RESUMEN
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.
RESUMEN
Philadelphia chromosome-positive B cell acute lymphoblastic leukemia (B-ALL), characterized by the BCR::ABL1 fusion gene, remains a poor prognosis cancer needing new therapeutic approaches. Transcriptomic profiling identified up-regulation of oncogenic transcription factors ERG and c-MYC in BCR::ABL1 B-ALL with ERG and c-MYC required for BCR::ABL1 B-ALL in murine and human models. Profiling of ERG- and c-MYC-dependent gene expression and analysis of ChIP-seq data established ERG and c-MYC coordinate a regulatory network in BCR::ABL1 B-ALL that controls expression of genes involved in several biological processes. Prominent was control of ribosome biogenesis, including expression of RNA polymerase I (POL I) subunits, the importance of which was validated by inhibition of BCR::ABL1 cells by POL I inhibitors, including CX-5461, that prevents promoter recruitment and transcription initiation by POL I. Our results reveal an essential ERG- and c-MYC-dependent transcriptional network involved in regulation of metabolic and ribosome biogenesis pathways in BCR::ABL1 B-ALL, from which previously unidentified vulnerabilities and therapeutic targets may emerge.
Asunto(s)
Proteínas de Fusión bcr-abl , Leucemia-Linfoma Linfoblástico de Células Precursoras B , Regulador Transcripcional ERG , Animales , Humanos , Ratones , Proteínas de Fusión bcr-abl/genética , Proteínas de Fusión bcr-abl/metabolismo , Proteínas de Fusión bcr-abl/uso terapéutico , Redes Reguladoras de Genes , Leucemia-Linfoma Linfoblástico de Células Precursoras B/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras B/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras , Factores de Transcripción/genética , Regulador Transcripcional ERG/genéticaRESUMEN
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.
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Antineoplásicos/farmacología , Nucléolo Celular/efectos de los fármacos , Daño del ADN , Neoplasias/terapia , ADN Ribosómico/genética , HumanosRESUMEN
Ribosome biogenesis and protein synthesis are fundamental rate-limiting steps for cell growth and proliferation. The ribosomal proteins (RPs), comprising the structural parts of the ribosome, are essential for ribosome assembly and function. In addition to their canonical ribosomal functions, multiple RPs have extra-ribosomal functions including activation of p53-dependent or p53-independent pathways in response to stress, resulting in cell cycle arrest and apoptosis. Defects in ribosome biogenesis, translation, and the functions of individual RPs, including mutations in RPs have been linked to a diverse range of human congenital disorders termed ribosomopathies. Ribosomopathies are characterized by tissue-specific phenotypic abnormalities and higher cancer risk later in life. Recent discoveries of somatic mutations in RPs in multiple tumor types reinforce the connections between ribosomal defects and cancer. In this article, we review the most recent advances in understanding the molecular consequences of RP mutations and ribosomal defects in ribosomopathies and cancer. We particularly discuss the molecular basis of the transition from hypo- to hyper-proliferation in ribosomopathies with elevated cancer risk, a paradox termed "Dameshek's riddle." Furthermore, we review the current treatments for ribosomopathies and prospective therapies targeting ribosomal defects. We also highlight recent advances in ribosome stress-based cancer therapeutics. Importantly, insights into the mechanisms of resistance to therapies targeting ribosome biogenesis bring new perspectives into the molecular basis of cancer susceptibility in ribosomopathies and new clinical implications for cancer therapy.
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Enfermedades Genéticas Congénitas/genética , Terapia Molecular Dirigida , Neoplasias/genética , Proteínas Ribosómicas/genética , Puntos de Control del Ciclo Celular/genética , Proliferación Celular/genética , Enfermedades Genéticas Congénitas/terapia , Humanos , Mutación/genética , Neoplasias/terapia , Proteínas Ribosómicas/uso terapéutico , Ribosomas/genéticaRESUMEN
BACKGROUND: Intrinsic and acquired drug resistance represent fundamental barriers to the cure of high-grade serous ovarian carcinoma (HGSC), the most common histological subtype accounting for the majority of ovarian cancer deaths. Defects in homologous recombination (HR) DNA repair are key determinants of sensitivity to chemotherapy and poly-ADP ribose polymerase inhibitors. Restoration of HR is a common mechanism of acquired resistance that results in patient mortality, highlighting the need to identify new therapies targeting HR-proficient disease. We have shown promise for CX-5461, a cancer therapeutic in early phase clinical trials, in treating HR-deficient HGSC. METHODS: Herein, we screen the whole protein-coding genome to identify potential targets whose depletion cooperates with CX-5461 in HR-proficient HGSC. RESULTS: We demonstrate robust proliferation inhibition in cells depleted of DNA topoisomerase 1 (TOP1). Combining the clinically used TOP1 inhibitor topotecan with CX-5461 potentiates a G2/M cell cycle checkpoint arrest in multiple HR-proficient HGSC cell lines. The combination enhances a nucleolar DNA damage response and global replication stress without increasing DNA strand breakage, significantly reducing clonogenic survival and tumour growth in vivo. CONCLUSIONS: Our findings highlight the possibility of exploiting TOP1 inhibition to be combined with CX-5461 as a non-genotoxic approach in targeting HR-proficient HGSC.
