RESUMEN
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.
RESUMEN
Pancreatic carcinoma lacks effective therapeutic strategies resulting in poor prognosis. Transcriptional dysregulation due to alterations in KRAS and MYC affects initiation, development, and survival of this tumor type. Using patient-derived xenografts of KRAS- and MYC-driven pancreatic carcinoma, we show that coinhibition of topoisomerase 1 (TOP1) and bromodomain-containing protein 4 (BRD4) synergistically induces tumor regression by targeting promoter pause release. Comparing the nascent transcriptome with the recruitment of elongation and termination factors, we found that coinhibition of TOP1 and BRD4 disrupts recruitment of transcription termination factors. Thus, RNA polymerases transcribe downstream of genes for hundreds of kilobases leading to readthrough transcription. This occurs during replication, perturbing replisome progression and inducing DNA damage. The synergistic effect of TOP1 + BRD4 inhibition is specific to cancer cells leaving normal cells unaffected, highlighting the tumor's vulnerability to transcriptional defects. This preclinical study provides a mechanistic understanding of the benefit of combining TOP1 and BRD4 inhibitors to treat pancreatic carcinomas addicted to oncogenic drivers of transcription and replication.
Asunto(s)
Neoplasias Pancreáticas , Factores de Transcripción , Humanos , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Línea Celular Tumoral , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Neoplasias Pancreáticas/tratamiento farmacológico , Neoplasias Pancreáticas/genética , Proteínas Proto-Oncogénicas p21(ras)/genética , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Transcripción Genética , ADN-Topoisomerasas de Tipo I/metabolismo , Neoplasias PancreáticasRESUMEN
TOP1 CAD-seq enables mapping of TOP1 sites of covalent engagement with DNA. The procedure depends upon enrichment of DNA-covalent adducts using chaotropic salts and immunoprecipitation with an antibody specific for TOP1. Here, we describe a step-by-step protocol compatible with Illumina sequencing and bioinformatic pipeline for preliminary data analysis. Compared to other approaches for the genomic study of topoisomerases, TOP1 CAD-seq provides information about active TOP1 engaged on the DNA, taking advantage of low background due to absence of crosslinking. For complete details on the use and execution of this protocol, please refer to Das et al. (2022).
Asunto(s)
Aductos de ADN , ADN , ADN-Topoisomerasas de Tipo I/genética , HumanosRESUMEN
High-intensity transcription and replication supercoil DNA to levels that can impede or halt these processes. As a potent transcription amplifier and replication accelerator, the proto-oncogene MYC must manage this interfering torsional stress. By comparing gene expression with the recruitment of topoisomerases and MYC to promoters, we surmised a direct association of MYC with topoisomerase 1 (TOP1) and TOP2 that was confirmed in vitro and in cells. Beyond recruiting topoisomerases, MYC directly stimulates their activities. We identify a MYC-nucleated "topoisome" complex that unites TOP1 and TOP2 and increases their levels and activities at promoters, gene bodies, and enhancers. Whether TOP2A or TOP2B is included in the topoisome is dictated by the presence of MYC versus MYCN, respectively. Thus, in vitro and in cells, MYC assembles tools that simplify DNA topology and promote genome function under high output conditions.
