(PQD1) Evolution of vemurafenib resistance in circulating melanoma cells

摘要 黑色素瘤是美国第六大常见癌症，每年导致近8000人死亡。 虽然黑色素瘤在早期被发现是可以治疗的，但一旦疾病进展并成为 转移性肿瘤在5年内几乎都是致命的，因此显然需要新的治疗方案。 循环肿瘤细胞(CTCs)是从原发或转移性肿瘤中脱落的癌细胞，最终 播种新的癌变部位。在黑色素瘤中，CTC被发现是导致大部分侵袭性的原因 转移性疾病的性质，包括对分子靶向治疗的抵抗。因为沙门氏菌的毒性 单个CTC各不相同，我们假设单个CTC中独特的基因表达特征是 耐药性的来源。具体地说，我们假设BRAF抑制剂维莫拉非尼施加了一种 对黑色素瘤CTCs的选择性压力会导致耐药性。 解开维莫拉非尼耐药的潜在机制需要进行全面的分析。 在抗药性演变过程中，个体CTC的数量。我们最近将技术进步到了这样一个程度，我们 可以很好地分离罕见黑色素瘤CTCs，并通过单细胞RNA-seq在转录水平上分析单个CTCs。 通过我们的创新方法，我们可以识别黑色素瘤CTC中不同的遗传程序，每个程序都具有独特的 有可能对分子靶向治疗产生抵抗力。我们建议准确地注释特定的 单个CTC中的基因表达特征对维莫拉非尼耐药进化的影响。 黑色素瘤具有遗传异质性和临床可获得性，因此代表了一种 制定用于评估异质性影响的分析方法的最佳癌症类型 关于耐药进化的转录输出。我们的调查小组非常适合执行 所需的技术开发和数据生成。从历史上看，黑色素瘤CTC很难 与世隔绝，因此是一个研究的挑战。然而，我们已经克服了这一技术障碍，最近 确定了从血液中收集黑色素瘤CTCs的最佳条件。对于这项提议，我们寻求 利用我们的经验、资源和能力通过以下方式开发和实施转录特征分析 维莫拉非尼治疗前后黑色素瘤患者分离的单个CTC的rna-seq。我们 因此将定义转录输出以响应维莫拉非尼治疗，通过实验验证它们的作用 在建立维莫拉非尼抗性从而确定关键基因作为新的潜在靶点 维莫拉非尼耐药治疗。

ABSTRACT Melanoma is the sixth most common cancer in the United States, causing almost 8000 deaths annually. While melanoma is treatable when caught in the early stages, once the disease has progressed and becomes metastatic it is almost universally fatal within 5 years, so new therapeutic options are clearly needed. Circulating tumor cells (CTCs) are cancer cells shed from a primary or metastatic tumor and eventually seed new cancerous sites. In melanoma, CTCs have been found to be responsible for much of the aggressive nature of metastatic disease, including resistance to molecularly-targeted therapies. Because the virulence of individual CTCs varies, we hypothesize that unique gene expression signatures in individual CTCs is the source of drug resistance. Specifically, we hypothesize that the BRAF inhibitor vemurafenib imposes a selective pressure on melanoma CTCs which results in resistance. Unraveling the underlying mechanisms of vemurafenib resistance will require comprehensive analysis of individual CTCs during the evolution of resistance. We recently advanced technology to the point where we can robustly isolate rare melanoma CTCs and transcriptionally profile individual CTCs by single cell RNA-seq. With our innovative approaches, we can identify distinct genetic programs in melanoma CTC each with unique potential to acquire resistance to molecularly-targeted therapies. We propose to precisely annotate specific gene expression signatures in individual CTCs to the evolution of vemurafenib resistance. Melanomas are both genetically heterogeneous and clinically accessible and therefore represent an optimal cancer type for the development of analytic methods to assess the effects heterogeneous transcriptional outputs on the evolution of drug resistance. Our investigative team is ideally suited to carry out the technology development and data generation required. Historically, melanoma CTCs have been difficult to isolate and therefore a challenge to study. However, we have overcome this technical hurdle and recently determined optimal conditions for collecting melanoma CTCs from the bloodstream. For this proposal, we seek to leverage our experience, resources and capabilities to develop and implement transcriptional profiling by RNA-seq from individual CTCs isolated from melanoma patients before and after vemurafenib treatment. We will thus define transcriptional outputs in response to vemurafenib treatment, experimentally validate their roles in establishing vemurafenib resistance and thereby identify key genes as potential targets for novel vemurafenib-resistance therapy.