Targeting Gq pathway in uveal melanoma

项目总结： 葡萄膜黑色素瘤(UM)是美国最常见的眼内癌，约占 占所有黑色素瘤的5%。大约50%的UM患者发生转移，主要转移到肝脏 死亡率为100%。UM缺乏与其他黑色素瘤类型相同的BRAF、NRAS、NF1和KIT突变。 相反，超过90%的Gaq家族成员GNAQ或GNA11存在体细胞激活突变，其中 其余的携带也作用于Gaq信号通路的基因突变，如CYSLTR2，一个Gaq- 与GPCR偶联，磷脂酶Cb4(PLCb4)是Gaq的直接效应因子。因此，UM的基因定义是 激活Gaq途径的突变。尽管在其他黑色素瘤亚型上取得了戏剧性的成功，但免疫 检查点抑制剂和靶向治疗未能证明UM的临床益处，导致 迫切需要开发新的有效的治疗方案。CYSLTR2-&GT；Gaq-&GT；PLCB-&GT；蛋白激酶 C(PKC)模块是一个线性信号级联，驱动UM细胞的基本MAPK信号转导 增殖，使Gaq途径成为这种破坏性疾病靶向治疗的首要靶点。虽然两者都是 GNAQ/11和PKC抑制剂在体外非常有效地抑制UM细胞的增殖/存活，靶向是 GNAQ/11或PKC单独治疗UM肝转移的疗效有限。理解对人类的抵抗 抑制Gaq通路对于开发在特定信号中起作用的新策略是至关重要的 一个结构性激活的Gaq途径的背景。我们的初步数据显示GNAQ/11抑制或PKC 抑制会导致Gaq偶联受体EDNRB的强烈上调，当遇到其配体时 肝脏环境中的EDN1可以导致MAPK的重新激活，从而导致对Gaq途径的抵抗 UM中的抑制作用。在这项建议中，我们将评估阻断内皮素信号转导将增加治疗 利用新开发的基因技术直接或下游靶向致癌Gaq信号的有效性 UM肝转移的工程化和异种移植模型。而内皮素信号转导通路的作用 黑素细胞发育和黑色素瘤进展是有据可查的，反馈的性质是 上调EDNRB的表达/信号转导尚不清楚。我们将使用一组候选对象 方法、RNAseq和磷酸化蛋白质组学剖析UM细胞和UM的潜在机制 黑素细胞。我们的初步数据还表明，GNA11的次级突变可以赋予对 GAQ抑制。在这项建议中，我们将扩大作为基础的机制的新兴格局 适应性和获得性对Gaq通路抑制的抵抗，并确定合理的治疗组合以改善 转移性尿道炎的治疗效果。使用全基因组CRISPR/Cas9组合合成致死率 筛选和磷酸化蛋白质组筛选我们确定了一条脂质合成途径，这条途径对于人的生存至关重要 带有Gaq突变的细胞，我们将探索这一全新的UM治疗靶点。

Project summary: Uveal melanoma (UM) is the most common intraocular cancer in the United States and accounts for about 5% of all kinds of melanomas. Approximate 50% of UM patients develop metastases, predominantly to the liver with 100% mortality. UM lacks mutations in BRAF, NRAS, NF1 and KIT common to other melanoma types. Instead, over 90% harbor somatic activating mutations in the Gaq family members GNAQ or GNA11, with the remainder carrying mutations of genes also acting in the Gaq signaling pathway such as CYSLTR2, a Gaq- coupled GPCR, phospholipases C b4 (PLCb4), a direct effector of Gaq. Therefore, UM is genetically defined by activating mutations of the Gaq pathway. Despite dramatic successes in other melanoma subtypes, immune checkpoint inhibitors and targeted therapies have failed to demonstrate clinical benefits in UM, leading to an urgent need to develop novel and effective therapeutic regimens. The CYSLTR2->Gaq->PLCb-> protein kinase C (PKC) module is a linear signaling cascade that drives the essential MAP-kinase (MAPK) signaling for UM cell proliferation, making Gaq pathway the prime target for targeted therapy for this devasting disease. Although both GNAQ/11 and PKC inhibitors are very effective to suppress UM cell proliferation/survival in vitro, targeting either GNAQ/11 or PKC alone has shown limited efficacy in UM liver metastasis. Understanding the resistance to the Gaq pathway inhibition is of paramount importance to develop new strategies that work in the specific signaling context of a constitutively activated Gaq pathway. Our preliminary data show either GNAQ/11 inhibition or PKC inhibition yields strong upregulation of the Gaq-coupled receptor EDNRB, which when encountering its ligand EDN1 from the liver environment can lead to reactivation of MAPK, thus driving resistance to Gaq pathway inhibition in UM. In this proposal, we will evaluate that blocking endothelin signaling will increase the therapeutic efficacy of targeting oncogenic Gaq signaling, directly or downstream, using newly developed genetically engineered and xenograft models of UM metastatic to the liver. While the role of endothelin signaling in melanocyte development and melanoma progression is well documented, the nature of the feedback that upregulates EDNRB expression/signaling is not understood. We will utilize a combination of candidate approaches, RNAseq and phospho-proteomics to dissect the underlying mechanisms in UM cells and in melanocytes. Our preliminary data also show that secondary mutations in GNA11 can confer resistance to the Gaq inhibition. In this proposal, we will expand the emerging landscape of the mechanism underlying the adaptive and acquired resistance to Gaq pathway inhibition and identify rational therapy combinations to improve the therapeutic efficacy for metastatic UM. Using a combined genome-wide CRISPR/Cas9 synthetic lethality screen and phospho-proteomic screen we identified a lipid synthesis pathway that is essential for the survival of cells with Gaq mutation, which we will explore as entirely novel therapeutic target for UM.