Novel functions of Pyruvate kinase M2 in DNA double-strand break repair

描述(由申请人提供)： 胶质母细胞瘤(GBM)患者平均只存活15个月。肿瘤对放射和其他形式的治疗的抵抗是GBM治疗的主要挑战。GBM对放射治疗(RT)耐受的机制尚不清楚，缺乏选择性地使GBM对RT敏感而不影响正常脑组织的药物。丙酮酸激酶M2(PKM2)是GBM肿瘤细胞增殖的关键酶，在肿瘤细胞中高表达，在正常脑组织中低水平表达。我们的实验室发现了一个新的信号网络，它将核PKM2功能与同源重组(HR)介导的DNA双链断裂(DSB)修复和GBM肿瘤细胞对辐射诱导的细胞毒性的抗性联系起来。此外，我们的初步数据显示，PKM2在辐射后积聚在细胞核中，并与关键的HR限速蛋白CtlP相互作用。同时，共济失调-毛细血管扩张突变(ATM)是主要的DNA损伤反应蛋白激酶，它使PKM2磷酸化，并调节辐射诱导的PKM2核聚集和PKM2抑制的HR DSB修复。因此，我们假设，除了控制胞浆糖酵解代谢外，核PKM2还响应ATM新的上游调控，通过增强CtIP导向的HR促进辐射诱导的DSB的修复，从而决定辐射后GBM细胞的命运。一系列体外和体内实验被用来验证我们的假设：目的1将确定CtIP是否是PKM2-促进受辐射的GBM细胞的HR DSB修复和存活的关键下游功能靶点。目的2将确定ATM如何调节PKM2在放射治疗后HR DSB修复和随后的GBM细胞存活中的作用。目的3在体外和体内实验中，确定靶向依赖于PKM2的核HR修复是否选择性地使GBM肿瘤细胞对DNA损伤敏感，同时避免非癌性脑细胞的损伤。

DESCRIPTION (provided by applicant): Glioblastoma (GBM) patients only survive an average of 15 months. Tumor resistance to radiation and other forms therapy is the leading challenge in GBM treatment. The mechanisms underlying GBM resistance to radiation therapy (RT) remain poorly understood and agents that selectively sensitize GBM to RT while sparing normal brain tissue are lacking. Pyruvate kinase M2 (PKM2) is the key cytoplasmic glycolytic enzyme, which is critical GBM tumor cell proliferation and expresses highly in cancer cells but minimally in normal brain tissue. Our laboratory has discovered a novel signaling network which connects the nuclear PKM2 function with homologous recombination (HR)-mediated repair of DNA double-strand breaks (DSBs) and GBM tumor cell resistance to radiation-induced cytotoxicity. Furthermore, our preliminary data revealed that PKM2 accumulates in the nucleus following irradiation and interacts with the critical HR rate-limiting protein, CtlP. Meanwhile, ataxia-telangiectasia mutated (ATM), the prime DNA-damage response protein kinase, phosphorylates PKM2 and regulates radiation-induced PKM2 nuclear accumulation and PKM2-depedent HR DSB repair. Therefore, we hypothesize that, in addition to controlling cytosolic glycolytic metabolism, nuclear PKM2 responds to novel upstream regulation by ATM to dictate the fate of irradiated GBM cells by promoting the repair of radiation-induced DSBs through enhanced CtIP-directed HR. A series of in vitro and in vivo experiments are proposed to test our hypotheses: Aim 1 will determine whether CtIP is a critical downstream functional target in PKM2-promotion of HR DSB repair and survival of irradiated GBM cells. Aim 2 will determine how ATM regulates PKM2 in HR DSB repair and subsequent GBM cell survival following radiation treatment. Aim 3 will determine whether targeting nuclear PKM2-dependent HR repair selectively sensitizes GBM tumor cells to DNA damage while sparing noncancerous brain cells in vitro and in vivo.