A phase 2b double-blind placebo-controlled randomized clinical trial of SB-061, an aggrecan mimetic, in patients with symptomatic knee osteoarthritis.

OBJECTIVES: To evaluate the efficacy and safety of intra-articular injections of a novel aggrecan mimetic, SB-061, in subjects with knee osteoarthritis (OA). METHODS: This was a randomized, placebo-controlled, double-blind phase II study comparing intra-articular injections of SB-061 with placebo (isotonic saline) for 52 weeks, administered at baseline, Wk 16, and Wk 32. Eligible subjects had a KL grade of 2 or 3 on X-ray of the target knee and a Western Ontario McMaster Universities Osteoarthritis Index (WOMAC) pain score ≥20 out of 50 at screening and baseline visits. Subjects having any other knee condition were excluded. Use of analgesics was prohibited, except for rescue medication. The primary endpoint was change from baseline (CFB) in WOMAC pain at Week 8. Secondary endpoints were CFB in WOMAC function and total, ICOAP, Patient Global Assessment, and 20-meter walk test. Exploratory endpoints included structural CFB in magnetic resonance imaging entities. RESULTS: A total of 288 subjects were randomized to SB-061 (n = 145) or placebo (n = 143), and 252 (87.5%) completed injections. The groups were comparable at baseline. The primary endpoint was not met, as no significant difference in the CFB of the WOMAC pain score at Week 8 between groups was observed, nor at any other time point during the study. Similarly, neither of the secondary or exploratory endpoints indicated any significant difference between groups. The frequency and type of adverse events were similar between groups. SB-061 was well-tolerated. CONCLUSION: Intra-articular injections of SB-061 administered at baseline, Week 16, and Week 32, over one year in subjects with knee OA, were safe but did not show any statistically significant effect on knee pain nor on other symptomatic or structural entities compared to placebo. TRIAL REGISTRATION NUMBER EUDRACT NO: 2019-004515-31.

The Efficacy and Safety of Multiple Dose Regimens of Kudzu (Pueraria lobata) Root Extract on Bone and Cartilage Turnover and Menopausal Symptoms.

Background: Menopause is associated with detrimental changes in turnover of bone and cartilage and a variety of symptoms with negative impact on the quality of life. Naturally occurring isoflavones from Radix Pueraria lobata, Kudzu root, may possess chondroprotective and symptom-relieving properties, but efficacy and safety of dosing and dose frequencies required for pharmacological action is unclear. Purpose: This clinical trial evaluates the efficacy on bone and cartilage turnover, menopausal symptoms, and safety of five dose regimens of Kudzu root extract administered either once, twice or three times daily in women with at least mild menopausal symptoms. Materials and Methods: Fifty postmenopausal women were randomized equally into five different dose regimen groups of Kudzu root extract in a four-week, parallel group, open-label, single-center, exploratory study design. Biomarkers CTX-I and CTX-II reflecting bone and cartilage degradation, respectively, were assessed in blood samples and 24-h urine samples. Change from baseline in the Menopause Rating Scale (MRS) and subscales was evaluated. Safety endpoints were frequency of adverse events, changes in hematology and safety chemistry data, vital signs and electrocardiogram. Results: Fifty women (Age 54.2 years, SD: 2.9) were randomized. After 4 weeks of treatment, biomarkers of bone resorption and cartilage degradation were statistically significantly reduced from baseline levels in the group receiving two capsules three times a day, serum/urine CTX-I (-18.4%, 95% CI: -8.1 to -27.5, p = 0.001/-34.2%, 95% CI: -21.6 to -44.7, p < 0.0001), urine CTX-II (-17.4% 95% CI: -2.5 to -30.0, p = 0.02). The observed effects were consistent across study groups but appeared to favour three times daily dosing. Four weeks of treatment led to statistically significant reductions in the MRS Total Score (p < 0.0001-0.03) in four out of five treatment groups. Kudzu root extract was well tolerated in all dose regimens, and no serious adverse events were reported. Conclusion: The results indicate that Kudzu extract may possess beneficial effects on bone and cartilage health and may be a promising natural alternative to existing treatments for menopausal symptoms. Kudzu root extract was well tolerated for short-term treatment of mild to severe menopausal symptoms in women in all tested doses and dose frequencies.

epiG: statistical inference and profiling of DNA methylation from whole-genome bisulfite sequencing data.