Asunto(s)
ADN-Topoisomerasas de Tipo II/metabolismo , Neoplasias/enzimología , Proteínas de Unión a Poli-ADP-Ribosa/metabolismo , Proteínas Proto-Oncogénicas c-myc/metabolismo , Transcripción Genética , Animales , Replicación del ADN , ADN-Topoisomerasas de Tipo I/genética , ADN-Topoisomerasas de Tipo I/metabolismo , ADN-Topoisomerasas de Tipo II/genética , ADN de Neoplasias/biosíntesis , ADN de Neoplasias/genética , ADN Superhelicoidal/biosíntesis , ADN Superhelicoidal/genética , Activación Enzimática , Regulación Neoplásica de la Expresión Génica , Células HCT116 , Humanos , Células K562 , Complejos Multienzimáticos , Neoplasias/genética , Neoplasias/patología , Proteínas de Unión a Poli-ADP-Ribosa/genética , Regiones Promotoras Genéticas , Unión Proteica , Proteínas Proto-Oncogénicas c-myc/genética , RatasRESUMEN
Deregulated expression of MYC family oncogenes occurs frequently in human cancer and is often associated with aggressive disease and poor prognosis. While MYC is a highly warranted target, it has been considered "undruggable," and no specific anti-MYC drugs are available in the clinic. We recently identified molecules named MYCMIs that inhibit the interaction between MYC and its essential partner MAX. Here we show that one of these molecules, MYCMI-7, efficiently and selectively inhibits MYC:MAX and MYCN:MAX interactions in cells, binds directly to recombinant MYC, and reduces MYC-driven transcription. In addition, MYCMI-7 induces degradation of MYC and MYCN proteins. MYCMI-7 potently induces growth arrest/apoptosis in tumor cells in a MYC/MYCN-dependent manner and downregulates the MYC pathway on a global level as determined by RNA sequencing. Sensitivity to MYCMI-7 correlates with MYC expression in a panel of 60 tumor cell lines and MYCMI-7 shows high efficacy toward a collection of patient-derived primary glioblastoma and acute myeloid leukemia (AML) ex vivo cultures. Importantly, a variety of normal cells become G1 arrested without signs of apoptosis upon MYCMI-7 treatment. Finally, in mouse tumor models of MYC-driven AML, breast cancer, and MYCN-amplified neuroblastoma, treatment with MYCMI-7 downregulates MYC/MYCN, inhibits tumor growth, and prolongs survival through apoptosis with few side effects. In conclusion, MYCMI-7 is a potent and selective MYC inhibitor that is highly relevant for the development into clinically useful drugs for the treatment of MYC-driven cancer. Significance: Our findings demonstrate that the small-molecule MYCMI-7 binds MYC and inhibits interaction between MYC and MAX, thereby hampering MYC-driven tumor cell growth in culture and in vivo while sparing normal cells.
Asunto(s)
Neuroblastoma , Animales , Ratones , Humanos , Proteína Proto-Oncogénica N-Myc/genética , Línea Celular Tumoral , Neuroblastoma/tratamiento farmacológico , Proliferación Celular , Ciclo CelularRESUMEN
As cells enter mitosis, chromatin compacts to facilitate chromosome segregation yet remains transcribed. Transcription supercoils DNA to levels that can impede further progression of RNA polymerase II (RNAPII) unless it is removed by DNA topoisomerase 1 (TOP1). Using ChIP-seq on mitotic cells, we found that TOP1 is required for RNAPII translocation along genes. The stimulation of TOP1 activity by RNAPII during elongation allowed RNAPII clearance from genes in prometaphase and enabled chromosomal segregation. Disruption of the TOP1-RNAPII interaction impaired RNAPII spiking at promoters and triggered defects in the post-mitotic transcription program. This program includes factors necessary for cell growth, and cells with impaired TOP1-RNAPII interaction are more sensitive to inhibitors of mTOR signaling. We conclude that TOP1 is necessary for assisting transcription during mitosis with consequences for growth and gene expression long after mitosis is completed. In this sense, TOP1 ensures that cellular memory is preserved in subsequent generations.
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Proliferación Celular , Ensamble y Desensamble de Cromatina , Neoplasias Colorrectales/enzimología , ADN-Topoisomerasas de Tipo I/metabolismo , Fase G1 , Mitosis , ARN Polimerasa II/metabolismo , Transcripción Genética , Proliferación Celular/efectos de los fármacos , Secuenciación de Inmunoprecipitación de Cromatina , Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/patología , ADN-Topoisomerasas de Tipo I/genética , Fase G1/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica , Células HCT116 , Humanos , Inhibidores mTOR/farmacología , Mitosis/efectos de los fármacos , ARN Polimerasa II/genéticaRESUMEN
Topoisomerases are essential for the replication of herpesviruses but the mechanisms by which the viruses hijack the cellular enzymes are largely unknown. We found that topoisomerase-II (TOP2) is a substrate of the Epstein-Barr virus (EBV) ubiquitin deconjugase BPLF1. BPLF1 co-immunoprecipitated and deubiquitinated TOP2, and stabilized SUMOylated TOP2 trapped in cleavage complexes (TOP2ccs), which halted the DNA damage response to TOP2-induced double strand DNA breaks and promoted cell survival. Induction of the productive virus cycle in epithelial and lymphoid cell lines carrying recombinant EBV encoding the active enzyme was accompanied by TOP2 deubiquitination, accumulation of TOP2ccs and resistance to Etoposide toxicity. The protective effect of BPLF1 was dependent on the expression of tyrosyl-DNA phosphodiesterase 2 (TDP2) that releases DNA-trapped TOP2 and promotes error-free DNA repair. These findings highlight a previously unrecognized function of BPLF1 in supporting a non-proteolytic pathway for TOP2ccs debulking that favors cell survival and virus production.
