Promising Epigenetic Target Research Articles

Covalent-Fragment Screening of BRD4 Identifies a Ligandable Site Orthogonal to the Acetyl-Lysine Binding Sites.

BRD4, a member of the bromodomain and extraterminal domain (BET) family, has emerged as a promising epigenetic target in cancer and inflammatory disorders. All reported BET family ligands bind within the bromodomain acetyl-lysine binding sites and competitively inhibit BET protein interaction with acetylated chromatin. Alternative chemical probes that act orthogonally to the highly conserved acetyl-lysine binding sites may exhibit selectivity within the BET family and avoid recently reported toxicity in clinical trials of BET bromodomain inhibitors. Here, we report the first identification of a ligandable site on a bromodomain outside the acetyl-lysine binding site. Inspired by our computational prediction of hotspots adjacent to nonhomologous cysteine residues within the C-terminal BRD4 bromodomain (BRD4-BD2), we performed a midthroughput mass spectrometry screen to identify cysteine-reactive fragments that covalently and selectively modify BRD4. Subsequent mass spectrometry, NMR, and computational docking analyses of electrophilic fragment hits revealed a novel ligandable site near Cys356 that is unique to BRD4 among human bromodomains. This site is orthogonal to the BRD4-BD2 acetyl-lysine binding site as Cys356 modification did not impact binding of the pan-BET bromodomain inhibitor JQ1 in fluorescence polarization assays nor an acetylated histone peptide in AlphaScreen assays. Finally, we tethered our top-performing covalent fragment to JQ1 and performed NanoBRET assays to provide proof of principle that this orthogonal site can be covalently targeted in intact human cells. Overall, we demonstrate the potential of targeting sites orthogonal to bromodomain acetyl-lysine binding sites to develop bivalent and covalent inhibitors that displace BRD4 from chromatin.

HDAC8 Maintain Cytoskeleton Integrity Via Homologous Recombination and Represent a Novel Therapeutic Target in Multiple Myeloma

Multiple Myeloma (MM) is a plasma cell malignancy vulnerable to epigenetic intervention, with histone deacetylases (HDACs) emerging as the most promising epigenetic targets in combination with current anti-myeloma agents. Pan-HDAC inhibitors are effective as therapeutic agents both in preclinical and clinical setting; however, there is an increasing emphasis on understanding the biological and molecular roles of individual HDACs to limit toxicities observed with pan-HDAC inhibitors. Based on correlation with patient outcome in three independent myeloma datasets, we have evaluated the functional role of HDAC8, a member of Class I HDAC isoenzymes, in MM. Unlike other isoforms, there is limited information about molecular and epigenomic functions of HDAC8. We have previously confirmed expression of HDAC8 in a large panel of MM cell lines, where it is localized predominantly to cytoplasm. Moreover, genetic and pharmacological modulation of HDAC8 with RNAi and specific inhibitor PCI-34051 resulted in a significant inhibition of myeloma cell proliferation and decrease in colony formation (p<.001). HDAC8 inhibition led to an increase in the ongoing spontaneous and radiation-induced DNA damage in MM cells by affecting DNA repair via the homologous recombination (HR) pathway, suggesting a novel function of HDAC8 in promoting HR and DNA repair in MM cells. Using laser micro-irradiation in MM1S and U2OS cells, we observed HDAC8 recruitment to DSBs sites and its co-localization with Rad51 and Scm3, a member of cohesin complex. A transcriptomic analysis of HDAC8 knock-down cells also shows perturbation of number of cytoskeleton-related genes confirming significant role of HAD8 in cytoskeleton rearrangement in MM. Mass-spectrometry analysis to identify the HDAC8 substrates in MM cells is currently ongoing. Classical pan-HDACi, such as SAHA (vorinostat), bind to HDAC8 with substantially diminished activity (IC50 = 2 μM), reflecting a unique binding site of this isoform. To discover and validate new small molecules with HDAC8 subtype selectivity, we have explored the efficacy of OJI-1, a novel selective and potent HDAC8 inhibitor (IC50 = 0.8 nM) with modest inhibition of HDAC6 (1200 nM). Treatment with OJI-1 selectively impact cell viability of a large panel of MM cell lines (n=20) in a time and dose dependent manner, while sparing healthy donors PBMC both in resting and activated state (n=3). The significantly higher IC50 observed in PBMCs suggests a favorable therapeutic index. Western blotting analysis confirmed target selectivity with significant time and dose dependent decrease in H3 and H4 acetylation in MM cells treated with OJI-1. Moreover, pharmacological inhibition of HDAC8 specifically inhibited HR but not non-homologous end joining. These data suggest that targeting of HDAC8 using OJI-1 could be effective treatment approach in MM. Based on molecular data combination studies and in vivo evaluation are ongoing. In conclusion, our results provide insight into the role of HDAC8 in DNA stability and cell growth and viability which can be exploited in future for therapeutic application alone and in combination in MM. Disclosures Munshi: Takeda: Consultancy; Janssen: Consultancy; Amgen: Consultancy; Abbvie: Consultancy; Janssen: Consultancy; Celgene: Consultancy; Takeda: Consultancy; Adaptive: Consultancy; Amgen: Consultancy; Adaptive: Consultancy; Abbvie: Consultancy; Oncopep: Consultancy; Oncopep: Consultancy; Celgene: Consultancy.

