Targeting defective spliceosomal pathway in myeloid malignancies

摘要 MDS患者样品的全外显子组和靶向测序已经导致发现一组基因（SF 3B 1， U2 AF 1、SRSF 2、ZRSR 2和LUC 7 L2）编码突变体RNA剪接因子，其改变 MDS进展过程中造血细胞克隆进化中的蛋白同种型。随后的研究发现， 这些剪接因子突变在AML和实体瘤中的发生。临床数据还显示， 这些基因的杂合错义突变或半合缺失大多是相互排斥的 这意味着在MDS细胞中，剪接调节中的多种蛋白质的突变可能是致命的。迄今为止，只有一个 靶向SF 3B 1的化合物H3 B-8800正处于治疗MDS和其他骨髓恶性肿瘤的I期临床试验中。 没有靶向其他突变剪接因子的药物处于临床前开发阶段，如果其他突变因子 可能是潜在的药物靶点。其中，U2 AF 1突变发生在早期MDS克隆中， 是白血病进展的不良预后特征。研究显示U2 AF 1 S34 F造血细胞和 转基因小鼠模型对靶向SF 3B 1的sudemycin敏感。最近，U2 AF 1 wt被建议用于 是含有U2 AF 1突变的癌细胞生存的单倍必需基因。我们的核心假设是 U2 AF 1 wt抑制可诱导对U2 AF 1 S34 F克隆的合成致死，这取决于U2 AF 1 wt的形成， 生存我们的基本原理是阻断U2 AF 1/U2 AF 2结合可能会损害U2 AF 1 wt功能。使用 基于片段的文库筛选，我们已经鉴定了抑制U2 AF 1/U2 AF 2结合的命中化合物 在K562-U2 AF 1 S34 F和携带U2 AF 1的人原代细胞中表现出选择性生长抑制 突变，而不是它们的野生型对应物。我们的目标是开发一种新的 U2 AF 1抑制剂的基础上打击和评估MDS/AML的综合致死性的治疗概念 疾病模型我们的长期目标是开发小分子疗法，以靶向有缺陷的癌细胞。 在剪接体途径中。为了实现我们的目标，我们设计了两个具体目标：1）优化 U2 AF 1抑制剂的计算机辅助设计与化学合成相结合; 2）研究U2 AF 1抑制剂的治疗作用 使用体外和体内U2 AF 1 mut MDS/AML模型观察U2 AF 1抑制剂的作用。在目标1中，我们将改进 基于初步构效关系和选择性的U2 AF 1抑制剂的活性和选择性 通过整合化学合成、计算机辅助设计、生化分析和协同分析， 晶体结构测定在目标2中，我们将评估我们的U2 AF 1抑制剂在MDS/AML中的活性 细胞模型，分析用U2 AF 1抑制剂处理的细胞系中的转录组和剪接模式变化 并确定我们的抑制剂对异种移植小鼠模型中红细胞生成的影响。我们的应用程序 创新性和重要性，因为它建立在我们发现U2 AF 1的基础上，U2 AF 1是一种新兴的新靶点 与骨髓恶性肿瘤的转化相关，并有可能建立一个新的 本发明提供了一种通过靶向突变体U2 AF 1细胞治疗MDS/AML的治疗平台。

Abstract Whole-exome and targeted sequencing of MDS patients’ samples have led to discover a set of genes (SF3B1, U2AF1, SRSF2, ZRSR2 and LUC7L2) encoding mutant RNA splicing factors that alter expression patterns of protein isoforms in clonal evolution of hematopoietic cells during MDS progression. Subsequent studies found occurrences of these splicing factor mutations in AML and solid tumors. Clinical data also showed heterozygous missense mutations or hemizygous deletions of these genes are mostly mutually exclusive implying that mutations in multiple proteins in splicing regulation may be lethal in MDS cells. To date, only one compound, H3B-8800, to target SF3B1 is in phase I clinical trial to treat MDS and other myeloid malignancies. No agents targeting other mutated splicing factors are in pre-clinical development and if other mutant factors can be potential drug targets is unknown. Among them, U2AF1 mutations occurred in early MDS clones and are poor prognostic features for progression to leukemia. Studies showed U2AF1S34F hematopoietic cells and transgenic mouse models are sensitive to sudemycin that targets SF3B1. Recently, U2AF1wt was suggested to be a haplo-essential gene for the survival of cancer cells containing U2AF1 mutation. Our central hypothesis is that U2AF1wt inhibition may induce synthetic lethality to U2AF1S34F clones that depends on U2AF1wt for survival. Our rationale is that blockade of U2AF1/U2AF2 association may impair U2AF1wt function. Using fragment-based library screening, we have identified a hit compound that inhibited the U2AF1/U2AF2 binding and exhibited selective growth inhibition in K562-U2AF1S34F and human primary cells carrying U2AF1 mutations but not their wild-type counterparts. Our objective of this proposal is to develop a new class of U2AF1 inhibitors based on the hit and assess the therapeutic concept of synthetic lethality in MDS/AML disease models. Our long-term goal is to develop small-molecule therapeutics to target cancer cells defective in the spliceosome pathway. To achieve our goal, we have devised two specific aims: 1) Optimization of U2AF1 inhibitors by integrating computer-aided design with chemical syntheses; 2) Study the therapeutic effects of U2AF1 inhibitors using in vitro and in vivo U2AF1mut MDS/AML models. In Aim 1, we will improve the activity and selectivity of U2AF1 inhibitors based on the preliminary structure activity relationship and selectivity data of SF1-8 by integrating chemical syntheses, computer-aided design, biochemical assays and the co- crystal structure determination. In Aim 2, we will assess the activities of our U2AF1 inhibitors in MDS/AML cellular models, analyze transcriptome and splicing pattern changes in cell lines treated with U2AF1 inhibitors and determine the effects of our inhibitors on erythropoiesis in xenograft mouse models. Our application is innovative and significant, because it builds on our discovery of the hit to U2AF1, an emerging novel target associated with the transformation of myeloid malignancies, and has the potential to establish a new therapeutic platform to treat MDS/AML by pharmacologically targeting mutant U2AF1 cells.