Small-molecule-induced polymerization triggers degradation of BCL6.

Effective and sustained inhibition of non-enzymatic oncogenic driver proteins is a major pharmacological challenge. The clinical success of thalidomide analogues demonstrates the therapeutic efficacy of drug-induced degradation of transcription factors and other cancer targets1-3, but a substantial subset of proteins are resistant to targeted degradation using existing approaches4,5. Here we report an alternative mechanism of targeted protein degradation, in which a small molecule induces the highly specific, reversible polymerization of a target protein, followed by its sequestration into cellular foci and subsequent degradation. BI-3802 is a small molecule that binds to the Broad-complex, Tramtrack and Bric-à-brac (BTB) domain of the oncogenic transcription factor B cell lymphoma 6 (BCL6) and leads to the proteasomal degradation of BCL66. We use cryo-electron microscopy to reveal how the solvent-exposed moiety of a BCL6-binding molecule contributes to a composite ligand-protein surface that engages BCL6 homodimers to form a supramolecular structure. Drug-induced formation of BCL6 filaments facilitates ubiquitination by the SIAH1 E3 ubiquitin ligase. Our findings demonstrate that a small molecule such as BI-3802 can induce polymerization coupled to highly specific protein degradation, which in the case of BCL6 leads to increased pharmacological activity compared to the effects induced by other BCL6 inhibitors. These findings open new avenues for the development of therapeutic agents and synthetic biology.

ALT-FISH quantifies alternative lengthening of telomeres activity by imaging of single-stranded repeats.

Alternative lengthening of telomeres (ALT) occurs in ∼10% of cancer entities. However, little is known about the heterogeneity of ALT activity since robust ALT detection assays with high-throughput in situ readouts are lacking. Here, we introduce ALT-FISH, a method to quantitate ALT activity in single cells from the accumulation of single-stranded telomeric DNA and RNA. It involves a one-step fluorescent in situ hybridization approach followed by fluorescence microscopy imaging. Our method reliably identified ALT in cancer cell lines from different tumor entities and was validated in three established models of ALT induction and suppression. Furthermore, we successfully applied ALT-FISH to spatially resolve ALT activity in primary tissue sections from leiomyosarcoma and neuroblastoma tumors. Thus, our assay provides insights into the heterogeneity of ALT tumors and is suited for high-throughput applications, which will facilitate screening for ALT-specific drugs.

TelomereHunter - in silico estimation of telomere content and composition from cancer genomes.

BACKGROUND: Establishment of telomere maintenance mechanisms is a universal step in tumor development to achieve replicative immortality. These processes leave molecular footprints in cancer genomes in the form of altered telomere content and aberrations in telomere composition. To retrieve these telomere characteristics from high-throughput sequencing data the available computational approaches need to be extended and optimized to fully exploit the information provided by large scale cancer genome data sets. RESULTS: We here present TelomereHunter, a software for the detailed characterization of telomere maintenance mechanism footprints in the genome. The tool is implemented for the analysis of large cancer genome cohorts and provides a variety of diagnostic diagrams as well as machine-readable output for subsequent analysis. A novel key feature is the extraction of singleton telomere variant repeats, which improves the identification and subclassification of the alternative lengthening of telomeres phenotype. We find that whole genome sequencing-derived telomere content estimates strongly correlate with telomere qPCR measurements (r = 0.94). For the first time, we determine the correlation of in silico telomere content quantification from whole genome sequencing and whole genome bisulfite sequencing data derived from the same tumor sample (r = 0.78). An analogous comparison of whole exome sequencing data and whole genome sequencing data measured slightly lower correlation (r = 0.79). However, this is considerably improved by normalization with matched controls (r = 0.91). CONCLUSIONS: TelomereHunter provides new functionality for the analysis of the footprints of telomere maintenance mechanisms in cancer genomes. Besides whole genome sequencing, whole exome sequencing and whole genome bisulfite sequencing are suited for in silico telomere content quantification, especially if matched control samples are available. The software runs under a GPL license and is available at https://www.dkfz.de/en/applied-bioinformatics/telomerehunter/telomerehunter.html .

