Dissecting the evolution of targeted therapy resistance in BRAFV600E-mutant cancer

项目摘要 BRAFV 600 E突变是最常见的BRAF突变，存在于约3%的肺腺癌中 和50%的黑色素瘤，并提出了重大的疾病负担。虽然RAF抑制剂，单独或组合 使用MEK抑制剂，提高了生存率，最终出现耐药性，患者在治疗中取得进展。更 迫切需要有效的治疗方法，特别是对于晚期疾病患者。我们以前的工作 表明引起耐药性改变的传播，如BRAFV 600 E扩增，是由于 治疗施加的适应性阈值不足，其中适应性阈值是指屏障亚克隆必须 克服持续增长。这表明，由多种药物组成的联合治疗将是 更有效地我们设计了一种间歇性RAF、MEK和ERK抑制剂方案， 在一组肺腺癌和黑色素瘤患者来源的异种移植物（PDX）模型中，即使是那些 已知的对单一药剂产生耐药性的改变。该项目的目标是评估 对间歇性三种药物组合的反应，并阐明可能促进肿瘤克隆相互作用的肿瘤克隆相互作用。 抵抗力的进化具体而言，在目标1中，我将a）评估对 PDX模型中的间歇性三种药物组合; B）通过深度靶向 测序;和c）使用新的单细胞DNA评估治疗对肿瘤克隆结构的影响 测序在目的2中，使用BRAFV 600 E扩增作为模型并荧光条形码化单细胞克隆抗体， 扩展作为我的实验系统，我计划a）确定支持 抗性克隆的生长和B）前瞻性地跟踪治疗对肿瘤克隆组成的影响。这些 研究将提高对靶向治疗耐药性演变的决定因素的理解， 设计更有效的治疗方法，以及确定一种治疗组合， 一些BRAFV 600 E突变型癌症。 !

PROJECT SUMMARY BRAFV600E mutation, the most common BRAF mutation, is found in approximately 3% of lung adenocarcinomas and 50% of melanomas and presents a significant disease burden. While RAF inhibitors, alone or in combination with MEK inhibitors, have improved survival, resistance eventually arises and patients progress on therapy. More effective treatments are urgently needed, especially for patients with advanced disease. Our previous work showed that propagation of resistance-causing alterations, such as BRAFV600E-amplification, is due to an inadequate fitness threshold imposed by therapy, where fitness threshold refers to the barrier subclones must overcome for continued growth. This suggests that a combination therapy consisting of multiple-drugs would be more effective. We designed an intermittent RAF, MEK, and ERK inhibitor regimen that inhibited tumor growth in a panel of lung adenocarcinoma and melanoma patient-derived xenograft (PDX) models, even those with known resistance-causing alterations to single agents. The goal of this project is to assess the durability of response to the intermittent three-drug combination, and to elucidate tumor clonal interactions that may facilitate the evolution of resistance. Specifically, in Aim 1, I will a) evaluate the long-term treatment response to the intermittent three-drug combination in PDX models; b) identify resistance mechanisms through deep targeted sequencing; and c) evaluate the effect of treatment on tumor clonal architecture using novel single cell DNA sequencing. In Aim 2, using BRAFV600E-amplification as a model and fluorescently barcoded single cell clonal expansions as my experimental system, I plan to a) identify clonal interaction mechanisms that support the growth of resistant clones and b) prospectively track the effect of therapy on tumor clonal composition. These studies will improve understanding of the determinants of evolution of targeted therapy resistance and enable the design of more effective treatments, as well as identify a therapeutic combination that may be curative for some BRAFV600E-mutant cancers. !