The embryonic transcription factor TBXT (brachyury) drives chordoma, a spinal neoplasm without effective drug therapies. TBXT's regulatory network is poorly understood, and strategies to disrupt its activity for therapeutic purposes are lacking. We developed designed ankyrin repeat proteins that block TBXT-DNA binding (T-DARPins). In chordoma cells, T-DARPins reduced cell cycle progression, spheroid formation, and tumor growth in mice and induced signs of senescence and differentiation. Transcriptomic and proteomic analyses identified gene networks involved in cell cycle regulation, embryonic cell identity, and interferon response and revealed features of regulome components, such as susceptibility to pharmacologic inhibition and the fine-tuning of TBXT downstream effectors through IGFBP3. Finally, we found high interferon signaling in chordoma cell lines and patient tumors, which was promoted by TBXT and associated with sensitivity to JAK2 inhibitors. These findings demonstrate the potential of DARPins for probing nuclear proteins to understand the regulatory networks of transcription factor-driven cancers, including entry points for therapies that warrant testing in patients.

Supplementary Figure S1. Effects of anti-estrogens on cell proliferation in MCF-7 #Wt, #TamR and #FulvR cells.

Supplementary Figure S6. Immunohistochemical stainings of ERRalpha in primary ERalpha-positive breast carcinomas on a tissue microarray (n=1041).

Supplementary Figure S2. Chromatin immunoprecipitation (ChIP).

Supplementary Material and Methods, Figure Legends S1-6. Supplementary Material and Methods, Figure Legends S1-6.

Supplementary Figure S3. ChIP-Seq validation by qRT-PCR.

Supplementary Figure S4. AIB1 in tamoxifen- and fulvestrant-resistant breast cancer cells.

Supplementary Figure S5. Co-administration of anti-estrogens upon lentiviral-mediated ERRα knockdown.

Supplementary Tables S1-10. Supplementary Table S1: Clinico-pathological features of primary treatment-naive and relapsed tamoxifen-resistant breast carcinomas assessed by routine pathological evaluation. Supplementary Table S2: Antibodies for Western blot analyses, ChIP and immunohisto-chemistry. Supplementary Table S3: Primer sequences for qRT-PCR (5'-3'). Supplementary Table S4 : p-values. Supplementary Table S5: Top differentially expressed transcripts between MCF-7 #Wt and #TamR. Supplementary Table S6: ERalpha, ERRalpha and AIB1 target genes identified by ChIP-Seq and gene expression profiling (EP) in MCF-7 #Wt vs. # TamR cells. Supplementary Table S7: ERalpha and ERRalpha target genes identified by ChIP-Seq and gene expression profiling in MCF-7 cells. Supplemetary Table S8: Enrichment of functional categories for ERalpha and ERRalpha target genes in #TamR. Supplementary Table S9: Pairwise relationship between ERRalpha and co-variables in ERalpha-positive breast carcinomas (tumor set III, n=1041). Supplementary Table S10: Effects of co-variables on patient survival in tamoxifen-treated primary breast tumors (GSE9893) stratified to grade and PR status.

Chromothripsis is a form of genome instability by which a presumably single catastrophic event generates extensive genomic rearrangements of one or a few chromosomes. Widely assumed to be an early event in tumor development, this phenomenon plays a prominent role in tumor onset. In this study, an analysis of chromothripsis in 252 human breast cancers from two patient cohorts (149 metastatic breast cancers, 63 untreated primary tumors, 29 local relapses, and 11 longitudinal pairs) using whole-genome and whole-exome sequencing reveals that chromothripsis affects a substantial proportion of human breast cancers, with a prevalence over 60% in a cohort of metastatic cases and 25% in a cohort comprising predominantly luminal breast cancers. In the vast majority of cases, multiple chromosomes per tumor were affected, with most chromothriptic events on chromosomes 11 and 17 including, among other significantly altered drivers, CCND1, ERBB2, CDK12, and BRCA1. Importantly, chromothripsis generated recurrent fusions that drove tumor development. Chromothripsis-related rearrangements were linked with univocal mutational signatures, with clusters of point mutations due to kataegis in close proximity to the genomic breakpoints and with the activation of specific signaling pathways. Analyzing the temporal order of events in tumors with and without chromothripsis as well as longitudinal analysis of chromothriptic patterns in tumor pairs offered important insights into the role of chromothriptic chromosomes in tumor evolution. SIGNIFICANCE: These findings identify chromothripsis as a major driving event in human breast cancer.

