Supplemental Table 2. Primer sequences for genotyping, qualitative reverse transcriptase-PCR, and oligonucleotides for cloning.

Ki67 staining and single-cell RNA sequencing

Immunohistochemistry of EHE allograft tumors

Gross and microscopic pathology of lymphoma samples

Supplemental Table 3. Single cell RNA sequencing of GEMM EHE tumors Differentially expressed genes between normal endothelial cells and Cdkn2a WT and KO tumors by single cell RNA sequencing. Functional annotations with the gene ontology sets are displayed for the Cdkn2a KO vs WT comparison, graphically displayed in Figure 3D. Transcripts used for the EHE tumor gene set are similarly displayed.

Supplemental Table 4. Bulk RNA sequencing of EHE cell lines Differential gene expression between EHE cell lines and endothelial cells via DESEQ2. Overlap in transcripts that are overexpressed by EHE cells (LogFC2>2 and FDR<0.05) are listed. These overlaps were used to generate the Venn diagram in Figure 4G.

Supplemental Table 1. Mouse Short Tandem Repeat (STR) loci for all three cell lines Short tandem repeat analysis of each EHE cell line and using both mouse and human loci. Human defining loci were undetected in all three cell lines.

Figure S2. Localization of TC following Dina + MG132 treatment

Abstract Purpose: There are no effective treatment options for patients with aggressive epithelioid hemangioendothelioma (EHE) driven by the TAZ–CAMTA1 (TC) fusion gene. Here, we aimed to understand the regulation of TC using pharmacologic tools and identify vulnerabilities that can potentially be exploited for the treatment of EHE. Experimental Design: TC is a transcriptional coregulator; we hypothesized that compounds that reduce TC nuclear levels, either through translocation of TC to the cytoplasm, or through degradation, would render TC less oncogenic. TC localization was monitored using immunofluorescence in an EHE tumor cell line. Two target-selective libraries were used to identify small molecules that reduce TC localization in the nucleus. The ability of the shortlisted hits to affect cell viability, apoptosis, and tumorigenesis was also evaluated. Results: Basal TC remained “immobile” in the nucleus; administration of cyclin-dependent kinase (CDK) inhibitors such as CGP60474 and dinaciclib (Dina) mobilized TC. “Mobile” TC shuttled between the nucleus and cytoplasm; however, it was eventually degraded through proteasomes. This dramatically suppressed the levels of TC-regulated transcripts and cell viability, promoted apoptosis, and reduced the area of metastatic lesions in the allograft model of EHE. We specifically identified that the inhibition of CDK9, a transcriptional CDK, destabilizes TC. Conclusions: The CDK inhibitor Dina exhibited antitumorigenic properties both in vitro and in vivo in EHE models. Dina has been rigorously tested in clinical trials and displayed an acceptable toxicity profile. Therefore, there is a potential therapeutic window for repurposing Dina for the treatment of EHE.

Figure S1. Dose-response curves of triptonide, triptolide, and dBET6

Abstract Purpose: Epithelioid hemangioendothelioma (EHE) is a vascular sarcoma caused by the WWTR1(TAZ)–CAMTA1 (TC) gene fusion. This fusion gene has been observed in almost all reported EHE cases and functions as a constitutively activated TAZ. Sequencing of human tumors has, however, identified additional secondary mutations in approximately 50% of EHE, most commonly the loss of tumor suppressor CDKN2A. In this study, the effect of loss of CDKN2A in EHE tumorigenesis was evaluated. Experimental Design: Mice bearing a conditional TC allele were paired with a conditional Cdkn2a knockout allele and an endothelial-specific Cre. Histologic characterization and single-cell RNA-seq of the resultant tumors were performed. EHE cell lines were established through ex vivo culture of tumor cells and evaluated for sensitivity to TEAD inhibition and trametinib. Results: Loss of Cdkn2a within EHE was associated with more aggressive disease, as displayed by earlier tumor-related morbidity/mortality and enhanced tumor cell proliferation. As no previous EHE cell lines exist, we attempted, successfully, to expand EHE tumor cells ex vivo and produced the first EHE cell lines. These cell lines are “addicted” to the TC oncoprotein, replicate the EHE transcriptional profile, and generate EHE tumors when injected into immunodeficient mice. Conclusions: CDKN2A loss enhances the tumorigenicity of EHE in vivo and enabled the generation of the first cell lines of this disease. These cell lines replicate key facets of the human disease phenotype. Therefore, these cell lines and allograft tumors generated after implantation serve as robust model systems for therapeutic testing of compounds directed at either EHE or other TAZ-driven cancers.

Background: Epithelioid Hemangioendothelioma (EHE) is the second most common vascular sarcoma and contains a TAZ-CAMTA1 gene fusion (TC) in >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.

PURPOSE:A consistent genetic alteration in vascular cancer epithelioid hemangioendothelioma (EHE) is the t(1;3)(p36;q25) chromosomal translocation, which generates a WWTR1(TAZ)-CAMTA1 (TC) fusion gene. TC is a transcriptional coactivator that drives EHE. Here, we aimed to identify the TC transcriptional targets and signaling mechanisms that underlie EHE tumorigenesis.EXPERIMENTAL DESIGN:We used NIH3T3 cells transformed with TC (NIH3T3/TC) as a model system to uncover TC-dependent oncogenic signaling. These cells proliferated in an anchorage-independent manner in suspension and soft agar. The findings of the cell-based studies were validated in a xenograft model.RESULTS:We identified connective tissue growth factor (CTGF) as a tumorigenic transcriptional target of TC. We show that CTGF binds to integrin αIIbβ3, which is essential for sustaining the anchorage-independent proliferation of transformed NIH3T3/TC cells. NIH3T3/TC cells also have enhanced Ras and MAPK signaling, and the activity of these pathways is reduced upon CTGF knockdown, suggesting that CTGF signaling occurs via the Ras-MAPK cascade. Further, pharmacologic inhibition of MAPK signaling through PD 0325901 and trametinib abrogated TC-driven anchorage-independent growth. Likewise, for tumor growth in vivo, NIH3T3/TC cells require CTGF and MAPK signaling. NIH3T3/TC xenograft growth was profoundly reduced upon CTGF knockdown and after trametinib treatment.CONCLUSIONS:Collectively, our results demonstrated that CTGF and the Ras-MAPK signaling cascade are essential for TC-mediated tumorigenesis. These studies provided the preclinical rationale for SARC033 (NCI 10015-NCT03148275), a nonrandomized, open-label, phase II study of trametinib in patients with unresectable or metastatic EHE.

Epithelioid hemangioendothelioma (EHE) is a genetically homogenous vascular sarcoma that is a paradigm for TAZ dysregulation in cancer. EHE harbors a WWTR1 (TAZ)- CAMTA1 gene fusion in >90% of cases, 45% of which have no other genetic alterations. In this study, we used a first of its kind approach to target the Wwtr1-Camta1 gene fusion to the Wwtr1 locus, to develop a conditional EHE mouse model whereby Wwtr1-Camta1 is controlled by the endogenous transcriptional regulators upon Cre activation. These mice develop EHE tumors that are indistinguishable from human EHE clinically, histologically, immunohistochemically, and genetically. Overall, these results demonstrate unequivocally that TAZ-CAMTA1 is sufficient to drive EHE formation with exquisite specificity, as no other tumor types were observed. Furthermore, we fully credential this unique EHE mouse model as a valid preclinical model for understanding the role of TAZ dysregulation in cancer formation and for testing therapies directed at TAZ-CAMTA1, TAZ, and YAP/TAZ signaling.