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While genotoxic chemotherapeutic agents are among the most effective tools to combat cancer, they are often associated with severe adverse effects caused by indiscriminate DNA damage in non-tumor tissue as well as increased risk of secondary carcinogenesis. This study builds on our previous work demonstrating that the RNA Polymerase I (Pol I) transcription inhibitor CX-5461 elicits a non-canonical DNA damage response and our discovery of a critical role for Topoisomerase 2α (Top2α) in the initiation of Pol I-dependent transcription. Here, we identify Top2α as a mediator of CX-5461 response in the murine Eµ-Myc B lymphoma model whereby sensitivity to CX-5461 is dependent on cellular Top2α expression/activity. Most strikingly, and in contrast to canonical Top2α poisons, we found that the Top2α-dependent DNA damage induced by CX-5461 is preferentially localized at the ribosomal DNA (rDNA) promoter region, thereby highlighting CX-5461 as a loci-specific DNA damaging agent. This mechanism underpins the efficacy of CX-5461 against certain types of cancer and can be used to develop effective non-genotoxic anticancer drugs. [ABSTRACT FROM AUTHOR]

RNA polymerase I (Pol I) is responsible for synthesizing the three largest eukaryotic ribosomal RNAs (rRNAs), which form the backbone of the ribosome. Transcription by Pol I is required for cell growth and, therefore, is subject to complex and intricate regulatory mechanisms. To accomplish this robust regulation, the cell engages a series of trans-acting transcription factors. One such factor, high mobility group protein 1 (Hmo1), has long been established as a trans-acting factor for Pol I in Saccharomyces cerevisiae; however, the mechanism by which Hmo1 promotes rRNA synthesis has not been defined. Here, we investigated the effect of the deletion of HMO1 on transcription elongation by Pol I in vivo. We determined that Hmo1 is an important activator of transcription elongation, and without this protein, Pol I accumulates across rDNA in a sequence-specific manner. Our results demonstrate that Hmo1 promotes efficient transcription elongation by rendering Pol I less sensitive to pausing in the G-rich regions of rDNA. [ABSTRACT FROM AUTHOR]

Simple Summary: Building ribosomes for cellular protein translation is a massive, energy-consuming undertaking. The process is executed by all cells to replenish the relevant pools of proteins required for cellular functions. Cancer cells are strikingly dependent on this activity, as they need continuous protein synthesis for sustained proliferation and growth. Ribosome biogenesis requires the activities of three RNA polymerases, which transcribe essential RNAs that make up the backbone of the ribosome, and hundreds of proteins, which provide structural and functional support. Of the three RNA polymerases, RNA polymerase I executes a critical and rate-limiting step by transcribing three key ribosomal RNAs. Pol I transcription is pervasively deregulated in cancers, enabling unlimited protein synthesis. Here, we review this enzyme and provide examples of current efforts to target Pol I transcription therapeutically. RNA polymerase I is a highly processive enzyme with fast initiation and elongation rates. The structure of Pol I, with its in-built RNA cleavage ability and incorporation of subunits homologous to transcription factors, enables it to quickly and efficiently synthesize the enormous amount of rRNA required for ribosome biogenesis. Each step of Pol I transcription is carefully controlled. However, cancers have highjacked these control points to switch the enzyme, and its transcription, on permanently. While this provides an exceptional benefit to cancer cells, it also creates a potential cancer therapeutic vulnerability. We review the current research on the regulation of Pol I transcription, and we discuss chemical biology efforts to develop new targeted agents against this process. Lastly, we highlight challenges that have arisen from the introduction of agents with promiscuous mechanisms of action and provide examples of agents with specificity and selectivity against Pol I. [ABSTRACT FROM AUTHOR]