HDAC8 Mediates Homologous Recombination and Cytoskeleton Integrity in Myeloma with Potential Impact on Cell Growth and Survival

The chromatin structure and histone modifications have emerged as essential epigenetic layers affecting gene expression changes in cancer. In recent years, both clinical and preclinical studies have confirmed that MM is vulnerable to epigenetic intervention, with histone deacetylases (HDACs) emerging as the most promising epigenetic targets. Although Pan-HDAC inhibitors are effective as therapeutic agents, there is an increasing emphasis on understanding the biological and molecular roles of individual HDACs.

Synthesis of reversible PAD4 inhibitors via copper-catalyzed C−H arylation of benzimidazole

PAD4 is a promising epigenetic drug target for various cancers and immune diseases. In this work, we applied a Cu-catalyzed C–H arylation reaction of N-heteroarene to the synthesis of complex non-covalent PAD4 inhibitors bearing a bi-heteroaryl pharmacophore. This strategy allowed us to access various analogs of C2-aryl substituted benzimidazoles from a common benzimidazole core and easily accessible aryl iodides. Preliminary SAR studies revealed the indole motif of GSK-484 is critical to its activity. Replacing the N-cyclopropylmethyl group to N-benzyl group on the indole ring of GSK-484 resulted in more than 5-fold increase in cell killing efficacy against 4T1 cell line.

LSD1 inhibition by tranylcypromine derivatives interferes with GFI1-mediated repression of PU.1 target genes and induces differentiation in AML.

LSD1 has emerged as a promising epigenetic target in the treatment of acute myeloid leukemia (AML). We used two murine AML models based on retroviral overexpression of Hoxa9/Meis1 (H9M) or MN1 to study LSD1 loss of function in AML. The conditional knockout of Lsd1 resulted in differentiation with both granulocytic and monocytic features and increased ATRA sensitivity and extended the survival of mice with H9M-driven AML. The conditional knockout led to an increased expression of multiple genes regulated by the important myeloid transcription factors GFI1 and PU.1. These include the transcription factors GFI1B and IRF8. We also compared the effect of different irreversible and reversible inhibitors of LSD1 in AML and could show that only tranylcypromine derivatives were capable of inducing a differentiation response. We employed a conditional knock-in model of inactive, mutant LSD1 to study the effect of only interfering with LSD1 enzymatic activity. While this was sufficient to initiate differentiation, it did not result in a survival benefit in mice. Hence, we believe that targeting both enzymatic and scaffolding functions of LSD1 is required to efficiently treat AML. This finding as well as the identified biomarkers may be relevant for the treatment of AML patients with LSD1 inhibitors.

Overexpression of SMYD3 in Ovarian Cancer is Associated with Ovarian Cancer Proliferation and Apoptosis via Methylating H3K4 and H4K20.

