Studies on thymidylate synthase as a tumor-promoting oncogene for development of new allosteric inhibitors for cancer treatment

 描述（由申请人提供）：在广泛的人类癌症中观察到胸苷酸合酶（TS）的过表达，其中其与晚期疾病和对市售TS抑制剂的耐药性相关。我的实验室证明，TS的激活表达，DNA合成和修复的必需酶，在促进肿瘤发生中起着直接的作用。这是一个意想不到的观察结果，因为之前认为TS水平升高反映了肿瘤细胞生长的被动生物标志物。我们的观察改变了TS作为生物标志物的范式，对癌症治疗具有重要意义，因为它重新关注TS作为肿瘤促进信号的重要性。虽然我们发表的数据表明TS在促进肿瘤发生中起着积极的作用，但问题仍然存在，TS升高是否与其他常见的遗传病变合作，以增强肿瘤的过程。为了解决这个问题，我们开发了一种新的基因工程小鼠模型（GEMM）与Ink 4a/Arf-/-突变和过表达的人TS（指定hTS/Ink 4a/Arf-/-）。我们首次发现Ink 4a/Arf基因敲除小鼠的TS激活与肿瘤生长加速、肿瘤体积增大和疾病进展阶段相关。我们还发现，活化的TS促进先前建立的肿瘤的生长和转移，而不是诱导新的肿瘤谱的发展。Ink 4a/Arf是人类肿瘤中最常见的突变位点之一，其产物编码G1期阻滞所需的肿瘤抑制蛋白。该提议的目的是：i）确定TS过表达如何促进hTS/Ink 4a/Arf-/-小鼠中的致瘤性。我们假设TS过表达通过解除复制因子和核苷酸库比率的调节来驱动DNA复制应激，从而导致在缺乏INK 4a/ARF检查点的情况下双链断裂和基因组不稳定。ii）开发新的有效的TS变构抑制剂类似物。我们发现了一种新的策略，利用高TS亚基协同性来鉴定TS的小分子抑制剂。靶向TS二聚体界面与变构抑制剂“过度稳定”二聚体结构，导致亚基协同性丧失和TS抑制 酶的功能我们将确定有效的衍生物，以防止或逆转限制当前治疗有效性的耐药性。我们的新的TS变构抑制剂不引起TS过表达，因此这些新的小分子可以克服TS过表达相关的抗性的问题，和iii）在我们的具有活化的hTS的新GEMM中测试变构抑制剂单独和与常规TS抑制剂组合的抗肿瘤活性。总之，该研究项目提出了一种控制TS介导的肿瘤发生的新方法，其目标是减少耐药性并优化癌症治疗中的TS抑制。确定TS过度表达如何促进致瘤性和新型TS抑制剂的开发将为靶向TS进行治疗干预和癌症治疗提供新的焦点。

 DESCRIPTION (provided by applicant): Overexpression of thymidylate synthase (TS) is observed in a wide range of human cancers where it is associated with advanced disease and resistance to commercially available TS inhibitors. My Lab demonstrated that activated expression of TS, an essential enzyme for DNA synthesis and repair, plays a direct role in promoting tumorigenesis. This was an unexpected observation as elevated TS levels were previously believed to reflect a passive biomarker for tumor cell growth. Our observation changed the paradigm of TS as a biomarker and has great significance for cancer treatment as it refocuses attention on the importance of TS as a tumor-promoting signal. While our published data proposed that TS plays an active role in promoting tumorigenesis, questions remain whether elevated TS cooperates with other common genetic lesions to enhance the neoplastic process. To address this question we developed a new genetically engineered mouse model (GEMM) with Ink4a/Arf-/- mutation and overexpression of human TS (designated hTS/Ink4a/Arf-/-). We showed for the first time that TS activation in Ink4a/Arf null mice is associated with accelerated tumor growth, larger tumor size and more advanced stage of the disease. We also showed that activated TS promote the growth and metastasis of previously established tumors rather than inducing development of a new tumor spectrum. Ink4a/Arf is one of the most commonly mutated loci in human cancer and its products encode tumor suppressor proteins required for G1 arrest. The objectives of this proposal are: i) to determine how TS overexpression promotes tumorigenicity in hTS/Ink4a/Arf-/-mice. We hypothesize that TS overexpression drives DNA replication stress by deregulating replication factors and nucleotide pool ratios that result in double strands break and genomic instability in the absence of INK4a/ARF checkpoints. ii) to develop new potent TS allosteric inhibitor analogues. We have discovered a new strategy that takes advantage of high TS subunit cooperativity to identify small molecule inhibitors of TS. Targeting TS dimer interface with allosteric inhibitors "overstabilize" the dimer structure resulting in loss of subunit cooperativity and inhibition of TS enzymatic function. We will identify potent derivatives to prevent or reverse drug resistance that limits the effectiveness of current therapy. Our new TS allosteric inhibitors do not cause TS overexpression thus these new small molecules may overcome the problem of TS overexpression associated resistance and iii) to test anti-tumor activity of allosteric inhibitors alone and in combination with conventional TS inhibitors in our new GEMM with activated hTS. In summary, this research project proposes a new approach to control TS mediated tumorigenesis with the goal to reduce drug resistance and optimize TS inhibition in cancer treatment. Determination of how TS overexpression promotes tumorigenicity and the development of novel TS inhibitors will provide a new focus toward targeting TS for therapeutic intervention and for cancer treatment.