One-carbon (C1) metabolism is compartmentalized between the cytosol and mitochondria with the mitochondrial C1 pathway as the major source of glycine and C1 units for cellular biosynthesis. Expression of mitochondrial C1 genes including SLC25A32, serine hydroxymethyl transferase (SHMT) 2, 5,10-methylene tetrahydrofolate dehydrogenase 2, and 5,10-methylene tetrahydrofolate dehydrogenase 1-like was significantly elevated in primary epithelial ovarian cancer (EOC) specimens compared to normal ovaries. 5-Substituted pyrrolo[3,2-d]pyrimidine antifolates (AGF347, AGF359, AGF362) inhibited proliferation of cisplatin sensitive (A2780, CaOV3, IGROV1) and resistant (A2780-E80, SKOV3) EOC cells. In SKOV3 and A2780-E80 cells, colony formation was inhibited. AGF347 induced apoptosis in SKOV3 cells. In IGROV1 cells, AGF347 was transported by folate receptor (FR) α. AGF347 was also transported into IGROV1 and SKOV3 cells by the proton-coupled folate transporter (SLC46A1) and the reduced folate carrier (SLC19A1). AGF347 accumulated to high levels in the cytosol and mitochondria of SKOV3 cells. By targeted metabolomics with [2,3,3-2H]L-serine, AGF347, AGF359 and AGF362 inhibited SHMT2 in the mitochondria. In the cytosol, SHMT1 and de novo purine biosynthesis (i.e., glycinamide ribonucleotide formyltransferase, 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase) were targeted; AGF359 also inhibited thymidylate synthase. Antifolate treatments of SKOV3 cells depleted cellular glycine, mitochondrial NADH and glutathione, and showed synergistic in vitro inhibition toward SKOV3 and A2780-E80 cells when combined with cisplatin. In vivo studies with subcutaneous SKOV3 EOC xenografts in SCID mice confirmed significant antitumor efficacy of AGF347. Collectively, our studies demonstrate a unique metabolic vulnerability in EOC involving mitochondrial and cytosolic C1 metabolism that offers a promising new platform for therapy.

中文翻译：

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线粒体和细胞质一碳代谢是顺铂耐药卵巢癌的目标代谢脆弱性

一碳 (C1) 代谢在细胞质和线粒体之间划分，线粒体 C1 途径是细胞生物合成甘氨酸和 C1 单位的主要来源。与对照组相比，原发性上皮性卵巢癌（EOC）标本中线粒体 C1 基因（包括 SLC25A32、丝氨酸羟甲基转移酶（SHMT）2、5,10-亚甲基四氢叶酸脱氢酶 2 和 5,10-亚甲基四氢叶酸脱氢酶 1 样）的表达显着升高。正常卵巢。5-取代的吡咯并[3,2-d]嘧啶抗叶酸剂（AGF347、AGF359、AGF362）抑制顺铂敏感（A2780、CaOV3、IGROV1）和耐药（A2780-E80、SKOV3）EOC 细胞的增殖。在 SKOV3 和 A2780-E80 细胞中，集落形成受到抑制。AGF347 诱导 SKOV3 细胞凋亡。在 IGROV1 细胞中，AGF347 由叶酸受体 (FR) α 转运。AGF347 还通过质子耦合叶酸转运蛋白 (SLC46A1) 和还原叶酸载体 (SLC19A1) 转运到 IGROV1 和 SKOV3 细胞中。AGF347 在 SKOV3 细胞的细胞质和线粒体中积累至高水平。通过[2,3,3-2H]L-丝氨酸靶向代谢组学，AGF347、AGF359和AGF362抑制线粒体中的SHMT2。在胞质溶胶中，SHMT1和从头嘌呤生物合成（即甘氨酰胺核糖核苷酸甲酰基转移酶、5-氨基咪唑-4-甲酰胺核糖核苷酸甲酰基转移酶）被靶向；AGF359 还抑制胸苷酸合酶。SKOV3 细胞的抗叶酸治疗耗尽了细胞甘氨酸、线粒体 NADH 和谷胱甘肽，并且与顺铂联合使用时，在体外显示出对 SKOV3 和 A2780-E80 细胞的协同抑制作用。SCID 小鼠皮下 SKOV3 EOC 异种移植物的体内研究证实了 AGF347 具有显着的抗肿瘤功效。总的来说，我们的研究证明了 EOC 中独特的代谢脆弱性，涉及线粒体和胞质 C1 代谢，这为治疗提供了一个有前途的新平台。