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

 描述(申请人提供)：胸苷酸合酶(TS)的过度表达在多种人类癌症中观察到，它与晚期疾病和对商用TS抑制剂的耐药性有关。我的实验室证明，TS的激活表达在促进肿瘤发生方面起着直接作用，TS是DNA合成和修复的关键酶。这是一个意想不到的观察结果，因为升高的TS水平以前被认为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，并对癌症治疗具有重要意义，因为它重新将注意力集中在TS作为促肿瘤信号的重要性上。虽然我们发表的数据表明TS在促进肿瘤发生中发挥了积极的作用，但问题仍然存在，TS升高是否与其他常见的遗传损伤协同作用来增强肿瘤的发生过程。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)，该模型具有Ink4a/Arf-/-突变和人类TS的过表达(命名为HTS/Ink4a/Arf-/-)。我们首次表明，Ink4a/Arf基因缺失小鼠的TS激活与肿瘤生长加速、肿瘤体积更大和疾病更晚期相关。我们还表明，激活的TS促进了先前已建立的肿瘤的生长和转移，而不是诱导新的肿瘤谱的发展。Ink4a/Arf是人类癌症中最常见的突变基因之一，其产物编码G1期停滞所需的肿瘤抑制蛋白。这项提案的目的是：i)确定TS过度表达如何促进HTS/Ink4a/Arf-/-小鼠的致瘤性。我们假设，在没有INK4a/ARF检查点的情况下，TS的过度表达通过解除对复制因子和核苷酸池比率的调节，导致双链断裂和基因组不稳定，从而驱动DNA复制应激。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亚单位的协作性来识别TS的小分子抑制剂。以变构抑制剂为靶点的TS二聚体界面“过度稳定”二聚体结构，导致亚基协同作用丧失并抑制TS 酶功能。我们将确定有效的衍生物来预防或逆转限制当前治疗效果的耐药性。我们的新TS变构抑制剂不会导致TS过度表达，因此这些新的小分子可以克服TS过度表达与耐药性相关的问题，以及iii)在我们具有激活的HTS的新GEMM中，测试变构抑制剂单独和与传统TS抑制剂联合使用的抗肿瘤活性。综上所述，本研究项目提出了一种控制TS介导的肿瘤发生的新方法，目的是减少肿瘤治疗中的耐药性，优化TS的抑制作用。确定TS过度表达如何促进肿瘤发生，以及开发新型TS抑制剂，将为针对TS的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.