RESUMEN
We identified apilimod as an antiproliferative compound by high-throughput screening of clinical-stage drugs. Apilimod exhibits exquisite specificity for phosphatidylinositol-3-phosphate 5-kinase (PIKfyve) lipid kinase and has selective cytotoxic activity in B-cell non-Hodgkin lymphoma (B-NHL) compared with normal cells. Apilimod displays nanomolar activity in vitro, and in vivo studies demonstrate single-agent efficacy as well as synergy with approved B-NHL drugs. Using biochemical and knockdown approaches, and discovery of a kinase domain mutation conferring resistance, we demonstrate that apilimod-mediated cytotoxicity is driven by PIKfyve inhibition. Furthermore, a critical role for lysosome dysfunction as a major factor contributing to apilimod's cytotoxicity is supported by a genome-wide CRISPR screen. In the screen, TFEB (master transcriptional regulator of lysosomal biogenesis) and endosomal/lysosomal genes CLCN7, OSTM1, and SNX10 were identified as important determinants of apilimod sensitivity. These findings thus suggest that disruption of lysosomal homeostasis with apilimod represents a novel approach to treat B-NHL.
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Linfoma de Células B/tratamiento farmacológico , Morfolinas/uso terapéutico , Inhibidores de las Quinasa Fosfoinosítidos-3 , Inhibidores de Proteínas Quinasas/uso terapéutico , Triazinas/uso terapéutico , Antineoplásicos , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Evaluación Preclínica de Medicamentos/métodos , Endosomas/efectos de los fármacos , Endosomas/genética , Ensayos Analíticos de Alto Rendimiento , Humanos , Hidrazonas , Lisosomas/efectos de los fármacos , Lisosomas/genética , Fosfatidilinositol 3-Quinasas , PirimidinasRESUMEN
DNA double-strand breaks (DSBs) with 5' adducts are frequently formed from many nucleic acid processing enzymes, in particular DNA topoisomerase 2 (TOP2). The key intermediate of TOP2 catalysis is the covalent complex (TOP2cc), consisting of two TOP2 subunits covalently linked to the 5' ends of the nicked DNA. In cells, TOP2ccs can be trapped by cancer drugs such as etoposide and then converted into DNA double-strand breaks (DSBs) that carry adducts at the 5' end. The repair of such DSBs is critical to the survival of cells, but the underlying mechanism is still not well understood. We found that etoposide-induced DSBs are efficiently resected into 3' single-stranded DNA in cells and the major nuclease for resection is the DNA2 protein. DNA substrates carrying model 5' adducts were efficiently resected in Xenopus egg extracts and immunodepletion of Xenopus DNA2 also strongly inhibited resection. These results suggest that DNA2-mediated resection is a major mechanism for the repair of DSBs with 5' adducts.
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Aductos de ADN , Roturas del ADN de Doble Cadena , Reparación del ADN , ADN-Topoisomerasas de Tipo II/metabolismo , Animales , Línea Celular Tumoral , Proliferación Celular , Roturas del ADN de Doble Cadena/efectos de los fármacos , ADN-Topoisomerasas de Tipo II/genética , ADN de Cadena Simple , Etopósido/toxicidad , Expresión Génica , Técnicas de Silenciamiento del Gen , Humanos , ARN Interferente Pequeño/genética , XenopusRESUMEN
G protein-coupled receptor kinases (GRKs) are important regulators of G protein-coupled receptor function and mediate receptor desensitization, internalization, and signaling. While GRKs also interact with and/or phosphorylate many other proteins and modify their function, relatively little is known about the cellular localization of endogenous GRKs. Here we report that GRK5 co-localizes with γ-tubulin, centrin, and pericentrin in centrosomes. The centrosomal localization of GRK5 is observed predominantly at interphase and although its localization is not dependent on microtubules, it can mediate microtubule nucleation of centrosomes. Knockdown of GRK5 expression leads to G2/M arrest, characterized by a prolonged G2 phase, which can be rescued by expression of wild type but not catalytically inactive GRK5. This G2/M arrest appears to be due to increased expression of p53, reduced activity of aurora A kinase and a subsequent delay in the activation of polo-like kinase 1. Overall, these studies demonstrate that GRK5 is localized in the centrosome and regulates microtubule nucleation and normal cell cycle progression.
