RESUMO
Immunomodulatory imide drugs (IMiDs) are central components of therapy for multiple myeloma (MM). IMiDs bind cereblon (CRBN), an adaptor for the CUL4-DDB1-RBX1 E3 ligase to change its substrate specificity and induce degradation of 'neosubstrate' transcription factors that are essential to MM cells. Mechanistic studies to date have largely focussed on mediators of therapeutic activity and insight into clinical IMiD toxicities is less developed. We adopted BioID2-dependent proximity labelling (BioID2-CRBN) to characterise the CRBN interactome in the presence and absence of various IMiDs and the proteasome inhibitor, bortezomib. We aimed to leverage this technology to further map CRBN interactions beyond what has been achieved by conventional proteomic techniques. In support of this approach, analysis of cells expressing BioID2-CRBN following IMiD treatment displayed biotinylation of known CRBN interactors and neosubstrates. We observed that bortezomib alone significantly modifies the CRBN interactome. Proximity labelling also suggested that IMiDs augment the interaction between CRBN and proteins that are not degraded, thus designating 'neointeractors' distinct from previously disclosed 'neosubstrates'. Here we identify Non-Muscle Myosin Heavy Chain IIA (MYH9) as a putative CRBN neointeractor that may contribute to the haematological toxicity of IMiDs. These studies provide proof of concept for proximity labelling technologies in the mechanistic profiling of IMiDs and related E3-ligase-modulating drugs.
RESUMO
Preclinical tumor models with native tissue microenvironments provide essential tools to understand how heterogeneous tumor phenotypes relate to drug response. Here we present syngeneic graft models of aggressive, metastasis-prone histopathology-specific NSCLC tumor types driven by KRAS mutation and loss of LKB1 (KL): adenosquamous carcinoma (ASC) and adenocarcinoma (AC). We show that subcutaneous injection of primary KL; ASC cells results in squamous cell carcinoma (SCC) tumors with high levels of stromal infiltrates, lacking the source heterogeneous histotype. Despite forming subcutaneous tumors, intravenously injected KL;AC cells were unable to form lung tumors. In contrast, intravenous injection of KL;ASC cells leads to their lung re-colonization and lesions recapitulating the mixed AC and SCC histopathology, tumor immune suppressive microenvironment and oncogenic signaling profile of source tumors, demonstrating histopathology-selective phenotypic dominance over genetic drivers. Pan-ERBB inhibition increased survival, while selective ERBB1/EGFR inhibition did not, suggesting a role of the ERBB network crosstalk in resistance to ERBB1/EGFR. This immunocompetent NSCLC lung colonization model hence phenocopies key properties of the metastasis-prone ASC histopathology, and serves as a preclinical model to dissect therapy responses and metastasis-associated processes.
Assuntos
Adenocarcinoma , Carcinoma Adenoescamoso , Carcinoma Pulmonar de Células não Pequenas , Carcinoma de Células Escamosas , Neoplasias Pulmonares , Humanos , Carcinoma Adenoescamoso/genética , Neoplasias Pulmonares/genética , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patologia , Adenocarcinoma/patologia , Receptores ErbB/genética , Microambiente TumoralRESUMO
Accurate control of gene expression is essential for normal development and dysregulation of transcription underpins cancer onset and progression. Similar to cell cycle regulation, RNA polymerase II-driven transcription can be considered as a unidirectional multistep cycle, with thousands of unique transcription cycles occurring in concert within each cell. Each transcription cycle comprises recruitment, initiation, pausing, elongation, termination and recycling stages that are tightly controlled by the coordinated action of transcriptional cyclin-dependent kinases and their cognate cyclins as well as the opposing activity of transcriptional phosphatases. Oncogenic dysregulation of transcription can entail defective control of gene expression, either at select loci or more globally, impacting a large proportion of the genome. The resultant dependency on the core-transcriptional machinery is believed to render 'transcriptionally addicted' cancers sensitive to perturbation of transcription. Based on these findings, small molecules targeting transcriptional cyclin-dependent kinases and associated proteins hold promise for the treatment of cancer. Here, we utilize the transcription cycles concept to explain how dysregulation of these finely tuned gene expression processes may drive tumorigenesis and how therapeutically beneficial responses may arise from global or selective transcriptional perturbation. This conceptual framework helps to explain tumour-selective transcriptional dependencies and facilitates the rational design of combination therapies.
