Targeting defective spliceosomal pathway in myeloid malignancies

摘要 对MDS患者样本的全外显子组和靶向测序导致发现了一组基因(SF3B1， U2AF1、SRSF2、ZRSR2和LUC7L2)编码突变的RNA剪接因子，改变其表达模式 MDS进展过程中造血细胞克隆进化中的蛋白质异构体。后续研究发现 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子缺失大多是相互排斥的。 这意味着剪接调控中多个蛋白质的突变在MDS细胞中可能是致命的。到目前为止，只有一个 靶向SF3B1的化合物H3B-8800正在进行治疗MDS和其他髓系恶性肿瘤的I期临床试验。 没有针对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 能否成为潜在的药物靶点尚不清楚。其中，U2AF1突变发生在早期MDS克隆和 是进展为白血病的不良预后特征。研究表明U2AF1S34F造血细胞和 转基因小鼠模型对靶向SF3B1的苏迪霉素敏感。最近，U2AF1wt被建议 是含有U2AF1突变的癌细胞生存所必需的单倍体基因。我们的中心假设是 抑制U2AF1wt可诱导依赖U2AF1wt的U2AF1S34F克隆的合成致死 生死存亡。我们的理论基础是，阻断U2AF1/U2AF2关联可能会损害U2AF1wt的功能。vbl.使用 基于片段的文库筛选，我们已经鉴定出一个抑制U2AF1/U2AF2结合的HIT化合物 对K562-U2AF1S34F和携带U2AF1的人原代细胞具有选择性生长抑制作用 突变，但不是野生型。我们这项提议的目标是发展一类新的 基于HIT的U2AF1抑制剂和评估MDS/AML合成致死性的治疗概念 疾病模型。我们的长期目标是开发针对有缺陷的癌细胞的小分子疗法 在剪接体途径中。为了实现我们的目标，我们制定了两个具体目标：1)优化 将计算机辅助设计与化学合成相结合的U2AF1抑制剂；2)研究治疗 U2AF1抑制剂在体外和体内对U2AF1mut MDS/AML模型的作用在目标1中，我们将改进 基于初步构效关系和选择性的U2AF1抑制剂的活性和选择性 通过将化学合成、计算机辅助设计、生化分析和计算机辅助分析相结合，获得SF1-8的数据。 晶体结构测定。在目标2中，我们将评估我们的U2AF1抑制剂在MDS/AML中的活性 细胞模型，分析U2AF1抑制剂处理的细胞系转录组和剪接模式的变化 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。我们的应用程序是 创新和意义重大，因为它建立在我们发现U2AF1的命中的基础上，U2AF1是一个新兴的新目标 与髓系恶性肿瘤的转化有关，并有可能建立一种新的 通过药物靶向突变的U2AF1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.