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• W2565967540 abstract "Soft-tissue sarcoma (STS) is a rare type of tumor that accounts for approximately 1 % of adult cancers. STS tumors frequently recur or result in distant metastasis after surgical resection. Cytotoxic chemotherapy, the current “gold standard”, is often confronted with development of resistance, leading to patient survival periods below 15 months. Therefore, further insight into new therapies and mechanisms leading to drug resistance in sarcoma is required to improve therapeutic success. In the present study, we analyzed drug resistance in vitro using (i) a genome-wide assay for identification of drug resistance-associated genetic alterations and (ii) generation of isogenic cell lines using gene editing approaches to functionally characterize the genetic alterations. To address drug response and resistance in sarcoma, we have established a genome-wide insertional mutagenesis screening approach. To this end, a panel of STS cell lines is screened for the sensitivity towards drugs currently used for standard therapy of STS. Sensitive cell lines will be treated with transforming lentiviral vectors to identify genes involved in drug resistance. The genetic alterations potentially involved in acquired resistance mechanisms will be functionally characterized using isogenic cell lines created by gene editing approaches. Towards this end, we have successfully employed transcription activator-like effector nucleases and CRISPR/Cas9 system RNA guided endonucleases targeting the BRAF locus to introduce the previously described V600E mutation in 293T cells, as a “proof of concept”. Analysis of the target site showed that 31 % of the sequences were positive for the mutation. The mutation was also confirmed in single cell clones by tetra-primer ARMS-PCR and western blot using an antibody specific for V600E mutated B-Raf. To test the method in a model relevant to STS, we applied gene editing to introduce a previously undescribed KRT8 mutation found in STS patients into a sarcoma cell line. For this mutation, wild type and D10A-mutated Cas9 expressing constructs were generated to allow use of single nucleases and paired nickases. We applied single stranded oligodeoxynucleotides as donor template for KRT8. Using high-throughput sequencing, we detected successful recombination of the mutated KRT8 gene locus in 0.6 % of the treated STS cells. We are using improved guide RNA and donor design as well as compounds affecting non-homologous end-joining and homologous recombination to improve efficiency of successful recombination. To summarize, we successfully edited wild-type BRAF to BRAF V600E in 293T cells and showed that gene editing of KRT8 is possible in sarcoma cells. The methodology will be further optimized to characterize genes involved in resistance to therapy identified in the genome-wide screens. In addition, our gene editing approaches will also be used to model genetic alterations identified in STS patients by next generation sequencing. Our findings will provide interesting insights in nominating drugs for combinatorial therapy to circumvent drug resistance in STS." @default.
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• W2565967540 date "2016-05-01" @default.
• W2565967540 modified "2023-10-18" @default.
• W2565967540 title "134. Gene Editing Approaches for Investigating Therapy-Resistance in Soft-Tissue Sarcoma" @default.
• W2565967540 doi "https://doi.org/10.1016/s1525-0016(16)32943-4" @default.
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