RESUMEN
Emerging evidence indicates that alternative splicing plays a critical role in cancer progression through abnormal expression or mutation of splicing factors. Small-molecule splicing modulators have recently attracted considerable attention as a novel class of cancer therapeutics. CDC-like kinases (CLKs) are central to exon recognition in mRNA splicing and CLK inhibitors exhibit anti-tumour activities. Most importantly, molecular mechanism-based combination strategies for cancer therapy must be considered. However, it remains unclear whether CLK inhibitors modulate expression and splicing of apoptosis-related genes, and whether CLK inhibitors enhance cytotoxicity in combination with apoptosis inducers. Here we report an appropriate mechanism-based drug combination approach. Unexpectedly, we found that the CLK inhibitor T3 rapidly induced apoptosis in A2780 cells and G2/M cell cycle arrest in HCT116 cells. Regardless of the different phenotypes of the two cancer cell types, T3 decreased the levels of anti-apoptotic proteins (cIAP1, cIAP2, XIAP, cFLIP and Mcl-1) for a short period of exposure and altered the splicing of the anti-apoptotic MCL1L and CFLAR isoform in A2780 and HCT116 cells. In contrast, other members of the Bcl-2 family (i.e., Bcl-xL and Bcl-2) were resistant to T3-induced expression and splicing modulation. T3 and a Bcl-xL/Bcl-2 inhibitor synergistically induced apoptosis. Taken together, the use of a CLK inhibitor is a novel therapeutic approach to sensitise cancer cells to Bcl-xL/Bcl-2 inhibitors.
Asunto(s)
Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-bcl-2/antagonistas & inhibidores , Empalme Alternativo/efectos de los fármacos , Antineoplásicos/química , Apoptosis/genética , Proteína Reguladora de Apoptosis Similar a CASP8 y FADD/metabolismo , Puntos de Control del Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Sinergismo Farmacológico , Fase G2/efectos de los fármacos , Humanos , Diana Mecanicista del Complejo 2 de la Rapamicina/metabolismo , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/genética , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacocinética , Precursores del ARN/genética , Precursores del ARN/metabolismoRESUMEN
Replication stress (RS) is a cancer hallmark; chemotherapeutic drugs targeting RS are widely used as treatments for various cancers. To develop next-generation RS-inducing anticancer drugs, cell division cycle 7 (CDC7) has recently attracted attention as a target. We have developed an oral CDC7-selective inhibitor, TAK-931, as a candidate clinical anticancer drug. TAK-931 induced S phase delay and RS. TAK-931-induced RS caused mitotic aberrations through centrosome dysregulation and chromosome missegregation, resulting in irreversible antiproliferative effects in cancer cells. TAK-931 exhibited significant antiproliferative activity in preclinical animal models. Furthermore, in indication-seeking studies using large-scale cell panel data, TAK-931 exhibited higher antiproliferative activities in RAS-mutant versus RAS-wild-type cells; this finding was confirmed in pancreatic patient-derived xenografts. Comparison analysis of cell panel data also demonstrated a unique efficacy spectrum for TAK-931 compared with currently used chemotherapeutic drugs. Our findings help to elucidate the molecular mechanisms for TAK-931 and identify potential target indications.
Asunto(s)
Antineoplásicos/farmacología , Proteínas de Ciclo Celular/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Pirazolonas/farmacología , Pirimidinas/farmacología , Animales , Línea Celular Tumoral , Proliferación Celular , Separación Celular , Supervivencia Celular , Centrosoma/efectos de los fármacos , Aberraciones Cromosómicas/efectos de los fármacos , Biología Computacional , Resistencia a Antineoplásicos/efectos de los fármacos , Ensayos de Selección de Medicamentos Antitumorales , Femenino , Células HeLa , Humanos , Concentración 50 Inhibidora , Estimación de Kaplan-Meier , Ratones , Ratones Endogámicos BALB C , Mitosis/efectos de los fármacos , Modelos Animales , Mutación , Trasplante de Neoplasias , Neoplasias Pancreáticas/tratamiento farmacológico , Unión Proteica , Inhibidores de Proteínas Quinasas/farmacología , Proteómica , Resultado del Tratamiento , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
General control nonderepressible 2 (GCN2) plays a major role in the cellular response to amino acid limitation. Although maintenance of amino acid homeostasis is critical for tumor growth, the contribution of GCN2 to cancer cell survival and proliferation is poorly understood. In this study, we generated GCN2 inhibitors and demonstrated that inhibition of GCN2 sensitizes cancer cells with low basal-level expression of asparagine synthetase (ASNS) to the antileukemic agent l-asparaginase (ASNase) in vitro and in vivo. We first tested acute lymphoblastic leukemia (ALL) cells and showed that treatment with GCN2 inhibitors rendered ALL cells sensitive to ASNase by preventing the induction of ASNS, resulting in reduced levels of de novo protein synthesis. Comprehensive gene-expression profiling revealed that combined treatment with ASNase and GCN2 inhibitors induced the stress-activated MAPK pathway, thereby triggering apoptosis. By using cell-panel analyses, we also showed that acute myelogenous leukemia and pancreatic cancer cells were highly sensitive to the combined treatment. Notably, basal ASNS expression at protein levels was significantly correlated with sensitivity to combined treatment. These results provide mechanistic insights into the role of GCN2 in the amino acid response and a rationale for further investigation of GCN2 inhibitors for the treatment of cancer.
