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It has been recognized for decades that ERBB signaling is important in prostate cancer, but targeting ERBB receptors as a therapeutic strategy for prostate cancer has been ineffective clinically. However, we show here that membranous HER3 protein is commonly highly expressed in lethal prostate cancer, associating with reduced time to castration resistance (CR) and survival. Multiplex immunofluorescence indicated that the HER3 ligand NRG1 is detectable primarily in tumor-infiltrating myelomonocytic cells in human prostate cancer; this observation was confirmed using single-cell RNA sequencing of human prostate cancer biopsies and murine transgenic prostate cancer models. In castration-resistant prostate cancer (CRPC) patient-derived xenograft organoids with high HER3 expression as well as mouse prostate cancer organoids, recombinant NRG1 enhanced proliferation and survival. Supernatant from murine bone marrow–derived macrophages and myeloid-derived suppressor cells promoted murine prostate cancer organoid growth in vitro, which could be reversed by a neutralizing anti-NRG1 antibody and ERBB inhibition. Targeting HER3, especially with the HER3-directed antibody–drug conjugate U3-1402, exhibited antitumor activity against HER3-expressing prostate cancer. Overall, these data indicate that HER3 is commonly overexpressed in lethal prostate cancer and can be activated by NRG1 secreted by myelomonocytic cells in the tumor microenvironment, supporting HER3-targeted therapeutic strategies for treating HER3-expressing advanced CRPC.Significance:HER3 is an actionable target in prostate cancer, especially with anti-HER3 immunoconjugates, and targeting HER3 warrants clinical evaluation in prospective trials.

Supplementary Figure S1

Supplementary methods and Supplementary figure legends - Tracked Changes

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.

SPOP mutation identified in this cohort.

Purpose: CHD1 deletions and SPOP mutations frequently cooccur in prostate cancer with lower frequencies reported in castration-resistant prostate cancer (CRPC). We monitored CHD1 expression during disease progression and assessed the molecular and clinical characteristics of CHD1-deleted/SPOP-mutated metastatic CRPC (mCRPC).Experimental Design: We identified 89 patients with mCRPC who had hormone-naive and castration-resistant tumor samples available: These were analyzed for CHD1, PTEN, and ERG expression by IHC. SPOP status was determined by targeted next-generation sequencing (NGS). We studied the correlations between these biomarkers and (i) overall survival from diagnosis; (ii) overall survival from CRPC; (iii) duration of abiraterone treatment; and (iv) response to abiraterone. Relationship with outcome was analyzed using Cox regression and log-rank analyses.Results: CHD1 protein loss was detected in 11 (15%) and 13 (17%) of hormone-sensitive prostate cancer (HSPC) and CRPC biopsies, respectively. Comparison of CHD1 expression was feasible in 56 matched, same patient HSPC and CRPC biopsies. CHD1 protein status in HSPC and CRPC correlated in 55 of 56 cases (98%). We identified 22 patients with somatic SPOP mutations, with six of these mutations not reported previously in prostate cancer. SPOP mutations and/or CHD1 loss was associated with a higher response rate to abiraterone (SPOP: OR, 14.50 P = 0.001; CHD1: OR, 7.30, P = 0.08) and a longer time on abiraterone (SPOP: HR, 0.37, P = 0.002, CHD1: HR, 0.50, P = 0.06).Conclusions: SPOP-mutated mCRPCs are strongly enriched for CHD1 loss. These tumors appear highly sensitive to abiraterone treatment. Clin Cancer Res; 24(22); 5585–93. ©2018 AACR.

Supplementary figure and table legends

Supplementary Figure from JMJD6 Is a Druggable Oxygenase That Regulates AR-V7 Expression in Prostate Cancer

Supplementary Table from JMJD6 Is a Druggable Oxygenase That Regulates AR-V7 Expression in Prostate Cancer

