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Supplementary Table S1 and Fig S1 and S2. Supplementary Table 1: qPCR primers used in this study; Supplementary Figure S1: Structures of compounds screened in Figure 1A and 1B; Supplementary Figure S2: Proximity ligation assay (PLA) analyzing SIN3A-MAD interactions

Supplementary Table S2. List of genes with expression regulated by selamectin treatment

Triple-negative breast cancers (TNBC) lacking estrogen, progesterone, and HER2 receptors account for 10% to 20% of breast cancer and are indicative of poor prognosis. The development of effective treatment strategies therefore represents a pressing unmet clinical need. We previously identified a molecularly targeted approach to target aberrant epigenetics of TNBC using a peptide corresponding to the SIN3 interaction domain (SID) of MAD. SID peptide selectively blocked binding of SID-containing proteins to the paired α-helix (PAH2) domain of SIN3, resulting in epigenetic and transcriptional modulation of genes associated with epithelial–mesenchymal transition (EMT). To find small molecule inhibitor (SMI) mimetics of SID peptide, we performed an in silico screen for PAH2 domain–binding compounds. This led to the identification of the avermectin macrocyclic lactone derivatives selamectin and ivermectin (Mectizan) as candidate compounds. Both selamectin and ivermectin phenocopied the effects of SID peptide to block SIN3–PAH2 interaction with MAD, induce expression of CDH1 and ESR1, and restore tamoxifen sensitivity in MDA-MB-231 human and MMTV-Myc mouse TNBC cells in vitro. Treatment with selamectin or ivermectin led to transcriptional modulation of genes associated with EMT and maintenance of a cancer stem cell phenotype in TNBC cells. This resulted in impairment of clonogenic self-renewal in vitro and inhibition of tumor growth and metastasis in vivo. Underlining the potential of avermectins in TNBC, pathway analysis revealed that selamectin also modulated the expression of therapeutically targetable genes. Consistent with this, an unbiased drug screen in TNBC cells identified selamectin-induced sensitization to a number of drugs, including those targeting modulated genes. Mol Cancer Ther; 14(8); 1824–36. ©2015 AACR.

Significance STAT3 (signal transducer and activator of transcription 3) is a master transcription factor that organizes cellular responses to cytokines and growth factors and is implicated in inflammatory disorders. STAT3 is a well-recognized therapeutic target for human cancer and inflammatory disorders, but how its function is regulated in a cell type–specific manner has been a major outstanding question. We discovered that Stat3 imposes self-directed regulation through controlling transcription of its own regulator homeodomain-interacting protein kinase 2 ( Hipk2 ) in a T helper 17 (Th17) cell–specific manner. Our validation of the functional importance of the Stat3–Hipk2 axis in Th17 cell development in the pathogenesis of T cell–induced colitis in mice suggests an approach to therapeutically treat inflammatory bowel diseases that currently lack a safe and effective therapy.

Summary THAP1 (THAP [Thanatos-associated protein] domain-containing, apoptosis-associated protein 1) is a ubiquitously expressed member of a family of transcription factors with highly conserved DNA-binding and protein-interacting regions. Mutations in THAP1 cause dystonia, DYT6, a neurologic movement disorder. THAP1 downstream targets and the mechanism via which it causes dystonia are largely unknown. Here, we show that wild-type THAP1 regulates embryonic stem cell (ESC) potential, survival, and proliferation. Our findings identify THAP1 as an essential factor underlying mouse ESC survival and to some extent, differentiation, particularly neuroectodermal. Loss of THAP1 or replacement with a disease-causing mutation results in an enhanced rate of cell death, prolongs Nanog, Prdm14, and/or Rex1 expression upon differentiation, and results in failure to upregulate ectodermal genes. ChIP-Seq reveals that these activities are likely due in part to indirect regulation of gene expression., Highlights • Wild-type THAP1 regulates ESC potential, survival, and proliferation • THAP1 is essential for ESC differentiation, particularly neuroectodermal • Thap1C54Y or ΔExon2 ESCs prolong expression of pluripotent genes upon differentiation • Thap1C54Y or ΔExon2 EBs show increased cell death and abnormal differentiation, Ehrlich and colleagues identified THAP1, mutations of which cause dystonia (DYT6), as an essential regulator of ESC survival, proliferation, and differentiation. As THAP1 mutations result in a neurologic disease, this work examined the effects of a causative mutation, C54Y, and an essentially null allele, ΔExon2, on neuronal differentiation.