Background: Epigenetic regulation has been verified as a key mechanism in tumorigenesis. SET and MYND domain-containing protein 3 (SMYD3), a histone methyltransferase, is a promising epigenetic therapeutic target and is overexpressed in numerous human tumors. SMYD3 can promote oncogenic progression by methylating lysines to integrate cytoplasmic kinase signaling cascades or by methylating histone lysines to regulate specific gene transcription. However, the exact role of SMYD3 in the progression of ovarian cancer is still unknown.Methods: Immunohistochemistry was employed to test SMYD3 expression in ovarian cancer tissues from clinical patients. CCK-8 assay, Real-time cell analysis (RTCA), colony formation assay, cell cycle and apoptosis tested by Flow cytometer were employed to test the effects of SMYD3 on cell proliferation and apoptosis in ovarian cancer cell lines. A PCR array was used to identify the downstream targets of SMYD3. And, PCR and Western blot were used to verify their expression. The binding of SMYD3 on the promoter of target genes were tested by ChIP assays. We also use nude mice subcutaneous tumor model and patient-derived xenograft (PDX) model to investigate the tumor promotive function of SMYD3 in vivo.Results: SMYD3 expression was higher in ovarian cancer tissues and cell lines than in normal ovarian epithelial tissue and human ovarian surface epithelial cells (HOSEpiC). After silencing SMYD3, the proliferation of ovarian cancer cells was significantly inhibited in vitro. In addition, the SMYD3-specific small-molecule inhibitor BCI-121 suppressed ovarian cancer cell proliferation. Downregulation of SMYD3 led to S phase arrest and increased the cell apoptosis rate. Furthermore, a PCR array revealed that SMYD3 knockdown caused the upregulation of the cyclin-dependent kinase (CDK) inhibitors CDKN2A (p16INK4), CDKN2B (p15INK4B), CDKN3 and CDC25A, which may be responsible for the S phase arrest. In addition, the upregulation of CD40LG and downregulation of BIRC3 may explain the increased cell apoptosis rate after silencing SMYD3. We also discovered that SMYD3 bound on the promoter of CDKN2A and down-regulated its expression by triple-methylating H4K20. In addition, SMYD3 bound on the promoter of BIRC3 and up-regulated its expression by triple-methylating H3K4. Finally, knocking down SMYD3 could inhibit ovarian cancer growth in nude mice subcutaneous tumor model and PDX model.Conclusion: Our results demonstrated that SMYD3 was overexpressed in ovarian cancer and contributes to the regulation of tumor proliferation and apoptosis via SMYD3-H4K20me3-CDKN2A pathway and SMYD3-H3K4me3-BIRC3 pathway. Thus, SMYD3 is a promising epigenetic therapeutic target for ovarian cancer.

GLP overexpression is associated with poor prognosis in Chronic Lymphocytic Leukemia and its inhibition induces leukemic cell death.

Background Heterodimeric methyltransferases GLP (EHMT1/KMT1D) and G9a (EHMT2/KMT1C) are two closely related enzymes that promote the monomethylation and dimethylation of histone H3 lysine 9. Dysregulation of their activity has been implicated in several types of human cancer. Patients and methods Here, in order to investigate whether GLP/G9a exerts any impact on Chronic Lymphocytic Leukemia (CLL), GLP/G9a expression levels were assessed in a cohort of 50 patients and the effects of their inhibition were verified for the viability of CLL cells. Also, qRT-PCR was used to investigate the transcriptional levels of GLP/G9a in CLL patients. In addition, patient samples were classified according to ZAP-70 protein expression by flow cytometry and according to karyotype integrity by cytogenetics analysis. Finally, a selective small molecule inhibitor for GLP/G9a was used to ascertain whether these methyltransferases influenced the viability of MEC-1 CLL cell lineage. Results mRNA analysis revealed that CLL samples had higher levels of GLP, but not G9a, when compared to non-leukemic controls. Interestingly, patients with unfavorable cytogenetics showed higher expression levels of GLP compared to patients with favorable karyotypes. More importantly, GLP/G9a inhibition markedly induced cell death in CLL cells. Conclusion Taken together, these results indicate that GLP is associated with a worse prognosis in CLL, and that the inhibition of GLP/G9a influences CLL cell viability. Altogether, the present data demonstrate that these methyltransferases can be potential markers for disease progression, as well as a promising epigenetic target for CLL treatment and the prevention of disease evolution.

Multimodal HDAC Inhibitors with Improved Anticancer Activity

Histone deacetylases (HDACs) play a significant role in the proliferation and dissemination of cancer and represent promising epigenetic drug targets. The HDAC inhibitor vorinostat featuring a zinc-binding hydroxamate fragment was already clinically approved. However, HDAC inhibitors containing hydroxamic acids are often hampered by acquired or intrinsic drug resistance and may lead to enhanced tumor aggressiveness. In order to overcome these drawbacks of hydroxamate HDAC inhibitors, a series of multimodal derivatives of this compound class, including such with different zinc-binding groups, was recently developed and showed promising anticancer activity. This review provides an overview of the chemistry and pleiotropic anticancer modes of action of these conceptually new HDAC inhibitors.

Lsd1 inhibition induces differentiation and decreases leukemic stem cell frequency in Hoxa9/Meis1-driven AML

Lysine specific demethylase 1 (LSD1) represents a promising epigenetic target in the treatment of Acute Myeloid Leukemia (AML) and inhibition of LSD1 has been shown to facilitate a response of AML cells to all-trans retinoic acid (ATRA). In this project, we have now modeled the effect of pharmacological or genetic inactivation of LSD1 in two different murine leukemia models based on the retroviral overexpression of either Hoxa9/Meis1 (H/M) or MN1. We transformed bone marrow cells from conditional LSD1 knock-out (KO) mice.

