Non-coding RNAs at the interface of aberrant NF-kB signals and lymphomagenesis

描述(由申请人提供)： 弥漫性大B细胞淋巴瘤(DLBCL)是成人最常见的淋巴系统恶性肿瘤。尽管最近取得了进展，但这些患者中仍有很大一部分死于他们的疾病。DLBCL最具侵袭性的亚型是激活的B细胞(ABC)样DLBCL，其特征是NF-B途径的结构性激活。在大约一半的病例中，核因子-βB的异常激活是由于这一途径的正负调控因子的体细胞突变所致。然而，在很大一部分核因子-B成瘾的DLBCL中，这些异常的分子基础尚不清楚。MicroRNAs(MiRNAs)是一类非蛋白质编码基因，通过与靶基因的3‘端非编码区配对，诱导RNA切割和/或翻译抑制来调节人类转录组。在DLBCL中，miRNAs经常被破坏，提示它们可能在这些肿瘤中发挥结构性激活NF-kB信号的作用。同样，来自其他生物系统的证据表明，miRNAs和NF-B途径之间存在广泛的相互作用。这一建议的首要目标是系统地检验这一新的假设，即在DLBCL的致命性亚型中，miRNAs和NF-B途径成员之间相互作用的遗传和表观遗传干扰有助于这些信号的结构性激活。更具体地说，我们将：1.鉴定并功能验证DLBCL相关miR-125a/b与miR-18a、miR-19a/b与肿瘤抑制基因之间的直接相互作用，以及负的NF-B调控因子TNFAIP3；2.鉴定并表征直接靶向阳性NF-B调控因子CARD11、MYD88、CD79B、TRAF2、TRAF5和TAK1的miRNAs；3.确定Lin28b和let-7 miRNA家族在表观遗传驱动的自我维持环中的作用，该环可以构成DLBCL中的NF-B。为了实现这项研究计划的目标，我们将广泛使用体外和体内的转基因DLBCL细胞系，以及原发淋巴瘤样本。简而言之，miRNA的表达和功能将通过逆转录病毒系统(功能增益)通过其前体序列的稳定表达来调节，或者通过“miRNA海绵”来调节，“miRNA海绵”将miRNA从内源性结合位点隔离出来，导致显著的功能敲除(功能丧失)。此外，稳定的异位表达和shRNA介导的Lin28b敲除将以互补的方式用于定义表观遗传驱动的正反馈环的存在，该环在DLBCL中保持NF-B的结构性活性。在整个研究过程中，在体外产生相关结果的模型将在具有非侵入性成像能力的人类淋巴瘤异种移植模型中得到验证。最后，这些结果的相关性将在最关键的背景下，即原发肿瘤，通过测量miRNAs、靶基因表达和核因子-B活性的特征来证实。识别当被干扰时激活核因子-B信号的miRNAs将阐明多种致癌和抑癌miRNAs的功能，并促进我们对正常和恶性淋巴细胞生物学的理解。这些发现将阐明很大一部分的分子基础 DLBCL对核因子-kB信号“上瘾”，这是一个需要解决的重要知识缺口，因为它与DLBCL的致命亚集有关。此外，由于高核因子-kB活性不是ABC-DLBCL独有的，而是与多种成熟B细胞恶性肿瘤和上皮癌的发病机制有关，我们的数据可能具有广泛的相关性，并最终使大量患者受益。最后，随着通过治疗意图抑制和恢复miRNA水平的潜力正在成为现实，我们的研究可能为在癌症中测试这一概念提供一个蓝图。

DESCRIPTION (provided by applicant): Diffuse Large B-cell lymphoma (DLBCL) is the most common lymphoid malignancy in adults. Despite recent progress, a large fraction of these patients still die of their disease. The hallmar of the most aggressive subtype of DLBCL, Activated B-cell (ABC)-like DLBCL, is constitutive activation of the NF-¿B pathway. In approximately half of these cases, aberrant NF-¿B activation results from somatic mutations in negative and positive regulators of this pathway. However, in a large proportion of NF-¿B-addicted DLBCLs the molecular basis for these abnormalities is unknown. MicroRNAs (miRNAs) are non-protein coding genes that regulate the human transcriptome by pairing to the 3'UTR of target genes, inducing RNA cleavage and/or translational inhibition. MiRNAs are frequently disrupted in DLBCL, suggesting that they may play a role in constitutively activating NF-kB signals in these tumors. Concordantly, evidence from other biological systems indicates that there is an extensive interplay between miRNAs and the NF-¿B pathway. The overarching objective of this proposal is to systematically test the novel hypothesis that genetic and epigenetic disruption of the interactions between miRNAs and members of the NF-¿B pathway contributes to the constitutive activation of these signals in the fatal subtypes of DLBCL. More specifically, we will: 1. Characterize and functionally validate the direct interaction between the DLBCL-relevant miR-125a/b and miR-18a, miR-19a/b with the tumor suppressor gene, and negative NF-¿B regulator, TNFAIP3; 2. Identify and characterize the miRNAs that directly target the positive NF-¿B regulators CARD11, MYD88, CD79B, TRAF2, TRAF5 and TAK1; 3. Define the role of the Lin28b and the let-7 miRNA family in an epigenetically-driven self-sustained loop that could constitutively activate NF-¿B in DLBCL. To achieve the goals of this research proposal, we will make extensive use of genetically modified DLBCL cell lines, in vitro and in vivo, and primary lymphoma samples. In brief, miRNA expression and function will be modulated with stable expression of their precursor sequence via a retrovirus system (gain-of-function), or with "miRNA sponges", constructs that sequester the miRNAs away from endogenous binding sites resulting in a significant functional knockdown (loss-of-function). Furthermore, stable ectopic expression and shRNA-mediated knockdown of LIN28B will be used in complementary fashion to define the presence of an epigenetically-driven positive feedback loop that maintains NF-¿B constitutive active in DLBCL. Throughout this study, the models that yield relevant result in vitro will be validated in a xenograft model o human lymphoma with non-invasive imaging capability. Finally, the relevance of these results will be confirmed in the most critical setting, primary tumors, via measurement of miRNAs, target genes expression and characterization of NF-¿B activity. The identification of miRNAs that when disrupted activate NF-¿B signals will clarify the function of multiple oncogenic and tumor suppressive miRNAs and advance our understanding of normal and malignant lymphocyte biology. These findings will elucidate the molecular basis for a substantial fraction of DLBCLs that are "addicted" to NF-kB signals, an important knowledge gap to be addressed as it relates to the fatal subsets of DLBCLs. Further, since high NF-kB activity is not exclusive of ABC-DLBCL, but contributes to the pathogenesis of multiple mature B-cell malignancies as well as epithelial cancer, our data is likely to have broad relevance and eventually benefit a large patient population. Finally, as the potential of inhibiting and restoring miRNA levels with therapeutic intent is becoming a reality, our studies may provide a blueprint for testing this concept in cancer.