A Strategy for Reactivating p53 in Cancer Stem Cells Using a Novel HdmX Inhibitor

描述(由申请人提供)：近年来，由于我们对驱动癌症发展的信号通路的理解取得了重大进展，肿瘤学家可供选择的治疗方法数量急剧增加。一个关键的细胞周期检查点调节因子是P53肿瘤抑制因子，它在正常细胞中受到HdmX和Hdm2的抑制。毫不奇怪，在大多数人类癌症中，由于Hdm2或HdmX的突变或过度表达，P53被灭活了。鉴于它最近被描述在抑制癌症干细胞(CSC)转录谱中的作用，这是三阴性乳腺癌(TNBC)亚型的标志，我们提出了一种旨在重新激活P53的新策略，以造福于TNBC患者。使用TNBC模型，我们的初步研究发现在CSC人群中存在高水平的HdmX蛋白。目前，DNA损伤化疗是TNBC的一线治疗方法。然而，这种方法的副作用是显著的，而且往往是剂量有限的，因为DNA损伤无法激活突变的p53或从HdmX充分释放野生型p53。此外，与其他乳腺癌亚型相比，CSC的比例增加，这使得快速复发成为一个重要问题。目前在临床试验中用于重新激活P53的最先进的分子包括Hdm2拮抗剂Nutlin-3和突变型P53重新激活化合物Prima-1。然而，Nutlin-3特异性地干扰Hdm2-P53，而不是HdmX-P53的相互作用。因此，最初对Nutlin-3的兴奋已经被观察到HdmX水平高的肿瘤对Nutlin-3治疗耐药的观察所抑制。我们认为TNBC CSC不会对Nutlin-3或其他Hdm2特异性分子产生反应。为了确定HdmX特异性抑制物来帮助对抗TNBC，我们建立了一种检测高水平HdmX表达情况下P53激活的方法，并进行了高通量筛选。我们在这里描述了一种名为CTX1的HdmX选择性抑制剂的发现。该方案的假设是HdmX是TNBC中CSC的关键调节因子，联合抑制HdmX和Hdm2将有效地杀死TNBC CSC，防止肿瘤生长。我们为当前的研究设定了两个目标。在AIM 1中，我们将确定CTX1介导的生长抑制在TNBC中的机制和有效性。我们将测试CTX1/Nutlin-3联合治疗对HdmX和Hdm2的抑制是否导致野生型P53的持续激活以及突变型P53被Prima-1重新激活。在AIM 2中，我们将使用体外产生的CSC和从原发三阴性乳腺癌分离的CSC建立异种移植模型，研究CTX1和Nutlin-3联合治疗对TNBC干细胞的抑制作用。综上所述，CTX1是一个很好的化合物，因为它表现出高抗癌活性，高特异性的HdmX，以及与Hdm2抑制剂Nutlin-3的协同作用。此外，由于TNBC患者转移疾病的风险增加，对当前化疗方案的反应较差，复发时间较短，总体存活率较低，因此他们是可以从P53-再激活联合治疗中受益最大的理想候选者，如下所述。

DESCRIPTION (provided by applicant): The number of treatment options available to oncologists has dramatically increased in recent years due to major advances in our understanding of the signaling pathways that drive cancer development. A critical cell cycle checkpoint regulator is the p53 tumor suppressor, which is kept in check in normal cells by HdmX and Hdm2. Not surprisingly, p53 is inactivated in most human cancers, either by mutation or overexpression of Hdm2 or HdmX. Given its recently described role in suppressing a cancer stem cell (CSC) transcriptional profile, which is a hallmark of triple-negative breast cancer (TNBC) subtypes, we are proposing a new strategy aimed at reactivating p53 for the benefit of TNBC patients. Using a TNBC model, our preliminary studies have identified high levels of HdmX protein in the CSC population. Currently, DNA-damaging chemotherapy is the first-line therapy for TNBC. However, the side effects from this approach are significant and often dose limiting because DNA damage is unable to activate mutant p53 or sufficiently release wild-type p53 from HdmX. Moreover, an increased proportion of CSC compared to other breast cancer subtypes makes rapid recurrence a significant issue. The most advanced molecules currently in clinical trials for reactivating p53 include the Hdm2 antagonist Nutlin-3 and the mutant p53 reactivating compound PRIMA-1. However, Nutlin-3 specifically disrupts Hdm2-p53 but not HdmX-p53 interactions. Thus, the initial excitement over Nutlin-3 has been dampened by observations that tumors harboring high levels of HdmX are resistant to Nutlin-3 treatment. We propose TNBC CSC will not respond to Nutlin-3, or other Hdm2-specific molecules. Towards identifying HdmX- specific inhibitors to help combat TNBC, we established an assay to detect p53 activation in the presence of high-level HdmX expression and performed a high-throughput screen. We describe here the discovery of a selective inhibitor of HdmX named CTX1. The hypothesis of this proposal is that HdmX is a key regulator of CSC in TNBC, and that combined suppression of HdmX and Hdm2 will efficiently kill TNBC CSC and prevent tumor growth. We have devised two aims for the current study. In AIM 1, we will determine the mechanism and efficacy of CTX1-mediated growth suppression in TNBC. We will test whether suppression of both HdmX and Hdm2 by combined CTX1/Nutlin-3 therapy results in the sustained activation of wild-type p53, as well as mutant p53 reactivated by PRIMA-1. In AIM 2, we will examine the suppression of TNBC stem cells by combination therapy involving CTX1 and Nutlin-3 using xenograft models of CSC generated in vitro and CSC isolated from primary triple-negative breast cancers. In summary, CTX1 is an excellent compound for further studies as it exhibits high anti-cancer activity, high specificity for HdmX, and a synergy with the Hdm2 inhibitor Nutlin-3. Moreover, because TNBC patients have an increased risk of metastatic disease, respond poorly to current chemotherapeutic regimens, have a short time to recurrence, and poor overall survival, they are the ideal candidates who could benefit most from combined p53-reactivation therapy, as described here.