Dual Targeting of DNA Repair and p53 pathways for treatment of brain cancer

描述（由申请人提供）：开发治疗多形性胶质母细胞瘤（GBM）的有效策略仍然是儿科和成人患者面临的重大挑战。一些改进，如放疗和替莫唑胺（TMZ）治疗，已经提高了生存率。然而，脑肿瘤患者的预后仍然很差，这主要是由于这些恶性肿瘤通过调节p53调节的控制细胞存活的信号通路获得化疗耐药的能力。我们的长期目标是开发治疗策略，使药物和放射耐药脑肿瘤对治疗敏感。在本研究中，我们将研究一种针对HDM2/p53网络和DNA修复的新型联合疗法的疗效。通过小分子抑制剂nutlin3抑制HDM2与关键信号分子p53、p73a和hif1a的相互作用，可以调节它们的下游效应功能。根据所研究的细胞类型，暴露于HDM2拮抗剂可导致细胞周期阻滞、衰老、凋亡、迁移减少和VEGF产生的衰减。TMZ和辐射与nutlin3联合在多大程度上可以调节这些关键的细胞内靶点还没有研究。我们的数据表明，nutlin3可以显著增强TMZ和辐射介导的胶质母细胞瘤细胞的体外毒性。此外，nutlin3还在异位异种移植模型中增强tmz介导的胶质母细胞瘤细胞杀伤。我们的总体目标是开发有效的治疗策略，杀死脑肿瘤细胞而不是正常细胞。为了进行药物疗效研究，将在NOD/SCID/IL2Rnull小鼠中建立异位和正位胶质母细胞瘤。将使用一组已建立的胶质母细胞瘤细胞系和早期传代胶质母细胞瘤原代培养物，它们在EGFR基因扩增、p53状态（野生型或突变型）、HDM2状态、MGMT表达以及对TMZ和辐照的敏感性方面存在差异。实时生物发光成像将用于连续监测胶质母细胞瘤随时间的进展。我们的中心假设是，nutlin3通过干扰hdm2介导的关键信号分子的调节，增强了TMZ和/或辐射诱导的DNA损伤反应，并导致体内胶质母细胞瘤细胞死亡增加。为了验证这一假设，提出了以下具体目标：1)制定治疗方案并验证暴露于DNA损伤剂时通过抑制hdm2蛋白相互作用介导的细胞内靶标调节；2)在体内评估调节hdm2依赖性信号以提高TMZ和/或辐射介导的DNA损伤的治疗效果的结果。3)采用颅内GBM异种移植模型结合连续实时生物发光成像，监测调节hdm2依赖性信号与TMZ和/或辐射介导的DNA损伤联合的治疗效果。本文研究的治疗策略将使用临床相关的体内模型和新的多靶向方法，并有可能提高GBM患者的治疗效果和生活质量。

DESCRIPTION (provided by applicant): Development of efficacious strategies for treatment of glioblastoma multiforme (GBM) remains a significant challenge in both pediatric and adult patients. Some improvements, such as treatment with radiation and temozolomide (TMZ), have led to increased survival. However, the prognosis for brain tumor patients remains poor, and is largely due to the ability of these malignancies to acquire chemoresistance by modulation of p53-regulated signaling pathways that control cell survival. Our long-term goal is to develop therapeutic strategies that sensitize drug- and radiation-resistant brain tumors to therapy. In this proposal, we will investigate the efficacy of a novel combination therapy that targets the HDM2/p53 network and DNA repair. Inhibition of HDM2 interactions with key signaling molecules-p53, p73a, and HIF1a-by the small molecule inhibitor, nutlin3, can modulate their downstream effector function. Depending on the cell type studied, exposure to the HDM2 antagonist can lead to cell cycle arrest, senescence, apoptosis, decreased migration, and attenuation of VEGF production. To what extent TMZ and radiation in combination with nutlin3 can modulate these critical intracellular targets has not been studied. Our data indicate that nutlin3 can significantly potentiate TMZ- and radiation-mediated cytotoxicity in glioblastoma cells in vitro. In addition, nutlin3 also enhanced TMZ-mediated glioblastoma cell kill in an ectopic xenograft model. Our overall objective is to develop efficacious treatment strategies that kill brain tumor cells but not normal cells. For drug efficacy studies, ectopic and orthotopic glioblastomas will be established in NOD/SCID/IL2Rnull mice. A panel of established glioblastoma cell lines and early passaged glioblastoma primary cultures that differ in EGFR gene amplification, p53 status (wild-type or mutant), HDM2 status, MGMT expression, and sensitivities to TMZ and irradiation will be utilized. Real-time bioluminescence imaging will be utilized to serially monitor glioblastoma progression over time. Our central hypothesis is that nutlin3 potentiates the TMZ- and/or radiation-induced DNA damage response by perturbing HDM2-mediated regulation of key signaling molecules, and leads to increased glioblastoma cell death in vivo. To test this hypothesis, the following specific aims are proposed: 1) Develop therapeutic regimens and validate intracellular target modulation mediated by inhibition of HDM2-protein interactions during exposure to DNA-damaging agents 2) Assess in vivo the outcome of modulating HDM2-dependent signaling to increase therapeutic efficacy of TMZ- and/or radiation- mediated DNA damage. 3) Employ intracranial GBM xenograft models in combination with serial real- time bioluminescence imaging to monitor therapeutic impact of modulating HDM2-dependent signaling in combination with TMZ- and/or radiation-mediated DNA damage. The treatment strategies investigated here will use clinically relevant in vivo models and novel multi-targeting approaches and have the potential to improve treatment efficacy and quality of life for patients with GBM. PUBLIC HEALTH RELEVANCE: The central hypothesis is that the HDM2 antagonist, nutlin3, potentiates TMZ and/or radiation-induced DNA damage response by perturbing HDM2-mediated regulation of key signaling molecules and leads to increased glioblastoma cell death in vivo. The underlying mechanisms of glioblastoma cell kill will be determined and ectopic and orthotopic brain tumor models will be used to evaluate in vivo efficacy.