A High-Throughput Model for Human Melanoma

黑色素瘤研究的重大进展导致了新的疗法，这些疗法显著地改善了 黑色素瘤的治疗和总体存活率。然而，许多患者仍然死于这种疾病，甚至更多。 超过一半的黑色素瘤死亡是由于脑转移。作为克服这一挑战的手段，我们 开发了一种新的小鼠模型，允许黑素细胞在出生后传递感兴趣的基因。vbl.使用 来自人类黑色素瘤样本的基因组和蛋白质组数据，我们测试了几种基因组合 在体内诱发转移性黑色素瘤的能力。突变型PI3K/AKT通路的过度激活 BRAF和CDKN2A缺失导致完全外显的自发性黑色素瘤的发展和 平均存活时间仅为6周。这些小鼠中的大多数也发生了多发性肺转移和 大脑。这个模型从遗传学、组织学和转移部位上模拟了人类疾病。重要的是 该模型系统为进一步研究黑色素瘤的转移机制提供了有力的平台。 这个网络的阐明将使识别代表潜在治疗的关键通路节点成为可能 破坏这一进程的目标。我们使用下一代测序和蛋白质组学分析非转移性 和转移性肿瘤样本，以确定与黑色素瘤脑转移有关的AKT1下游靶点。 我们的数据显示，表达激活的AKT1的肿瘤显示出高水平的焦点黏附(FA)因子 和磷酸化粘着斑激酶(P-FAK)。此外，突变的AKT1表达增加了侵袭性和 AKT或FAK的药理抑制可降低这种作用。此外，PTEN的丢失，这也导致 在FAK活性升高的情况下，协同激活的AKT1进一步增强体内脑转移。尽管 这些有希望的初步发现，在理解FAK是否以及如何推广方面仍然存在关键差距 黑色素瘤脑转移以及AKT1-FAK-PTEN轴是否可以作为治疗的靶点 这种病。我们的长期目标是利用这一知识开发新的黑色素瘤疗法并改善 病人的结果。根据这一目标，我们产生了一系列最先进的研究工具 并组建了一支才华横溢的科学家和临床医生团队，他们有着卓有成效的协作记录 并对拟议目标的结果进行批判性评估。利用这些独特的资源，我们建议测试 假设：1)AKT1、→、FAK信号通路在黑色素瘤的发生发展中起重要作用 2)联合抑制BRAFV600E、MEK和FAK优于标准护理靶向治疗 3)PTEN表达缺失导致FAK持续磷酸化，从而促进肿瘤的发育。 黑色素瘤脑转移的可能性。成功完成本提案中的目标将对 通过为转化为临床试验奠定基础，为患者提供新的更好的治疗方法 有脑转移的人和那些有高风险发展脑转移的人。

Significant advances in melanoma research have resulted in new therapies that have remarkably improved the management and overall survival of melanoma. However, many patients still succumb to the disease and more than half of all melanoma deaths are due to brain metastases. As a means to overcome this challenge, we developed a novel mouse model that allows postnatal delivery of genes of interest to melanocytes. Using genomic and proteomic data from human melanoma samples, we tested several gene combinations for their ability to induce metastatic melanoma in vivo. Hyperactivation of the PI3K/AKT pathway in the context of mutant BRAF and CDKN2A loss resulted in the development of spontaneous melanoma with full penetrance and a mean survival of only 6 weeks. The majority of these mice also developed multiple metastases to the lungs and brain. This model mimics the human disease genetically, histologically, and by sites of metastasis. Importantly, this model system provides a powerful platform to further study the mechanisms of melanoma metastasis. Elucidation of this network will allow the identification of key pathway nodes that represent potential therapeutic targets to disrupt this process. We used next generation sequencing and proteomics analysis of non-metastatic and metastatic tumor samples to identify downstream targets of AKT1 implicated in melanoma brain metastasis. Our data showed that tumors expressing activated AKT1 displayed elevated levels of focal adhesion (FA) factors and phosphorylated focal adhesion kinase (P-FAK). In addition, mutant AKT1 expression increased invasion and this was reduced by pharmacological inhibition of either AKT or FAK. Moreover, loss of PTEN, which also results in increased FAK activity, cooperated with activated AKT1 to further enhance brain metastasis in vivo. Despite these promising preliminary findings, a critical gap still remains in understanding whether and how FAK promotes melanoma brain metastasis, and whether the AKT1-FAK-PTEN axis can be exploited as a therapeutic target in this disease. Our long-term goal is to use this knowledge to develop novel melanoma therapies and improve patient outcomes. Pursuant to this goal, we have generated a diverse collection of state-of-the-art research tools and assembled a talented team of scientists and clinicians with a track record of productive collaboration to carry out and critically evaluate the results of the proposed aims. Using these unique resources, we propose to test the following hypotheses: 1) AKT1→ FAK signaling is essential for the development of melanoma brain metastases; 2) Combined inhibition of BRAFV600E, MEK, and FAK is superior to standard of care targeted therapy and; 3) Loss of PTEN expression results in sustained FAK phosphorylation, which enhances the development of melanoma brain metastases. Successful completion of the aims in this proposal will significantly impact the field by laying the groundwork for translation into clinical trials, leading to new and better treatments for patients with brain metastases and those who are at high risk of developing brain metastases.