Targeting EWS/FLI1-driven pathways to improve therapeutic gains in Ewing's Sarcom

描述(申请人提供)：尤文氏肉瘤家族肿瘤(ESFT)是一种骨质和软组织的高度恶性的实体肿瘤，最常影响儿童和青少年，是年轻人中第二常见的骨恶性肿瘤。目前的治疗方法包括化疗和放射治疗相结合的方法。然而，有两个重要的问题需要考虑：a)虽然ESFT一般对治疗有反应，但总体治愈率很低，因为这些肿瘤侵袭性很强，患者经常出现转移疾病；b)年轻患者暴露在大剂量化疗和/或放射之下，往往与各种不利的健康影响有关，在某些情况下，这些影响要在治疗完成后多年才会发展起来。因此，治疗策略需要最大限度地提高治愈率，同时将患者的不良后遗症降至最低。在初步研究中，我们直接针对EWS/FLI-1，这是已知的导致大多数ESFT恶性特性的转录因子，使用旨在单独或与化疗药物和/或放射联合阻断其活性的分子工具。虽然体外实验和小鼠异种移植实验证明这些工具有效地延缓了肿瘤的生长，但很明显，同时靶向作用于EWS/FLI-1下游的额外分子对于最大限度地发挥抗肿瘤活性并允许使用较低的治疗剂量是必要的，从而尽可能减少负面的晚期效应。在这种背景下，这项建议的主要目标是利用我们实验室最近建立的一种新的EWS/FLI-1驱动的途径(EWS/FLI-1-[Caveolin-&gt；1-Snail-E-cadherin])来使ESFT细胞对治疗敏感，使用电离辐射(IR)作为一种模型抗肿瘤药物，它不受特异性相关问题的困扰，这些问题经常使化疗药物的研究复杂化。由于肿瘤周围正常组织中与IR相关的远期效应的程度与IR剂量直接相关，因此在儿童癌症中使用尽可能低的剂量来治愈肿瘤是很重要的。以EWS细胞作为ESFT的原型，我们的中心假设是：(A)靶向小窝蛋白-1(CAV1)本身和/或其与ESFT分子病理相关的信号或调节蛋白(如磷脂酶D2和蛋白激酶C1)的相互作用将使EWS细胞对IR更加敏感，以及(B)通过同时靶向其他也可使EWS细胞辐射增敏的途径的成分，如聚(ADP-核糖)聚合酶(PARP)，这种反应可能会进一步改善。由于我们已经确定CAV1是EWS/FLI-1的直接转录靶点，也是EWS细胞致瘤性和化疗反应的关键决定因素，靶向CAV1信号可能具有双重作用：通过低剂量IR增强对EWS细胞的杀伤作用，并下调其肿瘤特性。公共卫生关系：尤文肉瘤家族的肿瘤(ESFT)是一种恶性程度极高的实质性骨和软组织癌症，最常影响儿童和青少年，是年轻人中第二常见的骨恶性肿瘤。有两个重要问题需要考虑：a)尽管采取了积极的治疗策略，如大剂量化疗结合手术和/或延长放射治疗，但患有较大原发肿瘤或转移疾病的ESFT患者的预后仍然很差；大约50%的患者最终复发，即使在五年后，超过30%的局部疾病患者和大约80%的转移患者由于疾病进展而死亡；以及b)年轻患者暴露于大剂量化疗和/或放射治疗经常与各种不良健康影响有关，在某些情况下，这些不良健康影响在治疗完成多年后才会发展。因此，治疗策略需要最大限度地提高治愈率，同时将患者的不良后遗症降至最低。在这种情况下，我们建议使用尤文的肉瘤细胞作为ESFT的原型，并将辐射作为一种模型抗癌药物，以设计使ESFT细胞对治疗敏感的方案。具体地说，我们将使用最先进的技术来阻断我们实验室建立的由EWS/FLI-1蛋白驱动的新途径{EWS/FLI-1-&gt；[CAV1-PKC1-PLD2]-mTOR}的分子成分的产生和/或活性，已知EWS/FLI-1蛋白与ESFT的恶性特性有关。我们的初步研究有力地表明，该途径的组成部分，如CAV1和PKC1，有助于使ESFT细胞对化疗和放射耐药。我们的方法将首先描述每个通路组件的作用机制及其相互关系，然后单独或联合针对它们来提高ESFT细胞和肿瘤的治疗反应。PARP-1抑制剂的治疗也将包括在我们的实验方案中。PARP-1是我们部门以前显示的一种有助于ESFT抗辐射的分子。

DESCRIPTION (provided by applicant): Tumors of the Ewing's sarcoma family (ESFT) are solid, highly malignant neoplasms of the bone and soft tissues that most often affect children and adolescents, being the second most common bone malignancies among young adults. Current treatment includes a combined modality with chemotherapy and radiotherapy. However, there are two important problems to consider: a) although ESFT are generally responsive to treatment, the overall cure rate is low because these tumors are very aggressive and patients frequently present with metastatic disease; and b) exposure of young patients to high doses of chemotherapy and/or radiation is frequently associated with a variety of adverse health effects that in some cases do not develop until many years after treatment completion. Consequently, treatment strategies are needed to maximize curability while simultaneously minimizing adverse late effects for the patients. In preliminary studies, we directly targeted EWS/FLI-1, the transcription factor known to be responsible for the malignant properties of most ESFT, using molecular tools designed to block its activity, either alone or in combination with chemotherapeutic drugs and/or radiation. Although experiments in vitro and in mouse xenografts demonstrated that these tools efficiently delayed tumor growth, it became clear that simultaneous targeting of additional molecules that act downstream of EWS/FLI-1 would be necessary to maximize antitumor activity and to allow the use of lower therapeutic doses, thus minimizing negative late effects as much as possible. In this context, the main objective of this proposal is to exploit a novel EWS/FLI-1-driven pathway recently established in our laboratory (EWS/FLI-1-[Caveolin->1-Snail-E-cadherin]) to sensitize ESFT cells to therapy, using