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

描述(申请人提供)：癌症一直是人类疾病相关死亡的主要原因，但其主要的非手术治疗一直是化疗和放射治疗，这两种方法毒性很大，会产生严重的副作用。此外，癌症患者的存活率几乎没有改善。因此，尽管具有挑战性，但开发更有效和特定的分子靶向疗法仍然非常重要。最重要的抑癌基因p53的失活是一种与癌症高度相关的分子变化，其基因在约50%的人类癌症中发生突变，而在其余50%携带野生型TP53的癌症中，其活性或水平往往显著降低。P53的失活主要是由于其两个主要抑制子MDM2和MDMX的负反馈调节，它们在肿瘤中过度表达，形成一个复合体，介导P53泛素化和降解，并直接抑制P53的活性。SIRT1进一步促进了这种负调控，SIRT1在一些癌症中高度表达，因为这种脱乙酰基酶对P53的脱乙酰化有利于MDM2/MDMX介导的该蛋白的泛素化。因此，通过靶向SIRT1在癌症中重新激活P53可以被用来筛选小分子以开发抗癌治疗。事实上，我们最近的工作已经发现了一种名为Inauhzin(INZ)的小分子，它可以抑制SIRT1的活性，并诱导P53乙酰化、水平和活性，导致含P53的人肺癌和结肠癌细胞依赖于P53的凋亡和衰老，并抑制异种肺癌和结肠癌的生长。我们对INZ的进一步研究意外地发现了另一个靶点IMPDH2，它也在人类癌症中高度表达。我们以前的研究表明，抑制这种酶通过降低核干素(NS)的水平而导致核糖体应激，核干素是rRNA加工所必需的。与此一致，INZ还与IMPDH2结合并降低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SIRT1和IMPDH2来激活P53，从而通过依赖P53的机制消除癌细胞。我们将通过解决两个具体目标来检验这一假设。1.确定INZ是否通过抑制IMPDH2和下调NS而诱导核糖体应激和激活P53。由于我们最近报道了INZ抑制SIRT1活性，在这里，我们将通过验证INZ作为IMPDH2的特异性抑制剂来确定INZ是否针对IMPDH2，巩固INZ抑制IMPDH2是否降低NS水平并诱导随后的核糖体应激，以及确定RPL11和RPL5是否对INZ诱导的p53激活至关重要。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。为此，我们将测试我们新合成的INZ衍生物，特别是有效的INZ-14，方法是进一步对其进行修饰，在我们建立的生化、细胞和动物肿瘤模型系统中对其进行表征，并在异种移植和原位肿瘤模型系统中测试INZ-14与阿霉素或放射治疗的协同作用。

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.