Using Single Cell Analysis to identify therapeutic vulnerabilities in leptomeningeal melanoma metastases

项目摘要 这项研究计划的长期目标是开发治疗策略， 改善播散性黑色素瘤患者的预后。黑色素瘤是一种高度 转移性肿瘤，具有扩散到皮肤、肺和脑的倾向。在约5%的 在一些病例中，患者还发生软脑膜黑素瘤转移（LMM：例如， 黑色素瘤细胞浸润到软脑膜、蛛网膜和 脑脊液），其预后甚至更差，中位生存期为8-10 周软脑膜很可能是黑色素瘤细胞的“避难所” 逃避靶向治疗和免疫治疗。目前，没有治疗策略 已经确定了对LMM有效的方法。黑色素瘤是遗传性的 异质性，表明不同的遗传克隆/突变模式决定了 哪些细胞会回到大脑和软脑膜。这方面的进展有限 由于从软脑膜获得标本的技术困难， 脑脊液（CSF）和蛛网膜下腔。最近我们组开始了 常规收集LMM患者的CSF，我们已经证明其含有 循环黑素瘤细胞（循环肿瘤细胞：CTC）。我们还建立 从接受超说明书治疗的患者中获得系列CSF样本的方案 BRAF-MEK抑制剂治疗LMM。用于分离和扩增的方法 来自单细胞的RNA和DNA已经变得可用，并且非常适合于 分析罕见的细胞群，例如在CSF中发现的细胞群。总体 该R21的目标是确定CTC的遗传和转录谱， LMM患者，并确定这些肿瘤的新的治疗靶点。我们将使用 Fluidigm C1平台进行复杂的单细胞RNA和DNA分析， 定义LMM CTC和匹配的黑素瘤标本之间的克隆关系 从其他网站。然后，我们将收集LMM患者的连续CSF标本 正在接受靶向治疗，并将决定治疗如何推动 克隆选择将进行验证实验，其中BRAF突变体LMM 将细胞原位植入小鼠的软脑膜中， 基于达拉非尼-曲美替尼和靶向药物的个性化药物组合 与我们识别的可行突变/途径进行对比。这些研究预计 为脑和软脑膜黑色素瘤的生物学提供重要的新见解 转移并形成临床相关药物开发的基础 组合。

Project summary The long-term goal of this research program is to develop therapeutic strategies that improve the outcome of patients with disseminated melanoma. Melanoma is a highly metastatic tumor, with a propensity to disseminate to the skin, lungs and brain. In ~5% of cases, patients also develop leptomeningeal melanoma metastases (LMM: e.g. melanoma cell infiltration into the pia mater, the arachnoid membranes and cerebrospinal fluid) which have an even worse prognosis and a median survival of 8-10 weeks. It is likely that the leptomeninges represent “sanctuary sites” for melanoma cells that evade both targeted and immune therapies. At this time, no therapeutic strategies have been identified that are effective against LMM. Melanomas are genetically heterogeneous, suggesting that distinct genetic clones/patterns of mutation determine which cells home to the brain and leptomeninges. Progress in this area has been limited due to the technical difficulties of obtaining specimens from the leptomeninges, cerebrospinal fluid (CSF) and sub-arachnoid space. Recently our group has begun the routine collection of CSF from patients with LMM, which we have shown to contain circulating melanoma cells (circulating tumor cells: CTCs). We have also established protocols to obtain serial CSF samples from patients undergoing off-label treatment BRAF-MEK inhibitor treatment for LMM. Methods for the isolation and amplification of RNA and DNA from single cells have become available and are ideally suited for analyzing rare populations of cells, such as those found in the CSF. The overarching goal of this R21 is to define the genetic and transcriptional profiles of the CTCs from patients with LMM and to identify novel therapeutic targets for these tumors. We will use the Fluidigm C1 platform to perform sophisticated single cell RNA and DNA analysis to define the clonal relationship between LMM CTCs and matched melanoma specimens from other sites. We will then collect serial CSF specimens from LMM patients undergoing treatment with targeted therapy and will determine how treatment drives clonal selection. Validation experiments will be performed in which BRAF-mutant LMM cells are orthotopically implanted into the leptomeninges of mice and treated with personalized drug combinations based upon dabrafenib-trametinib and drugs targeted against the actionable mutations/pathways that we identify. These studies are expected to provide critical new insights into the biology of brain and leptomeningeal melanoma metastases and form the groundwork for the development of clinically relevant drug combinations.