Project description:Mutations of the fibroblast growth factor receptor (FGFR) family members are frequently observed in metastatic bladder cancer. The development of erdafitinib, a pan-FGFR inhibitor, provides a significant therapeutic advance in bladder cancer, but resistance still limits its efficacy. In this study, we perform an unbiased whole-genome CRISPR-Cas9 synthetic lethal screen on FGFR-mutant bladder cancer cell lines treated with erdafitinib-targeted therapy and identify SRM as a critical contributor to erdafitinib resistance. In polyamine metabolism, SRM catalyzes the production of spermidine, which subsequently promotes the hypusination of eukaryotic translation factor 5A (eIF5A). Moreover, we demonstrate that hypusinated eIF5A (eIF5AHyp) facilitates the efficient translation of HMGA2, which in turn promotes EGFR expression. Notably, pharmacologic inhibition of SRM using MCHA enhances the efficacy of erdafitinib both in vitro and in vivo. Together, these results offer evidence of the synthetic lethality between SRM inhibition and erdafitinib, suggesting that combination treatment is a promising therapeutic strategy to overcome erdafitinib resistance for FGFR-mutant bladder cancer.
Project description:Mutations of the fibroblast growth factor receptor (FGFR) family members are frequently observed in metastatic bladder cancer. The development of erdafitinib, a pan-FGFR inhibitor, provides a significant therapeutic advance in bladder cancer, but resistance still limits its efficacy. In this study, we perform an unbiased whole-genome CRISPR-Cas9 synthetic lethal screen on FGFR-mutant bladder cancer cell lines treated with erdafitinib-targeted therapy and identify SRM as a critical contributor to erdafitinib resistance. In polyamine metabolism, SRM catalyzes the production of spermidine, which subsequently promotes the hypusination of eukaryotic translation factor 5A (eIF5A). Moreover, we demonstrate that hypusinated eIF5A (eIF5AHyp) facilitates the efficient translation of HMGA2, which in turn promotes EGFR expression. Notably, pharmacologic inhibition of SRM using MCHA enhances the efficacy of erdafitinib both in vitro and in vivo. Together, these results offer evidence of the synthetic lethality between SRM inhibition and erdafitinib, suggesting that combination treatment is a promising therapeutic strategy to overcome erdafitinib resistance for FGFR-mutant bladder cancer.
Project description:RIP-chip-SRM : a New Combinatorial Large Scale Approach Identifies a Set of Translationally Regulated bantam/miR 58 Targets in C. elegans RNA binding protein immunopurification + microarray + targeted protein quantification via Selected Reaction Monitoring This SuperSeries is composed of the SubSeries listed below.
Project description:RIP-chip-SRM : a New Combinatorial Large Scale Approach Identifies a Set of Translationally Regulated bantam/miR 58 Targets in C. elegans RNA binding protein immunopurification + microarray + targeted protein quantification via Selected Reaction Monitoring This SuperSeries is composed of the following subset Series: GSE32941: Purification of TAP::ALG-1 complexes and associated RNA from mixed-stage TAP::ALG-1 transgenic (WS4303) and wild-type (N2) animals GSE32942: Microarray analysis of TAP::ALG-1 associated RNAs isolated from synchronized 'wild-type' animals and 'mir-58' mutants GSE32943: Expression analysis of WS4303 (wild type) vs WS5041 (mir-58 mutants) total mRNA Refer to individual Series
Project description:Protein turnover represents an important mechanism in the functioning of cells, with deregulated synthesis and degradation of proteins implicated in many diseased states. Therefore, proteomics strategies to measure, with high confidence, turnover rates are of vital importance to understanding many biological processes. In this study, the more widely used approach of non-targeted precursor ion signal intensity (MS1) quantification is compared to selected reaction monitoring (SRM), a data acquisition strategy that records data for specific peptides, to determine if improved quantitative data would be obtained using a targeted quantification approach. Using mouse liver as a model system, turnover measurement of four tricarboxylic acid cycle proteins was performed using both MS1 and SRM quantification strategies. SRM outperformed MS1 in terms of sensitivity and selectivity of measurement, allowing more confident determination of protein turnover rates. SRM data is acquired using cheaper and more widely available tandem quadrupole mass spectrometers, making the approach accessible to a larger number of researchers than MS1 quantification, which is best performed on high mass resolution instruments. SRM acquisition is ideally suited to focussed studies where the turnover of tens of proteins is measured, making it applicable in determining the dynamics of proteins complexes and complete metabolic pathways.
Project description:Human filamin C (FLNc) is a target of the protease calpain at Y2625. To confirm the dependency of calpain cleavage from FLNc phosphorylation at position S2623/S2624 by PKCa, FLNc was overexpressed in HEK293 cells and cells were treated with PMA to stimulated kinase activity, with Gö6976 to inhibit kinase activity or mock-treated with DMSO. Subsequently, recombinant calpain-1 and GluC were used and the resulting peptide TVTSSSSRGSSY was monitored by SRM analyses.