Additional Information on molecular analyses determining CHD1 and SPOP status

Sample overview

Endocrine resistance (EnR) in advanced prostate cancer is fatal. EnR can be mediated by androgen receptor (AR) splice variants, with AR splice variant 7 (AR-V7) arguably the most clinically important variant. In this study, we determined proteins key to generating AR-V7, validated our findings using clinical samples, and studied splicing regulatory mechanisms in prostate cancer models. Triangulation studies identified JMJD6 as a key regulator of AR-V7, as evidenced by its upregulation with in vitro EnR, its downregulation alongside AR-V7 by bromodomain inhibition, and its identification as a top hit of a targeted siRNA screen of spliceosome-related genes. JMJD6 protein levels increased (P < 0.001) with castration resistance and were associated with higher AR-V7 levels and shorter survival (P = 0.048). JMJD6 knockdown reduced prostate cancer cell growth, AR-V7 levels, and recruitment of U2AF65 to AR pre-mRNA. Mutagenesis studies suggested that JMJD6 activity is key to the generation of AR-V7, with the catalytic machinery residing within a druggable pocket. Taken together, these data highlight the relationship between JMJD6 and AR-V7 in advanced prostate cancer and support further evaluation of JMJD6 as a therapeutic target in this disease.Significance:This study identifies JMJD6 as being critical for the generation of AR-V7 in prostate cancer, where it may serve as a tractable target for therapeutic intervention.

Combined SPOP/ CHD1 status and time/ response to abiraterone.

It has been recognized for decades that ERBB signaling is important in prostate cancer, but targeting ERBB receptors as a therapeutic strategy for prostate cancer has been ineffective clinically. However, we show here that membranous HER3 protein is commonly highly expressed in lethal prostate cancer, associating with reduced time to castration resistance (CR) and survival. Multiplex immunofluorescence indicated that the HER3 ligand NRG1 is detectable primarily in tumor-infiltrating myelomonocytic cells in human prostate cancer; this observation was confirmed using single-cell RNA sequencing of human prostate cancer biopsies and murine transgenic prostate cancer models. In castration-resistant prostate cancer (CRPC) patient-derived xenograft organoids with high HER3 expression as well as mouse prostate cancer organoids, recombinant NRG1 enhanced proliferation and survival. Supernatant from murine bone marrow–derived macrophages and myeloid-derived suppressor cells promoted murine prostate cancer organoid growth in vitro, which could be reversed by a neutralizing anti-NRG1 antibody and ERBB inhibition. Targeting HER3, especially with the HER3-directed antibody–drug conjugate U3-1402, exhibited antitumor activity against HER3-expressing prostate cancer. Overall, these data indicate that HER3 is commonly overexpressed in lethal prostate cancer and can be activated by NRG1 secreted by myelomonocytic cells in the tumor microenvironment, supporting HER3-targeted therapeutic strategies for treating HER3-expressing advanced CRPC. Significance: HER3 is an actionable target in prostate cancer, especially with anti-HER3 immunoconjugates, and targeting HER3 warrants clinical evaluation in prospective trials.

Background Better blood tests to elucidate the behaviour of metastatic castration-resistant prostate cancer (mCRPC) are urgently needed to drive therapeutic decisions. Plasma cell-free DNA (cfDNA) comprises normal and circulating tumour DNA (ctDNA). Low-pass whole-genome sequencing (lpWGS) of ctDNA can provide information on mCRPC behaviour. Objective To validate and clinically qualify plasma lpWGS for mCRPC. Design, setting, and participants Plasma lpWGS data were obtained for mCRPC patients consenting to optional substudies of two prospective phase 3 trials (FIRSTANA and PROSELICA). In FIRSTANA, chemotherapy-naïve patients were randomised to treatment with docetaxel (75 mg/m2) or cabazitaxel (20 or 25 mg/m2). In PROSELICA, patients previously treated with docetaxel were randomised to 20 or 25 mg/m2 cabazitaxel. lpWGS data were generated from 540 samples from 188 mCRPC patients acquired at four different time points (screening, cycle 1, cycle 4, and end of study). Outcome measurements and statistical analysis lpWGS data for ctDNA were evaluated for prognostic, response, and tumour genomic measures. Associations with response and survival data were determined for tumour fraction. Genomic biomarkers including large-scale transition (LST) scores were explored in the context of prior treatments. Results and limitations Plasma tumour fraction was prognostic for overall survival in univariable and stratified multivariable analyses (hazard ratio 1.75, 95% confidence interval 1.08–2.85; p = 0.024) and offered added value compared to existing biomarkers (C index 0.722 vs 0.709; p = 0.021). Longitudinal changes were associated with drug response. PROSELICA samples were enriched for LSTs (p = 0.029) indicating genomic instability, and this enrichment was associated with prior abiraterone and enzalutamide treatment but not taxane or radiation therapy. Higher LSTs were correlated with losses of RB1/RNASEH2B, independent of BRCA2 loss. Conclusions Plasma lpWGS of ctDNA describes CRPC behaviour, providing prognostic and response data of clinical relevance. The added prognostic value of the ctDNA fraction over established biomarkers should be studied further. Patient summary We studied tumour DNA in blood samples from patients with prostate cancer. We found that levels of tumour DNA in blood were indicative of disease prognosis, and that changes after treatment could be detected. We also observed a “genetic scar” in the results that was associated with certain previous treatments. This test allows an assessment of tumour activity that can complement existing tests, offer insights into drug response, and detect clinically relevant genetic changes., Take Home Message Tumour fraction, independent from cell-free DNA concentration and other clinical variables, is prognostic, with low-pass whole-genome sequencing profiles detecting abiraterone/enzalutamide-induced emerging genomic instability that is likely to be acquired as a result of tumour development.

