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.

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. Design: Mice bearing a conditional TC allele were paired with a conditional Cdkn2a knockout allele and an endothelial-specific Cre. Histological characterization and single-cell RNA sequencing of the resultant tumors was 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. Conclusion: CDKN2A loss enhances the tumorigenicity of EHE in vivo and enabled the generation of. the first cell lines of this disease. These cell line 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 either directed at EHE or at other TAZ-driven cancers.

Hey2 gene mutations in both humans and mice have been associated with multiple cardiac defects. However, the currently reported localization of Hey2 in the ventricular compact zone cannot explain the wide variety of cardiac defects. Furthermore, it was reported that, in contrast to other organs, Notch doesn’t regulate Hey2 in the heart. To determine the expression pattern and the regulation of Hey2, we used novel methods including RNAscope and a Hey2CreERT2 knockin line to precisely determine the spatiotemporal expression pattern and level of Hey2 during cardiac development. We found that Hey2 is expressed in the endocardial cells of the atrioventricular canal and the outflow tract, as well as at the base of trabeculae, in addition to the reported expression in the ventricular compact myocardium. By disrupting several signaling pathways that regulate trabeculation and/or compaction, we found that, in contrast to previous reports, Notch signaling and Nrg1/ErbB2 regulate Hey2 expression level in myocardium and/or endocardium, but not its expression pattern: weak expression in trabecular myocardium and strong expression in compact myocardium. Instead, we found that FGF signaling regulates the expression pattern of Hey2 in the early myocardium, and regulates the expression level of Hey2 in a Notch1 dependent manner.

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 co-activator 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, pharmacological 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 non-randomized, open-label, phase 2 study of trametinib in patients with unresectable or metastatic EHE.

<p>Validation of the EHE cell lines</p>