RESUMEN
The diversity of structural variants (SVs) in melanoma and how they impact oncogenesis are incompletely known. We performed harmonized analysis of SVs across melanoma histologic and genomic subtypes, and we identified distinct global properties between subtypes. These included the frequency and size of SVs and SV classes, their relation to chromothripsis events, and the impact on cancer-related genes of SVs that alter topologically associated domain (TAD) boundaries. Following our prior identification of double-stranded break repair deficiency in a subset of triple-wild-type cutaneous melanoma, we identified MRE11 and NBN loss-of-function SVs in melanomas with this mutational signature. Experimental knockouts of MRE11 and NBN, followed by olaparib cell viability assays in melanoma cells, indicated that dysregulation of each of these genes may cause sensitivity to PARP inhibitors in cutaneous melanomas. Broadly, harmonized analysis of melanoma SVs revealed distinct global genomic properties and molecular drivers, which may have biological and therapeutic impact.
Asunto(s)
Melanoma , Melanoma/genética , Melanoma/patología , Melanoma/metabolismo , Humanos , Línea Celular Tumoral , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/patología , Neoplasias Cutáneas/metabolismo , Carcinogénesis/genética , Proteína Homóloga de MRE11/genética , Proteína Homóloga de MRE11/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Ftalazinas/farmacología , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Variación Estructural del Genoma/genética , Piperazinas/farmacología , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacologíaRESUMEN
Extrachromosomal circular DNA (eccDNA) are present within all eukaryotic organisms and actively contribute to gene expression changes. MicroDNA (200-1000bp) are the most abundant type of eccDNA and can amplify tRNA, microRNA, and novel si-like RNA sequences. Due to the heterogeneity of microDNA and the limited technology to directly quantify circular DNA molecules, the specific DNA repair pathways that contribute to microDNA formation have not been fully elucidated. Using a sensitive and quantitative assay that quantifies eight known abundant microDNA, we report that microDNA levels are dependent on resection after double-strand DNA break (DSB) and repair by Microhomology Mediated End Joining (MMEJ). Further, repair of DSB without resection by canonical Non-Homologous End Joining (c-NHEJ) diminishes microDNA formation. MicroDNA levels are induced locally even by a single site-directed DSB, suggesting that excision of genomic DNA by two closely spaced DSB is not necessary for microDNA formation. Consistent with all this, microDNA levels accumulate as cells undergo replication in S-phase, when DNA breaks and repair are elevated, and microDNA levels are decreased if DNA synthesis is prevented. Thus, formation of microDNA occurs during the repair of endogenous or induced DNA breaks by resection-based DNA repair pathways.
Asunto(s)
Daño del ADN , Reparación del ADN por Unión de Extremidades , ADN Circular/genética , Replicación del ADN , Células HEK293 , Células HeLa , HumanosRESUMEN
DNA double-strand breaks (DSBs) are highly cytotoxic lesions that can lead to chromosome rearrangements, genomic instability and cell death. Consequently, cells have evolved multiple mechanisms to efficiently repair DSBs to preserve genomic integrity. We have developed a DSB repair assay system, designated CDDR (CRISPR-Cas9-based Dual-fluorescent DSB Repair), that enables the detection and quantification of DSB repair outcomes in mammalian cells with high precision. CDDR is based on the introduction and subsequent resolution of one or two DSB(s) in an intrachromosomal fluorescent reporter following the expression of Cas9 and sgRNAs targeting the reporter. CDDR can discriminate between high-fidelity (HF) and error-prone non-homologous end-joining (NHEJ), as well as between proximal and distal NHEJ repair. Furthermore, CDDR can detect homology-directed repair (HDR) with high sensitivity. Using CDDR, we found HF-NHEJ to be strictly dependent on DNA Ligase IV, XRCC4 and XLF, members of the canonical branch of NHEJ pathway (c-NHEJ). Loss of these genes also stimulated HDR, and promoted error-prone distal end-joining. Deletion of the DNA repair kinase ATM, on the other hand, stimulated HF-NHEJ and suppressed HDR. These findings demonstrate the utility of CDDR in characterizing the effect of repair factors and in elucidating the balance between competing DSB repair pathways.