T-helper 17 (Th17) cells have important functions in adaptor immunity and have also been implicated in inflammatory disorders. The bromodomain and extraterminal domain (BET) family proteins regulate gene transcription during lineage-specific differentiation of naive CD4+ T cells to produce mature T-helper cells. Inhibition of acetyl-lysine binding of the BET proteins by pan-BET bromodomain (BrD) inhibitors, such as JQ1, broadly affects differentiation of Th17, Th1, and Th2 cells that have distinct immune functions, thus limiting their therapeutic potential. Whether these BET proteins represent viable new epigenetic drug targets for inflammatory disorders has remained an unanswered question. In this study, we report that selective inhibition of the first bromodomain of BET proteins with our newly designed small molecule MS402 inhibits primarily Th17 cell differentiation with a little or almost no effect on Th1 or Th2 and Treg cells. MS402 preferentially renders Brd4 binding to Th17 signature gene loci over those of housekeeping genes and reduces Brd4 recruitment of p-TEFb to phosphorylate and activate RNA polymerase II for transcription elongation. We further show that MS402 prevents and ameliorates T-cell transfer-induced colitis in mice by blocking Th17 cell overdevelopment. Thus, selective pharmacological modulation of individual bromodomains likely represents a strategy for treatment of inflammatory bowel diseases.

The importance of BET protein BRD4 in gene transcription is well recognized through the study of chemical modulation of its characteristic tandem bromodomain (BrD) binding to lysine-acetylated histones and transcription factors. However, while monovalent inhibition of BRD4 by BET BrD inhibitors such as JQ1 blocks growth of hematopoietic cancers, it is much less effective generally in solid tumors. Here, we report a thienodiazepine-based bivalent BrD inhibitor, MS645, that affords spatially constrained tandem BrD inhibition and consequently sustained repression of BRD4 transcriptional activity in blocking proliferation of solid-tumor cells including a panel of triple-negative breast cancer (TNBC) cells. MS645 blocks BRD4 binding to transcription enhancer/mediator proteins MED1 and YY1 with potency superior to monovalent BET inhibitors, resulting in down-regulation of proinflammatory cytokines and genes for cell-cycle control and DNA damage repair that are largely unaffected by monovalent BrD inhibition. Our study suggests a therapeutic strategy to maximally control BRD4 activity for rapid growth of solid-tumor TNBC cells.

Histones and their modifications play an important role in the regulation of gene transcription. Numerous modifications, such as acetylation, phosphorylation, methylation, ubiquitination, and SUMOylation, have been described. These modifications almost always co-occur and thereby increase the combinatorial complexity of post-translational modification detection. The domains that recognize these histone modifications often occur in tandem in the context of larger proteins and complexes. The presence of multiple modifications can positively or negatively regulate the binding of these tandem domains, influencing downstream cellular function. Alternatively, these tandem domains can have novel functions from their independent parts. Here we summarize structural and functional information known about major tandem domains and their histone binding properties. An understanding of these interactions is key for the development of epigenetic therapy.

