Understanding and Overcoming Resistance to BRAF/MEK Kinase Inhibitors in Melanoma

绝大多数(80%)BRAFV600E/K黑色素瘤患者最初对高反应性 特异性BRAFV600E/K抑制剂作为单一疗法或BRAF/MEK抑制剂联合治疗， 但在不同的水平。尽管如此，几乎所有的患者在三个月到两年后都会复发 他们的肿瘤细胞中携带着BRAF突变。在此应用程序中，我们重点介绍 BRAFV600E/K黑色素瘤内先天小种群的生物动力学 耐药或在治疗后迅速适应，并导致最终复发。在第一个目标中，我们将 BRAF和MEK处理突变型BRAFV600E/K黑色素瘤细胞的动态观察 抑制剂组合。我们已经确定了一个缓慢循环的亚群，它是动态的 随着细胞在最初的处理中存活，然后持续存在，最后随着获得性细胞的再生而发生变化 对多种药物产生抗药性。我们将在体外和体内模型中跟踪人类的细胞 使用条形码和单细胞RNA分析检测黑色素瘤。这些研究不仅将确定 次要亚群(S)在药物治疗下是随机进化还是分级进化，但 也将为我们提供有关它们先前存在的信息和/或恶性细胞 可塑性是肿瘤内药物反应异质性的主要原因。在第二个目标中， 我们正在检验这样一种假设，即内在抗性细胞亚群呈现出新的 治疗机会，如果有针对性，将特别抑制耐药性的发生。我们的 使用CRISPR/Cas9的初步研究使我们能够从功能上剖析这些通路 形成了这些稀有的亚群，并分别形成了允许这些 要重新编程的子种群。我们将在3D培养系统和活体小鼠身上验证HITS 模特们。我们希望以理性的方式针对抵抗进程的不同方面 设计的方法(在体外，然后在临床前研究中)将能够减少耐药性 在接受BRAF/MEK抑制剂治疗后发病。

The vast majority (>80%) of patients with BRAFV600E/K melanomas initially respond to highly specific BRAFV600E/K inhibitors as monotherapy or BRAF/MEK inhibitor combination therapy, but at varying levels. Nearly all patients relapse after three months to two years, despite that all of their tumor cells are carrying the BRAF mutations. In this application, we focus on the biological dynamics of small populations within BRAFV600E/K melanomas that are a priori resistant or rapidly adapt upon treatment and drive eventual relapse. In the first aim we will follow the dynamics of mutant BRAFV600E/K melanoma cells treated with BRAF and MEK inhibitor combinations. We have identified a slow-cycling subpopulation that is dynamically changing as the cells survive the initial treatment, then persist and finally regrow with acquired resistance to a variety of drugs. We will follow the cells in in vitro and in vivo models of human melanoma using bar codes and single cell RNA analyses. These studies will not only determine whether minor subpopulation(s) evolve under drug treatment stochastically or hierarchically, but also will provide us with information on their pre-existence and/or whether malignant cell plasticity is the major reason for intra-tumor heterogeneity to drug responses. In the second aim, we are testing the hypothesis that intrinsically resistant subpopulations of cells present new therapeutic opportunities that, if targeted, will specifically inhibit the onset of resistance. Our preliminary studies using CRISPR/Cas9 have allowed us to functionally dissect the pathways that form these rare subpopulations and, separately, the pathways that allow those subpopulations to reprogram. We will validate hits in 3D culture systems and in in vivo mouse models. We expect that targeting separate aspects of the resistance process in a rationally designed way (in vitro and then in preclinical studies) will enable the reduction of resistance onset upon BRAF/MEK inhibitor therapy.