Analysis of APL-Fusion Retinoic Acid Receptor Proteins

描述（由申请人提供）：急性早幼粒细胞白血病（APL）的特点是在早幼粒细胞阶段被阻断的恶性细胞选择性扩增，具有特异性染色体易位t(15;17)(q22;q21)，并且对全反式维甲酸（ATRA）治疗有有效的临床反应。除了产生pml - rar α融合蛋白的t（15;17）外，APL患者中还发现了另外四种变异染色体易位：t（11;17）、t（5;17）、t（11;17）和del(17)，它们分别产生plzf - rar α、npm - rar α、numa - rar α和stat5b - rar α融合蛋白。具有pml - rarα、npm - rarα或numa - rarα融合蛋白的APL疾病对ATRA有反应，而具有plzf - rarα或statsdb - rarα的APL疾病对ATRA有抗性。大量证据表明，这些rar α融合受体通过干扰视黄酸受体（RARs）的功能而引起白血病。转基因小鼠研究表明，xrar α（其中X是rar α可选择的融合伙伴之一：PML， PLZF， NPM， NuMA或STATSb）在APL的发病机制中起关键作用。然而，x - rarα融合蛋白是如何引起白血病的，以及对ATRA反应的分子和细胞基础尚不完全清楚。rar α基因参与每一种apl易位的事实表明，正常rar α功能的破坏对疾病的发病至关重要。我们和其他人已经证明，每个x - rrα融合蛋白都结合rxrα （rrα转录活性必需的异源二聚化伙伴）和核受体（NR）共调节因子，如辅激活因子（CoA）和辅抑制因子（CoR）。利用cfp标记的构建体和FRAP技术，我们证明了两个含有rarα的融合蛋白pml - rarα和numa - rarα具有不同于rarα的核定位模式和核迁移率，从而改变了rxrα的核定位和迁移率。这些发现支持了我们的假设，即与野生型RARalpha相比，X-RARalpha改变了APL融合蛋白的核定位和移动性，以及核受体共调节因子的核定位和移动性，这有助于APL的发病机制。转录动力学的实时可视化将在三个具体目标中进行探索：目的1将进一步表征与野生型rar α相比，未配体或配体x - rar α融合蛋白在核内组织、动力学和与其他共调节因子相互作用方面的差异。Aim II将在集成lac操作符阵列上确定未配体或配体xrar α蛋白与野生型rar α蛋白对染色质结构的影响，以及x - rar α蛋白与染色质修饰共调节因子之间的不同相互作用。Aim III将使用含有驱动RFP-pts报告基因的稀有集成阵列的细胞，确定lac操作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，并可能被证明是新的APL治疗的快速通量药物筛选试验的一部分。在本提案中建立的系统也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.