Asunto(s)
Bioensayo/métodos , Sistemas CRISPR-Cas/genética , Roturas del ADN de Doble Cadena , Reparación del ADN , Colorantes Fluorescentes/química , Genes Reporteros , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Línea Celular Tumoral , ADN Ligasa (ATP)/metabolismo , Enzimas Reparadoras del ADN/metabolismo , Proteínas de Unión al ADN/metabolismo , Células HEK293 , HumanosRESUMEN
OBJECTIVE: To understand role of barrier molecules in melanomas. BACKGROUND: We have reported poor patient survival and low immune infiltration of melanomas that overexpress a set of genes that include filaggrin (FLG), dystonin (DST), junction plakoglobin (JUP), and plakophilin-3 (PKP3), and are involved in cell-cell adhesions. We hypothesized that these associations are causal, either by interfering with immune cell infiltration or by enhancing melanoma cell growth. METHODS: FLG and DST were knocked out by CRISPR/Cas9 in human DM93 and murine B16-F1 melanoma cells. PKP3 and JUP were overexpressed in murine B16-AAD and human VMM39 melanoma cells by lentiviral transduction. These cell lines were evaluated in vitro for cell proliferation and in vivo for tumor burden, immune composition, cytokine expression, and vascularity. RESULTS: Immune infiltrates were not altered by these genes. FLG/DST knockout reduced proliferation of human DM93 melanoma in vitro, and decreased B16-F1 tumor burden in vivo. Overexpression of JUP, but not PKP3, in B16-AAD significantly increased tumor burden, increased VEGF-A, reduced IL-33, and enhanced vascularity. CONCLUSIONS: FLG and DST support melanoma cell growth in vitro and in vivo. Growth effects of JUP were only evident in vivo, and may be mediated, in part, by enhancing angiogenesis. In addition, growth-promoting effects of FLG and DST in vitro suggest that these genes may also support melanoma cell proliferation through angiogenesis-independent pathways. These findings identify FLG, DST, and JUP as novel therapeutic targets whose down-regulation may provide clinical benefit to patients with melanoma.
Asunto(s)
Biomarcadores de Tumor/metabolismo , Distonina/metabolismo , Proteínas de Filamentos Intermediarios/metabolismo , Melanoma/patología , Neovascularización Patológica/metabolismo , gamma Catenina/metabolismo , Animales , Línea Celular Tumoral , Proliferación Celular , Citocinas/metabolismo , Proteínas Filagrina , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Humanos , Melanoma/inmunología , Melanoma/metabolismo , Ratones , Ratones Endogámicos C57BLRESUMEN
Chemoresistance is driven by unique regulatory networks in the genome that are distinct from those necessary for cancer development. Here, we investigate the contribution of enhancer elements to cisplatin resistance in ovarian cancers. Epigenome profiling of multiple cellular models of chemoresistance identified unique sets of distal enhancers, super-enhancers (SE), and their gene targets that coordinate and maintain the transcriptional program of the platinum-resistant state in ovarian cancer. Pharmacologic inhibition of distal enhancers through small-molecule epigenetic inhibitors suppressed the expression of their target genes and restored cisplatin sensitivity in vitro and in vivo. In addition to known drivers of chemoresistance, our findings identified SOX9 as a critical SE-regulated transcription factor that plays a critical role in acquiring and maintaining the chemoresistant state in ovarian cancer. The approach and findings presented here suggest that integrative analysis of epigenome and transcriptional programs could identify targetable key drivers of chemoresistance in cancers. SIGNIFICANCE: Integrative genome-wide epigenomic and transcriptomic analyses of platinum-sensitive and -resistant ovarian lines identify key distal regulatory regions and associated master regulator transcription factors that can be targeted by small-molecule epigenetic inhibitors.
