Abstract 3578: Alternative RNA splicing and protein products in leukemia outcome

Abstract

Abstract Tumors and leukemias have dramatically increased levels of aberrant RNA splicing, which generates a large population of transcripts that could encode variant proteins. This increased complexity of RNA products could be due to “noisy” splicing that is increased in transformed cells, but has no functional consequence. Alternatively, the variant RNAs and the proteins they encode could actively contribute to the transformed phenotype. For example, alternative RNA splicing produces variant forms of Myb proteins whose expression correlate with poor survival. Recurrent mutations in RNA splicing factors have been detected in some leukemias, and overexpression of some RNA splicing factors can transform cells. But there is still little direct evidence linking increased levels of alternative RNA splicing to leukemogenesis or transformation. We are testing the hypothesis that increased levels of alternative RNA splicing leads to the synthesis of aberrant protein products that contribute to the transformed phenotype. To test this, we are developing a whole molecule “deep” RNA sequencing approach that will capture the full-length structures of individual whole transcripts and predict the proteins they would encode. The long-term goal is to use the new assay to analyze gene expression and alternative RNA splicing of all transcripts in a well-defined cohort of high-risk childhood B-progenitor ALL patients who routinely fail the standard therapies. We will compare whether RNA levels or predicted protein levels are better predictors of outcome, as a test of whether the encoded variant proteins are contributing to the development of leukemia. As a starting point, we have conducted conventional RNA sequencing using Human Embryonic Kidney (HEK) 293T and Jurkat T-cells, using the Ion Torrent Proton sequencing platform followed by statistical analysis with the R-based EdgeR package. We identified more than 1600 differentially expressed genes. We compared the RNA-seq results to Affymetrix microarray gene expression profiles for the same samples and found relatively similar gene expression patterns. These data sets serve as the benchmark for development of the new single-molecule sequencing assay. With the improved assay we have obtained full-length sequence information for most expressed genes starting with as little as 1 ng of total RNA. We are currently developing the method further along with the bioinformatics, computational and statistical tools to analyze the single-molecule data. If increased alternative splicing is found to play a role in tumor development, these studies could identify novel and potentially “druggable” targets for the development of new therapies or interventions for these patients. Citation Format: Hideaki Suzuki. Alternative RNA splicing and protein products in leukemia outcome. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3578. doi:10.1158/1538-7445.AM2014-3578