Endocrine resistance (EnR) in advanced prostate cancer is fatal. EnR can be mediated by androgen receptor (AR) splice variants, with AR splice variant 7 (AR-V7) arguably the most clinically important variant. In this study, we determined proteins key to generating AR-V7, validated our findings using clinical samples, and studied splicing regulatory mechanisms in prostate cancer models. Triangulation studies identified JMJD6 as a key regulator of AR-V7, as evidenced by its upregulation with in vitro EnR, its downregulation alongside AR-V7 by bromodomain inhibition, and its identification as a top hit of a targeted siRNA screen of spliceosome-related genes. JMJD6 protein levels increased (P < 0.001) with castration resistance and were associated with higher AR-V7 levels and shorter survival (P = 0.048). JMJD6 knockdown reduced prostate cancer cell growth, AR-V7 levels, and recruitment of U2AF65 to AR pre-mRNA. Mutagenesis studies suggested that JMJD6 activity is key to the generation of AR-V7, with the catalytic machinery residing within a druggable pocket. Taken together, these data highlight the relationship between JMJD6 and AR-V7 in advanced prostate cancer and support further evaluation of JMJD6 as a therapeutic target in this disease. Significance: This study identifies JMJD6 as being critical for the generation of AR-V7 in prostate cancer, where it may serve as a tractable target for therapeutic intervention.

Background: We, and others, have shown that responses to therapies in metastatic castration-resistant prostate cancer (mCRPC) can be monitored using plasma cell-free DNA (cfDNA), and that this can be both quantitative and qualitative. In this study, we explored the utility of low pass whole genome sequencing (lpWGS) of cfDNA in mCRPC patients treated on two large prospectively collected Phase III trials of taxane chemotherapy, FIRSTANA (NCT01308567) and PROSELICA (NCT01308580). Methods: Plasma samples collated from the FIRSTANA and PROSELICA trials were evaluated. cfDNA was isolated and analysed with lpWGS using the Illumina NovaSeq 6000. Reads were aligned using BWA-MEM and quality controlled using Picard and FASTQC. Whole-genome copy number aberration (CNA) profiles were generated using ichorCNA, which also estimated tumor purity and ploidy. Log-rank tests were used for univariable survival comparisons, and Cox proportional hazard models were used for multivariable survival analysis. Results: Overall, lp-WGS data was generated from 528 samples acquired at three different timepoints (Baseline [SCR and C1], C4 and EOS), representing 188 unique patients. We observed CNA profiles in line with emergent mCRPC subtypes including highly aberrant, large-scale events which have been linked to homologous-repair deficient tumors. We found that CNAs in this cohort matched other published studies with frequent amplifications of AR (~30%), copy-gains of PI3KCA (~45%) and copy-loss of NKX3-1 (~75%), RB1 (~70%) and PTEN (~50%). We did not observe changes in baseline tumor purity or ploidy between the FIRSTANA and PROSELICA cohorts. Following calculation of a large-scale transition (LST) score (measuring CNA breakpoints), we observed that this was significantly elevated (Wilcox test, p=0.0073) in cases that had prior exposure to second line androgen deprivation (abiraterone or enzalutamide). Baseline tumor purity values were associated with several mCRPC clinical variables including ECOG performance status. High lpWGS tumor purity values were univariably associated with poorer overall survival in both FIRSTANA and PROSELICA cohorts (log-rank test p=0.0021 and 0.0009), and were also associated with significantly poorer RPFS and PSAPFS. Tumor purity was also associated (HR 1.66, p=0.036) with survival when assessed using multivariable models alongside other mCRPC clinical variables, including cell-free DNA concentration. Further, longitudinal changes in tumor purity on treatment can be illustrative of drug responses. Conclusions: In our analysis of this prospectively collected clinical trial data, we show mCRPC CNA profiles in cfDNA, and that these CNAs may be induced by prior aggressive treatments. We also found that tumor purity estimation can serve as a robust, competitive indicator of both overall survival and, assessed longitudinally, shows clear association with drug response. We envision that these techniques will enable improved strategies for monitoring mCRPC patients. Citation Format: George Seed, Semini Sumanasuriya, Claudia Bertan, Harry Parr, Gemma Fowler, Rossitza Christova, Lorna Pope, Jane Goodall, Maryou Lambros, Pasquale Rescigno, Penelope Flohr, Suzanne Carreira, Wei Yuan, Mustapha Chadjaa, Sandrine Mace, Johann De Bono. Discovery of genomic correlates and tumor purity as an independent clinical factor of poor outcome in advanced prostate cancer lpWGS CNA data [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr LB-270.