Assuntos
Neoplasias , Transcrição Gênica , Quinases Ciclina-Dependentes/genética , Quinases Ciclina-Dependentes/metabolismo , Humanos , Neoplasias/genética , Oncogenes , RNA Polimerase II/genética , RNA Polimerase II/metabolismoRESUMO
To separate causal effects of histone acetylation on chromatin accessibility and transcriptional output, we used integrated epigenomic and transcriptomic analyses following acute inhibition of major cellular lysine acetyltransferases P300 and CBP in hematological malignancies. We found that catalytic P300/CBP inhibition dynamically perturbs steady-state acetylation kinetics and suppresses oncogenic transcriptional networks in the absence of changes to chromatin accessibility. CRISPR-Cas9 screening identified NCOR1 and HDAC3 transcriptional co-repressors as the principal antagonists of P300/CBP by counteracting acetylation turnover kinetics. Finally, deacetylation of H3K27 provides nucleation sites for reciprocal methylation switching, a feature that can be exploited therapeutically by concomitant KDM6A and P300/CBP inhibition. Overall, this study indicates that the steady-state histone acetylation-methylation equilibrium functions as a molecular rheostat governing cellular transcription that is amenable to therapeutic exploitation as an anti-cancer regimen.
Assuntos
Biocatálise , Histonas/metabolismo , Oncogenes , Transcrição Gênica , Fatores de Transcrição de p300-CBP/metabolismo , Acetilação , Linhagem Celular , Cromatina/metabolismo , Proteínas Correpressoras/metabolismo , Sequência Conservada , Evolução Molecular , Redes Reguladoras de Genes , Genoma , Histona Desacetilases/metabolismo , Humanos , Cinética , Metilação , Modelos Biológicos , RNA Polimerase II/metabolismoRESUMO
Gene expression by RNA polymerase II (RNAPII) is tightly controlled by cyclin-dependent kinases (CDKs) at discrete checkpoints during the transcription cycle. The pausing checkpoint following transcription initiation is primarily controlled by CDK9. We discovered that CDK9-mediated, RNAPII-driven transcription is functionally opposed by a protein phosphatase 2A (PP2A) complex that is recruited to transcription sites by the Integrator complex subunit INTS6. PP2A dynamically antagonizes phosphorylation of key CDK9 substrates including DSIF and RNAPII-CTD. Loss of INTS6 results in resistance to tumor cell death mediated by CDK9 inhibition, decreased turnover of CDK9 phospho-substrates, and amplification of acute oncogenic transcriptional responses. Pharmacological PP2A activation synergizes with CDK9 inhibition to kill both leukemic and solid tumor cells, providing therapeutic benefit in vivo. These data demonstrate that fine control of gene expression relies on the balance between kinase and phosphatase activity throughout the transcription cycle, a process dysregulated in cancer that can be exploited therapeutically.
Assuntos
Quinase 9 Dependente de Ciclina/metabolismo , Terapia de Alvo Molecular , Neoplasias/tratamento farmacológico , Neoplasias/genética , Proteína Fosfatase 2/metabolismo , Proteínas de Ligação a RNA/metabolismo , Transcrição Gênica , Proteínas Supressoras de Tumor/metabolismo , Animais , Linhagem Celular Tumoral , Quinase 9 Dependente de Ciclina/antagonistas & inibidores , Resistencia a Medicamentos Antineoplásicos/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos Endogâmicos NOD , Fosforilação , Ligação Proteica , RNA Polimerase II/química , RNA Polimerase II/metabolismo , Especificidade por SubstratoRESUMO
Internal tandem duplication of the FMS-like tyrosine kinase 3 gene (FLT3-ITD) occurs in 30% of all acute myeloid leukemias (AML). Limited clinical efficacy of FLT3 inhibitors highlights the need for alternative therapeutic modalities in this subset of disease. Using human and murine models of FLT3-ITD-driven AML, we demonstrate that FLT3-ITD promotes serine synthesis and uptake via ATF4-dependent transcriptional regulation of genes in the de novo serine biosynthesis pathway and neutral amino acid transport. Genetic or pharmacologic inhibition of PHGDH, the rate-limiting enzyme of de novo serine biosynthesis, selectively inhibited proliferation of FLT3-ITD AMLs in vitro and in vivo. Moreover, pharmacologic inhibition of PHGDH sensitized FLT3-ITD AMLs to the standard-of-care chemotherapeutic cytarabine. Collectively, these data reveal novel insights into FLT3-ITD-induced metabolic reprogramming and reveal a targetable vulnerability in FLT3-ITD AML. SIGNIFICANCE: FLT3-ITD mutations are common in AML and are associated with poor prognosis. We show that FLT3-ITD stimulates serine biosynthesis, thereby rendering FLT3-ITD-driven leukemias dependent upon serine for proliferation and survival. This metabolic dependency can be exploited pharmacologically to sensitize FLT3-ITD-driven AMLs to chemotherapy.This article is highlighted in the In This Issue feature, p. 1307.