Jumonji domain-containing protein 6 (JMJD6) is a member of the Jumonji C family of Fe(II) and 2-oxoglutarate (2OG) dependent oxygenases. It possesses unique bi-functional oxygenase activities, acting as both an arginine demethylase and a lysyl-hydroxylase. JMJD6 has been reported to be over-expressed in oral, breast, lung, and colon cancers and plays important roles in regulation of transcription through interactions with transcription regulator BRD4, histones, U2AF65, Luc7L3, and SRSF11. Here, we report a structural mechanism revealed by NMR of JMJD6 recognition by the extraterminal (ET) domain of BRD4 in that a JMJD6 peptide (Lys84-Asn96) adapts an α-helix when bound to the ET domain. This intermolecular recognition is established through JMJD6 interactions with the conserved hydrophobic core of the ET domain, and reinforced by electrostatic interactions of JMJD6 with residues in the inter-helical α1-α2 loop of the ET domain. Notably, this mode of ligand recognition is different from that of ET domain recognition of NSD3, LANA of herpesvirus, and integrase of MLV, which involves formation of an intermolecular amphipathic two- or three- strand antiparallel β sheet. Furthermore, we demonstrate that the association between the BRD4 ET domain and JMJD6 likely requires a protein conformational change induced by single-stranded RNA binding.

The BET proteins are major transcriptional regulators and have emerged as new drug targets, but their functional distinction has remained elusive. In this study, we report that the BET family members Brd2 and Brd4 exert distinct genomic functions at genes whose transcription they co-regulate during mouse T helper 17 (Th17) cell differentiation. Brd2 is associated with the chromatin insulator CTCF and the cohesin complex to support cis-regulatory enhancer assembly for gene transcriptional activation. In this context, Brd2 binds the transcription factor Stat3 in an acetylation-sensitive manner and facilitates Stat3 recruitment to active enhancers occupied with transcription factors Irf4 and Batf. In parallel, Brd4 temporally controls RNA polymerase II (Pol II) processivity during transcription elongation through cyclin T1 and Cdk9 recruitment and Pol II Ser2 phosphorylation. Collectively, our study uncovers both separate and interdependent Brd2 and Brd4 functions in potentiating the genetic program required for Th17 cell development and adaptive immunity.

The human IgG repertoire contains endogenous antibodies against beta amyloid (Aβ) that may be relevant to the pathogenesis and treatment of Alzheimer's disease. There have been widely disparate estimates of the levels of these antibodies in human plasma. We identify factors that have contributed to these disparities and describe improved methods for measuring anti-Aβ antibodies in blood. These methods include isolating immunoglobulin by thiophilic chromatography and using chaotropic salts to dislodge weakly bound antibodies without significantly reducing the binding of specific anti-Aβ antibodies. Using these methods, we show that human blood contains polyvalent IgG antibodies that bind to Aβ with relatively low avidity and specificity, as well as IgG antibodies that bind to linear and conformational epitopes on amyloid monomers and aggregates with moderate to high avidity.

// Nidhi Bansal 1 , Kevin Petrie 2 , Rossitza Christova 2,* , Chi-Yeh Chung 3,* , Boris A. Leibovitch 1 , Louise Howell 2 , Veronica Gil 2 , Yordan Sbirkov 2 , EunJee Lee 4 , Joanna Wexler 1 , Edgardo V. Ariztia 1 , Rajal Sharma 1 , Jun Zhu 4 , Emily Bernstein 5 , Ming-Ming Zhou 1 , Arthur Zelent 6 , Eduardo Farias 1 and Samuel Waxman 1 1 Division of Hematology and Oncology, The Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, New York, USA 2 Division of Clinical Studies, Institute of Cancer Research, Sutton, United Kingdom 3 Department of Oncological Sciences, Department of Genetics and Genomic Sciences, Graduate School of Biomedical Sciences, Icahn School of Medicine at Mount Sinai, New York, USA 4 Genetics and Genomic Science, Icahn School of Medicine at Mount Sinai, New York, USA 5 Department of Oncological Sciences, Graduate School of Biomedical Sciences, Icahn School of Medicine at Mount Sinai, New York, USA 6 Division of Hemato-Oncology, Department of Medicine, Sylvester Comprehensive Cancer Center, Miller School of Medicine, University of Miami, Florida, USA * These authors have contributed equally to this work Correspondence to: Samuel Waxman, email: // Keywords : epigenetics, SIN3, PF1, triple negative breast cancer, cancer stem cells Received : July 17, 2015 Accepted : September 24, 2015 Published : October 09, 2015 Abstract Triple negative breast cancer (TNBC) is characterized by a poorly differentiated phenotype and limited treatment options. Aberrant epigenetics in this subtype represent a potential therapeutic opportunity, but a better understanding of the mechanisms contributing to the TNBC pathogenesis is required. The SIN3 molecular scaffold performs a critical role in multiple cellular processes, including epigenetic regulation, and has been identified as a potential therapeutic target. Using a competitive peptide corresponding to the SIN3 interaction domain of MAD (Tat-SID), we investigated the functional consequences of selectively blocking the paired amphipathic α-helix (PAH2) domain of SIN3. Here, we report the identification of the SID-containing adaptor PF1 as a factor required for maintenance of the TNBC stem cell phenotype and epithelial-to-mesenchymal transition (EMT). Tat-SID peptide blocked the interaction between SIN3A and PF1, leading to epigenetic modulation and transcriptional downregulation of TNBC stem cell and EMT markers. Importantly, Tat-SID treatment also led to a reduction in primary tumor growth and disseminated metastatic disease in vivo . In support of these findings, knockdown of PF1 expression phenocopied treatment with Tat-SID both in vitro and in vivo . These results demonstrate a critical role for a complex containing SIN3A and PF1 in TNBC and provide a rational for its therapeutic targeting.