The study of epigenetic heterogeneity at the level of individual cells and in whole populations is the key to understanding cellular differentiation, organismal development, and the evolution of cancer. We develop a statistical method, epiG, to infer and differentiate between different epi-allelic haplotypes, annotated with CpG methylation status and DNA polymorphisms, from whole-genome bisulfite sequencing data, and nucleosome occupancy from NOMe-seq data. We demonstrate the capabilities of the method by inferring allele-specific methylation and nucleosome occupancy in cell lines, and colon and tumor samples, and by benchmarking the method against independent experimental data.

Heterogeneous patterns of DNA methylation-based field effects in histologically normal prostate tissue from cancer patients.

Prostate cancer (PC) diagnosis is based on histological evaluation of prostate needle biopsies, which have high false negative rates. Here, we investigated if cancer-associated epigenetic field effects in histologically normal prostate tissue may be used to increase sensitivity for PC. We focused on nine genes (AOX1, CCDC181 (C1orf114), GABRE, GAS6, HAPLN3, KLF8, MOB3B, SLC18A2, and GSTP1) known to be hypermethylated in PC. Using quantitative methylation-specific PCR, we analysed 66 malignant and 134 non-malignant tissue samples from 107 patients, who underwent ultrasound-guided prostate biopsy (67 patients had at least one cancer-positive biopsy, 40 had exclusively cancer-negative biopsies). Hypermethylation was detectable for all genes in malignant needle biopsy samples (AUC: 0.80 to 0.98), confirming previous findings in prostatectomy specimens. Furthermore, we identified a four-gene methylation signature (AOX1xGSTP1xHAPLN3xSLC18A2) that distinguished histologically non-malignant biopsies from patients with vs. without PC in other biopsies (AUC = 0.65; sensitivity = 30.8%; specificity = 100%). This signature was validated in an independent patient set (59 PC, 36 adjacent non-malignant, and 9 normal prostate tissue samples) analysed on Illumina 450 K methylation arrays (AUC = 0.70; sensitivity = 40.6%; specificity = 100%). Our results suggest that a novel four-gene signature may be used to increase sensitivity for PC diagnosis through detection of epigenetic field effects in histologically non-malignant prostate tissue samples.

Identifying aggressive prostate cancer foci using a DNA methylation classifier.

BACKGROUND: Slow-growing prostate cancer (PC) can be aggressive in a subset of cases. Therefore, prognostic tools to guide clinical decision-making and avoid overtreatment of indolent PC and undertreatment of aggressive disease are urgently needed. PC has a propensity to be multifocal with several different cancerous foci per gland. RESULTS: Here, we have taken advantage of the multifocal propensity of PC and categorized aggressiveness of individual PC foci based on DNA methylation patterns in primary PC foci and matched lymph node metastases. In a set of 14 patients, we demonstrate that over half of the cases have multiple epigenetically distinct subclones and determine the primary subclone from which the metastatic lesion(s) originated. Furthermore, we develop an aggressiveness classifier consisting of 25 DNA methylation probes to determine aggressive and non-aggressive subclones. Upon validation of the classifier in an independent cohort, the predicted aggressive tumors are significantly associated with the presence of lymph node metastases and invasive tumor stages. CONCLUSIONS: Overall, this study provides molecular-based support for determining PC aggressiveness with the potential to impact clinical decision-making, such as targeted biopsy approaches for early diagnosis and active surveillance, in addition to focal therapy.

Bivalent Regions of Cytosine Methylation and H3K27 Acetylation Suggest an Active Role for DNA Methylation at Enhancers.

The role of cytosine methylation in the structure and function of enhancers is not well understood. In this study, we investigate the role of DNA methylation at enhancers by comparing the epigenomes of the HCT116 cell line and its highly demethylated derivative, DKO1. Unlike promoters, a portion of regular and super- or stretch enhancers show active H3K27ac marks co-existing with extensive DNA methylation, demonstrating the unexpected presence of bivalent chromatin in both cultured and uncultured cells. Furthermore, our findings also show that bivalent regions have fewer nucleosome-depleted regions and transcription factor-binding sites than monovalent regions. Reduction of DNA methylation genetically or pharmacologically leads to a decrease of the H3K27ac mark. Thus, DNA methylation plays an unexpected dual role at enhancer regions, being anti-correlated focally at transcription factor-binding sites but positively correlated globally with the active H3K27ac mark to ensure structural enhancer integrity.

Top2 and Sgs1-Top3 Act Redundantly to Ensure rDNA Replication Termination.