Assuntos
Leucemia Mieloide Aguda/metabolismo , Serina/metabolismo , Tirosina Quinase 3 Semelhante a fms/metabolismo , Animais , Linhagem Celular Tumoral/metabolismo , Modelos Animais de Doenças , Humanos , Camundongos , Inibidores de Proteínas QuinasesRESUMO
Elevated ribosome biogenesis in oncogene-driven cancers is commonly targeted by DNA-damaging cytotoxic drugs. Our previous first-in-human trial of CX-5461, a novel, less genotoxic agent that specifically inhibits ribosome biogenesis via suppression of RNA polymerase I (Pol I) transcription, revealed single-agent efficacy in refractory blood cancers. Despite this clinical response, patients were not cured. In parallel, we demonstrated a marked improvement in the in vivo efficacy of CX-5461 in combination with PI3K/AKT/mTORC1 pathway inhibitors. Here, we reveal the molecular basis for this improved efficacy observed in vivo, which is associated with specific suppression of translation of mRNAs encoding regulators of cellular metabolism. Importantly, acquired resistance to this cotreatment is driven by translational rewiring that results in dysregulated cellular metabolism and induction of a cAMP-dependent pathway critical for the survival of blood cancers including lymphoma and acute myeloid leukemia. Our studies thus identify key molecular mechanisms underpinning the response of blood cancers to selective inhibition of ribosome biogenesis and define metabolic vulnerabilities that will facilitate the rational design of more effective regimens for Pol I-directed therapies.
Assuntos
Neoplasias/metabolismo , Biossíntese de Proteínas/genética , Biossíntese de Proteínas/fisiologia , Ribossomos/metabolismo , Transcrição Gênica/efeitos dos fármacos , Animais , Antineoplásicos/farmacologia , Benzotiazóis/farmacologia , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Humanos , Masculino , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Naftiridinas/farmacologia , Neoplasias/genética , Fosfatidilinositol 3-Quinases/metabolismo , Biossíntese de Proteínas/efeitos dos fármacos , Inibidores de Proteínas Quinases , RNA Polimerase I/metabolismo , RNA Mensageiro/metabolismo , RNA Ribossômico , Ribossomos/efeitos dos fármacos , TranscriptomaRESUMO
The RNA polymerase II (POLII)-driven transcription cycle is tightly regulated at distinct checkpoints by cyclin-dependent kinases (CDKs) and their cognate cyclins. The molecular events underpinning transcriptional elongation, processivity, and the CDK-cyclin pair(s) involved remain poorly understood. Using CRISPR-Cas9 homology-directed repair, we generated analog-sensitive kinase variants of CDK12 and CDK13 to probe their individual and shared biological and molecular roles. Single inhibition of CDK12 or CDK13 induced transcriptional responses associated with cellular growth signaling pathways and/or DNA damage, with minimal effects on cell viability. In contrast, dual kinase inhibition potently induced cell death, which was associated with extensive genome-wide transcriptional changes including widespread use of alternative 3' polyadenylation sites. At the molecular level, dual kinase inhibition resulted in the loss of POLII CTD phosphorylation and greatly reduced POLII elongation rates and processivity. These data define substantial redundancy between CDK12 and CDK13 and identify both as fundamental regulators of global POLII processivity and transcription elongation.
RESUMO
MCL-1 is one of the most frequently amplified genes in cancer, facilitating tumor initiation and maintenance and enabling resistance to anti-tumorigenic agents including the BCL-2 selective inhibitor venetoclax. The expression of MCL-1 is maintained via P-TEFb-mediated transcription, where the kinase CDK9 is a critical component. Consequently, we developed a series of potent small-molecule inhibitors of CDK9, exemplified by the orally active A-1592668, with CDK selectivity profiles that are distinct from related molecules that have been extensively studied clinically. Short-term treatment with A-1592668 rapidly downregulates RNA pol-II (Ser 2) phosphorylation resulting in the loss of MCL-1 protein and apoptosis in MCL-1-dependent hematologic tumor cell lines. This cell death could be attenuated by either inhibiting caspases or overexpressing BCL-2 protein. Synergistic cell killing was also observed between A-1592668 or the related analog A-1467729, and venetoclax in a number of hematologic cell lines and primary NHL patient samples. Importantly, the CDK9 inhibitor plus venetoclax combination was well tolerated in vivo and demonstrated efficacy superior to either agent alone in mouse models of lymphoma and AML. These data indicate that CDK9 inhibitors could be highly efficacious in tumors that depend on MCL-1 for survival or when used in combination with venetoclax in malignancies dependent on MCL-1 and BCL-2.