Triple-negative breast cancers (TNBC) lacking estrogen, progesterone, and HER2 receptors account for 10% to 20% of breast cancer and are indicative of poor prognosis. The development of effective treatment strategies therefore represents a pressing unmet clinical need. We previously identified a molecularly targeted approach to target aberrant epigenetics of TNBC using a peptide corresponding to the SIN3 interaction domain (SID) of MAD. SID peptide selectively blocked binding of SID-containing proteins to the paired α-helix (PAH2) domain of SIN3, resulting in epigenetic and transcriptional modulation of genes associated with epithelial–mesenchymal transition (EMT). To find small molecule inhibitor (SMI) mimetics of SID peptide, we performed an in silico screen for PAH2 domain–binding compounds. This led to the identification of the avermectin macrocyclic lactone derivatives selamectin and ivermectin (Mectizan) as candidate compounds. Both selamectin and ivermectin phenocopied the effects of SID peptide to block SIN3–PAH2 interaction with MAD, induce expression of CDH1 and ESR1, and restore tamoxifen sensitivity in MDA-MB-231 human and MMTV-Myc mouse TNBC cells in vitro. Treatment with selamectin or ivermectin led to transcriptional modulation of genes associated with EMT and maintenance of a cancer stem cell phenotype in TNBC cells. This resulted in impairment of clonogenic self-renewal in vitro and inhibition of tumor growth and metastasis in vivo. Underlining the potential of avermectins in TNBC, pathway analysis revealed that selamectin also modulated the expression of therapeutically targetable genes. Consistent with this, an unbiased drug screen in TNBC cells identified selamectin-induced sensitization to a number of drugs, including those targeting modulated genes. Mol Cancer Ther; 14(8); 1824–36. ©2015 AACR.

