VGLL2 and TEAD1 fusion proteins identified in human sarcoma drive YAP/TAZ-independent tumorigenesis by engaging EP300

Abstract

Studies on Hippo pathway regulation of tumorigenesis largely center on YAP and TAZ, the transcriptional co-regulators of TEADs. Here, we present an oncogenic mechanism involving VGLL and TEAD fusions that is Hippo pathway-related but YAP/TAZ-independent. We characterize two recurrent fusions, VGLL2-NCOA2 and TEAD1-NCOA2, recently identified in human spindle cell rhabdomyosarcoma. We demonstrate that in contrast to VGLL2 and TEAD1 the fusion proteins are potent activators of TEAD-dependent transcription, and the function of these fusion proteins does not require YAP/TAZ. Furthermore, we identify that VGLL2 and TEAD1 fusions engage specific epigenetic regulation by recruiting histone acetyltransferase EP300 to control TEAD-mediated transcriptional and epigenetic landscapes. We show that small-molecule EP300 inhibition can suppress fusion protein-induced oncogenic transformation both in vitro and in vivo in mouse models. Overall, our study reveals a molecular basis for VGLL involvement in cancer and provides a framework for targeting tumors carrying VGLL, TEAD, or NCOA translocations.

Keywords: EP300; HIPPO; NCOA2; TEAD; VGLL2; YAP; cancer biology; developmental biology; human.

Figure 1.. VGLL2-NCOA2 and TEAD1-NCOA2 Induce TEAD-mediated transcriptional activation.

(A) Schematic representation of protein structure of VGLL2, TEAD1, NCOA2, VGLL2-NCOA2, and TEAD1-NCOA2. Tondu motif (TDU), TEA DNA binding domain (TEA), YAP binding domain (YBD), basic Helix-Loop-Helix (bHLH), Per-Arnt-Sim domain (PAS), nuclear receptor interaction domain (NID), and transcriptional activation domain (TAD). Arrows point to the break points. (B) Immunoblot analysis of YAP^5SA, VGLL2-NCOA2, TEAD1-NCOA2, TAZ^4SA, TEAD1, VGLL2, and NCOA2 in HEK293T cells transfected with the expression constructs carrying the HA tag. The figure shows the representative results of three biological replicates. (C) YAP^5SA, VGLL2-NCOA2, TEAD1-NCOA2, TAZ^4SA, TEAD1, VGLL2, and NCOA2 induce transcriptional activation of TBS (TEAD-binding site)-luciferase reporter (TBS-Luc) in HEK293T cells. Data were expressed as mean ± SD. n=3. ****p<0.0001. (D) Heatmap showing expression levels of the core genes including CCN2, CCN1, ANKRD1, and AMOTL2 significantly regulated in HEK293T cells expressing YAP^5SA, VGLL2-NCOA2, and TEAD1-NCOA2. N=3. (E) mRNA expression levels of CCN2, ANKRD1, and CCN1 in HEK293T cells expressing YAP^5SA, VGLL2-NCOA2, TEAD1-NCOA2, TEAD1, VGLL2, or NCOA2. Data were expressed as mean ± SD. n=3; ****p<0.0001. NS, no significance.

Figure 1—figure supplement 1.. VGLL2-NCOA2 regulates TEAD-dependent reporter activity.

(A) Schematic representation of the exons and breaking points of the VGLL2, TEAD1, and NCOA2 genes involved in generating VGLL2-NCOA2 and TEAD1-NCOA2 gene arrangement. (B) EdU staining showing cell proliferation of HEK293T cells transfected with VGLL2-NCOA2 or TEAD1-NCOA2. Bar chart showing the percentage of EdU-positive cells. Scale bars, 200 μm. Data were expressed as mean ± SD. n=3; **p<0.01. (C) Immunoblot analysis of VGLL2-NCOA2, VGLL2-NCOA2^∆VGLL2, and VGLL2-NCOA2^∆NCOA2 in HEK293T cells transfected with the expression constructs carrying the V5 tag. (D) TBS-Luc reporter activity in HEK293T cells expressing VGLL2-NCOA2, VGLL2-NCOA2^∆VGLL2, and VGLL2-NCOA2^∆NCOA2. Data were expressed as mean ± SD. n=3. ****p<0.0001. (E) Immunoblot analysis of VGLL2-NCOA2-V5 in HEK293T cells transfected with different amount of expression constructs. (F) VGLL2-NCOA2 promotes the activation of TBS-Luc reporter in dose-dependent manner. Data were expressed as mean ± SD. n=3. ***p<0.001; ****p<0.0001. (G–H) Relative mRNA levels of CCN2, ANKRD1, and CCN1 in HEK293T cells expressing VGLL2-NCOA2 (G) or TEAD1-NCOA2 (H) with HA (5′ end), FLAG (3′ end), or V5 (3′ end) tag. Data were expressed as mean ± SD. n=3; NS, no significance.

Figure 1—figure supplement 2.. Analysis of VGLL2-NCOA2, TEAD1-NCOA2, and YAP^5SA -induced transcriptomes.

