Mechanisms of Resistance for Cancers with Fibroblast Growth Factor Receptor Gene Fusions
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Date
2015-05
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Publisher
The Ohio State University
Abstract
Fusions involving tyrosine kinase genes can activate pathways in cancers and are targets for inhibitors. Recently, novel kinase gene fusions involving fibroblast growth factor receptors (FGFR) have been described in patients. We will be using next generation sequencing techniques to understand resistance to tyrosine kinase inhibitors involved in cancer. Specifically, we study kinases that are activated through a gene rearrangement or fusion event. In this proposal, we developed cancer cell lines with FGFR-gene fusions that are resistant to inhibitors to study acquired resistance. To address our hypothesis that the tumors develop resistance with drug exposure, we treated RT-4, a bladder cancer cell line with FGFR3 fusion, chronically with clinically relevant FGFR inhibitors BGJ398 and ponatinib. We used Western blot analysis to corroborate FGFR inhibition in RT-4 cells with ponatinib and BGJ398. Then we assessed for resistance using cell viability assays (Cell Titer-Glo, Promega). Both the resistant cell lines (ponatinib and BGJ3989) displayed significantly higher (10-12-fold) IC50 values compared to the parental RT-4 cell line. Next, we evaluated proteins involved in FGFR signaling using Western blot analysis and observed upregulation of pAKT, pMEK and pERK in both BGJ398- and ponatinib-resistant cells compared to parental cells. In addition to Western blot analysis, we will be reviewing Reverse Phase Protein Array (RPPA) analysis to look at a wide range of proteins from other pathways, which can lead to resistance. Preliminary sequencing analysis shows upregulation in FGF19 ligand in ponatinib-resistant Rt-4 cells. After DNA and RNA sequencing data, we will be able to see any increases in copy number sequences and in expression. When all the information is collected, we will be able to develop theories about possible resistant mechanisms. These theories can be tested using a variety of knockdown methods. This project is important to understanding drug resistance, which could possibly be overcome with the administration of a combination of drugs.
Description
3rd place Denman Undergraduate Research Forum
Keywords
Cancer, Fusion, Resistance, FGFR