Clinical implications and molecular features of extracellular matrix networks in soft tissue sarcomas.

PURPOSE: The landscape of extracellular matrix (ECM) alterations in soft tissue sarcomas (STS) remains poorly characterised. We aimed to investigate the tumour ECM and adhesion signalling networks present in STS and their clinical implications. EXPERIMENTAL DESIGN: Proteomic and clinical data from 321 patients across 11 histological subtypes were analysed to define ECM and integrin adhesion networks. Subgroup analysis was performed in leiomyosarcomas (LMS), dedifferentiated liposarcomas (DDLPS) and undifferentiated pleiomorphic sarcomas (UPS). RESULTS: This analysis defined subtype-specific ECM profiles including enrichment of basement membrane proteins in LMS and ECM proteases in UPS. Across the cohort, we identified three distinct co-regulated ECM networks which are associated with tumour malignancy grade and histological subtype. Comparative analysis of LMS cell line and patient proteomic data identified the LCP1 cytoskeletal protein as a prognostic factor in LMS. Characterisation of ECM network events in DDLPS revealed three subtypes with distinct oncogenic signalling pathways and survival outcomes. Evaluation of the DDLPS subtype with the poorest prognosis nominates ECM remodelling proteins as candidate anti-stromal therapeutic targets. Finally, we define a proteoglycan signature which is an independent prognostic factor for overall survival in DDLPS and UPS. CONCLUSIONS: STS comprise heterogeneous ECM signalling networks and matrix-specific features have utility for risk stratification and therapy selection which could in future guide precision medicine in these rare cancers.

O-GlcNAc transferase inhibits KSHV propagation and modifies replication relevant viral proteins as detected by systematic O-GlcNAcylation analysis.

O-GlcNAcylation is an inducible, highly dynamic and reversible post-translational modification, mediated by a unique enzyme named O-linked N-acetyl-d-glucosamine (O-GlcNAc) transferase (OGT). In response to nutrients, O-GlcNAc levels are differentially regulated on many cellular proteins involved in gene expression, translation, immune reactions, protein degradation, protein-protein interaction, apoptosis and signal transduction. In contrast to eukaryotic cells, little is known about the role of O-GlcNAcylation in the viral life cycle. Here, we show that the overexpression of the OGT reduces the replication efficiency of Kaposi's sarcoma-associated herpesvirus (KSHV) in a dose-dependent manner. In order to investigate the global impact of O-GlcNAcylation in the KSHV life cycle, we systematically analyzed the 85 annotated KSHV-encoded open reading frames for O-GlcNAc modification. For this purpose, an immunoprecipitation (IP) strategy with three different approaches was carried out and the O-GlcNAc signal of the identified proteins was properly controlled for specificity. Out of the 85 KSHV-encoded proteins, 18 proteins were found to be direct targets for O-GlcNAcylation. Selected proteins were further confirmed by mass spectrometry for O-GlcNAc modification. Correlation of the functional annotation and the O-GlcNAc status of KSHV proteins showed that the predominant targets were proteins involved in viral DNA synthesis and replication. These results indicate that O-GlcNAcylation plays a major role in the regulation of KSHV propagation.

Expression and localization of axin 2 in colorectal carcinoma and its clinical implication.