Purpose: CHD1 deletions and SPOP mutations frequently cooccur in prostate cancer with lower frequencies reported in castration-resistant prostate cancer (CRPC). We monitored CHD1 expression during disease progression and assessed the molecular and clinical characteristics of CHD1-deleted/SPOP-mutated metastatic CRPC (mCRPC). Experimental Design: We identified 89 patients with mCRPC who had hormone-naive and castration-resistant tumor samples available: These were analyzed for CHD1, PTEN, and ERG expression by IHC. SPOP status was determined by targeted next-generation sequencing (NGS). We studied the correlations between these biomarkers and (i) overall survival from diagnosis; (ii) overall survival from CRPC; (iii) duration of abiraterone treatment; and (iv) response to abiraterone. Relationship with outcome was analyzed using Cox regression and log-rank analyses. Results: CHD1 protein loss was detected in 11 (15%) and 13 (17%) of hormone-sensitive prostate cancer (HSPC) and CRPC biopsies, respectively. Comparison of CHD1 expression was feasible in 56 matched, same patient HSPC and CRPC biopsies. CHD1 protein status in HSPC and CRPC correlated in 55 of 56 cases (98%). We identified 22 patients with somatic SPOP mutations, with six of these mutations not reported previously in prostate cancer. SPOP mutations and/or CHD1 loss was associated with a higher response rate to abiraterone (SPOP: OR, 14.50 P = 0.001; CHD1: OR, 7.30, P = 0.08) and a longer time on abiraterone (SPOP: HR, 0.37, P = 0.002, CHD1: HR, 0.50, P = 0.06). Conclusions: SPOP-mutated mCRPCs are strongly enriched for CHD1 loss. These tumors appear highly sensitive to abiraterone treatment. Clin Cancer Res; 24(22); 5585–93. ©2018 AACR.

Background Deletion of the chromatin remodeler chromodomain helicase DNA-binding protein 1 (CHD1) is a common genomic alteration found in human prostate cancers (PCas). CHD1 loss represents a distinct PCa subtype characterized by SPOP mutation and higher genomic instability. However, the role of CHD1 in PCa development in vivo and its clinical utility remain unclear. Patients and methods To study the role of CHD1 in PCa development and its loss in clinical management, we generated a genetically engineered mouse model with prostate-specific deletion of murine Chd1 as well as isogenic CHD1 wild-type and homozygous deleted human benign and PCa lines. We also developed patient-derived organoid cultures and screened patients with metastatic PCa for CHD1 loss. Results We demonstrate that CHD1 loss sensitizes cells to DNA damage and causes a synthetic lethal response to DNA damaging therapy in vitro, in vivo, ex vivo, in patient-derived organoid cultures and in a patient with metastatic PCa. Mechanistically, CHD1 regulates 53BP1 stability and CHD1 loss leads to decreased error-free homologous recombination (HR) repair, which is compensated by increased error-prone non-homologous end joining (NHEJ) repair for DNA double-strand break (DSB) repair. Conclusions Our study provides the first in vivo and in patient evidence supporting the role of CHD1 in DSB repair and in response to DNA damaging therapy. We uncover mechanistic insights that CHD1 modulates the choice between HR and NHEJ DSB repair and suggest that CHD1 loss may contribute to the genomic instability seen in this subset of PCas.