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División Celular/fisiología , Centrosoma/enzimología , Quinasa 5 del Receptor Acoplado a Proteína-G/metabolismo , Fase G2/fisiología , Transducción de Señal/fisiología , Aurora Quinasas , Membrana Celular/metabolismo , Quinasa 5 del Receptor Acoplado a Proteína-G/genética , Células HEK293 , Células HeLa , Humanos , Microtúbulos/metabolismo , Fosforilación/fisiología , Proteínas Serina-Treonina Quinasas/metabolismo , ARN Interferente Pequeño/genética , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
Acute myeloid leukemias (AML) harboring a constitutively active internal tandem duplication (ITD) mutation in the FMS-like kinase tyrosine kinase (FLT3) receptor are associated with poor patient prognosis. Despite initial clinical responses to FLT3 kinase inhibitors, patients eventually relapse. Mechanisms of resistance include the acquisition of secondary FLT3 mutations and protective stromal signaling within the bone marrow niche. Here we show that LAM-003, a prodrug of the heat shock protein 90 inhibitor LAM-003A, has cytotoxic activity against AML cell lines and primary samples harboring FLT3-ITD. LAM-003 regressed tumors in an MV-4-11 xenograft mouse model and extended survival in a MOLM-13 systemic model. LAM-003 displayed synergistic activity with chemotherapeutic drugs and FLT3 inhibitors, with the most robust synergy being obtained with venetoclax, a BCL-2 inhibitor. This finding was verified in a MOLM-13 systemic survival model in which the combination significantly prolonged survival compared with the single agents. Importantly, LAM-003 exhibited equipotent activity against FLT3 inhibitor-resistant mutants of FLT3, such as D835 or F691, in cytotoxic and FLT3 degradation assays. LAM-003 also retained potency in AML cells grown in stromal-conditioned media that were resistant to FLT3 inhibitors. Lastly, a genome-wide CRISPR screen revealed epigenetic regulators, including KDM6A, as determinants of LAM-003 sensitivity in AML cell lines, leading to the discovery of synergy with an EZH2 inhibitor. Collectively, these preclinical findings support the use of LAM-003 in FLT3-ITD patients with AML who no longer respond to FLT3 inhibitor therapy either as a single agent or in combination with drugs known to be active in AML.
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Antineoplásicos/farmacología , Resistencia a Antineoplásicos/genética , Duplicación de Gen , Leucemia Mieloide Aguda/genética , Inhibidores de Proteínas Quinasas/farmacología , Tirosina Quinasa 3 Similar a fms/genética , Animales , Antineoplásicos/uso terapéutico , Línea Celular Tumoral , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Sinergismo Farmacológico , Epigénesis Genética , Regulación Leucémica de la Expresión Génica/efectos de los fármacos , Humanos , Leucemia Mieloide Aguda/tratamiento farmacológico , Ratones , Mutación , Inhibidores de Proteínas Quinasas/uso terapéuticoRESUMEN
INTRODUCTION: Screening for synthetic lethality markers has demonstrated that the inhibition of the cell cycle checkpoint kinases WEE1 together with CHK1 drastically affects stability of the cell cycle and induces cell death in rapidly proliferating cells. Exploiting this finding for a possible therapeutic approach has showed efficacy in various solid and hematologic tumors, though not specifically tested in acute lymphoblastic leukemia. METHODS: The efficacy of the combination between WEE1 and CHK1 inhibitors in B and T cell precursor acute lymphoblastic leukemia (B/T-ALL) was evaluated in vitro and ex vivo studies. The efficacy of the therapeutic strategy was tested in terms of cytotoxicity, induction of apoptosis, and changes in cell cycle profile and protein expression using B/T-ALL cell lines. In addition, the efficacy of the drug combination was studied in primary B-ALL blasts using clonogenic assays. RESULTS: This study reports, for the first time, the efficacy of the concomitant inhibition of CHK1/CHK2 and WEE1 in ALL cell lines and primary leukemic B-ALL cells using two selective inhibitors: PF-0047736 (CHK1/CHK2 inhibitor) and AZD-1775 (WEE1 inhibitor). We showed strong synergism in the reduction of cell viability, proliferation and induction of apoptosis. The efficacy of the combination was related to the induction of early S-phase arrest and to the induction of DNA damage, ultimately triggering cell death. We reported evidence that the efficacy of the combination treatment is independent from the activation of the p53-p21 pathway. Moreover, gene expression analysis on B-ALL primary samples showed that Chek1 and Wee1 are significantly co-expressed in samples at diagnosis (Pearson r = 0.5770, p = 0.0001) and relapse (Pearson r= 0.8919; p = 0.0001). Finally, the efficacy of the combination was confirmed by the reduction in clonogenic survival of primary leukemic B-ALL cells. CONCLUSION: Our findings suggest that the combination of CHK1 and WEE1 inhibitors may be a promising therapeutic strategy to be tested in clinical trials for adult ALL.
