ABSTRACT
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
The heterogeneous composition of cellular transcriptomes poses a major challenge for detecting weakly expressed RNA classes, as they can be obscured by abundant RNAs. Although biochemical protocols can enrich or deplete specified RNAs, they are time-consuming, expensive and can compromise RNA integrity. Here we introduce RISER, a biochemical-free technology for the real-time enrichment or depletion of RNA classes. RISER performs selective rejection of molecules during direct RNA sequencing by identifying RNA classes directly from nanopore signals with deep learning and communicating with the sequencing hardware in real time. By targeting the dominant messenger and mitochondrial RNA classes for depletion, RISER reduces their respective read counts by more than 85%, resulting in an increase in sequencing depth of 47% on average for long non-coding RNAs. We also apply RISER for the depletion of globin mRNA in whole blood, achieving a decrease in globin reads by more than 90% as well as an increase in non-globin reads by 16% on average. Furthermore, using a GPU or a CPU, RISER is faster than GPU-accelerated basecalling and mapping. RISER's modular and retrainable software and intuitive command-line interface allow easy adaptation to other RNA classes. RISER is available at https://github.com/comprna/riser .
Subject(s)
RNA, Messenger , Sequence Analysis, RNA , Sequence Analysis, RNA/methods , Humans , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA, Long Noncoding/genetics , RNA/genetics , Software , Globins/genetics , High-Throughput Nucleotide Sequencing/methods , Deep Learning , Transcriptome , RNA, Mitochondrial/genetics , RNA, Mitochondrial/metabolismABSTRACT
The high rates of protein synthesis and processing render multiple myeloma (MM) cells vulnerable to perturbations in protein homeostasis. The induction of proteotoxic stress by targeting protein degradation with proteasome inhibitors (PIs) has revolutionized the treatment of MM. However, resistance to PIs is inevitable and represents an ongoing clinical challenge. Our first-in-human study of the selective inhibitor of RNA polymerase I transcription of ribosomal RNA genes, CX-5461, has demonstrated a potential signal for anti-tumor activity in three of six heavily pre-treated MM patients. Here, we show that CX-5461 has potent anti-myeloma activity in PI-resistant MM preclinical models in vitro and in vivo. In addition to inhibiting ribosome biogenesis, CX-5461 causes topoisomerase II trapping and replication-dependent DNA damage, leading to G2/M cell-cycle arrest and apoptotic cell death. Combining CX-5461 with PI does not further enhance the anti-myeloma activity of CX-5461 in vivo. In contrast, CX-5461 shows synergistic interaction with the histone deacetylase inhibitor panobinostat in both the Vk∗MYC and the 5T33-KaLwRij mouse models of MM by targeting ribosome biogenesis and protein synthesis through distinct mechanisms. Our findings thus provide strong evidence to facilitate the clinical development of targeting the ribosome to treat relapsed and refractory MM.
ABSTRACT
DNA methylation plays essential roles in regulating physiological processes, from tissue and organ development to gene expression and aging processes and has emerged as a widely used biomarker for the identification of body fluids and age prediction. Currently, methylation markers are targeted independently at specific CpG sites as part of a multiplexed assay rather than through a unified assay. Methylation detection is also dependent on divergent methodologies, ranging from enzyme digestion and affinity enrichment to bisulfite treatment, alongside various technologies for high-throughput profiling, including microarray and sequencing. In this pilot study, we test the simultaneous identification of age-associated and body fluid-specific methylation markers using a single technology, nanopore adaptive sampling. This innovative approach enables the profiling of multiple CpG marker sites across entire gene regions from a single sample without the need for specialized DNA preparation or additional biochemical treatments. Our study demonstrates that adaptive sampling achieves sufficient coverage in regions of interest to accurately determine the methylation status, shows a robust consistency with whole-genome bisulfite sequencing data, and corroborates known CpG markers of age and body fluids. Our work also resulted in the identification of new sites strongly correlated with age, suggesting new possible age methylation markers. This study lays the groundwork for the systematic development of nanopore-based methodologies in both age prediction and body fluid identification, highlighting the feasibility and potential of nanopore adaptive sampling while acknowledging the need for further validation and expansion in future research.