5070 Background: Antitumor efficacy of chemotherapeutic drugs docetaxel and cabazitaxel against hormone-sensitive and castration-resistant prostate cancer (CRPC) is well recognized. The identification and validation of minimally invasive biomarkers of taxane response remain of paramount importance. Methods: Serial pre-, during and post-treatment plasma samples were collected prospectively from two large Phase III clinical trials, FIRSTANA (NCT01308567) and PROSELICA (NCT01308580). Chemotherapy-naïve patients (pts) were treated with docetaxel (75 mg/m2) or cabazitaxel (20 or 25 mg/m2) in FIRSTANA, and pts previously treated with docetaxel received cabazitaxel (20 or 25 mg/m2) as second-line chemotherapy in PROSELICA. Plasma cell-free DNA (cfDNA) was extracted, low-pass whole genome sequencing (lp-WGS) libraries were prepared using the QIAGEN QiaSeq FX DNA library kit, and samples sequenced on the Illumina NovaSeq 6000. Targeted next-generation sequencing (NGS) on a custom-made AmpliSeq panel of 30 genes, pre-selected for putative roles in taxane resistance, was also performed. lp-WGS copy number (CN) profiles were generated using ichorCNA (v0.1.0) and targeted NGS variant calls derived using IonReporter software. Results: Overall, lp-WGS data was generated from 265 samples (99 pts; 51 treated on the FIRSTANA study, 48 treated on the PROSELICA study), acquired at three time-points (pre-, during and at progression). Average cfDNA input was 10 ng and mean coverage achieved was ~2X (SD 1.2X). We observed changes in lp-WGS tumor purity over time as a result of therapy; samples from non-responding pts exhibited significantly higher tumor purity values post-treatment compared with samples from responding pts (p = 0.02). Targeted NGS results were available from 294 pts (153 from FIRSTANA, 141 from PROSELICA), and changes in tumor purity were also associated with treatment response (p = 0.04). CN frequency of key CRPC genes was similar to previously reported datasets; several aberrant loci associated with response to taxane therapy. Conclusions: cfDNA lp-WGS may have clinical utility in the management of lethal prostate cancer. Funding: Sanofi. Clinical trial information: NCT01308580, NCT01308567.

Activation of the major histocompatibility complex (MHC) by interferon-gamma (IFN-γ) is a fundamental step in the adaptive immune response to pathogens. Here, we show that reorganization of chromatin loop domains in the MHC is evident within the first 30 min of IFN-γ treatment of fibroblasts, and that further dynamic alterations occur up to 6 h. These very rapid changes occur at genomic sites which are occupied by CTCF and are close to IFN-γ-inducible MHC genes. Early responses to IFN-γ are thus initiated independently of CIITA, the master regulator of MHC class II genes and prepare the MHC for subsequent induction of transcription.

// 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.

DNA-binding proteins play a critical role in many major cellular processes. The immunoprecipitation of protein complexes with associated DNA fragments, termed chromatin immunoprecipitation or ChIP, allows for the analysis of the binding of protein factors in their natural environment. It evolved from a confirmation assay to a discovery tool that really changed our understanding of the biology of living cells. With the widespread use of next-generation high-throughput sequencing technologies, factor binding can not only be assessed in an unbiased and genome-wide manner but can also be predicted and linked to gene expression and chromatin structure. This review summarizes the current advances of the ChIP-based approaches to decipher gene regulatory and epigenetic network in the cells. The limitations of the method are discussed and the future ChIP-based developments are explored.