Simple Summary: Cancer cells upregulate RNA polymerase I (Pol I) activity to increase ribosome abundance in support of rapid cell growth and proliferation. During the last decade, Pol I has emerged as a promising anti-cancer target. Eukaryotes express three closely related RNA polymerases (Pols I, II, and III), responsible for synthesis of all the genome-encoded RNA required by the cell. Effective therapeutic development requires that the treatment be selective for Pol I, without inhibition of Pols II or III. This study evaluates the specificity of the compound BMH-21 on transcription by Pols I, II, and III using purified in vitro transcription assays. These results reveal that Pol I is uniquely sensitive to inhibition by BMH-21, compared to Pols II and III. These findings support ongoing preclinical development of BMH-21 and its derivatives for potential therapeutic applications. Cancer cells require robust ribosome biogenesis to maintain rapid cell growth during tumorigenesis. Because RNA polymerase I (Pol I) transcription of the ribosomal DNA (rDNA) is the first and rate-limiting step of ribosome biogenesis, it has emerged as a promising anti-cancer target. Over the last decade, novel cancer therapeutics targeting Pol I have progressed to clinical trials. BMH-21 is a first-in-class small molecule that inhibits Pol I transcription and represses cancer cell growth. Several recent studies have uncovered key mechanisms by which BMH-21 inhibits ribosome biosynthesis but the selectivity of BMH-21 for Pol I has not been directly measured. Here, we quantify the effects of BMH-21 on Pol I, RNA polymerase II (Pol II), and RNA polymerase III (Pol III) in vitro using purified components. We found that BMH-21 directly impairs nucleotide addition by Pol I, with no or modest effect on Pols II and III, respectively. Additionally, we found that BMH-21 does not affect the stability of any of the Pols' elongation complexes. These data demonstrate that BMH-21 directly exploits unique vulnerabilities of Pol I. [ABSTRACT FROM AUTHOR]

Simple Summary: Cells need to produce ribosomes to sustain continuous proliferation and expand in numbers, a feature that is even more prominent in uncontrollably proliferating cancer cells. Certain cancer cell types are expected to depend more on ribosome biogenesis based on their genetic background, and this potential vulnerability can be exploited in designing effective, targeted cancer therapies. This review provides information on anti-cancer molecules that target the ribosome biogenesis machinery and indicates avenues for future research. 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. [ABSTRACT FROM AUTHOR]

Defects in coordinated ribosomal RNA (rRNA) transcription in the nucleolus cause cellular and organismal growth deficiencies. Bloom's syndrome, an autosomal recessive human disorder caused by mutated recQ-like helicase BLM, presents with growth defects suggestive of underlying defects in rRNA transcription. Our previous studies showed that BLM facilitates rRNA transcription and interacts with RNA polymerase I and topoisomerase I (TOP1) in the nucleolus. The mechanisms regulating localization of BLM to the nucleolus are unknown. In this study, we identify the TOP1-interaction region of BLM by co-immunoprecipitation of in vitro transcribed and translated BLM segments and show that this region includes the highly conserved nuclear localization sequence (NLS) of BLM. Biochemical and nucleolar co-localization studies using site-specific mutants show that two serines within the NLS (S1342 and S1345) are critical for nucleolar localization of BLM but do not affect the functional interaction of BLM with TOP1. Mutagenesis of both serines to aspartic acid (phospho-mimetic), but not alanine (phospho-dead), results in approximately 80% reduction in nucleolar localization of BLM while retaining the biochemical functions and nuclear localization of BLM. Our studies suggest a role for this region in regulating nucleolar localization of BLM via modification of the two serines within the NLS. [ABSTRACT FROM AUTHOR]

