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动态平衡在很大程度上仍是未知的。正常情况下 细胞，6-磷酸葡萄糖脱氢酶(6PGD)，氧化戊糖磷酸途径中的一种酶 (PPP)，将6-磷酸葡萄糖酸(6-PG)转化为5-磷酸核酮糖(Ru-5-P)并产生NADPH。 据报道，6PGD活性在几种癌症组织中上调，包括结直肠癌、宫颈癌、 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 6PGD活性在人类癌症中的激活及其在肿瘤发生发展中的重要作用 仍然不为人所知。我们发现K76和K294上的乙酰化增强了6PGD的激活，通常 在不同的人类癌细胞中观察到。稳定敲除癌细胞中的6PGD导致氧化减少 PPP通量和RNA/DNA生物合成。令人惊讶的是，6PGD基因敲除也导致NADPH/NADP下降 这表明6PGD在NADPH生产中起着其他物质无法弥补的重要作用 产生NADPH的酶。此外，具有6PGD基因敲除的癌细胞表现出较高的ROS水平和 生物合成异常，导致裸鼠移植瘤细胞增殖和肿瘤生长减少。我们是下一个 筛选并鉴定了一种新型的、选择性的小分子6PGD抑制剂。大黄素甲素治疗 或其衍生物S3有效地抑制多种人类癌细胞的增殖，而不会产生靶外效应。 大黄素甲素和S3也能有效抑制人原代白血病细胞的活性和增殖 毒性最小的患者。此外，S3还能显著抑制裸鼠移植瘤的生长 体内皮下注射人H1299肺癌或K562白血病细胞，毒性最小。 因此，我们假设赖氨酸乙酰化增强了6PGD的激活，从而促进了癌细胞的增殖 和肿瘤生长；6PGD因此是临床治疗的一个新的抗癌靶点。有趣的是，我们还 研究发现，6PGD基因敲除导致细胞内Ru-5-P(6PGD产物)水平下降，导致 激活AMP激活的蛋白激酶(AMPK)，从而抑制乙酰辅酶A羧基酶1 (Acc1)，从而促进脂肪生成。因此，除了PPP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， 除了6PGD在NADPH产生和氧化还原动态平衡中令人惊讶的关键作用外，6PGD对 癌症代谢与肿瘤生长。我们将用人类肺癌和白血病来检验这些假说。 (CML、AML和B-ALL)作为平台。提出了三个具体的目标：(1)检查赖氨酸是否 乙酰化对6PGD的激活、促进癌细胞代谢和肿瘤生长具有重要作用； 探讨6PGD如何将PPP、AMPK信号和脂肪生成与氧化还原调节协调在促进 肿瘤细胞代谢和肿瘤生长；以及(3)验证6PGD作为抗白血病靶点治疗白血病的作用。 用本实验室研制的6PGD小分子抑制剂对人白血病细胞进行体外和体内实验。

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tissues including colorectal cancers, cervical intraepithelial neoplasia and thyroid tumors, as well as leukemia (our unpublished data).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.