Asunto(s)
Cisplatino/farmacología , Resistencia a Antineoplásicos/genética , Elementos de Facilitación Genéticos , Regulación Neoplásica de la Expresión Génica , Neoplasias Ováricas/patología , Antineoplásicos/farmacología , Apoptosis , Biomarcadores de Tumor/genética , Proliferación Celular , Resistencia a Antineoplásicos/efectos de los fármacos , Epigenómica , Femenino , Humanos , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Ováricas/genética , Factor de Transcripción SOX9/genética , Factor de Transcripción SOX9/metabolismo , Transcriptoma , Células Tumorales CultivadasRESUMEN
Glioblastoma (GBM) is the most common primary brain malignancy and carries an extremely poor prognosis. Recent molecular studies revealed the CDK4/6-Rb-E2F axis and receptor tyrosine kinase (RTK) signaling to be deregulated in most GBM, creating an opportunity to develop more effective therapies by targeting both pathways. Using a phospho-RTK protein array, we found that both c-Met and TrkA-B pathways were significantly activated upon CDK4/6 inhibition in GBM cells. We therefore investigated the efficacy of combined CDK4/6 and c-Met/TrkA-B inhibition against GBM. We show that both c-Met and TrkA-B pathways transactivate each other, and targeting both pathways simultaneously results in more efficient pathway suppression. Mechanistically, inhibition of CDK4/6 drove NF-κB-mediated upregulation of hepatocyte growth factor, brain-derived neurotrophic factor, and nerve growth factor that in turn activated both c-Met and TrkA-B pathways. Combining the CDK4/6 inhibitor abemaciclib with the c-Met/Trk inhibitor altiratinib or the corresponding siRNAs induced apoptosis, leading to significant synergy against GBM. Collectively, these findings demonstrate that the activation of c-Met/TrkA-B pathways is a novel mechanism involved in therapeutic resistance of GBM to CDK4/6 inhibition and that dual inhibition of c-Met/Trk with CDK4/6 should be considered in future clinical trials.Significance: CDK4/6 inhibition in glioblastoma activates the c-Met and TrkA-B pathways mediated by NF-κB and can be reversed by a dual c-Met/Trk inhibitor. Cancer Res; 78(15); 4360-9. ©2018 AACR.
Asunto(s)
Neoplasias Encefálicas/metabolismo , Quinasa 4 Dependiente de la Ciclina/metabolismo , Quinasa 6 Dependiente de la Ciclina/metabolismo , Glioblastoma/metabolismo , Proteínas Proto-Oncogénicas c-met/metabolismo , Receptor trkA/metabolismo , Animales , Apoptosis/efectos de los fármacos , Neoplasias Encefálicas/tratamiento farmacológico , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Femenino , Glioblastoma/dietoterapia , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones SCID , Inhibidores de Proteínas Quinasas/farmacología , Transducción de Señal/efectos de los fármacosRESUMEN
The cullin RING E3 ubiquitin ligase 4 (CRL4) with its substrate receptor CDT2 (CRL4-CDT2) is emerging as a critical regulator of DNA replication through targeting CDT1, SET8, and p21 for ubiquitin-dependent proteolysis. The aberrant increased stability of these proteins in cells with inactivated CRL4-CDT2 results in DNA rereplication, which is deleterious to cells due to the accumulation of replication intermediates and stalled replication forks. Here, we demonstrate that CDT2 is overexpressed in head and neck squamous cell carcinoma (HNSCC), and its depletion by siRNA inhibits the proliferation of human papilloma virus-negative (HPV-ve) HNSCC cells primarily through the induction of rereplication. Treatment of HNSCC with the NEDD8-activating enzyme inhibitor pevonedistat (MLN4924), which inhibits all cullin-based ligases, induces significant rereplication and inhibits HNSCC cell proliferation in culture and HNSCC xenografts in mice. Pevonedistat additionally sensitizes HNSCC cells to ionizing radiation (IR) and enhances IR-induced suppression of xenografts in mice. Induction of rereplication via CDT2 depletion, or via the stabilization or activation of CDT1, also radiosensitizes HNSCC cells. Collectively, these results demonstrate that induction of rereplication represents a novel approach to treating radioresistant HNSCC tumors and suggest that pevonedistat may be considered as an adjuvant for IR-based treatments. Mol Cancer Ther; 17(2); 368-80. ©2017 AACRSee all articles in this MCT Focus section, "Developmental Therapeutics in Radiation Oncology."