Adenocarcinomas of the prostate consist of a collection of malignancies with distinct molecular underpinnings. Linking molecular background to clinical behaviour, i.e. tumour progression and response to therapy has not yet been fully established. This is in large measure due to a paucity of well-annotated patient cohorts, which cover the evolution from localized, hormone naïve to metastatic castration resistant prostate cancer. Deletions in the gene Chromodomain Helicase DNA Binding Protein 1 (CHD1) are among the most frequent genomic alterations in prostate cancer. CHD1 is a ATPase-dependent helicase mediating a variety of biological processes by modifying chromatin structure. In hormone sensitive prostate cancer, loss of CHD1 has been associated with increased genomic instability and aggressiveness. Here we monitor CHD1 status during the progression from hormone sensitive (HSPC) to castration resistant prostate cancer (CRPC). To molecularly stratify this patient subgroup we correlate CHD1 status with markers of prostate cancer including AR, ERG, PTEN and Ki67. Finally, we analyze the impact of alterations in CHD1 on outcome and response to therapy of CRPC patients. We retrospectively identified 52 patients of whom HSPC and CRPC tissue was available for immunohistochemical analysis of CHD1, ERG, PTEN, AR and Ki67 as well as for CHD1 fluorescence in situ hybridization. Relationship with outcome was analyzed using univariate Cox regression and log-rank analyses. By using Fluorescence In Situ Hybridization (FISH) and immunohistochemistry (IHC) in a cohort of 52 men who developed CRPC, we show that the frequency of loss of CHD1 does not change during disease progression (7.7% in HSPC vs 9.6% in CRPC). Homozygously deleted tumors demonstrate a tendency towards poorer prognosis but this observation lacks statistical power in our cohort. Interestingly, in patients that show CHD1 protein expression in their hormone naïve sample, a decrease of expression in the matched CRPC sample correlates with poorer overall survival (OS) (p = 0.03). Although preclinical data suggest a role for CHD1 in androgen receptor (AR) activity, CHD1 expression did not correlate with AR protein level. Additionally, Ki67 expression did not show correlation with CHD1 levels of expression. Further molecular stratification confirms a known mutual exclusive relationship with the expression of the ERG transcription factor (p = 0.048). Clinically, although decreased CHD1 protein expression during disease progression correlated with worse outcome (overall survival (p = 0.03) and survival from diagnosis of CRPC (p = 0.02)), no significant impact on survival after docetaxel or abiraterone treatment was measured.Here we monitor CHD1 deletion during prostate cancer progression. Our results show that this genomic marker defines a distinct molecular subgroup of prostate cancer. In addition, loss of CHD1 protein expression during disease progression is associated with poorer clinical outcome. Citation Format: Gunther Boysen, Daniel Nava Rodrigues, Joaquin Mateo, Rossitza Christova, Ruth Riisnaes, Mateus Crespo, Theresa MacDonald, Susana Miranda, Ines Figueiredo, Veronica Gil, Sara Aziz, Adam Sharp, Niven Mehra, Pasquale Rescigno, Juan M. Mosquera, Christopher E. Barbieri, Mark A. Rubin, Johann S. de Bono. Monitoring CHD1 during prostate cancer progression. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4976.

5014Background: Biomarkers are needed to guide CRPC treatment; we evaluated [cfDNA] pre- and post-TT, associating this with outcome. Methods: cfDNA was isolated and quantified from 1 mL of plasma, ...

The folding of chromatin into topologically constrained loop domains is essential for genomic function. We have identified genomic anchors that define the organization of chromatin loop domains across the human major histocompatibility complex (MHC). This locus contains critical genes for immunity and is associated with more diseases than any other region of the genome. Classical MHC genes are expressed in a cell type-specific pattern and can be induced by cytokines such as interferon-gamma (IFNG). Transcriptional activation of the MHC was associated with a reconfiguration of chromatin architecture resulting from the formation of additional genomic anchors. These findings suggest that the dynamic arrangement of genomic anchors and loops plays a role in transcriptional regulation.

Transcriptional activation of the major histocompatibility complex (MHC) by IFNγ is a key step in cell-mediated immunity. At an early stage of IFNγ induction, chromatin carrying the entire MHC locus loops out from the chromosome 6 territory. We show here that JAK/STAT signalling triggers this higher-order chromatin remodelling and the entire MHC locus becomes decondensed prior to transcriptional activation of the classical HLA class II genes. A single point mutation of STAT1 that prevents phosphorylation is sufficient to abolish chromatin remodelling, thus establishing a direct link between the JAK/STAT signalling pathway and human chromatin architecture. The onset of chromatin remodelling corresponds with the binding of activated STAT1 and the chromatin remodelling enzyme BRG1 at specific sites within the MHC, and is followed by RNA-polymerase recruitment and histone hyperacetylation. We propose that the higher-order chromatin remodelling of the MHC locus is an essential step to generate a transcriptionally permissive chromatin environment for subsequent activation of classical HLA genes.