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Melanoma is an aggressive neoplasm with increasing incidence that is classified by the NCI as a recalcitrant cancer, i.e., a cancer with poor prognosis, lacking progress in diagnosis and treatment. In addition to conventional therapy, melanoma treatment is currently based on targeting the BRAF/MEK/ERK signaling pathway and immune checkpoints. As drug resistance remains a major obstacle to treatment success, advanced therapeutic approaches based on novel targets are still urgently needed. We reasoned that the base excision repair enzyme thymine DNA glycosylase (TDG) could be such a target for its dual role in safeguarding the genome and the epigenome, by performing the last of the multiple steps in DNA demethylation. Here we show that TDG knockdown in melanoma cell lines causes cell cycle arrest, senescence, and death by mitotic alterations; alters the transcriptome and methylome; and impairs xenograft tumor formation. Importantly, untransformed melanocytes are minimally affected by TDG knockdown, and adult mice with conditional knockout of Tdg are viable. Candidate TDG inhibitors, identified through a high-throughput fluorescence-based screen, reduced viability and clonogenic capacity of melanoma cell lines and increased cellular levels of 5-carboxylcytosine, the last intermediate in DNA demethylation, indicating successful on-target activity. These findings suggest that TDG may provide critical functions specific to cancer cells that make it a highly suitable anti-melanoma drug target. By potentially disrupting both DNA repair and the epigenetic state, targeting TDG may represent a completely new approach to melanoma therapy.
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Inhibidores Enzimáticos/farmacología , Melanoma/patología , Timina ADN Glicosilasa/genética , Animales , Ciclo Celular/genética , Línea Celular Tumoral , Proliferación Celular/genética , Citosina/análogos & derivados , Citosina/metabolismo , Metilación de ADN , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Melanoma/tratamiento farmacológico , Melanoma/genética , Melanoma Experimental/genética , Melanoma Experimental/patología , Ratones Noqueados , Ratones SCID , Ratones Transgénicos , Terapia Molecular Dirigida/métodos , Timina ADN Glicosilasa/antagonistas & inhibidores , Timina ADN Glicosilasa/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
BACKGROUND: Despite the recent progress that has been made in the understanding and treatment of acute lymphoblastic leukemia (ALL), the outcome is still dismal in adult ALL cases. Several studies in solid tumors identified high expression of WEE1 kinase as a poor prognostic factor and reported its role as a cancer-conserving oncogene that protects cancer cells from DNA damage. Therefore, the targeted inhibition of WEE1 kinase has emerged as a rational strategy to sensitize cancer cells to antineoplastic compounds, which we evaluate in this study. METHODS: The effectiveness of the selective WEE1 inhibitor AZD-1775 as a single agent and in combination with different antineoplastic agents in B and T cell precursor ALL (B/T-ALL) was evaluated in vitro and ex vivo studies. The efficacy of the compound in terms of cytotoxicity, induction of apoptosis, and changes in gene and protein expression was assessed using different B/T-ALL cell lines and confirmed in primary ALL blasts. RESULTS: We showed that WEE1 was highly expressed in adult primary ALL bone marrow and peripheral blood blasts (n = 58) compared to normal mononuclear cells isolated from the peripheral blood of healthy donors (p = 0.004). Thus, we hypothesized that WEE1 could be a rational target in ALL, and its inhibition could enhance the cytotoxicity of conventional therapies used for ALL. We evaluated the efficacy of AZD-1775 as a single agent and in combination with several antineoplastic agents, and we elucidated its mechanisms of action. AZD-1775 reduced cell viability in B/T-ALL cell lines by disrupting the G2/M checkpoint and inducing apoptosis. These findings were confirmed in human primary ALL bone marrow and peripheral blood blasts (n = 15). In both cell lines and primary leukemic cells, AZD-1775 significantly enhanced the efficacy of several tyrosine kinase inhibitors (TKIs) such as bosutinib, imatinib, and ponatinib, and of chemotherapeutic agents (clofarabine and doxorubicin) in terms of the reduction of cell viability, apoptosis induction, and inhibition of proliferation. CONCLUSIONS: Our data suggest that WEE1 plays a role in ALL blast's survival and is a bona fide target for therapeutic intervention. These data support the evaluation of the therapeutic potential of AZD-1775 as chemo-sensitizer agent for the treatment of B/T-ALL.
