Efficacy of MS-275, a selective inhibitor of class I histone deacetylases, in human colon cancer models

N-(2-aminophenyl)-4-[N-(pyridine-3yl-methoxy-carbonyl) aminomethyl] benzamide (MS-275) is a second generation histone deacetylase (HDAC) inhibitor with significant anti-tumor efficacy currently in clinical development. We investigated the effect of MS-275 treatment on various colon cancer cell lines, as well as on mouse xenograft models derived from human colorectal cancer. MS-275 exerted strong anti-proliferative effects in five cell lines and increased the acetylation of histones 3 and 4. In vivo testing of the compound in eight different models of human colon cancer derived from primary colorectal cancers or from established cell lines revealed that five models were responders, two non-responders and one an anti-responder. Gene expression profiles were determined in order to identify genes and pathways differentially regulated upon MS-275 treatment in responder versus non-responder models. Principle component analysis revealed a correlation of the anti-tumor efficacy with the sub-clustering of the MS-275 treatment groups in 7 out of 8 models. Although the overall gene expression pattern was rather unique for each individual model, 129 genes were significantly up- and 58 genes significantly down-regulated in at least 2 out of 5 responder models in response to MS-275 treatment. We identified potential biomarkers for response to MS-275, such as PRA1, MYADM and PALM2-AKAP2 which were up-regulated in all responder models and down-regulated or unchanged in all non-responder models. Our results provide a starting point for the development of clinically relevant biomarkers for predicting a response to MS-275 and the understanding of the mode of action of this HDAC inhibitor

Synergism of Heat Shock Protein 90 and Histone Deacetylase Inhibitors in Synovial Sarcoma

Current systemic therapies have little curative benefit for synovial sarcoma. Histone deacetylase (HDAC) inhibitors and the heat shock protein 90 (Hsp90) inhibitor 17-AAG have recently been shown to inhibit synovial sarcoma in preclinical models. We tested combinations of 17-AAG with the HDAC inhibitor MS-275 for synergism by proliferation and apoptosis assays. The combination was found to be synergistic at multiple time points in two synovial sarcoma cell lines. Previous studies have shown that HDAC inhibitors not only induce cell death but also activate the survival pathway NF-κB, potentially limiting therapeutic benefit. As 17-AAG inhibits activators of NF-κB, we tested if 17-AAG synergizes with MS-275 through abrogating NF-κB activation. In our assays, adding 17-AAG blocks NF-κB activation by MS-275 and siRNA directed against histone deacetylase 3 (HDAC3) recapitulates the effects of MS-275. Additionally, we find that the NF-κB inhibitor BAY 11-7085 synergizes with MS-275. We conclude that agents inhibiting NF-κB synergize with HDAC inhibitors against synovial sarcoma

Investigation into the use of histone deacetylase inhibitor MS-275 as a topical agent for the prevention and treatment of cutaneous squamous cell carcinoma in an SKH-1 hairless mouse model

<div><p>Cutaneous squamous cell carcinomas are a common form of highly mutated keratinocyte skin cancers that are of particular concern in immunocompromised patients. Here we report on the efficacy of topically applied MS-275, a clinically used histone deacetylase inhibitor, for the treatment and management of this disease. At 2 mg/kg, MS-275 significantly decreased tumor burden in an SKH-1 hairless mouse model of UVB radiation-induced skin carcinogenesis. MS-275 was cell permeable as a topical formulation and induced histone acetylation changes in mouse tumor tissue. MS-275 was also effective at inhibiting the proliferation of patient derived cutaneous squamous cell carcinoma lines and was particularly potent toward cells isolated from a regional metastasis on an immunocompromised individual. Our findings support the use of alternative routes of administration for histone deacetylase inhibitors in the treatment of high-risk squamous cell carcinoma which may ultimately lead to more precise delivery and reduced systemic toxicity.</p></div

MS-275 (Entinostat) Promotes Radio-sensitivity in PAX3-FOXO1 Rhabdomyosarcoma cells

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma of childhood. About 25% of RMS expresses fusion oncoproteins such as PAX3/PAX7‐FOXO1 (fusion‐positive, FP) while fusion‐negative (FN)‐RMS harbors RAS mutations. Radiotherapy (RT) plays a crucial role in local control but metastatic RMS is often radio‐resistant. HDAC inhibitors (HDACi) radio‐sensitize different cancer cells types. Thus, we evaluated MS−275 (Entinostat), a Class I and IV HDACi, in combination with RT on RMS cells in vitro and in vivo. MS−275 reversibly hampered cell survival in vitro in FN‐RMS RD (RASmut) and irreversibly in FP‐RMS RH30 cell lines down‐regulating cyclin A, B, and D1, up‐regulating p21 and p27 and reducing ERKs activity, and c‐Myc expression in RD and PI3K/Akt/mTOR activity and N‐Myc expression in RH30 cells. Further, MS−275 and RT combination reduced colony formation ability of RH30 cells. In both cell lines, co‐treatment increased DNA damage repair inhibition and reactive oxygen species formation, down‐regulated NRF2, SOD, CAT and GPx4 anti‐oxidant genes and improved RT ability to induce G2 growth arrest. MS−275 inhibited in vivo growth of RH30 cells and completely prevented the growth of RT‐unresponsive RH30 xenografts when combined with radiation. Thus, MS−275 could be considered as a radio‐sensitizing agent for the treatment of intrinsically radio‐resistant PAX3‐FOXO1 RMS