Asunto(s)
ADN-Topoisomerasas de Tipo II/metabolismo , Infecciones por Virus de Epstein-Barr/metabolismo , Proteínas Reguladoras y Accesorias Virales/metabolismo , Células HEK293 , Células HeLa , HumanosRESUMEN
The structural maintenance of chromosome (SMC) complex cohesin mediates sister chromatid cohesion established during replication, and damage-induced cohesion formed in response to DSBs post-replication. The translesion synthesis polymerase Polη is required for damage-induced cohesion through a hitherto unknown mechanism. Since Polη is functionally associated with transcription, and transcription triggers de novo cohesion in Schizosaccharomyces pombe, we hypothesized that transcription facilitates damage-induced cohesion in Saccharomyces cerevisiae. Here, we show dysregulated transcriptional profiles in the Polη null mutant (rad30Δ), where genes involved in chromatin assembly and positive transcription regulation were downregulated. In addition, chromatin association of RNA polymerase II was reduced at promoters and coding regions in rad30Δ compared to WT cells, while occupancy of the H2A.Z variant (Htz1) at promoters was increased in rad30Δ cells. Perturbing histone exchange at promoters inactivated damage-induced cohesion, similarly to deletion of the RAD30 gene. Conversely, altering regulation of transcription elongation suppressed the deficient damage-induced cohesion in rad30Δ cells. Furthermore, transcription inhibition negatively affected formation of damage-induced cohesion. These results indicate that the transcriptional deregulation of the Polη null mutant is connected with its reduced capacity to establish damage-induced cohesion. This also suggests a linkage between regulation of transcription and formation of damage-induced cohesion after replication.
Asunto(s)
Proteínas de Ciclo Celular/biosíntesis , Proteínas Cromosómicas no Histona/biosíntesis , ADN Polimerasa Dirigida por ADN/genética , ARN Polimerasa II/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/enzimología , Transcripción Genética , Cromatina/metabolismo , ADN Polimerasa Dirigida por ADN/metabolismo , Regulación Enzimológica de la Expresión Génica , Regulación Fúngica de la Expresión Génica , Genes Fúngicos , Mutación , Regiones Promotoras Genéticas , ARN Polimerasa II/metabolismo , Saccharomyces cerevisiae/genética , TATA Box , CohesinasRESUMEN
Here, we present a strategy to map and quantify the interactions between Myc and chromatin using a calibrated Myc ChIP-seq approach. We recommend the use of an internal spike-in control for post-sequencing normalization to enable detection of broad changes in Myc binding as can occur under conditions with varied Myc abundance. We also highlight a range of bioinformatic analyses that can dissect the downstream effects of Myc binding. These methods include peak calling, mapping Myc onto an integrated metagenome, juxtaposing ChIP-seq data with matching RNA-seq data, and identifying gene ontologies enriched for genes with high Myc binding. Our aim is to provide a guided strategy, from cell harvest through to bioinformatic analysis, to elucidate the global effects of Myc on transcription.
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Secuenciación de Inmunoprecipitación de Cromatina/métodos , Inmunoprecipitación de Cromatina/métodos , Proteínas Proto-Oncogénicas c-myc/genética , Sitios de Unión , Cromatina/genética , Biología Computacional/métodos , ADN/genética , Proteínas de Unión al ADN , Genes myc , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Proteínas Proto-Oncogénicas c-myc/metabolismo , Análisis de Secuencia de ADN/métodosRESUMEN
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.
