Molecular approaches to overcome intrinsic drug resistance in BRAF and NRAS-mutan

描述(由申请人提供)：申请人是维斯塔尔研究所分子和细胞肿瘤发生项目的助理教授。她的总体目标是开发有效和持久的黑色素瘤治疗策略。这项建议服务于申请人的长期目标，即通过研究BRAF-V600E和NRAS突变黑色素瘤对靶向治疗的内在耐药机制来改善黑色素瘤患者的预后。候选人将在研究领域进行培训，这些领域对于培养研究利基市场至关重要，并成为一名独立的学术癌症研究人员。已经组建了一个高级科学顾问团队，通过在蛋白质组学、系统和计算生物学以及自噬等新研究领域提供培训和专业知识，正式指导申请者。指导团队致力于支持申请者发展成为一名全额资助的独立科学家。黑色素瘤是一种高度侵袭性的疾病，治疗选择有限。尽管BRAF抑制剂在治疗黑色素瘤方面取得了成功，但反应是短暂的，大多数患者都产生了耐药性。因此，了解、克服和预防耐药性是一项重大挑战。这项申请建议研究内在耐药的机制，这是一种重要的未得到满足的医疗需求，影响着一大批黑色素瘤患者，对他们来说，没有有效的治疗方法。该提案重点关注对BRAF抑制剂具有内在耐药性的NRAS突变黑色素瘤和BRAF-V6000E突变黑色素瘤。总体假设是，发现黑色素瘤内在耐药性的潜在机制将有助于设计更有效和更持久的治疗方法。申请人假设，抑制BRAF会导致系统信号网络的重新布线，导致受体酪氨酸激酶(RTK)激活，并减轻对BRAF的依赖。为了验证这一假设，将评估在BRAF抑制后生长在免疫低下小鼠(患者来源的异种移植物；PDX)中的黑色素瘤细胞系和患者来源的肿瘤样本中RTK的表达和激活。将进行机制研究，以确定抑制MAPK如何导致RTK激活。在这些研究的基础上，将在生长为3D肿瘤球体的黑色素瘤细胞和PDX模型中评估合理的药物组合策略。第二个工作假设是，通过识别关键的下游NRAS效应器，含有NRAS突变的黑色素瘤可以成为靶点。目的是确定在黑色素瘤细胞中沉默NRAS的生化和生物学后果，这些黑色素瘤细胞中含有这种癌基因的突变。为了实现这一目标，将采用候选和无偏见的蛋白质组学方法来鉴定对NRAS突变黑色素瘤的生存至关重要的NRAS效应因子。总而言之，这些知识对于开发消除几乎所有黑色素瘤细胞的新策略并为未来临床试验的设计提供信息将是至关重要的。

DESCRIPTION (provided by applicant): The applicant is an assistant professor at Molecular and Cellular Oncogenesis Program at the Wistar Institute. Her overall goal is to develop effective and long-lasting treatment strategies for melanoma. This proposal serves the applicant's long-term objective of improving outcomes for melanoma patients by investigating the mechanisms of intrinsic resistance to targeted therapy in BRAF-V600E and NRAS mutant melanomas. The candidate will pursue training in research areas that are essential to foster a research niche and become an independent academic cancer researcher. A team of senior scientific advisors has been assembled to formally mentor the applicant by providing training and expertise in new research areas including proteomics, systems and computational biology, and autophagy. The mentoring team is committed to support the development of the applicant into a fully funded independent scientist. Melanoma is a highly aggressive disease with limited therapeutic options. Despite the success of BRAF inhibitors for the treatment of melanoma, responses are transient and most patients develop resistance. Therefore, a major challenge is to understand, overcome, and prevent drug resistance. This application proposes to investigate the mechanisms of intrinsic drug resistance, an important unmet medical need affecting a large subset of melanoma patients, for whom no effective therapies are available. The proposal focuses on NRAS mutant melanomas and BRAF-V6000E mutant melanomas that are intrinsically resistant to BRAF inhibitors. The overall hypothesis is that uncovering the mechanisms underlying intrinsic drug resistance in melanoma will allow designing more effective and longer lasting therapies. The applicant postulates that inhibition of BRAF induces rewiring of the system's signaling networks leading to receptor tyrosine kinase (RTK) activation and attenuated dependency on BRAF. To test this hypothesis, the expression and activation of RTKs will be evaluated in melanoma cell lines and patient-derived tumor samples grown in immunocompromised mice (patient-derived xenografts; PDX) following BRAF inhibition. Mechanistic studies will be performed to determine how inhibition of MAPK leads to RTK activation. Based on these studies rational drug combination strategies will be evaluated in melanoma cells grown as 3D tumor spheroids and in PDX models. The second working hypothesis is that by identifying critical downstream NRAS effectors, melanomas harboring mutations in NRAS can be targeted. The objective is to define the biochemical and biological consequences of silencing NRAS in melanoma cells that harbor mutations in this oncogene. To achieve this goal a candidate and an unbiased proteomic based approaches will be undertaken to identify NRAS effectors that are essential for survival of NRAS mutant melanomas. Collectively, this knowledge will be critical to develop new strategies that eliminate nearly all melanoma cells and inform the design of future clinical trials.