Biology and Targeting of noncoding RNAs in AML

急性髓系白血病（AML）的预后仍然很差。因此，了解这些机制 调节AML的生物学对于开发针对该疾病的有效疗法是重要的。非随机 在50-55%的AML患者中鉴定出染色体异常。相比之下，约45-50%的AML 例细胞遗传学正常（CN-AML）。最近的工作已经确定了新的复发性基因突变CN- 急性髓细胞白血病其中，核磷蛋白（NPM 1）基因的突变，代表了最常见的遗传 CN-AML的改变。最近，一类新的非编码RNA（转录长于200个核苷酸） 长链非编码RNA（lncRNA）。虽然lncRNA有助于固体中的致癌作用， 肿瘤，它们在AML中的作用尚未得到表征。我们的研究小组最近发现了lncRNA HOXB-AS 3 在NPM 1突变（NPM 1 mut）CN-AML病例中，我们进一步表明， HOXB-AS 3敲低导致AML细胞系中原始细胞增殖和集落形成减少， 原发性AML患者体外。使用锁核酸（LNA）缺口聚物在体内沉默HOXB-AS 3导致： 相对于对照，治疗的患者来源的异种移植物（PDX）小鼠的存活率增加。比较 蛋白质组学鉴定了HOXB-AS 3的几种RNA结合蛋白伴侣，如EBP 1，它们与 与核糖体生物合成有关。进一步的实验表明，HOXB-AS 3与EBP 1结合并调节EBP 1的表达。 通过影响EBP 1和NPM 1复合物之间的相互作用，在AML中的核糖体生物发生。总之，我们的 初步数据支持我们的假设，即HOXB-AS 3在NPM 1 mut AML中起重要作用， 阻断HOXB-AS 3可能是NPM 1 mut AML的可行治疗靶点。总体目标是建议 研究HOXB-AS 3参与髓系白血病发生的机制， 如何治疗这种lncRNA我们将通过以下具体目标实现这一目标 (SA)具体目的1：阐明HOXB-AS 3促进白血病发生的机制 在AML。我们将进行体外和体内研究，以阐明HOXB-AS 3如何调节细胞增殖 2）具体目标2：研究体内药物代谢动力学 (PK)合成的纳米颗粒标记的LNA的药效学（PD）和抗白血病活性 使用AML的PDX模型针对HOXB-AS 3的gapmer。为此，我们将开展以下临床前研究： 在过表达HOXB-AS 3的AML的PDX模型中针对HOXB-AS 3的合成Tf-NP LNA缺口体， 评价：a）毒性; B）血浆PK和细胞内浓度; c）PD终点; d）PK/PD建模;和 e）功效在该项目完成后，我们将对lncRNA在以下方面的作用有更多的了解： NPM 1 mut AML，并将这些发现纳入未来的治疗策略， 成果。

The prognosis of acute myeloid leukemia (AML) is still very poor. Thus, understanding the mechanisms regulating the biology of AML is important for developing effective therapies for this disease. Non-random chromosomal abnormalities are identified in 50-55% of all AML patients. In contrast, about 45-50% of all AML cases are cytogenetically normal (CN-AML). Recent work has identified novel recurrent gene mutations in CN- AML. Among them, mutations of the nucleophosmin (NPM1) gene, represent the most common genetic alteration in CN-AML. Recently a novel class of noncoding RNAs (transcripts longer than 200 nucleotides) named long noncoding RNAs (lncRNAs) was discovered. While lncRNAs contribute to carcinogenesis in solid tumors, their role in AML has not been characterized. Our group recently identified the lncRNA HOXB-AS3 among the top up-regulated lncRNAs in NPM1 mutated (NPM1mut) CN-AML cases. We further showed that HOXB-AS3 knockdown leads to a decrease blast proliferation and colony formation in AML cell lines and primary AML patients in vitro. Silencing HOXB-AS3 in vivo using locked nucleic acid (LNA) gapmers resulted in an increased survival of treated patient derived xenograft (PDX) mice with respect to controls. Comparative proteomics identified several RNA binding protein partners of HOXB-AS3, such as EBP1, which are associated with ribosomal biogenesis. Further experiments indicated that HOXB-AS3 binds to EBP1 and regulates ribosomal biogenesis in AML by affecting the interactions between EBP1 and NPM1 complex. Altogether, our preliminary data supports our hypothesis that HOXB-AS3 plays an important role in NPM1mut AML, and blocking HOXB-AS3 may be a viable therapeutic target in NPM1mut AML. The overall goal of this is proposal is to dissect the mechanisms through which HOXB-AS3 contributes to myeloid leukemogenesis and to explore how to target therapeutically this lncRNA. We will accomplish this goal through the following Specific Aims (SA): 1) Specific Aim 1: To elucidate the mechanisms by which HOXB-AS3 promotes leukemogenesis in AML. We will perform in vitro and in vivo studies to elucidate how HOXB-AS3 modulates cell proliferation and ribosome biogenesis; 2) Specific Aim 2: To investigate in vivo the pharmacokinetic (PK), pharmacodynamics (PD) and anti-leukemic activity of a synthetic nanoparticle tagged LNA gapmer against HOXB-AS3 using PDX models of AML. In this aim we will conduct preclinical studies of synthetic Tf-NP LNA gapmer against HOXB-AS3 in PDX models of AML overexpressing HOXB-AS3 to evaluate: a) toxicity; b) plasma PK and intracellular concentrations; c) PD endpoints; d) PK/PD modeling; and e) efficacy At completion of this project, we will have an increased understanding of the role of lncRNAs in NPM1mut AML and incorporated these findings into future treatment strategies that may improve overall outcomes.