Mechanisms of Cytotoxicity and Radiosensitization for Antimetabolites

描述（由申请人提供）：吉西他滨是一种抗代谢物，在患者中具有广泛的实体瘤活性。此外，它还是一种强效的辐射致敏剂。在之前的资助期间，我们研究了吉西他滨对细胞毒性和放射致敏的重要代谢和修复途径。机制研究表明，由吉西他滨二磷酸介导的核糖核苷酸还原酶的抑制主要负责抑制DNA合成，而将类似物掺入DNA更有助于细胞毒性。此外，核糖核苷酸还原酶介导的脱氧核苷酸减少与放射增敏密切相关。我们测试了这样一种假设，即脱氧核苷酸的不平衡导致核苷酸错误地整合到DNA中，如果在照射前没有修复，就会导致放射致敏。结果表明，吉西他滨在DNA中产生错配，仅在放射致敏浓度下发生，并且仅在照射后持续存在。错配修复缺陷增加了DNA的错配，抑制了p53诱导的核糖核苷酸还原酶亚基p53R2，延长了脱氧核苷酸库的不平衡，吉西他滨的放射增敏作用增强了。相反，错配修复缺陷降低了吉西他滨的细胞毒性。我们现在提出，这些机制可以推广到其他产生脱氧核苷酸池改变并作为放射增敏剂的抗代谢物，如羟基脲（核糖核苷酸还原酶抑制剂）和氟脱氧尿苷、甲氨蝶呤和培美曲塞（胸苷酸合成酶抑制剂），从而产生抗代谢物放射增敏的第一个统一假设。我们还将评估shRNA介导的这些抗代谢物靶向酶抑制的细胞毒性和放射致敏性，并比较shRNA酶抑制与单独抗代谢物或电离辐射的体内抗肿瘤效果。新的数据表明，晚期发生的DNA双链断裂无法通过同源重组修复是吉西他滨放射致敏的原因。将评估产生放射致敏的DNA损伤和修复途径，其中可能包括ATM， ATR和同源重组。初步结果表明，用shRNA抑制胸苷酸合成酶或核糖核苷酸还原酶R2亚基在产生放射致敏方面至少与抗代谢物一样有效。机制研究将有助于发展抗代谢物和shrna的细胞毒性和放射致敏性的一般假设。体内动物研究将根据药物或shRNA的剂量来探索抗代谢物或shRNA的放射增敏作用，这些药物或shRNA将减少dNTPs并在肿瘤细胞DNA中产生错误结合的脱氧核苷酸。我们进一步假设，在体内抑制R2将产生更好的放射致敏性和更低的正常组织毒性。这些基于机制的研究有很大的潜力转化为临床，以改善患者的放射增敏方案。

DESCRIPTION (provided by applicant): Gemcitabine is an antimetabolite with broad solid tumor activity in patients. In addition, it is a potent radiation sensitizer. During the previous grant period, we investigated the metabolic and repair pathways important for cytotoxicity and radiosensitization with gemcitabine. Mechanistic studies demonstrated that inhibition of ribonucleotide reductase, mediated by the diphosphate of gemcitabine, was responsible primarily for inhibition of DNA synthesis, whereas incorporation of the analog into DNA contributed more to cytotoxicity. In addition, ribonucleotide reductase-mediated decrease in deoxynucleotides correlated strongly with radiosensitization. We tested the hypothesis that the imbalance in deoxynucleotides led to misincorporation of nucleotides into DNA which, if not repaired prior to irradiation, resulted in radiosensitization. Results demonstrated that gemcitabine produced mismatches in DNA, which occurred only at radiosensitizing concentrations and persisted only after irradiation. Radiosensitization with gemcitabine was enhanced by mismatch repair deficiency, which increased mismatches in DNA, and suppression of the p53-inducible ribonucleotide reductase subunit p53R2, which prolonged the deoxynucleotide pool imbalances. In contrast, mismatch repair deficiency decreased cytotoxicity with gemcitabine. We now propose that these mechanisms can be generalized to other antimetabolites that produce alterations in deoxynucleotide pools and function as radiosensitizers, such as hydroxyurea (ribonucleotide reductase inhibitor), and fluorodeoxyuridine, methotrexate and pemetrexed (thymidylate synthase inhibitors) to produce the first unifying hypothesis for antimetabolite radiosensitization. We will also evaluate cytotoxicity and radiosensitization of shRNA-mediated suppression of the enzymes targeted by these antimetabolites, and compare the in vivo antitumor efficacy of shRNA enzyme suppression with antimetabolites alone or with ionizing radiation. New data indicate that late- occurring DNA double strand breaks which are unable to be repaired by homologous recombination are responsible for radiosensitization with gemcitabine. The DNA damage and repair pathways, which may include ATM, ATR and homologous recombination, that produce radiosensitization will be evaluated. Preliminary results indicate that suppression of either thymidylate synthase or the R2 subunit of ribonucleotide reductase with shRNA is at least as effective as antimetabolites in producing radiosensitization. Mechanistic studies will aid in developing a general hypothesis for cytotoxicity and radiosensitization with the antimetabolites and shRNAs. In vivo animal studies will explore dosing antimetabolites or shRNA for radiosensitization based on the amount of drug or shRNA that will decrease dNTPs and produce misincorporated deoxynucleotides in tumor cell DNA. We further hypothesize that suppression of R2 in vivo will produce superior radiosensitization with lower normal tissue toxicity. These mechanism-based studies have high potential for translation to clinic to improve radiosensitization protocols for patients. PUBLIC HEALTH RELEVANCE: These studies propose to test the hypothesis that, for anticancer drugs which target enzymes required to supply compounds for replication of DNA (gemcitabine, fluorodeoxyuridine, hydroxyurea, methotrexate and pemetrexed), the drug-mediated imbalance in these compounds produces mistakes in DNA which are most harmful to the cancer cell when combined with radiotherapy. Furthermore, a novel approach to cancer therapy is proposed in which we will decrease the proteins that supply the required compounds for DNA replication, which we predict will result in anticancer activity alone or in combination with radiotherapy. Understanding the mechanisms responsible for the activity of these common anticancer drugs or the novel protein suppression approach will help us to optimize their use in patients, with the ultimate goal of improving tumor control while minimizing unwanted side effects in normal tissues.