RESUMEN
This study describes the identification and target deconvolution of small molecule inhibitors of oncogenic Yes-associated protein (YAP1)/TAZ activity with potent anti-tumor activity in vivo. A high-throughput screen (HTS) of 3.8 million compounds was conducted using a cellular YAP1/TAZ reporter assay. Target deconvolution studies identified the geranylgeranyltransferase-I (GGTase-I) complex as the direct target of YAP1/TAZ pathway inhibitors. The small molecule inhibitors block the activation of Rho-GTPases, leading to subsequent inactivation of YAP1/TAZ and inhibition of cancer cell proliferation in vitro. Multi-parameter optimization resulted in BAY-593, an in vivo probe with favorable PK properties, which demonstrated anti-tumor activity and blockade of YAP1/TAZ signaling in vivo.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Antineoplásicos , Proliferación Celular , Ensayos Analíticos de Alto Rendimiento , Transducción de Señal , Factores de Transcripción , Proteínas Señalizadoras YAP , Humanos , Factores de Transcripción/metabolismo , Factores de Transcripción/antagonistas & inhibidores , Proteínas Señalizadoras YAP/metabolismo , Antineoplásicos/farmacología , Antineoplásicos/química , Antineoplásicos/síntesis química , Transducción de Señal/efectos de los fármacos , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Adaptadoras Transductoras de Señales/antagonistas & inhibidores , Animales , Proliferación Celular/efectos de los fármacos , Ratones , Proteínas de Unión al GTP rho/metabolismo , Proteínas de Unión al GTP rho/antagonistas & inhibidores , Línea Celular Tumoral , Fosfoproteínas/metabolismo , Fosfoproteínas/antagonistas & inhibidores , Ensayos de Selección de Medicamentos Antitumorales , Transferasas Alquil y Aril/antagonistas & inhibidores , Transferasas Alquil y Aril/metabolismo , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/farmacología , Descubrimiento de Drogas , Ratones Desnudos , Aciltransferasas/antagonistas & inhibidores , Aciltransferasas/metabolismo , Fenotipo , Relación Estructura-Actividad , Proteínas Coactivadoras Transcripcionales con Motivo de Unión a PDZRESUMEN
The Hedgehog (Hh) pathway is required for cell-fate determination during the embryonic life, as well as cell growth and differentiation in the adult organism, where the inappropriate activation has been implicated in several cancers. Here we demonstrate that Hh signaling plays a significant role in growth and survival of multiple myeloma (MM) cells. We observed that CD138(+) MM cells express Hh genes and confirmed Smoothened (Smo)-dependent Hh signaling in MM using a novel synthetic Smo inhibitor, NVP-LDE225 (Novartis), which decreased MM cell viability by inducing specific down-regulation of Gli1 and Ptch1, hallmarks of Hh activity. In addition, we detected a nuclear localization of Gli1 in MM cells, which is completely abrogated by Forskolin, a Gli1-modulating compound, confirming Smo-independent mechanisms leading to Hh activation in MM. Finally, we identified that bone marrow stromal cells are a source of the Shh ligand, although they are resistant to the Hh inhibitor because of defective Smo expression and Ptch1 up-regulation. Further in vitro as well as in vivo studies showed antitumor efficacy of NVP-LDE225 in combination with bortezomib. Altogether, our data demonstrate activation of both canonical and noncanonical Hh pathway in MM, thus providing the rationale for testing Hh inhibitors in clinical trials to improve MM patient outcome.