LSD1 Inhibition Induces Differentiation and Reduces the Frequency of Leukemia-Initiating Cells in Hoxa9/Meis1-Induced Acute Myeloid Leukemia

The histone demethylating enzyme LSD1 has emerged as a promising epigenetic target in the treatment of acute myeloid leukemia (AML). Inhibition of LSD1 has been shown to induce differentiation and facilitate the responsiveness of AML cells to all-trans retinoic acid (ATRA). As it remains unclear how LSD1 inhibition induces differentiation, we have now used a murine leukemia model based on retroviral overexpression of Hoxa9 and Meis1 to study the effect of of both genetic inactivation as well as pharmacological inhibition of LSD1.We transformed bone marrow cells from conditional LSD1 knock-out (KO) mice to confirm the responsiveness of Hoxa9/Meis1-induced leukemias to an inhibition of LSD1. Marked granulocytic differentiation was observed in these cells upon induction of the LSD1 KO. The cells also showed a decrease in expression of c-Kit by 53 %. Furthermore the cells acquired the mature granulocytic marker Ly6G. We also found an increased sensitivity to a treatment with ATRA upon induction of the LSD1 KO. We then transplanted Hoxa9/Meis1-transformed cells from conditional LSD1 KO mice into lethally irradiated mice and induced the LSD1 KO 20 days after transplantation by treatment with tamoxifen. This extended the survival of mice from a median of 48 days for control-treated mice (N=5) to a median of 78 days for tamoxifen-treated mice (N=5) (p=0.0021).We then treated Hoxa9/Meis1-transformed myeloid progenitors with three irreversible (tranylcypromine, trans-N-((2-methoxypyridin-3-yl)methyl)-2-phenylcyclopropan-1-amine (ORY86, AW69), (1S,2R)-2-phenyl-N-(piperidin-4-ylmethyl)cyclopropanamine (AW84)) and three reversible LSD1 inhibitors (MS120 (=Namoline), MS142, SP2509) alone or in combination with ATRA. We observed signs of granulocytic differentiation with the irreversible inhibitors and a reduction in c-Kit expression from 92% to 25% after treatment with AW69. Colony formation of Hoxa9/Meis1-transformed myeloid progenitors was reduced by 70% compared to control cells after treatment with AW69 for 96 hrs. Treatment with AW69 and AW84 also enhanced the response of leukemia cells to ATRA by enhancing the anti-proliferative effect and by reducing colony formation by 99% in Hoxa9/Meis1 cells in the combination. The reversible LSD1 inhibitors exhibited only minor effects on differentiation even when used at effective concentrations.We also analyzed the effect of LSD1 inhibition on leukemia-initiating cell frequency. We therefore treated Hoxa9/Meis1-transformed myeloid progenitors with AW69 in vitro for a total of 96 hrs and then assessed the LIC frequency in a limiting-dilution assay. In this assay, AW69 induced a striking reduction in LIC frequency from 1/40 (95 % CI 1/10.7 to 1/175) to 1/3200 (95 % CI 1/835.5 to 1/12101) (p < 0.001).As the differentiating effect of LSD1 inhibition in our model was limited to the irreversible inhibitors we then wanted to investigate if the effect was due to interference with the enzymatic activity of LSD1. We therefore used the combination of Hoxa9 and Meis1 to transform cells from mice carrying a conditional knock-in (KI) of an enzymatically inactive form of LSD1 (LSD1mut) and assessed its consequence both in vitro and in vivo. Similar to the effect seen with the conditional KO we observed granulocytic differentiation upon induction of the conditional LSD1mut KI as well as a downregulation of c-Kit expression. However, we did not find a survival benefit in vivo upon induction of the conditional LSD1mut KI, but observed a more differentiated immunophenotype in the developing leukemias.In summary, LSD1 depletion or inhibition using tranylcypromine or its derivatives induces granulocytic differentiation, reduces LIC frequency and induces a survival benefit in Hoxa9/Meis1-driven AML. Our data further suggest that these effects of LSD1 inhibition are mediated by interfering with both enzymatic and scaffolding functions of LSD1. DisclosuresLübbert:Ratiopharm: Other: Study drug valproic acid; Janssen-Cilag: Other: Travel Funding, Research Funding; Celgene: Other: Travel Funding. Berg:Astellas: Other: Travel Funding; Alexion: Other: Travel Funding; Celgene: Other: Travel Funding.