(A) Volcano maps of RNA-seq data sets of HEK293T cells expressing VGLL2-NCOA2, TEAD1-NCOA2, or YAP^5SA. Red dots represent upregulated mRNAs. Blue dots represent downregulated mRNAs. p<0.05, Log₂FoldChange >1 or < –1. (B) Venn diagram showing the overlaps of differentially regulated genes identified by RNA-seq in HEK293T cells expressing VGLL2-NCOA2, TEAD1-NCOA2, or YAP^5SA. (C) KEGG pathway enrichment analysis of differentially regulated genes identified by RNA-seq in HEK293T cells expressing VGLL2-NCOA2, TEAD1-NCOA2 or YAP^5SA. ‘Hippo signaling pathway’ is highlighted in red.

Figure 2.. VGLL2-NCOA2 and TEAD1-NCOA2-induced transcription does not require YAP and TAZ.

(A) Co-IP assays showing VGLL2-NCOA2 binding to TEAD1 but not YAP^5SA. YAP^5SA-Flag or TEAD1-Flag was co-expressed with VGLL2-NCOA2-HA in HEK293T cells and immunoprecipitated with an anti-HA antibody. (B) VGLL2-NCOA2 binds to endogenous TEAD but not YAP/TAZ. Endogenous YAP/TAZ and TEAD proteins in HEK293T cells were detected by anti-YAP/TAZ and panTEAD antibodies, respectively. (C) Co-IP assays showing endogenous YAP/TAZ binding to TEAD1 but not TEAD1-NCOA2. TEAD1-Flag or TEAD1-NCOA2-Flag was expressed in HEK293T cells and immunoprecipitated with an anti-Flag antibody. Endogenous YAP/TAZ proteins were detected by anti-YAP/TAZ antibodies. (D) The activity of TBS-Luc reporter in HEK293T cells expressing YAP^5SA, VGLL2-NCOA2, or TEAD1-NCOA2, with TEAD inhibitor CP1 (5 μM) treatment or co-expression of TEAD-ENR repressor construct. Data were expressed as mean ± SD. n=3; ****p<0.0001. NS, no significance. (E) Schematic representation of TEAD-ENR. TEA DNA-binding domain (TEA) and Engrailed repressor domain (ENR). (F) Immunoblot analysis of TEAD-ENR expression in HEK293T cells. (G) Co-IP assays showing YAP/TAZ were not essential for VGLL2-NCOA2 binding to endogenous TEADs. VGLL2-NCOA2-HA was expressed in HEK293T cells with or without YAP/TAZ knockdown and immunoprecipitated with an anti-HA antibody. (H) Relative mRNA levels of CCN2, ANKRD1, and CCN1 in HEK293T cells with YAP/TAZ knockdown and expressing VGLL2-NCOA2 or TEAD1-NCOA2. Data were expressed as mean ± SD. n=3; ****p<0.0001.

Figure 3.. Characterization of VGLL2-NCOA2- and YAP-controlled transcriptional and chromatin landscapes.

(A) Intersection of ATAC-seq (n=2), RNA-seq (n=3), and CUT&RUN (n=2) datasets in HEK293T cells expressing VGLL2-NCOA2 or YAP^5SA. (B) Venn diagrams showing the overlaps of ATAC-seq peaks, CUT&RUN peaks, and differentially regulated genes from RNA-seq in HEK293T cells expressing VGLL2-NCOA2 or YAP^5SA. (C) KEGG pathway enrichment analysis of ATAC-seq peaks identified in HEK293T cells expressing VGLL2-NCOA2 or YAP^5SA. The ‘Hippo signaling pathway’ is highlighted in red. (D) Distribution of CUT&RUN binding sites for VGLL2-NCOA2 and YAP^5SA. (E) Motif enrichment analysis of VGLL2-NCOA2 and YAP^5SA CUT&RUN Peaks. (F) Genomic tracks showing VGLL2-NCOA2 and YAP^5SA occupancy at the CCN2, ANKRD1, and CCN1 loci.

Figure 3—figure supplement 1.. ATAC-seq and CUT&RUN data characterization in VGLL2-NCOA2 and YAP^5SA-expressing cells.

