The role of the SF3B1 splicing factor in chronic lymphocytic leukemia

描述(申请人提供)：通过大规模DNA测序，我们最近发现了核心剪接体因子SF3B1，它是慢性淋巴细胞白血病(CLL)的一个新的癌症基因。SF3B1在U2小核核糖核蛋白(U2 SnRNP)的催化核心发挥作用，U2小核核糖核蛋白是参与前mRNA剪接的重要RNA-蛋白质复合体。这种新的癌症基因的几个特征使其成为研究的高度优先事项。首先，SF3B1在CLL中高频率突变，所有突变都定位在一个离散的基因区，K700E有一半复发。其次，SF3B1的突变与del(11q)显著相关，del(11q)是一种与侵袭性疾病相关的细胞遗传学异常，但它是预后不良的独立预测标志。第三，与野生型SF3B1突变的样本相比，带有SF3B1突变的CLL样本在已知剪接体靶基因中显示出改变的前mRNA剪接。最后，SF3B1-K700E最近被确认为骨髓发育不良中的一种复发突变。SF3B1的突变和RNA剪接的调节可能代表了一种跨越血液系统恶性肿瘤的新的致癌过程，并且优先发生在侵袭性形式的CLL中。我们的假设是，突变的SF3B1产生错误拼接的前mRNAs，编码促进白血病发生的蛋白质。我们在功能上将SF3B1突变与CLL发病机制联系起来的策略是，首先确定SF3B1突变对B细胞存活和增殖的影响，以及最近我们发现对CLL至关重要的其他途径(炎症、Notch1和Wnt信号、DNA损伤/修复)(目标1)。其次，我们将确定这些细胞功能变化的调节机制(目标2)。由于SF3B1是RNA剪接所必需的，我们将研究SF3B1突变对剪接体内关键的蛋白质-蛋白质和蛋白质-RNA相互作用的影响，并使用RNA测序来全局识别由SF3B1突变产生的新的剪接变体。了解受SF3B1突变影响的关键细胞过程(根据目标1)将优先考虑候选剪接变体，并在功能上验证其对促进CLL的关键作用。由于SF3B1突变似乎在细胞谱系特定的背景下起作用，我们将在B细胞中进行所有研究 细胞系或原代正常B细胞；突变或野生型SF3B1是通过我们团队首创的新型生物分子传递或感染方法导入的。此外，我们将使用自然携带SF3B1突变的CLL样本来验证我们的发现。第三，为了确定SF3B1突变在驱动CLL中的作用，我们正在产生一个转基因的条件性敲入SF3B1-K700E小鼠(目标3)。当SF3B1-K700E的表达仅限于CD19 B细胞或与其他产生CLL的基因改变结合时，我们将检查这些小鼠以寻找克隆B细胞淋巴造血积聚的体内证据。拟议中的研究有望为一部小说提供批判性的见解 CLL潜在的剪接机制(与其他血癌有关)，预计将有助于改进治疗这种不治之症的策略，特别是考虑到剪接体抑制剂的开发。

DESCRIPTION (provided by applicant): Through large-scale DNA sequencing, we recently identified the core spliceosome factor, SF3B1, as a novel cancer gene in chronic lymphocytic leukemia (CLL). SF3B1 functions in the catalytic core of the U2 small nuclear ribonucleoprotein (U2 snRNP), an essential RNA-protein complex involved in pre-mRNA splicing. Several features of this new cancer gene make it a high priority for investigation. First, SF3B1 is mutated at a high frequency in CLL, with all mutations localizing to a discrete gene region, and with half recurrent at K700E. Second, mutation in SF3B1 is significantly associated with del(11q), a cytogenetic abnormality associated with aggressive disease, and yet, is an independent predictive marker of poor prognosis. Third, CLL samples with SF3B1 mutation demonstrate altered pre-mRNA splicing in known spliceosome target genes compared to samples with wildtype SF3B1. Finally, SF3B1-K700E was recently identified as a recurring mutation in myelodysplasia. Mutations in SF3B1 and modulation of RNA splicing are likely to represent a novel oncogenic process across hematologic malignancies and preferentially in aggressive forms of CLL. Our hypothesis is that mutated SF3B1 generates mis-spliced pre-mRNAs, encoding proteins that promote leukemogenesis. Our strategy for functionally linking SF3B1 mutation and CLL pathogenesis is to first define the effects of SF3B1 mutation on survival and proliferation of B cells, and on other pathways that we recently identified as critical to CLL (inflammation, Notch1 and Wnt signaling, DNA damage/repair) (Aim 1). Second, we will identify the mechanism by which these changes in cellular function are mediated (Aim 2). Since SF3B1 is essential for RNA splicing, we will examine the effects of SF3B1 mutation on critical protein-protein and protein-RNA interactions within the spliceosome, and use RNA sequencing to globally identify the novel splice variants generated by SF3B1 mutation. Understanding of the critical cellular processes affected by SF3B1 mutation (per Aim 1) will prioritize the candidate splice variants to be functionally validated as crucial to promoting CLL. Because SF3B1 mutation appears to function in a cell lineage specific context, we will carry out all studies in B cell lines or primary normal B cells; into which mutated or wildtype SF3B1 is introduced using novel biomolecule delivery or infection methods that our group has pioneered. Additionally, we will validate our findings using CLL samples naturally harboring SF3B1 mutations. Third, to definitively establish the role of SF3B1 mutation in driving CLL, we are generating a transgenic conditional knock-in SF3B1-K700E mouse (Aim 3). We will examine these mice for in vivo evidence of lymphohematopoietic accumulation of clonal B cells when SF3B1-K700E expression is restricted to CD19+ B cells alone or in combination with other CLL-generating genetic alterations. The proposed studies are anticipated to provide critical insights into a novel splicing mechanism underlying CLL (with implications for other blood cancers) that are expected to contribute to improved strategies to treat this incurable disease, especially given the development of spliceosome inhibitors.