Assuntos
Antineoplásicos/farmacologia , Quinase 9 Dependente de Ciclina/antagonistas & inibidores , Neoplasias Hematológicas , Inibidores de Proteínas Quinases/farmacologia , Animais , Apoptose/efeitos dos fármacos , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sinergismo Farmacológico , Humanos , Camundongos , Sulfonamidas/farmacologia , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Most non-small cell lung cancers (NSCLC) contain nontargetable mutations, including KRAS, TP53, or STK11/LKB1 alterations. By coupling ex vivo drug sensitivity profiling with in vivo drug response studies, we aimed to identify drug vulnerabilities for these NSCLC subtypes. Primary adenosquamous carcinoma (ASC) or adenocarcinoma (AC) cultures were established from KrasG12D/+;Lkb1fl/fl (KL) tumors or AC cultures from KrasG12D/+;p53fl/fl (KP) tumors. Although p53-null cells readily propagated as conventional cultures, Lkb1-null cells required conditional reprograming for establishment. Drug response profiling revealed short-term response to MEK inhibition, yet long-term clonogenic assays demonstrated resistance, associated with sustained or adaptive activation of receptor tyrosine kinases (RTK): activation of ERBBs in KL cultures, or FGFR in AC cultures. Furthermore, pan-ERBB inhibition reduced the clonogenicity of KL cultures, which was exacerbated by combinatorial MEK inhibition, whereas combinatorial MEK and FGFR inhibition suppressed clonogenicity of AC cultures. Importantly, in vivo studies confirmed KL-selective sensitivity to pan-ERBB inhibition, which correlated with high ERBB ligand expression and activation of ERBB receptors, implying that ERBB network activity may serve as a predictive biomarker of drug response. Interestingly, in human NSCLCs, phosphorylation of EGFR or ERBB3 was frequently detected in ASCs and squamous cell carcinomas. We conclude that analysis of in situ ERBB signaling networks in conjunction with ex vivo drug response profiling and biochemical dissection of adaptive RTK activities may serve as a valid diagnostic approach to identify tumors sensitive to ERBB network inhibition.
Assuntos
Receptores ErbB/antagonistas & inibidores , Neoplasias Pulmonares/genética , Mutação/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Receptores Proteína Tirosina Quinases/metabolismo , Transdução de Sinais , Animais , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/genética , Linhagem Celular Tumoral , Proliferação de Células , Ativação Enzimática , Receptores ErbB/metabolismo , Genótipo , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Camundongos , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismoRESUMO
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.
Assuntos
Benzotiazóis/farmacologia , Leucemia Mieloide Aguda/tratamento farmacológico , Naftiridinas/farmacologia , Células-Tronco Neoplásicas/enzimologia , Proteínas Pol1 do Complexo de Iniciação de Transcrição/antagonistas & inibidores , Transcrição Gênica/efeitos dos fármacos , Animais , Divisão Celular/efeitos dos fármacos , Divisão Celular/genética , Linhagem Celular Tumoral , Quinase 1 do Ponto de Checagem/genética , Quinase 1 do Ponto de Checagem/metabolismo , Quinase do Ponto de Checagem 2/genética , Quinase do Ponto de Checagem 2/metabolismo , Fase G2/efeitos dos fármacos , Fase G2/genética , Humanos , Leucemia Mieloide Aguda/epidemiologia , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos Mutantes , Células-Tronco Neoplásicas/patologia , Proteínas Pol1 do Complexo de Iniciação de Transcrição/genética , Proteínas Pol1 do Complexo de Iniciação de Transcrição/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
Lung cancers exhibit pronounced functional heterogeneity, confounding precision medicine. We studied how the cell of origin contributes to phenotypic heterogeneity following conditional expression of KrasG12D and loss of Lkb1 (Kras;Lkb1). Using progenitor cell-type-restricted adenoviral Cre to target cells expressing surfactant protein C (SPC) or club cell antigen 10 (CC10), we show that Ad5-CC10-Cre-infected mice exhibit a shorter latency compared with Ad5-SPC-Cre cohorts. We further demonstrate that CC10+ cells are the predominant progenitors of adenosquamous carcinoma (ASC) tumors and give rise to a wider spectrum of histotypes that includes mucinous and acinar adenocarcinomas. Transcriptome analysis shows ASC histotype-specific upregulation of pro-inflammatory and immunomodulatory genes. This is accompanied by an ASC-specific immunosuppressive environment, consisting of downregulated MHC genes, recruitment of CD11b+ Gr-1+ tumor-associated neutrophils (TANs), and decreased T cell numbers. We conclude that progenitor cell-specific etiology influences the Kras;Lkb1-driven tumor histopathology spectrum and histotype-specific immune microenvironment.