ionizing radiation (IR) as a model anti-neoplastic agent that does not suffer from the specificity- related problems that frequently complicate studies with chemotherapeutic drugs. Since the extent of IR related late effects in normal tissues surrounding the tumor are directly related to the IR dose, in pediatric cancers it is important to employ the lowest dose possible to cure the tumors. Using EWS cells as the ESFT prototype, our central hypothesis is that (a) targeting caveolin-1 (CAV1) itself and/or its interactions with signaling or regulatory proteins relevant for ESFT molecular pathobiology (such as phospholipase D2 and protein kinase C1) will render EWS cells more sensitive to IR, and (b) that this response may be further improved by simultaneously targeting components of other pathways also known to radiosensitize EWS cells, such as poly (ADP-ribose) polymerase (PARP). Because we already identified CAV1 as a direct transcriptional target of EWS/FLI-1 and a key determinant of the tumorigenicity and chemotherapeutic response of EWS cells, targeting CAV1 signaling may have a dual effect: enhancing killing of EWS cells by low-dose IR and down- regulating their neoplastic properties. PUBLIC HEALTH RELAVANCE: Tumors of the Ewing's sarcoma family (ESFT) are solid, highly malignant cancers of the bone and soft tissues that most often affect children and adolescents, being the second most common bone malignancies among young adults. There are two important problems to consider: a) despite aggressive treatment strategies, such as high-dose chemotherapy combined with surgery and/or extended radiotherapy, the prognosis for ESFT patients with large primary tumors or metastatic disease remains poor; about 50% of patients eventually relapse, even after five years, and more than 30% of patients with localized disease and about 80% of patients with metastases die due to disease progression; and b) exposure of young patients to high doses of chemotherapy and/or radiation is frequently associated with a variety of adverse health effects that in some cases do not develop until many years after treatment completion. Consequently, treatment strategies are needed to maximize curability while simultaneously minimizing adverse late effects for the patients. In this context, we propose to use Ewing's sarcoma cells as the ESFT prototype, and radiation as a model anti-cancer agent that does not suffer from the specificity-related problems that frequently complicate studies with chemotherapeutic drugs, to devise protocols to sensitize ESFT cells to therapy. Specifically, we will use state-of-the-art techniques to block the production and/or activity of molecular components of a novel pathway {EWS/FLI-1-> [CAV1-PKC1-PLD2]-mTOR} established in our laboratory as driven by the EWS/FLI-1 protein, which is known to be responsible for the malignant properties of ESFT. Our preliminary studies strongly indicate that components of this pathway such as CAV1 and PKC1 contribute to render ESFT cells resistant to chemotherapy and radiation. Our approach will, first, characterize the mechanism of action of each of the pathway components and their interrelationships, and then target them either individually or in combination to improve the therapeutic response of the ESFT cells and tumors. Treatments with inhibitors of PARP-1, a molecule previously shown in our Department to contribute to the radiation resistance of ESFT, will be included also in our experimental protocols.