Saccharomyces cerevisiae has approximately 200 copies of the 35S rDNA gene, arranged tandemly on chromosome XII. This gene is transcribed by RNA polymerase I (Pol I) and the 35S rRNA transcript is processed to produce three of the four rRNAs required for ribosome biogenesis. An intergenic spacer (IGS) separates each copy of the 35S gene and contains the 5S rDNA gene, the origin of DNA replication, and the promoter for the adjacent 35S gene. Pol I is a 14-subunit enzyme responsible for the majority of rRNA synthesis, thereby sustaining normal cellular function and growth. The A12.2 subunit of Pol I plays a crucial role in cleavage, termination, and nucleotide addition during transcription. Deletion of this subunit causes alteration of nucleotide addition kinetics and read-through of transcription termination sites. To interrogate both of these phenomena, we performed native elongating transcript sequencing (NET-seq) with an rpa12Δ strain of S. cerevisiae and evaluated the resultant change in Pol I occupancy across the 35S gene and the IGS. Compared to wild-type (WT), we observed template sequence-specific changes in Pol I occupancy throughout the 35S gene. We also observed rpa12Δ Pol I occupancy downstream of both termination sites and throughout most of the IGS, including the 5S gene. Relative occupancy of rpa12Δ Pol I increased upstream of the promoter-proximal Reb1 binding site and dropped significantly downstream, implicating this site as a third terminator for Pol I transcription. Collectively, these high-resolution results indicate that the A12.2 subunit of Pol I plays an important role in transcription elongation and termination. [ABSTRACT FROM AUTHOR]

Bud27 is a prefoldin-like, a member of the family of ATP-independent molecular chaperones that associates with RNA polymerases I, II, and III in Saccharomyces cerevisiae . Bud27 and its human ortholog URI perform several functions in the cytoplasm and the nucleus. Both proteins participate in the TOR signaling cascade by coordinating nutrient availability with gene expression, and lack of Bud27 partially mimics TOR pathway inactivation. Bud27 regulates the transcription of the three RNA polymerases to mediate the synthesis of ribosomal components for ribosome biogenesis through the TOR cascade. This work presents a high-copy suppression screening of the temperature sensitivity of the bud27 Δ mutant. It shows that Bud27 influences different TOR-dependent processes. Our data also suggest that Bud27 can impact some of these TOR-dependent processes: cell wall integrity and autophagy induction.

Simple Summary: CX-5461 is an RNA polymerase I inhibitor that is in clinical trials for both advanced hematological cancers and solid tumors. Experimentally, this drug has been shown to induce a p53-independent DNA damage response through ATM and ATR kinase, and has particular activity against chemoresistant tumors. The current study shows for the first time that CX-5461 treatment in ovarian cancer cells induces the release of cytoplasmic DNA that stimulates cGAS–STING signaling, leading to the production of IFN type I in both cancer cells and xenografts in vivo. Because the cGAS–STING pathway is a key mediator of the immune response against cancer cells, this novel finding may lead to utilization of RNA Pol I inhibitors in combination with checkpoint inhibition. Epithelial ovarian cancer (EOC) is the deadliest of the gynecologic malignancies, with an overall survival rate of <30%. Recent research has suggested that targeting RNA polymerase I (POL I) with small-molecule inhibitors may be a viable therapeutic approach to combating EOC, even when chemoresistance is present. CX-5461 is one of the most promising POL I inhibitors currently being investigated, and previous reports have shown that CX-5461 treatment induces DNA damage response (DDR) through ATM/ATR kinase. Investigation into downstream effects of CX-5461 led us to uncovering a previously unreported phenotype. Treatment with CX-5461 induces a rapid accumulation of cytosolic DNA. This accumulation leads to transcriptional upregulation of 'STimulator of Interferon Genes' (STING) in the same time frame, phosphorylation of IRF3, and activation of type I interferon response both in vitro and in vivo. This activation is mediated and dependent on cyclic GMP–AMP synthase (cGAS). Here, we show THAT CX-5461 leads to an accumulation of cytosolic dsDNA and thereby activates the cGAS–STING–TBK1–IRF3 innate immune pathway, which induces type I IFN. CX-5461 treatment-mediated immune activation may be a powerful mechanism of action to exploit, leading to novel drug combinations with a chance of increasing immunotherapy efficacy, possibly with some cancer specificity limiting deleterious toxicities. [ABSTRACT FROM AUTHOR]