Subject(s)
Aging , CpG Islands , DNA Methylation , Humans , CpG Islands/genetics , Pilot Projects , Genetic Markers , Aging/genetics , Adult , Nanopores , Middle Aged , Aged , Sequence Analysis, DNA , Male , Saliva/chemistry , Female , Young Adult , Nanopore Sequencing , Semen/chemistryABSTRACT
Mouse embryonic fibroblasts (MEFs) are primary fibroblasts purified from mouse embryos at a defined time post-fertilization. MEFs have versatile applications, including use as feeder cell layers or sources of untransformed primary cells for a variety of biological assays. MEFs are most commonly isolated between embryonic day (E)12.5 and E13.5 but can be isolated from embryos as early as E8.5 and as late as E15.5. The individual embryos are harvested by carefully removing uterine tissue, yolk sac, and placenta. The embryos are euthanized, and non-mesenchymal tissues, such as the fetal liver and heart, are removed before tissue homogenization. The remaining fetal tissue is homogenized by mechanical mincing using a sterile blade, followed by enzymatic digestion and resuspension. During tissue dissociation, the duration of trypsin-EDTA/DNase digestion and enzyme concentration are critical parameters to produce high-quality MEFs with the highest rates of cell viability and proliferation potential. MEFs can be cryopreserved at passage (P) 0 if >80% confluent, passaged for further expansion before freezing down, or directly utilized for downstream applications, i.e., preparation as feeder cell layers. Primary MEFs possess a limited proliferation capacity of â¼20 cell divisions, beyond which the percentage of senescent cells rapidly increases; thus, cultures should only be expanded/passaged to a maximum of P5. Critical for cell viability during cryopreservation and thawing of MEFs is the slow decrease in temperature when freezing, the rapid increase when thawing, the use of a cryoprotective agent, and an optimal cell density. While it is critical to generate high-quality MEFs to standardize and optimize preparation procedures and utilize fresh reagents, some variability in proliferation capacity and cell viability between MEF preparations remains. Thus, MEF preparation, culture, and cryopreservation procedures are continuously being optimized. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Purification, passaging, and expansion of MEFs Supporting Protocol: Cryopreservation and thawing of MEFs.
Subject(s)
Embryonic Stem Cells , Fibroblasts , Pregnancy , Female , Animals , Mice , Feeder Cells , Cryoprotective Agents , Cryopreservation/methodsABSTRACT
The survival rate of patients with osteosarcoma (OS) has not improved over the last 30 years. Mutations in the genes TP53, RB1 and c-Myc frequently occur in OS and enhance RNA Polymerase I (Pol I) activity, thus supporting uncontrolled cancer cell proliferation. We therefore hypothesised that Pol I inhibition may be an effective therapeutic strategy for this aggressive cancer. The Pol I inhibitor CX-5461 has demonstrated therapeutic efficacy in different cancers in pre-clinical and phase I clinical trials; thus, the effects were determined on ten human OS cell lines. Following characterisation using genome profiling and Western blotting, RNA Pol I activity, cell proliferation and cell cycle progression were evaluated in vitro, and the growth of TP53 wild-type and mutant tumours was measured in a murine allograft model and in two human xenograft OS models. CX-5461 treatment resulted in reduced ribosomal DNA (rDNA) transcription and Growth 2 (G2)-phase cell cycle arrest in all OS cell lines. Additionally, tumour growth in all allograft and xenograft OS models was effectively suppressed without apparent toxicity. Our study demonstrates the efficacy of Pol I inhibition against OS with varying genetic alterations. This study provides pre-clinical evidence to support this novel therapeutic approach in OS.
ABSTRACT
The nucleolar surveillance pathway monitors nucleolar integrity and responds to nucleolar stress by mediating binding of ribosomal proteins to MDM2, resulting in p53 accumulation. Inappropriate pathway activation is implicated in the pathogenesis of ribosomopathies, while drugs selectively activating the pathway are in trials for cancer. Despite this, the molecular mechanism(s) regulating this process are poorly understood. Using genome-wide loss-of-function screens, we demonstrate the ribosome biogenesis axis as the most potent class of genes whose disruption stabilizes p53. Mechanistically, we identify genes critical for regulation of this pathway, including HEATR3. By selectively disabling the nucleolar surveillance pathway, we demonstrate that it is essential for the ability of all nuclear-acting stresses, including DNA damage, to induce p53 accumulation. Our data support a paradigm whereby the nucleolar surveillance pathway is the central integrator of stresses that regulate nuclear p53 abundance, ensuring that ribosome biogenesis is hardwired to cellular proliferative capacity.