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Proteínas de Ciclo Celular/genética , Proteínas Nucleares/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Proteínas Tirosina Quinasas/genética , Línea Celular Tumoral , Humanos , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismoRESUMEN
Small molecule screens are widely used to prioritize pharmaceutical development. However, determining the pathways targeted by these molecules is challenging, since the compounds are often promiscuous. We present a network strategy that takes into account the polypharmacology of small molecules in order to generate hypotheses for their broader mode of action. We report a screen for kinase inhibitors that increase the efficacy of gemcitabine, the first-line chemotherapy for pancreatic cancer. Eight kinase inhibitors emerge that are known to affect 201 kinases, of which only three kinases have been previously identified as modifiers of gemcitabine toxicity. In this work, we use the SAMNet algorithm to identify pathways linking these kinases and genetic modifiers of gemcitabine toxicity with transcriptional and epigenetic changes induced by gemcitabine that we measure using DNaseI-seq and RNA-seq. SAMNet uses a constrained optimization algorithm to connect genes from these complementary datasets through a small set of protein-protein and protein-DNA interactions. The resulting network recapitulates known pathways including DNA repair, cell proliferation and the epithelial-to-mesenchymal transition. We use the network to predict genes with important roles in the gemcitabine response, including six that have already been shown to modify gemcitabine efficacy in pancreatic cancer and ten novel candidates. Our work reveals the important role of polypharmacology in the activity of these chemosensitizing agents.
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Algoritmos , Reparación del ADN/efectos de los fármacos , Bases de Datos Genéticas , Desoxicitidina/análogos & derivados , Epigénesis Genética/efectos de los fármacos , Transición Epitelial-Mesenquimal/efectos de los fármacos , Modelos Biológicos , Neoplasias Pancreáticas , Inhibidores de Proteínas Quinasas , Transcripción Genética/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular , Desoxicitidina/farmacocinética , Desoxicitidina/farmacología , Humanos , Neoplasias Pancreáticas/tratamiento farmacológico , Neoplasias Pancreáticas/metabolismo , Inhibidores de Proteínas Quinasas/farmacocinética , Inhibidores de Proteínas Quinasas/farmacología , GemcitabinaRESUMEN
We identified the PIKFYVE inhibitor apilimod as a potent and selective cytotoxic agent against B-cell non-Hodgkin lymphoma (B-NHL). Our data robustly establish PIKFYVE as the target through which apilimod kills B-NHL cells and show that apilimod-induced death in B-NHL is mediated by broad disruption of lysosome homeostasis characterized by lysosomal swelling, TFEB nuclear translocation, impaired maturation of lysosomal enzymes and incomplete autophagosome clearance. Furthermore, through genome-wide CRISPR knockout screening, we identified specific lysosomal genes (TFEB, CLCN7, OSTM1 and SNX10) as critical determinants of apilimod-induced cytotoxicity. Together these data highlight disruption of lysosome homeostasis through PIKFYVE inhibition as a novel anticancer mechanism in B-NHL and potentially other cancers.
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Linfocitos B/patología , Linfoma no Hodgkin/tratamiento farmacológico , Inhibidores de las Quinasa Fosfoinosítidos-3 , Inhibidores de Proteínas Quinasas/uso terapéutico , Linfocitos B/efectos de los fármacos , Linfocitos B/enzimología , Endosomas/metabolismo , Humanos , Linfoma no Hodgkin/enzimología , Linfoma no Hodgkin/patología , Lisosomas/metabolismo , Modelos Biológicos , Fosfatidilinositol 3-Quinasas/metabolismo , Inhibidores de Proteínas Quinasas/farmacologíaRESUMEN
It has been proposed that low activities of antioxidant enzymes in pancreatic beta cells may increase their susceptibility to autoimmune attack. We have therefore used the spontaneously diabetic BB/S rat model of type 1 diabetes to compare islet catalase and superoxide dismutase activities in diabetes-prone and diabetes-resistant animals. In parallel studies, we employed the RINm5F beta cell line as a model system (previously validated) to investigate whether regulation of antioxidant enzyme activity by inflammatory mediators (cytokines, nitric oxide) occurs at the gene or protein expression level. Diabetes-prone rat islets had high insulin content at the age used (58-65 days) but showed increased amounts of DNA damage when subjected to cytokine or hydrogen peroxide treatments. There was clear evidence of oxidative damage in freshly isolated rat islets from diabetes-prone animals and significantly lower catalase and superoxide dismutase activities than in islets from age-matched diabetes-resistant BB/S and control Wistar rats. The mRNA expression of antioxidant enzymes in islets from diabetes-prone and diabetes-resistant BB/S rats and in RINm5F cells, treated with a combination of cytokines or a nitric oxide donor, DETA-NO, was analysed semi-quantitatively by real time PCR. The mRNA expression of catalase was lower, whereas MnSOD expression was higher, in diabetes-prone compared to diabetes-resistant BB/S rat islets, suggesting regulation at the level of gene expression as well as of the activities of these enzymes in diabetes. The protein expression of catalase, CuZnSOD and MnSOD was assessed by Western blotting and found to be unchanged in DETA-NO treated cells. Protein expression of MnSOD was increased by cytokines in RINm5F cells whereas the expression of CuZnSOD was slightly decreased and the level of catalase protein was unchanged. We conclude that there are some changes, mostly upregulation, in protein expression but no decreases in the mRNA expression of catalase, CuZnSOD or MnSOD enzymes in beta cells treated with either cytokines or DETA-NO. The lower antioxidant enzyme activities observed in islets from diabetes-prone BB/S rats could be a factor in the development of disease and in susceptibility to DNA damage in vitro and could reflect islet alterations prior to immune attack or inherent differences in the islets of diabetes-prone animals, but are not likely to result from cytokine or nitric oxide exposure in vivo at that stage.
