Identifying a nodal point for G alpha q signaling in eye disease

描述（由申请方提供）：两种q类Gα蛋白（Gαq）GNAQ和GNA 11的激活突变是约80%葡萄膜黑色素瘤肿瘤发生的驱动因素。在患有Sturge-Weber综合征的患者中也发现了类似的GNAQ激活突变，Sturge-Weber综合征可以表现为青光眼和结膜、脉络膜、视网膜和巩膜外层的血管畸形。葡萄膜黑色素瘤是最常见的原发性眼部肿瘤，在大约50%的病例中，肿瘤会转移到其他器官，主要是肝脏。一旦转移，这种疾病总是致命的。激活GNAQ和GNA 11突变通过控制几种最近鉴定的信号传导途径来驱动葡萄膜黑色素瘤肿瘤发生，所述信号传导途径包括磷脂酶C-ERK/蛋白激酶C（PLC-ERK/PKC）和TRIO-RhoA/Rac 1途径，其激活MAPK/ERK和雅普以诱导AP 1和YAP-TEAD介导的转录。然而， 哪些Gαq蛋白激活多个下游通路尚未完全阐明。初步数据表明，小GT3 ARF 6可能作为激活的GNAQ/GNA 11的直接下游效应物，控制所有目前已知的致癌Gαq信号通路。其他初步数据也表明，Gαq可能通过ARF 6发出信号，通过促进β-连环蛋白从细胞膜重新定位到细胞核来激活β-连环蛋白信号，在细胞核中β-连环蛋白可以介导基因转录。初步数据也支持ARF 6在葡萄膜黑色素瘤中的体内作用。当ARF 6在葡萄膜黑色素瘤中被shRNA沉默或被小分子抑制剂抑制时，在原位异种移植小鼠模型中肿瘤的建立和生长被显著抑制。根据这些初步数据，将努力实现以下目标。在目的1中，我们将研究激活的Gαq蛋白是否通过激活葡萄膜黑色素瘤中的ARF 6诱导β-连环蛋白信号传导。我们将通过RNA干扰和小分子抑制选定的靶点来评估葡萄膜黑色素瘤中β-连环蛋白的细胞内定位、转录活性和功能。在目标2中，我们将阐明ARF 6在协调致癌Gαq的已知下游信号通路中的作用。将采用与目标1中使用的相同策略来确定ARF 6是否作为激活GNAQ/GNA 11突变的直接下游效应物，以控制（PLC-β 1/PKC）和TRIORho A/Rac 1途径以及AP-1和YAP-TEAD介导的转录。在目标3中，我们将通过使用人葡萄膜黑色素瘤的原位异种移植模型来评估ARF 6在肿瘤建立和生长中的体内功能。这些目标的成功完成将使我们能够确定ARF 6是否在G α q信号传导中发挥关键作用，从而为开发可用于治疗葡萄膜黑色素瘤和其他可能的Gα q相关疾病（如Sturge-Weber综合征）的药物提供有希望的治疗靶点。

DESCRIPTION (provided by applicant): Activating mutations in two Gα proteins of the q class (Gαq), GNAQ and GNA11, are the drivers of oncogenesis in approximately 80% of uveal melanomas. Similar activating mutations of GNAQ are also found in patients with Sturge-Weber syndrome, which can manifest itself as glaucoma and vascular malformations of the conjunctiva, choroid, retina, and episclera. Uveal melanoma is the most common primary ocular tumor, and in approximately 50% of the cases, the tumor will metastasize to other organs, primarily the liver. Once metastasized, the disease is invariably fatal. Activating GNAQ and GNA11 mutations drive uveal melanoma oncogenesis via the control of several recently identified signaling pathways, including phospholipase C-ß/protein kinase C (PLC-ß/PKC) and TRIO-RhoA/Rac1 pathways, which activate MAPK/ERK and YAP to induce AP1- and YAP-TEAD-mediated transcription. However, the mechanism(s) by which Gαq proteins activate multiple downstream pathways has not been completely elucidated. Preliminary data suggest that the small GTPase ARF6 may act as an immediate downstream effector of activated GNAQ/GNA11 to control all of the currently known oncogenic Gαq signaling pathways. Other preliminary data also suggest that Gαq may signal through ARF6 to activate ß-catenin signaling by promoting the relocalization of ß-catenin from the cell membrane to the nucleus where it can mediate gene transcription. Preliminary data also support the in vivo role of ARF6 in uveal melanoma. When ARF6 is silenced in uveal melanoma by shRNA or is inhibited with a small molecule inhibitor, tumor establishment and growth is significantly inhibited in an orthotopic xenograft mouse model. Based on these preliminary data, the following aims will be pursued. In Aim 1, we will investigate whether activated Gαq proteins induce ß-catenin signaling via activation of ARF6 in uveal melanoma. We will employ gene silencing via RNA interference and small molecule inhibition of selected targets to assess intracellular localization, transcriptional activity, and function of ß-catenin in uveal melanoma. In Aim 2, we will elucidate the role of ARF6 in orchestrating known downstream signaling pathways of oncogenic Gαq. The same strategies used in Aim 1 will be employed to determine whether ARF6 acts as an immediate downstream effector of activating GNAQ/GNA11 mutations to control (PLC-ß/PKC) and TRIORhoA/ Rac1 pathways and AP-1 and YAP-TEAD-mediated transcription. In Aim 3, we will assess the in vivo function of ARF6 in tumor establishment and growth by using orthotopic xenograft models of human uveal melanoma. The successful completion of these aims will allow us to determine whether ARF6 plays a critical role in Gq signaling, thus providing a promising therapeutic target for the development of drugs that could be used to treat uveal melanoma and possibly other Gαq-related disorders such as Sturge-Weber syndrome.