Asunto(s)
Aminoácidos/metabolismo , Asparaginasa/farmacología , Aspartatoamoníaco Ligasa/metabolismo , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas de Neoplasias/metabolismo , Neoplasias/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Aminoácidos/genética , Aspartatoamoníaco Ligasa/genética , Línea Celular Tumoral , Humanos , Proteínas de Neoplasias/genética , Neoplasias/enzimología , Neoplasias/genética , Neoplasias/patología , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismoRESUMEN
The CDK8/19 kinase module comprises a subcomplex that interacts with the Mediator complex and regulates gene expression through phosphorylation of transcription factors and Mediator subunits. Mediator complex subunits have been increasingly implicated in cancer and other diseases. Although high expression of CDK8/19 has been demonstrated in prostate cancer, its function has not been thoroughly examined. Here we report that CDK8/19 modulates the gene expression of cell cycle regulators and thereby maintains the proper G1/S transition in prostate cancer cells. We show that highly selective CDK8/19 inhibitors exerted anti-proliferative activity in prostate cancer cells both in vitro and in vivo. In CDK8/19 inhibitor-sensitive prostate cancer cells, the compounds reduced the population of G1 phase cells and elevated that of S phase cells through the modulation of G1/S transition regulators at the level of mRNA expression. Furthermore, the premature G1/S transition induced a DNA damage response that was followed by ATR-dependent and caspase-independent cell death. These findings suggest a novel role of CDK8/19 in transcription-mediated cell cycle control, albeit with possible contribution of other proteins inhibited by the compounds. Our data provide a rationale for further investigation of CDK8/19 inhibitors as a new therapeutic approach to prostate cancer.
RESUMEN
Panitumumab is a monoclonal antibody developed against the human epidermal growth factor receptor (EGFR). TAS-102 is a novel chemotherapeutic agent containing trifluridine (FTD) as the active cytotoxic component. Both panitumumab and TAS-102 have been approved for the treatment of metastatic colorectal cancer. In this study, we revealed the mechanism underlying the anticancer effects of panitumumab/TAS-102 combination using preclinical models. Panitumumab/FTD cotreatment showed additive antiproliferative effects in LIM1215 and synergistic antiproliferative effects in SW48 colon cancer cells. Consistent with the in vitro effects, panitumumab/TAS-102 combination caused tumor regression in LIM1215 and COL-01-JCK colon cancer patient-derived xenograft models. In LIM1215 cells, FTD induced extracellular signal-regulated kinase (ERK)/protein kinase B (AKT)/signal transducer and activator of transcription 3 (STAT3) phosphorylation and subsequent serine/threonine phosphorylation of EGFR, while it had no effects on EGFR tyrosine phosphorylation. Panitumumab and the tyrosine kinase inhibitor erlotinib reduced the basal level of EGFR tyrosine phosphorylation and reversed FTD-induced ERK/AKT/STAT3 and EGFR serine/threonine phosphorylation. These results suggested that FTD in combination with the basal activity of EGFR tyrosine kinase induced downstream prosurvival signaling through ERK/AKT/STAT3 phosphorylation. Collectively, we propose that panitumumab interacts with FTD by targeting EGFR-mediated adaptive responses, thereby exerting anticancer effects when used in combination with TAS-102. These preclinical findings provide a compelling rationale for evaluating the combination of anti-EGFR antibodies with TAS-102 against metastatic colorectal cancer.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Neoplasias del Colon/tratamiento farmacológico , Neoplasias del Colon/metabolismo , Receptores ErbB/antagonistas & inhibidores , Anticuerpos Monoclonales/farmacología , Línea Celular Tumoral , Neoplasias del Colon/patología , Combinación de Medicamentos , Ensayos de Selección de Medicamentos Antitumorales , Receptores ErbB/metabolismo , Humanos , Panitumumab , Pirrolidinas , Timina , Trifluridina/farmacología , Uracilo/análogos & derivados , Uracilo/farmacologíaRESUMEN