Asunto(s)
Proteínas Hedgehog/metabolismo , Mieloma Múltiple/metabolismo , Células Plasmáticas/metabolismo , Transducción de Señal , Animales , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Compuestos de Bifenilo/farmacología , Compuestos de Bifenilo/uso terapéutico , Ácidos Borónicos/farmacología , Ácidos Borónicos/uso terapéutico , Bortezomib , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica , Proteínas Hedgehog/genética , Humanos , Ratones , Ratones SCID , Mieloma Múltiple/tratamiento farmacológico , Mieloma Múltiple/genética , Mieloma Múltiple/patología , Receptores Patched , Receptor Patched-1 , Células Plasmáticas/efectos de los fármacos , Células Plasmáticas/patología , Pirazinas/farmacología , Pirazinas/uso terapéutico , Piridinas/farmacología , Piridinas/uso terapéutico , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/metabolismo , Receptores Acoplados a Proteínas G/antagonistas & inhibidores , Receptores Acoplados a Proteínas G/metabolismo , Receptor Smoothened , Sindecano-1/análisisRESUMEN
BACKGROUND: Isothiocyanates, a family of phytochemicals found in cruciferous vegetables, have cytotoxic effects against several types of tumor cells. Multiple myeloma is a fatal disease characterized by clonal proliferation of plasma cells in the bone marrow. The growing body of preclinical information on the anti-cancer activity of isothiocyanates led us to investigate their anti-myeloma properties. DESIGN AND METHODS: We evaluated the anti-myeloma activity of the isothiocyanates, sulforaphane and phenethyl isothiocyanate, on a panel of human myeloma cell lines as well as primary myeloma tumor cells. Cell viability, apoptosis, cell cycle alterations and cell proliferation were then analyzed in vitro and in a xenograft mouse model in vivo. The molecular sequelae of isothiocyanate treatment in multiple myeloma cells were evaluated by multiplex analyses using bead arrays and western blotting. RESULTS: We observed that sulforaphane and phenylethyl isothiocyanate have activity against myeloma cell lines and patients' myeloma cells both in vitro and in vivo using a myeloma xenograft mouse model. Isothiocyanates induced apoptotic death of myeloma cells; depletion of mitochondrial membrane potential; cleavage of PARP and caspases-3 and -9; as well as down-regulation of anti-apoptotic proteins including Mcl-1, X-IAP, c-IAP and survivin. Isothiocyanates induced G(2)/M cell cycle arrest accompanied by mitotic phosphorylation of histone H3. Multiplex analysis of phosphorylation of diverse components of signaling cascades revealed changes in MAPK activation; increased phosphorylation of c-jun and HSP27; as well as changes in the phosphorylation of Akt, and GSK3α/ß and p53. Isothiocyanates suppressed proliferation of myeloma cells alone and when co-cultured with HS-5 stromal cells. Sulforaphane and phenylethyl isothiocyanate enhanced the in vitro anti-myeloma activity of several conventional and novel therapies used in multiple myeloma. CONCLUSIONS: Our study shows that isothiocyanates have potent anti-myeloma activities and may enhance the activity of other anti-multiple myeloma agents. These results indicate that isothiocyanates may have therapeutic potential in multiple myeloma and provide the preclinical framework for future clinical studies of isothiocyanates in multiple myeloma.
Asunto(s)
Antineoplásicos/farmacología , Isotiocianatos/farmacología , Mieloma Múltiple/tratamiento farmacológico , Transducción de Señal/efectos de los fármacos , Tiocianatos/farmacología , Animales , Antineoplásicos/uso terapéutico , Antineoplásicos/toxicidad , Apoptosis/efectos de los fármacos , Ciclo Celular/efectos de los fármacos , Línea Celular , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Humanos , Isotiocianatos/uso terapéutico , Isotiocianatos/toxicidad , Ratones , Ratones SCID , Mieloma Múltiple/metabolismo , Células del Estroma/efectos de los fármacos , Sulfóxidos , Tiocianatos/uso terapéutico , Tiocianatos/toxicidad , Carga Tumoral/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Recurrence of multiple myeloma (MM) after therapy suggests the presence of tumor-initiating subpopulations. In our study, we performed flow cytometry-based Hoechst 33342 staining to evaluate the existence of a MM population with stem-like features known as side population (SP) cells. SP cells exhibit substantial heterogeneity in MM cell lines and primary MM cells; express CD138 antigen in MM cell lines; display higher mRNA expression and functional activity of ABCG2 transporter; and have a higher proliferation index compared with non-SP cells. We observed evidence for clonogenic potential of SP cells, as well as the ability of SP cells to regenerate original population. Moreover, SP cells revealed higher tumorigenicity compared with non-SP cells. Importantly, lenalidomide decreased the percentage and clonogenicity of SP cells, and also induced phosphorylation changes in Akt, GSK-3α/ß, MEK1, c-Jun, p53, and p70S6K in SP cells. Adherence to bone marrow stromal cells (BMSCs) increased the percentage, viability, and proliferation potential of SP cells. Lenalidomide and thalidomide abrogated this stimulatory effect of BMSCs and significantly decreased the percentage of SP cells. Our studies demonstrate a novel mechanism of action for lenalidomide, namely targeting SP fraction, providing the framework for new therapeutic strategies targeting subpopulations of MM cells including presumptive stem cells.