PURPOSE: Aberrant activation of the Wnt/ß-catenin pathway plays a major role in the development of colorectal carcinoma (CRC). Axin 2 is a key protein of this pathway and is upregulated in CRC. Here, we investigated RNA- and protein expression of axin 2 in CRC tissues at the single cell level. Moreover, the association of axin 2 with prognosis and survival was investigated in a large cohort of CRC patients (n = 280). METHODS: Localization and expression of axin 2 and ß-catenin was investigated using in situ hybridization and immunohistochemical staining. The quantitative expression levels of axin 2 were determined using RT-qPCR. The association of axin 2 expression with prognosis and survival of the patients was determined by statistical analysis (logrank test, Kaplan-Meier). RESULTS: Our results confirmed the upregulation of axin 2 in CRC and showed that it is broadly expressed in the cytoplasm of the tumor epithelial cells both, in the tumor center and at the invasion front. Axin 2 was rarely expressed by tumor stromal cells and only weakly by normal colonic epithelial cells. Staining of ß-catenin and axin 2 in consecutive CRC tissue sections revealed that nuclear translocation of ß-catenin in the tumor front was not associated with changes in the cytoplasmic localization of axin 2. Axin 2 did not show any association with proven prognostic factors or survival of the CRC patients. CONCLUSION: The generally increased expression of axin 2 in all tumor stages as compared to normal tissue suggests an initiating pathogenic function in the development of CRC.

Heterogeneity in the gene regulatory landscape of leiomyosarcoma.

Characterizing inter-tumor heterogeneity is crucial for selecting suitable cancer therapy, as the presence of diverse molecular subgroups of patients can be associated with disease outcome or response to treatment. While cancer subtypes are often characterized by differences in gene expression, the mechanisms driving these differences are generally unknown. We set out to model the regulatory mechanisms driving sarcoma heterogeneity based on patient-specific, genome-wide gene regulatory networks. We developed a new computational framework, PORCUPINE, which combines knowledge on biological pathways with permutation-based network analysis to identify pathways that exhibit significant regulatory heterogeneity across a patient population. We applied PORCUPINE to patient-specific leiomyosarcoma networks modeled on data from The Cancer Genome Atlas and validated our results in an independent dataset from the German Cancer Research Center. PORCUPINE identified 37 heterogeneously regulated pathways, including pathways representing potential targets for treatment of subgroups of leiomyosarcoma patients, such as FGFR and CTLA4 inhibitory signaling. We validated the detected regulatory heterogeneity through analysis of networks and chromatin states in leiomyosarcoma cell lines. We showed that the heterogeneity identified with PORCUPINE is not associated with methylation profiles or clinical features, thereby suggesting an independent mechanism of patient heterogeneity driven by the complex landscape of gene regulatory interactions.

The landscape of chromothripsis across adult cancer types.

Chromothripsis is a recently identified mutational phenomenon, by which a presumably single catastrophic event generates extensive genomic rearrangements of one or a few chromosome(s). Considered as an early event in tumour development, this form of genome instability plays a prominent role in tumour onset. Chromothripsis prevalence might have been underestimated when using low-resolution methods, and pan-cancer studies based on sequencing are rare. Here we analyse chromothripsis in 28 tumour types covering all major adult cancers (634 tumours, 316 whole-genome and 318 whole-exome sequences). We show that chromothripsis affects a substantial proportion of human cancers, with a prevalence of 49% across all cases. Chromothripsis generates entity-specific genomic alterations driving tumour development, including clinically relevant druggable fusions. Chromothripsis is linked with specific telomere patterns and univocal mutational signatures in distinct tumour entities. Longitudinal analysis of chromothriptic patterns in 24 matched tumour pairs reveals insights in the clonal evolution of tumours with chromothripsis.

Defective homologous recombination DNA repair as therapeutic target in advanced chordoma.

Chordomas are rare bone tumors with few therapeutic options. Here we show, using whole-exome and genome sequencing within a precision oncology program, that advanced chordomas (n = 11) may be characterized by genomic patterns indicative of defective homologous recombination (HR) DNA repair and alterations affecting HR-related genes, including, for example, deletions and pathogenic germline variants of BRCA2, NBN, and CHEK2. A mutational signature associated with HR deficiency was significantly enriched in 72.7% of samples and co-occurred with genomic instability. The poly(ADP-ribose) polymerase (PARP) inhibitor olaparib, which is preferentially toxic to HR-incompetent cells, led to prolonged clinical benefit in a patient with refractory chordoma, and whole-genome analysis at progression revealed a PARP1 p.T910A mutation predicted to disrupt the autoinhibitory PARP1 helical domain. These findings uncover a therapeutic opportunity in chordoma that warrants further exploration, and provide insight into the mechanisms underlying PARP inhibitor resistance.

