Dual Targeting of the p53 pathway for development of anti-cancer therapy

描述（由申请人提供）：癌症一直是人类疾病相关死亡的主要原因，然而，其主要的非手术治疗是化疗和放疗，这两种治疗都具有相当的毒性并引起严重的副作用。此外，癌症患者的生存率也没有多大改善。因此，尽管具有挑战性，但开发更有效和更特异的分子靶向疗法仍然非常重要。最重要的肿瘤抑制因子p53的失活是一种高度与癌症相关的分子改变，因为其基因在约50%的所有类型的人类癌症中突变，而其活性或水平通常在其余50%的携带野生型TP 53的癌症中显著降低。p53失活主要是由于其两个主要抑制因子MDM 2和MDMX的负反馈调节，这两个抑制因子在癌症中过表达并形成介导p53泛素化和降解以及直接抑制p53活性的复合物。SIRT 1在某些癌症中高度表达，进一步促进了这种负调控，因为这种脱乙酰酶对p53的脱乙酰化有利于MDM 2/MDMX介导的这种蛋白质的泛素化。因此，通过靶向SIRT 1重新激活癌症中的p53可用于筛选用于开发抗癌疗法的小分子。事实上，我们最近的工作已经确定了一种名为Inauhzin（INZ）的小分子，它抑制SIRT 1的活性并诱导p53乙酰化，水平和活性，导致p53依赖性细胞凋亡和含p53的人肺癌和结肠癌细胞衰老，并抑制异种移植肺和结肠肿瘤的生长。我们对INZ的进一步研究令人惊讶地发现了另一个靶点IMPDH 2，它在人类癌症中也高度表达。我们以前的研究表明，这种酶的抑制导致核糖体压力通过减少核干细胞（NS）的水平，这是必不可少的rRNA加工。与此一致，INZ还结合IMPDH 2并降低NS水平，导致核糖体应激-p53途径的激活。根据这些有趣的发现，我假设INZ 可以通过同时靶向SIRT 1和IMPDH 2激活p53，从而通过p53依赖性机制消除癌细胞。我们将通过两个具体目标来检验这一假设。1.确定INZ是否通过抑制IMPDH 2和下调NS诱导核糖体应激并激活p53。由于我们最近报道了INZ抑制SIRT 1活性，因此在此目的中，我们将通过验证INZ作为IMPDH 2的特异性抑制剂来确定INZ是否靶向IMPDH 2，巩固INZ对IMPDH 2的抑制是否降低NS水平并诱导随后的核糖体应激，并确定RPL 11和RPL 5是否对INZ诱导的p53活化至关重要。2.确定INZ-14（一种更有效的INZ类似物）在p53激活和肿瘤抑制中的作用。在这个目标中，我们将测试我们新合成的INZ衍生物，特别是有效的INZ-14，通过进一步修饰它，在我们建立的生物化学，细胞和动物肿瘤模型系统中表征它，并测试INZ-14与阿霉素或γ-辐射在异种移植和原位肿瘤模型系统中的协同作用。

DESCRIPTION (provided by applicant): Cancer has been the leading cause of disease-related deaths in human beings, yet, its major non-surgery treatments have been chemotherapy and radiotherapy, both of which are quite toxic and cause severe side effects. Also, the survival rates of cancer patients have had little improvement. Thus, it still remains remarkably important, though challenging, to develop more potent and specific molecule-targeted therapies. The inactivation of the most important tumor suppressor p53 is one highly cancer-related molecular alteration, as its gene is mutated in ~50% of all types of human cancers while its activity or leve is often markedly reduced in the remaining 50% of cancers that harbor wild type TP53. The p53 deactivation is primarily due to the negative feedback regulation by its two chief suppressors, MDM2 and MDMX, which are over expressed in cancers and form a complex that mediates p53 ubiquitination and degradation as well as inhibits p53 activity directly. This negative regulation s further facilitated by SIRT1, which is highly expressed in some cancers, as the deacetylation of p53 by this deacetylase favors MDM2/MDMX-mediated ubiquitination of this protein. Thus, re-activation of p53 in cancers by targeting SIRT1 can be utilized to screen small molecules for the development of an anti-cancer therapy. Indeed, our recent work has identified a small molecule named Inauhzin (INZ) that inhibits the activity of SIRT1 and induces p53 acetylation, level and activity, leading to p53-dependent apoptosis and senescence in p53-containing human lung and colon cancer cells and suppressing the growth of xenograft lung and colon tumors. Our further studying INZ surprisingly reveals another target, IMPDH2, which is also highly expressed in human cancers. Our previous study shows that inhibition of this enzyme causes ribosomal stress by reducing the level of nucleostemin (NS), which is essential for rRNA processing. Consistent with this, INZ also binds to IMPDH2 and reduces NS levels, leading to the activation of the ribosomal stress-p53 pathway. In light of these interesting findings, I hypothesize that INZ can activate p53 by simultaneously targeting SIRT1 and IMPDH2 and thus eliminate cancer cells via a p53-dependent mechanism. We will test this hypothesis by addressing two specific aims. 1. To determine if INZ induces ribosomal stress and activates p53 by inhibiting IMPDH2 and downregulating NS. Since we have recently reported that INZ inhibits SIRT1 activity, here in this aim we will determine if INZ targets IMPDH2 by verifying INZ as a specific inhibitor of IMPDH2, consolidating if inhibition of IMPDH2 by INZ reduces NS levels and induces consequent ribosomal stress, and determining if RPL11 and RPL5 are critical for INZ-induced p53 activation. 2. To determine the role of INZ-14, a more potent INZ analog, in p53 activation and tumor suppression. In this aim, we will test our newly synthesized INZ derivatives, particularly potent INZ-14, by further modifying it, characterizing it in our established biochemical, cellular and animal tumor model systems, and testing the cooperative effect of INZ-14 with doxorubicin or ¿-irradiation in xenograft and orthotopic tumor model systems.