Inhibition of G9a/GLP Complex Promotes Long-Term Potentiation and Synaptic Tagging/Capture in Hippocampal CA1 Pyramidal Neurons.

Epigenetic regulations play an important role in regulating the learning and memory processes. G9a/G9a-like protein (GLP) lysine dimethyltransferase complex controls a prominent histone H3 lysine9 dimethylation (H3K9me2) that results in transcriptional silencing of the chromatin. Here, we report that the inhibition of G9a/GLP complex by either of the substrate competitive inhibitors UNC 0638 or BIX 01294 reinforces protein synthesis-independent long-term potentiation (early-LTP) to protein synthesis-dependent long-term potentiation (late-LTP). The reinforcement effect was observed if the inhibitors were present during the induction of early-LTP and in addition when G9a/GLP complex inhibition was carried out by priming of synapses within an interval of 30 min before or after the induction of early-LTP. Surprisingly, the reinforced LTP by G9a/GLP complex inhibition was able to associate with a weak plasticity event from nearby independent synaptic populations, resulting in synaptic tagging/capture (STC). We have identified brain-derived neurotrophic factor (BDNF) as a critical plasticity protein that maintains G9a/GLP complex inhibition-mediated LTP facilitation and its STC. Our study reveals an epigenetic mechanism for promoting plasticity and associativity by G9a/GLP complex inhibition, and it may engender a promising epigenetic target for enhancing memory in neural networks.

Dominant Expression of DCLK1 in Human Pancreatic Cancer Stem Cells Accelerates Tumor Invasion and Metastasis.

Patients with pancreatic cancer typically develop tumor invasion and metastasis in the early stage. These malignant behaviors might be originated from cancer stem cells (CSCs), but the responsible target is less known about invisible CSCs especially for invasion and metastasis. We previously examined the proteasome activity of CSCs and constructed a real-time visualization system for human pancreatic CSCs. In the present study, we found that CSCs were highly metastatic and dominantly localized at the invading tumor margins in a liver metastasis model. Microarray and siRNA screening assays showed that doublecortin-like kinase 1 (DCLK1) was predominantly expressed with histone modification in pancreatic CSCs with invasive and metastatic potential. Overexpression of DCLK1 led to amoeboid morphology, which promotes the migration of pancreatic cancer cells. Knockdown of DCLK1 profoundly suppressed in vivo liver metastasis of pancreatic CSCs. Clinically, DCLK1 was overexpressed in the metastatic tumors in patients with pancreatic cancer. Our studies revealed that DCLK1 is essential for the invasive and metastatic properties of CSCs and may be a promising epigenetic and therapeutic target in human pancreatic cancer.

Investigation of EZH2 pathways for novel epigenetic treatment strategies in oropharyngeal cancer.

BackgroundIn recent decades, the incidence of oropharyngeal squamous cell carcinoma (OPSCC) has been rising worldwide as a result of increasing oncogenic human papillomavirus (HPV) infections in the oropharynx. EZH2 is an epigenetic regulatory protein associated with tumor aggressiveness and negative survival outcomes in several human cancers. We aimed to determine the role of EZH2 as a potential therapeutic epigenetic target in HPV-positive and negative OPSCC.MethodsThe expression of EZH2 was measured by immunohistochemistry (IHC) and droplet digital PCR (ddPCR) in 2 HPV-positive and 2 HPV-negative cell lines. The cell lines were then cultured and treated with one of 3 EZH2 epigenetic inhibitors (3-deazaneplanocin A, GSK-343 and EPZ005687) or DMSO (control). Following 2, 4 and 7 days of treatment, cells were analyzed and compared by gene expression, cell survival and proliferation assays.ResultsEZH2 targeting resulted in greater inhibition of growth and survival in HPV-positive compared to HPV-negative cells lines. The expression profile of genes important in OPSCC also differed according to HPV-positivity for Ki67, CCND1, MET and PTEN/PIK3CA, but remained unchanged for EGFR, CDKN2A and p53.ConclusionInhibition of EZH2 has anti-tumorigenic effects on OPSCC cells in culture that is more pronounced in HPV-positive cell lines. EZH2 is a promising epigenetic target for the treatment of OPSCC.

