The function of the PRMT5 methylosome in MTAP deleted cancers

项目摘要 开发新的癌症治疗方法的重大障碍包括发现新的靶点和新的治疗方法。 蛋白质口袋和界面适合于治疗开发。甲硫腺苷基因 磷酸化酶（MTAP）在25-50%的人中经常与肿瘤抑制基因CDKN 2A共缺失。 胰腺癌、胶质母细胞瘤和间皮瘤，其中迫切需要新的治疗方法。我们 发现MTAP缺失导致其底物甲硫腺苷（MTA）的积累，并且MTA 作为甲基转移酶PRMT 5的内源SAM竞争性抑制剂。这导致了“合成 MTAP缺失的肿瘤对PRMT 5的“致死”依赖性。不幸的是，由于他们的SAM合作， 由于其机制，目前的PRMT 5抑制剂对MTAP缺失的肿瘤没有选择性，而是广泛应用于肿瘤细胞。 抗增殖PRMT 5甲基化与3个衔接蛋白RIOK 1、pICln和COPR 5复合的底物。 值得注意的是，RIOK 1和pICln也是MTAP缺失的合成致死相互作用物。这些数据表明 PRMT 5活性及其与衔接子的相互作用是缺失MTAP的生存力所必需的 癌的我们已经发现了一个短肽序列，存在于每个衔接子中，它介导与 PRMT 5（PRMT 5结合基序或PBM）。我们解决了结合到PRMT 5的PBM的结构， 我们称之为PBM沟。由于衔接子是MTAP缺失细胞中生存力所需的，因此这种相互作用 位点可以提供一种新途径以获得不同的PRMT 5治疗剂。因此，我们将测试假设， PBM沟和PBM对于PRMT 5甲基转移酶功能和维持PRMT 5甲基转移酶活性是必需的。 细胞活力其目标是： 目的1：检测PRMT 5上PBM和PBM结合位点是否是甲基化所必需的 和用于维持MTAP缺陷型细胞中的细胞活力。 目的2：验证PRMT 5的PBM沟靶向治疗的有效性。 新的初步数据表明，我们已经确定的衔接蛋白募集机制， 介导某些但不是所有PRMT 5底物的甲基化。用基因工具和试剂 我们现在可以确定哪些底物是通过PBM介导的机制募集的 和/或是否存在独立于PBM募集的底物。A的主要候选人 通过独立机制潜在募集的底物是雄激素受体。在Aim 3我们将使用质谱蛋白质组学方法： 目的3：确定PBM沟介导PRMT 5底物甲基化的程度。

Project Summary Significant hurdles to developing new cancer therapeutics include the discovery of new targets and of novel protein pockets and interfaces amenable to therapeutic development. The gene methylthioadenosine phosphorylase (MTAP) is frequently co-deleted with the tumor suppressor gene CDKN2A in 25-50% of pancreas cancer, glioblastoma and mesothelioma, where there is a desperate need for new therapeutics. We found that MTAP deletion leads to accumulation of its substrate methylthioadenosine (MTA), and that MTA acts as an endogenous SAM-competitive inhibitor of the methyltransferase PRMT5. This leads to “synthetic lethal” dependence of MTAP deleted tumors on PRMT5. Unfortunately, due to their SAM-cooperative mechanism, current PRMT5 inhibitors have no selectivity for MTAP deleted tumors and instead are broadly anti-proliferative. PRMT5 methylates substrates in complex with 3 adaptor proteins RIOK1, pICln and COPR5. Notably, RIOK1 and pICln were also synthetic lethal interactors with MTAP deletion. These data suggest that PRMT5 activity AND its interaction with the adaptors are required for the viability of MTAP deleted cancers. We have discovered a short peptide sequence, present in each adaptor, which mediates binding to PRMT5 (the PRMT5 binding motif or PBM). We solved the structure of the PBM bound to PRMT5 at a site we term the PBM groove. Since the adaptors are required for viability in MTAP deleted cells this interaction site may provide a new route to distinct PRMT5 therapeutics. Hence, we will test the hypothesis that the PBM groove and the PBM are necessary for PRMT5 methyltransferase function and maintenance of cell viability. The aims are: Aim 1: To test whether the PBM and the PBM-binding site on PRMT5 are necessary for the methylation of PRMT5 substrates and for the maintenance of cell viability in MTAP-deficient cells. Aim 2: To test the therapeutic validity of targeting the PBM groove of PRMT5. New preliminary data suggests that the mechanism of adaptor protein recruitment that we have identified mediates the methylation of certain but not all PRMT5 substrates. With the genetic tools and reagents that we have developed we can now determine which substrates are recruited through the PBM-mediate mechanism and/or whether there are substrates that are recruited independently of the PBM. A prime candidate for a substrate potentially recruited through an independent mechanism is the androgen receptor. Therefore in Aim 3 we will use a mass spectrometry proteomic approach: Aim 3: To determine the extent to which the PBM groove mediates PRMT5 substrate methylation.