(A) Heatmaps of TEAD-motif containing ATAC-seq peaks in HEK293T cells expressing VGLL2-NCOA2 or YAP^5SA. (B) Distribution of ATAC-seq peaks in HEK293T cells expressing VGLL2-NCOA2 or YAP^5SA. For VGLL2-NCOA2 ATAC-seq peaks, Promoter (≤ 1 kb) 50.57%, promoter (1–2 kb) 6.59%, 5′ UTR 0.13%, 3' UTR 1.44%, exon 1.52%, intron 18.39%, and distal and Intergenic 21.35%. For YAP^5SA ATAC-seq peaks, promoter (≤ 1 kb) 51.4%, promoter (1–2 kb) 6.89%, 5′ UTR 0.13%, 3′ UTR 1.45%, exon 1.58%, intron 17.28%, and distal and intergenic 21.26%. n=2. (C) VGLL2-NCOA2 promotes chromatin accessibility. Scatter diagrams of ATAC-seq peaks show more upregulated (red) than downregulated (blue) in HEK293T cells transfected with VGLL2-NCOA2 or YAP^5SA. (D) Heatmaps of VGLL2-NCOA2 and YAP^5SA CUT&RUN peaks. (E) VGLL2-NCOA2 and YAP genomic occupancy. Scatter diagrams showing more upregulated CUT&RUN peaks (red) than downregulated CUT&RUN peaks (blue) of VGLL2-NCOA2 and YAP^5SA. (F) KEGG pathway enrichment analysis of VGLL2-NCOA2 and YAP^5SA CUT&RUN peaks. ‘Hippo signaling pathway’ is highlighted in red.

Figure 4.. VGLL2-NCOA2 and TEAD1-NCOA2 engage EP300 epigenetic regulators.

(A) Diagram showing the BioID proteomic analyses of BirA*-VGLL2-NCOA2, BirA*-TEAD1-NCOA2, BirA*-YAP^5SA, and BirA*-TAZ^4SA. (B) Co-IP assays showing endogenous EP300 binding to VGLL2-NCOA2 and TEAD1-NCOA2 but not YAP^5SA. (C) KEGG enrichment analysis of EP300 CUT&RUN peaks in control HEK293T cells and HEK293T cells expressing VGLL2-NCOA2 or TEAD1-NCOA2. The ‘Hippo signaling pathway’ is highlighted in red. (D) Motif enrichment analysis of EP300 CUT&RUN peaks in control HEK293T cells and HEK293T cells expressing VGLL2-NCOA2 or TEAD1-NCOA2. (E) Genomic tracks showing EP300 occupancy at the CCN1, ANKRD1, CCN2, and CREB3 loci in control HEK293T cells and HEK293T cells expressing VGLL2-NCOA2 or TEAD1-NCOA2.

Figure 5.. EP300 is required for VGLL2-NCOA2- and TEAD1-NCOA2-induced tumorigenesis in vitro.

(A) Co-IP assays showing the NCOA2 fusion part of VGLL2-NCOA2 was essential for EP300 binding. VGLL2-NCOA2-V5, VGLL2-NCOA2^∆NCOA2-V5, and VGLL2-NCOA2^∆VGLL2-V5 were expressed in HEK293T cells and immunoprecipitated using an anti-V5 antibody. Endogenous EP300 proteins were detected by anti-EP300 antibody. (B–D) mRNA levels of Ccn2, Ankrd1, and Ccn1 in C2C12 cells expressing YAP^5SA, VGLL2-NCOA2, or TEAD1-NCOA2 with or without treatment of A485 (5 μM). Data were expressed as mean ± SD. n=3; ****p<0.0001. NS, no significance. (E) Representative image of colony formation of C2C12 cells expressing YAP^5SA, VGLL2-NCOA2, or TEAD1-NCOA2 with or without treatment of A485 (5 μM) for 2 weeks. Scale bars, 100 μm. (F, G) Colony size (F) and number of colonies (G) formed by C2C12 cells expressing YAP^5SA, VGLL2-NCOA2, or TEAD1-NCOA2 with or without treatment of A485 (5 μM). Data were expressed as mean ± SD. **p<0.01; ****p<0.0001. NS, no significance.

Figure 6.. EP300 is essential for VGLL2-NCOA2- and TEAD1-NCOA2-induced tumorigenesis in vivo.

(A) Representative H&E and IHC staining of Desmin and Ki67 in C2C12-control allograft, C2C12-VGLL2-NCOA2 tumor allograft, and C2C12-TEAD1-NCOA2 tumor allograft. Scale bars, 200 μm. (B) Immunoblot analysis of VGLL2-NCOA2-FLAG and TEAD1-NCOA2-FLAG expression in C2C12 cells, detected by an anti-FLAG antibody. (C, D) Allograft leg volume of C2C12-VGLL2-NCOA2 (C) and C2C12-TEAD1-NCOA2 (D) after intramuscular injection into the leg of Nude mice with or without intraperitoneal injection of A485 (100 mg/kg). The error bars represent the mean leg volume ± SEM. n=6. ****p<0.0001. (E) Representative H&E and IHC staining of Ki67 in C2C12-VGLL2-NCOA2 and C2C12-TEAD1-NCOA2 tumor allografts with or without A485 (100 mg/kg) treatment. Scale bars, 200 μm. (F) Percentage of Ki67-positive cells in (E). Data were expressed as mean ± SD. n=6; ***p<0.001. (G, H) mRNA levels of Ccn2, Ankrd1, and Ccn1 in C2C12-VGLL2-NCOA2 and C2C12-TEAD1-NCOA2 tumor allografts with or without A485 (100 mg/kg) treatment. Data were expressed as mean ± SD. **p<0.01; ***p<0.001; ****p<0.0001.