Role of Receptor Tyrosine Kinase cross-talk in colorectal cancer

摘要： 背景：表皮生长因子受体(EGFR)是一种高表达的受体酪氨酸激酶(RTK)。 超过50%的结直肠癌(CRC)病例与转移和预后不良有关。 西妥昔单抗是一种靶向EGFR的单抗，被美国FDA批准用于治疗晚期 野生型KRAS CRC。然而，作为单一疗法，西妥昔单抗仅在约10%的患者中有效 耐药和耐药现象频繁出现。因此，迫切需要1)找出 最有可能对西妥昔单抗有反应(或无反应)的患者和2)制定治疗策略 这将防止耐药性和/或增强西妥昔单抗的反应。我们建议解决这些问题 通过积极追求我们的观察结果，增强了多个RTK的活动(例如 Met和Ron)产生新的和获得性西妥昔单抗耐药性，可通过以下方法克服 添加双MET/RON抑制剂Crizotinib。我们的中心假设是 RTK活性增加了结直肠癌对西妥昔单抗的耐药性，它可能是一个可行的治疗靶点。 在目标1中，我们将确定在CRC进展过程中RTK合作的机制 西妥昔单抗在结直肠癌细胞株和患者中的基因和化学修饰耐药 衍生异种移植物(PDX)和有机体(PDO)。在目标2中，我们将评估中断的影响 RTK在克服对EGFR导向抗体的耐药性方面的合作。在目标3中，我们将确定 合理的药物组合和患者群体分层以克服对EGFR的耐药性- 定向单抗。 研究设计：我们将使用我们的体外3D培养系统来培养大肠癌细胞株和PDO，并在 体内裸鼠移植瘤、PDX和免疫活性同基因小鼠结直肠癌模型 格兰特。我们还将采用补充方法(Transwell培养，肿瘤组织 微阵列、磷酸化RTK阵列和人类CRC样本)。关键蛋白 (EGFR、MET、RON、ERBB3、HGF、HGFL、NRG1等正负RTK和配体 调节子)将被操纵(过度表达、CRISPR介导的基因敲除、配体刺激、 以及基于化学和抗体的抑制)来表征个人的贡献。这些 实验将记录在亚细胞定位、形态和表型水平上，以 梳理出关键的差异。 这些研究将识别和表征新的西妥昔单抗耐药模式及其 在制定预防或克服耐药性的手段的同时，加强儿童在儿童权利委员会中的患病率。这项研究 将有助于优化精准医疗，并根据个人的反应/抵抗力对其进行分层 用于更好的CRC治疗和管理的配置文件。

ABSTRACT: Background: EGF receptor (EGFR) is a receptor tyrosine kinase (RTK) that is overexpressed in over 50% of colorectal cancer (CRC) cases where it is linked to metastasis and poor prognosis. Cetuximab, an EGFR-targeting monoclonal antibody is approved by US-FDA to treat advanced wild-type KRAS CRC. However, cetuximab, as monotherapy, is effective in only about 10% of CRCs and resistance frequently emerges. Thus, there is a pressing need 1) to identify those patients most likely to respond (or not respond) to cetuximab and 2) to devise treatment strategies that would prevent resistance and/or enhance cetuximab response. We propose to address these needs by aggressively pursuing our observations that enhanced activity of multiple RTKs (e.g. MET and RON) confers de novo and acquired cetuximab resistance, which may be overcome by addition of the dual MET/RON inhibitor, crizotinib. Our central hypothesis is that upregulation of RTK activity confers cetuximab resistance in CRC and that it may be a viable therapeutic target. In Aim 1 we will identify the mechanism of RTK cooperation during CRC progression and cetuximab resistance using genetic and chemical modifications in CRC cell lines and patient- derived xenografts (PDXs) and organoids (PDOs). In Aim 2 we will assess the impact of disrupting RTK cooperation in overcoming resistance to EGFR-directed antibodies. In Aim 3 we will identify rational drug combinations and stratify patient populations to overcome resistance to EGFR- directed mAbs. Study design: We will employ our in vitro 3D culture system for CRC cell lines and PDOs and in vivo nude mice xenografts, PDXs, and immune-competent syngeneic mouse CRC models in this grant. We will also employ complementary approaches (Transwell cultures, tumor tissue microarrays, phospho-RTK arrays, and human CRC samples) in relevant sections. Key proteins (EGFR, MET, RON, ERBB3, HGF, HGFL, NRG1 and other positive and negative RTK and ligand regulators) will be manipulated (overexpression, CRISPR-mediated knockout, ligand stimulation, and chemical and antibody-based inhibition) to characterize individual contribution. These experiments will be recorded at subcellular localization, morphological, and phenotypic levels to tease out key differences. These studies will identify and characterize new modes of cetuximab resistance and its prevalence in CRC, while devising means to prevent or overcoming the resistance. This research will help optimize precision medicine and stratify individuals based on their response/resistance profile for better CRC treatment and management.