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

项目概要/摘要 胃肠道间质瘤（GIST）是美国最常见的肉瘤。 伊马替尼是一种酪氨酸激酶抑制剂（TKI），靶向KIT癌基因，为精确性提供了原理证明 肿瘤学我们现在知道，GIST的发生也是由于PDGFRA，RAS/BRAF/NF 1，SDHA-D， 以及FGFR 1和ETV 6-NTRK 3融合物。因此，有几种GIST亚型，其中许多是 伊马替尼耐药。伊马替尼显著提高患者生存率，但未能治愈GIST，因为许多患者会发展为 停用伊马替尼后8-12个月复发。这些反复出现的问题提出了GIST是否 伊马替尼后的持久性通过未被认识的机制发生。我们假设KIT阴性肿瘤细胞， 缺乏KIT蛋白表达，代表TKI抗性GIST癌症干细胞（CSC），基于我们的初步研究， 数据表明：1）人KIT突变GIST细胞系具有自我更新、形成肿瘤球的KIT阴性细胞，和 具有原发性TKI抗性; 2）KIT和非KIT突变体切除的人GIST都具有KIT阴性细胞， TKI治疗后干细胞基因表达增加; 3）这些KITneg细胞过度表达 可药物靶点（即，Hedgehog、AMPK、NF-kB和AXL）。在这个项目中，我们建议 进一步表征和定位这些KITneg GIST CSC，以克服以前未认识到的问题， 临床相关，使用最先进的实验技术和先进的 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生长和TKI抗性。我们将决定自我更新， 在与FDA平行的一系列TKI治疗后，每个推定的CSC群体的分化潜力 批准的疗法。然后，我们将鉴定分离的TKI耐药KITneg的保守蛋白和途径， KIThi细胞。我们还将确定KITneg细胞中TKI耐药的常见机制， 治疗在目的2中，我们将表征来自以下的KITneg细胞的CSC特性和分子异质性： 原发性KIT突变型和非KIT突变型肿瘤。我们将确定肿瘤间/肿瘤内的自我更新， 分化和肿瘤球形成的主要推定CSC群体从GIST。因此，我们将 确定来自基因组多样性GIST的推定CSC中的保守途径。在目标3中，我们将验证 可用药的目标（例如，Hedgehog、AMPK、NF-kB和AXL），并利用这些途径根除KITneg 细胞在体外和体内。我们将用遗传学方法验证我们的发现，以确定这些目标是否是 需要保持活力。本项目将探讨TKI治疗和 基因组多样的GIST CSC，代表了克服疾病持续性和TKI的新靶点， 阻力我们的建议将确定新的药物靶点和药物杀死GIST CSC，从而将有 对单独使用或与伊马替尼联合使用新药临床试验设计的直接和显著影响 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.