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/patologia , Neoplasias Pulmonares/patologia , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Proteínas Quinases Ativadas por AMP , Animais , Arginase/genética , Arginase/metabolismo , Carcinoma Pulmonar de Células não Pequenas/imunologia , Carcinoma Pulmonar de Células não Pequenas/mortalidade , Humanos , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Estimativa de Kaplan-Meier , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/mortalidade , Camundongos , Camundongos Endogâmicos C57BL , Neutrófilos/metabolismo , Fenótipo , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Transcriptoma , Proteína Supressora de Tumor p53/metabolismo , Uteroglobina/genética , Uteroglobina/metabolismoRESUMO
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.
Assuntos
Antineoplásicos/farmacologia , Neoplasias da Próstata/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-pim-1/antagonistas & inibidores , RNA Polimerase I/antagonistas & inibidores , Transcrição Gênica/efeitos dos fármacos , Animais , Azepinas/farmacologia , Benzotiazóis/farmacologia , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Feminino , Humanos , Indóis/farmacologia , Masculino , Camundongos , Naftiridinas/farmacologia , PTEN Fosfo-Hidrolase/metabolismo , Próstata/efeitos dos fármacos , Próstata/metabolismo , Neoplasias da Próstata/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Transdução de Sinais/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto/métodosRESUMO
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.
Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Benzotiazóis/farmacologia , Naftiridinas/farmacologia , Inibidores da Síntese de Ácido Nucleico/farmacologia , RNA Polimerase I/antagonistas & inibidores , Transdução de Sinais , Animais , Apoptose , Crescimento Celular , Nucléolo Celular/metabolismo , Proliferação de Células , Cromatina/metabolismo , Ensaio Cometa , Dano ao DNA , DNA Ribossômico/genética , Fibroblastos/metabolismo , Neoplasias Hematológicas/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos C57BL , RNA Polimerase I/metabolismo , Proteína Supressora de Tumor p53/metabolismoRESUMO
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.
Assuntos
Benzotiazóis/administração & dosagem , DNA Ribossômico/antagonistas & inibidores , Everolimo/administração & dosagem , Linfoma de Células B/terapia , Naftiridinas/administração & dosagem , Proteínas Proto-Oncogênicas c-myc/genética , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Benzotiazóis/farmacologia , Sinergismo Farmacológico , Everolimo/farmacologia , Humanos , Linfoma de Células B/genética , Camundongos , Naftiridinas/farmacologia , Biossíntese de Proteínas/efeitos dos fármacos , Proto-Oncogene Mas , Transdução de Sinais/efeitos dos fármacos , Análise de Sobrevida , Transcrição Gênica/efeitos dos fármacos , Resultado do Tratamento , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The contribution of the nucleolus to cancer is well established with respect to its traditional role in facilitating ribosome biogenesis and proliferative capacity. More contemporary studies however, infer that nucleoli contribute a much broader role in malignant transformation. Specifically, extra-ribosomal functions of the nucleolus position it as a central integrator of cellular proliferation and stress signaling, and are emerging as important mechanisms for modulating how oncogenes and tumor suppressors operate in normal and malignant cells. The dependence of certain tumor cells to co-opt nucleolar processes to maintain their cancer phenotypes has now clearly been demonstrated by the application of small molecule inhibitors of RNA Polymerase I to block ribosomal DNA transcription and disrupt nucleolar function (Bywater et al., 2012 [1]). These drugs, which selectively kill tumor cells in vivo while sparing normal cells, have now progressed to clinical trials. It is likely that we have only just begun to scratch the surface of the potential of the nucleolus as a new target for cancer therapy, with "suppression of nucleolar stress" representing an emerging "hallmark" of cancer. This article is part of a Special Issue entitled: Role of the Nucleolus in Human Disease.
