Project 3: Therapeutic inhibition of splicing through inhibition of protein arginine methylation in leukemia

摘要 白血病通常表现为基因改变，导致表观基因组的失调。发掘潜力 表观遗传的脆弱性，我们最近在体外和体内进行了配对的shRNA筛查 急性白血病细胞系。结果表明，髓系白血病细胞和淋巴细胞性白血病细胞优先 依赖于蛋白质精氨酸甲基转移酶(PRMTs)，这是一种对精氨酸进行二甲基化的酶家族 许多蛋白质的残基。许多先前的研究已经确定PRMT5是一个有希望的治疗靶点。 在癌症中，这导致了一项正在进行的PRMT5抑制剂的I期临床试验，用于难治性癌症患者 实体瘤和非霍奇金淋巴瘤。然而，哪些PRMT的底物对抗癌作用最为关键？ PRMT抑制对肿瘤的影响尚不清楚，预测PRMT抑制反应的生物标志物 是非常需要的。 为了了解PRMT抑制剂的抗癌作用，我们还发现阻断PRMT 功能干扰RNA剪接，抑制对称性(由PRMT5介导)或不对称 二甲基精氨酸甲基化(由I型PRMTs引起)导致剪接体突变体的强烈选择性杀伤 白血病的发病率高于野生型(WT)白血病。此外，我们还观察到了以下因素的协同效应 将I型和II型PRMT抑制和/或核心剪接体功能抑制结合起来。因此，我们 假设PRMT抑制的主要细胞毒作用是剪接调控的结果。 我们将通过表征抑制I型PRMT或II型PRMT的影响来检验我们的假设 WT或剪接体突变体的前mRNA剪接、基因表达和甲基精氨酸蛋白质组 白血病细胞(目标1)。我们还将评估I型PRMTs、II型PRMTs和 剪接因子SF3B增强对髓系和淋巴系白血病细胞的毒性及其相互关系 这些抑制剂的有效性与各种剪接因子基因突变之间的关系(目标2)。最后，我们将确定 使用葛兰素史克小分子I/II期试验样本预测PRMT5抑制效果的生物标志物 PRMT5拮抗剂(AIM 3)，用于治疗难治性AML、CMML和MDS患者。 该项目将极大地提高对PRMT抑制剂疗效的分子基础的理解。 并将这些药物推向白血病的临床试验。

ABSTRACT Leukemias often display genetic alterations that result in dysregulation of the epigenome. To identify potential epigenetic vulnerabilities, we recently performed a paired in vitro and in vivo shRNA screen in a number of acute leukemia cell lines. The results showed that myeloid as well as lymphoid leukemia cells are preferentially dependent on protein arginine methyltransferase (PRMTs), a family of enzymes that dimethylate arginine residues of many proteins. A number of prior studies have identified PRMT5 as a promising therapeutic target in cancer, which has led to an ongoing phase I clinical trial of a PRMT5 inhibitor for patients with refractory solid tumors and Non-Hodgkin's lymphoma. However, which substrates of PRMTs are most critical for anti- cancer effects of PRMT inhibition remains unknown and biomarkers predicting response to PRMT inhibition are greatly needed. Toward understanding the anticancer effects of PRMT inhibitors, we have also found that blocking PRMT function perturbs RNA splicing, and that inhibiting either symmetric (mediated by PRMT5) or asymmetric dimethyl arginine methylation (by Type I PRMTs) results in strong preferential killing of spliceosomal mutant leukemias over their wild-type (WT) counterparts. Moreover, we have observed synergistic effects of combining both type I with type II PRMT inhibition and/or inhibition of core spliceosome function. We therefore hypothesize that the main cytotoxic effect of PRMT inhibition results from modulation of splicing. We will examine our hypothesis by characterizing the effects of inhibiting type I PRMT or type II PRMTs on pre-mRNA splicing, gene expression, and the methyl-arginine proteome in WT or spliceosomal-mutant leukemia cells (Aim 1). We will also evalute whether combining inhibitors of type I PRMTs, type II PRMTs, and the splicing factor SF3b enhances toxicity to myeloid and lymphoid leukemia cells, and the relationship between these inhibitors' efficacy and mutations in various splicing factor genes (Aim 2). Finally, we will identiy biomarkers of efficacy of PRMT5 inhibition using samples from a phase I/II trial of GSK's small molecule PRMT5 antagonist (Aim 3) for the treatment of patients with refractory AML, CMML, and MDS. This project stands to greatly improve understanding of the molecular basis for the efficacy of PRMT inhibitors in cancer and to advance these drugs toward clinical trials in leukemia.