Isolation and small molecule targeting of Ewing's Sarcoma stem cells

描述(由申请人提供)：“癌症干细胞假说”假定每个肿瘤内存在一个稀有的干细胞群体，该干细胞群体对化疗具有抵抗力，并负责产生和维持大量肿瘤群体。尤文肉瘤家族肿瘤(ESFT)是由癌症干细胞驱动的肿瘤预期治疗的典型反应。目前的治疗方法很可能是针对干细胞的后代(肿瘤的大部分)，而不是真正的癌症干细胞。因此，尽管大多数患者病情得到缓解，但复发是常见的。ESFT含有编码致癌转录的特征性易位t(11：22) 因素EWS-FLI1。EWS-FLI1被认为促进肿瘤的发生，因此抑制EWS-FLI1应该是一种有效的治疗策略。因为EWS-FLI1缺乏内在的酶活性，我们的药物抑制方法是通过靶向蛋白质-蛋白质相互作用。RNA解旋酶A是EWSFLI1的关键结合伙伴。我们已经确定了一个先导化合物YK-4-279，它可以阻止EWS-FLI1与RHA的相互作用。此外，我们的初步数据表明，具有干细胞样特性的ESFT细胞系中的一个亚群仍然表达EWS-FLI1。因此，EWS-FLI1有望成为ESFT干细胞的关键分子靶点。我们假设，一种阻断RHA与EWS-FLI1相互作用的药物将针对耐化疗的ESFT干细胞。这些研究将提供必要的临床前数据，证明使用一种专门针对ESFT干细胞的化合物进行临床试验是合理的。本研究的主要目的是开发一种针对尤文氏肉瘤干细胞(ESSC)的靶向治疗方法。我们正在提出一项旨在鉴定和表征ESSC的综合性、协作性研究计划，这将使我们能够开发一种有效的治疗策略，针对并消除患者体内的这些细胞。这项研究的第一个目的是识别和表征ESSC。我们将使用已提出的各种方法来鉴定癌症干细胞，包括乙醛脱氢酶的表达、染料外流、在非贴壁条件下形成球形聚集体的能力，以及特征基因的表达。基因表达谱将由假设驱动，并将专门用于测试尤文氏肉瘤是由间充质干细胞的肿瘤转化而来的假设。然后，我们将证明抑制EWS-FLI1/RHA相互作用对化疗耐药的ESSC是致命的。这项研究的最终目标是确定其他适合于小分子靶向的关键信号通路。这将解决仅抑制EWS-FLI1活性可能不足以消除ESSC的可能性。同时靶向多个信号通路可能是一种更有效的治疗策略。我们预计，在项目结束时，ESSC将被分离出来，并具有足够的特性，从而能够开发出可迅速转化为临床试验的靶向疗法，并极大地改善尤文氏肉瘤家族肿瘤患者的预后。

DESCRIPTION (provided by the applicant): The "Cancer Stem Cell Hypothesis" posits the existence of a scarce stem cell population within each tumor that is resistant to chemotherapy and is responsible for generating and maintaining the bulk tumor population. Ewing sarcoma family tumors (ESFT) typify the response to treatment expected of a tumor driven by cancer stem cells. Current therapies most likely target the stem cells' progeny (the bulk of the tumor) and spare the true cancer stem cells. Consequently, although the majority of patients achieve a remission, relapse is common. ESFT contains a characteristic translocation, t(11:22), which encodes the oncogenic transcription factor EWS-FLI1. EWS-FLI1 is thought to drive tumorigenesis, so inhibition of EWS-FLI1 should be an effective treatment strategy. Because EWS-FLI1 lacks intrinsic enzymatic activity, our approach to pharmacologic inhibition is through targeting protein-protein interactions. RNA Helicase A is a critical binding partner of EWSFLI1. We have identified a lead compound, YK-4-279, that blocks EWS-FLI1 interaction with RHA. Furthermore, our preliminary data suggest that a subpopulation within ESFT cell lines with stem-like properties still expresses EWS-FLI1. Therefore, EWS-FLI1 is expected to be a key molecular target in ESFT stem cells. We hypothesize that a drug that blocks the interaction of RHA with EWS-FLI1 will target chemotherapyresistant ESFT stem cells. These studies will provide the necessary preclinical data to justify clinical trials with a compound designed specifically to target ESFT stem cells. The primary objective of this study is to develop a targeted therapy for Ewing's sarcoma stem cells (ESSC). We are proposing an integrative, collaborative research program aimed at the identification and characterization of ESSC which will allow the development of an effective therapeutic strategy to target and eliminate these cells in patients. The first aim of the study is to identify and characterize ESSC. We will employ a variety of assays that have been proposed to identify cancer stem cells, including expression of aldehyde dehydrogenase, dye efflux, the ability to form spherical aggregates under nonadherent conditions, and the expression of characteristic genes. Gene expression profiling will be hypothesis-driven, and will be performed specifically to test the hypothesis that Ewing's sarcoma is derived from the neoplastic transformation of mesenchymal stem cells. We will then demonstrate that inhibition of the EWS-FLI1/RHA interaction is lethal to chemotherapy-resistant ESSC. The final aim of the study is to identify other key signaling pathways that are amenable to small molecule targeting. This will address the possibility that inhibition of EWS-FLI1 activity alone might be insufficient to eliminate ESSC. Simultaneous targeting of multiple signaling pathways is likely to be a substantially more effective therapeutic strategy. We anticipate that at the conclusion of the project, ESSC will have been isolated and sufficiently characterized to allow the development of targeted therapies that can be rapidly translated into clinical trials and dramatically improve the prognosis of patients with Ewing's sarcoma family tumors.