RESUMEN
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.
Asunto(s)
Antineoplásicos/uso terapéutico , Benzotiazoles/uso terapéutico , Neoplasias Hematológicas/tratamiento farmacológico , Neoplasias Hematológicas/genética , Naftiridinas/uso terapéutico , ARN Polimerasa I/metabolismo , Transcripción Genética/efectos de los fármacos , Adulto , Anciano , Antineoplásicos/administración & dosificación , Antineoplásicos/efectos adversos , Antineoplásicos/farmacocinética , Benzotiazoles/administración & dosificación , Benzotiazoles/efectos adversos , Benzotiazoles/farmacología , ADN Ribosómico/genética , Femenino , Neoplasias Hematológicas/diagnóstico , Neoplasias Hematológicas/metabolismo , Humanos , Masculino , Persona de Mediana Edad , Naftiridinas/administración & dosificación , Naftiridinas/efectos adversos , Naftiridinas/farmacología , Clasificación del Tumor , Estadificación de Neoplasias , Adulto JovenRESUMEN
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.
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Transformación Celular Neoplásica/genética , ADN Ribosómico/genética , Genes de ARNr , Predisposición Genética a la Enfermedad , Genoma , ARN Polimerasa II/genética , Línea Celular Tumoral , Cromatina/genética , Cromatina/metabolismo , Ensamble y Desensamble de Cromatina , Progresión de la Enfermedad , Epistasis Genética , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Humanos , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patologíaRESUMEN
Increased CDK4 activity occurs in the majority of melanomas and CDK4/6 inhibitors in combination with BRAF and MEK inhibitors are currently in clinical trials for the treatment of melanoma. We hypothesize that the timing of the addition of CDK4/6 inhibitors to the current BRAF and MEK inhibitor regime will impact on the efficacy of this triplet drug combination. The efficacy of BRAF, MEK and CDK4/6 inhibitors as single agents and in combination was assessed in human BRAF mutant cell lines that were treatment naïve, BRAF inhibitor tolerant or had acquired resistance to BRAF inhibitors. Xenograft studies were then performed to test the in vivo efficacy of the BRAF and CDK4/6 inhibitor combination. Melanoma cells that had developed early reversible tolerance or acquired resistance to BRAF inhibition remained sensitive to palbociclib. In drug-tolerant cells, the efficacy of the combination of palbociclib with BRAF and/or MEK inhibitors was equivalent to single agent palbociclib. Similarly, acquired BRAF inhibitor resistance cells lost efficacy to the palbociclib and BRAF combination. In contrast, upfront treatment of melanoma cells with palbociclib in combination with BRAF and/or MEK inhibitors induced either cell death or senescence and was superior to a BRAF plus MEK inhibitor combination. In vivo palbociclib plus BRAF inhibitor induced rapid and sustained tumor regression without the development of therapy resistance. In summary, upfront dual targeting of CDK4/6 and mutant BRAF signaling enables tumor cells to evade resistance to monotherapy and is required for robust and sustained tumor regression. Melanoma patients whose tumors have acquired resistance to BRAF inhibition are less likely to have favorable responses to subsequent treatment with the triplet combination of BRAF, MEK and CDK4/6 inhibitors.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Quinasas Quinasa Quinasa PAM/antagonistas & inhibidores , Melanoma/tratamiento farmacológico , Piperazinas/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas B-raf/antagonistas & inhibidores , Piridinas/farmacología , Animales , Línea Celular Tumoral , Quinasa 4 Dependiente de la Ciclina/antagonistas & inhibidores , Quinasa 6 Dependiente de la Ciclina/antagonistas & inhibidores , Resistencia a Antineoplásicos , Sinergismo Farmacológico , Femenino , Humanos , Indoles/administración & dosificación , Indoles/farmacología , Melanoma/enzimología , Ratones , Ratones SCID , Piperazinas/administración & dosificación , Inhibidores de Proteínas Quinasas/administración & dosificación , Piridinas/administración & dosificación , Sulfonamidas/administración & dosificación , Sulfonamidas/farmacología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Trypanosoma brucei relies on an essential Variant Surface Glycoprotein (VSG) coat for survival in the mammalian bloodstream. High VSG expression within an expression site body (ESB) is mediated by RNA polymerase I (Pol I), which in other eukaryotes exclusively transcribes ribosomal RNA genes (rDNA). As T. brucei is reliant on Pol I for VSG transcription, we investigated Pol I transcription inhibitors for selective anti-trypanosomal activity. The Pol I inhibitors