Targeting MBNL1-mediated alternative splicing in MLL-fusion leukemia

建议书摘要/摘要： 尽管在过去的几十年里，儿童急性白血病的预后有了显著的改善，但婴儿 白血病是一种特别侵袭性的疾病，即使用化疗也很难治愈。 强化和骨髓移植。这些白血病中的大多数携带MLL基因的重排， 这种现象也见于一些初治的急性髓系白血病(AML)以及与治疗相关的AML， 研究表明，这些白血病背后有一个共同的基因表达程序 不分血统。通过初步研究，我们发现MLL融合白血病高表达 MBNL1的水平，这是一种RNA结合蛋白，调节选择性RNA剪接。我们还确定了一个 MLL融合白血病特有的选择性剪接(AS)签名，并表明MBNL1缺失导致逆转 并损害MLL融合白血病细胞的生长。因此，我们假设MBNL1是一个关键的 调节MLL-融合AS程序，并通过MBNL1抑制导致该程序中断 白血病细胞死亡。为了检验这些假说，我们的具体目标是：1)确定机制 潜在的MLL融合白血病对MBNL1的依赖，以及2)小分子的优化特性 MBNL1抑制剂用于MLL融合白血病的治疗为了实现第一个目标，我们将描述变化的特征 在关键的细胞生长和死亡途径中，我们假设这些途径对基因的影响负有责任 击倒对手。我们还将使用CLIP-SEQ鉴定MLL融合白血病中新的MBNL1-mRNA相互作用 (用RNAseq进行免疫沉淀交联法)。对于第二个目标，我们已经展示了概念的证明 MBNL1小分子抑制剂可诱导MLL融合白血病细胞死亡。这种化合物需要 药效的优化，我们将通过应用药物化学原理来实现。此外，我们 已经利用它的结构特征和公布的晶体结构启动了一种硅胶屏幕 专有复合库。这项建议将增进我们对AS在 MLL融合白血病的发病机制，同时也直接导致了这种疾病的一流治疗。 申请人目前是辛辛那提儿童医院肿瘤科的讲师 医学中心，将执行这项研究计划，同时从事结构化教学和 接受申请表中所述导师小组的密切个人指导。这些科学家 在白血病的分子发病机制方面拥有丰富的专业知识。实验、指导和 这份职业发展计划中描述的结构化课堂作业将使申请者成功地 转变为独立研究人员和内科科学家，具有RNA结合作用方面的专业知识 白血病发病机制中的蛋白质。

Proposal Summary/Abstract: Though outcomes for pediatric acute leukemia have improved dramatically over the last few decades, infant leukemia is a particularly aggressive disease which remains difficult to cure even with chemotherapy intensification and bone marrow transplant. Most of these leukemias carry a rearrangement of the MLL gene, a phenomenon also seen in some de novo acute myeloid leukemias (AML) as well as therapy-related AML, and studies have demonstrated that a common gene expression program underlies these leukemias regardless of lineage. Through preliminary studies, we have found that MLL-fusion leukemias express high levels of MBNL1, an RNA binding protein that regulates alternative RNA splicing. We have also identified an alternative splicing (AS) signature unique to MLL-fusion leukemia, and show that MBNL1 loss causes reversion of this signature and impairs MLL-fusion leukemia cell growth. We thus hypothesize that MBNL1 is a critical regulator of an MLL-fusion specific AS program, and that disruption of this program via MBNL1 inhibition leads to leukemia cell death. To test these hypotheses, our specific aims are 1) to determine the mechanism underlying MLL-fusion leukemia dependence on MBNL1, and 2) optimize characteristics of a small-molecule MBNL1 inhibitor as a treatment for MLL-fusion leukemia. To achieve the first aim, we will characterize changes in key cell growth and death pathways which we hypothesize are responsible for the effects seen with genetic knockdown. We will also identify novel MBNL1-mRNA interactions in MLL-fusion leukemia using CLIP-seq (cross-linking immunoprecipitation with RNAseq). For the second aim, we have shown as a proof of concept that a small molecule inhibitor of MBNL1 can induce MLL-fusion leukemia cell death. This compound requires optimizations for potency, which we will achieve by applying medicinal chemistry principles. Furthermore, we have used its structural characteristics and published crystal structures to initiate an in silico screen of a proprietary compound library. This proposal will advance our understanding of the role of AS in the pathogenesis of MLL-fusion leukemia, while also directly leading to a first-in-class therapy for this disease. The applicant, who is currently an instructor in the Division of Oncology at Cincinnati Children’s Hospital Medical Center, will execute this research plan while simultaneously engaging in structured didactics and receiving close individual guidance from a panel of mentors as described in the application. These scientists possess significant expertise in the molecular pathogenesis of leukemia. The experiments, mentoring, and structured classwork described in this career development plan will position the applicant to successfully transition into an independent researcher and physician-scientist, with expertise in the role of RNA binding proteins in leukemia pathogenesis.