TAZ-CAMTA1 and YAP-TFE3 alter the TAZ/YAP transcriptome by recruiting the ATAC histone acetyltransferase complex.

Epithelioid hemangioendothelioma (EHE) is a vascular sarcoma that metastasizes early in its clinical course and lacks an effective medical therapy. The TAZ-CAMTA1 and YAP-TFE3 fusion proteins are chimeric transcription factors and initiating oncogenic drivers of EHE. A combined proteomic/genetic screen in human cell lines identified YEATS2 and ZZZ3, components of the Ada2a-containing histone acetyltransferase (ATAC) complex, as key interactors of both fusion proteins despite the dissimilarity of the C terminal fusion partners CAMTA1 and TFE3. Integrative next-generation sequencing approaches in human and murine cell lines showed that the fusion proteins drive a unique transcriptome by simultaneously hyperactivating a TEAD-based transcriptional program and modulating the chromatin environment via interaction with the ATAC complex. Interaction of the ATAC complex with both fusion proteins indicates that it is a key oncogenic driver and unifying enzymatic therapeutic target for this sarcoma. This study presents an approach to mechanistically dissect how chimeric transcription factors drive the formation of human cancers.

The proliferation of human cells is tightly regulated to ensure that enough cells are made to build and repair organs and tissues, while at the same time stopping cells from dividing uncontrollably and damaging the body. To get the right balance, cells rely on physical and chemical cues from their environment that trigger the biochemical signals that regulate two proteins called TAZ and YAP. These proteins control gene activity by regulating the rate at which genes are copied to produce proteins. If this process becomes dysregulated, cells can grow uncontrollably, causing cancer. In cancer cells, it is common to find TAZ and YAP fused to other proteins. In epithelioid hemangioendothelioma, a rare cancer that grows in the blood vessels, cancerous growth can be driven by a version of TAZ fused to the protein CAMTA1, or a version of YAP fused to the protein TFE3. While the role of TAZ and YAP in promoting gene activity is known, it is unclear how CAMTA1 and TFE3 contribute to cell growth becoming dysregulated. Merritt, Garcia et al. studied sarcoma cell lines to show that these two fusion proteins, TAZ-CAMTA1 and YAP-TFE3, change the pattern of gene activity seen in the cells compared to TAZ or YAP alone. An analysis of molecules that interact with the two fusion proteins identified a complex called ATAC as the cause of these changes. This complex adds chemical markers to DNA-packaging proteins, which control which genes are available for activation. The fusion proteins combine the ability of TAZ and YAP to control gene activity and the ability of CAMTA1 and TFE3 to make DNA more accessible, allowing the fusion proteins to drive uncontrolled cancerous growth. Similar TAZ and YAP fusion proteins have been found in other cancers, which can activate genes and potentially alter DNA packaging. Targeting drug development efforts at the proteins that complex with TAZ and YAP fusion proteins may lead to new therapies.

A comprehensive evaluation of Hippo pathway silencing in sarcomas.

TAZ and YAP are transcriptional coactivators negatively regulated by the Hippo pathway that have emerged as key oncoproteins in several cancers including sarcomas. We hypothesized that loss of expression of the Hippo kinases might be a mechanism of activating TAZ and YAP. By immunohistochemistry, TAZ/YAP activated clinical sarcoma samples demonstrated loss of MST1 (47%), MST2 (26%), LATS1 (19%), and LATS2 (27%). Western blot similarly demonstrated loss of MST1 (58%), MST2 (25%), and LATS2 (17%). Treatment with MG132 demonstrated an accumulation of MST2 in 25% of sarcoma cell lines, indicating that proteosomal degradation regulates MST2 expression. qRT-PCR in sarcoma cell lines demonstrated loss of expression of the Hippo kinases at the RNA level, most pronounced in MST1 (42%) and MST2 (25%). 5-azacytidine treatment in sarcoma cell lines modestly reversed expression of predominantly MST1 (8%) and MST2 (17%), indicating CpG island hypermethylation can silence expression of MST1 and MST2. Trichostatin A treatment reversed expression of MST1 (58%) and MST2 (67%), indicating histone deacetylation also plays a role in silencing expression of MST1 and MST2. Loss of expression of the Hippo kinases is frequent in sarcomas and is due to a variety of mechanisms including regulation at the post-translational level and epigenetic silencing.