quarfloxin (CX-3543), CX-5461, and BMH-21 are currently under investigation for treating cancer, as rapidly dividing cancer cells are particularly dependent on high levels of Pol I transcription compared with nontransformed cells. In T. brucei all three Pol I inhibitors have IC50 concentrations for cell proliferation in the nanomolar range: quarfloxin (155 nM), CX-5461 (279 nM) or BMH-21 (134 nM) compared with IC50 concentrations in the MCF10A human breast epithelial cell line (4.44 µM, 6.89 µM or 460 nM, respectively). T. brucei was therefore 29-fold more sensitive to quarfloxin, 25-fold more sensitive to CX-5461 and 3.4-fold more sensitive to BMH-21. Cell death in T. brucei was due to rapid inhibition of Pol I transcription, as within 15 minutes treatment with the inhibitors rRNA precursor transcript was reduced 97-98% and VSG precursor transcript 91-94%. Incubation with Pol I transcription inhibitors also resulted in disintegration of the ESB as well as the nucleolus subnuclear structures, within one hour. Rapid ESB loss following the block in Pol I transcription argues that the ESB is a Pol I transcription nucleated structure, similar to the nucleolus. In addition to providing insight into Pol I transcription and ES control, Pol I transcription inhibitors potentially also provide new approaches to treat trypanosomiasis.
Asunto(s)
Inhibidores Enzimáticos/farmacología , ARN Polimerasa I/antagonistas & inhibidores , Transcripción Genética/efectos de los fármacos , Tripanocidas/farmacología , Trypanosoma brucei brucei/efectos de los fármacos , Trypanosoma brucei brucei/enzimología , Tripanosomiasis Africana/tratamiento farmacológico , Concentración 50 InhibidoraRESUMEN
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.
Asunto(s)
Benzotiazoles/farmacología , Leucemia Mieloide Aguda/tratamiento farmacológico , Naftiridinas/farmacología , Células Madre Neoplásicas/enzimología , Proteínas del Complejo de Iniciación de Transcripción Pol1/antagonistas & inhibidores , Transcripción Genética/efectos de los fármacos , Animales , División Celular/efectos de los fármacos , División Celular/genética , Línea Celular Tumoral , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1)/genética , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1)/metabolismo , Quinasa de Punto de Control 2/genética , Quinasa de Punto de Control 2/metabolismo , Fase G2/efectos de los fármacos , Fase G2/genética , Humanos , Leucemia Mieloide Aguda/epidemiología , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patología , Ratones , Ratones Endogámicos NOD , Ratones Mutantes , Células Madre Neoplásicas/patología , Proteínas del Complejo de Iniciación de Transcripción Pol1/genética , Proteínas del Complejo de Iniciación de Transcripción Pol1/metabolismo , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
PURPOSE: The MYC oncogene is frequently overexpressed in prostate cancer. Upregulation of ribosome biogenesis and function is characteristic of MYC-driven tumors. In addition, PIM kinases activate MYC signaling and mRNA translation in prostate cancer and cooperate with MYC to accelerate tumorigenesis. Here, we investigate the efficacy of a single and dual approach targeting ribosome biogenesis and function to treat prostate cancer. EXPERIMENTAL DESIGN: The inhibition of ribosomal RNA (rRNA) synthesis with CX-5461, a potent, selective, and orally bioavailable inhibitor of RNA polymerase I (Pol I) transcription, has been successfully exploited therapeutically but only in models of hematologic malignancy. CX-5461 and CX-6258, a pan-PIM kinase inhibitor, were tested alone and in combination in prostate cancer cell lines, in Hi-MYC- and PTEN-deficient mouse models and in patient-derived xenografts (PDX) of metastatic tissue obtained from a patient with castration-resistant prostate cancer. RESULTS: CX-5461 inhibited anchorage-independent growth and induced cell-cycle arrest in prostate cancer cell lines at nanomolar concentrations. Oral administration of 50 mg/kg CX-5461 induced TP53 expression and activity and reduced proliferation (MKI67) and invasion (loss of ductal actin) in Hi-MYC tumors, but not in PTEN-null (low MYC) tumors. While 100 mg/kg CX-6258 showed limited effect alone, its combination with CX-5461 further suppressed proliferation and dramatically reduced large invasive lesions in both models. This rational combination strategy significantly inhibited proliferation and induced cell death in PDX of prostate cancer. CONCLUSIONS: Our results demonstrate preclinical efficacy of targeting the ribosome at multiple levels and provide a new approach for the treatment of prostate cancer. Clin Cancer Res; 22(22); 5539-52. ©2016 AACR.
