Analysis of APL-Fusion Retinoic Acid Receptor Proteins

描述(申请人提供)：急性早幼粒细胞白血病(APL)的特征是阻止在早幼粒细胞阶段的恶性细胞选择性扩张，携带特定的染色体易位t(15；17)(q22；q21)，以及对全反式维甲酸(ATRA)治疗的有效临床反应。除了产生PML-RARpha融合蛋白的t(15；17)外，在APL患者中还发现了其他四种染色体易位：t(11；17)、t(5；17)、t(11；17)和del(17)，它们分别产生PLZF-RARpha、NPM-RARpha、NUMA-RARpha和STAT5b-RARpha融合蛋白。具有PML-RARpha、NPM-RARpha或NUMA-RARpha融合蛋白的APL疾病对ATRA敏感，而具有PLZF-RARpha或STATSb-RARpha融合蛋白的APL疾病对ATRA耐药。大量证据表明，这些RARpha融合受体通过干扰维甲酸受体(RARs)的功能而导致白血病。转基因小鼠研究表明，XRARpha(其中X是可选的RARpha融合伙伴之一：PML、PLZF、NPM、NUMA或STATSb)在APL的发病机制中发挥关键作用。然而，X-RARpha融合蛋白是如何导致白血病的，以及对ATRA的反应的分子和细胞基础还不完全清楚。RARpha基因参与了每一种APL易位，这一事实表明正常的RARpha功能的破坏在疾病的发病机制中是至关重要的。我们和其他人已经证明，每个X-RARpha融合蛋白都与RXRpha结合，RXRpha是RARpha的异源二聚伙伴，对其转录活性是必不可少的，以及核受体(NR)辅助调节因子，如辅活化子(CoA)和辅阻遏子(COR)。利用CFP标记的构建物和FRAP技术，我们证明了两种含有RARpha的融合蛋白PML-RARpha和NUMA-RARpha具有不同于RARpha的核定位模式和核迁移能力，这改变了RXRpha的核定位和迁移能力。这些发现支持我们的假设，即与野生型RARpha相比，APL融合蛋白的核定位和迁移率的变化以及X-RARpha核受体共调节因子的核定位和迁移率的变化参与了APL的发病。转录动力学的实时可视化将在三个特定目标中进行探索：目的利用共聚焦荧光显微镜和FRAP分析，进一步表征未连接或连接的X-RARpha融合蛋白与野生型RARpha在核内组织、动力学以及与其他辅助调节因子相互作用方面的差异。目的II将确定未连接或连接的XRARpha蛋白与野生型RARpha蛋白对染色质结构的影响，以及X-RARpha蛋白与染色质修饰辅助调节因子在一个完整的Lac操作子阵列中的不同相互作用。目的III将利用含有驱动RFP-PTS报告的稀有整合阵列的细胞，确定在lac操控系统中确定的关键相互作用对实时基因转录的影响。这些实验将为APL的分子和细胞发病机制及其对ATRA的反应提供新的见解，并可能被证明是新APL治疗的快速通过药物筛选试验的一部分。这项提案中将建立的系统还将提供强大的工具和有用的信息，可以扩展到其他癌症研究。

DESCRIPTION (provided by applicant): Acute promyelocytic leukemia (APL) is characterized by selective expansion of malignant cells blocked at the promyelocytic stage bearing the specific chromosomal translocation t(15;17)(q22;q21) and an effective clinical response to all-trans retinoic acid (ATRA) treatment. In addition to t(15;17), which produces the fusion protein PML-RARalpha, four other variant chromosomal translocations also have been described in APL patients: t(11;17), t(5;17), t(11;17) and del(17), which produce PLZF-RARalpha, NPM-RARalpha, NuMA-RARalpha and STAT5b-RARalpha fusion proteins, respectively. APL disease with PML-RARalpha, NPM-RARalpha, or NuMA-RARalpha fusion proteins is responsive to ATRA, while APL disease with PLZF-RARalpha or STATSb-RARalpha is ATRA-resistant. A considerable amount of evidence has suggested that these RARalpha fusion receptors cause leukemias by interfering with the function of retinoic acid receptors (RARs). Transgenic mouse studies demonstrate that XRARalpha (where X is one of the alternative RARalpha fusion partners: PML, PLZF, NPM, NuMA or STATSb) plays a pivotal role in the pathogenesis of APL. However, how X-RARalpha fusion proteins cause leukemia, and the molecular and cellular basis for the response to ATRA, are not completely known. The fact that the RARalpha gene is involved in each of the APL-translocations suggests that disruption of normal RARalpha function is critical for disease pathogenesis. We and others have demonstrated that each of the X-RARalpha fusion proteins bind RXRalpha, a heterodimerization partner of RARalpha essential for its transcriptional activity, and nuclear receptor (NR) coregulators, such as coactivator (CoA) and corepressor (CoR). Using CFP-tagged constructs and FRAP technique, we demonstrated that two RARalpha-containing fusion proteins, PML-RARalpha and NuMA-RARalpha, have nuclear localization patterns and nuclear mobility distinct from RARalpha that alter the nuclear localization and mobility of RXRalpha. These findings support our hypothesis that alterations in nuclear localization and mobility of APL fusion proteins as well as changes in nuclear localization and mobility of nuclear receptor coregulators by X-RARalpha, compared to wild-type RARalpha, contribute to the pathogenesis of APL. The real-time visualization of transcription dynamics will be explored in three specific aims: Aim I will further characterize differences in intranuclear organization, dynamics and interactions with other coregulators among unliganded or liganded X-RARalpha fusion proteins compared to wild-type RARalpha using confocal fluorescent microscopy and FRAP analysis. Aim II will determine the effects of unliganded or liganded XRARalpha proteins versus wild-type RARalpha protein on chromatin structure, as well as the different interactions between X-RARalpha proteins and chromatin-modifying coregulators at an integrated lac operator array. Aim III will determine the effects of key interactions identified in the lac operator system on real time gene transcription using cells containing a RARE-integrated array that drives an RFP-pts reporter. These experiments will give new insight into the molecular and cellular pathogenesis of APL and its response to ATRA and may prove useful as part of a rapid throughput drug screening assay for new APL treatments. The systems to be established in this proposal will also provide powerful tools and useful information that could be extended to other cancer investigations.