HDAC I Inhibition in the Dorsal and Ventral Hippocampus Differentially Modulates Predator-Odor Fear Learning and Generalization

Although predator odors are ethologically relevant stimuli for rodents, the molecular pathways and contribution of some brain regions involved in predator odor conditioning remain elusive. Inhibition of histone deacetylases (HDACs) in the dorsal hippocampus has been shown to enhance shock-induced contextual fear learning, but it is unknown if HDACs have differential effects along the dorso-ventral hippocampal axis during predator odor fear learning. We injected MS-275, a class I HDAC inhibitor, bilaterally in the dorsal or ventral hippocampus of mice and found that it had no effects on innate anxiety in either region. We then assessed the effects of MS-275 at different stages of fear learning along the longitudinal hippocampal axis. Animals were injected with MS-275 or vehicle after context pre-exposure (pre-conditioning injections), when a representation of the context is first formed, or after exposure to coyote urine (post-conditioning injections), when the context becomes associated with predator odor. When MS-275 was administered after context pre-exposure, dorsally injected animals showed enhanced fear in the training context but were able to discriminate it from a neutral environment. Conversely, ventrally injected animals did not display enhanced learning in the training context but generalized the fear response to a neutral context. However, when MS-275 was administered after conditioning, there were no differences between the MS-275 and vehicle control groups in either the dorsal or ventral hippocampus. Surprisingly, all groups displayed generalization to a neutral context, suggesting that predator odor exposure followed by a mild stressor such as restraint leads to fear generalization. These results may elucidate distinct functions of the dorsal and ventral hippocampus in predator odor-induced fear conditioning as well as some of the molecular mechanisms underlying fear generalization

Anti-Leukemia Activity of MS-275 Histone Deacetylase Inhibitor Implicates 4-1BBL/4-1BB Immunomodulatory Functions

Histone deacetylase inhibitors (HDACi) have demonstrated promising therapeutic potential in clinical trials for hematological malignancies. HDACi, such as SAHA/Vorinostat, Trichostatin A, and MS-275 were found to induce apoptosis of leukemic blasts through activation of the death receptor pathway and transcriptional induction of the Tumor Necrosis Factor (TNF)-related pro-apoptotic family members, TRAIL and FasL. The impact of HDACi on TNF-related costimulatory molecules such as 4-1BB ligand (4-1BBL/TNFSF9) is however not known. Following exposure to SAHA/Vorinostat, Trichostatin A, and MS-275, transcript levels were determined by real time PCR in Jurkat, Raji and U937 cells. Treatment with HDACi up-regulated TNFSF9 gene expression in the three leukemia cell lines, yet to different extend and with distinct kinetics, which did not require de novo protein synthesis and was not associated with DNAse I hypersensitive chromatin remodeling. Transcriptional activity of TNFSF9 promoter-luciferase constructs was induced up to 12 fold by HDACi, and implication of Sp1/Sp3 transcription factors binding to functional GC-box elements was evidenced by reporter gene assays, site-directed mutagenesis, and electrophoretic mobility shift assays. Functionality of modulated target genes was assessed in allogeneic mixed leukocyte reaction experiments. MS-275- and to a lesser extent Trichostatin A- and SAHA-treated Raji cells significantly up regulated T lymphocytes proliferation which was reduced by about 50% by a 4-1BB blocking recombinant protein, while MS-275- but neither Trichostatin A- nor SAHA-treated cells up-regulated IFNγ secretion by T lymphocytes. Our results identify 4-1BBL/4-1BB as a downstream target of HDACi, especially of MS-275 anti-leukemia action in vitro. Thus, HDACi such as MS-275 displaying dual TNF-dependent proapoptotic and costimulatory activities might be favored for inclusion in HDACi-based anti-cancer therapeutic strategies

HDAC1 inhibition by MS-275 in mesothelial cells limits cellular invasion and promotes MMT reversal

Peritoneal fibrosis is a pathological alteration of the peritoneal membrane occurring in a variety of conditions including peritoneal dialysis (PD), post-surgery adhesions and peritoneal metastases. The acquisition of invasive and pro-fibrotic abilities by mesothelial cells (MCs) through induction of MMT, a cell-specific form of EMT, plays a main role in this process. Aim of this study was to evaluate possible effects of histone deacetylase (HDAC) inhibitors, key components of the epigenetic machinery, in counteracting MMT observed in MCs isolated from effluent of PD patients. HDAC inhibitors with different class/isoform selectivity have been used for pharmacological inhibition. While the effect of other inhibitors was limited to a partial E-cadherin re-expression, MS-275, a HDAC1-3 inhibitor, promoted: (i) downregulation of mesenchymal markers (MMP2, Col1A1, PAI-1, TGFβ1, TGFβRI) (ii) upregulation of epithelial markers (E-cadherin, Occludin), (iii) reacquisition of an epithelial-like morphology and (iv) marked reduction of cellular invasiveness. Results were confirmed by HDAC1 genetic silencing. Mechanistically, MS-275 causes: (i) increase of nuclear histone H3 acetylation (ii) rescue of the acetylation profile on E-cadherin promoter, (iii) Snail functional impairment. Overall, our study, pinpointing a role for HDAC1, revealed a new player in the regulation of peritoneal fibrosis, providing the rationale for future therapeutic opportunities