In eukaryotes, ribosome biogenesis is driven by the synthesis of the ribosomal RNA (rRNA) by RNA polymerase I (Pol-I) and is tightly linked to cell growth and proliferation. The 3D-structure of the rDNA promoter plays an important, yet not fully understood role in regulating rRNA synthesis. We hypothesized that DNA intercalators/groove binders could affect this structure and disrupt rRNA transcription. To test this hypothesis, we investigated the effect of a number of compounds on Pol-I transcription in vitro and in cells. We find that intercalators/groove binders are potent inhibitors of Pol-I specific transcription both in vitro and in cells, regardless of their specificity and the strength of its interaction with DNA. Importantly, the synthetic ability of Pol-I is unaffected, suggesting that these compounds are not targeting post-initiating events. Notably, the tested compounds have limited effect on transcription by Pol-II and III, demonstrating the hypersensitivity of Pol-I transcription. We propose that stability of pre-initiation complex and initiation are affected as result of altered 3D architecture of the rDNA promoter, which is well in line with the recently reported importance of biophysical rDNA promoter properties on initiation complex formation in the yeast system. [ABSTRACT FROM AUTHOR]

The nucleoli are subdomains of the nucleus that form around actively transcribed ribosomal RNA (rRNA) genes. They serve as the site of rRNA synthesis and processing, and ribosome assembly. There are 400–600 copies of rRNA genes (rDNA) in human cells and their highly repetitive and transcribed nature poses a challenge for DNA repair and replication machineries. It is only in the last 7 years that the DNA damage response and processes of DNA repair at the rDNA repeats have been recognized to be unique and distinct from the classic response to DNA damage in the nucleoplasm. In the last decade, the nucleolus has also emerged as a central hub for coordinating responses to stress via sequestering tumor suppressors, DNA repair and cell cycle factors until they are required for their functional role in the nucleoplasm. In this review, we focus on features of the rDNA repeats that make them highly vulnerable to DNA damage and the mechanisms by which rDNA damage is repaired. We highlight the molecular consequences of rDNA damage including activation of the nucleolar DNA damage response, which is emerging as a unique response that can be exploited in anti-cancer therapy. In this review, we focus on CX-5461, a novel inhibitor of Pol I transcription that induces the nucleolar DNA damage response and is showing increasing promise in clinical investigations. [ABSTRACT FROM AUTHOR]

Cockayne syndrome (CS) is a developmental disorder with symptoms that are typical for the aging body, including subcutaneous fat loss, alopecia, and cataracts. Here, we show that in the cells of CS patients, RNA polymerase I transcription and the processing of the pre-rRNA are disturbed, leading to an accumulation of the 18S-E intermediate. The mature 18S rRNA level is reduced, and isolated ribosomes lack specific ribosomal proteins of the small 40S subunit. Ribosomal proteins are susceptible to unfolding and the CS cell proteome is heat-sensitive, indicating misfolded proteins and an error-prone translation process in CS cells. Pharmaceutical chaperones restored impaired cellular proliferation. Therefore, we provide evidence for severe protein synthesis malfunction, which together with a loss of proteostasis constitutes the underlying pathophysiology in CS. [ABSTRACT FROM AUTHOR]

Nucleoli form around actively transcribed ribosomal RNA (rRNA) genes (rDNA), and the morphology and location of nucleolus-associated genomic domains (NADs) are linked to the RNA Polymerase I (Pol I) transcription status. The number of rDNA repeats (and the proportion of actively transcribed rRNA genes) is variable between cell types, individuals and disease state. Substantial changes in nucleolar morphology and size accompanied by concomitant changes in the Pol I transcription rate have long been documented during normal cell cycle progression, development and malignant transformation. This demonstrates how dynamic the nucleolar structure can be. Here, we will discuss how the structure of the rDNA loci, the nucleolus and the rate of Pol I transcription are important for dynamic regulation of global gene expression and genome stability, e.g., through the modulation of long-range genomic interactions with the suppressive NAD environment. These observations support an emerging paradigm whereby the rDNA repeats and the nucleolus play a key regulatory role in cellular homeostasis during normal development as well as disease, independent of their role in determining ribosome capacity and cellular growth rates. [ABSTRACT FROM AUTHOR]

