Abstract 3108: Modeling translocation driven tumors with human induced pluripotent stem cells (hiPSCs) using CRISPR-Cas9: Pseudomyogenic hemangioendothelioma as a proof of principle
Authors: IJzendoorn David G. van , Hil Francijna van den , Szuhai Karoly , Bovée Judith V. , Orlova Valeria V. 
Published: August 2018

Abstract
Within the group of vascular tumors balanced chromosomal translocations often drive tumorigenesis. For pseudomyogenic hemangioendothelioma (PHE) no cell line is available, therefore to model this tumor of endothelial origin, we induced a t(7;19)(q22;q13) chromosomal translocation leading to a SERPINE1-FOSB gene fusion in “healthy” human induced Pluripotent Stem Cells (hiPSCs). The hiPSC line was derived using Sendai virus (SeV)-based reprogramming and has been fully characterized. To create a model for PHE we used CRISPR-Cas9 to induce double-stranded breaks in SERPINE1 and FOSB. Using a repair template we introduced a removable neomycin resistance cassette to facilitate selection of cells with translocation. Droplet digital PCR (ddPCR) showed that the wild-type allele of both SERPINE1 and FOSB was mutated in 50% of the cells, and 12% of cells had SERPINE1-FOSB translocations. Screening of single cell-derived clones resulted in 2 clones harboring the correct chromosomal translocation out of 73 screened colonies. Fluorescence in situ hybridization (FISH) using dual-fusion probes confirmed presence of translocation in hiPSCs. Next, isogenic “healthy” and SERPINE1-FOSB targeted hiPSC clones were used to derive endothelial cells (ECs). SERPINE1-FOSB fusion had no effect on differentiation efficiency of ECs from hiPSCs. Fluorescence-activated cell sorting (FACS) confirmed comparable expression of endothelial-specific markers, such as Ve-cadherin, PECAM1, and VEGFR2. Interestingly, expression of CD105 and VEGFR3 that were shown to be elevated in tumor associated endothelial cells were increased in ECs derived from SERPINE1-FOSB targeted hiPSCs compared with ECs derived from “healthy” hiPSCs. Functional assessment of hiPSC-derived ECs showed increased proliferation, tube formation and a decrease in endothelial barrier function in the presence of a SERPINE1-FOSB fusion. Real-time PCR confirmed elevated expression of FOSB (5.9 log2 fold), CD105 (1.9 log2 fold), as well as known pro-angiogenic and pro-inflammatory cytokines, such as VEGF (2.2 log2 fold), IL6 (1.2 log2 fold) and IL8 (2.9 log2 fold). In summary, we demonstrate SERPINE1-FOSB translocation can be introduced into hiPSCs. Importantly, hiPSC-derived ECs that harbor the SERPINE1-FOSB translocation display abnormal tumor endothelium-like phenotype and therefore could serve as an excellent model to study the pathogenesis of PHE. This method could also be potentially interesting to introduce other translocation-driven tumors where no cell lines are available for the discovery of novel targeted therapy and deeper understanding of tumorigenesis.
Citation Format: David G. van IJzendoorn, Francijna van den Hil, Karoly Szuhai, Judith V. Bovée, Valeria V. Orlova. Modeling translocation driven tumors with human induced pluripotent stem cells (hiPSCs) using CRISPR-Cas9: Pseudomyogenic hemangioendothelioma as a proof of principle [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3108.