Biology of major folate transporters and STING signaling in cancer

摘要 在哺乳动物细胞中，环二核苷酸（CDN）作为一种突出的危险信号，被先天性免疫缺陷细胞（免疫缺陷细胞）所感知。 免疫传感器，干扰素基因刺激物（STING）。STING的激活启动了信号级联反应， 最终诱导I型干扰素（IFN）和各种细胞因子，从而促进强有力的 炎症反应。哺乳动物细胞能够在环鸟苷的催化下产生自己的CDN 一磷酸腺苷一磷酸（GMP-AMP）合酶（cGAS），其被双链腺苷酸（GMP-AMP）激活。 合成真核生物的cDNA，2′3′-cGAMP。除了细胞内CDN，STING激活CDN可以 来源于细胞外来源，例如治疗性CDN、由肿瘤细胞产生的cGAMP和来自肿瘤细胞的CDN。 胞外细菌cGAS-STING途径在抗肿瘤免疫中的作用支持STING的使用 激动剂作为癌症治疗剂。受2 '3'-cGAMP激活人STING启动发现的启发 合成了稳定的下游IFN信号传导，STING活化剂（主要是2′3′-cGAMP类似物）。 STING的直接药理学激活显示出通过T细胞驱动的肿瘤抑制肿瘤生长。 回归机制和增强免疫原性。在荷瘤小鼠中施用cGAMP还 增强了免疫检查点抑制剂和放疗的治疗效果。根据结果， 在小鼠中的CDN类似物方面，启动了两项I期临床试验用于STING激动剂的肿瘤内递送 （ADU-S100和MK-1454）对实体瘤和淋巴瘤的作用。尽管STING具有令人印象深刻的抗肿瘤活性， 尽管在小鼠中使用了激动剂，但来自此类激动剂的临床试验的初步结果表明在人类中的功效较低。 CDN的进口，出口和降解之间的监管相互作用仍然是一个重要的， 未来研究的有趣领域。外源性2′3′-cGAMP和合成CDN，包括研究用 用于癌症免疫治疗的新药（IND）2′3′-CDAS，通过还原的 叶酸载体（RFC; SLC 19 A1）激活靶细胞中的STING，以及质子偶联叶酸转运蛋白（PCFT; SLC 46 A1）增强这种应答。RFC和PCFT是人体组织中叶酸的主要易化转运蛋白 和肿瘤。PCFT与RFC的独特之处在于它具有酸性而不是中性的最佳pH值。RFC和PCFT 它们具有相同的底物，例如临床上使用的甲氨蝶呤和培美曲塞，但也显示出它们自己独特的底物 特殊性本课题组对RFC和PCFT的结构、功能和调控进行了广泛的研究。在此R 03 应用，我们探索RFC的独特生物学，以及PCFT，和STING激动剂在癌症中，目标是 进一步了解这些促进性转运蛋白对CDN的吸收，并通过扩展， 治疗应用。我们在目标1中提出研究RFC和PCFT的CDN摄取机制， 生理条件。在目标2中，我们将研究RFC与CDN摄取相关的分子调节， 包括转录表观遗传学，以及蛋白质-蛋白质相互作用在调节RFC中的潜在作用。 我们提出的研究因其新奇和临床转化潜力而与众不同。

ABSTRACT In mammalian cells, cyclic dinucleotides (CDNs) act as prominent danger signals that are sensed by the innate immune sensor, stimulator of interferon genes (STING). Activation of STING elicits a signaling cascade that culminates in the induction of type I interferons (IFNs) and various cytokines, thereby promoting a powerful inflammatory response. Mammalian cells are able to produce their own CDNs, catalyzed by the cyclic guanosine monophosphate–adenosine monophosphate (GMP-AMP) synthase (cGAS), which is activated by double-strand DNAs to synthesize the eukaryotic CDN, 2′3′-cGAMP. Beyond intracellular CDNs, STING-activating CDNs can originate from extracellular sources, such as therapeutic CDNs, cGAMP produced by tumor cells, and CDNs from extracellular bacteria. The role of the cGAS-STING pathway in anti-tumor immunity supports the use of STING agonists as cancer therapeutics. Inspired by the discovery that 2′3′-cGAMP activates human STING to initiate robust downstream IFN signaling, STING-activating agents (mostly 2′3′-cGAMP analogues) were synthesized. Direct pharmacologic activation of STING was shown to restrict tumor growth by a T cell-driven tumor regression mechanism and to enhance immunogenicity. Administration of cGAMP in tumor-bearing mice also potentiated the therapeutic effects of immune-checkpoint inhibitors and radiotherapy. Based on results with CDN analogs in mice, two phase I clinical trials were initiated for the intra-tumoral delivery of STING agonists (ADU-S100 and MK-1454) to solid tumors and lymphomas. Despite the impressive anti-tumor activity of STING agonists in mice, initial results from clinical trials of such agonists have indicated a lower efficacy in humans. The regulatory interplay among the import, export and degradation of CDNs remains an important and interesting area for future research. Exogenous 2′3′-cGAMP and synthetic CDNs, including the investigational new drug (IND) 2′3′-CDAS used in cancer immunotherapy, traverse the cell membrane through the reduced folate carrier (RFC; SLC19A1) to activate STING in target cells, and the proton-coupled folate transporter (PCFT; SLC46A1) enhances such response. RFC and PCFT are major facilitative transporters of folate in human tissues and tumors. PCFT is unique from RFC in that it has an acidic rather than neutral pH optimum. RFC and PCFT share substrates such as clinically used methotrexate and pemetrexed but also show their own unique substrate specificities. Our group has studied RFC and PCFT structure, function and regulation extensively. In this R03 application, we explore the unique biology of RFC, as well as PCFT, and STING agonists in cancer, with a goal of further understanding CDN uptake by these facilitative transporters and, by extension, improving therapeutic applications. We propose in Aim 1 to study mechanisms of CDN uptake by RFC and PCFT under physiological conditions. In Aim 2, we will study the molecular regulation of RFC in relation to CDN uptake, including transcriptional epigenetics, and the potential role of protein-protein interactions in regulating RFC. Our proposed studies are distinctive for their novelty and potential for clinical translation.