Abstract 931: Loss of CDKN2A in epithelioid hemangioendothelioma enhances tumorigenicity and imparts cellular immortality, thereby facilitating the generation of the first cell lines of this disease
Authors: Seavey Caleb Nathaniel , Ma Shuang , Hallett Andrea , Pobbati Ajaybabu V. , Che Kepeng , Li Shuo , Burtscher Ashley , Kanai Ryan , Lamar John M. , Rubin Brian P. 
Published: June 2022

Abstract
Background: Epithelioid Hemangioendothelioma (EHE) is the second most common vascular sarcoma and contains a TAZ-CAMTA1 gene fusion (TC) in &gt;90% of cases, and concurrent work demonstrates that endothelial expression of this oncogene at physiologic levels is sufficient to induce EHE tumorigenesis in mice. A major impediment to EHE research is the lack of EHE cell lines, and to date efforts to establish primary cultures of EHE cells have been unsuccessful. As 57% of human EHEs contain secondary genetic alterations, most commonly CDKN2A variants, that are associated with later stage disease, we hypothesized that these alterations mechanistically enhance EHE tumorigenesis. We further hypothesized that loss of CDKN2A in EHE cells will allow for tumor cell outgrowth in vitro and generation of the first EHE cell lines.
Methods: Mice bearing our previously generated TC allele, whereby the endogenous Taz allele can be conditionally replaced with a TC transgene under the action of Cre, were paired with a conditional Cdkn2a knockout allele and an endothelial specific Cre (Cdh5-CreERT2). Histological characterization and single cell RNA sequencing of the resultant tumors was performed. Tumors were dissociated and the resultant tumor cells were grown ex vivo as cell lines. We characterized the cell lines using genomic, immunocytochemistry and RNA Seq methodologies. Expanded cells were then reimplanted into NSG mice.
Results: When compared to Cdkn2a WT murine EHE tumors, tumors containing a mono- or bi-allelic deletion of Cdkn2a have a greater proliferative rate as displayed by greater cellularity, and increased number of Ki67+ cells. While CDKN2a variant tumors displayed enhanced proliferative capacity, they maintained the characteristic EHE histologic phenotype and transcriptional profile. Tumor cells were successfully grown ex vivo and the resultant cell lines maintained an endothelial morphology in vitro, expressed the TC oncoprotein, and had a transcriptional profile characteristic of both murine and human EHE. These cell lines were successfully cultured to greater than 40 passages. Finally, in mouse models, these cell lines generate local tumors when injected subcutaneously, metastatic lung tumors when injected intravenously, and induce peritoneal sarcomatosis when injected intraperitoneally in an orthotopic model. In each of these sites, the ensuing tumors maintained the EHE histologic phenotype.
Conclusions: We were able to demonstrate that CDKN2A loss enhances the tumorigenicity of EHE in vivo. Further, we were able to generate the first cell lines of this disease which replicate several facets of the human disease and therefore serve as a robust model system for therapeutic testing of compounds, which if successful can be used to treat EHE or other TAZ-dependent cancers.
Citation Format: Caleb Nathaniel Seavey, Shuang Ma, Andrea Hallett, Ajaybabu V. Pobbati, Kepeng Che, Shuo Li, Ashley Burtscher, Ryan Kanai, John M. Lamar, Brian P. Rubin. Loss of CDKN2A in epithelioid hemangioendothelioma enhances tumorigenicity and imparts cellular immortality, thereby facilitating the generation of the first cell lines of this disease [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 931.