Novel approaches to enhance tumor cell cytotoxicity of alkylating agents

描述(申请人提供)：胶质母细胞瘤是美国癌症相关死亡的主要原因，每年约有17,000例新诊断的脑癌病例。不幸的是，治疗仍然不足，死亡率很高。烷化剂替莫唑胺(TMZ)在治疗胶质母细胞瘤方面的成功有限。然而，与许多烷基化试剂一样，抗药性限制了其疗效。碱基切除修复(BER)途径通过修复超过80%的TMZ诱导的碱基损伤来提供对TMZ的显著抵抗。因此，可以合理地预期BER对TMZ提供显著水平的抵抗，因此可以通过阻塞或中断修复从而促进BER故障来获得增强的TMZ效果。这个项目的总体目标是利用策略来规避对TMZ的耐药性，以增强这种烷化剂的细胞毒性和潜在的疗效。DNA聚合酶ss(Polss)作为BER中的限速酶，在DNA损伤后促进修复和维持细胞存活方面起着重要作用。因此，抑制Polss将增强TMZ的反应。具体地说，我们将描述一种新的Polss(赖氨酸二甲基化)调节机制，该机制可用于通过促进细胞毒性BER中间体的积累来抑制Polss和增强烷化剂诱导的细胞死亡(目标1)。我们假设Polss抑制或BER故障信号是通过多聚(ADP)核糖(PAR)的合成和NAD+/ATP的耗尽而实现的，这个过程需要PARP1和PARP2的激活，并受PARG酶的调节。我们发现，BER失败诱导的细胞死亡是由于PARP1/PARP2 BER传感器复合体介导的PAR合成增加导致的能量(NAD+&ATP)耗尽，这表明对TMZ的反应可以通过增加PAR合成或耗尽细胞NAD+合成(目标2)和/或解除BER酶PARG的调节(目标3)来增强。总体而言，我们将检验BER通路是TMZ耐药性的决定因素的假设，因此选择性地靶向BER通路将显著增强TMZ的疗效。在我们的三个具体目标中，我们的目标都是识别并从功能上表征控制细胞对烷化剂反应的BER途径的关键元件，目的是增加TMZ诱导的胶质瘤细胞的细胞毒性。相关性：拟议的研究将提供应对烷基化诱导的DNA损伤和细胞死亡的新的生物标志物，并提供可用于增强反应的机制的洞察力。从这些研究中获得的见解有可能确定辅助治疗的新靶点，包括BER抑制剂和NAD+生物合成调节剂，它们可以与烷化剂结合以提高抗癌药物的疗效。 公共卫生相关性： 项目简介胶质母细胞瘤是美国癌症相关死亡的主要原因，每年约有17,000例新诊断的脑癌病例。不幸的是，治疗仍然不足，死亡率很高。烷化剂替莫唑胺(TMZ)在治疗胶质母细胞瘤方面的成功有限。然而，与许多烷基化试剂一样，活跃的DNA修复途径产生的耐药性限制了其疗效。总体而言，我们将检验碱基切除修复(BER)途径是TMZ耐药性的决定因素的假设，因此选择性地靶向关键的BER途径蛋白将显著提高TMZ的疗效。这项拟议的研究将提供应对替莫唑胺诱导的DNA损伤和细胞死亡的新的生物标志物，并提供对可用于增强反应的机制的洞察，并为辅助治疗确定新的靶点。

DESCRIPTION (provided by applicant): Glioblastoma is a major cause of cancer related death in the United States, with approximately 17,000 new cases of brain cancer diagnosed annually. Unfortunately, therapy remains inadequate and the mortality rate is high. Limited success in the treatment of glioblastoma has been demonstrated with the alkylating agent Temozolomide (TMZ). However, as with many alkylating agents, drug resistance has limited its efficacy. The base excision repair (BER) pathway provides significant resistance to TMZ by repairing greater than 80% of the TMZ-induced base lesions. As such, it is reasonable to expect that BER provides a significant level of resistance to TMZ and therefore enhanced TMZ efficacy may be obtained by blocking or interrupting repair and thereby promoting BER failure. The overall goals of this project are to utilize strategies to circumvent resistance to TMZ to enhance the cytotoxicity and potentially the efficacy of this alkylating agent. As the rate-limiting enzyme in BER, DNA polymerase ss (Polss) is important to facilitate repair and to maintain cell survival following DNA damage. Therefore, inhibition of Polss will enhance TMZ response. Specifically, we wil characterize a novel regulatory mechanism of Polss (lysine di-methylation) that can be exploited to inhibit Polss and enhance alkylating agent-induced cell death by enhancing the accumulation of cytotoxic BER intermediates (Aim 1). We posit that Polss inhibition or BER failure signals via poly(ADP)ribose (PAR) synthesis and NAD+/ATP depletion by a process that requires activation of PARP1 & PARP2 and is regulated by the enzyme PARG. We find that BER failure- induced cell death results from energy (NAD+ & ATP) depletion due to elevated PAR synthesis mediated by the PARP1/PARP2 BER sensor complex, suggesting that the response to TMZ can be enhanced via increased PAR synthesis or depletion of cellular NAD+ synthesis (Aim 2) and/or deregulation of the BER enzyme PARG (Aim 3). Overall, we will test the hypothesis that the BER pathway is a determinant of resistance to TMZ and therefore selectively targeting the BER pathway will significantly enhance TMZ efficacy. In each of our three specific aims, our goals are to identify and functionally characterize key elements of the BER pathway that control cellular responses to alkylating agents with the goal of increasing TMZ-induced cytotoxicity in cells from glioma tumors. Relevance: The proposed studies will provide new biomarkers of response to alkylation-induced DNA damage and cell death and provide insight into mechanisms that can be exploited to enhance response. Insights gained from these studies have the potential to identify novel targets for adjuvant therapies including BER inhibitors and NAD+ biosynthesis modulators that can be combined with alkylators to improve anticancer drug efficacy. PUBLIC HEALTH RELEVANCE: Project Narrative Glioblastoma is a major cause of cancer related death in the United States, with approximately 17,000 new cases of brain cancer diagnosed annually. Unfortunately, therapy remains inadequate and the mortality rate is high. Limited success in the treatment of glioblastoma has been demonstrated with the alkylating agent temozolomide (TMZ). However, as with many alkylating agents, drug resistance from active DNA repair pathways limits its efficacy. Overall, we will test the hypothesis that the base excision repair (BER) pathway is a determinant of resistance to TMZ and therefore selectively targeting critical BER pathway proteins will significantly enhance TMZ efficacy. The proposed studies will provide new biomarkers of response to temozolomide-induced DNA damage and cell death and provide insight into mechanisms that can be exploited to enhance response and to identify novel targets for adjuvant therapies.