Signaling and Targeting of 6-Phosphogluconate Dehydrogenase in Human Cancers

摘要： 癌细胞如何协调合成代谢生物合成和氧化还原稳态仍然是未知的。正常 细胞，6-磷酸葡萄糖酸脱氢酶（6PGD），氧化戊糖磷酸途径中的酶 (PPP)将6-磷酸葡萄糖酸盐（6-PG）转化为5-磷酸核酮糖（Ru-5-P）并产生NADPH。 已经报道了在几种癌组织中上调的6PGD活性，包括结肠直肠癌、宫颈癌、乳腺癌、乳腺癌和乳腺癌。 上皮内瘤变和甲状腺肿瘤，以及白血病（我们未发表的数据）。然而，6PGD是如何 以及6PGD活性在发病机制和肿瘤发展中是否重要 仍然未知。我们发现K76和K294的乙酰化增强了6PGD的活化，并且通常是 在不同的人类癌细胞中观察到。癌细胞中6PGD的稳定敲低导致氧化还原性降低。 PPP通量和RNA/DNA生物合成。令人惊讶的是，6PGD敲低也会导致NADPH/NADP+降低， 比率，表明6PGD在NADPH生产中的重要作用，不能通过其他方法补偿。 NADPH生成酶。此外，具有6PGD敲低的癌细胞显示升高的ROS水平， 异常生物合成，导致异种移植裸鼠中细胞增殖和肿瘤生长减少。我们接下来 筛选并鉴定Physcion为新型选择性小分子6PGD抑制剂。Physcion治疗 或其衍生物S3有效地抑制多种人类癌细胞中的细胞增殖，而没有脱靶效应。 Physcion和S3也能有效抑制人白血病原代细胞的增殖和细胞活力 患者毒性最小。此外，S3显著降低异种移植裸鼠中的肿瘤生长 皮下注射人H1299肺癌或K562白血病细胞，体内毒性最小。 因此，我们假设赖氨酸乙酰化增强了6PGD的激活，从而促进癌细胞增殖 和肿瘤生长; 6PGD因此代表了临床治疗中的新抗癌靶点。有趣的是，我们也 发现敲低6PGD导致细胞内Ru-5-P（6PGD产物）水平降低，导致 AMP活化蛋白激酶（AMPK）的活化，其随后抑制乙酰辅酶A羧化酶1 （ACC 1）和因此脂肪生成。因此，除了公私伙伴关系和 核苷酸生物合成，6PGD提供了PPP，AMPK信号和脂肪生成之间的新联系， 沿着6-PGD在NADPH产生和氧化还原稳态中令人惊讶的关键作用， 癌症代谢和肿瘤生长。我们将用人类肺癌和白血病来检验这些假设 (CML、AML和B-ALL）作为平台。提出了三个具体目的：（1）检测赖氨酸是否 乙酰化对于6PGD活化和促进癌细胞代谢和肿瘤生长是重要的;（2） 探索6PGD如何连接PPP，AMPK信号和脂肪生成，以协调氧化还原调节，促进 癌细胞代谢和肿瘤生长;和（3）验证6PGD作为治疗白血病的抗白血病靶点。 用本实验室研制的6-PGD小分子抑制剂对人白血病细胞进行了体外和体内研究。

Abstract: How cancer cells coordinate anabolic biosynthesis and redox homeostasis remains largely unknown. In normal cells, 6-phosphogluconate dehydrogenase (6PGD), an enzyme in the oxidative pentose phosphate pathway (PPP), converts 6-phosphogluconate (6-PG) to ribulose 5-phosphate (Ru-5-P) and produces NADPH. Upregulated 6PGD activity has been reported in several cancer tissues including colorectal cancers, cervical intraepithelial neoplasia and thyroid tumors, as well as leukemia (our unpublished data). However, how 6PGD is activated in human cancers and whether 6PGD activity is important in pathogenesis and tumor development remain unknown. We found that acetylation at K76 and K294 enhances 6PGD activation and is commonly observed in diverse human cancer cells. Stable knockdown of 6PGD in cancer cells results in reduced oxidative PPP flux and RNA/DNA biosynthesis. Surprisingly, 6PGD knockdown also causes decreased NADPH/NADP+ ratio, suggesting an important role for 6PGD in NADPH production that cannot be compensated by other NADPH-producing enzymes. Moreover, cancer cells with 6PGD knockdown show elevated ROS levels and aberrant biosynthesis, leading to reduced cell proliferation and tumor growth in xenograft nude mice. We next screened and identified Physcion as a novel, selective small molecule 6PGD inhibitor. Treatment with Physcion or its derivative S3 effectively inhibits cell proliferation in diverse human cancer cells with no off-target effect. Physcion and S3 also effectively inhibit cell viability and proliferation of primary leukemia cells from human patients with minimal toxicity. Furthermore, S3 significantly reduces tumor growth in xenograft nude mice subcutaneously injected with human H1299 lung cancer or K562 leukemia cells with minimal toxicity in vivo. Thus, we hypothesize that lysine acetylation enhances 6PGD activation, which promotes cancer cell proliferation and tumor growth; 6PGD thus represents a novel anti-cancer target in clinical treatment. Intriguingly, we also found that knockdown of 6PGD results in decreased intracellular levels of Ru-5-P (6PGD product), leading to activation of AMP-activated protein kinase (AMPK), which subsequently inhibits acetyl-CoA carboxylase 1 (ACC1) and consequently lipogenesis. Thus, in addition to the well-established connection between PPP and nucleotide biosynthesis, 6PGD provides a novel link between PPP, AMPK signaling and lipogenesis, which, along with the surprisingly crucial role for 6PGD in NADPH production and redox homeostasis, is important for cancer metabolism and tumor growth. We will test these hypotheses using human lung cancer and leukemias (CML, AML and B-ALL) as platforms. Three Specific Aims were proposed (1) To examine whether lysine acetylation is important for 6PGD activation and promotion of cancer cell metabolism and tumor growth; (2) To explore how 6PGD links PPP, AMPK signaling and lipogenesis to coordinate with redox regulation in promoting cancer cell metabolism and tumor growth; and (3) To validate 6PGD as an anti-leukemia target in treatment of human leukemia cells in vitro and in vivo using 6PGD small molecule inhibitors developed in our laboratory.