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Antioxidantes/metabolismo , Catalasa/metabolismo , Diabetes Mellitus Tipo 1/enzimología , Islotes Pancreáticos/enzimología , Superóxido Dismutasa/metabolismo , Animales , Catalasa/genética , Línea Celular , Citocinas/farmacología , Diabetes Mellitus Tipo 1/genética , Modelos Animales de Enfermedad , Insulina/biosíntesis , Nitratos/sangre , Nitritos/sangre , ARN Mensajero/metabolismo , Ratas , Ratas Endogámicas BB , Superóxido Dismutasa/genéticaRESUMEN
Pixantrone is a novel aza-anthracenedione active against aggressive lymphoma and is being evaluated for use against various hematologic and solid tumors. The drug is an analog of mitoxantrone, but displays less cardiotoxicity than mitoxantrone or the more commonly used doxorubicin. Although pixantrone is purported to inhibit topoisomerase II activity and intercalate with DNA, exact mechanisms of how it induces cell death remain obscure. Here we evaluated the effect of pixantrone on a panel of solid tumor cell lines to understand its mechanism of cell killing. Initial experiments with pixantrone showed an apparent discrepancy between its anti-proliferative effects in MTS assays (short-term) compared with clonogenic assays (long-term). Using live cell videomicroscopy to track the fates of cells, we found that cells treated with pixantrone underwent multiple rounds of aberrant cell division before eventually dying after approximately 5 d post-treatment. Cells underwent abnormal mitosis in which chromosome segregation was impaired, generating chromatin bridges between cells or within cells containing micronuclei. While pixantrone-treated cells did not display γH2AX foci, a marker of DNA damage, in the main nuclei, such foci were often detected in the micronuclei. Using DNA content analysis, we found that pixantrone concentrations that induced cell death in a clonogenic assay did not impede cell cycle progression, further supporting the lack of canonical DNA damage signaling. These findings suggest pixantrone induces a latent type of DNA damage that impairs the fidelity of mitosis, without triggering DNA damage response or mitotic checkpoint activation, but is lethal after successive rounds of aberrant division.
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Isoquinolinas/farmacología , Mitosis/efectos de los fármacos , Inhibidores de Topoisomerasa II/farmacología , Apoptosis , Línea Celular Tumoral , Proliferación Celular , Segregación Cromosómica , Daño del ADN , Ensayos de Selección de Medicamentos Antitumorales , HumanosRESUMEN
5-Fluorouracil (FU) is a halogenated nucleobase analog that is widely used in chemotherapy. Here we show that 5-hydroxymethyl-2'-deoxyuridine (hmUdR) synergistically enhances the activity of FU in cell lines derived from solid tumors but not normal tissues. While the cytotoxicity of FU and hmUdR was not directly related to the amount of the modified bases incorporated into cellular DNA, incubation with this combination resulted in dramatic increase in the number of single strand breaks in replicating cancer cells, leading to NAD-depletion as consequence of poly(ADP-ribose) synthesis and S phase arrest. Cell death resulting from the base/nucleoside combination did not occur by apoptosis, autophagy or necroptosis. Instead, the cells die via necrosis as a result of NAD depletion. The FU-related nucleoside analog, 5-fluoro-2'-deoxyuridine, also displayed synergy with hmUdR, whereas hmUdR could not be replaced by 5-hydroxymethyluracil. Among other 5-modified deoxyuridine analogs tested, 5-formyl-2'-deoxyuridine and, to a lesser extent, 5-hydroxy-2'-deoxyuridine, also acted synergistically with FU, whereas 5-hydroxyethyl-2'-deoxyuridine did not. Together, our results have revealed an unexpected synergistic interaction between deoxyuridine analogs and FU in a cancer cell-specific manner, and suggest that these novel base/nucleoside combinations could be developed into improved FU-based chemotherapies.