Subject(s)
Proto-Oncogene Proteins c-mdm2 , Tumor Suppressor Protein p53 , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Protein p53/metabolism , Proto-Oncogene Proteins c-mdm2/genetics , Proto-Oncogene Proteins c-mdm2/metabolism , Signal Transduction/genetics , Cell Nucleolus/metabolism , Ribosomal Proteins/genetics , Ribosomal Proteins/metabolismABSTRACT
BACKGROUND: Uterine leiomyosarcoma is a rare aggressive smooth muscle cancer with poor survival rates. RNA Polymerase I (Pol I) activity is elevated in many cancers supporting tumour growth and prior studies in uterine leiomyosarcoma revealed enlarged nucleoli and upregulated Pol I activity-related genes. This study aimed to investigate the anti-tumour potential of CX-5461, a Pol I transcription inhibitor currently being evaluated in clinical trials for several cancers, against the human uterine leiomyosarcoma cell line, SK-UT-1. METHODS: SK-UT-1 was characterised using genome profiling and western blotting. The anti-tumour effects of CX-5461 were investigated using cell proliferation assays, expression analysis using qRT-PCR, and BrdU/PI based cell cycle analysis. RESULTS: Genetic analysis of SK-UT-1 revealed mutations in TP53, RB1, PTEN, APC and TSC1 & 2, all potentially associated with increased Pol I activity. Protein expression analysis showed dysregulated p53, RB1 and c-Myc. CX-5461 treatment resulted in an anti-proliferation response, G2 phase cell-cycle arrest and on-target activity demonstrated by reduced ribosomal DNA transcription. CONCLUSIONS: SK-UT-1 was confirmed as a representative model of uterine leiomyosarcoma and CX-5461 has significant potential as a novel adjuvant for this rare cancer.
Subject(s)
Benzothiazoles , Leiomyosarcoma , Naphthyridines , Uterine Neoplasms , Benzothiazoles/pharmacology , Cell Line, Tumor , Cell Proliferation/drug effects , Enzyme Inhibitors/pharmacology , Female , Humans , Leiomyosarcoma/drug therapy , Leiomyosarcoma/genetics , Leiomyosarcoma/metabolism , Naphthyridines/pharmacology , RNA Polymerase I/antagonists & inhibitors , RNA Polymerase I/metabolism , Signal Transduction/drug effects , Uterine Neoplasms/drug therapy , Uterine Neoplasms/genetics , Uterine Neoplasms/metabolismABSTRACT
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.
Subject(s)
Genetic Loci/genetics , Ribosomes/genetics , Animals , Cell Cycle/genetics , Cell Nucleolus/genetics , Genome/genetics , Genomic Instability/genetics , Homeostasis/genetics , Humans , Transcription, Genetic/geneticsABSTRACT
Hyperactivation of RNA polymerase I (Pol I) transcription of ribosomal RNA (rRNA) genes (rDNA) is a key determinant of growth and proliferation and a consistent feature of cancer cells. We have demonstrated that inhibition of rDNA transcription by the Pol I transcription inhibitor CX-5461 selectively kills tumor cells in vivo. Moreover, the first-in human trial of CX-5461 has demonstrated CX-5461 is well-tolerated in patients and has single-agent anti-tumor activity in hematologic malignancies. However, the mechanisms underlying tumor cell sensitivity to CX-5461 remain unclear. Understanding these mechanisms is crucial for the development of predictive biomarkers of response that can be utilized for stratifying patients who may benefit from CX-5461. The rDNA repeats exist in four different and dynamic chromatin states: inactive rDNA can be either methylated silent or unmethylated pseudo-silent; while active rDNA repeats are described as either transcriptionally competent but non-transcribed or actively transcribed, depending on the level of rDNA promoter methylation, loading of the essential rDNA chromatin remodeler UBF and histone marks status. In addition, the number of rDNA repeats per human cell can reach hundreds of copies. Here, we tested the hypothesis that the number and/or chromatin status of the rDNA repeats, is a critical determinant of tumor cell sensitivity to Pol I therapy. We systematically examined a panel of ovarian cancer (OVCA) cell lines to identify rDNA chromatin associated biomarkers that might predict sensitivity to CX-5461. We demonstrated that an increased proportion of active to inactive rDNA repeats, independent of rDNA copy number, determines OVCA cell line sensitivity to CX-5461. Further, using zinc finger nuclease genome editing we identified that reducing rDNA copy number leads to an increase in the proportion of active rDNA repeats and confers sensitivity to CX-5461 but also induces genome-wide instability and sensitivity to DNA damage. We propose that the proportion of active to inactive rDNA repeats may serve as a biomarker to identify cancer patients who will benefit from CX-5461 therapy in future clinical trials. The data also reinforces the notion that rDNA instability is a threat to genomic integrity and cellular homeostasis.
