Signaling and Targeting of 6-Phosphogluconate Dehydrogenase in Human Cancers

描述（由申请人提供）：癌细胞如何协调合成代谢生物合成和氧化还原稳态仍然很大程度上未知。在正常细胞中，6-磷酸葡萄糖酸脱氢酶（6PGD）是氧化戊糖磷酸途径（PPP）中的一种酶，可将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。6PGD活性上调已在几种癌症组织中被报道，包括结直肠癌、宫颈上皮内瘤变、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活性在肿瘤的发病和发展中是否重要，仍然是未知的。我们发现K76和K294的乙酰化增强了6PGD的激活，并且在多种人类癌细胞中普遍观察到。癌细胞中6PGD的稳定敲低导致氧化PPP通量和RNA/DNA生物合成减少。令人惊讶的是，6PGD敲低还会导致NADPH/NADP+比例降低，这表明6PGD在NADPH产生中发挥了重要作用，而其他NADPH产生酶无法弥补这一作用。此外，6PGD敲低的癌细胞表现出ROS水平升高和生物合成异常，导致异种移植裸鼠细胞增殖和肿瘤生长减少。接下来，我们筛选并鉴定了physion作为一种新型的、选择性的小分子6PGD抑制剂。用physion或其衍生物S3治疗可有效抑制多种人类癌细胞的细胞增殖，无脱靶效应。Physcion和S3还能有效抑制人类患者原发性白血病细胞的细胞活力和增殖，毒性很小。此外，S3在皮下注射人H1299肺癌细胞或K562白血病细胞的异种移植裸鼠中显著降低肿瘤生长，体内毒性很小。因此，我们假设赖氨酸乙酰化可以增强6PGD的激活，从而促进癌细胞的增殖和肿瘤的生长；因此，6PGD在临床治疗中代表了一个新的抗癌靶点。有趣的是，我们还发现6PGD的敲除导致细胞内Ru-5-P （6PGD产物）水平的降低，导致amp激活的蛋白激酶（AMPK）的激活，其随后抑制乙酰辅酶a羧化酶1 (ACC1)，从而导致脂肪生成。因此，除了PPP和核苷酸生物合成之间的既定联系外，6PGD还在PPP、AMPK信号传导和脂肪生成之间提供了一种新的联系，这与6PGD在NADPH产生和氧化还原稳态中惊人的关键作用一起，对癌症代谢和肿瘤生长很重要。我们将以人类肺癌和白血病（CML， AML和B-ALL）为平台验证这些假设。我们提出了三个特定的目的(1)研究赖氨酸乙酰化是否在6PGD激活和促进癌细胞代谢和肿瘤生长中起重要作用；(2)探索6PGD如何将PPP、AMPK信号和脂肪生成联系起来，协同氧化还原调控，促进癌细胞代谢和肿瘤生长；(3)利用本实验室研制的6PGD小分子抑制剂，在体外和体内验证6PGD作为抗白血病靶点治疗人白血病细胞。

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tissues including colorectal cancers, cervical intraepithelial neoplasia and thyroid tumors, as well as leukemia (our unpublished data).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