Abstract 499: Characterization of the cell death mechanism after silencing of PAX3-FOXO1 in alveolar rhabdomyosarcoma using a CRISPR-Cas mediated knockout approach

Abstract

Abstract Chromosomal translocations leading to the generation of PAX-FOXO1 fusion oncogenes are found in about 80 percent of all alveolar Rhabdomyosarcoma (aRMS). Recent whole genome sequencing studies revealed that apart from these translocations mutation frequency is generally very low in aRMS, with no other genetic changes detected in some cases. aRMS tumor cells depend on the fusion protein and exhibit cell cycle arrest or undergo cell death upon blocking of its activity. Taken together this strongly suggests that the PAX-FOXO1 fusion represents the tumor initiating event and that it remains the most important tumorigenic factor for aRMS maintenance at later tumor stages. However, the molecular mechanisms involved in tumorigenesis downstream of PAX3-FOXO1 are only partially understood yet, including the ones involved in oncogene addiction. We therefore aimed to further characterize the molecular events leading to cell death after silencing of PAX3-FOXO1. Towards this aim, we used an inducible shRNA system to silence PAX3-FOXO1 in different aRMS cell lines. Reduction of PAX3-FOXO1 below a threshold of about 20 percent of its basal level efficiently induced Caspase-dependent cell death. CRISPR-Cas mediated knock-out of individual members of the apoptotic cascade was then used to further characterize the involved apoptotic mechanism. BH3-only proteins including Bim were found as relevant sensors detecting cellular stress after silencing of PAX3-FOXO1. This data suggests that combined interference with PAX-FOXO1 activity and anti-apoptotic Bcl-2 family members might accelerate tumor cell death. Citation Format: Marco Wachtel, Felix Niggli, Beat Schäfer. Characterization of the cell death mechanism after silencing of PAX3-FOXO1 in alveolar rhabdomyosarcoma using a CRISPR-Cas mediated knockout approach. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 499. doi:10.1158/1538-7445.AM2015-499