ASPH Targeted Therapy for Chondrosarcoma

项目总结/摘要 该应用的长期目标是改善患有以下疾病的患者的生存率和生活质量： 软骨肉瘤是一种原发性恶性骨肿瘤，迫切需要更有效的治疗。五个- 软骨肉瘤是成人最常见的骨肉瘤，其年生存率低至10- 25%，其中大多数 死于肺转移的病人传统的细胞毒化疗对 软骨肉瘤所以目前的治疗标准是手术切除没有任何改善， 软骨肉瘤的治愈率。本提案的目标是系统地评估 有希望的新治疗策略，靶向天冬氨酸β-羟化酶（ASPH），一种跨膜蛋白， 通常在胚胎发育期间表达，但在出生后不表达。我们发现ASPH是一种 在软骨肉瘤中表达。在一些癌中，ASPH通过Notch途径信号传导以影响肿瘤细胞的增殖。 基质金属蛋白酶（MMPs）和血管内皮生长因子（VEGF）的表达，支持 细胞增殖、侵袭和转移。我们有数据表明，ASPH的功能类似于 软骨肉瘤ASPH在>90%的软骨肉瘤中高度表达，活化的Notch和多种 基质金属蛋白酶和高ASPH表达与肿瘤分级和不良长期生存率呈正相关。抑制 Notch是癌症治疗中长期追求但尚未实现的目标。本提案中的工作将 通过确定ASPH和Notch之间的关系，将ASPH作为潜在的治疗靶点， 软骨肉瘤首先，将使用CRISPR-Cas9调节人软骨肉瘤细胞系中的ASPH水平 基因编辑，然后用经证实的ASPH小分子抑制剂（SMI）治疗。变化 增殖、侵袭、凋亡和Notch信号传导将被用作ASPH信号传导的机制读数 体外Notch信号将在这些ASPH调节的细胞系中被操纵，具有功能的获得和丧失 评估Notch在ASPH信号传导中的作用的方法（Aim 1）。第二，ASPH和Notch在 将在人软骨肉瘤细胞系中评价化疗抗性，并且SMI和 将测试增强多柔比星功效的ASPH靶向抗体-药物缀合物（ADC）（目的2）。最后， 将使用小鼠软骨肉瘤模型在体内评价靶向ASPH的三种不同方法： ASPH的SMI抑制、SMI +多柔比星和靶向与美坦辛连接的ASPH的抗体-药物缀合物 (Aim 3）。这些目标的完成将为软骨肉瘤的生物学提供重要的见解， 确定ASPH的治疗靶向是否有潜力作为治疗以下疾病的有效策略： 这些恶性肿瘤该结果也可能具有更广泛的影响，并适用于其他类型的患者。 骨和软组织肉瘤。

PROJECT SUMMARY/ABSTRACT The long-term objective of this application is to improve the survival and quality of life for patients with chondrosarcoma, a primary malignant bone tumor desperately in need of more effective treatments. The five- year survival for chondrosarcoma, the most common bone sarcoma in adults, is an abysmal 10-25%, with most patients succumbing to pulmonary metastases. Conventional cytotoxic chemotherapy has no effect on chondrosarcoma, so the current standard of care is surgical resection. There has been no improvement in the cure rate for chondrosarcoma in the last several decades. The goal of this proposal is to systematically evaluate promising new treatment strategies that target aspartate β-hydroxylase (ASPH), a transmembrane protein that is normally expressed during embryonic development, but not after birth. We have found that ASPH is re- expressed in chondrosarcoma. In some carcinomas, ASPH signals through the Notch pathway to influence the expression of matrix metalloproteinases (MMPs) and vascular endothelial growth factor (VEGF), which support cell proliferation, invasion, and metastasis. We have data suggesting that ASPH functions similarly in chondrosarcoma. ASPH is highly expressed in >90% of chondrosarcomas, as are activated Notch and multiple MMPs, and high ASPH expression correlates positively with tumor grade and poor long-term survival. Inhibition of Notch is a long sought after but, as of yet, unachieved goal in cancer treatment. The work in this proposal will advance ASPH as a potential therapeutic target by determining the relationship between ASPH and Notch in chondrosarcoma. First, ASPH levels in human chondrosarcoma cell lines will be modulated using CRISPR-Cas9 gene editing, followed by treatment with a proven small molecule inhibitor (SMI) of ASPH. Changes in proliferation, invasion, apoptosis, and Notch signaling will be used as mechanistic readouts of ASPH signaling in vitro. Notch signaling will be manipulated in these ASPH-modulated cell lines with gain and loss of function approaches to evaluate the role of Notch in ASPH signaling (Aim 1). Second, the role of ASPH and Notch in chemotherapy resistance will be evaluated in human chondrosarcoma cell lines, and the ability of the SMI and an ASPH-targeted antibody-drug conjugate (ADC) to enhance doxorubicin efficacy will be tested (Aim 2). Finally, three different approaches to targeting ASPH will be evaluated in vivo using mouse chondrosarcoma models: SMI inhibition of ASPH, SMI + doxorubicin, and an antibody-drug conjugate targeting ASPH linked to emtansine (Aim 3). The completion of these aims will provide important insight into the biology of chondrosarcoma and determine whether the therapeutic targeting of ASPH has potential as an effective strategy for the treatment of these malignancies. The results may also have wider impact and be applicable to patients with other types of bone and soft tissue sarcomas.