Targeting Drug-Resistant Cancer Stem Cell Niches of Gastrointestinal Stromal Tumor

项目摘要/摘要 胃肠道间质瘤(GIST)是美国最常见的肉瘤。 针对KIT癌基因的酪氨酸激酶抑制剂(TKI)伊马替尼为精密度提供了原理证明 肿瘤学。我们现在知道，GIST的发生也是由于PDGFRA、RAS/BRAF/NF1、SDHA-D、 以及FGFR1和ETV6-NTRK3融合。因此，有几个GIST子类型，其中许多是 对伊马替尼耐药。伊马替尼显著提高了患者的存活率，但由于许多患者的病情发展，未能治愈GIST 停用伊马替尼8-12个月后≈复发。这些反复引发了这样一个问题：GIST是否 伊马替尼后的持久性通过一种不为人知的机制发生。我们假设KITneg肿瘤细胞，它 缺乏KIT蛋白表达，代表耐TKI GIST肿瘤干细胞(CSCs) 数据表明：1)人KIT突变型GIST细胞系拥有可自我更新、形成肿瘤球体的KITneg细胞，以及 具有原代TKI抗性；2)KIT和非KIT突变体切除的人GIST均具有KITneg细胞和 在TKI治疗后干细胞基因表达增加；以及3)这些KITneg细胞过度表达 涉及CSC生物学的可药物靶点(即Hedgehog、AMPK、NF-kB和Axl)。在这个项目中，我们建议 为了进一步确定这些KITneg GIST CSC的特征和目标，以克服这一以前未被认识到的问题，以及 临床相关，使用最先进的实验技术和先进的TKI耐药性来源 计算分析方法。在目标1中，我们将定义KITneg细胞的分子和功能特性 以确定它们如何促进GIST增长和TKI抵抗。我们将确定自我更新和 在平行FDA-TKI系列治疗后，每个假定的CSC群体的分化潜力 批准的治疗方法。然后我们将鉴定耐TKI的KITneg和KITneg的保守蛋白和途径。 基提细胞。我们还将在KITneg细胞中确定TKI耐药的共同机制 治疗。在目标2中，我们将表征KITneg细胞的CSC特性和分子异质性。 原发KIT突变和非KIT突变肿瘤。我们将确定肿瘤间/瘤内的自我更新， GIST原代CSC群体的分化和肿瘤球体的形成。因此，我们将 从基因组不同的GIST中确定推测的CSCs中的保守通路。在目标3中，我们将验证 可药物靶标(如Hedgehog、AMPK、NF-kB和Axl)，并利用这些途径根除KITneg 细胞在体外和体内。我们将用遗传方法验证我们的发现，以确定这些靶点是否 是维持生存所必需的。该项目将探索经TKI治疗的和 基因多样性的GIST CSCs，代表了克服疾病持久性和TKI的新靶点- 抵抗。我们的提案将确定新的药物靶点和药物来杀死GIST CSCs，因此将拥有 对新药(S)单独或与伊马替尼联合治疗的临床试验设计立即产生重大影响 治愈GIST。

PROJECT SUMMARY/ABSTRACT Gastrointestinal Stromal Tumor (GIST) is the most common sarcoma in the U.S. The treatment of GIST with imatinib, a tyrosine kinase inhibitor (TKI), that targets the KIT oncogene, provides proof of principle for precision oncology. We now understand that GISTs also occur due to mutations in PDGFRA, RAS/BRAF/NF1, SDHA-D, as well as FGFR1 and ETV6-NTRK3 fusions. Thus, there are several GIST subtypes and many of these are imatinib resistant. Imatinib markedly improves patient survival, but fails to cure GIST as many patients develop recurrences ≈8-12 months after stopping imatinib. These recurrences raise the question as to whether GIST persistence post-imatinib occurs via an unappreciated mechanism. We hypothesize that KITneg tumor cells, which lack KIT protein expression, represent TKI-resistant GIST cancer stem cells (CSCs) based on our preliminary data that: 1) human KIT mutant GIST cell lines possess KITneg cells that self-renew, form tumorspheres, and have primary TKI-resistance; 2) both KIT and non-KIT mutant resected human GISTs possess KITneg cells and have increased expression of stem cell genes following TKI therapy; and 3) these KITneg cells overexpress druggable targets (i.e., Hedgehog, AMPK, NF-kB, and AXL) involved in CSC biology. In this project, we propose to further characterize and target these KITneg GIST CSCs to overcome this previously unappreciated, and clinically relevant, source of TKI-resistance using state-of-the-art experimental techniques and advanced computational analysis methods. In Aim 1, we will define the molecular and functional properties of KITneg cells to determine how they promote GIST growth and TKI resistance. We will determine the self-renewal and differentiation potential of each putative CSC population following serial TKI treatments that parallel FDA- approved therapies. We will then identify conserved proteins and pathways of isolated TKI-resistant KITneg and KIThi cells. We will also determine common mechanisms of TKI-resistance amongst KITneg cells following serial therapies. In Aim 2, we will characterize the CSC properties and molecular heterogeneity of KITneg cells from primary KIT mutant and non-KIT mutant tumors. We will determine the inter-/intra-tumoral self-renewal, differentiation, and tumorsphere formation for primary putative CSC populations from GISTs. Thus, we will determine conserved pathways in putative CSCs from genomically diverse GISTs. In Aim 3, we will validate druggable targets (e.g., Hedgehog, AMPK, NF-kB, and AXL) and exploit these pathways for eradication of KITneg cells in vitro and in vivo. We will validate our findings with genetic approaches to determine if these targets are required to maintain viability. This project will explore the commonalities and differences across TKI-treated and genomically diverse GIST CSCs, which represent a novel target for overcoming disease persistence and TKI- resistance. Our proposal will identify novel drug targets and agents for killing GIST CSCs, and thus will have immediate and significant impact on clinical trial design of new agent(s) alone or in combination with imatinib for curing GIST.