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Polyunsaturated fats are the main target for lipid peroxidation and subsequent formation of mutagenic metabolites, but diets high in saturated fats are more strongly associated with adverse health effects. We show that the common saturated fatty acid, palmitic acid, is a potent inducer of DNA damage in an insulin-secreting cell line, and in primary human fibroblasts. Damage is not associated with upregulation of inducible nitric oxide synthase, but is prevented by two different antioxidants, alpha-lipoic acid and 3,3'-methoxysalenMn(III) (EUK134), which also partly prevent palmitic acid-induced apoptosis and growth inhibition. Since mutagenic metabolites can be formed from peroxidation of polyunsaturated fatty acids, co-administration of palmitic and a polyunsaturated fatty acid might be particularly harmful. Palmitic acid-induced DNA damage is instead prevented by linoleic acid, which is acting here as a protective agent against oxidative stress, rather than as a source of mutagenic metabolites. These results illustrate the complexity of the relationship of dietary fat intake to genotoxicity.
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Antioxidantes/farmacología , Apoptosis/efectos de los fármacos , Daño del ADN/efectos de los fármacos , Grasas de la Dieta/efectos adversos , Ácido Linoleico/farmacología , Ácido Palmítico/efectos adversos , Células Cultivadas , Etilenodiaminas/farmacología , Fibroblastos , Humanos , Compuestos Organometálicos/farmacología , Estrés Oxidativo , Ácido Palmítico/antagonistas & inhibidores , Salicilatos/farmacologíaRESUMEN
The chaperone HSP70 promotes the survival of cells exposed to many different types of stresses, and is also potently anti-apoptotic. The major stress-induced form of this protein, HSP70-1, is overexpressed in a number of human cancers, yet is negligibly expressed in normal cells. Silencing of the gene encoding HSP70-1 (HSPA1A) is cytotoxic to transformed but not normal cells. Therefore, HSP70 is considered to be a promising cancer drug target, and there has been active interest in the identification and characterization of HSP70 inhibitors for cancer therapy. Because HSP70 behaves in a relatively non-specific manner in the control of protein folding, to date there are no reliably-identified "clients" of this protein, nor is there consensus as to what the phenotypic effects of HSP70 inhibitors are on a cancer cell. Here for the first time we compare three recently-identified HSP70 inhibitors, PES-Cl, MKT-077, and Ver-155008, for their ability to impact some of the known and reported functions of this chaperone; specifically, the ability to inhibit autophagy, to influence the level of HSP90 client proteins, to induce cell cycle arrest, and to inhibit the enzymatic activity of the anaphase-promoting complex/cyclosome (APC/C). We report that all three of these compounds can inhibit autophagy and cause reduced levels of HSP90 client proteins; however, only PES-Cl can inhibit the APC/C and induce G 2/M arrest. Possible reasons for these differences, and the implications for the further development of these prototype compounds as anti-cancer agents, are discussed.
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Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Autofagia/efectos de los fármacos , Puntos de Control del Ciclo Celular/efectos de los fármacos , Proteínas HSP70 de Choque Térmico/antagonistas & inhibidores , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Línea Celular Tumoral , Proteínas HSP70 de Choque Térmico/metabolismo , Humanos , Nucleósidos de Purina/farmacología , Piridinas/farmacología , Sulfonamidas/farmacología , Tiazoles/farmacologíaRESUMEN
Inhibitors of the DNA damage checkpoint kinase, Chk1, are highly effective as chemo- and radio-sensitizers in preclinical studies but are not well-tolerated by patients. We exploited the promiscuous nature of kinase inhibitors to screen 9 clinically relevant kinase inhibitors for their ability to sensitize pancreatic cancer cells to a sub-lethal concentration of gemcitabine. Bosutinib, dovitinib, and BEZ-235 were identified as sensitizers that abrogated the DNA damage checkpoint. We further characterized bosutinib, an FDA-approved Src/Abl inhibitor approved for chronic myelogenous leukemia. Unbeknownst to us, we used an isomer (Bos-I) that was unknowingly synthesized and sold to the research community as "authentic" bosutinib. In vitro and cell-based assays showed that both the authentic bosutinib and Bos-I inhibited DNA damage checkpoint kinases Chk1 and Wee1, with Bos-I showing greater potency. Imaging data showed that Bos-I forced cells to override gemcitabine-induced DNA damage checkpoint arrest and destabilized stalled replication forks. These inhibitors enhanced sensitivity to the DNA damaging agents' gemcitabine, cisplatin, and doxorubicin in pancreatic cancer cell lines. The in vivo efficacy of Bos-I was validated using cells derived directly from a pancreatic cancer patient's tumor. Notably, the xenograft studies showed that the combination of gemcitabine and Bos-I was significantly more effective in suppressing tumor growth than either agent alone. Finally, we show that the gatekeeper residue in Wee1 dictates its sensitivity to the 2 compounds. Our strategy to screen clinically relevant kinase inhibitors for off-target effects on cell cycle checkpoints is a promising approach to re-purpose drugs as chemosensitizers.
