Targeting RNA polymerase I transcription: Uncovering long non-coding RNAS-mediated mechanisms and small molecule inhibitor for cancer therapy

Abstract

RNA Polymerase I (Pol I) plays a pivotal role in ribosome biogenesis by transcribing ribosomal DNA (rDNA) into ribosomal RNA (rRNA), a critical step regulating protein synthesis, cell division, growth, proliferation, differentiation, and apoptosis [1]. Notably, hyperactive Pol I transcription and upregulation of Pol I transcription machinery are common aberrations in cancer. In addition, N6-methyladenosine modification in 18S rRNA, has shown to be a key factor in tumorigenesis and chemoresistance [2]. However, the complex molecular processes, particularly those involving regulatory RNAs, that drive the hyperactivation of Pol I transcription in cancer remain poorly understood. The Pol I enzyme relies on class-specific transcription factors, including Selectivity Factor (SL1) and Upstream Binding Factor (UBF) and RRN3, for initiation. SL1 consists of a TATA-binding protein (TBP) and four TBP-associated factors (TAF1A-D), and is critical in pre-initiation complex (PIC) formation by recognizing and binding to the rDNA core promoter. Critical interactions between SL1 and UBF enhance the stability of UBF binding at the promoter, facilitating the recruitment of the Pol Iβ complex to the transcription start site via RRN3 [3]. Aberrant upregulation of Pol I-specific transcription factors and hyperactive Pol I transcription are common in cancer, promoting various hallmarks of cancer, including therapeutic resistance [4]. Our previous work has identified a novel post-transcriptional regulatory pathway involving microRNAs and circular RNA that contributes to the upregulation of PIC components in lung adenocarcinoma (LUAD) [5]. Recent studies have implied RRN3 as a pivotal regulator of cancer cell proliferation and tumor growth. Remarkably, silencing RRN3 in breast cancer cells significantly impeded rRNA transcription and cell proliferation, which were restored by RRN3 overexpression, demonstrating the essential role of RRN3 in cancer cell growth [6]. Similarly, RRN3 inhibition in pancreatic cancer xenograft models markedly reduced rRNA transcription and tumor burden [7]. These studies underscore its potential as a vulnerable target for cancer therapy This thesis aims to elucidate novel molecular mechanisms and therapeutic strategies to inhibit Pol I transcription under specific aims as below. A. Investigate the molecular mechanisms underlying long non-coding RNAs (lncRNA)-mediated regulation of Pol I transcription in cancer cells. B. Identify and characterize small molecule inhibitors that specifically target RRN3 for the inhibition of Pol I transcription. Results A. Investigate the molecular mechanisms and therapeutic potential of lncRNA-mediated regulation of Pol I transcription in cancer cells. Despite the established role of Pol I transcription in cancer, the underlying regulatory mechanisms remain poorly understood. A recent study from our lab showed miRNA-mediated post-transcriptional regulation, leading to the upregulation of Pol I transcription in cancer [5]. However, novel regulatory mechanisms involving other non-coding RNAs resulting in the upregulation of Pol I transcription in LUAD remain largely unexplored. Mounting evidence highlights the crucial role of lncRNAs in cancer [8, 9]. This part of the study aims to elucidate lncRNA-mediated molecular mechanisms underlying Pol I transcription and investigate the therapeutic potential of targeting lncRNA-Pol I transcription axis in cancer. This study reveals LINC01116 as a critical regulator of Pol I transcription in LUAD, scaffolding essential transcription factors TAF1A and TAF1D to the rDNA promoter. Through RNA immunoprecipitation, RNA-FISH, and Chromatin isolation by RNA purification (ChIRP) assays, we demonstrated LINC01116 direct interaction with SL1 subunits, nucleolar localization, and association with transcriptionally active rDNA promoter. Functional analyses showed that LINC01116 overexpression enhances SL1 recruitment, Pol I transcription, and 47S rRNA synthesis, while knockdown reduces SL1 promoter assembly and rRNA synthesis. Notably, specific inhibition of Pol I transcription completely abrogated LINC01116-mediated oncogenic phenotypes. This underscores the Pol I transcription dependency of LINC01116 oncogenic functions, establishing a causal link between LINC01116-driven Pol I transcription activation and its tumorigenic effects in lung adenocarcinoma. In addition, c-Myc was identified as a key upstream activator of LINC01116 expression, linking this lncRNA to broader oncogenic signaling networks. These findings highlight the therapeutic potential of targeting the Pol I transcription machinery, particularly the LINC01116-SL1 axis, in LUAD, offering novel strategies for cancer treatment. B. Identify and characterize small molecule inhibitors that specifically target RRN3 for the inhibition of Pol I transcription. Pol I transcription inhibition offers a promising cancer therapeutic strategy [10], but existing inhibitors are limited by specificity, toxicity, and mutagenic properties [11, 12]. Novel, highly specific, and potent inhibitors are needed to tap the full therapeutic potential of Pol I inhibition. In this study, we have developed a novel small molecule, NSH76, as a potent and selective inhibitor of RRN3. NSH76 specifically inhibited RRN3 recruitment to the rDNA promoter, significantly inhibiting Pol I transcription without affecting Pol II transcription. The selective inhibition of Pol I transcription by NSH76 resulted in significant antiproliferative effects in cancer cells, while normal cells were minimally affected. Cancer cells treated with NSH76 exhibited G1 arrest, activation of apoptosis, and upregulation of the p53 pathway, underscoring its ability to trigger anti-cancer response. Furthermore, NSH76 demonstrated potent cytotoxic effects in cisplatin and doxorubicin-resistant to A549 cells. Notably, NSH76 showed a favorable safety profile, as it did not induce DNA damage or related genotoxicity, which are major limitations of current Pol I inhibitors. These findings highlight the advantages of NSH76 over existing Pol I inhibitors. In conclusion, our results suggest that NSH76-mediated inhibition of RRN3 represents a novel, selective, and effective strategy for targeting Pol I transcription in cancer.