Investigating the NRAS 5’ UTR as a Target for Small Molecules

Neuroblastoma RAS (NRAS) is an oncogene that is deregulated and highly mutated in cancers including melanomas and acute myeloid leukemias. Constitutively activated NRAS induces the MAPK and AKT signaling pathways and leads to uncontrolled proliferation and cell growth, making it an attractive target for small molecule inhibition. Like all RAS-family proteins, it has proven difficult to identify small molecules that directly inhibit the protein. An alternative approach would involve targeting the NRAS mRNA. The 5′ untranslated region (5′ UTR) of the NRAS mRNA is reported to contain a G-quadruplex (G4) that regulates the translation of NRAS mRNA. Stabilizing the G4 structure in NRAS by small molecules could reduce NRAS protein expression in cancer cells by impacting translation. Here we report a novel class of small molecule that binds to the G4 structure located in the 5′ UTR of the NRAS mRNA. We used a small molecule microarray (SMM) screen to identify molecules that selectively bind to the NRAS-G4. Biophysical studies demonstrated that compound 18 binds reversibly to the NRAS-G4 structure with submicromolar affinity. A Luciferase based reporter assay indicated that 18 inhibits the translation of NRAS via stabilizing the NRAS-G4 in vitro but showed only moderate effects on the NRAS levels in cellulo. Rapid Amplification of cDNA Ends (RACE), RT-PCR analysis on 14 different NRAS-expressing cell lines, coupled with analysis of publicly available CAGE seq experiments, revealed that predominant NRAS transcript does not possess the G4 structure. Further analysis of published rG4 and G4 sequencing data indicated the presence of G4 structure in the promoter region of NRAS gene (DNA) but not in the mRNA. Thus, although the NRAS transcript lacks a G4 in many cell lines the broader concept of targeting folded regions within 5’ UTRs to control translation remains a highly attractive strategy. RNA-seq, SHAPE-MaP.

神经母细胞瘤RAS基因（Neuroblastoma RAS, NRAS）是一类致癌基因，在黑色素瘤、急性髓系白血病等多种癌症中存在表达失调与高频突变的特征。组成型激活的NRAS可激活丝裂原活化蛋白激酶（Mitogen-Activated Protein Kinase, MAPK）与AKT信号通路，引发细胞不受控增殖与异常生长，使其成为小分子抑制剂研发的理想靶点。与所有RAS家族蛋白一致，直接靶向NRAS蛋白的小分子抑制剂开发已被证实极具挑战。另一种可行的干预策略为靶向NRAS mRNA。
研究表明，NRAS mRNA的5'非翻译区（5′ untranslated region, 5′ UTR）含有可调控NRAS翻译过程的G-四链体（G-quadruplex, G4）结构。通过小分子稳定NRAS mRNA的G4结构，可通过干扰翻译过程降低癌细胞内NRAS蛋白的表达水平。本研究报道了一类全新的小分子化合物，可特异性结合NRAS mRNA 5' UTR区域的G4结构。研究人员通过小分子微阵列（small molecule microarray, SMM）筛选，鉴定出可选择性结合NRAS-G4的化合物。生物物理实验证实，化合物18可与NRAS-G4结构以亚微摩尔亲和力可逆结合。基于荧光素酶（Luciferase）的报告基因实验显示，化合物18在体外可通过稳定NRAS-G4结构抑制NRAS翻译，但在细胞内对NRAS蛋白水平的抑制效果仅为中等程度。
对14株表达NRAS的细胞系开展cDNA末端快速扩增（Rapid Amplification of cDNA Ends, RACE）与逆转录PCR（RT-PCR）分析，并结合公开的CAGE测序（CAGE-seq）实验数据，结果显示，主流NRAS转录本并不含有G4结构。对已发表的rG4与G4测序数据的进一步分析表明，G4结构存在于NRAS基因的启动子区域（DNA层面），而非mRNA层面。综上，尽管多数细胞系中的NRAS转录本并不含G4结构，但靶向5' UTR内折叠区域以调控翻译的核心策略仍具备极高的研究价值。本研究涉及的实验技术包括RNA测序（RNA-seq）与选择性2'-羟基酰化分析高通量测序（SHAPE-MaP）。