REGULATION OF UVEAL MELANOMA CELL FATE BY THE PKC PATHWAY VIA MITF

 描述（由申请人提供）：葡萄膜黑色素瘤（UM）是成人中最常见的眼内恶性肿瘤，对所有可用的全身化疗均难治，因此，转移时普遍致命，因此对这种孤儿病的新型、有效、靶向治疗的需求未得到满足。G（alpha）q和G（alpha）11的体细胞激活突变以相互排斥的模式存在于约80%的UM中，激活PKC通路并作为真正的致癌驱动因子发挥作用。由于O 'Malley实验室先前曾报道类固醇受体共激活因子-3（SRC-3），一种具有强效生长促进活性的转录共激活因子，通过PKC介导的磷酸化作用在免疫后稳定，因此我们决定建立在SRC-3研究的经验基础上，并检查SRC-3在UM中的作用。我们的初步研究表明，G（α）诱导的致癌信号是由蛋白激酶C（PKC）α/蛋白激酶D（PKD）途径介导的，并导致SRC-3的稳定，然后共同定位在染色质上，并与黑素细胞的关键转录因子小眼相关转录因子（MITF）合作，以驱动致癌信号。G（alpha）q突变型UM细胞在体外和体内的增殖/存活都非常依赖SRC-3。PKC的小分子抑制剂（SMI）引起SRC-3蛋白的消耗并在G（alpha）q突变的UM细胞中发挥抗癌活性，而SRC-3表达的恢复挽救了已经用G（alpha）q/PKC抑制剂处理的UM细胞的活力。基于这些最新的发现，我们的核心假设是，突变的G（α）蛋白触发PKC介导的细胞内致癌信号，稳定SRC-3，然后作为转录因子MITF的共激活因子。此外，SRC-3/MITF的药理学抑制将发挥有效的抗癌活性，从而为UM患者提供创新的治疗机会。我们的提案为开发这种高致命性孤儿病的创新疗法提供了路线图。我们的目的是阐明SRC-3在UM病理生理学和抵抗系统治疗中的作用（特别是PKC抑制剂），并使用大量UM细胞系、UM细胞的原代培养物和患者活组织检查将其确立为UM中的治疗靶标;研究SRC-3和MITF之间的相互作用，确定它们的转录靶基因，确定MITF和SRC- 3如何调控这些转录靶点，并剖析它们在UM中的功能意义;将新鉴定的SRC-3/MITF途径SMI的活性定义为UM的新治疗方法，特别是用于使用体外和小鼠UM模型（作为单一疗法和组合方案）对靶向疗法具有抗性的疾病。我们的目标是为UM开发新的靶向治疗药物，特别强调合理设计的组合方法，以克服治疗耐药性。总的来说，我们的研究策略将提高我们对UM病理生理学的理解，并提供新的靶点和治疗药物。

 DESCRIPTION (provided by applicant): Uveal melanoma (UM), the most common intraocular malignancy in adults, is uniformly refractory to all available systemic chemotherapies, and, as a result, is universally lethal when metastatic, creating an unmet need for novel, effective, targeted therapies for this orphan disease. Somatic activating mutations in G(alpha)q and G(alpha)11, present in a mutually exclusive pattern in ~80% of UMs, activate the PKC pathway and function as bona fide oncogenic drivers. As the O'Malley lab had previously reported that steroid receptor coactivator-3 (SRC-3), a transcriptional coactivator with potent growth-promoting activity, is post-translationally stabilized via PKC-mediated phosphorylation, we decided to build upon our experience in SRC-3 studies and examine the role of SRC-3 in UM. Our preliminary studies suggest that G(alpha)-induced oncogenic signaling is mediated by the protein kinase C (PKC)alpha/protein kinase D (PKD) pathway, and leads to stabilization of SRC-3, which then co-localizes on chromatin and co-operates with microphthalmia-associated transcription factor (MITF), a critical transcription factor for melanocytes, to drive oncogenic signaling. G(alpha)q-mutant UM cells are exquisitely dependent on SRC-3 for proliferation/survival in vitro and in vivo. Small molecule inhibitors (SMIs) of PKC cause depletion of SRC-3 protein and exert anticancer activity in G(alpha)q-mutant UM cells, while restoration of SRC-3 expression rescues the viability of UM cells that have been treated with G(alpha)q/PKC inhibition. Based on these recent findings, our core hypothesis is that mutant G(alpha) proteins trigger PKC-mediated intracellular oncogenic signaling that stabilizes SRC-3, which then functions as a coactivator for the transcription factor MITF. Moreover, pharmacological inhibition of SRC-3/MITF will exert potent anticancer activity, thus providing an innovative therapeutic opportunity for UM patients. Our proposal provides the roadmap for the development of such innovative therapies for this highly lethal orphan disease. Our aims are to elucidate the role of SRC-3 in UM pathophysiology and resistance to systemic therapy (in particular PKC inhibitors), and to establish it as a therapeutic target in UM, using a large panel of UM cell lines, primary cultures of UM cells and patient biopsies; examine the cooperation between SRC-3 and MITF, define their transcriptional target genes, determine how these transcriptional targets are regulated by MITF and SRC- 3, and dissect their functional significance in UM; define the activity of newly identified SRC-3/MITF pathway SMIs as a novel therapeutic approach for UM, in particular for disease resistant to targeted therapies using in vitro and in mouse UM models (both as monotherapies and in combination regimens). Our goal is to develop novel targeted therapeutics for UM, with particular emphasis on rationally-designed combinatorial approaches, in order to overcome resistance to treatment. Collectively, our research strategy will enhance our understanding of UM pathophysiology and provide novel targets and therapeutic agents.