Abstract Endocrine treatment regimens for breast cancer that target the estrogen receptor-α (ERα) are effective, but acquired resistance remains a limiting drawback. One mechanism of acquired resistance that has been hypothesized is functional substitution of the orphan receptor estrogen-related receptor-α (ERRα) for ERα. To examine this hypothesis, we analyzed ERRα and ERα in recurrent tamoxifen-resistant breast tumors and conducted a genome-wide target gene profiling analysis of MCF-7 breast cancer cell populations that were sensitive or resistant to tamoxifen treatment. This analysis uncovered a global redirection in the target genes controlled by ERα, ERRα, and their coactivator AIB1, defining a novel set of target genes in tamoxifen-resistant cells. Beyond differences in the ERα and ERRα target gene repertoires, both factors were engaged in similar pathobiologic processes relevant to acquired resistance. Functional analyses confirmed a requirement for ERRα in tamoxifen- and fulvestrant-resistant MCF-7 cells, with pharmacologic inhibition of ERRα sufficient to partly restore sensitivity to antiestrogens. In clinical specimens (n = 1041), increased expression of ERRα was associated with enhanced proliferation and aggressive disease parameters, including increased levels of p53 in ERα-positive cases. In addition, increased ERRα expression was linked to reduced overall survival in independent tamoxifen-treated patient cohorts. Taken together, our results suggest that ERα and ERRα cooperate to promote endocrine resistance, and they provide a rationale for the exploration of ERRα as a candidate drug target to treat endocrine-resistant breast cancer. Cancer Res; 75(4); 720–31. ©2015 AACR.