Integrative genomic and transcriptomic analysis of leiomyosarcoma.

Leiomyosarcoma (LMS) is an aggressive mesenchymal malignancy with few therapeutic options. The mechanisms underlying LMS development, including clinically actionable genetic vulnerabilities, are largely unknown. Here we show, using whole-exome and transcriptome sequencing, that LMS tumors are characterized by substantial mutational heterogeneity, near-universal inactivation of TP53 and RB1, widespread DNA copy number alterations including chromothripsis, and frequent whole-genome duplication. Furthermore, we detect alternative telomere lengthening in 78% of cases and identify recurrent alterations in telomere maintenance genes such as ATRX, RBL2, and SP100, providing insight into the genetic basis of this mechanism. Finally, most tumors display hallmarks of "BRCAness", including alterations in homologous recombination DNA repair genes, multiple structural rearrangements, and enrichment of specific mutational signatures, and cultured LMS cells are sensitive towards olaparib and cisplatin. This comprehensive study of LMS genomics has uncovered key biological features that may inform future experimental research and enable the design of novel therapies.

PD-L1 (CD274) copy number gain, expression, and immune cell infiltration as candidate predictors for response to immune checkpoint inhibitors in soft-tissue sarcoma.

Soft-tissue sarcomas (STS) are rare malignancies that account for 1% of adult cancers and comprise more than 50 entities. Current therapeutic options for advanced-stage STS are limited. Immune checkpoint inhibitors targeting the PD-1/PD-L1 signaling axis are being explored as new treatment modality in STS; however, the determinants of response to these agents are largely unknown. Using the sarcoma data set of The Cancer Genome Altas (TCGA) and an independent cohort of untreated high-grade STS, we analyzed DNA copy number status and mRNA expression of PD-L1 in a total of 335 STS cases. Copy number gains (CNG) were detected in 54 TCGA cases (21.1%), of which 21 (8.2%) harbored focal PD-L1 CNG and that were most prevalent in myxofibrosarcoma (35%) and undifferentiated pleomorphic sarcoma (34%). In the untreated high-grade STS cohort, we detected CNG in six cases (7.6%). Analysis of co-amplified genes identified a 5.6-Mb core region comprising 27 genes, including JAK2. Patients with PD-L1 CNG had higher PD-L1 expression compared with STS without CNG (fold change, 1.8; p = 0.02), an effect that was most pronounced in the setting of focal PD-L1 CNG (fold change, 3.0; p = 0.0027). STS with PD-L1 CNG showed a significantly higher mutational load compared with tumors with a diploid PD-L1 locus (median number of mutated genes; 58 vs. 40; p = 3.6E-06), and PD-L1 CNG were associated with inferior survival (HR = 1.82; p = 0.025). In contrast, T-cell infiltrates quantified by mRNA expression of CD3Z were associated with improved survival (HR = 0.88; p = 0.024) and consequently influenced the prognostic power of PD-L1 CNG, with low CD3Z levels conferring poor survival in cases with PD-L1 CNG (HR = 1.8; p = 0.049). These data demonstrate that PD-L1 GNG and elevated expression of PD-L1 occur in a substantial proportion of STS, have prognostic impact that is modulated by T-cell infiltrates, and thus warrant investigation as response predictors for immune checkpoint inhibition.

Targeting Fibroblast Growth Factor Receptor 1 for Treatment of Soft-Tissue Sarcoma.