Homology modeling of parasite histone deacetylases to guide the structure-based design of selective inhibitors

Histone deacetylases (HDACs) are promising epigenetic targets for the treatment of various diseases, including cancer and neurodegenerative disorders. There is evidence that they can also be addressed to treat parasitic infections. Recently, the first X-ray structure of a parasite HDAC was published, Schistosoma mansoni HDAC8, giving structural insights into its inhibition. However, most of the targets from parasites of interest still lack this structural information. Therefore, we prepared homology models of relevant parasitic HDACs and compared them to human and S. mansoni HDACs. The information about known S. mansoni HDAC8 inhibitors and compounds that affect the growth of Trypanosoma, Leishmania and Plasmodium species was used to validate the models by docking and molecular dynamics studies. Our results provide analysis of structural features of parasitic HDACs and should be helpful for selecting promising candidates for biological testing and for structure-based optimisation of parasite-specific inhibitors.

Abstract 3035: HDAC8 is recruited to DNA double strand breaks sites and affects the homologous recombination efficiency in multiple myeloma

Abstract Epigenomic changes have emerged as an important player of cellular regulation and understanding of pathogenesis of Multiple Myeloma (MM) as well as of other cancers. In recent years, both clinical and preclinical studies have confirmed that MM is vulnerable to epigenetic intervention, with histone deacetylases (HDACs) emerging as the most promising epigenetic targets. Although Pan-HDAC inhibitors are effective as therapeutic agents, there is increasing emphasis on understanding the biological and molecular roles of individual HDACs. Here we evaluated the role of HDAC8, a member of Class I HDAC isoenzymes in MM. First, we evaluated the expression of HDAC8 in 172 newly-diagnosed MM patients from the IFM myeloma dataset and observed HDAC8 overexpression as well as its significant correlation with poor survival outcome (P&amp;lt;0.0015). We further evaluated the expression of HDAC8 in HMCLs (probe ID_223909-s_at, 223345_at) and confirmed the high expression and its cytoplasmic and nuclear localization in all MM cells lines studied. The HDAC8 depletion (lentiviral-shRNA)in HMCLs resulted in significant inhibition of proliferation of MM cells as measured by 3[H]-thymidine assay, and as decrease in colony formation evaluated after 3 weeks post transfection (P&amp;lt;.001). We observed similar cell growth inhibition using PCI-34051, a small molecule HDAC8 inhibitor. Interestingly, the combination of HDAC8 inhibitor with melphalan or bendamustine enhanced the anti-MM effects of the DNA damaging agents (all p&amp;lt;0.01). Immunoblotting analysis using a panel of 15 antibodies for DNA damage response (DDR) pathway confirmed increased levels of DNA damage in OPM2 and MM1S cells lacking HDAC8. Consistent with this observation HDAC8 depletion led to decreased homologous recombination (HR) activity as measured by plasmid-based assay. We performed singe cell electrophoresis (Comet-assay) and observed decreased repair of DSBs after IR in OPM2-HDAC8 depleted cells as well as after pharmacologic inhibition of HDAC8. Importantly, using laser micro-irradiation in myeloma and U2OS cells, we observed HDAC8 recruitment to DSBs sites. The HDAC8 was co-localized and co-immunoprecipitated with Rad51 after IR, and with Scm3, member of cohesion complex suggesting its relation with cytoskeleton. In MM1s cells containing a stably integrated Rad51-luciferase reporter construct, the addition of HDAC8 inhibitor suppressed Rad51, confirming the immunoblotting findings. A mass spectromentry-based analysis identified the HDAC8-interacting complexes with cohesion- (cohesin subunit SA-2, Condensin-2) and DDR-key components (Mre11a, XRCC1, Rad50). In conclusion, our results demonstrate impact of epigenomic change on DNA integrity through connection between HDAC8 and DNA damage response pathway, and provide insights into the effect of HDAC8 on DNA stability and cell growth and survival that may have therapeutic implications in MM. Citation Format: Maria Gkotzamanidou, Masood Shammas, Jesús Martín Sánchez, Mehmet Kemal Samur, Stephane Minvielle, Florence Magrangeas, Herve Avet-Loiseau, Athanasios-Meletios Dimopoulos, Kenneth C. Anderson, Nikhil C. Munshi. HDAC8 is recruited to DNA double strand breaks sites and affects the homologous recombination efficiency in multiple myeloma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3035. doi:10.1158/1538-7445.AM2015-3035

Observed bromodomain flexibility reveals histone peptide- and small molecule ligand-compatible forms of ATAD2.