Effect of MS-275 combined with cisplatin on ErbB3-overexpressing human bladder transitional cell carcinoma, and its clinical prospect

摘要 第一部分MS-275联合顺铂对ErbB3过表达膀胱尿路上皮癌细胞的杀伤作用及机制研究 目的 ErbB3是表皮生长因子受体（ErbB/HER）家族的一员，其在肿瘤的发生发展过程中起着重要的作用。我们的前期研究通过慢病毒转染的方式获得了ErbB3过表达的人膀胱尿路上皮癌（BTCC）细胞株，并发现ErbB3过表达的人膀胱尿路上皮癌细胞株会对顺铂（DDP）等化疗药物产生耐药。有研究表明MS-275可抑制ErbB3蛋白在ErbB2过表达的人乳腺癌细胞中的表达并促进其凋亡。本研究使用MS-275及DDP单药和联合用药处理，观察MS-275和DDP对ErbB3过表达的人膀胱尿路上皮癌细胞株的杀伤...Abstract Part one: Effect of MS-275 combined with cisplatin on ErbB3-overexpressing human bladder transitional cell carcinoma, and its underlying mechanism. Objective: ErbB3 is a member of the epidermal growth factor receptor（ErbB/HER）family, which plays an important role in the development and progression of the tumor. Our previous study obtained the ErbB3-overexpressing human bladder uroth...学位：医学硕士院系专业：医学院_外科学学号：2452014115355

Site-specific quantification of lysine acetylation in the N-terminal tail of histone H4 using a double-labelling, targeted UHPLC MS/MS approach

We developed a targeted liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the site-specific quantification of lysine acetylation in the N-terminal region of histone H4 by combining chemical derivatization at the protein and peptide levels with digestion using chymotrypsin and trypsin. Unmodified epsilon-amino groups were first modified with propionic acid anhydride and the derivatized protein digested with trypsin and chymotrypsin. The newly formed peptide N-termini were subjected to a second derivatization step with d(6)- (heavy) or d(0)- (light) acetic acid anhydride. Samples were mixed at different ratios and peptides monitored by multiple reaction monitoring (MRM) LC-MS/MS. The method was validated in terms of linearity (R (2) a parts per thousand yenaEuro parts per thousand 0.94), precision (RSD a parts per thousand currency signaEuro parts per thousand 10 %), and accuracy (a parts per thousand currency sign27 %) and used to assess the effect of the histone deacetylase (HDAC) inhibitors SAHA and MS-275 in the murine macrophage-like cell line RAW 264.7. SAHA and MS-275 showed site-specific effects on the acetylation levels of K5 and K8 with the K5(Ac)-K8 and K5-K8(Ac) peptides increasing 2.5-fold and 5-fold upon treatment with SAHA and MS-275, respectively. Assessing lysine acetylation in a site-specific manner is important for gaining a better understanding of the effects of HDAC inhibitors and for clarifying disease mechanisms where lysine acetylation plays a role

Regulation of ROCK1 via Notch1 during breast cancer cell migration into dense matrices

Background The behaviour of tumour cells depends on factors such as genetics and the tumour microenvironment. The latter plays a crucial role in normal mammary gland development and also in breast cancer initiation and progression. Breast cancer tissues tend to be highly desmoplastic and dense matrix as a pre-existing condition poses one of the highest risk factors for cancer development. However, matrix influence on tumour cell gene expression and behaviour such as cell migration is not fully elucidated. Results We generated high-density (HD) matrices that mimicked tumour collagen content of 20 mg/cm3 that were ~14-fold stiffer than low-density (LD) matrix of 1 mg/cm3. Live-cell imaging showed breast cancer cells utilizing cytoplasmic streaming and cell body contractility for migration within HD matrix. Cell migration was blocked in the presence of both the ROCK inhibitor, Y-27632, and the MMP inhibitor, GM6001, but not by the drugs individually. This suggests roles for ROCK1 and MMP in cell migration are complicated by compensatory mechanisms. ROCK1 expression and protein activity, were significantly upregulated in HD matrix but these were blocked by treatment with a histone deacetylase (HDAC) inhibitor, MS-275. In HD matrix, the inhibition of ROCK1 by MS-275 was indirect and relied upon protein synthesis and Notch1. Inhibition of Notch1 using pooled siRNA or DAPT abrogated the inhibition of ROCK1 by MS-275. Conclusion Increased matrix density elevates ROCK1 activity, which aids in cell migration via cell contractility. The upregulation of ROCK1 is epigenetically regulated in an indirect manner involving the repression of Notch1. This is demonstrated from inhibition of HDACs by MS-275, which caused an upregulation of Notch1 levels leading to blockade of ROCK1 expression