Introduction: Dearth of clinically validated drug targets is a major challenge in Triple Negative Breast Cancer (TNBC) that limits the treatment options to chemotherapy with intense cytotoxic consequences. We previously identified PAH2 domain of Sin3 protein as potential therapeutic target in TNBC and shown that protein-protein interactions of Sin3 via its PAH-2 domain can be disrupted by decoys (Mad1-SID peptide/small molecule inhibitors) designed based on the Sin3 interaction domain (SID) of transcription factor Mad1. Chromatin regulator Pf1 shown to be overexpressed in breast cancer and interacting with PAH2 domain of Sin3 can also be dissociated from Sin3 complex by Mad1-SID. In this study we identify Pf1 to be a significant contributor to the oncogenic phenotype associated with TNBC. Methods: Pf1 knockdown lines were generated by stably transfecting MDA-MB-231 cells with Pf1-shRNA or scr-shRNA. Cells were cultured in 2D and assayed for cancer stem cell (CSC) markers and ALDH activity by flow cytometry. For colony morphogenesis cells were cultured in 3D Matrigel. qRT-PCR were performed to assay expression of Nanog, Sox2 and Oct4. Fluorescence anisotropy and co-immunoprecipitation assays were used to test Sin3-Mad1 and Sin3-Pf1 interaction in the presence or absence of Mad1-SID and/or Pf1-SID. Results: MDA-MB-231 cells transfected with Pf1-shRNA had over two fold reduced ability to form colonies in 3D Matrigel cultures, with ∼95% of colonies non-invasive in contrast to the invasive star-like colonies formed by cells transfected with Scr-shRNA. Additionally reduction in Pf1 level was accompanied by a ∼1.5-fold reduction (p Conclusion: PAH2 domain of Sin3 and its interaction with Pf1 is potential drug target and Pf-1SID-mediated disruption of Sin3-Pf1 complex as translation relevance for first site-specific epigenetic therapy for TNBC. Citation Format: Nidhi Bansal, Joanna Wexler, Yeon-jin Kwon, Elena C. Gil, Boris Leibovitch, Rajal Sharma, Arthur Zelent, Ming-Ming Zhou, Eduardo Farias, Samuel Waxman. Targeting Sin3-Pf1 complex: Novel site-specific epigenetic therapy for triple negative breast cancer. [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 1989. doi:10.1158/1538-7445.AM2015-1989

INTRODUCTION: Sin3 is a complex scaffolding protein that participates in epigenetic regulation involved in a variety of biological processes, including chromatin remodeling, development, differentiation, cell cycle and survival. Paired Amphipathic domain 2 (PAH2) domain of Sin3 was shown to interact with Sin3 interacting domain (SID)-containing transcription factors, such as Mxd1, KLF10/11, and REST. Previously, we reported that SID transcripts and decoy peptides disrupt the interactions between Sin3-PAH2 domain and SID-containing transcription factors and induce epigenetic reprogramming in TNBC. RESULTS AND CONCLUSION: Here we show that fourteen SMIs mimicking SID decoys were identified by in silico computation from a chemical library composed of 115,000 compounds. We confirmed that selamectin and ivermectin are SID decoys using duo-link assays. Our NMR 15N-HSQC spectroscopy binding results further demonstrated ivermectin, which shares the same binding site as selamectin, directly interacted with PAH2. Moreover, selamectin inhibited Sin3 repression activity as measured by mammalian two-hybrid and in vitro GST pull-down assays. Notably, ivermectin was approved by Food and Drug Administration (FDA) to treat small animals and human. However, the anti-tumorigenic roles for these SMIs had not been investigated in TNBC in vitro and in vivo. Remarkably, these SMIs did not affect growth in 2-dimensional (2D) clonogenecity assay, but significantly suppressed growth in 3D matrigel and tumorsphere cultures. Moreover, selamectin significantly suppressed Myc mammary tumor growth in vivo more than 50 percent without any cytotoxicity. Selamectin and ivermectin induced re-expression of CDH1 and ESR1, restoring tamoxifen sensitivity. Treatment with these compounds inhibited in vitro invasion as measured by Boyden chamber in MCF7/ADR and MDA-MB-231 cells. Selamectin and ivermectin were shown to alleviate multidrug resistance and enhance doxorubicin and paclitaxel cytotoxicity in MCF7/ADR cells, which is unrelated to disruption of Sin3-PAH2. Based on further studies of the Sin3A complexes using selamectin and ivermectin, we seek to identify novel SMIs with better efficiency for targeting Sin3 complex and treating TNBCs in the future. Citation Format: Yeon-Jin Kwon, Boris A. Leibovitch, Lei Zeng, Mihaly Mezei, Rossitza Christova, Shuai Yang, Rajal Sharma, Edgardo Aritzia, nidhi bansal, Ming-Ming Zhou, Authur Zelent, Eduardo Farias, Samuel Waxman. Selamectin and ivermectin are small molecule inhibitors that interfere with Sin3A-PAH2 function and exert anti-tumor activity in triple-negative breast cancer. [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 807. doi:10.1158/1538-7445.AM2014-807