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Antineoplásicos/farmacología , Puntos de Control del Ciclo Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Reposicionamiento de Medicamentos , Resistencia a Antineoplásicos/efectos de los fármacos , Neoplasias Pancreáticas/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Quinasas/metabolismo , Transducción de Señal/efectos de los fármacos , Compuestos de Anilina/farmacología , Animales , Proteínas de Ciclo Celular/antagonistas & inhibidores , Proteínas de Ciclo Celular/metabolismo , Línea Celular Tumoral , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1) , Cisplatino/farmacología , Daño del ADN , Replicación del ADN , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacología , Relación Dosis-Respuesta a Droga , Doxorrubicina/farmacología , Humanos , Masculino , Ratones Endogámicos C57BL , Ratones SCID , Modelos Moleculares , Nitrilos/farmacología , Proteínas Nucleares/antagonistas & inhibidores , Proteínas Nucleares/metabolismo , Neoplasias Pancreáticas/enzimología , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patología , Inhibidores de Proteínas Quinasas/química , Proteínas Quinasas/química , Proteínas Quinasas/genética , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Proteínas Tirosina Quinasas/metabolismo , Quinolinas/farmacología , Factores de Tiempo , Ensayos Antitumor por Modelo de Xenoinjerto , GemcitabinaRESUMEN
HuR (ELAV1), an RNA-binding protein abundant in cancer cells, primarily resides in the nucleus, but under specific stress (e.g., gemcitabine), HuR translocates to the cytoplasm in which it tightly modulates the expression of mRNA survival cargo. Here, we demonstrate for the first time that stressing pancreatic ductal adenocarcinoma (PDA) cells by treatment with DNA-damaging anticancer agents (mitomycin C, oxaliplatin, cisplatin, carboplatin, and a PARP inhibitor) results in HuR's translocation from the nucleus to the cytoplasm. Importantly, silencing HuR in PDA cells sensitized the cells to these agents, whereas overexpressing HuR caused resistance. HuR's role in the efficacy of DNA-damaging agents in PDA cells was, in part, attributed to the acute upregulation of WEE1 by HuR. WEE1, a mitotic inhibitor kinase, regulates the DNA damage repair pathway, and therapeutic inhibition of WEE1 in combination with chemotherapy is currently in early phase trials for the treatment of cancer. We validate WEE1 as a HuR target in vitro and in vivo by demonstrating (i) direct binding of HuR to WEE1's mRNA (a discrete 56-bp region residing in the 3' untranslated region) and (ii) HuR siRNA silencing and overexpression directly affects the protein levels of WEE1, especially after DNA damage. HuR's positive regulation of WEE1 increases γ-H2AX levels, induces Cdk1 phosphorylation, and promotes cell-cycle arrest at the G2-M transition. We describe a novel mechanism that PDA cells use to protect against DNA damage in which HuR posttranscriptionally regulates the expression and downstream function of WEE1 upon exposure to DNA-damaging agents.
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Carcinoma Ductal Pancreático/genética , Proteínas de Ciclo Celular/genética , Daño del ADN/fisiología , Proteínas ELAV/fisiología , Proteínas Nucleares/genética , Neoplasias Pancreáticas/genética , Proteínas Tirosina Quinasas/genética , Interferencia de ARN , Animales , Antineoplásicos/farmacología , Carcinoma Ductal Pancreático/metabolismo , Proteínas de Ciclo Celular/metabolismo , Regulación Neoplásica de la Expresión Génica , Humanos , Ratones , Proteínas Nucleares/metabolismo , Neoplasias Pancreáticas/metabolismo , Transporte de Proteínas/efectos de los fármacos , Proteínas Tirosina Quinasas/metabolismo , Células Tumorales CultivadasRESUMEN
DNA damaging agents, including those used in the clinic, activate cell cycle checkpoints, which blocks entry into mitosis. Given that checkpoint override results in cell death via mitotic catastrophe, inhibitors of the DNA damage checkpoint are actively being pursued as chemosensitization agents. Here we explored the effects of gemcitabine in combination with Chk1 inhibitors in a panel of pancreatic cancer cell lines and found variable abilities to override the S phase checkpoint. In cells that were able to enter mitosis, the chromatin was extensively fragmented, as assessed by metaphase spreads and Comet assay. Notably, electron microscopy and high-resolution light microscopy showed that the kinetochores and centromeres appeared to be detached from the chromatin mass, in a manner reminiscent of mitosis with unreplicated genomes (MUGs). Cell lines that were unable to override the S phase checkpoint were able to override a G2 arrest induced by the alkylator MMS or the topoisomerase II inhibitors doxorubicin or etoposide. Interestingly, checkpoint override from the topoisomerase II inhibitors generated fragmented kinetochores (MUGs) due to unreplicated centromeres. Our studies show that kinetochore and centromere fragmentation is a defining feature of checkpoint override and suggests that loss of cell viability is due in part to acentric genomes. Furthermore, given the greater efficacy of forcing cells into premature mitosis from topoisomerase II-mediated arrest as compared with gemcitabine-mediated arrest, topoisomerase II inhibitors maybe more suitable when used in combination with checkpoint inhibitors.
