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

摘要 白血病通常表现出导致表观基因组失调的遗传改变。以识别潜在 表观遗传的脆弱性，我们最近进行了配对的体外和体内的shRNA筛选在一些 急性白血病细胞系结果表明，髓系和淋巴样白血病细胞优先 依赖于蛋白质精氨酸甲基转移酶（PRMTs），一种使精氨酸二甲基化的酶家族 许多蛋白质的残基。许多先前的研究已经将PRMT 5确定为有希望的治疗靶点 在癌症中，这导致了正在进行的PRMT 5抑制剂用于难治性癌症患者的I期临床试验。 实体瘤和非霍奇金淋巴瘤。然而，PRMT的哪些底物对于抗- PRMT抑制的癌症效应仍然未知，并且预测对PRMT抑制的反应的生物标志物 非常需要。 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，并抑制对称（由PRMT 5介导）或不对称 二甲基精氨酸甲基化（通过I型PRMT）导致剪接体突变体的强烈优先杀伤 与野生型（WT）白血病相比。此外，我们还观察到了 结合I型和II型PRMT抑制和/或核心剪接体功能抑制。因此我们 假设PRMT抑制的主要细胞毒性作用来自剪接的调节。 我们将通过描述抑制I型PRMT或II型PRMT对细胞增殖的影响来检验我们的假设。 野生型或剪接体突变体中前mRNA剪接、基因表达和甲基精氨酸蛋白质组 白血病细胞（Aim 1）。我们还将评估是否将I型PRMT、II型PRMT和I型PRMT的抑制剂联合使用。 剪接因子SF 3b增强对髓系和淋巴样白血病细胞的毒性， 这些抑制剂的功效与各种剪接因子基因的突变之间的关系（Aim 2）。最后，我们将识别 使用来自GSK小分子药物I/II期试验的样品的PRMT 5抑制功效的生物标志物 PRMT 5拮抗剂（Aim 3）用于治疗难治性AML、CMML和MDS患者。 该项目将大大提高对PRMT抑制剂功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.