THYMIDYLATE SYNTHASE IN HEAD AND NECK CANCER

本研究的目的是改进头部选择性化疗。 和颈部(HN)鳞状细胞癌(SCC)通过靶向叶酸- 依赖酶胸苷合成酶(TS)。当前的抗叶酸疗法 甲氨蝶呤(MTX；MTX)用于HNSCC，但经常对 这种二氢叶酸还原酶(DHFR)的化疗方案 使用了抑制剂，从而限制了应答率和长期 幸存者。然而，由于HNSCC的叶酸代谢很脆弱， 与MTX无关的抗叶酸可能具有更好的治疗效果。叶酸- 依赖酶TS，因为它是唯一的从头来源 用于DNA合成的胸苷核苷酸，是新的关键靶点 抗叶酸。我们建议探索细胞/生化药理学 三种新的TS导向的抑制剂在体外人HNSCC模型中的应用。这些 数据将被用来优化设计对小鼠的治疗研究 在这个互动RO1的项目2中(Y.Rustum博士)，并定义关键 应在体内监测的对这些药物的反应决定因素 (项目2)。这一目标将通过以下具体目标来实现： 1.鉴定人HNSCC细胞系以开发模型系统 研究TS抑制剂。FaDu，来自人类咽鳞状细胞癌，A253，来自 人颈部表皮样癌的细胞系将被使用，因为它们 表示对甲氨蝶呤的固有敏感性(FaDu)和阻力(A253) 短暂暴露，如临床上发生的。他们选择的另一个因素 A253和FaDu产生分化较好和分化较低的 分别在裸鼠体内发生鳞状细胞癌，因此可以使用 活体研究(项目2)。生长特性，克隆 补充剂的效率和效果将在培养过程中进行评估，如 单层和多细胞球体(MS)。 2.明确人HNSCC模型的细胞和生化药理学 和正常小鼠肠道D1694、AG331和1843U89(三种叶酸- 相关的TS抑制剂)作为单一药物。与集中度/日程相关 药物作为单药对HNSCC细胞生长的单层抑制作用 将被完全定义；克隆存活率将被衡量为 恰如其分。这些药物的作用机理将用 分离的酶(TS、叶基多谷氨酸合成酶和DHFR)，代谢物 全细胞运输/代谢和叶酸的保护和测量 确定应对措施的关键决定因素。其他参数将 根据结果进行调查。HNSCC MS中的类似研究， 将会被执行。单层和MS研究的结果将是 与体内结果相关(项目2)。具体比较研究 利用正常小鼠肠道上皮细胞进行选择性探索 这些药物的毒性。TS抑制剂和精选药物的有用组合 代理商将在续签中接受研究。 3.选择对每个TS具有获得性抗性的HNSCC模型的子系 并鉴定其耐药表型。HNSCC的子线 对药物具有抗药性的模型将在单层中使用这两种方法进行选择 持续和间歇性药物暴露，因为暴露时间表可能 影响抗性频率和/或抗性表型。 将对抗性亚系进行生化/细胞学研究 亲代细胞。HNSCC MS模型中的抗性研究将是 按照类似的协议启动。两者在体外的抗药性 模型将与体内的耐药性相关(项目2)。

The goal of this research is to improve selective chemotherapy for head and neck (HN) squamous cell carcinoma (SCC) by targeting the folate- dependent enzyme thymidylate synthase (TS). Current antifolate therapy (methotrexate; MTX) is used in HNSCC, but resistance often occurs to chemotherapy regimens in which this dihydrofolate reductase (DHFR) inhibitor is employed thus limiting both response rate and long-term survivors. However, since folate metabolism is vulnerable in HNSCC, antifolates unrelated to MTX may be therapeutically superior. The folate- dependent enzyme TS, because of its role as the only de novo source of thymidine nucleotides for DNA synthesis, is a key target for new antifolates. We propose to explore the cellular/biochemical pharmacology of three new TS-directed inhibitors in in vitro human HNSCC models. These data will be used to optimally design therapeutic studies in mice proposed in Project 2 of this Interactive RO1 (Dr. Y. Rustum) and to define crucial determinants of response to these drugs that should be monitored in vivo (Project 2). This goal will be addressed through these Specific Aims: 1. Characterize human HNSCC cell lines to develop model systems for studying TS inhibitors. FaDu, from a human pharynx SCC, and A253, from a human epidermoid carcinoma of the neck, cell lines will be used since they represent intrinsic sensitivity (FaDu) and resistance (A253) to MTX in brief exposures, such as occur clinically. Another factor in their choice is that A253 and FaDu produce well-differentiated and less differentiated squamous cell carcinomas, respectively, in nude mice and thus can be used in in vivo studies (Project 2). Growth characteristics, cloning efficiency, and effects of supplements will be assessed during culture as monolayers and as multicell spheroids (MS). 2. Define the cellular and biochemical pharmacology in human HNSCC models and normal murine intestine of D1694, AG331, and 1843U89 (three folate- related TS inhibitors) as single agents. Concentration/schedule dependence of monolayer HNSCC cell growth inhibition by the drugs as single agents will be fully defined; clonogenic survival will be measured as appropriate. The mechanism of action of the drugs will be studied using isolated enzymes (TS, folylpolyglutamate synthetase, and DHFR), metabolite protection, and measurement of whole cell transport/metabolism and folate pools to identify crucial determinants of response. Other parameters would be investigated as dictated by results. Analogous studies in HNSCC MS, would be undertaken. Results from monolayer and MS studies will be correlated with in vivo results (Project 2). Specific comparative studies using normal mouse intestinal epithelium will be used to explore selective toxicity of these drugs. Useful combinations of TS inhibitors and selected agents would be studied in a renewal. 3. Select sublines of the HNSCC models with acquired resistance to each TS inhibitor and characterize the resistance phenotype. Sublines of HNSCC models resistant to the drugs will be selected in monolayers using both continuous and intermittent drug exposure, since the exposure schedule may influence resistance frequency and/or resistance phenotype. Biochemical/cellular study of resistant sublines will be performed as for parental cells. Studies of resistance in the HNSCC MS model would be initiated following a similar protocol. Resistance in both in vitro models will be correlated with resistance in vivo (Project 2).