RESUMEN
Alternative lengthening of telomeres (ALT) supports telomere maintenance in 10-15% of cancers, thus representing a compelling target for therapy. By performing anti-cancer compound library screen on isogenic cell lines and using extrachromosomal telomeric C-circles, as a bona fide marker of ALT activity, we identify a receptor tyrosine kinase inhibitor ponatinib that deregulates ALT mechanisms, induces telomeric dysfunction, reduced ALT-associated telomere synthesis, and targets, in vivo, ALT-positive cells. Using RNA-sequencing and quantitative phosphoproteomic analyses, combined with C-circle level assessment, we find an ABL1-JNK-JUN signalling circuit to be inhibited by ponatinib and to have a role in suppressing telomeric C-circles. Furthermore, transcriptome and interactome analyses suggest a role of JUN in DNA damage repair. These results are corroborated by synergistic drug interactions between ponatinib and either DNA synthesis or repair inhibitors, such as triciribine. Taken together, we describe here a signalling pathway impacting ALT which can be targeted by a clinically approved drug.
Asunto(s)
Transducción de Señal , Telómero , Supervivencia Celular , Transducción de Señal/efectos de los fármacos , Regulación de la Expresión Génica , Reparación del ADN , Replicación del ADN , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Humanos , Animales , Ratones , Línea Celular TumoralRESUMEN
Recurrent loss-of-function mutations of spliceosome gene, ZRSR2, occur in myelodysplastic syndromes (MDS). Mutation/loss of ZRSR2 in human myeloid cells primarily causes impaired splicing of the U12-type introns. In order to further investigate the role of this splice factor in RNA splicing and hematopoietic development, we generated mice lacking ZRSR2. Unexpectedly, Zrsr2-deficient mice developed normal hematopoiesis with no abnormalities in myeloid differentiation evident in either young or ≥1-year old knockout mice. Repopulation ability of Zrsr2-deficient hematopoietic stem cells was also unaffected in both competitive and non-competitive reconstitution assays. Myeloid progenitors lacking ZRSR2 exhibited mis-splicing of U12-type introns, however, this phenotype was moderate compared to the ZRSR2-deficient human cells. Our investigations revealed that a closely related homolog, Zrsr1, expressed in the murine hematopoietic cells, but not in human cells contributes to splicing of U12-type introns. Depletion of Zrsr1 in Zrsr2 KO myeloid cells exacerbated retention of the U12-type introns, thus highlighting a collective role of ZRSR1 and ZRSR2 in murine U12-spliceosome. We also demonstrate that aberrant retention of U12-type introns of MAPK9 and MAPK14 leads to their reduced protein expression. Overall, our findings highlight that both ZRSR1 and ZRSR2 are functional components of the murine U12-spliceosome, and depletion of both proteins is required to accurately model ZRSR2-mutant MDS in mice.
Asunto(s)
Síndromes Mielodisplásicos , Empalme del ARN , Ribonucleoproteínas , Factor de Empalme U2AF , Animales , Intrones , Ratones , Mutación , Síndromes Mielodisplásicos/genética , Ribonucleoproteínas/genética , Ribonucleoproteínas/metabolismo , Empalmosomas/genética , Factor de Empalme U2AF/genética , Factor de Empalme U2AF/metabolismoRESUMEN
Proteasome inhibitors, such as bortezomib and carfilzomib, have shown efficacy in anti-cancer therapy in hematological diseases but not in solid cancers. Here, we found that liposarcomas (LPS) are susceptible to proteasome inhibition, and identified drugs that synergize with carfilzomib, such as selinexor, an inhibitor of XPO1-mediated nuclear export. Through quantitative nuclear protein profiling and phospho-kinase arrays, we identified potential mode of actions of this combination, including interference with ribosome biogenesis and inhibition of pro-survival kinase PRAS40. Furthermore, by assessing global protein levels changes, FADS2, a key enzyme regulating fatty acids synthesis, was found down-regulated after proteasome inhibition. Interestingly, SC26196, an inhibitor of FADS2, synergized with carfilzomib. Finally, to identify further combinational options, we performed high-throughput drug screening and uncovered novel drug interactions with carfilzomib. For instance, cyclosporin A, a known immunosuppressive agent, enhanced carfilzomib's efficacy in vitro and in vivo. Altogether, these results demonstrate that carfilzomib and its combinations could be repurposed for LPS clinical management.
