Rational design of anti-cancer therapeutics harnessing the synthetic lethality of methionine metabolism and arginine methyltransferases

提案摘要 甲硫氨酸腺苷转移酶2 α（MAT 2A）和蛋白质精氨酸甲基转移酶5（PRMT 5）是 具有MTAP缺失的合成致死性的癌症靶点，并且在临床上具有几种候选药物， 针对MTAP-/-癌症的试验。MTAP在约15%的人类癌症中缺失，并编码代谢产物。 酶5 '-甲硫腺苷磷酸化酶，唯一的酶在人类负责回收的 甲硫腺苷（MTA）转化为甲硫氨酸。MAT 2A合成S-腺苷甲硫氨酸（SAM），甲基 甲基转移酶反应的供体底物。PRMT 5利用SAM作为底物并被MTA抑制， 并且培养物中的MTAP-/-细胞显示升高的MTA水平。胶质母细胞瘤肿瘤的体内观察 然而，MTAP-/-并不总是导致肿瘤MTA水平增加，这是由于MTA流出到 基质MTAP感受态细胞。此外，MTAP缺失在结直肠癌中很少发生（约2%） （CRC），排除了MAT 2A和PRMT 5抑制剂用于大多数CRC。施拉姆实验室 先前解决了MTAP的过渡态（TS）结构，并合成了一种有效的小分子抑制剂 甲硫基-DADMe-免疫球蛋白-A（MTDIA），其概括了MTA在细胞内积累的体外作用， 组织中MTDIA已被证明在包括CRC在内的几种癌症模型中抑制肿瘤生长，并且与 通过升高MTA水平降低PRMT 5活性。我们建议将MTDIA用于 与MAT 2A抑制剂AG-270组合，目前处于I期临床试验中，以利用其合成致死性 PRMT 5为目标。我们将测试MTDIA的安全性、靶点参与和抗癌功效， 在ApcMin/+和CRC患者来源的异种移植物（PDX）小鼠中与AG-270的组合。以确定 为了研究PRMT 5的抗癌作用机制，我们将探讨PRMT 5的上游和下游作用。 活动我们将对相关代谢物、组蛋白和蛋白质进行肿瘤代谢组学定量- 使用免疫组织化学和蛋白质组学技术进行精氨酸甲基化表征。我们还将 使用单细胞RNA测序分析基因表达变化，以确定联合治疗 改变肿瘤的结构和生长最后，我们将求解PRMT 5的过渡态结构，目标是 为开发新型过渡态类似物抑制剂奠定了基础。这项工作将扩大 在使用MAT 2A和PRMT 5抑制剂时，超过约15%的MTAP缺失的癌症，并提供 MTDIA用于临床试验的途径。

Proposal Abstract Methionine adenosyltransferase 2 alpha (MAT2A) and protein arginine methyltransferase 5 (PRMT5) are cancer targets that are synthetically lethal with MTAP deletions and have several drug candidates in clinical trials targeting MTAP-/- cancers. MTAP is deleted in ~15% of human cancers and encodes the metabolic enzyme 5’-methylthioadenosine phosphorylase, the sole enzyme in humans responsible for recycling of methylthioadenosine (MTA) to methionine. MAT2A synthesizes S-adenosyl methionine (SAM), the methyl donor substrate for methyltransferase reactions. PRMT5 utilizes SAM as a substrate and is inhibited by MTA, and MTAP-/- cells in culture demonstrate elevated MTA levels. In vivo observations of glioblastoma tumors suggest however, that MTAP-/- does not always lead to increased tumoral MTA levels due to MTA efflux into matrix MTAP-competent cells. Additionally, MTAP deletions are a rare (~2%) occurrence in colorectal cancers (CRCs), precluding MAT2A and PRMT5 inhibitors’ use for most CRCs. The Schramm laboratory has previously solved the transition state (TS) structure of MTAP and synthesized a potent small molecule inhibitor methylthio-DADMe-immucillin-A (MTDIA) that recapitulates the in vitro effects of MTA accumulation within tissues. MTDIA has been shown to inhibit tumor growth in several cancer models, including CRC, and is linked to a decrease in PRMT5 activity through elevation of MTA levels. We propose that MTDIA be used in combination with MAT2A inhibitor AG-270, currently in Phase I clinical trials, to harness their synthetic lethality by targeting PRMT5. We will test the safety, target engagement, and anti-cancer efficacy of MTDIA in combination with AG-270 in ApcMin/+ and CRC patient-derived xenograft (PDX) mice. To determine mechanisms of anti-cancer effects, we will probe the upstream and downstream effects related to PRMT5 activity. We will perform tumor metabolomic quantification of relevant metabolites and histone and protein- arginine methylation characterization using immunohistochemistry and proteomic techniques. We will also profile the gene expression changes using single-cell RNA sequencing to determine how combination therapy alters tumor architecture and growth. Finally, we will solve the transition state structure of PRMT5 with the goal of laying the foundations for development of novel transition state analogue inhibitors. This work will expand upon the use of MAT2A and PRMT5 inhibitors beyond the ~15% of MTAP-deleted cancers and provide avenues for MTDIA to be used in clinical trials.