Molecular mechanisms of GBM radioresistance and strategies for radiosensitization

描述(申请人提供)：多形性胶质母细胞瘤(GBM)肿瘤是30-50岁成年人癌症相关死亡的第三大原因，尽管它们在美国每年报告的所有新癌症病例中所占比例不到1.5%。尽管积极的治疗包括手术切除、放射治疗和化疗，但在过去的40年里，非常高的GBM死亡率(两年时为90%)相对保持不变。然而，通过随机试验确定了切除后放疗的明显生存优势，显示在接受了接近大脑最大耐受剂量的电离辐射后，GBM患者的中位生存期从大约6个月提高到10-12个月。具有里程碑意义的Stupp研究也说明了辐射在治疗GBM中所起的核心作用，该研究表明，同时进行辐射和替莫唑胺可以进一步将中位生存期从12.1个月提高到14.6个月。因此，放射治疗仍然是GBM治疗的主要手段，Stupp研究的成功，虽然不大，但提供了进一步改善GBM放射治疗的希望。治疗上的任何改进都需要对GBM辐射抵抗的基础有更多的机械性理解。GBM治疗缺乏改善的部分原因是缺乏可用于描述GBM中发生的遗传变化对辐射抗性的影响的适当遗传模型。最近由癌症基因组图谱网络绘制的GBM基因组图谱显示，这些肿瘤从根本上改变了基因组，发生了许多突变，基因拷贝数增加和丢失，甲基化改变。在存在于GBM基因组版图中的无数基因改变中，有5个关键的基因改变占主导地位：Ink4a、Arf、P53或PTEN的丢失以及EGFR的扩增(特别是成分活性的EGFRvIII)。目前尚不清楚这些关键的遗传变异中的哪一种可能导致治疗耐药。了解这些病变单独或联合对基底膜辐射抵抗的贡献及其潜在机制(S)将对开发更有效的治疗方法至关重要。为此，我们计划使用条件性基因敲除小鼠模型，在该模型中，这些关键的遗传损伤可以通过CRE-ERT2系统引入培养中的星形胶质细胞和神经干细胞(NSCs)和小鼠的大脑。我们建议研究这些基因变化如何影响辐射诱导的DNA双链断裂(DSB)修复机制。根据我们的初步结果，我们假设EGFRvIII-PI3K-Akt轴和DNA修复酶DNA-PKcs之间的串扰是GBM辐射抵抗的基础，这种联系可以作为有效放射治疗的靶点。具体地说，我们建议：1.确认GBM辐射抗性是由关键的GBM特异性遗传损伤之间的相互作用授予的。2.验证Akt对DNA-PKcs的磷酸化促进辐射诱导的DNA损伤修复。3.检测Akt-DNA-PKcs是否是可靶向提高GBMS放射治疗效果的易损结节。

DESCRIPTION (provided by applicant): Glioblastoma Multiforme (GBM) tumors are the third leading cause of cancer-related death among adults aged 30 - 50 years, even though they account for less than 1.5% of all new cancer cases reported in the United States each year. The very high mortality rate of GBMs (>90% at 2 years) has remained relatively unchanged over the past 40 years despite aggressive therapy that includes surgical resection, radiotherapy and chemotherapy. However, a clear survival advantage of post-resection radiation has been established by randomized trials, showing that the median survival of GBM patients was improved, from approximately 6 months to 10-12 months, following near-maximal brain-tolerated doses of ionizing radiation. The central role that radiation plays in treating GBM is also illustrated by the landmark Stupp study showing that concurrent radiation and Temozolomide can further increase median survival from 12.1 to 14.6 months. Thus, radiation remains the mainstay of GBM therapy, and the success of the Stupp study, though modest, offers hope that radiotherapy of GBMs can be further improved. Any improvement in therapy would require a more mechanistic understanding of the basis of GBM radioresistance. The lack of improvement in GBM treatment is partly due to a paucity of appropriate genetic models that can be used to delineate the effects of genetic changes that occur in GBMs on radioresistance. The recent mapping of the GBM genome by the Cancer Genome Atlas Network revealed that these tumors have radically altered genomes with many mutations, gene copy number gains and losses, and methylation changes. Amongst the myriad genetic alterations that populate the GBM genomic landscape, 5 key genetic alterations dominate: loss of Ink4a, Arf, p53, or PTEN and amplification of EGFR (especially, the constitutively active EGFRvIII). Which of these key genetic aberrations may confer therapeutic resistance remains unclear. Understanding the contribution of these lesions, singly and in combination, to GBM radioresistance along with the underlying mechanism(s) will be of paramount importance in developing more effective therapeutic modalities. Towards this goal, we plan to use conditional knock out mouse models wherein these key genetic lesions can be introduced in astrocytes and neural stem cells (NSCs) both in culture and in the mouse brain using the Cre-ERT2 system. We propose to examine how these genetic changes impact on mechanisms for the repair of radiation-induced DNA double-strand breaks (DSBs). Based upon our preliminary results, we hypothesize that cross talk between the EGFRvIII-PI3K-Akt axis and the DNA repair enzyme, DNA-PKcs, underlies the radioresistance of GBMs and that this connection can be targeted for effective radiotherapy. Specifically, we propose: 1. To confirm that GBM radioresistance is conferred by interactions between key GBM-specific genetic lesions. 2. To validate that phosphorylation of DNA-PKcs by Akt promotes the repair of radiation-induced DNA breaks. 3. To test whether Akt-DNA-PKcs is a vulnerable node that can be targeted to improve radiotherapy of GBMs.