Asunto(s)
Ciclopentanos/uso terapéutico , Pirimidinas/uso terapéutico , Fármacos Sensibilizantes a Radiaciones/uso terapéutico , Carcinoma de Células Escamosas de Cabeza y Cuello/tratamiento farmacológico , Animales , Ciclopentanos/farmacología , Femenino , Silenciador del Gen , Humanos , Ratones , Ratones Desnudos , Pirimidinas/farmacología , Fármacos Sensibilizantes a Radiaciones/farmacología , Carcinoma de Células Escamosas de Cabeza y Cuello/patología , Transfección , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Purpose: Glioblastoma (GBM) is a deadly brain tumor marked by dysregulated signaling and aberrant cell-cycle control. Molecular analyses have identified that the CDK4/6-Rb-E2F axis is dysregulated in about 80% of GBMs. Single-agent CDK4/6 inhibitors have failed to provide durable responses in GBM, suggesting a need to combine them with other agents. We investigate the efficacy of the combination of CDK4/6 inhibition and mTOR inhibition against GBM.Experimental Design: Preclinical in vitro and in vivo assays using primary GBM cell lines were performed.Results: We show that the CDK4/6 inhibitor palbociclib suppresses the activity of downstream mediators of the mTOR pathway, leading to rebound mTOR activation that can be blocked by the mTOR inhibitor everolimus. We further show that mTOR inhibition with everolimus leads to activation of the Ras mediator Erk that is reversible with palbociclib. The combined treatment strongly disrupts GBM metabolism, resulting in significant apoptosis. Further increasing the utility of the combination for brain cancers, everolimus significantly increases the brain concentration of palbociclib.Conclusions: Our findings demonstrate that the combination of CDK4/6 and mTOR inhibition has therapeutic potential against GBM and suggest it should be evaluated in a clinical trial. Clin Cancer Res; 23(22); 6958-68. ©2017 AACR.
Asunto(s)
Quinasa 4 Dependiente de la Ciclina/antagonistas & inhibidores , Quinasa 6 Dependiente de la Ciclina/antagonistas & inhibidores , Glioblastoma/metabolismo , Glioblastoma/patología , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Animales , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Caspasa 3/metabolismo , Caspasa 7/metabolismo , Puntos de Control del Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Sinergismo Farmacológico , Everolimus/farmacología , Femenino , Glioblastoma/tratamiento farmacológico , Humanos , Ratones , Modelos Biológicos , Piperazinas/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Piridinas/farmacología , Transducción de Señal/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The cullin-based CRL4-CDT2 ubiquitin ligase is emerging as a master regulator of cell proliferation. CRL4-CDT2 prevents re-initiation of DNA replication during the same cell cycle "rereplication" through targeted degradation of CDT1, SET8 and p21 during S-phase of the cell cycle. We show that CDT2 is overexpressed in cutaneous melanoma and predicts poor overall and disease-free survival. CDT2 ablation inhibited a panel of melanoma cell lines through the induction of SET8- and p21-dependent DNA rereplication and senescence. Pevonedistat (MLN4924), a specific inhibitor of the NEDD8 activating enzyme (NAE), inhibits the activity of cullin E3 ligases, thereby stabilizing a vast number of cullin substrates and resulting in cancer cell inhibition in vitro and tumor suppression in nude mice. We demonstrate that pevonedistat is effective at inhibiting the proliferation of melanoma cell lines in vitro through the induction of rereplication-dependent permanent growth arrest as well as through a transient, non-rereplication-dependent mechanism. CRISPR/Cas9-mediated heterozygous deletion of CDKN1A (encoding p21) or SET8 in melanoma cells demonstrated that the rereplication-mediated cytotoxicity of pevonedistat is mediated through preventing the degradation