Asunto(s)
Ácido Graso Desaturasas/genética , Carioferinas/genética , Liposarcoma/tratamiento farmacológico , Oligopéptidos/farmacología , Receptores Citoplasmáticos y Nucleares/genética , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Bortezomib/farmacología , Línea Celular Tumoral , Núcleo Celular/efectos de los fármacos , Resistencia a Antineoplásicos/genética , Sinergismo Farmacológico , Ácido Graso Desaturasas/antagonistas & inhibidores , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Hidrazinas/farmacología , Liposarcoma/genética , Liposarcoma/patología , Piperazinas/farmacología , Complejo de la Endopetidasa Proteasomal/efectos de los fármacos , Inhibidores de Proteasoma/farmacología , Triazoles/farmacología , Proteína Exportina 1RESUMEN
BACKGROUND & AIMS: We investigated the transcriptome of esophageal squamous cell carcinoma (ESCC) cells, activity of gene regulatory (enhancer and promoter regions), and the effects of blocking epigenetic regulatory proteins. METHODS: We performed chromatin immunoprecipitation sequencing with antibodies against H3K4me1, H3K4me3, and H3K27ac and an assay for transposase-accessible chromatin to map the enhancer regions and accessible chromatin in 8 ESCC cell lines. We used the CRC_Mapper algorithm to identify core regulatory circuitry transcription factors in ESCC cell lines, and determined genome occupancy profiles for 3 of these factors. In ESCC cell lines, expression of transcription factors was knocked down with small hairpin RNAs, promoter and enhancer regions were disrupted by CRISPR/Cas9 genome editing, or bromodomains and extraterminal (BET) family proteins and histone deacetylases (HDACs) were inhibited with ARV-771 and romidepsin, respectively. ESCC cell lines were then analyzed by whole-transcriptome sequencing, immunoprecipitation, immunoblots, immunohistochemistry, and viability assays. Interactions between distal enhancers and promoters were identified and verified with circular chromosome conformation capture sequencing. NOD-SCID mice were given injections of modified ESCC cells, some mice where given injections of HDAC or BET inhibitors, and growth of xenograft tumors was measured. RESULTS: We identified super-enhancer-regulated circuits and transcription factors TP63, SOX2, and KLF5 as core regulatory factors in ESCC cells. Super-enhancer regulation of ALDH3A1 mediated by core regulatory factors was required for ESCC viability. We observed direct interactions between the promoter region of TP63 and functional enhancers, mediated by the core regulatory circuitry transcription factors. Deletion of enhancer regions from ESCC cells decreased expression of the core regulatory circuitry transcription factors and reduced cell viability; these same results were observed with knockdown of each core regulatory circuitry transcription factor. Incubation of ESCC cells with BET and HDAC disrupted the core regulatory circuitry program and the epigenetic modifications observed in these cells; mice given injections of HDAC or BET inhibitors developed smaller xenograft tumors from the ESCC cell lines. Xenograft tumors grew more slowly in mice given the combination of ARV-771 and romidepsin than mice given either agent alone. CONCLUSIONS: In epigenetic and transcriptional analyses of ESCC cell lines, we found the transcription factors TP63, SOX2, and KLF5 to be part of a core regulatory network that determines chromatin accessibility, epigenetic modifications, and gene expression patterns in these cells. A combination of epigenetic inhibitors slowed growth of xenograft tumors derived from ESCC cells in mice.
Asunto(s)
Epigénesis Genética , Neoplasias Esofágicas/genética , Carcinoma de Células Escamosas de Esófago/genética , Regulación Neoplásica de la Expresión Génica , Factores de Transcripción de Tipo Kruppel/genética , Factores de Transcripción SOXB1/genética , Factores de Transcripción/genética , Transcripción Genética , Proteínas Supresoras de Tumor/genética , Animales , Antineoplásicos/farmacología , Línea Celular Tumoral , Proliferación Celular , Ensamble y Desensamble de Cromatina , Epigénesis Genética/efectos de los fármacos , Neoplasias Esofágicas/tratamiento farmacológico , Neoplasias Esofágicas/metabolismo , Neoplasias Esofágicas/patología , Carcinoma de Células Escamosas de Esófago/tratamiento farmacológico , Carcinoma de Células Escamosas de Esófago/metabolismo , Carcinoma de Células Escamosas de Esófago/patología , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Inhibidores de Histona Desacetilasas/farmacología , Humanos , Factores de Transcripción de Tipo Kruppel/metabolismo , Ratones Endogámicos NOD , Ratones SCID , Proteínas/antagonistas & inhibidores , Proteínas/metabolismo , Factores de Transcripción SOXB1/metabolismo , Factores de Transcripción/metabolismo , Transcripción Genética/efectos de los fármacos , Transcriptoma , Carga Tumoral , Proteínas Supresoras de Tumor/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Some cancer cells elongate their telomeres through the ALT (alternative lengthening of telomeres) pathway, which is based on homologous recombination for the addition of telomere repeats without telomerase activity. General control non-derepressible 5 (GCN5) and P300/CBP-associated factor (PCAF), two homologous lysine acetyltransferases, exert opposite effects on the ALT pathway, inhibiting or favoring it respectively. Here we show that ALT cells are particularly sensitive to the inhibition of acetyltransferases activities using Anacardic Acid (AA). AA treatment recapitulates the effect of PCAF knockdown on several ALT features, suggesting that AA decreased the ALT mechanism through the inhibition of lysine transferase activity of PCAF, but not that of GCN5. Furthermore, AA specifically sensitizes human ALT cells to radiation as compared to telomerase-positive cells suggesting that the inhibition of lysine acetyltransferases activity may be used to increase the radiotherapy efficiency against ALT cancers.
