Dr. Margarete Fischer-Bosch Institute of Clinical Pharmacology, Stuttgart, Germany.
University of Tuebingen, Tuebingen, Germany.
Clin Transl Med. 2022 Jun;12(6):e883. doi: 10.1002/ctm2.883.
The metabolic enzyme nicotinamide-N-methyltransferase (NNMT) is highly expressed in various cancer entities, suggesting tumour-promoting functions. We systematically investigated NNMT expression and its metabolic interactions in clear cell renal cell carcinoma (ccRCC), a prominent RCC subtype with metabolic alterations, to elucidate its role as a drug target.
NNMT expression was assessed in primary ccRCC (n = 134), non-tumour tissue and ccRCC-derived metastases (n = 145) by microarray analysis and/or immunohistochemistry. Findings were validated in The Cancer Genome Atlas (kidney renal clear cell carcinoma [KIRC], n = 452) and by single-cell analysis. Expression was correlated with clinicopathological data and survival. Metabolic alterations in NNMT-depleted cells were assessed by nontargeted/targeted metabolomics and extracellular flux analysis. The NNMT inhibitor (NNMTi) alone and in combination with the inhibitor 2-deoxy-D-glucose for glycolysis and BPTES (bis-2-(5-phenylacetamido-1,3,4-thiadiazol-2-yl)ethyl-sulfide) for glutamine metabolism was investigated in RCC cell lines (786-O, A498) and in two 2D ccRCC-derived primary cultures and three 3D ccRCC air-liquid interface models.
NNMT protein was overexpressed in primary ccRCC (p = 1.32 × 10 ) and ccRCC-derived metastases (p = 3.92 × 10 ), irrespective of metastatic location, versus non-tumour tissue. Single-cell data showed predominant NNMT expression in ccRCC and not in the tumour microenvironment. High NNMT expression in primary ccRCC correlated with worse survival in independent cohorts (primary RCC-hazard ratio [HR] = 4.3, 95% confidence interval [CI]: 1.5-12.4; KIRC-HR = 3.3, 95% CI: 2.0-5.4). NNMT depletion leads to intracellular glutamine accumulation, with negative effects on mitochondrial function and cell survival, while not affecting glycolysis or glutathione metabolism. At the gene level, NNMT-depleted cells upregulate glycolysis, oxidative phosphorylation and apoptosis pathways. NNMTi alone or in combination with 2-deoxy-D-glucose and BPTES resulted in inhibition of cell viability in ccRCC cell lines and primary tumour and metastasis-derived models. In two out of three patient-derived ccRCC air-liquid interface models, NNMTi treatment induced cytotoxicity.
Since efficient glutamine utilisation, which is essential for ccRCC tumours, depends on NNMT, small-molecule NNMT inhibitors provide a novel therapeutic strategy for ccRCC and act as sensitizers for combination therapies.
代谢酶烟酰胺-N-甲基转移酶（NNMT）在各种癌症实体中高度表达，提示其具有促进肿瘤的功能。我们系统地研究了 NNMT 在透明细胞肾细胞癌（ccRCC）中的表达及其代谢相互作用，ccRCC 是一种具有代谢改变的突出的 RCC 亚型，以阐明其作为药物靶点的作用。
通过微阵列分析和/或免疫组织化学评估原发性 ccRCC（n=134）、非肿瘤组织和 ccRCC 衍生转移灶（n=145）中的 NNMT 表达。在 The Cancer Genome Atlas（肾透明细胞癌 [KIRC]，n=452）和单细胞分析中验证了这些发现。表达与临床病理数据和生存相关。通过非靶向/靶向代谢组学和细胞外通量分析评估 NNMT 耗尽细胞中的代谢改变。在 RCC 细胞系（786-O、A498）以及两个 2D ccRCC 衍生的原代培养物和三个 3D ccRCC 气液界面模型中，单独研究了 NNMT 抑制剂（NNMTi）以及与 2-脱氧-D-葡萄糖（用于糖酵解）和 BPTES（双-[2-（5-苯乙酰氨基-1,3,4-噻二唑-2-基）乙基]-硫醚）（用于谷氨酰胺代谢）的联合治疗。
NNMT 蛋白在原发性 ccRCC（p=1.32×10-4）和 ccRCC 衍生的转移灶（p=3.92×10-4）中过度表达，与非肿瘤组织相比，无论转移部位如何。单细胞数据显示 ccRCC 中 NNMT 的表达主要，而肿瘤微环境中没有。原发性 ccRCC 中高 NNMT 表达与独立队列中的生存不良相关（原发性 RCC-风险比 [HR] = 4.3，95%置信区间 [CI]：1.5-12.4；KIRC-HR = 3.3，95%CI：2.0-5.4）。NNMT 耗竭导致细胞内谷氨酰胺积累，对线粒体功能和细胞存活产生负面影响，而不影响糖酵解或谷胱甘肽代谢。在基因水平上，NNMT 耗尽的细胞上调糖酵解、氧化磷酸化和细胞凋亡途径。NNMTi 单独或与 2-脱氧-D-葡萄糖和 BPTES 联合使用可抑制 ccRCC 细胞系和原代肿瘤及转移衍生模型中的细胞活力。在三个患者衍生的 ccRCC 气液界面模型中的两个模型中，NNMTi 治疗诱导了细胞毒性。
由于 ccRCC 肿瘤所需的有效谷氨酰胺利用依赖于 NNMT，因此小分子 NNMT 抑制剂为 ccRCC 提供了一种新的治疗策略，并作为联合治疗的增敏剂。