Faithful DNA replication with correct termination is essential for genome stability and transmission of genetic information. Here we have investigated the potential roles of Topoisomerase II (Top2) and the RecQ helicase Sgs1 during late stages of replication. We find that cells lacking Top2 and Sgs1 (or Top3) display two different characteristics during late S/G2 phase, checkpoint activation and accumulation of asymmetric X-structures, which are both independent of homologous recombination. Our data demonstrate that checkpoint activation is caused by a DNA structure formed at the strongest rDNA replication fork barrier (RFB) during replication termination, and consistently, checkpoint activation is dependent on the RFB binding protein, Fob1. In contrast, asymmetric X-structures are formed independent of Fob1 at less strong rDNA replication fork barriers. However, both checkpoint activation and formation of asymmetric X-structures are sensitive to conditions, which facilitate fork merging and progression of replication forks through replication fork barriers. Our data are consistent with a redundant role of Top2 and Sgs1 together with Top3 (Sgs1-Top3) in replication fork merging at rDNA barriers. At RFB either Top2 or Sgs1-Top3 is essential to prevent formation of a checkpoint activating DNA structure during termination, but at less strong rDNA barriers absence of the enzymes merely delays replication fork merging, causing an accumulation of asymmetric termination structures, which are solved over time.

Hypermethylation of the GABRE~miR-452~miR-224 promoter in prostate cancer predicts biochemical recurrence after radical prostatectomy.

PURPOSE: Available tools for prostate cancer diagnosis and prognosis are suboptimal and novel biomarkers are urgently needed. Here, we investigated the regulation and biomarker potential of the GABRE∼miR-452∼miR-224 genomic locus. EXPERIMENTAL DESIGN: GABRE/miR-452/miR-224 transcriptional expression was quantified in 80 nonmalignant and 281 prostate cancer tissue samples. GABRE∼miR-452∼miR-224 promoter methylation was determined by methylation-specific qPCR (MethyLight) in 35 nonmalignant, 293 prostate cancer [radical prostatectomy (RP) cohort 1] and 198 prostate cancer tissue samples (RP cohort 2). Diagnostic/prognostic biomarker potential of GABRE∼miR-452∼miR-224 methylation was evaluated by ROC, Kaplan-Meier, uni- and multivariate Cox regression analyses. Functional roles of miR-224 and miR-452 were investigated in PC3 and DU145 cells by viability, migration, and invasion assays and gene-set enrichment analysis (GSEA) of posttransfection transcriptional profiling data. RESULTS: GABRE∼miR-452∼miR-224 was significantly downregulated in prostate cancer compared with nonmalignant prostate tissue and had highly cancer-specific aberrant promoter hypermethylation (AUC = 0.98). Functional studies and GSEA suggested that miR-224 and miR-452 inhibit proliferation, migration, and invasion of PC3 and DU145 cells by direct/indirect regulation of pathways related to the cell cycle and cellular adhesion and motility. Finally, in uni- and multivariate analyses, high GABRE∼miR-452∼miR-224 promoter methylation was significantly associated with biochemical recurrence in RP cohort 1, which was successfully validated in RP cohort 2. CONCLUSION: The GABRE∼miR-452∼miR-224 locus is downregulated and hypermethylated in prostate cancer and is a new promising epigenetic candidate biomarker for prostate cancer diagnosis and prognosis. Tumor-suppressive functions of the intronic miR-224 and miR-452 were demonstrated in two prostate cancer cell lines, suggesting that epigenetic silencing of GABRE∼miR-452∼miR-224 may be selected for in prostate cancer.

DNA methylation signatures for prediction of biochemical recurrence after radical prostatectomy of clinically localized prostate cancer.