RESUMEN
Squamous cell carcinomas (SCCs) are aggressive malignancies. Previous report demonstrated that master transcription factors (TFs) TP63 and SOX2 exhibited overlapping genomic occupancy in SCCs. However, functional consequence of their frequent co-localization at super-enhancers remains incompletely understood. Here, epigenomic profilings of different types of SCCs reveal that TP63 and SOX2 cooperatively and lineage-specifically regulate long non-coding RNA (lncRNA) CCAT1 expression, through activation of its super-enhancers and promoter. Silencing of CCAT1 substantially reduces cellular growth both in vitro and in vivo, phenotyping the effect of inhibiting either TP63 or SOX2. ChIRP analysis shows that CCAT1 forms a complex with TP63 and SOX2, which regulates EGFR expression by binding to the super-enhancers of EGFR, thereby activating both MEK/ERK1/2 and PI3K/AKT signaling pathways. These results together identify a SCC-specific DNA/RNA/protein complex which activates TP63/SOX2-CCAT1-EGFR cascade and promotes SCC tumorigenesis, advancing our understanding of transcription dysregulation in cancer biology mediated by master TFs and super-enhancers.
Asunto(s)
Carcinoma de Células Escamosas/genética , Elementos de Facilitación Genéticos , ARN Largo no Codificante/genética , Factores de Transcripción SOXB1/genética , Factores de Transcripción/genética , Proteínas Supresoras de Tumor/genética , Animales , Carcinoma de Células Escamosas/patología , Línea Celular Tumoral , Receptores ErbB/genética , Receptores ErbB/metabolismo , Regulación Neoplásica de la Expresión Génica , Humanos , Quinasas Quinasa Quinasa PAM/genética , Quinasas Quinasa Quinasa PAM/metabolismo , Ratones Endogámicos NOD , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Regiones Promotoras Genéticas , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Factores de Transcripción SOXB1/metabolismo , Factores de Transcripción/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The clinical management of metastatic colorectal cancer (mCRC) faces major challenges. Here, we show that nilotinib, a clinically approved drug for chronic myeloid leukaemia, strongly inhibits human CRC cell invasion in vitro and reduces their metastatic potential in intrasplenic tumour mouse models. Nilotinib acts by inhibiting the kinase activity of DDR1, a receptor tyrosine kinase for collagens, which we identified as a RAS-independent inducer of CRC metastasis. Using quantitative phosphoproteomics, we identified BCR as a new DDR1 substrate and demonstrated that nilotinib prevents DDR1-mediated BCR phosphorylation on Tyr177, which is important for maintaining ß-catenin transcriptional activity necessary for tumour cell invasion. DDR1 kinase inhibition also reduced the invasion of patient-derived metastatic and circulating CRC cell lines. Collectively, our results indicate that the targeting DDR1 kinase activity with nilotinib may be beneficial for patients with mCRC.
Asunto(s)
Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/patología , Receptor con Dominio Discoidina 1/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-bcr/metabolismo , Receptores de Colágeno/metabolismo , Animales , Receptor con Dominio Discoidina 1/genética , Células HCT116 , Células HEK293 , Humanos , Ratones , Fosfoproteínas/metabolismo , Proteínas Tirosina Quinasas/genética , Proteínas Proto-Oncogénicas c-bcr/genética , Pirimidinas/farmacología , Receptores de Colágeno/genética , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genéticaRESUMEN
Cancer cells can use a telomerase-independent mechanism, known as alternative lengthening of telomeres (ALT), to elongate their telomeres. General control non-derepressible 5 (GCN5) and P300/CBP-associated factor (PCAF) are two homologous acetyltransferases that are mutually exclusive subunits in SAGA-like complexes. Here, we reveal that down regulation of GCN5 and PCAF had differential effects on some phenotypic characteristics of ALT cells. Our results suggest that GCN5 is present at telomeres and opposes telomere recombination, in contrast to PCAF that may indirectly favour them in ALT cells.
