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

• 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
• 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

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%）BRAFV 600 E/K黑色素瘤患者最初对高浓度的 作为单一疗法或BRAF/MEK抑制剂组合疗法的特异性BRAFV 600 E/K抑制剂， 但是在不同的水平上。几乎所有的患者在三个月到两年后都会复发，尽管所有的 携带BRAF突变。在本应用程序中，我们重点关注 BRAFV 600 E/K黑色素瘤内小群体的生物学动力学是先验的 耐药或快速适应治疗并最终导致复发。在第一个目标中，我们将 跟踪用BRAF和MEK处理的突变BRAFV 600 E/K黑素瘤细胞的动力学 抑制剂组合。我们已经确定了一个缓慢循环的亚群， 随着细胞在初始处理中存活而变化，然后持续存在，最后随着获得性的再生而再生。 对多种药物的耐药性。我们将在体外和体内模型中跟踪细胞， 黑色素瘤使用条形码和单细胞RNA分析。这些研究不仅将确定 次要亚群是否在药物治疗下随机或分层演变，但 还将为我们提供关于它们的预先存在和/或是否恶性细胞的信息。 可塑性是肿瘤内对药物反应的异质性的主要原因。第二个目标， 我们正在检验这样一个假设，即内在抗性细胞亚群呈现新的 治疗的机会，如果有针对性，将具体抑制耐药性的发作。我们 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， 重新编程的亚群。我们将在3D培养系统和体内小鼠中验证命中 模型我们希望，以合理的方式针对抵抗进程的各个方面， 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抑制剂治疗后起效。