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

 描述(申请人提供)：葡萄膜黑色素瘤(UM)是成人最常见的眼内恶性肿瘤，对所有可用的全身化疗都是无效的，因此，在转移时是普遍致命的，创造了对这种孤儿疾病的新的、有效的、有针对性的治疗的未得到满足的需求。G(α)Q和G(α)11的体细胞激活突变在约80%的UM中以互斥模式存在，激活PKC途径，并作为真正的致癌驱动因素发挥作用。由于O‘Malley实验室以前曾报道过类固醇受体辅助激活因子-3(SRC-3)，一种具有强大的生长促进活性的转录辅助激活因子，是通过PKC介导的磷酸化在翻译后稳定的，我们决定根据我们在SRC-3研究中的经验，探讨SRC-3在UM中的作用。我们的初步研究表明，G(α)诱导的致癌信号是通过蛋白激酶C(PKC)α/蛋白激酶D(PKD)途径介导的，并导致SRC-3稳定，然后SRC-3共定位在染色质上，并与黑素细胞的关键转录因子小眼球相关转录因子(MITF)合作驱动致癌信号。G(α)q突变的UM细胞在体外和体内的增殖/存活都依赖于SRC-3。在G(α)q突变的UM细胞中，PKC的小分子抑制物(SMI)导致SRC-3蛋白缺失并发挥抗癌活性，而恢复SRC-3的表达则挽救了经G(α)Q/PKC抑制处理的UM细胞的生存能力。基于这些最近的发现，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的药理抑制将发挥强大的抗癌活性，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度致命的孤儿疾病的创新疗法提供了路线图。我们的目标是通过大量UM细胞系、UM细胞原代培养和患者活检，阐明SRC-3在UM病理生理学和对全身治疗(尤其是PKC抑制剂)的耐药性中的作用，并将其作为UM的治疗靶点；研究SRC-3和MITF之间的合作，定义它们的转录靶基因，确定这些转录靶点是如何被MITF和SRC-3调控的，并剖析它们在UM中的功能意义；将新发现的SRC-3/MITF途径SMIs的活性定义为UM的一种新的治疗方法，特别是在体外和在小鼠UM模型中(作为单一疗法和联合疗法)对靶向疗法产生的疾病耐药性。我们的目标是为UM开发新的靶向疗法，特别强调合理设计的组合方法，以克服治疗耐药性。总而言之，我们的研究战略将增强我们对UM病理生理学的理解，并提供新的靶点和治疗剂。

 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.