Novel approaches to enhance tumor cell cytotoxicity of alkylating agents

描述（由申请人提供）：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。烷基化剂替莫唑胺（TMZ）治疗胶质母细胞瘤的成功率有限。然而，与许多烷基化剂一样，耐药限制了其疗效。碱基切除修复（BER）途径通过修复超过80%的TMZ诱导的碱基病变，提供了对TMZ的显著抗性。因此，我们可以合理地预期，BER对TMZ提供了相当程度的抗性，因此可以通过阻断或中断修复从而促进BER失效来增强TMZ功效。该项目的总体目标是利用策略来规避对TMZ的抗性，以增强该烷基化剂的细胞毒性和潜在功效。DNA聚合酶（Polss）作为BER中的限速酶，对促进DNA损伤后的细胞修复和维持细胞存活具有重要意义。因此，抑制Polss会增强TMZ反应。具体来说，我们将描述一种新的Polss（赖氨酸二甲基化）调节机制，该机制可以通过增强细胞毒性BER中间体的积累来抑制Polss并增强烷基化剂诱导的细胞死亡（Aim 1）。我们假设Polss抑制或BER失效信号是通过聚（ADP）核糖（PAR）合成和NAD+/ATP消耗来传递的，这一过程需要激活PARP1和PARP2，并由PARG酶调节。我们发现，由于PARP1/PARP2 BER传感器复合物介导的PAR合成升高导致能量（NAD+和ATP）耗竭，导致BER失败诱导的细胞死亡，这表明对TMZ的反应可以通过增加PAR合成或消耗细胞NAD+合成（Aim 2）和/或解除BER酶PARG （Aim 3）来增强。总之，我们将验证BER途径是TMZ耐药的决定因素的假设，因此选择性靶向BER途径将显著提高TMZ的疗效。在我们的三个具体目标中，我们的目标是识别和功能表征BER途径的关键要素，这些要素控制细胞对烷基化剂的反应，目的是增加tmz诱导的胶质瘤细胞毒性。相关性：拟议的研究将提供对烷基化诱导的DNA损伤和细胞死亡反应的新生物标志物，并提供可用于增强反应的机制的见解。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，它们可以与烷基化剂结合以提高抗癌药物的疗效。

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.