Asunto(s)
Estudios de Asociación Genética , Homeostasis del Telómero/genética , Telómero/genética , Factores de Transcripción p300-CBP/genética , Ciclo Celular/genética , Línea Celular Tumoral , Proliferación Celular , Expresión Génica , Técnicas de Silenciamiento del Gen , Inestabilidad Genómica , Humanos , Cuerpos de Inclusión Intranucleares/genética , Cuerpos de Inclusión Intranucleares/metabolismo , Leucemia Promielocítica Aguda/genética , Leucemia Promielocítica Aguda/metabolismo , Leucemia Promielocítica Aguda/patología , Unión Proteica , Intercambio de Cromátides Hermanas , Translocación GenéticaRESUMEN
Cancer cells rely on telomerase or the alternative lengthening of telomeres (ALT) pathway to overcome replicative mortality. ALT is mediated by recombination and is prevalent in a subset of human cancers, yet whether it can be exploited therapeutically remains unknown. Loss of the chromatin-remodeling protein ATRX associates with ALT in cancers. Here, we show that ATRX loss compromises cell-cycle regulation of the telomeric noncoding RNA TERRA and leads to persistent association of replication protein A (RPA) with telomeres after DNA replication, creating a recombinogenic nucleoprotein structure. Inhibition of the protein kinase ATR, a critical regulator of recombination recruited by RPA, disrupts ALT and triggers chromosome fragmentation and apoptosis in ALT cells. The cell death induced by ATR inhibitors is highly selective for cancer cells that rely on ALT, suggesting that such inhibitors may be useful for treatment of ALT-positive cancers.
Asunto(s)
Antineoplásicos/farmacología , Pirazinas/farmacología , Sulfonas/farmacología , Homeostasis del Telómero , Telómero/efectos de los fármacos , Telómero/metabolismo , Apoptosis , Proteínas de la Ataxia Telangiectasia Mutada/antagonistas & inhibidores , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Ciclo Celular , Línea Celular Tumoral , ADN Helicasas/genética , ADN Helicasas/metabolismo , Técnicas de Silenciamiento del Gen , Glioma/tratamiento farmacológico , Glioma/genética , Células HeLa , Recombinación Homóloga , Humanos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Osteosarcoma/tratamiento farmacológico , Osteosarcoma/genética , Proteína de la Leucemia Promielocítica , ARN no Traducido/genética , ARN no Traducido/metabolismo , Proteína de Replicación A/metabolismo , Telomerasa/metabolismo , Telómero/genética , Proteína 2 de Unión a Repeticiones Teloméricas/metabolismo , Factores de Transcripción/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Proteína Nuclear Ligada al Cromosoma XRESUMEN
Glioblastoma multiforme is the most aggressive primary tumor of the central nervous system. Glioma stem cells (GSCs), a small population of tumor cells with stem-like properties, are supposedly responsible for glioblastoma multiforme relapse after current therapies. In approximately thirty percent of glioblastoma multiforme tumors, telomeres are not maintained by telomerase but through an alternative mechanism, termed alternative lengthening of telomere (ALT), suggesting potential interest in developing specific therapeutic strategies. However, no preclinical model of ALT glioma was available until the isolation of TG20 cells from a human ALT glioma. Herein, we show that TG20 cells exhibit a high level of telomeric recombination but a stable karyotype, indicating that their telomeres retain their protective function against chromosomal instability. TG20 cells possess all of the characteristic features of GSCs: the expression of neural stem cell markers, the generation of intracerebral tumors in NOD-SCID-IL2Rγ (NSG) mice as well as in nude mice, and the ability to sustain serial intracerebral transplantations without expressing telomerase, demonstrating the stability of the ALT phenotype in vivo. Furthermore, we also demonstrate that 360B, a G-quadruplex ligand of the pyridine derivative series that impairs telomere replication and mitotic progression in cancer cells, prevents the development of TG20 tumors. Together, our results show that intracerebral grafts of TG20 cells in immunodeficient mice constitute an efficient preclinical model of ALT glioblastoma multiforme and that G-quadruplex ligands are a potential therapy for this specific type of tumor.