ABSTRACT
RNA polymerase I (Pol I) transcription of ribosomal RNA genes (rDNA) is tightly regulated downstream of oncogenic pathways, and its dysregulation is a common feature in cancer. We evaluated CX-5461, the first-in-class selective rDNA transcription inhibitor, in a first-in-human, phase I dose-escalation study in advanced hematologic cancers. Administration of CX-5461 intravenously once every 3 weeks to 5 cohorts determined an MTD of 170 mg/m2, with a predictable pharmacokinetic profile. The dose-limiting toxicity was palmar-plantar erythrodysesthesia; photosensitivity was a dose-independent adverse event (AE), manageable by preventive measures. CX-5461 induced rapid on-target inhibition of rDNA transcription, with p53 activation detected in tumor cells from one patient achieving a clinical response. One patient with anaplastic large cell lymphoma attained a prolonged partial response and 5 patients with myeloma and diffuse large B-cell lymphoma achieved stable disease as best response. CX-5461 is safe at doses associated with clinical benefit and dermatologic AEs are manageable. SIGNIFICANCE: CX-5461 is a first-in-class selective inhibitor of rDNA transcription. This first-in-human study establishes the feasibility of targeting this process, demonstrating single-agent antitumor activity against advanced hematologic cancers with predictable pharmacokinetics and a safety profile allowing prolonged dosing. Consistent with preclinical data, antitumor activity was observed in TP53 wild-type and mutant malignancies.This article is highlighted in the In This Issue feature, p. 983.
Subject(s)
Antineoplastic Agents/therapeutic use , Benzothiazoles/therapeutic use , Hematologic Neoplasms/drug therapy , Hematologic Neoplasms/genetics , Naphthyridines/therapeutic use , RNA Polymerase I/metabolism , Transcription, Genetic/drug effects , Adult , Aged , Antineoplastic Agents/administration & dosage , Antineoplastic Agents/adverse effects , Antineoplastic Agents/pharmacokinetics , Benzothiazoles/administration & dosage , Benzothiazoles/adverse effects , Benzothiazoles/pharmacology , DNA, Ribosomal/genetics , Female , Hematologic Neoplasms/diagnosis , Hematologic Neoplasms/metabolism , Humans , Male , Middle Aged , Naphthyridines/administration & dosage , Naphthyridines/adverse effects , Naphthyridines/pharmacology , Neoplasm Grading , Neoplasm Staging , Young AdultABSTRACT
The three-dimensional organization of the genome contributes to its maintenance and regulation. While chromosomal regions associate with nucleolar ribosomal RNA genes (rDNA), the biological significance of rDNA-genome interactions and whether they are dynamically regulated during disease remain unclear. rDNA chromatin exists in multiple inactive and active states and their transition is regulated by the RNA polymerase I transcription factor UBTF. Here, using a MYC-driven lymphoma model, we demonstrate that during malignant progression the rDNA chromatin converts to the open state, which is required for tumor cell survival. Moreover, this rDNA transition co-occurs with a reorganization of rDNA-genome contacts which correlate with gene expression changes at associated loci, impacting gene ontologies including B-cell differentiation, cell growth and metabolism. We propose that UBTF-mediated conversion to open rDNA chromatin during malignant transformation contributes to the regulation of specific gene pathways that regulate growth and differentiation through reformed long-range physical interactions with the rDNA.