TAZ and YAP are frequently activated oncoproteins in sarcomas.

TAZ (WWTR1) and YAP are transcriptional coactivators and oncoproteins inhibited by the Hippo pathway. Herein we evaluate 159 sarcomas representing the most prevalent sarcoma types by immunohistochemistry for expression and activation (nuclear localization) of TAZ and YAP. We show that 50% of sarcomas demonstrate activation of YAP while 66% of sarcomas demonstrate activated TAZ. Differential activation of TAZ and YAP are identified in various sarcoma types. At an RNA level, expression of WWTR1 or YAP1 predicts overall survival in undifferentiated pleomorphic sarcoma and dedifferentiated liposarcoma. Immunohistochemistry demonstrates that TAZ and YAP expression and activation are positively correlated with grade in the well-differentiated liposarcoma to dedifferentiated liposarcoma tumor progression sequence as well as conventional chondrosarcomas. TAZ and YAP are constitutively activated oncoproteins in sarcoma cell lines. Knock-down of TAZ and YAP demonstrate differential activity for the two proteins. Verteporfin decreases colony formation in soft agar as well as CTGF expression in sarcoma cell lines harboring activated TAZ and YAP.

Human eosinophils express the high affinity IgE receptor, FcεRI, in bullous pemphigoid.

Bullous pemphigoid (BP) is an autoimmune blistering disease mediated by autoantibodies targeting BP180 (type XVII collagen). Patient sera and tissues typically have IgG and IgE autoantibodies and elevated eosinophil numbers. Although the pathogenicity of the IgE autoantibodies is established in BP, their contribution to the disease process is not well understood. Our aims were two-fold: 1) To establish the clinical relationships between total and BP180-specific IgE, eosinophilia and other markers of disease activity; and 2) To determine if eosinophils from BP patients express the high affinity IgE receptor, FcεRI, as a potential mechanism of action for IgE in BP. Our analysis of 48 untreated BP patients revealed a correlation between BP180 IgG and both BP180 IgE and peripheral eosinophil count. Additionally, we established a correlation between total IgE concentration and both BP180 IgE levels and eosinophil count. When only sera from patients (n = 16) with total IgE ≥ 400 IU/ml were analyzed, BP180 IgG levels correlated with disease severity, BP230 IgG, total circulating IgE and BP180 IgE. Finally, peripheral eosinophil count correlated more strongly with levels of BP180 IgE then with BP180 IgG. Next, eosinophil FcεRI expression was investigated in the blood and skin using several methods. Peripheral eosinophils from BP patients expressed mRNA for all three chains (α, ß and γ) of the FcεRI. Surface expression of the FcεRIα was confirmed on both peripheral and tissue eosinophils from most BP patients by immunostaining. Furthermore, using a proximity ligation assay, interaction of the α- and ß-chains of the FcεRI was observed in some biopsy specimens, suggesting tissue expression of the trimeric receptor form in some patients. These studies provide clinical support for the relevance of IgE in BP disease and provide one mechanism of action of these antibodies, via binding to the FcεRI on eosinophils.

Missing the target: characterization of bullous pemphigoid patients who are negative using the BP180 enzyme-linked immunosorbant assay.

