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.

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.

Targeted therapies are effective cancer treatments when accompanied by accurate diagnostic tests that can help identify patients that will respond to those therapies. The YAP/TAZ-TEAD axis is activated and plays a causal role in several cancer types, and TEAD inhibitors are currently in early-phase clinical trials in cancer patients. However, a lack of a reliable way to identify tumors with YAP/TAZ-TEAD activation for most cancer types makes it difficult to determine which tumors will be susceptible to TEAD inhibitors. Here, we used a combination of RNA-seq and bioinformatic analysis of metastatic melanoma cells to develop a YAP/TAZ gene signature. We found that the genes in this signature are TEAD-dependent in several melanoma cell lines, and that their expression strongly correlates with YAP/TAZ activation in human melanomas. Using DepMap dependency data, we found that this YAP/TAZ signature was predictive of melanoma cell dependence upon YAP/TAZ or TEADs. Importantly, this was not limited to melanoma because this signature was also predictive when tested on a panel of over 1000 cancer cell lines representing numerous distinct cancer types. Our results suggest that YAP/TAZ gene signatures like ours may be effective tools to predict tumor cell dependence upon YAP/TAZ-TEAD, and thus potentially provide a means to identify patients likely to benefit from TEAD inhibitors.

Background/Objectives: Epithelioid Hemangioendothelioma (EHE) is an ultra-rare, metastatic vascular sarcoma with limited therapeutic options. The hallmark of EHE is a chromosomal translocation that produces the WWTR1-CAMTA1 gene fusion, encoding the aberrant transcriptional regulator TAZ-CAMTA1. Given its central role in the EHE initiation and progression, TAZ-CAMTA1 represents a compelling therapeutic target. Methods and Results: In this study, we identified AMP-activated protein kinase (AMPK) as one of several proteins capable of repressing the TAZ-CAMTA1 transcriptional activity in NIH3T3 and HEK293 cell lines. The pharmacologic activation of AMPK inhibited the proliferation of EHE cell lines without inducing apoptosis; however, in contrast to the NIH3T3 and HEK293 models, AMPK activation in EHE cells unexpectedly increased the TAZ-CAMTA1 expression and activity. Notably, elevated TAZ-CAMTA1 expression was also associated with reduced EHE cell growth, suggesting that the induction of TAZ-CAMTA1 may be one mechanism by which AMPK suppresses EHE growth. Additionally, we found that AMPK inhibits mTOR activity and that direct mTOR inhibition also suppresses EHE cell growth. Conclusions: Together, these findings demonstrate that AMPK activation impairs EHE viability through dual mechanisms: by promoting TAZ-CAMTA1 expression and by inhibiting mTOR signaling. This work highlights AMPK as a potential therapeutic target in EHE and supports the growing body of evidence favoring mTOR inhibitors as promising treatments for this rare cancer.

Epithelioid Hemangioendothelioma (EHE) is an ultra-rare, metastatic vascular sarcoma with limited therapeutic options. The hallmark of EHE is a chromosomal trans-location that produces the WWTR1-CAMTA1 gene fusion, encoding the aberrant transcriptional regulator TAZ-CAMTA1. Given its central role in EHE initiation and progression, TAZ-CAMTA1 represents a compelling therapeutic target. In this study, we identified AMP-activated protein kinase (AMPK) as one of several proteins capable of repressing TAZ-CAMTA1 transcriptional activity in NIH3T3 and HEK293 cell lines. Pharmacologic activation of AMPK inhibited the growth of EHE cell lines; however, in contrast to the NIH3T3 and HEK293 models, AMPK activation in EHE cells unexpectedly increased TAZ-CAMTA1 expression and activity. Notably, elevated TAZ-CAMTA1 expression was also associated with reduced EHE cell growth, suggesting that induction of TAZ-CAMTA1 may be one mechanism by which AMPK sup-presses EHE growth. Additionally, we found that AMPK inhibits mTOR activity, and that direct mTOR inhibition also suppresses EHE cell growth. Together, these findings demonstrate that AMPK activation impairs EHE viability through dual mechanisms: by promoting TAZ-CAMTA1 expression and by inhibiting mTOR signaling. This work highlights AMPK as a potential therapeutic target in EHE and supports the growing body of evidence favoring mTOR inhibitors as promising treatments for this rare cancer.

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.