Subject(s)
Cell Transformation, Neoplastic/genetics , DNA, Ribosomal/genetics , Genes, rRNA , Genetic Predisposition to Disease , Genome , RNA Polymerase II/genetics , Cell Line, Tumor , Chromatin/genetics , Chromatin/metabolism , Chromatin Assembly and Disassembly , Disease Progression , Epistasis, Genetic , Gene Expression Profiling , Gene Expression Regulation , Humans , Neoplasms/genetics , Neoplasms/metabolism , Neoplasms/pathologyABSTRACT
The nucleolus is a dynamic subnuclear compartment that has a number of different functions, but its primary role is to coordinate the production and assembly of ribosomes. For well over 100 years, pathologists have used changes in nucleolar number and size to stage diseases such as cancer. New information about the nucleolus' broader role within the cell is leading to the development of drugs which directly target its structure as therapies for disease. Traditionally, it has been difficult to develop high-throughput image analysis pipelines to measure nucleolar changes due to the broad range of morphologies observed. In this study, we describe a simple high-content image analysis algorithm using Harmony software (PerkinElmer), with a PhenoLOGIC™ machine-learning component, that can measure and classify three different nucleolar morphologies based on nucleolin and fibrillarin staining ("normal," "peri-nucleolar rings" and "dispersed"). We have utilized this algorithm to determine the changes in these classes of nucleolar morphologies over time with drugs known to alter nucleolar structure. This approach could be further adapted to include other parameters required for the identification of new therapies that directly target the nucleolus.
Subject(s)
Cell Nucleolus/pathology , High-Throughput Screening Assays , A549 Cells , Algorithms , Cell Nucleolus/metabolism , Humans , Machine Learning , Oxidative Stress , Software , Tumor Cells, CulturedABSTRACT
Over the last decade, our appreciation of the importance of the nucleolus for cellular function has progressed from the ordinary to the extraordinary. We no longer think of the nucleolus as simply the site of ribosome production, or a dynamic subnuclear body noted by pathologists for its changes in size and shape with malignancy. Instead, the nucleolus has emerged as a key controller of many cellular processes that are fundamental to normal cell homeostasis and the target for dysregulation in many human diseases; in some cases, independent of its functions in ribosome biogenesis. These extra-nucleolar or new functions, which we term "non-canonical" to distinguish them from the more traditional role of the nucleolus in ribosome synthesis, are the focus of this review. In particular, we explore how these non-canonical functions may provide novel insights into human disease and in some cases new targets for therapeutic development.
Subject(s)
Cell Nucleolus/metabolism , Ribosomes/metabolism , Humans , Neoplasms/metabolism , Nervous System Diseases/metabolism , Organelle BiogenesisABSTRACT
Waldenström macroglobulinaemia (WM) is an indolent mature B cell lymphoma characterised by an infiltrate of heterogeneous B cells and hypersecretion of IgM. There are two distinct cellular populations that can be distinguished on morphology and immunophenotyping within the bone marrow. The predominant lymphoplasmacytic compartment arises at an earlier stage in ontogeny, and is responsible for the cytopenias noted during the symptomatic phase of the disease. This population is ably targeted by B cell immunodepletion. The smaller plasma cell compartment has been shown to be monotypic and to carry the MYD88L265P mutation noted in >90% of WM. Further, pathogenic IgM levels tend to correlate better with plasma cell burden as compared to lymphoplasmacytic cell burden within the bone marrow. B cell immunodepletion does not eradicate the plasma cell compartment resulting in persistent IgM levels and poor complete remission rates. In this review we discuss the different cellular compartments in WM and highlight evidence regarding the significance and impending function of the plasma cell compartment in WM. We suggest detection of plasma cells be incorporated into routine diagnostic algorithms, and highlight the need to trial incorporation of plasma cell depleting therapy into treatment algorithms to deepen responses and improve clinical outcomes.
Subject(s)
Bone Marrow/pathology , Immunophenotyping , Plasma Cells/pathology , Waldenstrom Macroglobulinemia/therapy , Animals , B-Lymphocytes/immunology , B-Lymphocytes/pathology , Bone Marrow/immunology , Humans , Mutation/genetics , Mutation/immunology , Plasma Cells/immunology , Waldenstrom Macroglobulinemia/diagnosis , Waldenstrom Macroglobulinemia/immunologyABSTRACT
Pancreatic ductal adenocarcinoma is a devastating disease with a poor prognosis regardless of stage. To date the mainstay of therapy for advanced disease has been chemotherapy with little incremental improvements in outcome. Despite extensive research investigating new treatment options the current practices continue to utilise fluorouracil or gemcitabine containing combinations. The need for novel therapeutic approaches is mandated by the ongoing poor survival rates associated with this disease. One such approach may include manipulation of ribosome biogenesis and the nucleolar stress response, which has recently been applied to haematological malignancies such as lymphoma and prostate cancer with promising results. This review will focus on the current therapeutic options for pancreatic ductal adenocarcinoma and the complexities associated with developing novel treatments, with a particular emphasis on the role of the nucleolus as a treatment strategy.
