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SRP084270
Homo sapiens
16 Downloadable Samples
Illumina HiSeq 2500
Description
ERG activity was blocked using YK-4-279 in three subcutaneously implanted ERG+ (LuCaP 23.1, 86.2, and 35) and one ERG- (LuCaP 96) PDX. Tumor volume (TV), body weight (BW), serum prostate specific antigen (PSA), and overall survival (OS) were compared to vehicle treated controls. Changes in gene expression were assessed by RNASeq and tissue microarrays were constructed to assess necrosis, proliferation, apoptosis, microvessel density, and ERG expression. Overall design: RNA sequencing of tumors from from 16 animals (2 control, 2 treated from each of four patient derived xenograft lines) using Illumina HiSeq 2500.
Publication Title
Inhibition of ERG Activity in Patient-derived Prostate Cancer Xenografts by YK-4-279.
Sample Metadata Fields
Sex, Treatment, Subject
View Samples- Homo sapiens
- 18 Downloadable Samples
Affymetrix Human Exon 1.0 ST Array [probe set (exon) version (huex10st)
Description
The synthesis and processing of mRNA, from transcription to translation initiation, often requires splicing of intragenic material. The final mRNA composition varies based upon proteins that modulate splice site selection. EWS-FLI1 is an Ewing sarcoma (ES) oncogene with an interactome that we demonstrate to have multiple partners in spliceosomal complexes. We evaluate EWS-FLI1 upon post-transcriptional gene regulation using both exon array and RNA-seq. Genes that potentially regulate oncogenesis including CLK1, CASP3, PPFIBP1, and TERT validate as alternatively spliced by EWS-FLI1. EWS-FLI1 also alters splicing by directly binding to known splicing factors including DDX5, hnRNPK, and PRPF6. Reduction of EWS-FLI1 produces an isoform of g-TERT that has increased telomerase activity compared to WT TERT. The small molecule YK-4-279 is an inhibitor of EWS-FLI1 oncogenic function that disrupts specific protein interactions including DDX5 and RNA helicase A (RHA) that alters RNA splicing ratios. As such, YK-4-279 validates the splicing mechanism of EWS-FLI1 showing alternatively spliced gene patterns that significantly overlap with EWS-FLI1 reduction and WT human mesenchymal stem cells. Exon array analysis of 75 ES patient samples show similar isoform expression patterns to cell line models expressing EWS-FLI1, supporting the clinical relevance of our findings. These experiments establish systemic alternative splicing as an oncogenic process modulated by EWS-FLI1. EWS-FLI1 modulation of mRNA splicing may provide insight into the contribution of splicing towards oncogenesis, and reciprocally, EWS-FLI1 interactions with splicing proteins may inform the splicing code.
Publication Title
Oncogenic fusion protein EWS-FLI1 is a network hub that regulates alternative splicing.
Sample Metadata Fields
Specimen part, Cell line
View Samples- Homo sapiens
- 3 Downloadable Samples
- Illumina HiSeq 2500
Description
A chimeric fusion between the RNA binding protein EWS and the ETS family transcription factor FLI1 (EWS-FLI1), created from a chromosomal translocation, is implicated in driving the majority of Ewing sarcomas (ES) by modulation of transcription and alternative splicing. The small molecule YK-4-279 inhibits EWS-FLI1 function and induces apoptosis. We tested 69 anti-cancer drugs in combination with YK-4-279 and found that vinca alkaloids exhibited synergy with YK-4-279 in five ES cell lines. The combination of YK-4-279 and vincristine reduced tumor burden and increased survival in mice bearing ES xenografts. We determined that independent drug-induced events converged to cause this synergistic therapeutic effect. YK-4-279 rapidly induced G2/M arrest, increased the abundance of cyclin B1, and decreased EWS-FLI1–mediated expression of microtubule-associated proteins, which rendered cells more susceptible to microtubule depolymerization by vincristine. YK-4-279 reduced the expression of the EWS-FLI1 target gene encoding ubiquitin ligase UBE2C, and this in part contributed to the increase in cyclin B1. Biochemical assays revealed that YK-4-279 also increased the abundance of proapoptotic isoforms of MCL1 and BCL2, presumably through inhibition of alternative splicing by EWS-FLI1, thus promoting cell death in response to vincristine. Thus a combination of vincristine and YK-4-279 might be therapeutically effective in ES patients. Overall design: Examination of mRNA profiles of TC32 on knockdown of EWS-FLI1 or treatment with YK-4-279: 3 samples Total: 1 TC32 WT Control, 1 TC32 shEF, 1 TC32 YK
Publication Title
Inhibition of the oncogenic fusion protein EWS-FLI1 causes G<sub>2</sub>-M cell cycle arrest and enhanced vincristine sensitivity in Ewing's sarcoma.
Sample Metadata Fields
Cell line, Subject
View Samples