The role of the SF3B1 splicing factor in chronic lymphocytic leukemia

描述（由申请人提供）：通过大规模DNA测序，我们最近确定了核心剪接体因子SF 3B 1，作为慢性淋巴细胞白血病（CLL）的一种新的癌症基因。SF 3B 1在U2小核核糖核蛋白（U2 snRNP）的催化核心中起作用，U2 snRNP是参与前mRNA剪接的必需RNA-蛋白质复合物。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，SF 3B 1在CLL中以高频率突变，所有突变定位于离散的基因区域，并且在K700 E处有一半复发。第二，SF 3B 1突变与del（11 q）显著相关，del（11 q）是一种与侵袭性疾病相关的细胞遗传学异常，但仍是预后不良的独立预测标志物。第三，与具有野生型SF 3B 1的样品相比，具有SF 3B 1突变的CLL样品证明已知剪接体靶基因中的前mRNA剪接改变。最后，SF 3B 1-K700 E最近被确定为骨髓增生异常中的复发突变。SF 3B 1的突变和RNA剪接的调节可能代表了血液恶性肿瘤中的一种新的致癌过程，并优先出现在侵袭性CLL中。我们的假设是突变的SF 3B 1产生错误剪接的前mRNA，编码促进白血病发生的蛋白质。我们在功能上将SF 3 B1突变与CLL发病机制联系起来的策略是首先确定SF 3 B1突变对B细胞存活和增殖的影响，以及对我们最近确定为对CLL至关重要的其他途径（炎症、Notch 1和Wnt信号传导、DNA损伤/修复）的影响（目的1）。其次，我们将确定这些细胞功能变化的介导机制（目的2）。由于SF 3B 1是RNA剪接所必需的，我们将研究SF 3B 1突变对剪接体内关键蛋白质-蛋白质和蛋白质-RNA相互作用的影响，并使用RNA测序来全面鉴定SF 3B 1突变产生的新剪接变体。了解受SF 3B 1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，以在功能上验证其对促进CLL至关重要。由于SF 3 B1突变似乎在细胞谱系特异性背景下起作用，因此我们将在B中进行所有研究 细胞系或原代正常B细胞;使用我们小组开创的新型生物分子递送或感染方法将突变或野生型SF 3 B1引入其中。此外，我们将使用天然携带SF 3B 1突变的CLL样本验证我们的发现。第三，为了明确确定SF 3B 1突变在驱动CLL中的作用，我们正在产生转基因条件性敲入SF 3B 1-K700 E小鼠（Aim 3）。我们将检查这些小鼠的克隆B细胞的淋巴造血积累的体内证据时，SF 3B 1-K700 E表达仅限于CD 19 + B细胞单独或与其他CLL生成的遗传改变的组合。预计拟议中的研究将为一部小说提供重要的见解 CLL潜在的剪接机制（对其他血液癌症的影响），预计将有助于改善治疗这种不治之症的策略，特别是考虑到剪接体抑制剂的发展。

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.