Subject(s)
Carcinoma, Pancreatic Ductal/therapy , Pancreatic Neoplasms/therapy , Humans , Organelle Biogenesis , RibosomesABSTRACT
Despite the development of novel drugs, the prospects for many patients with acute myeloid leukemia (AML) remain dismal. This study reveals that the selective inhibitor of RNA polymerase I (Pol I) transcription, CX-5461, effectively treats aggressive AML, including mixed-lineage leukemia-driven AML, and outperforms standard chemotherapies. In addition to the previously characterized mechanism of action of CX-5461 (ie, the induction of p53-dependent apoptotic cell death), the inhibition of Pol I transcription also demonstrates potent efficacy in p53null AML in vivo. This significant survival advantage in both p53WT and p53null leukemic mice treated with CX-5461 is associated with activation of the checkpoint kinases 1/2, an aberrant G2/M cell-cycle progression and induction of myeloid differentiation of the leukemic blasts. The ability to target the leukemic-initiating cell population is thought to be essential for lasting therapeutic benefit. Most strikingly, the acute inhibition of Pol I transcription reduces both the leukemic granulocyte-macrophage progenitor and leukemia-initiating cell (LIC) populations, and suppresses their clonogenic capacity. This suggests that dysregulated Pol I transcription is essential for the maintenance of their leukemia-initiating potential. Together, these findings demonstrate the therapeutic utility of this new class of inhibitors to treat highly aggressive AML by targeting LICs.
Subject(s)
Benzothiazoles/pharmacology , Leukemia, Myeloid, Acute/drug therapy , Naphthyridines/pharmacology , Neoplastic Stem Cells/enzymology , Pol1 Transcription Initiation Complex Proteins/antagonists & inhibitors , Transcription, Genetic/drug effects , Animals , Cell Division/drug effects , Cell Division/genetics , Cell Line, Tumor , Checkpoint Kinase 1/genetics , Checkpoint Kinase 1/metabolism , Checkpoint Kinase 2/genetics , Checkpoint Kinase 2/metabolism , G2 Phase/drug effects , G2 Phase/genetics , Humans , Leukemia, Myeloid, Acute/epidemiology , Leukemia, Myeloid, Acute/genetics , Leukemia, Myeloid, Acute/pathology , Mice , Mice, Inbred NOD , Mice, Mutant Strains , Neoplastic Stem Cells/pathology , Pol1 Transcription Initiation Complex Proteins/genetics , Pol1 Transcription Initiation Complex Proteins/metabolism , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Protein p53/metabolismABSTRACT
RNA polymerase I (Pol I)-mediated transcription of the ribosomal RNA genes (rDNA) is confined to the nucleolus and is a rate-limiting step for cell growth and proliferation. Inhibition of Pol I by CX-5461 can selectively induce p53-mediated apoptosis of tumour cells in vivo. Currently, CX-5461 is in clinical trial for patients with advanced haematological malignancies (Peter Mac, Melbourne). Here we demonstrate that CX-5461 also induces p53-independent cell cycle checkpoints mediated by ATM/ATR signaling in the absence of DNA damage. Further, our data demonstrate that the combination of drugs targeting ATM/ATR signaling and CX-5461 leads to enhanced therapeutic benefit in treating p53-null tumours in vivo, which are normally refractory to each drug alone. Mechanistically, we show that CX-5461 induces an unusual chromatin structure in which transcriptionally competent relaxed rDNA repeats are devoid of transcribing Pol I leading to activation of ATM signaling within the nucleoli. Thus, we propose that acute inhibition of Pol transcription initiation by CX-5461 induces a novel nucleolar stress response that can be targeted to improve therapeutic efficacy.
