Biological Implications and Translational Applications of HDMX Inhibition

HDMX抑制项目包括广泛的研究努力，并且其根源在于两年前发表的工作（参见Bernal等人Cancer Cell 2010，18，411）。在那里，我们已经证明SAH-p53-8，一种基于p53反式激活结构域的碳氢化合物钉合肽，能够在过表达HDM 2或HDMX的细胞中重新激活p53通路。虽然这一发现在SAH-p53-8是迄今为止公开的唯一能够破坏p53-HDM 2和p53-HDMX蛋白复合物的化合物的背景下具有重要意义，这种能力使我们能够阐明一种机制框架，用于确定哪些癌细胞将对单一药剂HDM 2或HDMX抑制敏感，并通过HDM 2和HDMX的协同靶向克服抗性细胞中的p53抑制。HDMX。这项工作发表后不久，我们就开始与比利时鲁汶天主教大学分子癌症生物学实验室的Jean-Christophe Marine博士团队合作。Marine博士的研究需要确定导致黑素细胞恶性转化为黑色素瘤的致病因子。Marine博士的研究已经确定黑素细胞特异性HDMX过表达与Ras突变合作，促进黑色素瘤的表现。此外，他们的研究表明，95%的黑色素瘤具有野生型内源性p53，三分之二过表达HDMX。我们证明了我们在以前的工作中发现的分子蓝图确实适用于黑素瘤。结果直接转化为p53转录研究和海洋实验室研究的黑色素瘤动物模型。这项工作更重要的发现是，HDMX直接导致转移性黑色素瘤对传统化疗治疗（如顺铂、美法仑和达卡巴嗪）的耐药性。为了使传统的化学治疗剂起作用，全功能的p53信号传导系统是诱导细胞凋亡所必需的;然而，HDMX的隔离阻断了p53功能，从而赋予癌细胞对细胞毒性剂的抗性。我们已经表明，用SAH-p53-8抑制HDMX使黑色素瘤细胞对DNA损伤剂的治疗敏感，潜在地为转移性黑色素瘤的治疗提供了另外的途径。这项工作最近已发表（参见Genmbarska等人，Nature Medicine，2012，18，1239）。通过合作努力，也在进行侧重于其他癌症的类似研究。我们一直在与荷兰莱顿大学的Aart Jochemsen博士的小组合作，研究HDMX抑制对葡萄膜黑色素瘤研究和治疗的适用性。这项工作最近提交出版。在将我们的研究扩展到更多的转化应用方面，我们扩大了我们的研究努力，以反映我们发现的适用性。鉴于新发现的HDMX抑制在耐药性背景下的重要性，我们已经开始与国家转化科学推进中心（NCATS）合作开展一项计划，以进行高通量荧光偏振筛选，以找到p53-HDMX相互作用的选择性抑制剂。我们目前正在验证该检测方法的高通量，同时在SAIC蛋白表达实验室的帮助下扩大研究所需试剂的生产。

The HDMX inhibition project encompasses a broad spectrum of research endeavors and has its roots on work that has published two years ago (see Bernal, et al. Cancer Cell 2010, 18, 411). There we have demonstrated that SAH-p53-8, a hydrocarbon stapled peptide based on the transactivation domain of p53, is capable of reactivating the p53 pathway in cells that overexpress either HDM2 or HDMX. While this finding is significant in the context that SAH-p53-8 is the only compound disclosed to date that is capable of disrupting p53-HDM2 and p53-HDMX protein complexes, this capability enabled us to elucidate a mechanistic framework for determining which cancer cells will be susceptible to single agent HDM2 or HDMX inhibition and overcoming p53 suppression in a resistant cell through synergistic targeting of HDM2 and HDMX. Soon after the publication of this work, we embarked on a collaboration with the group of Dr. Jean-Christophe Marine from the Laboratory for Molecular Cancer Biology at the Catholic University of Leuven in Belgium. Dr. Marine's research entails the determination of the causative agents responsible for the malignant transformation of melanocytes into melanoma. Dr. Marine's research has determined melanocyte specific overexpression of HDMX cooperates with mutations in Ras to promote the manifestation of melanoma. Moreover, their research has shown that 95% of all melanomas have wild type endogenous p53 and two-thirds overexpress HDMX. We demonstrated that the molecular blueprint we uncovered in our previous work indeed holds for melanoma. The results translate directly to the p53 transcription studies and the melanoma animal models studied in the Marine lab. The more significant discovery from this work is that HDMX is directly responsible for the resistance of metastatic melanoma towards traditional chemotherapeutic treatments such as cisplatin, melphalan and dacarbazine. In order for traditional chemotherapeutic agents to work, a fully functional p53 signaling system is necessary to induce apoptosis; however, sequestration by HDMX blocks p53 function thereby conferring upon the cancer cell resistance to cytotoxic agents. We have shown that inhibition of HDMX with SAH-p53-8 sensitizes melanoma cells to treatment with DNA damaging agents, potentially providing an additional avenue for the treatment of metastatic melanoma. This work has been recently published (see Gembarska, et al. Nature Medicine, 2012, 18, 1239). Similar studies focusing on other cancers are also being undertaken through collaborative efforts. We have been working with the group of Dr. Aart Jochemsen at the University of Leiden in the Netherlands on the applicability of HDMX inhibition towards the study and treatment of uveal melanoma. This work was recently submitted for publication. In expanding our research to more translational applications, we have expanded our research efforts to reflect the applicability of our discoveries. Given the newly found importance of HDMX inhibition in the context of drug resistance, we have begun a program in collaboration with the National Center for Advancing Translational Sciences (NCATS) to conduct a high throughput fluorescence polarization screen to find selective inhibitors of the p53-HDMX interaction. We are currently in process of validating the assay for high throughput while scaling up the production of the necessary reagents for the study with the help of the Protein Expression Laboratory at SAIC.