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

摘要 EGFR导向的酪氨酸激酶抑制剂治疗EGFR突变型非小细胞肺癌 (TKI的，例如吉非替尼)已经使胸部肿瘤学发生了革命性的变化。然而，患者对吉非替尼产生了耐药性 两年(平均)，目前尚不清楚吉非替尼是否预先存在于一个小的亚 治疗群体-幼稚细胞，或在治疗过程中获得的。最近，恩格尔曼实验室公布了 体外获得对吉非替尼的耐药性发生在表观遗传学上不同的“持续者”群体中 细胞。这种持续状态可能定义了TKI-难治性癌症的前驱细胞，但它的分子特征和 活体意义仍有待确定。这项提案的目标是发现 持续状态，开发工具在体内研究这种状态，并制定策略以防止 宿存细胞的积累。在目标1中，我们将确定两名患者是否出现持续状态- EGFR突变型非小细胞肺癌衍生异种移植模型的建立这两种模式都很天真，我们估计 经过3周的吉非替尼治疗后，剩下的肿瘤将由周围细胞组成。我们将演出这两个节目 RNASEQ和CHIP-SEQ在移植的未用药或宿存组织上的表达，并将重点放在EMT- 相关或其他表观遗传途径在推测的旁系细胞中上调。上调的路径将是 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在 间充质转化(EMT)在推动永续状态发展中的作用。我们已经确定EMT 发生在五个患者来源的EGFR突变细胞系中，这些突变细胞系表达几个EMT相关转录因子。 我们将通过工程方法确定这些转录因子是否与吉非替尼诱导的EMT有关。 击倒每个基因的细胞系。然后，我们将比较吉非替尼诱导的EMT和耐药时间 等基因亲本与基因敲除系，首先在体外，然后在PDX中。在《目标3》中，我们将筛选 防止对吉非替尼产生耐药性的化合物。我们为期8周的药物筛选新平台已经确定了两个 候选化合物(CDK7和FGFR抑制剂)，缺乏单药疗效，但防止耐药 与吉非替尼联合应用。我们将在3D球体培养和异种移植小鼠模型中验证HIT，以及 研究这些HITS的作用机制。这里提出的工作是对 癌症治疗--我们意识到治疗可能会将进化轨迹推向获得性 抵抗。我们将研究这种进化的潜在机制，重点是EMT。我们将探索 PDX模型中周围组织染色质和转录状态的变化，将为药物筛选提供新的途径 对新出现的耐药性进行独特的长期研究的平台。这项技术可能在各种不同的 获得性耐药性限制患者生存的癌症类型。

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.