Drug Tolerant Persisters in EGFR-mutant Non-small Cell Lung Cancer: Epigenetic Landscape and Therapeutic Targeting

EGFR 突变非小细胞肺癌持续存在耐药性：表观遗传景观和治疗靶向

• 批准号: 9393667
• 负责人: Sana Raoof
• 金额: $ 3.76万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-07-01 至 2020-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9393667
• 关键词: Apoptosis; Automobile Driving; Awareness; Biological; Cancer Etiology; Cancer Patient; Cell Line; Cell Survival; Cell Therapy; Cells; Cessation of life; ChIP-seq; Chromatin; Clinical; Development; Disease model; Dropout; Drug Combinations; Drug Targeting; Drug resistance; Engineering; Environment; Epidermal Growth Factor Receptor; Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitor; Epigenetic Process; Erlotinib; Evolution; Failure; Fibroblast Growth Factor Receptors; Gefitinib; Gene Expression Profile; Generations; Genes; Genetic Transcription; Goals; Gold; In Vitro; Longitudinal Studies; Maintenance; Malignant Neoplasms; Modeling; Molecular; Mutate; Mutation; Non-Small-Cell Lung Carcinoma; Pathway interactions; Patient Care; Patients; Pharmaceutical Preparations; Pharmacology; Play; Population; Preclinical Drug Evaluation; Publishing; Refractory; Residual Tumors; Resistance; Resistance development; Role; Signal Pathway; Techniques; Testing; Therapeutic; Thoracic Oncology; Time; Tissues; Toxic effect; Tyrosine Kinase Inhibitor; Work; Xenograft Model; Xenograft procedure; base; cancer cell; cancer therapy; cancer type; chemotherapy; chromatin modification; drug candidate; epithelial to mesenchymal transition; genetic evolution; genetic resistance; in vitro Model; in vivo; inhibitor/antagonist; interest; knock-down; mouse model; mutant; non-genetic; novel; novel therapeutics; prevent; programs; resistance mechanism; small hairpin RNA; standard of care; synergism; therapeutic target; tool; transcription factor; transcriptome sequencing; tumor

Abstract Treatment of EGFR-mutant non-small cell lung cancer (NSCLC) with EGFR-directed tyrosine kinase inhibitors (TKI’s, e.g. gefitinib) has revolutionized thoracic oncology. However, patients develop resistance to gefitinib in two years (on average), and it remains unclear whether gefitinib resistance is pre-existing in a small sub- population of treatment-naïve cells, or acquired during therapy. Recently, the Engelman lab published that acquisition of resistance to gefitinib in vitro arises among an epigenetically distinct population of “persister” cells. This persister state may define the precursors of TKI-refractory cancer, but its molecular features and in vivo significance remain to be determined. The goals of this proposal are to discover the determinants of the persister state, to develop tools to study this state in vivo, and to develop strategies to prevent the accumulation of persister cells. In Aim 1, we will determine whether the persister state occurs in two patient- derived xenograft (PDX) models of EGFR-mutant NSCLC. Both models are TKI-naïve, and we estimate that after 3 weeks of gefitinib treatment, the remaining tumor will consist of persister cells. We will perform both RNAseq and ChIP-seq on explanted undrugged or persister tissue, and focus our analysis on whether EMT- related or other epigenetic pathways are upregulated in putative persister cells. Upregulated pathways will be investigated in the drug screen proposed in Aim 3. In Aim 2, we will investigate the role of the Epithelial to Mesenchymal Transition (EMT) in driving the development of a persister state. We have established that EMT occurs in five patient-derived EGFR-mutant cell lines, which express several EMT-related transcription factors. We will determine whether these transcription factors are responsible for gefitinib-induced EMT by engineering knockdown cell lines for each gene. We will then compare both gefitinib-induced EMT and time-to-resistance in the isogenic parental vs. knockdown lines, first in vitro and then in PDXs. In Aim 3, we will screen for compounds that prevent resistance to gefitinib. Our novel platform for 8-week drug screening has identified two candidate compounds (CDK7 and FGFR inhibitors) that lack single-agent efficacy but prevent resistance in combination with gefitinib. We will validate hits in 3D spheroid cultures and in xenograft mouse models, and investigate the mechanism of action of these hits. The work proposed here responds to a paradigm shift in cancer therapy—one in which we are aware that therapy may drive an evolutionary trajectory towards acquired resistance. We will investigate potential mechanisms of this evolution, focusing on EMT. We will explore the chromatin and transcriptional state of persister tissue in PDX models, and will offer a novel drug-screening platform for uniquely long term studies of emerging resistance. This technique may be useful in a variety of cancer types in which acquired resistance limits patient survival.

摘要 EGFR靶向酪氨酸激酶抑制剂治疗EGFR突变型非小细胞肺癌 (TKI例如吉非替尼）已经彻底改变了胸部肿瘤学。然而，患者对吉非替尼产生耐药性， 两年（平均），目前还不清楚吉非替尼耐药性是否预先存在于一个小的亚群中， 未经治疗的细胞群，或在治疗期间获得的细胞群。最近，恩格尔曼实验室发表文章称， 体外获得对吉非替尼的耐药性是在表观遗传学上不同的"持久" 细胞这种持续状态可能定义了TKI难治性癌症的前体，但其分子特征和预后可能与TKI治疗有关。 体内意义仍有待确定。本提案的目标是发现 持续状态，开发工具来研究这种状态在体内，并制定战略，以防止 持续细胞的积累。在目标1中，我们将确定持续状态是否发生在两个患者中- EGFR突变型NSCLC的衍生异种移植（PDX）模型。这两个模型都是TKI-naïve，我们估计， 吉非替尼治疗3周后，剩余的肿瘤将由持续存在的细胞组成。我们将执行两者 RNAseq和ChIP-seq对未染色或持久组织的分析，并将我们的分析集中在EMT- 相关的或其它表观遗传途径在推定的持留细胞中被上调。上调的途径将是 在目标3中提出的药物筛选中进行了研究。在目标2中，我们将研究上皮细胞对 间充质转化（EMT）在驱动持续状态的发展。我们已经确定急救人员 发生在五个患者来源的EGFR突变细胞系，表达几个EMT相关的转录因子。 我们将通过基因工程的方法来确定这些转录因子是否与吉非替尼诱导的EMT有关 每个基因的敲低细胞系。然后，我们将比较吉非替尼诱导的EMT和耐药时间。 首先在体外，然后在PDX中的等基因亲本与敲低系。在目标3中，我们将筛选 预防吉非替尼耐药性的化合物。我们用于8周药物筛选的新平台已经确定了两个 候选化合物（CDK7和FGFR抑制剂）缺乏单一药物功效，但可防止耐药性， 与吉非替尼联合使用。我们将在3D球体培养物和异种移植小鼠模型中验证命中， 研究这些命中的作用机制。这里提出的工作响应了一个范式转变， 癌症治疗-我们意识到治疗可能会推动一个进化的轨迹， 阻力我们将研究这种演变的潜在机制，重点是EMT。我们将探讨 染色质和转录状态，并将提供一个新的药物筛选 为新出现的耐药性提供独特的长期研究平台。该技术可用于多种应用中。 获得性耐药性限制患者生存的癌症类型。