Showing papers by "Jason W. H. Wong published in 2015"

WDR5 Supports an N-Myc Transcriptional Complex That Drives a Protumorigenic Gene Expression Signature in Neuroblastoma

Abstract: MYCN gene amplification in neuroblastoma drives a gene expression program that correlates strongly with aggressive disease. Mechanistically, trimethylation of histone H3 lysine 4 (H3K4) at target gene promoters is a strict prerequisite for this transcriptional program to be enacted. WDR5 is a histone H3K4 presenter that has been found to have an essential role in H3K4 trimethylation. For this reason, in this study, we investigated the relationship between WDR5-mediated H3K4 trimethylation and N-Myc transcriptional programs in neuroblastoma cells. N-Myc upregulated WDR5 expression in neuroblastoma cells. Gene expression analysis revealed that WDR5 target genes included those with MYC-binding elements at promoters such as MDM2. We showed that WDR5 could form a protein complex at the MDM2 promoter with N-Myc, but not p53, leading to histone H3K4 trimethylation and activation of MDM2 transcription. RNAi-mediated attenuation of WDR5 upregulated expression of wild-type but not mutant p53, an effect associated with growth inhibition and apoptosis. Similarly, a small-molecule antagonist of WDR5 reduced N-Myc/WDR5 complex formation, N-Myc target gene expression, and cell growth in neuroblastoma cells. In MYCN-transgenic mice, WDR5 was overexpressed in precancerous ganglion and neuroblastoma cells compared with normal ganglion cells. Clinically, elevated levels of WDR5 in neuroblastoma specimens were an independent predictor of poor overall survival. Overall, our results identify WDR5 as a key cofactor for N-Myc-regulated transcriptional activation and tumorigenesis and as a novel therapeutic target for MYCN-amplified neuroblastomas.

Lynch Syndrome Associated with Two MLH1 Promoter Variants and Allelic Imbalance of MLH1 Expression

Abstract: Lynch syndrome is a hereditary cancer syndrome caused by a constitutional mutation in one of the mismatch repair genes. The implementation of predictive testing and targeted preventative surveillance is hindered by the frequent finding of sequence variants of uncertain significance in these genes. We aimed to determine the pathogenicity of previously reported variants (c.-28A>G and c.-7C>T) within the MLH1 5′untranslated region (UTR) in two individuals from unrelated suspected Lynch syndrome families. We investigated whether these variants were associated with other pathogenic alterations using targeted high-throughput sequencing of the MLH1 locus. We also determined their relationship to gene expression and epigenetic alterations at the promoter. Sequencing revealed that the c.-28A>G and c.-7C>T variants were the only potentially pathogenic alterations within the MLH1 gene. In both individuals, the levels of transcription from the variant allele were reduced to 50% compared with the wild-type allele. Partial loss of expression occurred in the absence of constitutional epigenetic alterations within the MLH1 promoter. We propose that these variants may be pathogenic due to constitutional partial loss of MLH1 expression, and that this may be associated with intermediate penetrance of a Lynch syndrome phenotype. Our findings provide further evidence of the potential importance of noncoding variants in the MLH1 5′UTR in the pathogenesis of Lynch syndrome.

Systematic Screening of Promoter Regions Pinpoints Functional Cis-Regulatory Mutations in a Cutaneous Melanoma Genome

Abstract: With the recent discovery of recurrent mutations in the TERT promoter in melanoma, identification of other somatic causal promoter mutations is of considerable interest. Yet, the impact of sequence variation on the regulatory potential of gene promoters has not been systematically evaluated. This study assesses the impact of promoter mutations on promoter activity in the whole-genome sequenced malignant melanoma cell line COLO-829. Combining somatic mutation calls from COLO-829 with genome-wide chromatin accessibility and histone modification data revealed mutations within promoter elements. Interestingly, a high number of potential promoter mutations ( n = 23) were found, a result mirrored in subsequent analysis of TCGA whole-melanoma genomes. The impact of wild-type and mutant promoter sequences were evaluated by subcloning into luciferase reporter vectors and testing their transcriptional activity in COLO-829 cells. Of the 23 promoter regions tested, four mutations significantly altered reporter activity relative to wild-type sequences. These data were then subjected to multiple computational algorithms that score the cis -regulatory altering potential of mutations. These analyses identified one mutation, located within the promoter region of NDUFB9 , which encodes the mitochondrial NADH dehydrogenase (ubiquinone) 1 beta subcomplex 9, to be recurrent in 4.4% (19 of 432) of TCGA whole-melanoma exomes. The mutation is predicted to disrupt a highly conserved SP1/KLF transcription factor binding motif and its frequent co-occurrence with mutations in the coding sequence of NF1 supports a pathologic role for this mutation in melanoma. Taken together, these data show the relatively high prevalence of promoter mutations in the COLO-829 melanoma genome, and indicate that a proportion of these significantly alter the regulatory potential of gene promoters. Implications: Genomic-based screening within gene promoter regions suggests that functional cis -regulatory mutations may be common in melanoma genomes, highlighting the need to examine their role in tumorigenesis. Mol Cancer Res; 13(8); 1218–26. ©2015 AACR . This article is featured in Highlights of This Issue, [p. 1161][1] [1]: /lookup/volpage/13/1161?iss=8

Overexpression of ERG in cord blood progenitors promotes expansion and recapitulates molecular signatures of high ERG leukemias.

