Ubiquitin-Proteolytic Control of HOXA9 in Leukemogenesis

描述（由申请人提供）：HOX同源结构域（HD）蛋白是造血的关键调节因子。涉及某些HOX蛋白（如HOXA9）的异常表达或染色体易位与急性髓性白血病（AML）的发病机制有关。我们对造血过程中细胞内HOX蛋白水平和活性如何被控制的理解目前仅限于转录调控和信号转导。关于调控HOX造血调节因子丰度的翻译后控制机制以及这些调节的功能意义，我们知之甚少。这项研究的长期目标是了解泛素依赖蛋白降解如何调节正常和恶性造血。该应用的中心假设是cullin 4A （CUL-4A）泛素连接酶通过靶向降解关键的造血调节因子来控制造血发育。这一假设是在强有力的初步数据基础上提出的，这些数据表明CUL-4A靶向HOXA9降解，并调节髓细胞分化和成熟。小鼠造血特异性敲除CUL-4A基因导致骨髓祖细胞和外周血白细胞扩增增加。本研究旨在确定CUL-4A依赖性蛋白水解控制HOXA9和染色体易位衍生的NUP98-HOXA9融合的生化机制，并阐明CUL-4A在抑制白血病转化中的功能意义。我们有独特的准备来进行拟议的研究，因为我们最近产生了一个抗CUL-4A的HOXA9突变体，并开发了条件CUL-4A敲除小鼠和CUL-4A siRNA来消除或调节CUL-4A活性。我们还优化了基于慢病毒和逆转录病毒的基因传递系统，用于初级造血干细胞和祖细胞的有效转导。我们建议将周鹏波博士实验室的生化和分子遗传学方法与马尔科姆·摩尔博士实验室在体外和体内造血分析方面的专业知识结合起来，以实现以下具体目标：(1)确定cul - 4a依赖性泛素化和HOXA9降解的生化机制。(2)阐明CUL-4A降解HOXA9在AML发病机制中的功能意义。(3)通过白血病性NUP98- hoxa9融合确定CUL-4A耐药的分子基础，并评估CUL-4A消融对NUP98- hoxa9诱导白血病小鼠模型的影响。由于关于蛋白质降解在白血病发生中的作用的信息很少，因此成功完成这一提议将代表着理解一种新的控制关键造血调节因子功能的翻译后机制的重大进展，并为未来正常和恶性造血中靶向蛋白质降解的研究提供框架。

DESCRIPTION (provided by applicant): The HOX homeodomain (HD) proteins are key regulators of hematopoiesis. Aberrant expression or chromosomal translocations involving certain HOX proteins, such as HOXA9, have been implicated in the pathogenesis of acute myeloid leukemia (AML). Our understanding of how the intracellular levels and activities of the HOX proteins are controlled during hematopoiesis is current limited to transcriptional regulation and signal transduction. Little is known about the posttranslational control mechanisms that govern the abundance of the HOX hematopoietic regulators and the functional significance for such regulations. The long-term goal of this study is to understand how ubiquitin-dependent protein degradation regulates normal and malignant hematopoiesis. The central hypothesis of the application is that the cullin 4A (CUL-4A) ubiquitin-ligase controls hematopoietic development through targeted degradation of key hematopoietic regulators. The hypothesis has been formulated on the basis of strong preliminary data, which demonstrated that CUL-4A targets HOXA9 for degradation, and regulates myeloid differentiation and maturation. Hematopoietic-specific knockout of the CUL-4A gene in mice led to an increased expansion of bone marrow progenitor cells and peripheral blood leukocytes. This proposal seeks to determine the biochemical mechanisms underlying the CUL-4A-dependent proteolytic control of HOXA9 and the chromosomal translocation-derived NUP98-HOXA9 fusion, and to elucidate the functional significance of CUL-4A in suppressing leukemic transformation. We are uniquely prepared to undertake the proposed research, since we have recently generated a CUL-4A-resistant HOXA9 mutant, and developed conditional CUL-4A knockout mice and CUL-4A siRNA to eliminate or modulate CUL-4A activity. We have also optimized lentiviral- and retroviral-based gene delivery systems for efficient transduction in primary hematopoietic stem and progenitor cells. We propose to combine the biochemical and molecular genetic approaches in Dr. Pengbo Zhou's lab and the expertise in ex vivo and in vivo hematopoietic analysis in Dr. Malcolm Moore's lab to address the following specific aims: (1) to define the biochemical mechanisms underlying CUL-4A-dependent ubiquitination and degradation of HOXA9. (2) to elucidate the functional significance of HOXA9 degradation by CUL-4A in the pathogenesis of AML. (3) to determine the molecular basis for CUL-4A resistance by the leukemogenic NUP98-HOXA9 fusion and to assess the impact of CUL-4A ablation in the mouse model of NUP98- HOXA9-induced leukemia. Since little information is available regarding the roles of protein degradation during leukemogenesis, successful completion of this proposal will represent a significant advance in understanding a novel posttranslational mechanism that governs the functions of key hematopoietic regulators, and provide a framework for future investigations of targeted protein degradation in normal and malignant hematopoiesis.