Assuntos
Nucléolo Celular/genética , DNA Ribossômico/metabolismo , Neoplasias/genética , RNA Polimerase I/metabolismo , Benzotiazóis/farmacologia , Transformação Celular Neoplásica/genética , DNA Ribossômico/genética , Genes myc/genética , Humanos , Naftiridinas/farmacologia , Neoplasias/patologia , RNA Polimerase I/antagonistas & inibidores , RNA Polimerase I/genética , Ribossomos/genética , Ribossomos/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
Pharmacological strategies capable of directly targeting MYC are elusive. Previous studies have shown that MYC-driven lymphomagenesis is associated with mammalian target of rapamycin (mTOR) activation and a MYC-evoked DNA damage response (DDR) transduced by phosphatidylinositol-3-kinase (PI3K)-related kinases (DNA-PK, ATM, and ATR). Here we report that BEZ235, a multitargeted pan-PI3K/dual-mTOR inhibitor, potently killed primary Myc-driven B-cell lymphomas and human cell lines bearing IG-cMYC translocations. Using pharmacologic and genetic dissection of PI3K/mTOR signaling, dual DDR/mTORC1 inhibition was identified as a key mediator of apoptosis. Moreover, apoptosis was initiated at drug concentrations insufficient to antagonize PI3K/mTORC2-regulated AKT phosphorylation. p53-independent induction of the proapoptotic BH3-only protein BMF was identified as a mechanism by which dual DDR/mTORC1 inhibition caused lymphoma cell death. BEZ235 treatment induced apoptotic tumor regressions in vivo that correlated with suppression of mTORC1-regulated substrates and reduced H2AX phosphorylation and also with feedback phosphorylation of AKT. These mechanistic studies hold important implications for the use of multitargeted PI3K inhibitors in the treatment of hematologic malignancies. In particular, the newly elucidated role of PI3K-related DDR kinases in response to PI3K inhibitors offers a novel therapeutic opportunity for the treatment of hematologic malignancies with an MYC-driven DDR.
Assuntos
Apoptose/efeitos dos fármacos , Imidazóis/farmacologia , Linfoma de Células B/prevenção & controle , Quinolinas/farmacologia , Animais , Western Blotting , Linhagem Celular , Dano ao DNA/efeitos dos fármacos , Receptor com Domínio Discoidina 1 , Relação Dose-Resposta a Droga , Citometria de Fluxo , Histonas/metabolismo , Humanos , Linfoma de Células B/genética , Linfoma de Células B/patologia , Alvo Mecanístico do Complexo 1 de Rapamicina , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Complexos Multiproteicos/antagonistas & inibidores , Complexos Multiproteicos/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-myc/genética , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Receptores Proteína Tirosina Quinases/metabolismo , Análise de Sobrevida , Serina-Treonina Quinases TOR/antagonistas & inibidores , Serina-Treonina Quinases TOR/metabolismo , Células Tumorais CultivadasRESUMO
The dysregulation of PI3K/AKT/mTORC1 signalling and/or hyperactivation of MYC are observed in a high proportion of human cancers, and together they form a 'super signalling' network mediating malignancy. A fundamental downstream action of this signalling network is up-regulation of ribosome biogenesis and subsequent alterations in the patterns of translation and increased protein synthesis, which are thought to be critical for AKT/MYC-driven oncogenesis. We have demonstrated that AKT and MYC cooperate to drive ribosomal DNA (rDNA) transcription and ribosome biogenesis, with AKT being essential for rDNA transcription and in vitro survival of lymphoma cells isolated from a MYC-driven model of B-cell lymphoma (Eµ-Myc) [Chan JC et al., (2011) Science Signalling 4, ra56]. Here we show that the allosteric AKT inhibitor MK-2206 rapidly and potently antagonizes rDNA transcription in Eµ-Myc B-cell lymphomas in vivo, and this is associated with a rapid reduction in indicators of disease burden, including spleen weight and the abundance of tumour cells in both the circulation and lymph nodes. Extended treatment of tumour-bearing mice with MK-2206 resulted in a significant delay in disease progression, associated with increased B-cell lymphoma apoptosis. Our findings suggest that malignant diseases characterized by unrestrained ribosome biogenesis may be vulnerable to therapeutic strategies that target the PI3K/AKT/mTORC1/MYC growth control network.