Abstract: High expression of the ETS family transcription factor ERG is associated with poor clinical outcome in acute myeloid leukemia (AML) and acute T-cell lymphoblastic leukemia (T-ALL). In murine models, high ERG expression induces both T-ALL and AML. However, no study to date has defined the effect of high ERG expression on primary human hematopoietic cells. In the present study, human CD34+ cells were transduced with retroviral vectors to elevate ERG gene expression to levels detected in high ERG AML. RNA sequencing was performed on purified populations of transduced cells to define the effects of high ERG on gene expression in human CD34+ cells. Integration of the genome-wide expression data with other data sets revealed that high ERG drives an expression signature that shares features of normal hematopoietic stem cells, high ERG AMLs, early T-cell precursor-ALLs and leukemic stem cell signatures associated with poor clinical outcome. Functional assays linked this gene expression profile to enhanced progenitor cell expansion. These results support a model whereby a stem cell gene expression network driven by high ERG in human cells enhances the expansion of the progenitor pool, providing opportunity for the acquisition and propagation of mutations and the development of leukemia.

The search for cis-regulatory driver mutations in cancer genomes

Abstract: With the advent of high-throughput and relatively inexpensive whole-genome sequencing technology, the focus of cancer research has begun to shift toward analyses of somatic mutations in non-coding cis-regulatory elements of the cancer genome. Cis-regulatory elements play an important role in gene regulation, with mutations in these elements potentially resulting in changes to the expression of linked genes. The recent discoveries of recurrent TERT promoter mutations in melanoma, and recurrent mutations that create a super-enhancer regulating TAL1 expression in T-cell acute lymphoblastic leukaemia (T-ALL), have sparked significant interest in the search for other somatic cis-regulatory mutations driving cancer development. In this review, we look more closely at the TERT promoter and TAL1 enhancer alterations and use these examples to ask whether other cis-regulatory mutations may play a role in cancer susceptibility. In doing so, we make observations from the data emerging from recent research in this field, and describe the experimental and analytical approaches which could be adopted in the hope of better uncovering the true functional significance of somatic cis-regulatory mutations in cancer.

Single nucleotide polymorphism array profiling identifies distinct chromosomal aberration patterns across colorectal adenomas and carcinomas.

Abstract: The progression of benign colorectal adenomas into cancer is associated with the accumulation of chromosomal aberrations. Even though patterns and frequencies of chromosomal aberrations have been well established in colorectal carcinomas, corresponding patterns of aberrations in adenomas are less well documented. The aim of this study was to profile chromosomal aberrations across colorectal adenomas and carcinomas to provide a better insight into key changes during tumor initiation and progression. Single nucleotide polymorphism array analysis was performed on 216 colorectal tumor/normal matched pairs, comprising 60 adenomas and 156 carcinomas. While many chromosomal aberrations were specific to carcinomas, those with the highest frequency in carcinomas (amplification of chromosome 7, 13q, and 20q; deletion of 17p and chromosome 18; LOH of 1p, chromosome 4, 5q, 8p, 17p, chromosome 18, and 20p) were also identified in adenomas. Hierarchical clustering using chromosomal aberrations revealed three distinct subtypes. Interestingly, these subtypes were only partially dependent on tumor staging. A cluster of colorectal cancer patients with frequent chromosomal deletions had the least favorable prognosis, and a number of adenomas (n = 9) were also present in the cluster suggesting that, at least in some tumors, the chromosomal aberration pattern is determined at a very early stage of tumor formation. Finally, analysis of LOH events revealed that copy-neutral/gain LOH (CN/G-LOH) is frequent (>10%) in carcinomas at 5q, 11q, 15q, 17p, chromosome 18, 20p, and 22q. Deletion of the corresponding region is sometimes present in adenomas, suggesting that LOH at these loci may play an important role in tumor initiation.

Bioorthogonal labelling of 3-nitrotyrosine in peptides and proteins through diazotisation mediated azidation

Abstract: A new chemical derivatisation method to functionalise peptide and protein bound 3-nitrotyrosine residues is described. By transforming 3-nitrotyrosine to 3-azidotyrosine, the method enables ‘click chemistry’-based labelling of the 3-nitrotyrosine residues and provides new opportunities for the study of this post-translational modification in biological samples.