Asunto(s)
Antineoplásicos/farmacología , Mieloma Múltiple , Células Madre Neoplásicas/efectos de los fármacos , Talidomida/análogos & derivados , Transportadoras de Casetes de Unión a ATP/genética , Inhibidores de la Angiogénesis/farmacología , Células de la Médula Ósea/citología , Células de la Médula Ósea/efectos de los fármacos , Células de la Médula Ósea/fisiología , División Celular/efectos de los fármacos , Fraccionamiento Celular , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Ensayo de Unidades Formadoras de Colonias , Resistencia a Antineoplásicos , Humanos , Lenalidomida , Mieloma Múltiple/tratamiento farmacológico , Mieloma Múltiple/patología , Mieloma Múltiple/fisiopatología , Recurrencia Local de Neoplasia/prevención & control , Células Madre Neoplásicas/patología , Transducción de Señal/efectos de los fármacos , Células del Estroma/citología , Células del Estroma/efectos de los fármacos , Células del Estroma/fisiología , Sindecano-1/metabolismo , Talidomida/farmacologíaRESUMEN
We have developed a tumor vaccine in which patient-derived myeloma cells are chemically fused with autologous dendritic cells (DCs) such that a broad spectrum of myeloma-associated antigens are presented in the context of DC-mediated costimulation. We have completed a phase 1 study in which patients with multiple myeloma underwent serial vaccination with the DC/multiple myeloma fusions in conjunction with granulocyte-macrophage colony-stimulating factor. DCs were generated from adherent mononuclear cells cultured with granulocyte-macrophage colony-stimulating factor, interleukin-4, and tumor necrosis factor-α and fused with myeloma cells obtained from marrow aspirates. Vaccine generation was successful in 17 of 18 patients. Successive cohorts were treated with 1 × 10(6), 2 × 10(6), and 4 × 10(6) fusion cells, respectively, with 10 patients treated at the highest dose level. Vaccination was well tolerated, without evidence of dose-limiting toxicity. Vaccination resulted in the expansion of circulating CD4 and CD8 lymphocytes reactive with autologous myeloma cells in 11 of 15 evaluable patients. Humoral responses were documented by SEREX (Serologic Analysis of Recombinant cDNA Expression Libraries) analysis. A majority of patients with advanced disease demonstrated disease stabilization, with 3 patients showing ongoing stable disease at 12, 25, and 41 months, respectively. Vaccination with DC/multiple myeloma fusions was feasible and well tolerated and resulted in antitumor immune responses and disease stabilization in a majority of patients.
Asunto(s)
Vacunas contra el Cáncer/uso terapéutico , Células Dendríticas/inmunología , Inmunoterapia/métodos , Mieloma Múltiple/terapia , Adulto , Anciano , Anticuerpos Antiprotozoarios/inmunología , Antígenos de Protozoos/inmunología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Vacunas contra el Cáncer/inmunología , Fusión Celular , Femenino , Humanos , Masculino , Persona de Mediana Edad , Mieloma Múltiple/inmunologíaRESUMEN
The transformation from monoclonal gammopathy of undetermined significance (MGUS) to multiple myeloma (MM) is thought to be associated with changes in immune processes. We have therefore used serologic analysis of recombinant cDNA expression library to screen the sera of MGUS patients to identify tumor-associated antigens. A total of 10 antigens were identified, with specific antibody responses in MGUS. Responses appeared to be directed against intracellular proteins involved in cellular functions, such as apoptosis (SON, IFT57/HIPPI), DNA and RNA binding (ZNF292, GPATCH4), signal transduction regulators (AKAP11), transcriptional corepressor (IRF2BP2), developmental proteins (OFD1), and proteins of the ubiquitin-proteasome pathway (PSMC1). Importantly, the gene responsible for the oral-facial-digital type I syndrome (OFD1) had response in 6 of 29 (20.6%) MGUS patients but 0 of 11 newly diagnosed MM patients. Interestingly, 3 of 11 (27.2%) MM patients after autologous stem cell transplantations showed responses to OFD1. We have confirmed T-cell responses against OFD1 in MGUS and observed down-regulation of GLI1/PTCH1 and p-beta-catenin after OFD1 knock-down with specific siRNA, suggesting its functional role in the regulation of Hh and Wnt pathways. These findings demonstrate OFD1 as an important immune target and highlight its possible role in signal transduction and tumorigenesis in MGUS and MM.