Ribosomal RNA synthesis is the rate-limiting step in ribosome biogenesis. In eukaryotes, RNA polymerase I (Pol I) is responsible for transcribing the ribosomal DNA genes that reside in the nucleolus. Aberrations in Pol I activity have been linked to the development of multiple cancers and other genetic diseases. Therefore, it is key that we understand the mechanisms of Pol I transcription. Recent studies have demonstrated that there are many differences between Pol I transcription in yeast and mammals. Our goal is to highlight the similarities and differences between the polymerase-associated factors (PAFs) in yeast and mammalian cells. We focus on the PAF heterodimer A49/34 in yeast and PAF53/49 in mammals. Recent studies have demonstrated that while the structures between the yeast and mammalian orthologs are very similar, they may function differently during Pol I transcription, and their patterns of regulation are different. [ABSTRACT FROM AUTHOR]

Ribosome biogenesis is a complex process that is responsible for the formation of ribosomes and ultimately global protein synthesis. The first step in this process is the synthesis of the ribosomal RNA in the nucleolus, transcribed by RNA Polymerase I. Historically, abnormal nucleolar structure is indicative of poor cancer prognoses. In recent years, it has been shown that ribosome biogenesis, and rDNA transcription in particular, is dysregulated in cancer cells. Coupled with advancements in screening technology that allowed for the discovery of novel drugs targeting RNA Polymerase I, this transcriptional machinery is an increasingly viable target for cancer therapies. In this review, we discuss ribosome biogenesis in breast cancer and the different cellular pathways involved. Moreover, we discuss current therapeutics that have been found to affect rDNA transcription and more novel drugs that target rDNA transcription machinery as a promising avenue for breast cancer treatment. [ABSTRACT FROM AUTHOR]

RNA polymerases (Pols) I, II, and III collectively synthesize most of the RNA in a eukaryotic cell. Transcription by Pols I, II, and III is regulated by hundreds of trans-acting factors. One such protein, Spt4, has been previously identified as a transcription factor that influences both Pols I and II. Spt4 forms a complex with Spt5, described as the Spt4/5 complex (or DSIF in mammalian cells). This complex has been shown previously to directly interact with Pol I and potentially affect transcription elongation. The previous literature identified defects in transcription by Pol I when SPT4 was deleted, but the necessary tools to characterize the mechanism of this effect were not available at the time. Here, we use a technique called Native Elongating Transcript Sequencing (NET-seq) to probe for the global occupancy of Pol I in wild-type (WT) and spt4△ Saccharomyces cerevisiae (yeast) cells at single nucleotide resolution in vivo. Analysis of NET-seq data reveals that Spt4 promotes Pol I processivity and enhances transcription elongation through regions of the ribosomal DNA that are particularly G-rich. These data suggest that Spt4/5 may directly affect transcription elongation by Pol I in vivo. [ABSTRACT FROM AUTHOR]

The nucleolus organizes around the sites of transcription by RNA polymerase I (RNA Pol I). rDNA transcription by this enzyme is the key step of ribosome biogenesis and most of the assembly and maturation processes of the ribosome occur co-transcriptionally. Therefore, disturbances in rRNA transcription and processing translate to ribosomal malfunction. Nucleolar malfunction has recently been described in the classical progeria of childhood, Hutchinson–Gilford syndrome (HGPS), which is characterized by severe signs of premature aging, including atherosclerosis, alopecia, and osteoporosis. A deregulated ribosomal biogenesis with enlarged nucleoli is not only characteristic for HGPS patients, but it is also found in the fibroblasts of "normal" aging individuals. Cockayne syndrome (CS) is also characterized by signs of premature aging, including the loss of subcutaneous fat, alopecia, and cataracts. It has been shown that all genes in which a mutation causes CS, are involved in rDNA transcription by RNA Pol I. A disturbed ribosomal biogenesis affects mitochondria and translates into ribosomes with a reduced translational fidelity that causes endoplasmic reticulum (ER) stress and apoptosis. Therefore, it is speculated that disease-causing disturbances in the process of ribosomal biogenesis may be more common than hitherto anticipated. [ABSTRACT FROM AUTHOR]