Biological Implications and Translational Applications of HDMX Inhibition

HDMX抑制项目涵盖了广泛的研究努力，其根源是两年前发表的工作(见Beral等人)。癌细胞2010，18,411)。在那里，我们已经证明了SAH-P53-8，一种基于P53反式激活结构域的碳氢化合物结合的多肽，能够在过度表达Hdm2或HDMX的细胞中重新激活P53途径。虽然这一发现具有重要意义，因为SAH-P53-8是迄今为止唯一能够破坏P53-Hdm2和P53-HDMX蛋白质复合体的化合物，这一能力使我们能够阐明一个机制框架，用于确定哪些癌细胞将对单一药物Hdm2或HDMX的抑制敏感，并通过协同靶向Hdm2和HDMX来克服耐药细胞中的P53抑制。在这项工作发表后不久，我们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。马林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素。马林博士的研究确定，黑素细胞特异性的HDMX过度表达与RAS基因突变协同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P53，三分之二的黑色素瘤过表达HDMX。我们证明了我们在之前的工作中发现的分子蓝图确实适用于黑色素瘤。这些结果直接转化为P53转录研究和海洋实验室研究的黑色素瘤动物模型。从这项工作中更有意义的发现是，HDMX直接导致转移性黑色素瘤对传统化疗药物如顺铂、马法兰和达卡巴津的耐药性。为了让传统化疗药物发挥作用，一个功能齐全的p53信号系统是诱导细胞凋亡所必需的；然而，HDMX的隔离作用阻止了p53的功能，从而增加了癌细胞对细胞毒药的耐药性。我们已经证明，用SAH-P53-8抑制HDMX可以使黑色素瘤细胞对DNA损伤剂治疗敏感，这可能为转移性黑色素瘤的治疗提供了另一条途径。这项工作最近出版了(见格布斯卡等人)。《自然医学》，2012，18,1239)。通过合作，针对其他癌症的类似研究也在进行中。我们一直在与荷兰莱顿大学的Aart Johemsen博士团队合作，研究HDMX抑制在葡萄膜黑色素瘤研究和治疗中的适用性。这部作品最近被提交出版。在将我们的研究扩展到更多翻译应用的过程中，我们已经扩展了我们的研究努力，以反映我们发现的适用性。鉴于新发现的HDMX抑制在耐药背景下的重要性，我们与国家先进翻译科学中心(NCATS)合作启动了一项计划，进行高通量荧光偏振筛选，以寻找选择性的p53-HDMX相互作用抑制剂。我们目前正在验证高通量分析，同时在上海工商大学蛋白质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.