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

了解并克服黑色素瘤对 BRAF/MEK 激酶抑制剂的耐药性

基本信息

批准号：
10307107
负责人：
Meenhard F Herlyn
金额：
$ 49.38万
依托单位：
UNIVERSITY OF PENNSYLVANIA
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-12-13 至 2024-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10307107
关键词：
Affect BRAF gene Bar Codes Biological Biology CRISPR/Cas technology Categories Cell Line Cells Combined Modality Therapy Complement DNA Development Distant Drug resistance Epigenetic Process Evolution Gene Expression Genes Growth Human Image Immunotherapy In Vitro Individual Libraries MAP2K1 gene MAP3K1 gene MEKs Melanoma Cell Minor Modeling Molecular Molecular Target Mutation Nature Organ Pathway interactions Patients Pattern Pharmaceutical Preparations Pharmacotherapy Phenotype Population RNA RNA analysis Relapse Resistance Resistance Process Resistance development Signal Pathway Specimen System Testing Therapeutic Time Transitional Cell Neoplasm Treatment outcome Tumor Cell Line Validation base cancer cell cancer drug resistance cellular transduction genome-wide human model in vivo in vivo Model inhibitor inhibitor therapy kinase inhibitor melanoma mouse model mutant neoplastic cell novel novel strategies novel therapeutic intervention novel therapeutics patient derived xenograft model preclinical study rational design relapse patients repository resistance mechanism response single-cell RNA sequencing targeted treatment three dimensional cell culture transcriptomics treatment response treatment strategy tumor tumor heterogeneity

项目摘要

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.

绝大多数 (>80%) BRAFV600E/K 黑色素瘤患者最初对高反应特定 BRAFV600E/K 抑制剂作为单一疗法或 BRAF/MEK 抑制剂联合疗法，但程度不同。几乎所有患者都会在三个月到两年后复发，尽管如此他们的肿瘤细胞携带 BRAF 突变。在此应用程序中，我们重点关注 BRAFV600E/K 黑色素瘤中小群体的生物动力学是先验的耐药或在治疗后迅速适应并最终导致复发。在第一个目标中，我们将追踪经 BRAF 和 MEK 处理的突变 BRAFV600E/K 黑色素瘤细胞的动态抑制剂组合。我们已经确定了一个缓慢循环的亚群，它是动态变化的随着细胞在最初的治疗中存活下来，然后持续存在并最终重新生长，细胞会发生变化对多种药物产生耐药性。我们将在人类体外和体内模型中跟踪细胞使用条形码和单细胞 RNA 分析来诊断黑色素瘤。这些研究不仅将决定次要亚群在药物治疗下是随机进化还是分层进化，但是还将向我们提供有关其先前存在和/或是否为恶性细胞的信息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，我们正在测试这样的假设：本质上具有抗药性的细胞亚群呈现出新的如果有针对性的治疗机会，将专门抑制耐药性的发生。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这些罕见的亚群，以及分别允许这些亚群的途径重新编程的亚群。我们将在 3D 培养系统和体内小鼠体内验证命中结果模型。我们期望以合理的方式针对抵抗过程的各个方面设计的方法（体外然后在临床前研究）将能够减少耐药性 BRAF/MEK 抑制剂治疗后起病。