Asunto(s)
Antineoplásicos/farmacología , Neoplasias de la Próstata/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas c-pim-1/antagonistas & inhibidores , ARN Polimerasa I/antagonistas & inhibidores , Transcripción Genética/efectos de los fármacos , Animales , Azepinas/farmacología , Benzotiazoles/farmacología , Puntos de Control del Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Femenino , Humanos , Indoles/farmacología , Masculino , Ratones , Naftiridinas/farmacología , Fosfohidrolasa PTEN/metabolismo , Próstata/efectos de los fármacos , Próstata/metabolismo , Neoplasias de la Próstata/metabolismo , Proteínas Proto-Oncogénicas c-myc/metabolismo , Transducción de Señal/efectos de los fármacos , Ensayos Antitumor por Modelo de Xenoinjerto/métodosRESUMEN
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.
Asunto(s)
Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Benzotiazoles/farmacología , Naftiridinas/farmacología , Inhibidores de la Síntesis del Ácido Nucleico/farmacología , ARN Polimerasa I/antagonistas & inhibidores , Transducción de Señal , Animales , Apoptosis , Aumento de la Célula , Nucléolo Celular/metabolismo , Proliferación Celular , Cromatina/metabolismo , Ensayo Cometa , Daño del ADN , ADN Ribosómico/genética , Fibroblastos/metabolismo , Neoplasias Hematológicas/metabolismo , Humanos , Ratones , Ratones Endogámicos C57BL , ARN Polimerasa I/metabolismo , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
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).
Asunto(s)
Regulación de la Expresión Génica , Inestabilidad Genómica , Proteínas del Complejo de Iniciación de Transcripción Pol1/metabolismo , ARN Polimerasa II/genética , ARN Polimerasa I/genética , Transcripción Genética , Animales , Sitios de Unión , Línea Celular Transformada , Cromatina/metabolismo , Inmunoprecipitación de Cromatina , Biología Computacional , Daño del ADN , Técnicas de Silenciamiento del Gen , Secuenciación de Nucleótidos de Alto Rendimiento , Histonas/genética , Humanos , Ratones , Familia de Multigenes , Células 3T3 NIH , Nucleosomas/metabolismo , Proteínas del Complejo de Iniciación de Transcripción Pol1/genética , Unión Proteica , Sitio de Iniciación de la TranscripciónRESUMEN
We have investigated the potential for the p16-cyclin D-CDK4/6-retinoblastoma protein pathway to be exploited as a therapeutic target in melanoma. In a cohort of 143 patients with primary invasive melanoma, we used fluorescence in situ hybridization to detect gene copy number variations (CNVs) in CDK4, CCND1, and CDKN2A and immunohistochemistry to determine protein expression. CNVs were common in melanoma, with gain of CDK4 or CCND1 in 37 and 18% of cases, respectively, and hemizygous or homozygous loss of CDKN2A in 56%. Three-quarters of all patients demonstrated a CNV in at least one of the three genes. The combination of CCND1 gain with either a gain of CDK4 and/or loss of CDKN2A was associated with poorer melanoma-specific survival. In 47 melanoma cell lines homozygous loss, methylation or mutation of CDKN2A gene or loss of protein (p16(INK) (4A) ) predicted sensitivity to the CDK4/6 inhibitor PD0332991, while RB1 loss predicted resistance.