PURPOSE: Diagnostic and prognostic tools for prostate cancer (PC) are suboptimal, causing overtreatment of indolent PC and risk of delayed treatment of aggressive PC. Here, we identify six novel candidate DNA methylation markers for PC with promising diagnostic and prognostic potential. METHODS: Microarray-based screening and bisulfite sequencing of 20 nonmalignant and 29 PC tissue specimens were used to identify new candidate DNA hypermethylation markers for PC. Diagnostic and prognostic potential was evaluated in 35 nonmalignant prostate tissue samples, 293 radical prostatectomy (RP) samples (cohort 1, training), and 114 malignant RP samples (cohort 2, validation) collected in Denmark, Switzerland, Germany, and Finland. Sensitivity and specificity for PC were evaluated by receiver operating characteristic analyses. Correlations between DNA methylation levels and biochemical recurrence were assessed using log-rank tests and univariate and multivariate Cox regression analyses. RESULTS: Hypermethylation of AOX1, C1orf114, GAS6, HAPLN3, KLF8, and MOB3B was highly cancer specific (area under the curve, 0.89 to 0.98). Furthermore, high C1orf114 methylation was significantly (P < .05) associated with biochemical recurrence in multivariate analysis in cohort 1 (hazard ratio [HR], 3.10; 95% CI, 1.89 to 5.09) and was successfully validated in cohort 2 (HR, 3.27; 95% CI, 1.17 to 9.12). Moreover, a significant (P < .05) three-gene prognostic methylation signature (AOX1/C1orf114/HAPLN3), classifying patients into low- and high-methylation subgroups, was trained in cohort 1 (HR, 1.91; 95% CI, 1.26 to 2.90) and validated in cohort 2 (HR, 2.33; 95% CI, 1.31 to 4.13). CONCLUSION: We identified six novel candidate DNA methylation markers for PC. C1orf114 hypermethylation and a three-gene methylation signature were independent predictors of time to biochemical recurrence after RP in two PC patient cohorts.

MRX protects fork integrity at protein-DNA barriers, and its absence causes checkpoint activation dependent on chromatin context.

To address how eukaryotic replication forks respond to fork stalling caused by strong non-covalent protein-DNA barriers, we engineered the controllable Fob-block system in Saccharomyces cerevisiae. This system allows us to strongly induce and control replication fork barriers (RFB) at their natural location within the rDNA. We discover a pivotal role for the MRX (Mre11, Rad50, Xrs2) complex for fork integrity at RFBs, which differs from its acknowledged function in double-strand break processing. Consequently, in the absence of the MRX complex, single-stranded DNA (ssDNA) accumulates at the rDNA. Based on this, we propose a model where the MRX complex specifically protects stalled forks at protein-DNA barriers, and its absence leads to processing resulting in ssDNA. To our surprise, this ssDNA does not trigger a checkpoint response. Intriguingly, however, placing RFBs ectopically on chromosome VI provokes a strong Rad53 checkpoint activation in the absence of Mre11. We demonstrate that proper checkpoint signalling within the rDNA is restored on deletion of SIR2. This suggests the surprising and novel concept that chromatin is an important player in checkpoint signalling.

DNA Topoisomerases maintain promoters in a state competent for transcriptional activation in Saccharomyces cerevisiae.

To investigate the role of DNA topoisomerases in transcription, we have studied global gene expression in Saccharomyces cerevisiae cells deficient for topoisomerases I and II and performed single-gene analyses to support our findings. The genome-wide studies show a general transcriptional down-regulation upon lack of the enzymes, which correlates with gene activity but not gene length. Furthermore, our data reveal a distinct subclass of genes with a strong requirement for topoisomerases. These genes are characterized by high transcriptional plasticity, chromatin regulation, TATA box presence, and enrichment of a nucleosome at a critical position in the promoter region, in line with a repressible/inducible mode of regulation. Single-gene studies with a range of genes belonging to this group demonstrate that topoisomerases play an important role during activation of these genes. Subsequent in-depth analysis of the inducible PHO5 gene reveals that topoisomerases are essential for binding of the Pho4p transcription factor to the PHO5 promoter, which is required for promoter nucleosome removal during activation. In contrast, topoisomerases are dispensable for constitutive transcription initiation and elongation of PHO5, as well as the nuclear entrance of Pho4p. Finally, we provide evidence that topoisomerases are required to maintain the PHO5 promoter in a superhelical state, which is competent for proper activation. In conclusion, our results reveal a hitherto unknown function of topoisomerases during transcriptional activation of genes with a repressible/inducible mode of regulation.

A Flp-nick system to study repair of a single protein-bound nick in vivo.

We present the Flp-nick system, which allows introduction of a protein-bound nick at a single genomic site in Saccharomyces cerevisiae and thus mimics a stabilized topoisomerase I-DNA cleavage complex. We took advantage of a mutant Flp recombinase that can introduce a nick at a specific Flp recombinase recognition target site that has been integrated in the yeast genome. The genetic requirement for cells to cope with this insult is the same as for cells treated with camptothecin, which traps topoisomerase I-DNA cleavage complexes genome-wide. Hence, a single protein-bound nick is enough to kill cells if functional repair pathways are lacking. The Flp-nick system can be used to dissect repair, checkpoint and replication fork management pathways activated by a single genomic insult, and it allows the study of events at the damage site, which so far has been impossible to address.