Preventing histone recognition by bromodomains emerges as an attractive therapeutic approach in cancer. Overexpression of ATAD2 (ATPase family AAA domain-containing 2 isoform A) in cancer cells is associated with poor prognosis making the bromodomain of ATAD2 a promising epigenetic therapeutic target. In the development of an invitro assay and identification of small molecule ligands, we conducted structure-guided studies which revealed a conformationally flexible ATAD2 bromodomain. Structural studies on apo-, peptide-and small molecule-ATAD2 complexes (by co-crystallization) revealed that the bromodomain adopts a 'closed', histone-compatible conformation and a more 'open' ligand-compatible conformation of the binding site respectively. An unexpected conformational change of the conserved asparagine residue plays an important role in driving the peptide-binding conformation remodelling. We also identified dimethylisoxazole-containing ligands as ATAD2 binders which aided in the validation of the invitro screen and in the analysis of these conformational studies.

HDAC8 Mediates Homologous Recombination and Cytoskeleton Integrity in Myeloma with Potential Impact on Cell Growth and Survival

Epigenomic changes have become an important component of cellular regulation and ultimately, of our understanding of oncogenomics in Multiple Myeloma (MM) as well as in other cancers. In recent years, both clinical and preclinical studies have confirmed that MM is vulnerable to epigenetic intervention, with histone deacetylases (HDACs) emerging as the most promising epigenetic targets. Although Pan-HDAC inhibitors are effective as therapeutic agents, there is increasing emphasis on understanding the biological and molecular roles of individual HDACs. Here we have evaluated the role of HDAC8, a member of Class I HDAC isoenzymes in MM.First, we evaluated the expression of HDAC8 in 172 newly-diagnosed MM patients from the IFM myeloma dataset and observed HDAC8 overexpression as well as its significant correlation with poor survival outcome (p<0.0015). We further evaluated the expression of HDAC8 in HMCLs (probe ID_223909-s_at, 223345_at) and confirmed the high expression and its cytoplasmic and nuclear localization in all six MM cells lines studied (MM1S, OPM2, RPMI8226, U266, MOLP8 and NCI-H929) and in primary bone marrow plasma cells (CD138+) from newly diagnosed MM patients (N=3).To address the functional role of HDAC8 in MM biology and to evaluate its potency as therapeutic target, we used a lentiviral-shRNA delivery system for HDAC8-knockdown in MM1S and OPM2 myeloma cells. The HDAC8 depletion in HMCLs resulted in significant inhibition of proliferation of MM at 1 week as measured by 3[H]-thymidine assay, and as decrease in colony formation evaluated after 3 weeks post transfection (p<.001). We observed similar cell growth inhibition using PCI-34051, a small molecule HDAC8 inhibitor. Interestingly, the combination of HDAC8 inhibitor with melphalan or bendamustine enhanced the anti-MM effects of the DNA damaging agents (all p<0.01) and was confirmed to be synergistic using Calcusyn software. Immunoblotting using a panel of 15 antibodies for DNA damage response (DDR) pathway proteins (including γH2Ax, pATM, pATR, pBRCA1, pBRCA2, pCHK2, pCHK1, ku70, RPA70, 53BP1, DNA-PKs, pP53) confirmed increased levels of DNA damage in OPM2 and MM1S cells with HDAC8 depletion. In consistence with this observation HDAC8 knockdown led to decreased homologous recombination (HR) activity as measured by a transient direct repeat DsRED-GFP/I-SceI plasmid-based assay. We performed singe cell electrophoresis under neutral conditions (comet-assay) in OPM2 and MM1S after HDAC8 depletion with or without exposure to gamma irradiation (γ-IR), and in OPM2 and MM1S cells treated and untreated with HDAC8 inhibitor in combination with γ-IR and observed decreased repair of DSBs after γ-IR measured following HDAC8 knockdown as well as following treatment of the cells with HDAC8 inhibitor. Importantly, using laser micro-irradiation in myeloma and U2OS cells, we observed HDAC8 recruitment to DSBs sites. Moreover, the HDAC8 protein was co-localized and co-immunoprecipitated with Rad51 after IR, and with Scm3, member of cohesion complex after mitotic sychronization, suggesting its relation with cytoskeleton. We confirmed the significant alteration in expression of cohesion complex members SMC1 and RAD21 after HDAC8 depletion and re-overexpression in MM cells. In MM1s cells containing a stably integrated Rad51-luciferase reporter construct, the addition of HDAC8 inhibitor resulted in a decrease in Rad51 promoter activity, confirming the immunoblotting findings. An ongoing mass spectromentry-based analysis is expected to identify thoroughly the HDAC8-interacting proteins.In conclusion, our results demonstrate an impact of aberrant epigenome on DNA integrity through connection between HDAC8 and DDR pathway, and provide insights into the effect of HDAC8 on cellular growth and survival with potent therapeutic implications in MM. DisclosuresAnderson:Celgene: Consultancy; Sanofi-Aventis: Consultancy; Onyx: Consultancy; Acetylon: Scientific Founder, Scientific Founder Other; Oncoprep: Scientific Founder Other; Gilead Sciences: Consultancy.

