Specific Inhibition of NOTCH2 Signaling in Bladder Cancer

转移性膀胱癌（BC）患者预后较差。个体靶向治疗似乎是一种假定的策略，以扩展治疗选择，超越全身化疗和免疫治疗。Peter Black博士的研究小组发现NOTCH2 （N2）是尿路上皮BC的致癌基因，在原位异种移植小鼠模型中，使用特异性的N2靶向抗体抑制肿瘤生长和转移。此外，在BC省的Cancer Genome Atlas 2017队列中，N2 RNA过表达与生存率低相关。有趣的是，NOTCH1 （N1）信号被证明在BC中发挥肿瘤抑制作用。Black博士的团队在N2受体上发现了两个可用药的位点，这两个位点都与N1分子受体的结构明显不同。因此，我们打算开展一项旨在鉴定和评估不干扰BC中其他Notch受体的新型高选择性N2抑制剂的项目。我们将从ZINC15和ChEMBL等公开数据库中对约1000万种化合物进行基于结构的虚拟筛选，利用计算机辅助药物设计程序和化学信息学技术确定最有希望用于选择性N2抑制的小分子化合物。下一步，我们将在基于细胞的荧光素酶实验中，使用notch特异性反应质粒，通过体外筛选N2抑制来选择化合物。化合物的稳定性和代谢降解将在微粒体不稳定性测定中进行评估。N2抑制剂的抗肿瘤作用将在BC细胞系中进一步分析，利用细胞生长、细胞周期进展、迁移和侵袭体外实验。剂量参数、化合物降解、毒性以及对肿瘤生长和转移的影响将在原位BC异种移植小鼠体内模型中进行评估。重要的是，到目前为止，还没有针对Notch信号的治疗方法被批准用于患者护理。小分子N2特异性抑制剂不仅可以绕过非特异性pan-Notch抑制剂常见的副作用，还可以克服单克隆抗体的缺点，如抗体降解和低组织渗透。总之，我们的翻译方法可能会导致一种高度特异性的小分子N2抑制剂，能够为转移性BC患者提供新的治疗选择。

The prognosis of patients with metastatic bladder cancer (BC) is poor. Individual targeted therapies seem to be a putative strategy in order to extend therapeutic options beyond systemic chemo- and immunotherapy. The group of Dr. Peter Black identified NOTCH2 (N2) as an oncogene in urothelial BC and both tumor growth and metastasis were suppressed using a specific N2-targeting antibody in an orthotopic xenograft mouse model. Furthermore, N2 RNA overexpression was correlated with poor survival in The Cancer Genome Atlas 2017 cohort in BC. Intriguingly, NOTCH1 (N1) signaling was demonstrated to play a tumor-suppressive role in BC. Two druggable sites on the N2 receptor were identified by Dr. Black’s group, which were both significantly dissimilar to the N1 molecular receptor configuration. Therefore, we intend to conduct a project aimed to identify and evaluate novel highly selective N2 inhibitors that do not interfere with other Notch receptors in BC.We will perform a structure-based virtual screening with ~10 million compounds from publicly available databases such as ZINC15 and ChEMBL, utilize computer-aided drug design programs and chemoinformatic techniques to identify the most promising small molecular compounds for selective N2 inhibition. In the next step, we will select compounds by in vitro screening of N2 inhibition using a Notch-specific responsive plasmid in a cell-based luciferase assay. Compound stability and metabolic degradation will be assessed in a microsomal instability assay. Antineoplastic effects of N2 inhibitors will be further analysed in BC cell lines, utilizing cell growth, cell-cycle progression, migration and invasion in vitro assays. Dosing parameters, degradation of compounds and toxicity as well as impact on tumor growth and metastasis will be assessed in an orthotopic BC xenograft mouse model in vivo.Importantly, there are no therapies targeting Notch signaling approved for patient care so far. A small molecule N2 specific inhibitor might bypass not only common side effects seen in unspecific pan-Notch inhibitors, but also disadvantages of monoclonal antibodies like antibody degradation and low tissue penetration. Taken together, our translational approach might lead to a highly specific small molecule N2 inhibitor with the capability to offer novel therapeutic options for patients with metastatic BC.