Purpose: Altered FGFR1 signaling has emerged as a therapeutic target in epithelial malignancies. In contrast, the role of FGFR1 in soft-tissue sarcoma (STS) has not been established. Prompted by the detection and subsequent therapeutic inhibition of amplified FGFR1 in a patient with metastatic leiomyosarcoma, we investigated the oncogenic properties of FGFR1 and its potential as a drug target in patients with STS.Experimental Design: The frequency of FGFR1 amplification and overexpression, as assessed by FISH, microarray-based comparative genomic hybridization and mRNA expression profiling, SNP array profiling, and RNA sequencing, was determined in three patient cohorts. The sensitivity of STS cell lines with or without FGFR1 alterations to genetic and pharmacologic FGFR1 inhibition and the signaling pathways engaged by FGFR1 were investigated using viability assays, colony formation assays, and biochemical analysis.Results: Increased FGFR1 copy number was detected in 74 of 190 (38.9%; cohort 1), 13 of 79 (16.5%; cohort 2), and 80 of 254 (31.5%; cohort 3) patients. FGFR1 overexpression occurred in 16 of 79 (20.2%, cohort 2) and 39 of 254 (15.4%; cohort 3) patients. Targeting of FGFR1 by RNA interference and small-molecule inhibitors (PD173074, AZD4547, BGJ398) revealed that the requirement for FGFR1 signaling in STS cells is dictated by FGFR1 expression levels, and identified the MAPK-ERK1/2 axis as critical FGFR1 effector pathway.Conclusions: These data identify FGFR1 as a driver gene in multiple STS subtypes and support FGFR1 inhibition, guided by patient selection according to the FGFR1 expression and monitoring of MAPK-ERK1/2 signaling, as a therapeutic option in this challenging group of diseases. Clin Cancer Res; 23(4); 962-73. ©2016 AACR.

Dental age assessment (DAA): a study of a Caucasian population at the 13 year threshold.

UNLABELLED: The purpose of this study was to develop a Reference Data Set for Dental Age Assessment at the 13 year old threshold in Caucasian children. PATIENTS, MATERIALS AND METHODS: The Reference Data Set comprised 5187 re-used Dental Panoramic Tomographs (DPTs) between the ages of 11-15 years, from both the Eastman Dental Hospital and King's College Dental Hospital archives. Tooth Development Stages were recorded for the left maxillary and mandibular teeth and all four permanent third molars (Demirjian et al., 1973, Demirjian 1978). A separate Study Sample of DPTs, comprising 50 males and 50 females aged between 10 and 16 years was collected to test the accuracy of the method. Summary Data was generated for the individual Tooth Development Stages which consisted of the number (n-tds), mean (x), standard deviation (sd) and the standard error (se). By using the mathematical techniques of meta-analysis, this data was used to estimate the age of each subject in the Study Sample. The estimated Dental Age derived was compared to the gold standard of Chronological Age. RESULTS: The mean difference between the Chronological Age and Dental Age was determined to be -0.1 years (-1.2 months) for males and 0.05 years (-0.6 months) for females. CONCLUSION: Dental Age was reliably estimated at the 13 year threshold.

Reverse transfected cell microarrays in infectious disease research.

Several human pathogenic viruses encode large genomes with often more than 100 genes. Viral pathogenicity is determined by carefully orchestrated co-operative activities of several different viral genes which trigger the phenotypic functions of the infected cells. Systematic analyses of these complex interactions require high-throughput transfection technology. Here we have provided a laboratory manual for the reverse transfected cell microarray (RTCM; alternative name: cell chip) as a high-throughput transfection procedure, which has been successfully applied for the systematic analyses of single and combination effects of genes encoded by the human herpesvirus-8 on the NF-kappaB signal transduction pathway. In order to quantitatively determine the effects of viral genes in transfected cells, protocols for the use of GFP as an indicator gene and for indirect immunofluorescence staining of cellular target proteins have been included. RTCM provides a useful methodological approach to investigate systematically combination effects of viral genes on cellular functions.