of p21 and SET8 and is essential for melanoma suppression in nude mice. By contrast, pevonedistat-induced transient growth suppression was independent of p21 or SET8, and insufficient to inhibit tumor growth in vivo. Pevonedistat additionally synergized with the BRAF kinase inhibitor PLX4720 to inhibit BRAF melanoma, and suppressed PLX4720-resistant melanoma cells. These findings demonstrate that the CRL4-CDT2-SET8/p21 degradation axis is the primary target of inhibition by pevonedistat in melanoma and suggest that a broad patient population may benefit from pevonedistat therapy. RESEARCH IN CONTEXT: The identification of new molecular targets and effective inhibitors is of utmost significance for the clinical management of melanoma. This study identifies CDT2, a substrate receptor for the CRL4 ubiquitin ligase, as a prognostic marker and therapeutic target in melanoma. CDT2 is required for melanoma cell proliferation and inhibition of CRL4(CDT2) by pevonedistat suppresses melanoma in vitro and in vivo through the induction of DNA rereplication and senescence through the stabilization of the CRL4(CDT2) substrates p21 and SET8. Pevonedistat also synergizes with vemurafenib in vivo and suppresses vemurafenib-resistant melanoma cells. These findings show a significant promise for targeting CRL4(CDT2) therapeutically.
Asunto(s)
Antineoplásicos/farmacología , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Ciclopentanos/farmacología , N-Metiltransferasa de Histona-Lisina/metabolismo , Melanoma/metabolismo , Proteínas Nucleares/metabolismo , Pirimidinas/farmacología , Ubiquitina-Proteína Ligasas/metabolismo , Animales , Antineoplásicos/uso terapéutico , Puntos de Control del Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular , Senescencia Celular/efectos de los fármacos , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Ciclopentanos/uso terapéutico , Modelos Animales de Enfermedad , Femenino , Expresión Génica , Silenciador del Gen , Genes ras , N-Metiltransferasa de Histona-Lisina/genética , Humanos , Estimación de Kaplan-Meier , Melanoma/tratamiento farmacológico , Melanoma/genética , Melanoma/mortalidad , Ratones , Mutación , Proteínas Nucleares/genética , Pronóstico , Estabilidad Proteica/efectos de los fármacos , Proteínas Proto-Oncogénicas B-raf/genética , Pirimidinas/uso terapéutico , Interferencia de ARN , Ubiquitina-Proteína Ligasas/antagonistas & inhibidores , Ubiquitina-Proteína Ligasas/genética , Ubiquitinación , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
PURPOSE: To test the hypothesis that small molecule targeting of nucleophosmin 1 (NPM1) represents a rational approach for radiosensitization. METHODS AND MATERIALS: Wilde-type and NPM1-deficient mouse embryo fibroblasts (MEFs) were used to determine whether radiosensitization produced by the small molecule YTR107 was NPM1 dependent. The stress response to ionizing radiation was assessed by quantifying pNPM1, γH2AX, and Rad51 foci, neutral comet tail moment, and colony formation. NPM1 levels in a human-derived non-small-cell lung cancer (NSCLC) tissue microarray (TMA) were determined by immunohistochemistry. YTR107-mediated radiosensitization was assessed in NSCLC cell lines and xenografts. RESULTS: Use of NPM1-null MEFs demonstrated that NPM1 is critical for DNA double- strand break (DSB) repair, that loss of NPM1 increases radiation sensitivity, and that YTR107-mediated radiosensitization is NPM1 dependent. YTR107 was shown to inhibit NPM1 oligomerization and impair formation of pNPM1 irradiation-induced foci that colocalized with γH2AX foci. Analysis of the TMA demonstrated that NPM1 is overexpressed in subsets of NSCLC. YTR107 inhibited DNA DSB repair and radiosensitized NSCLC lines and xenografts. CONCLUSIONS: These data demonstrate that YTR107-mediated targeting of NPM1 impairs DNA DSB repair, an event that increases radiation sensitivity.