Abstract A009: NR2F1 inhibits Gaq/11-YAP-TAZ signaling and limits reactivation of uveal melanoma disseminated cancer cells via induction of dormancy

Introduction: At the time of diagnosis, less than 5% of uveal melanoma (UM) patients have evidence of metastasis. However, up to 50% of UM patients will succumb to advanced metastatic in liver. UM cancer cells can persist undetected in liver for prolonged periods mainly and often take years-to-decades to grow out and do so without recurrence at the primary site. However, the mechanisms underlying the prolonged dormancy and why the liver is a preferred site for survival and eventual regrowth are a long-standing medical mystery. Results: GNAQ/11 are the main driving mutations in UM that activate downstream oncogenic YAP signaling. Using Gaq/11mut /BAPwt human uveal melanoma lines, we reveal that NR2F1, a neural cell commitment regulator is the main driver of regulating dormancy program. RNA-seq of rare UM disseminated cancer cells (DCCs) in livers identified that dormant DCCs upregulate the expression of neural differentiation genes, including NR2F1, while silencing Gaq downstream YAP/TAZ and cell cycle gene expression compared to proliferative cell clusters. Targeted pharmacological inhibition of Gaq/11mut signaling using YM-254890, an inhibitor of Gaq/11 resulted in NR2F1 upregulation and robust UM growth arrest. In contrast, inhibition of NR2F1 either by siRNA or CRISPR/cas9 upregulated YAP1 and its downstream targets at both transcript and protein levels along with increased colony growth in 3D culture. Importantly, UM solitary DCCs in nude mouse upregulated NR2F1 and in vivo CRISPR KO of NR2F1 results in dormant UM DCC awakening and massive metastatic growth in the murine liver. Immunofluorescence of UM patients’ liver metastatic lesions showed high expression of NR2F1 in solitary DCCs compared to the tumor mass. Chromatin-immunoprecipitation assays revealed that NR2F1 binds to the YAP1 promoter and regulates its gene expression. OMM1.3 CRISPR/Cas9 KO of NR2F1 cells showed increased recruitment of H3K4me3 and H3K27ac histone marks onto the YAP1 promoter while overexpression of NR2F1 decreased the recruitment of histone activating marks. Co-immunoprecipitation assay showed increased YAP1-TEAD1 complex formation in NR2F1 KO OMM1.3 cells while overexpression of NR2F1 reduced the abundance of this complex. Summary: Our work suggests that NR2F1 is required to maintain the single cell state of UM cells in the liver by inhibiting the YAP signally pathway. This study provides insight into the previously unrecognized mechanism of uveal melanoma dormancy opening