Subject(s)
Ataxia Telangiectasia Mutated Proteins/metabolism , Benzothiazoles/pharmacology , Naphthyridines/pharmacology , Nucleic Acid Synthesis Inhibitors/pharmacology , RNA Polymerase I/antagonists & inhibitors , Signal Transduction , Animals , Apoptosis , Cell Enlargement , Cell Nucleolus/metabolism , Cell Proliferation , Chromatin/metabolism , Comet Assay , DNA Damage , DNA, Ribosomal/genetics , Fibroblasts/metabolism , Hematologic Neoplasms/metabolism , Humans , Mice , Mice, Inbred C57BL , RNA Polymerase I/metabolism , Tumor Suppressor Protein p53/metabolismABSTRACT
UNLABELLED: Ribosome biogenesis and protein synthesis are dysregulated in many cancers, with those driven by the proto-oncogene c-MYC characterized by elevated Pol I-mediated ribosomal rDNA transcription and mTORC1/eIF4E-driven mRNA translation. Here, we demonstrate that coordinated targeting of rDNA transcription and PI3K-AKT-mTORC1-dependent ribosome biogenesis and protein synthesis provides a remarkable improvement in survival in MYC-driven B lymphoma. Combining an inhibitor of rDNA transcription (CX-5461) with the mTORC1 inhibitor everolimus more than doubled survival of Eµ-Myc lymphoma-bearing mice. The ability of each agent to trigger tumor cell death via independent pathways was central to their synergistic efficacy. CX-5461 induced nucleolar stress and p53 pathway activation, whereas everolimus induced expression of the proapoptotic protein BMF that was independent of p53 and reduced expression of RPL11 and RPL5. Thus, targeting the network controlling the synthesis and function of ribosomes at multiple points provides a potential new strategy to treat MYC-driven malignancies. SIGNIFICANCE: Treatment options for the high proportion of cancers driven by MYC are limited. We demonstrate that combining pharmacologic targeting of ribosome biogenesis and mTORC1-dependent translation provides a remarkable therapeutic benefit to Eµ-Myc lymphoma-bearing mice. These results establish a rationale for targeting ribosome biogenesis and function to treat MYC-driven cancer.
Subject(s)
Benzothiazoles/administration & dosage , DNA, Ribosomal/antagonists & inhibitors , Everolimus/administration & dosage , Lymphoma, B-Cell/therapy , Naphthyridines/administration & dosage , Proto-Oncogene Proteins c-myc/genetics , Animals , Antineoplastic Combined Chemotherapy Protocols/administration & dosage , Antineoplastic Combined Chemotherapy Protocols/pharmacology , Benzothiazoles/pharmacology , Drug Synergism , Everolimus/pharmacology , Humans , Lymphoma, B-Cell/genetics , Mice , Naphthyridines/pharmacology , Protein Biosynthesis/drug effects , Proto-Oncogene Mas , Signal Transduction/drug effects , Survival Analysis , Transcription, Genetic/drug effects , Treatment Outcome , Xenograft Model Antitumor AssaysABSTRACT
Mechanisms to coordinate programs of highly transcribed genes required for cellular homeostasis and growth are unclear. Upstream binding transcription factor (UBTF, also called UBF) is thought to function exclusively in RNA polymerase I (Pol I)-specific transcription of the ribosomal genes. Here, we report that the two isoforms of UBTF (UBTF1/2) are also enriched at highly expressed Pol II-transcribed genes throughout the mouse genome. Further analysis of UBTF1/2 DNA binding in immortalized human epithelial cells and their isogenically matched transformed counterparts reveals an additional repertoire of UBTF1/2-bound genes involved in the regulation of cell cycle checkpoints and DNA damage response. As proof of a functional role for UBTF1/2 in regulating Pol II transcription, we demonstrate that UBTF1/2 is required for recruiting Pol II to the highly transcribed histone gene clusters and for their optimal expression. Intriguingly, lack of UBTF1/2 does not affect chromatin marks or nucleosome density at histone genes. Instead, it results in increased accessibility of the histone promoters and transcribed regions to micrococcal nuclease, implicating UBTF1/2 in mediating DNA accessibility. Unexpectedly, UBTF2, which does not function in Pol I transcription, is sufficient to regulate histone gene expression in the absence of UBTF1. Moreover, depletion of UBTF1/2 and subsequent reduction in histone gene expression is associated with DNA damage and genomic instability independent of Pol I transcription. Thus, we have uncovered a novel role for UBTF1 and UBTF2 in maintaining genome stability through coordinating the expression of highly transcribed Pol I (UBTF1 activity) and Pol II genes (UBTF2 activity).