High grade gliomas (HGGs) are characterized by resistance to radiotherapy and chemotherapy. Targeting Rad51-dependent homologous recombination repair may be an effective target for chemo- and radiosensitization. In this study we assessed the role of Rad51-dependent repair on sensitivity to radiation and temozolomide (TMZ) as single agents or in combination. Repair protein levels in established glioma cell lines, early passage glioblastoma multiforme (GBM) cell lines, and normal human astrocytes (NHAs) were measured using western blot. Viability and clonogenic survival assays were used to measure the effects of Rad51 knockdown with radiation (XR) and TMZ. Immunocytochemistry was used to evaluate kinetics of Rad51 and γ-H2AX repair foci. Immunohistochemistry was used to assess Rad51 protein levels in glioma specimens. Repair proteins including Rad51 are upregulated in HGG cells compared with NHA. Established glioma cell lines show a dose-dependent increase in Rad51 foci formation after XR and TMZ. Rad51 levels are inversely correlated with radiosensitivity, and downregulation markedly increases the cytotoxicity of TMZ. Rad51 knockdown also promotes more residual γ-H2AX foci 24 h after combined treatment. Newly established GBM cell lines also have high Rad51 levels and are extremely sensitive to Rad51 knockdown. Clinical samples from recently resected gliomas of varying grades demonstrate that Rad51 levels do not correlate with tumor grade. Rad51-dependent repair makes a significant contribution to DNA repair in glioma cells and contributes to resistance to both XR and TMZ. Agents targeting Rad51-dependent repair would be effective adjuvants in standard combination regimens.
Genomic gain of the proto-oncogene transcription factor gene MYCN is associated with poor prognosis in several childhood cancers. Here we present a comprehensive copy number analysis of MYCN in Wilms tumour (WT), demonstrating that gain of this gene is associated with anaplasia and with poorer relapse-free and overall survival, independent of histology. Using whole exome and gene-specific sequencing, together with methylation and expression profiling, we show that MYCN is targeted by other mechanisms, including a recurrent somatic mutation, P44L, and specific DNA hypomethylation events associated with MYCN overexpression in tumours with high risk histologies. We describe parallel evolution of genomic copy number gain and point mutation of MYCN in the contralateral tumours of a remarkable bilateral case in which independent contralateral mutations of TP53 also evolve over time. We report a second bilateral case in which MYCN gain is a germline aberration. Our results suggest a significant role for MYCN dysregulation in the molecular biology of Wilms tumour. We conclude that MYCN gain is prognostically significant, and suggest that the novel P44L somatic variant is likely to be an activating mutation.
Background: Wilms tumours (WTs) are characterised by several hallmarks that suggest epimutations such as aberrant DNA methylation are involved in tumour progression: loss of imprinting at 11p15, lack of recurrent mutations and formation of nephrogenic rests (NRs), which are lesions of retained undifferentiated embryonic tissue that can give rise to WTs. Methods: To identify such epimutations, we performed a comprehensive methylome analysis on 20 matched trios of micro-dissected WTs, NRs and surrounding normal kidneys (NKs) using Illumina Infinium HumanMethylation450 Bead Chips and functionally validated findings using RNA sequencing. Results: Comparison of NRs with NK revealed prominent tissue biomarkers: 629 differentially methylated regions, of which 55% were hypermethylated and enriched for domains that are bivalent in embryonic stem cells and for genes expressed during development (P = 2.49 × 10
Background: Wilms tumor is the most common pediatric renal malignancy and there is a clinical need for a molecular biomarker to assess treatment response and predict relapse. The known mutated genes in this tumor type show low mutation frequencies, whereas aberrant methylation at 11p15 is by far the most common aberration. We therefore analyzed the epigenome, rather than the genome, to identify ubiquitous tumor-specific biomarkers.
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