Inhibition of bladder cancer invasion by Sp1-mediated BTG2 expression via inhibition of DNA methyltransferase 1.

Significantly lower endogenous expression of B-cell translocation gene 2 (BTG2) was observed in human muscle-invasive bladder cancers (MIBC) than matched normal tissues and non-muscle invasive bladder cancers (NMIBC). BTG2 expression was inversely correlated with increased expression of the DNA methyltransferases DNMT1 and DNMT3a in MIBC, but not NMIBC, suggesting a potential role for BTG2 expression in muscle invasion of bladder cancer. Over 90% of tumor tissues revealed strong methylation at CpG islands of the BTG2 gene, compared with no methylation in the normal tissues, implying epigenetic regulation of BTG2 expression in bladder carcinogenesis. By using EJ bladder cancer cells and the demethylating agent decitabine, transcription of BTG2 was shown to be up-regulated by inhibiting DNMT1 expression via modification at CpG islands. DNMT1 binding to the BTG2 gene further regulated BTG2 expression by chromatin remodeling, such as H3K9 dimethylation and H3K4 trimethylation, and Sp1 activation. Induced BTG2 expression significantly reduced EJ cell tumorigenesis and invasiveness together with induction of G2 /M arrest. These results demonstrate an important role for the BTG2(/TIS21/PC3) gene in the progression of bladder cancers, and suggest that BTG2(/TIS21/PC3) is a promising epigenetic target for prevention of muscle invasion in human bladder cancers.

Structural insights into binding of small molecule inhibitors to Enhancer of Zeste Homolog 2.

Enhancer of Zeste Homolog 2 (EZH2) is a SET domain protein lysine methyltransferase (PKMT) which has recently emerged as a chemically tractable and therapeutically promising epigenetic target, evidenced by the discovery and characterization of potent and highly selective EZH2 inhibitors. However, no experimental structures of the inhibitors co-crystallized to EZH2 have been resolved, and the structural basis for their activity and selectivity remains unknown. Considering the need to minimize cross-reactivity between prospective PKMT inhibitors, much can be learned from understanding the molecular basis for selective inhibition of EZH2. Thus, to elucidate the binding of small-molecule inhibitors to EZH2, we have developed a model of its fully-formed cofactor binding site and used it to carry out molecular dynamics simulations of protein-ligand complexes, followed by molecular mechanics/generalized born surface area calculations. The obtained results are in good agreement with biochemical inhibition data and reflect the structure-activity relationships of known ligands. Our findings suggest that the variable and flexible post-SET domain plays an important role in inhibitor binding, allowing possibly distinct binding modes of inhibitors with only small variations in their structure. Insights from this study present a good basis for design of novel and optimization of existing compounds targeting the cofactor binding site of EZH2.

Molecular dynamics simulations indicate an induced-fit mechanism for LSD1/CoREST-H3-histone molecular recognition

BackgroundLysine Specific Demethylase (LSD1 or KDM1A) in complex with its co-repressor protein CoREST catalyzes the demethylation of the H3 histone N-terminal tail and is currently one of the most promising epigenetic targets for drug discovery against cancer and neurodegenerative diseases. Models of non-covalent binding, such as lock and key, induced-fit, and conformational selection could help explaining the molecular mechanism of LSD1/CoREST-H3-histone association, thus guiding drug discovery and design efforts. Here, we quantify the extent to which LSD1/CoREST substrate binding is consistent with these hypothetical models using LSD1/CoREST conformational ensembles obtained through extensive explicit solvent molecular dynamics (MD) simulations.ResultsWe find that an induced-fit model is the most representative of LSD1/CoREST-H3-histone non-covalent binding and accounts for the local conformational changes occurring in the H3-histone binding site. We also show that conformational selection – despite in principle not ruled out by this finding – is minimal, and only relevant when global properties are considered, e.g. the nanoscale motion of the LSD1/CoREST clamp.ConclusionThe induced-fit mechanism revealed by our MD simulation study will aid the inclusion of protein dynamics for the discovery and design of LSD1 inhibitors targeting the H3-histone binding region. On a general basis, our study indicates the importance of using multiple metrics or selection schemes when testing alternative hypothetical mechanistic models of non-covalent binding.