Signaling and Targeting of 6-Phosphogluconate Dehydrogenase in Human Cancers

描述(申请人提供)：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不清楚。在正常细胞中，6-磷酸葡萄糖脱氢酶(6PGD)是磷酸戊糖氧化途径(PPP)中的一种酶，它将6-磷酸葡萄糖(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在人类癌症中是如何被激活的，以及6PGD活性是否在肿瘤的发病机制和肿瘤发生中起重要作用仍不清楚。我们发现K76和K294的乙酰化增强了6PGD的激活，并且在不同的人类癌细胞中普遍观察到。在癌细胞中稳定地敲除6PGD会导致氧化PPP通量和RNA/DNA生物合成减少。令人惊讶的是，6PGD基因敲除还导致NADPH/NADP+比值下降，这表明6PGD在NADPH产生中起着其他NADPH产生酶无法弥补的重要作用。此外，6PGD基因敲除的癌细胞表现出ROS水平升高和生物合成异常，导致裸鼠移植瘤细胞增殖和肿瘤生长减少。接下来，我们筛选并鉴定了一种新型的、选择性的小分子6PGD抑制剂。用大黄素甲醚或其衍生物S3治疗可以有效地抑制不同人类癌细胞的增殖，而不会产生任何偏离效应。大黄素甲素和S3还有效地抑制了人类患者原代白血病细胞的细胞活性和增殖，毒性最小。此外，S3可显著抑制裸鼠皮下注射人H1299肺癌或K562白血病细胞的肿瘤生长，且毒性最小。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的生长，因此6PGD在临床治疗中是一个新的抗癌靶点。有趣的是，我们还发现，敲除6PGD导致细胞内Ru-5-P(6PGD产物)水平降低，导致AMP激活的蛋白激酶(AMPK)激活，从而抑制乙酰辅酶A羧基酶1(ACC1)，从而抑制脂肪生成。因此，除了PPP和核苷酸生物合成之间已经确立的联系外，6PGD还在PPP、AMPK信号和脂肪生成之间提供了一种新的联系，与6PGD在NADPH产生和氧化还原动态平衡中令人惊讶的关键作用一起，对癌症代谢和肿瘤生长至关重要。我们将使用人类肺癌和白血病(CML、AML和B-ALL)作为平台来测试这些假设。我们提出了三个具体的目标：(1)研究赖氨酸乙酰化是否对6PGD的激活和促进癌细胞代谢和肿瘤生长起重要作用；(2)探讨6PGD如何将PPP、AMPK信号和脂肪生成与氧化还原调节相结合，促进癌细胞代谢和肿瘤生长；(3)利用本实验室开发的6PGD小分子抑制剂，在体内外验证6PGD作为抗白血病靶点的作用。

DESCRIPTION (provided by applicant): 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tissues including colorectal cancers, cervical intraepithelial neoplasia and thyroid tumors, as well as leukemia (our unpublished data).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-taret effect. Physcion and S3 also effectively inhibit cell viability and proliferation of primary leukema 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.