Targeting an RNA Binding Protein Network in Acute Myeloid Leukemia

RNA结合蛋白(RNAbindingProteins，RBPs)是转录和翻译的重要调节因子，经常不受调控 在癌症中。我们最近系统地询问了一些人类癌症患者的RBP依赖性。 针对490个经典的RNA结合结构域的全面CRISPR/Cas9结构域筛选 限制性商业惯例。这揭示了一个在急性髓系白血病(AML)中上调的物理相互作用的RBPs网络 对维持生理RNA剪接和急性髓系白血病生存至关重要。一个关键成员的基因靶向 在这个网络中，RBM39抑制了盒式磁带外显子的包含，并促进了内含子在mRNAs中的保留 对HOXA9目标以及AML优先要求的其他限制性商业惯例进行编码。幸运的是，它最近 已有报道称，一类经临床验证的抗癌磺胺类化合物(包括药物 Indisulam和E7820)作为其主要的细胞作用机制介导RBM39的降解。这 通过这些化合物与CUL4/DDB1的DCAF15适配蛋白之间的新的相互作用而发生 以RBM39为新底物的泛素连接酶复合体。这种化合物对急性髓系白血病细胞的作用 由于RNA剪接的干扰，体外和体内都产生了类似的致命细胞效应。的影响 RBM39剪接缺失导致剪接体突变体AML优先致死，提供了一种新的策略 用于治疗携带调节剪接的限制性商业惯例反复突变的AML。 总体而言，这些数据确定RBM39是功能上和物理上相互作用的RBP网络的核心 急性髓系白血病的表达高于正常的造血祖细胞，是急性髓系白血病维持所必需的。尽管如此 我们还不了解RBM39在恶性肿瘤中的细胞和上下文特定作用的基础 与正常的造血细胞相比。我们也不了解RBM39丢失的确切机制 结果急性髓系白血病被根除。我们假设RBM39在恶性和非恶性疾病中是不同的需要。 正常的造血细胞，可能需要不同的取决于准确的阶段 造血，以及白血病的启动和维持所需要的。这些假设 将在两个具体目标中加以解决。目标1将确定RBM39在正常和 恶性血液病。为此，我们将利用一种新的遗传模型来检测RBM39的基因缺失。 以细胞和时间特异性的方式在体内严格剖析RBM39在恶性肿瘤和正常中的作用 造血干、祖细胞群体。目标2将确定细胞类型和 RBM39在正常和恶性造血细胞中的疾病特异性作用。在这个目标中，我们将比较 直接RNA结合靶点及RBM39缺失对正常和恶性造血细胞剪接的影响 细胞。此外，我们将通过以下事实来评估RBM39在转录延伸方面的潜在小说 复合体，经初步研究确定为RBM39的相互作用因子。

RNA-binding proteins (RBPs) are essential modulators of transcription and translation frequently dysregulated in cancer. We recently systematically interrogated RBP dependencies in a number of human cancers using a comprehensive CRISPR/Cas9 domain-focused screen targeting RNA-binding domains (RBDs) of 490 classical RBPs. This uncovered a network of physically interacting RBPs upregulated in acute myeloid leukemia (AML) and crucial for maintaining physiological RNA splicing and AML survival. Genetic targeting of one key member of this network, RBM39, repressed cassette exon inclusion and promoted intron retention within mRNAs encoding HOXA9 targets as well as in other RBPs preferentially required in AML. Fortuitously, it has recently been described that a class of clinically validated anti-cancer sulfonamide compounds (including the drugs indisulam and E7820) mediate RBM39 degradation as their dominant cellular mechanism of action. This occurs via novel interactions between these compounds and the DCAF15 adapter protein of the CUL4/Ddb1 ubiquitin ligase complex with RBM39 as a neo-substrate. Treatment of AML cells with such compounds in vitro and in vivo resulted in similar lethal cellular effects due to perturbations in RNA splicing. The effects of RBM39 loss on splicing resulted in preferential lethality of spliceosomal mutant AML, providing a novel strategy for treatment of AML bearing recurrent mutations in RBPs that regulated splicing. Overall these data identify RBM39 as central to a network of functionally and physically interacting RBPs upregulated in AML over normal hematopoietic precursors and required for AML maintenance. Despite these insights we do not yet understand the basis for the cell- and context-specific roles of RBM39 in malignant versus normal hematopoietic cells. We also do not understand the exact mechanisms by which RBM39 loss results in eradication of AML. We hypothesize that RBM39 is differentially required in malignant versus normal hematopoietic cells, may be differentially required depending on the precise stage of hematopoiesis, and will be required for leukemia initiation as well as maintenance. These hypotheses will be addressed in two Specific Aims. Aim 1 will determine the biological role of RBM39 in normal and malignant hematopoiesis. In this Aim, we will utilize a novel genetic model for genetic deletion of RBM39 in vivo in a cell- and time-specific manner to rigorously dissect the roles of RBM39 in malignant versus normal hematopoietic stem and progenitor cell populations. Aim 2 will identify the mechanistic basis for cell-type and disease-specific roles for RBM39 in normal and malignant hematopoietic cells. In this Aim we will compare the direct RNA binding targets and effects of RBM39 loss on splicing across normal and malignant hematopoietic cells. In addition, we will evaluate a potential novel for RBM39 in transcriptional elongation by the FACT complex, identified by our preliminary studies as an interactor of RBM39.