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

项目总结 BRAFV600E突变是最常见的BRAF突变，在大约3%的肺腺癌中发现 和50%的黑色素瘤，并带来重大的疾病负担。而RAF抑制剂，单独或联合使用 使用MEK抑制剂，可以提高存活率，最终产生耐药性，并使患者在治疗方面取得进展。更多 迫切需要有效的治疗，特别是对晚期疾病的患者。我们之前的工作 表明引起抗性改变的传播，如BRAFV600E扩增，是由于 治疗施加的不充分的健康阈值，其中健康阈值是指亚克隆必须 克服困难，实现持续增长。这表明由多种药物组成的联合疗法将是 更有效。我们设计了一种间歇性的RAF、MEK和ERK抑制方案来抑制肿瘤生长 在一组肺腺癌和黑色素瘤患者来源的异种移植(PDX)模型中，即使是那些 已知的耐药性-导致单一药物的改变。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 抗药性的演变。具体地说，在目标1中，我将a)评估长期治疗对 PDX模型中的间歇性三药联合；b)通过深度靶向确定耐药机制 测序；以及c)使用新的单细胞DNA评估治疗对肿瘤克隆构筑的影响 测序。在目标2中，以BRAFV600E扩增为模型，荧光条形码单细胞克隆 扩展作为我的实验系统，我计划a)识别支持 耐药克隆的生长和b)前瞻性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，并使 设计更有效的治疗方法，以及确定可能治愈的治疗组合 一些BRAFV600E突变的癌症。 好了！

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. !