RNA polymerase I inhibition : mechanism and exploitation in cancer treatment

Cancer is an umbrella term for diseases characterized by uncontrollably proliferating abnormal cells that often have also gained the ability to spread and invade other tissues. It is one of the leading causes of death worldwide and the second-leading cause of death in Sweden. Chemotherapy is a commonly used treatment approach, where the drugs preferentially target cellular processes needed for cancer cell proliferation, leading to cancer cell growth arrest or death. Albeit a potent tool in managing cancer, the overall success rate remains low for certain cancer types, highlighting the need to identify new chemotherapeutic targets and strategies. Ribosome biogenesis (RiBi), a fundamental process that supplies cells with ribosomes, represents an emerging target, with several cancer types relying on high RiBi rates to maintain high proliferation rates. Small-molecule-mediated RiBi inhibition induces nucleolar stress, a cellular response resulting in cell cycle arrest, and apoptosis, often dependent on p53. Pre-clinical studies have shown promising results in a variety of cancer types; however, the compounds available are limited, and their mechanistic details are yet to be explored. Thus, the characterization of cancer-specific biological effects of RiBi inhibition, together with the identification of new RiBi targets and inhibitors, may expand the therapeutic promise of this strategy, accelerate the clinical development of drug candidates and potentially facilitate the selection of patients who might benefit from the clinical use of RiBi inhibitors in the future. The primary aim of the Thesis was to study: 1. the pharmacological inhibition of RiBi focusing on RNA polymerase I (Pol I), and repurposing of clinically approved drugs with underappreciated RiBi-inhibitory effects for cancer treatment 2. the effects of Pol I inhibition in high-grade gliomas (HGG) and identify synergistic treatment strategies to prevent potential resistance development 3. alternative druggable RiBi-associated protein targets In Paper I, we identified an FDA-approved antimalarial drug, amodiaquine, with previously unknown Pol I inhibitory effects. We designed and synthesized a chemical analog with comparable efficacy to limit potential toxicity and demonstrated the effectiveness of the analog series in a panel of colorectal cancer cell lines. In Paper II, we reported the relevance and effectiveness of RiBi as a target in HGG, uncovered a novel cellular response to nucleolar stress, mediated by the Fibroblast Growth Factor 2 (FGF2)- Fibroblast Growth factor receptor 1 (FGFR1) signaling axis, and proposed a highly synergistic combination with FGFR inhibitors to limit glioma cell growth. In Paper III, we further characterized the functional role of the DEAD-Box Helicase and Exon Junction Complex protein, eIF4A3, and suggested its relevance as a target for drug discovery, showing its involvement in RiBi and highlighting its association with tumor aggressiveness

Actionable cancer vulnerability due to translational arrest, p53 aggregation and ribosome biogenesis stress evoked by the disulfiram metabolite CuET.

We would like to thank M.Oren (Weizmann Institute of Science) for kindly providing the MDM2 antibodies, the core facility for Bioinformatics and Expression Analysis (BEA, Karolinska, Huddinge) for assisting in massive parallel sequencing and computational infrastructure, as well as E Dratkiewicz, AS Nilsson, and JF Martinez for excellent technical assistance.Drug repurposing is a versatile strategy to improve current therapies. Disulfiram has long been used in the treatment of alcohol dependency and multiple clinical trials to evaluate its clinical value in oncology are ongoing. We have recently reported that the disulfiram metabolite diethyldithiocarbamate, when combined with copper (CuET), targets the NPL4 adapter of the p97VCP segregase to suppress the growth of a spectrum of cancer cell lines and xenograft models in vivo. CuET induces proteotoxic stress and genotoxic effects, however important issues concerning the full range of the CuET-evoked tumor cell phenotypes, their temporal order, and mechanistic basis have remained largely unexplored. Here, we have addressed these outstanding questions and show that in diverse human cancer cell models, CuET causes a very early translational arrest through the integrated stress response (ISR), later followed by features of nucleolar stress. Furthermore, we report that CuET entraps p53 in NPL4-rich aggregates leading to elevated p53 protein and its functional inhibition, consistent with the possibility of CuET-triggered cell death being p53-independent. Our transcriptomics profiling revealed activation of pro-survival adaptive pathways of ribosomal biogenesis (RiBi) and autophagy upon prolonged exposure to CuET, indicating potential feedback responses to CuET treatment. The latter concept was validated here by simultaneous pharmacological inhibition of RiBi and/or autophagy that further enhanced CuET's tumor cytotoxicity, using both cell culture and zebrafish in vivo preclinical models. Overall, these findings expand the mechanistic repertoire of CuET's anti-cancer activity, inform about the temporal order of responses and identify an unorthodox new mechanism of targeting p53. Our results are discussed in light of cancer-associated endogenous stresses as exploitable tumor vulnerabilities and may inspire future clinical applications of CuET in oncology, including combinatorial treatments and focus on potential advantages of using certain validated drug metabolites, rather than old, approved drugs with their, often complex, metabolic profiles.This work was funded by the following grants: the Swedish Cancer Society (grant number: 170176), the Swedish Research Council (VR-MH 2014-46602-117891-30), Novo Nordisk Foundation (NNF20OC0060590), Danish National Research Foundation (project CARD, DNRF 125), the Danish Cancer Society (R204-A12617-B153), DFF 1026-00241B (all granted to JB), and the Grant agency of the Czech Republic: GACR 20-28685S (granted to ZS and MM). Open access funding provided by Karolinska Institute.S

Targeting Ribosome Biogenesis in Cancer: Lessons Learned and Way Forward

Rapid growth and unrestrained proliferation is a hallmark of many cancers. To accomplish this, cancer cells re-wire and increase their biosynthetic and metabolic activities, including ribosome biogenesis (RiBi), a complex, highly energy-consuming process. Several chemotherapeutic agents used in the clinic impair this process by interfering with the transcription of ribosomal RNA (rRNA) in the nucleolus through the blockade of RNA polymerase I or by limiting the nucleotide building blocks of RNA, thereby ultimately preventing the synthesis of new ribosomes. Perturbations in RiBi activate nucleolar stress response pathways, including those controlled by p53. While compounds such as actinomycin D and oxaliplatin effectively disrupt RiBi, there is an ongoing effort to improve the specificity further and find new potent RiBi-targeting compounds with improved pharmacological characteristics. A few recently identified inhibitors have also become popular as research tools, facilitating our advances in understanding RiBi. Here we provide a comprehensive overview of the various compounds targeting RiBi, their mechanism of action, and potential use in cancer therapy. We discuss screening strategies, drug repurposing, and common problems with compound specificity and mechanisms of action. Finally, emerging paths to discovery and avenues for the development of potential biomarkers predictive of therapeutic outcomes across cancer subtypes are also presented