Targeting Raf in Melanoma: Mechanisms for Effect & Resistance & Opportunities for

一些证据表明，丝氨酸/苏氨酸激酶B-raf可能是一个有吸引力的药物靶点， 在黑色素瘤的发展和许多raf抑制剂（如索拉非尼，CHIR-265，SB 590885），现在 可用于实验室和临床测试。体外暴露于索拉非尼导致半胱天冬酶激活 以及凋亡诱导因子（AIF）在一些但不是全部黑素瘤细胞系中的核转位， 提示黑色素瘤细胞对由raf抑制剂诱导的细胞凋亡的易感性可能是遗传性的， 测定用30个黑色素瘤细胞系进行了一系列基因表达谱实验， 索拉非尼和CHIR-265被提议来解决这个假设。索拉非尼的临床活性有限 除非药物与化疗剂一起给予，在这种情况下，高达40%的反应率 在广泛预治疗的患者中观察到。我们已经观察到索拉非尼激活了几个 补偿性存活途径（即JNK和p53），破坏药物的致死性。封锁 这些途径与药理学抑制剂或siRNA使黑色素瘤细胞对索拉非尼诱导的 凋亡索拉非尼抑制黑色素瘤细胞中的STATS，这种抑制有助于细胞凋亡 由药物引起的。我们还观察到，缺乏DMA甲基转移酶-1， 参与RASSF 1A和其他基因的表观遗传沉默，使黑色素瘤细胞系对 索拉非尼。本申请的目标之一是确定这些体外效应是否可以在 来自接受RAF抑制剂的患者的肿瘤异种移植物和肿瘤样本。这样的结果将提供一个 raf抑制剂与p53拮抗剂（例如EL 625）、STATS或DMA联合使用的有力依据 甲基转移酶抑制剂（如地西他滨）在黑色素瘤的治疗，我们建议测试的概念， 这几年的补助金尽管索拉非尼和CHIR-265抑制的激酶重叠，但它们之间存在差异。 这些差异可赋予后者药剂上级抗肿瘤活性。这个应用程序的目标之一 是CHIR-265的I期试验的设计和执行，其扩展阶段将评估其 临床潜力。在本申请中提出的研究将评估抗肿瘤和抗血管生成的药物组合物。 该药物在黑色素瘤患者中的作用，并提供关于如何最佳地 与其他靶向治疗相结合，以最大限度地利用这类新药的潜力。

Several lines of evidence suggest that the serine/threonine kinase B-raf might be an attractive target for drug development in melanoma and numerous raf inhibitors (e.g. sorafenib, CHIR-265, SB590885) are now available for laboratory and clinical testing. In vitro exposure to sorafenib leads to the activation of caspases and the nuclear translocation of Apoptosis-lnducing Factor (AIF) in some, but not all, melanoma cell lines, suggesting that the susceptibility of melanoma cells to apoptosis induced by raf inhibitors may be genetically determined. A series of gene expression profiling experiments with 30 melanoma cell lines treated with sorafenib and CHIR-265 is proposed to address this hypothesis. The clinical activity of sorafenib is limited unless the drug is given with chemotherapeutic agents, in which case response rates as high as 40% have been observed in extensively pretreated patients. We have observed that sorafenib activates several compensatory survival pathways (i.e. JNK and p53) that undermine the lethality of the drug. The blockade of these pathways with pharmacologic inhibitors or siRNAs sensitizes melanoma cells to sorafenib-induced apoptosis. Sorafenib inhibits STATS in melanoma cells and this inhibition contributes to the apoptosis induced by the drug. We have also observed that the absence of DMA methyltransferase-1, an enzyme involved in the epigenetic silencing of RASSF1A and other genes, sensitizes melanoma cell lines to sorafenib. One of the goals of this application is to determine if these in vitro effects can be duplicated in tumor xenografts and tumor specimens from patients receiving a raf inhibitor. Such a result would provide a strong rationale for the combination of a raf inhibitor with a p53 antagonist (e.g. EL625), a STATS or DMA methyltransferase inhibitor (e.g. decitabine) in the treatment of melanoma, a concept we propose to test in the last years of this grant. Although the kinases inhibited by sorafenib and CHIR-265 overlap, there are differences that may confer superior antitumor activity on the latter agent. One of the goals of this application is the design and execution of a Phase I trial of CHIR-265, the extension phase of which will assess its clinical potential. The studies proposed in this application will assess the antitumor and antiangiogenic effects of this agent in melanoma patients and provide information as to how a raf inhibitor might be optimally combined with other targeted therapies to best exploit the potential of this novel class of drugs.