacetylase Activity □V
2006Co-Authors: Roman J Cabrero, Ralph Mazitschek, James E Bradner, Juan M Serrador, Olga Barreiro, Maria Mittelbrunn, Salvador Naranjo-suárez, Noa Martín-cófreces, Miguel Vicente-manzanares, Jesús ÁvilaAbstract:In this work, the role of HDAC6, a type II histone deacetylase with tubulin deacetylase activity, in lymphocyte polarity, motility, and transmigration was explored. HDAC6 was localized at dynamic subcellular structures as leading lamellipodia and the uropod in migrating T-cells. However, HDAC6 activity did not appear to be involved in the polarity of migrating lymphocytes. Overexpression of HDAC6 in freshly isolated lymphocytes and T-cell lines increased the lymphocyte migration mediated by chemokines and their transendothelial migration under shear flow. Accordingly, the knockdown of HDAC6 expression in T-cells diminished their chemotactic capability. Additional experiments with HDAC6 inhibitors (trichostatin, Tubacin), other structural related molecules (nilTubacin, MAZ-1391), and HDAC6 dead mutants showed that the deacetylase activity of HDAC6 was not involved in the modulatory effect of this molecule on cell migration. Our results indicate that HDAC6 has an important role in the chemotaxis of T-lymphocytes, which is independent of its tubulin deacetylase activity. This article was published online ahead of print in MBC in Pres
Robert H Henning - One of the best experts on this subject based on the ideXlab platform.
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activation of histone deacetylase 6 induces contractile dysfunction through derailment of α tubulin proteostasis in experimental and human atrial fibrillation
Circulation, 2014Co-Authors: Deli Zhang, Artavazd Tadevosyan, Roelien A M Meijering, Femke Hoogstraberends, Gunseli Cubukcuoglu Deniz, Serkan Durdu, A R Akar, Ody C M Sibon, Stanley Nattel, Robert H HenningAbstract:Background—Atrial fibrillation (AF) is characterized by structural remodeling, contractile dysfunction, and AF progression. Histone deacetylases (HDACs) influence acetylation of both histones and cytosolic proteins, thereby mediating epigenetic regulation and influencing cell proteostasis. Because the exact function of HDACs in AF is unknown, we investigated their role in experimental and clinical AF models. Methods and Results—Tachypacing of HL-1 atrial cardiomyocytes and Drosophila pupae hearts significantly impaired contractile function (amplitude of Ca2+ transients and heart wall contractions). This dysfunction was prevented by inhibition of HDAC6 (Tubacin) and sirtuins (nicotinamide). Tachypacing induced specific activation of HDAC6, resulting in α-tubulin deacetylation, depolymerization, and degradation by calpain. Tachypacing-induced contractile dysfunction was completely rescued by dominant-negative HDAC6 mutants with loss of deacetylase activity in the second catalytic domain, which bears α-tubul...
