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

建议书摘要 蛋氨酸腺苷转移酶2α(MAT2A)和蛋白精氨酸甲基转移酶5(PRMT5)分别为 MTAP缺失具有合成致命性且临床上有几种候选药物的癌症靶点 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 酶5‘-甲基硫代腺苷磷酸化酶，人类体内唯一负责循环的酶 甲硫腺苷(MTA)转化为蛋氨酸。MAT2A合成S-腺苷甲硫氨酸 甲基转移酶反应的供体底物。PRMT5利用SAM作为底物并被MTA抑制， 培养的MTAP-/-细胞表现出较高的MTA水平。胶质母细胞瘤的活体观察 然而，提示MTAP-/-并不总是导致肿瘤MTA水平的增加，因为MTA流出到 为MTAP活性细胞提供基质。此外，MTAP缺失在结直肠癌中很少见(~2%) (CRCs)，排除了MAT2A和PRMT5抑制剂对大多数CRC的使用。施拉姆实验室有 以前解决了MTAP的过渡态(TS)结构，合成了一种有效的小分子缓蚀剂 甲硫基-DADMe-免疫粘菌素-A(MTDIA)概述了MTA在体内蓄积的体外效应 纸巾。MTDIA已被证明在包括结直肠癌在内的几种癌症模型中抑制肿瘤生长，并且与 通过提高MTA水平导致PRMT5活性下降。我们建议将MTDIA用于 与目前处于I期临床试验的MAT2A抑制剂AG-270联合使用，以利用它们的合成致命性 通过瞄准PRMT5。我们将测试MTDIA的安全性、靶向性和抗癌效果 在ApcMin/+和CRC患者来源的异种移植(PDX)小鼠中联合使用AG-270。要确定 抗癌作用机制，我们将探讨与PRMT5相关的上下游作用 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量化- 应用免疫组织化学和蛋白质组学技术鉴定精氨酸甲基化。我们还将 利用单细胞RNA测序分析基因表达的变化，以确定联合治疗的方式 改变肿瘤的结构和生长。最后，我们将解决PRMT5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。这项工作将会扩大 在超过15%的MTAP缺失型癌症中使用MAT2A和PRMT5抑制剂，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.