Asunto(s)
Centrómero/metabolismo , Mitosis , Animales , Antineoplásicos/toxicidad , Apoptosis/efectos de los fármacos , Células CHO , Línea Celular Tumoral , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1) , Ensayo Cometa , Cricetinae , Cricetulus , Daño del ADN/efectos de los fármacos , ADN-Topoisomerasas de Tipo II/química , ADN-Topoisomerasas de Tipo II/metabolismo , Desoxicitidina/análogos & derivados , Desoxicitidina/toxicidad , Doxorrubicina/toxicidad , Etopósido/toxicidad , Puntos de Control de la Fase G2 del Ciclo Celular/efectos de los fármacos , Células HeLa , Humanos , Cinetocoros/metabolismo , Proteínas Quinasas/química , Proteínas Quinasas/metabolismo , Puntos de Control de la Fase S del Ciclo Celular/efectos de los fármacos , Estaurosporina/análogos & derivados , Estaurosporina/farmacología , Inhibidores de Topoisomerasa II/farmacología , GemcitabinaRESUMEN
Proper completion of mitosis requires the concerted effort of multiple RhoGEFs. Here we show that leukemia-associated RhoGEF (LARG), a RhoA-specific RGS-RhoGEF, is required for abscission, the final stage of cytokinesis, in which the intercellular membrane is cleaved between daughter cells. LARG colocalizes with α-tubulin at the spindle poles before localizing to the central spindle. During cytokinesis, LARG is condensed in the midbody, where it colocalizes with RhoA. HeLa cells depleted of LARG display apoptosis during cytokinesis with unresolved intercellular bridges, and rescue experiments show that expression of small interfering RNA-resistant LARG prevents this apoptosis. Moreover, live cell imaging of LARG-depleted cells reveals greatly delayed fission kinetics in abscission in which a population of cells with persistent bridges undergoes apoptosis; however, the delayed fission kinetics is rescued by Aurora-B inhibition. The formation of a Flemming body and thinning of microtubules in the intercellular bridge of cells depleted of LARG is consistent with a defect in late cytokinesis, just before the abscission event. In contrast to studies of other RhoGEFs, particularly Ect2 and GEF-H1, LARG depletion does not result in cytokinetic furrow regression nor does it affect internal mitotic timing. These results show that LARG is a novel and temporally distinct RhoGEF required for completion of abscission.
Asunto(s)
Citocinesis , Factores de Intercambio de Guanina Nucleótido Rho/metabolismo , Apoptosis , Aurora Quinasa B/metabolismo , Células HeLa , Humanos , Cinética , Mitosis , Mutación/genética , Unión Proteica , ARN Interferente Pequeño/metabolismo , Factores de Intercambio de Guanina Nucleótido Rho/química , Factores de Intercambio de Guanina Nucleótido Rho/genética , Relación Estructura-Actividad , Tirosina/metabolismo , Proteína de Unión al GTP rhoA/metabolismoRESUMEN
The stress-induced HSP70 is an ATP-dependent molecular chaperone that plays a key role in refolding misfolded proteins and promoting cell survival following stress. HSP70 is marginally expressed in nontransformed cells, but is greatly overexpressed in tumor cells. Silencing HSP70 is uniformly cytotoxic to tumor but not normal cells; therefore, there has been great interest in the development of HSP70 inhibitors for cancer therapy. Here, we report that the HSP70 inhibitor 2-phenylethynesulfonamide (PES) binds to the substrate-binding domain of HSP70 and requires the C-terminal helical "lid" of this protein (amino acids 573-616) to bind. Using molecular modeling and in silico docking, we have identified a candidate binding site for PES in this region of HSP70, and we identify point mutants that fail to interact with PES. A preliminary structure-activity relationship analysis has revealed a derivative of PES, 2-(3-chlorophenyl) ethynesulfonamide (PES-Cl), which shows increased cytotoxicity and ability to inhibit autophagy, along with significantly improved ability to extend the life of mice with pre-B-cell lymphoma, compared with the parent compound (P = 0.015). Interestingly, we also show that these HSP70 inhibitors impair the activity of the anaphase promoting complex/cyclosome (APC/C) in cell-free extracts, and induce G2-M arrest and genomic instability in cancer cells. PES-Cl is thus a promising new anticancer compound with several notable mechanisms of action.