Asunto(s)
Autoantígenos/inmunología , Mieloma Múltiple/inmunología , Paraproteinemias/inmunología , Proteínas/inmunología , Anciano , Anciano de 80 o más Años , Proteínas Reguladoras de la Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/inmunología , Autoantígenos/genética , Proteínas Portadoras/genética , Proteínas Portadoras/inmunología , ADN Complementario/genética , Proteínas de Unión al ADN , Femenino , Humanos , Masculino , Mieloma Múltiple/genética , Mieloma Múltiple/terapia , Proteínas Nucleares/genética , Proteínas Nucleares/inmunología , Paraproteinemias/genética , Paraproteinemias/terapia , Receptores Patched , Receptor Patched-1 , Proteínas/genética , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/inmunología , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/inmunología , Transducción de Señal/inmunología , Trasplante de Células Madre , Linfocitos T/inmunología , Factores de Transcripción , Trasplante Autólogo , beta Catenina/genética , beta Catenina/inmunologíaRESUMEN
The interaction of multiple myeloma (MM) cells with the bone marrow (BM) milieu plays a crucial role in MM pathogenesis. Stromal cell-derived factor-1 (SDF1) regulates homing of MM cells to the BM. In this study, we examined the role of RhoA and Rac1 GTPases in SDF1-induced adhesion and chemotaxis of MM. We found that both RhoA and Rac1 play key roles in SDF1-induced adhesion of MM cells to BM stromal cells, whereas RhoA was involved in chemotaxis and motility. Furthermore, both ROCK and Rac1 inhibitors reduced SDF1-induced polymerization of actin and activation of LIMK, SRC, FAK, and cofilin. Moreover, RhoA and Rac1 reduced homing of MM cells to BM niches. In conclusion, we characterized the role of RhoA and Rac1 GTPases in SDF1-induced adhesion, chemotaxis, and homing of MM cells to the BM, providing the framework for targeting RhoA and Rac1 GTPases as novel MM therapy.
Asunto(s)
Adhesión Celular , Quimiocina CXCL12/fisiología , Quimiotaxis , Mieloma Múltiple/patología , Proteína de Unión al GTP rac1/fisiología , Proteína de Unión al GTP rhoA/fisiología , Animales , Médula Ósea , Proteínas del Citoesqueleto/metabolismo , Humanos , Ratones , Ratones SCID , Células del Estroma , Células Tumorales CultivadasRESUMEN
THE PARADIGM FOR THE TREATMENT OF MONOCLONAL GAMMOPATIES HAS DRAMATICALLY CHANGED: therapeutic options in multiple myeloma (MM) have evolved from the introduction of melphalan and prednisone in the 1960s, high-dose chemotherapy and stem cell transplantation in the late 1980s and 1990s, to the rapid introduction of small novel molecules within the last seven years. Based on the understanding of the complex interaction of the MM cells with the bone marrow microenvironment and the signaling pathways that are dysregulated in this process, a number of novel therapeutic agents are now available. Specifically, three novel agents with a specific-targeted anti-MM activity, have been FDA-approved for the treatment of this disease, namely Bortezomib, thalidomide, and lenalidomide which are now all playing a key role in the treatment of MM. The success of targeted therapy in MM has since led to the development and investigation of more than 30 new compounds in this disease and in other plasma cell dyscrasias such as Waldenström's macroglobulinemia and primary amyloidosis, both in the preclinical settings and as part of clinical trials.