Mutations in succinate dehydrogenase B (SDHB) gene are frequently observed in several tumors and associated with poor prognosis in these tumors. Therefore, drugs effective for SDHB-deficient tumors could fulfill an unmet medical need. In addition, such drugs would have an advantage in that selection of patients with SDHB-mutant cancer could increase the probability of success in clinical trials. Currently, however, the characteristics of SDHB-deficient cancers are not completely understood. Here, we established SDHB knockout cancer cell lines from human colon cancer HCT116 cells using the clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 knockout system, and clarified its metabolic characteristics.In the SDHB knockout cells, succinate was accumulated and fumarate was decreased. The oxygen consumption rate was decreased while the extracellular acidification rate was increased in the SDHB knockout cells. Accordingly, an enhanced glycolysis pathway in the SDHB knockout cells was demonstrated by metabolomics analysis. Tracer experiments showed bidirectional metabolic flow in the tricarboxylic acid (TCA) cycle, possibly to maintain the necessary amounts of metabolites in the SDHB knockout cells. The proliferation of SDHB knockout cells was suppressed by a glycolysis inhibitor but not by a mitochondrial inhibitor. Additionally, partial dependence on glutaminolysis was observed in the SDHB knockout cells. Compound screening revealed that a bromodomain and extra-terminal (BET) inhibitor, which downregulated c-Myc, suppressed the growth of the SDHB knockout cells more potently than that of control cells. These findings provide an understanding of the metabolic characteristics of SDHB-deficient cancer and its vulnerabilities, which may lead to new therapeutic options.
Asunto(s)
Antineoplásicos/farmacología , Neoplasias/tratamiento farmacológico , Neoplasias/genética , Succinato Deshidrogenasa/genética , Antineoplásicos/uso terapéutico , Azepinas/farmacología , Azepinas/uso terapéutico , Benzodiazepinas/farmacología , Benzodiazepinas/uso terapéutico , Sistemas CRISPR-Cas , Ciclo del Ácido Cítrico , Deshidroepiandrosterona/farmacología , Fumaratos/metabolismo , Técnicas de Inactivación de Genes , Glucosafosfato Deshidrogenasa/antagonistas & inhibidores , Glucólisis , Células HCT116 , Compuestos Heterocíclicos de 4 o más Anillos/farmacología , Compuestos Heterocíclicos de 4 o más Anillos/uso terapéutico , Humanos , Isoenzimas/antagonistas & inhibidores , L-Lactato Deshidrogenasa/antagonistas & inhibidores , Lactato Deshidrogenasa 5 , Metabolómica , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Mutación , Neoplasias/patología , Consumo de Oxígeno , Fenformina/farmacología , Fosfoglicerato-Deshidrogenasa/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-myc/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-myc/metabolismo , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Ácido Succínico/metabolismo , Triazoles/farmacología , Triazoles/uso terapéuticoRESUMEN
Ionizing radiation induces DNA double-strand breaks (DSBs). Mammalian cells repair DSBs through multiple pathways, and the repair pathway that is utilized may affect cellular radiation sensitivity. In this study, we examined effects on cellular radiosensitivity resulting from functional alterations in homologous recombination (HR). HR was inhibited by overexpression of the forkhead-associated (FHA) domain-mutated NBS1 (G27D/R28D: FHA-2D) protein in HeLa cells or in hamster cells carrying a human X-chromosome. Cells expressing FHA-2D presented partially (but significantly) HR-deficient phenotypes, which were assayed by the reduction of gene conversion frequencies measured with a reporter assay, a decrease in radiation-induced Mre11 foci formation, and hypersensitivity to camptothecin treatments. Interestingly, ectopic expression of FHA-2D did not increase the frequency of radiation-induced somatic mutations at the HPRT locus, suggesting that a partial reduction of HR efficiency has only a slight effect on genomic stability. The expression of FHA-2D rendered the exponentially growing cell population slightly (but significantly) more sensitive to ionizing radiation. This radiosensitization effect due to the expression of FHA-2D was enhanced when the cells were irradiated with split doses delivered at 24-h intervals. Furthermore, enhancement of radiation sensitivity by split dose irradiation was not seen in contact-inhibited G0/G1 populations, even though the cells expressed FHA-2D. These results suggest that the FHA domain of NBS1 might be an effective molecular target that can be used to induce radiosensitization using low molecular weight chemicals, and that partial inhibition of HR might improve the effectiveness of cancer radiotherapy.