Endocrine treatment regimens for breast cancer that target the estrogen receptor-a (ERa) are effective, but acquired resistance remains a limiting drawback. One mechanism of acquired resistance that has been hypothesized is functional substitution of the orphan receptor estrogen-related receptor-a (ERRa) for ERa. To examine this hypothesis, we analyzed ERRa and ERa in recurrent tamoxifen-resistant breast tumors and conducted a genome-wide target gene profiling analysis of MCF-7 breast cancer cell populations that were sensitive or resistant to tamoxifen treatment. This analysis uncovered a global redirection in the target genes controlled by ERa, ERRa, and their coactivator AIB1, defining a novel set of target genes in tamoxifen-resistant cells. Beyond differences in the ERa and ERRa target gene repertoires, both factors were engaged in similar pathobiologic processes relevant to acquired resistance. Functional analyses confirmed a requirement for ERRa in tamoxifenand fulvestrant-resistant MCF-7 cells, with pharmacologic inhibition of ERRa sufficient to partly restore sensitivity to antiestrogens. In clinical specimens (n 1⁄4 1041), increased expression of ERRa was associated with enhanced proliferation and aggressive disease parameters, including increased levels of p53 in ERa-positive cases. In addition, increased ERRa expression was linked to reduced overall survival in independent tamoxifen-treated patient cohorts. Taken together, our results suggest that ERa and ERRa cooperate to promote endocrine resistance, and they provide a rationale for the exploration of ERRa as a candidate drug target to treat endocrine-resistant breast cancer. Cancer Res; 75(4); 720–31. 2015 AACR. Introduction The ligand-activated transcription factor ERa is a key driver of the breast cancer phenotype in around 70% of patients (1). Accordingly, endocrine treatment modalities targeting ERa, such as the selective estrogen receptormodulator (SERM) tamoxifen or downregulator (SERD) fulvestrant (Fulv; SERM) constitute the basis for therapeutic intervention in ERa-positive tumors (2). Fulvestrant as a pure ERa antagonist and tamoxifen as partial antagonist counteract the pro–proliferative and antiapoptotic stimuli classically induced by estrogens (3). Beyond the widespread improvements by these treatment regimens, the frequent onset of antiestrogen resistance remains a major limitation (4), underlining the clinical need for alternative drug targets. A compelling body of evidence suggests involvement of another nuclear hormone receptor, estrogen-related receptor-a (ERRa), in the pathogenesis of breast cancer. Increased ERRa expression was found in mammary tumors and correlated with an impaired disease-free and overall patient survival (5, 6). The pathophysiologic relevance of ERRa has been further demonstrated in vivo, as ERRa inhibition in xenograft systems reduces breast tumor growth, and in HER-2/neu–driven breast cancer mouse models, ERRa knockout delays tumor formation (7, 8). Despite the structural relationship, ERa and ERRa share only 33% homology in their ligand-binding domains, resulting in the insensitivity of ERRa to classical ERa ligands such as estrogen and tamoxifen (9–11). Because of the lack of known natural ligands, ERRa is classified as orphan receptor, whereas its transcriptional activity can be abrogated by small-molecule inhibitors (i.e., XCT790; ref. 12). ERRa and ERa possess a high sequence homology in their central DNA-binding domains (68%), and therefore each recognize the others cognate-binding motif (9, 13). Identification of a subset of common target genes (e.g., pS2; refs. 14, 15), raised the hypothesis that ERRa bypasses the requirement for ERa in endocrine-resistant breast cancers and fuels resistance. This concept has been corroborated by the interplay of ERRa and known determinants of antiestrogen resistance such as HER-2/neu and the coactivator AIB1 (6, 8, 16). As a versatile nuclear hormone Division of Molecular Genetics, German Cancer Research Center (DKFZ), Heidelberg,Germany. InstituteofPathology,UniversityMedical Center Hamburg-Eppendorf, Hamburg, Germany. Foundation PATH—Patients' Tumor Bank of Hope, Munich, Germany. Institute of Pathology, University Hospital Cologne, Cologne, Germany. Genomics Core Facility, European Molecular Biology Laboratory (EMBL), Heidelberg, Germany. Molecular Biology of Breast Cancer, University Women's Clinic, Heidelberg, Germany. Breast Cancer Group, Cardiff University,Cardiff, UnitedKingdom. Institute of Pathology, University of Heidelberg, Heidelberg, Germany. Gynecologic Oncology, National Center for Tumor Diseases, University of Heidelberg, Heidelberg, Germany. Phenex Pharmaceuticals AG, Heidelberg, Germany. Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). Corresponding Author: Peter Lichter, Division of Molecular Genetics, German Cancer Research Center (DKFZ), Im Neuenheimer Feld 580, Heidelberg 69120, Germany. Phone: 0049-6221-424619; Fax: 0049-6221-424639; E-mail: peter.lichter@dkfz.de doi: 10.1158/0008-5472.CAN-14-0652 2015 American Association for Cancer Research. Cancer Research Cancer Res; 75(4) February 15, 2015 720 on May 4, 2017. © 2015 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from Published OnlineFirst February 2, 2015; DOI: 10.1158/0008-5472.CAN-14-0652