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

Triple Negative Breast cancer (TNBC) is an aggressive subtype of breast cancer associated with early recurrence and poor prognosis. The treatment options are limited due to lack of expression of common drug targets: estrogen receptor (ER), Progesterone receptor (PR) and Epidermal growth factor receptor 2 (Her2). Epigenetic programs can generate aberrant transcription contributing to TNBC progression; however the dynamic and reversible nature of epigenetic changes offers the possibility to reprogram cancer cells to re-express targets that can render TNBC sensitive to targeted therapies like tamoxifen. Envisioning such ‘epidrugs’, we previously published that targeting PAH2 domain of the master transcriptional scaffold Sin3 by stable expression of 13-mer peptide corresponding to a specific motif called SID (mSin3A interaction domain) disrupts its interaction with a small group of SID-containing transcription factors. This interference reverts the expression of important breast cancer-associated genes and impairs tumor growth in vivo. We have now extended our study towards the evaluation of a cell penetrating SID peptide (pSID) in in vitro and in vivo models to establish parameters for the design of targeted epigenetic therapy for TNBC. pSID co-localizes with Sin3A and interference with PAH2-mediated Sin3A functions by pSID is shown by disruption of Sin3A-MAD1 interactions in Co-IP and Duo-Link assays. pSID treatment in MDA-MB 231 cells results in functional re-expression of CDH1 and ER along with increased H3K4 and decreased H3K27 methylation on their promoters. We also show reduction in the tumorsphere formation by pSID-pretreated MDA-MB-231 cells indicating possible epigenetic reprogramming of tumor initiating stem cells towards a differentiated phenotype. Support to this hypothesis is added by the 50% reduction in tumor growth and re-expression of CDH1 observed in FVB mice injected with pSID-pretreated MMTV-myc cells. Moreover, microarray expression analysis indicates pSID-induced EMT reversal, increased cell adhesion and reduced cell migration. Intriguingly, upon further dissection of the mechanism of epigenetic regulation by pSID we show dissociation of two important chromatin readers/modifiers from the Sin3 complex: histone H3K4Me3/2 demethylase JARID1B and H3K4Me0 binding PHD-like domain containing protein PF1; both with significantly correlated overexpression in invasive breast carcinoma. We also observe loss of recruitment of JARID1B but not Sin3A from the CDH1 promoter. Currently studies are underway to understand the cooperative role between JARID1B and PF1 in potentiating the aberrant transcription regulation by Sin3 at important breast cancer-associated promoters that can be selectively reprogrammed by SID decoys. We believe this selectivity can limit the otherwise adverse affects that may be observed by the use of generic HDAC inhibitors and demethylating agents. Citation Format: Rossitza Christova, Kevin Petrie, Nidhi Bansal, Boris Leibovitch, Louise Howell, Veronica Gil, Ming-Ming Zhou, Edgardo Ariztia, Eduardo Farias, Arthur Zelent, Samuel Waxman. Targeted PF1, JARID1B inhibition induces epigenetic reprogramming 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 411. doi:10.1158/1538-7445.AM2014-411

When eukaryotic cells enter mitosis, transcription is abruptly silenced. Earlier studies indicated that most transcription factors and RNA polymerase II (RNAP II) are displaced when chromatin is condensed into mitotic chromosomes. A more recent study suggested that hitherto unidentified factors might 'bookmark' previously active genes for rapid reactivation after cell division. Here we used chromatin immunoprecipitation (ChIP) assays to examine the association of TFIID, TFIIB, NC2 and RNAP II with various gene promoters in asynchronous and mitotic human cell populations. We show that TFIID and TFIIB can remain associated with active gene promoters during mitosis whereas RNA polymerase II is displaced, and also that NC2, originally identified as ubiquitous repressor of transcription, is associated with active gene promoters in asynchronous cell populations and is displaced from some, but not all, genes in mitotic cells. Consistent with the remarkable stability of TFIID-promoter complexes observed in vitro, our data suggest that these complexes can withstand condensation of chromatin into transcriptionally silent chromosomes. Stable TFIID-promoter complexes are therefore implicated in the propagation of cell-type-specific gene expression patterns through cell division.