BACKGROUND: Bullous pemphigoid (BP) is an autoimmune blistering disease characterized by autoantibodies specific for the 180-kd BP antigen-2 (BP180) (also termed "type XVII collagen") protein. The BP180 enzyme-linked immunosorbent assay (ELISA) is specific for the immunodominant NC16A domain of the protein. However, we and others have observed patients whose reactivity to BP180 is exclusive of the NC16A domain (referred to henceforth as non-NC16A BP). OBJECTIVE: We sought to determine the incidence of non-NC16A BP and identify regions of reactivity within the BP180 protein. METHODS: Sera from 51 patients who met the clinical and histologic criteria for BP were screened for NC16A reactivity by ELISA. Sera that were negative by ELISA were screened for IgG reactivity to an epidermal extract, recombinant BP180 protein, and subregions of BP180, by immunoblot. Demographic and clinical data were also collected on all patients. RESULTS: Four sera (7.8%) were negative using the BP180 ELISA but positive for IgG reactivity to the extracellular domain of BP180. Further mapping identified 4 regions outside of NC16A recognized by these sera: amino acid (AA) 1280 to 1315, AA 1080 to 1107, AA 1331 to 1404, and AA 1365 to 1413. One of these sera also had IgE specific for NC16A. One patient had an atypical presentation with lesions limited to the lower aspect of the legs and scarring of the nail beds. LIMITATIONS: The small total number of patients with non-NC16A BP limits the identification of demographic or clinical correlates. CONCLUSION: It is significant that 7.8% of sera from patients with new BP react to regions of BP180 exclusively outside of NC16A and, thus, would not be identified using the currently available BP180 ELISA.

Glycosylation contributes to variability in expression of murine cytomegalovirus m157 and enhances stability of interaction with the NK-cell receptor Ly49H.

NK cell-mediated resistance to murine cytomegalovirus (MCMV) is controlled by allelic Ly49 receptors, including activating Ly49H (C57BL/6 strain) and inhibitory Ly49I (129 strain), which specifically recognize MCMV m157, a glycosylphosphatidylinositol-linked protein with homology to MHC class I. Although the Ly49 receptors retain significant homology to classic carbohydrate-binding lectins, the role of glycosylation in ligand binding is unclear. Herein, we show that m157 is expressed in multiple, differentially N-glycosylated isoforms in m157-transduced or MCMV-infected cells. We used site-directed mutagenesis to express single and combinatorial asparagine (N)-to-glutamine (Q) mutations at N178, N187, N213, and N267 in myeloid and fibroblast cell lines. Progressive loss of N-linked glycans led to a significant reduction of total cellular m157 abundance, although all variably glycosylated m157 isoforms were expressed at the cell surface and retained the capacity to activate Ly49H(B6) and Ly49I(129) reporter cells and Ly49H(+) NK cells. However, the complete lack of N-linked glycans on m157 destabilized the m157-Ly49H interaction and prevented physical transfer of m157 to Ly49H-expressing cells. Thus, glycosylation on m157 enhances expression and binding to Ly49H, factors that may impact the interaction between NK cells and MCMV in vivo where receptor-ligand interactions are more limiting.

Squamous cell carcinoma cells differentially stimulate NK cell effector functions: the role of IL-18.

Tumor cells stimulate natural killer (NK) cell effector functions, but the regulation of cytokine secretion and cytolysis is incompletely understood. We tested whether oral and pharyngeal squamous cell carcinoma cell lines differentially stimulated NK cell interferon-gamma (IFN-gamma) secretion and cytolysis using a clone of the NK-92-transformed human NK cell line, NK92.35. SCC-4 and SCC-25 cells, but not FaDu or Cal 27 cells, stimulated robust NK92.35 IFN-gamma secretion. All four carcinoma cell lines were lysed by NK92.35 cells. These findings indicate that carcinoma cells differentially stimulate NK cell IFN-gamma secretion and cytolysis. In Transwell experiments, a combination of SCC-4 or SCC-25 cell soluble factors and contact with FaDu cells synergistically stimulated NK92.35 cell IFN-gamma secretion. Stimulatory SCC-4 cells constitutively secreted IL-18, a cytokine that potently augments IFN-gamma secretion by T cells and NK cells. In contrast, poorly stimulatory FaDu cells produced little or no IL-18, but synergized with recombinant IL-18 to stimulate NK92.35 IFN-gamma secretion. mAb to IL-18 or IL-18 receptor diminished SCC-4-stimulated IFN-gamma secretion by NK92.35 cells and by nontransformed NK cells. Thus, IL-18 was necessary for optimal carcinoma stimulation of NK cell IFN-gamma secretion. In vivo, oral and upper aerodigestive tract epithelia and carcinomas produced IL-18, but one squamous cell carcinoma had heterogeneous IL-18 expression. Thus IL-18 production can account for squamous cell carcinoma differential stimulation of NK cell effector functions in vitro and may be important for stimulation of NK cells in vivo.