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Benzotiazoles/farmacología , Cistadenocarcinoma Seroso/tratamiento farmacológico , Daño del ADN/efectos de los fármacos , Recombinación Homóloga , Naftiridinas/farmacología , Neoplasias Ováricas/tratamiento farmacológico , ARN Polimerasa I/antagonistas & inhibidores , Inhibidores de Topoisomerasa I/farmacología , Topotecan/farmacología , Animales , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Cistadenocarcinoma Seroso/genética , Cistadenocarcinoma Seroso/patología , Replicación del ADN/efectos de los fármacos , Resistencia a Antineoplásicos/genética , Sinergismo Farmacológico , Quimioterapia Combinada , Femenino , Puntos de Control de la Fase G1 del Ciclo Celular , Genes BRCA2 , Humanos , Puntos de Control de la Fase M del Ciclo Celular , Ratones , Ratones Endogámicos NOD , Ratones SCID , Clasificación del Tumor , Neoplasias Ováricas/genética , Neoplasias Ováricas/patología , Inhibidores de Poli(ADP-Ribosa) Polimerasas/uso terapéutico , Interferencia de ARN , ARN Polimerasa I/genéticaRESUMEN
Acquired resistance to PARP inhibitors (PARPi) is a major challenge for the clinical management of high grade serous ovarian cancer (HGSOC). Here, we demonstrate CX-5461, the first-in-class inhibitor of RNA polymerase I transcription of ribosomal RNA genes (rDNA), induces replication stress and activates the DNA damage response. CX-5461 co-operates with PARPi in exacerbating replication stress and enhances therapeutic efficacy against homologous recombination (HR) DNA repair-deficient HGSOC-patient-derived xenograft (PDX) in vivo. We demonstrate CX-5461 has a different sensitivity spectrum to PARPi involving MRE11-dependent degradation of replication forks. Importantly, CX-5461 exhibits in vivo single agent efficacy in a HGSOC-PDX with reduced sensitivity to PARPi by overcoming replication fork protection. Further, we identify CX-5461-sensitivity gene expression signatures in primary and relapsed HGSOC. We propose CX-5461 is a promising therapy in combination with PARPi in HR-deficient HGSOC and also as a single agent for the treatment of relapsed disease.
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Benzotiazoles/farmacología , Cistadenocarcinoma Seroso/tratamiento farmacológico , Daño del ADN , Naftiridinas/farmacología , Neoplasias Ováricas/tratamiento farmacológico , Animales , Línea Celular Tumoral , Cistadenocarcinoma Seroso/genética , Cistadenocarcinoma Seroso/metabolismo , Replicación del ADN/efectos de los fármacos , Resistencia a Antineoplásicos , Inhibidores Enzimáticos/farmacología , Femenino , Xenoinjertos , Recombinación Homóloga , Humanos , Ratones , Ratones Endogámicos NOD , Ratones Noqueados , Ratones SCID , Modelos Biológicos , Neoplasias Ováricas/genética , Neoplasias Ováricas/metabolismo , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , ARN Polimerasa I/antagonistas & inhibidores , TranscriptomaRESUMEN
Cyclin-dependent kinase 4/6 (CDK4/6) inhibitors are an established treatment in estrogen receptor-positive breast cancer and are currently in clinical development in melanoma, a tumor that exhibits high rates of CDK4 activation. We analyzed melanoma cells with acquired resistance to the CDK4/6 inhibitor palbociclib and demonstrate that the activity of PRMT5, a protein arginine methyltransferase and indirect target of CDK4, is essential for CDK4/6 inhibitor sensitivity. By indirectly suppressing PRMT5 activity, palbociclib alters the pre-mRNA splicing of MDM4, a negative regulator of p53, leading to decreased MDM4 protein expression and subsequent p53 activation. In turn, p53 induces p21, leading to inhibition of CDK2, the main kinase substituting for CDK4/6 and a key driver of resistance to palbociclib. Loss of the ability of palbociclib to regulate the PRMT5-MDM4 axis leads to resistance. Importantly, combining palbociclib with the PRMT5 inhibitor GSK3326595 enhances the efficacy of palbociclib in treating naive and resistant models and also delays the emergence of resistance. Our studies have uncovered a mechanism of action of CDK4/6 inhibitors in regulating the MDM4 oncogene and the tumor suppressor, p53. Furthermore, we have established that palbociclib inhibition of the PRMT5-MDM4 axis is essential for robust melanoma cell sensitivity and provide preclinical evidence that coinhibition of CDK4/6 and PRMT5 is an effective and well-tolerated therapeutic strategy. Overall, our data provide a strong rationale for further investigation of novel combinations of CDK4/6 and PRMT5 inhibitors, not only in melanoma but other tumor types, including breast, pancreatic, and esophageal carcinoma.
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Proteínas de Ciclo Celular/metabolismo , Quinasa 4 Dependiente de la Ciclina/antagonistas & inhibidores , Quinasa 6 Dependiente de la Ciclina/antagonistas & inhibidores , Melanoma/metabolismo , Piperazinas/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Proteína-Arginina N-Metiltransferasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Piridinas/farmacología , Proteínas de Ciclo Celular/genética , Quinasa 2 Dependiente de la Ciclina/genética , Quinasa 2 Dependiente de la Ciclina/metabolismo , Quinasa 4 Dependiente de la Ciclina/genética , Quinasa 4 Dependiente de la Ciclina/metabolismo , Quinasa 6 Dependiente de la Ciclina/genética , Quinasa 6 Dependiente de la Ciclina/metabolismo , Resistencia a Antineoplásicos , Células HEK293 , Humanos , Células MCF-7 , Melanoma/tratamiento farmacológico , Melanoma/genética , Melanoma/patología , Proteína-Arginina N-Metiltransferasas/antagonistas & inhibidores , Proteína-Arginina N-Metiltransferasas/genética , Proteínas Proto-Oncogénicas/genética , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
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.
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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.