RESUMEN
Glucocorticoids have been widely used in the treatment of multiple myeloma (MM) both as single agents and in combination with other drugs. However, primary or acquired glucocorticoid resistance occurs in most cases. It was recently reported that R-etodolac induced in vitro cytotoxicity in MM cell lines and in primary MM cells, as well as synergistically enhanced dexamethasone (Dex)-induced apoptosis in Dex-sensitive MM.1S cells. This study examined the in vitro and in vivo effects of combination treatment with R-etodolac and Dex on Dex-resistant OPM1 cells. Treatment with R-etodolac and Dex was found to enhance cytotoxicity, inhibit nuclear factor kappaB activity via upregulation of IkappaBalpha, as well as enhance Dex-induced caspase activation and poly (ADP)-ribose polymerase cleavage in OPM1 cells. R-etodolac also enhanced Dex cytotoxicity in patient MM cells that were resistant to glucocorticoids. The in vivo anti-tumour effect of this combination on MM cells was evaluated by using severe combined immunodeficient mice engrafted with OPM1. Treatment with R-etodolac or Dex alone did not induce a significant reduction of tumour volume; in contrast, combination treatment with R-etodolac and Dex induced significant synergistic inhibition of tumour growth. These data indicate that R-etodolac overcomes resistance to Dex in glucocorticoid-resistant MM cells, providing the framework for clinical trials of R-etodolac combined with Dex, to improve patient outcome in MM.
Asunto(s)
Antiinflamatorios no Esteroideos/uso terapéutico , Dexametasona/uso terapéutico , Etodolaco/uso terapéutico , Glucocorticoides/uso terapéutico , Mieloma Múltiple/tratamiento farmacológico , Animales , Apoptosis/efectos de los fármacos , Caspasas/metabolismo , Línea Celular Tumoral , Sinergismo Farmacológico , Quimioterapia Combinada , Ensayo de Cambio de Movilidad Electroforética , Humanos , Proteínas I-kappa B/metabolismo , Immunoblotting , Ratones , Ratones SCID , Mieloma Múltiple/metabolismo , Mieloma Múltiple/patología , Poli(ADP-Ribosa) Polimerasas/metabolismo , Trasplante HeterólogoRESUMEN
Germ-line mutations in the breast cancer susceptibility BRCA1 gene account for approximately half of hereditary breast cancer cases and most of breast/ovarian cancer cases. We speculated whether breast hereditary cancers might be differentially sensitive to antitumor agents such as the mitotic spindle poisons Vinca alcaloid vinorelbine (VNR) and the taxoid docetaxel (DOC), which are commonly used in the treatment of breast cancer. We investigated the sensitivity of the BRCA1-mutated HCC1937 (derived from a BRCA1 related hereditary tumor) and BRCA1 competent MCF-7 and MDA-MB468 sporadic breast cancer cell lines to these drugs. We found that HCC1937 cells were significantly more sensitive to VNR as compared to MCF-7 or MDA-MB468 cells. Instead, BRCA1-mutated breast cancer cells exposed to DOC showed similar sensitivity as compared to BRCA1-competent MCF-7 or were less sensitive than MDA-MB468. In order to assess the role of BRCA1 in this specific pattern of chemosensitivity, we transfected the BRCA1-mutated HCC1937 cells with a full-length BRCA1 cDNA and the stable clone (HCC1937/WTBRCA1) was exposed to both drugs. Full-length BRCA1 transfection led to a significant induction of resistance to VNR, whereas only a weak but not significant increase of sensitivity to DOC was detected. Moreover, VNR induced apoptotic cell death and cytoskeletal rearrangements in HCC1937 cells. We further investigated whether a defective targeting of mitotic spindle by the mutated BRCA1 gene product might be involved in the differential sensitivity to VNR. We demonstrated that mutated BRCA1 was indeed capable of co-localizing with alpha-tubulin in the mitotic spindle, suggesting therefore that different mechanisms should account for these effects. In conclusion, our data suggest that BRCA1-mutated tumors might be differentially sensitive to anti-microtubule agents, supporting the rationale for clinical trials to improve the outcome of hereditary breast cancer patients by tailored treatments.