S100 proteins comprise a family of calcium-modulated proteins that have recently been associated with epithelial tumours. We examined the expression of two members of this family, S100A8 and S100A9, together with the S100 receptor RAGE in human prostate adenocarcinomas using histochemical staining procedures. S100A8, S100A9, and RAGE were up-regulated in prostatic intraepithelial neoplasia and preferentially in high-grade adenocarcinomas, whereas benign tissue was negative or showed weak expression of the proteins. The analysis of S100A9 in patient serum additionally revealed significantly elevated S100A9 serum levels in cancer patients compared to BPH (benign prostatic hyperplasia) patients or healthy individuals [1]. In cell culture experiments S100A8 and S100A9 were identified as extracellular factors which are able to induce MAP kinase and NF-κB signalling pathways and to stimulate the migration of prostate cells [2]. Thus, S100A8 and S100A9 are linked to the activation of important features of prostate cancer cells. Furthermore, S100A8 and S100A9 may represent novel players in prostate cancer development or progression, which may prove useful for future diagnostic and/or therapeutic approaches. [1] Hermani et al. (2005) Clin. Cancer Res. 11, 5146-52 [2] Hermani et al. (2006) Exp. Cell Res. 312, 184-97 Allelic silencing at 13q14.3: a novel oncogenic mechanism Daniel Mertens, Stephan Wolf, Cora Mund, Dirk Kienle , Sibylle Ohl, Petra Schroeter, Frank Lyko, Hartmut Döhner, Stephan Stilgenbauer, Peter Lichter 1) Department of Molecular Genetics and 2) Department of Epigenetics, DKFZ, Heidelberg, Germany; 3) Department of Internal Medicine III, University of Ulm, Germany INTRODUCTION: Genomic material from chromosomal band 13q14.3 distal to RB1 is recurrently lost in a variety of human neoplasms. Lack of point mutations in candidate tumor suppressor genes and downregulation of these genes in tumors indicate an epigenetic pathomechanism localized in the critical region. AIM: Characterization of the epigenetic tumor suppressor mechanism localized in 13q14.3. METHODS: Candidate tumor suppressor genes are down regulated by more than a factor of two in tumors with loss of one copy of the critical region. In addition, the presence of large non-coding RNA genes in 13q14.3 is reminiscent of imprinted regions where only one gene copy is active. Therefore we tested candidate tumor suppressor genes for monoallelic expression in healthy probands using single nucleotide polymorphisms and sequencing of RTPCR products. Genotyping parents of these probands allowed allocation of the parental origin of either gene copy. In addition, we performed FISH experiments to measure replication timing of the two copies of the critical region to find out whether they are functionally different. As transcriptional activity and replication timing are effectuated by chromatin packaging, we used combined bisulfite-restriction (COBRA) analyses and bisulfite sequencing to assess DNA methylation of the critical region. Treatment of cultured cells with inhibitors of DNA-methyltransferases and histone-deacetylases allowed functional correlation of chromatin modification with expression of candidate tumor suppressor genes localized in the critical region. RESULTS: In line with an imprinting mechanism, we find that the two copies of the critical region replicate asynchronously, suggesting differential chromatin packaging of the two copies of 13q14.3. In addition, we could detect monoallelic silencing of genes localized in the critical region and expression of one gene copy only. However, expression originated from either the maternal or paternal copy, excluding an imprinting mechanism. DNA methylation analyses showed one of the CpG islands of the region to be methylated. Demethylation of DNA and histone hyperacetylation induced biallelic expression, while replication timing was not affected. CONCLUSIONS: We propose that differential replication timing represents an early epigenetic mark that distinguishes the two copies of 13q14.3, resulting in differential chromatin packaging and monoallelic expression. This has profound effects for the tumor suppressor mechanism localized in 13q14.3: Deletion of the single active copy of the region at 13q14.3, which is detected in more than 50% of CLL tumors, or point mutations only in the active gene copies will suffice for complete loss of tumor suppressor function, as the remaining gene copies are epigentically silenced. Thus, we provide a model for the pathomechanism of 13q14.3 in CLL by the interaction of genetic lesions and epigenetic silencing. Development of Array-based Assay for High-resolution DNA-Methylation Profiling of B-cell Chronic Lymphocytic Leukemia E.A. Moskalyov, V. Beier, A.T. Eprintsev, I.A. Vorobjev, E.A. Nikitin, J.D. Hoheisel 1 Division of Functional Genome Analysis, Deutsches Krebsforschungszentrum, Heidelberg, Germany, 2 Biology Faculty, Department of Biochemistry, Voronezh State University, Voronezh, Russia, 3 Hematology Research Center of Russia, Moscow, Russia.

Genomic material from chromosome band 13q14.3 distal to the retinoblastoma locus is recurrently lost in a variety of human neoplasms, indicating an as-yet-unidentified tumor-suppressor mechanism. No pathogenic mutations have been found in the minimally deleted region until now. However, in B cell chronic lymphocytic leukemia tumors with loss of one copy of the critical region, respective candidate tumor-suppressor genes are down-regulated by a factor >2, which would be expected by a normal gene-dosage effect. This finding points to an epigenetic pathomechanism. We find that the two copies of the critical region replicate asynchronously, suggesting differential chromatin packaging of the two copies of 13q14.3. Although we also detect monoallelic silencing of genes localized in the critical region, monoallelic expression originates from either the maternal or paternal copy, excluding an imprinting mechanism. DNA methylation analyses revealed one CpG island of the region to be methylated. DNA demethylation of this CpG island and global histone hyperacetylation induced biallelic expression, whereas replication timing was not affected. We propose that differential replication timing represents an early epigenetic mark that distinguishes the two copies of 13q14.3, resulting in differential chromatin packaging and monoallelic expression. Accordingly, deletion of the single active copy of 13q14.3 results in significant down-regulation of the candidate genes and loss of function, providing a model for the interaction of genetic lesions and epigenetic silencing at 13q14.3 in B cell chronic lymphocytic leukemia.