(PQD1) Evolution of vemurafenib resistance in circulating melanoma cells

描述（由申请人提供）：黑色素瘤是美国第六大常见癌症，每年造成近8000人死亡。虽然黑色素瘤在早期阶段是可以治疗的，但一旦疾病进展并转移，几乎普遍会在5年内致命，因此显然需要新的治疗选择。循环肿瘤细胞（CTC）是从原发性或转移性肿瘤脱落的癌细胞，并最终播种新的癌位点。在黑色素瘤中，已经发现CTC是导致转移性疾病的大部分侵袭性的原因，包括对分子靶向疗法的抗性。由于单个CTC的毒力不同，我们假设单个CTC中独特的基因表达特征是耐药性的来源。具体而言，我们假设BRAF抑制剂维罗非尼对黑色素瘤CTC施加选择性压力，导致耐药性。揭示维罗非尼耐药的潜在机制需要在耐药演变过程中对单个CTC进行全面分析。我们最近将技术发展到可以通过单细胞RNA-seq稳健地分离罕见的黑色素瘤CTC并对单个CTC进行转录分析的程度。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，每个程序都具有获得对分子靶向治疗耐药性的独特潜力。我们建议精确注释单个CTC中的特定基因表达特征以演变维罗非尼耐药性。黑色素瘤是遗传异质性和临床上可获得的，因此代表了用于开发分析方法以评估异质性转录输出对耐药性演变的影响的最佳癌症类型。我们的调查团队非常适合进行所需的技术开发和数据生成。从历史上看，黑色素瘤CTC难以分离，因此是研究的挑战。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CTC的最佳条件。对于这项提案，我们寻求利用我们的经验，资源和能力，通过RNA-seq开发和实施从维罗非尼治疗前后从黑色素瘤患者中分离的个体CTC的转录谱分析。因此，我们将定义响应于维罗非尼治疗的转录输出，实验验证它们在建立维罗非尼耐药性中的作用，从而确定关键基因作为新型维罗非尼耐药性治疗的潜在靶点。

DESCRIPTION (provided by applicant): 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 signature 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.