Asunto(s)
Antineoplásicos/administración & dosificación , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Benzamidas/administración & dosificación , Ácidos Borónicos/administración & dosificación , Proteínas HSP90 de Choque Térmico/antagonistas & inhibidores , Mieloma Múltiple/tratamiento farmacológico , Pirazinas/administración & dosificación , Pirazoles/administración & dosificación , Animales , Apoptosis , Bortezomib , Línea Celular , Línea Celular Tumoral , Proliferación Celular , Supervivencia Celular , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Ratones , Ratones SCID , Proteínas de Unión al GTP Monoméricas/metabolismo , Mutación , Trasplante de Neoplasias , Epitelio Pigmentado de la Retina/citologíaRESUMEN
Here we report that targeting casein kinase 1-α1 (CSNK1α1) is a potential novel treatment strategy in multiple myeloma (MM) therapy distinct from proteasome inhibition. CSNK1α1 is expressed in all the tested MM cell lines and patient MM cells, and is not altered during bortezomib-triggered cytotoxicity. Inhibition of CSNK1α1 kinase activity in MM cells with targeted therapy D4476 or small hairpin RNAs triggers cell G0/G1-phase arrest, prolonged G2/M phase and apoptosis. D4476 also induced cytotoxicity in bortezomib-resistant MM cells and enhanced bortezomib-triggered cytotoxicity. CSNK1α1 signaling pathways include CDKN1B, P53 and FADD; gene signatures involved included interferon-α, tumor necrosis factor-α and LIN9. In addition, reduction of Csnk1α1 prevents cMYC/KRAS12V transformation of BaF3 cells independent of interleukin-3. Impartially, reducing Csnk1α1 prevented development of cMYC/KRAS12V-induced plasmacytomas in mice, suggesting that CSNK1α1 may be involved in MM initiation and progression. Our data suggest that targeting CSNK1α1, alone or combined with bortezomib, is a potential novel therapeutic strategy in MM. Moreover, inhibition of CSNK1α1 may prevent the progression of monoclonal gammopathy of undetermined significance to MM.
Asunto(s)
Caseína Quinasa Ialfa/fisiología , Mieloma Múltiple/metabolismo , Células Plasmáticas/citología , Animales , Apoptosis , Ácidos Borónicos/química , Bortezomib , Ciclo Celular , Línea Celular Tumoral , Supervivencia Celular , Progresión de la Enfermedad , Perfilación de la Expresión Génica , Humanos , Interleucina-3/metabolismo , Lentivirus/genética , Ratones , Gammopatía Monoclonal de Relevancia Indeterminada/prevención & control , Mieloma Múltiple/terapia , Plasmacitoma/terapia , Complejo de la Endopetidasa Proteasomal/metabolismo , Pirazinas/química , Transducción de SeñalRESUMEN
Bruton's tyrosine kinase (Btk) modulates B-cell development and activation and has an important role in antibody production. Interestingly, Btk may also affect human osteoclast (OC) function; however, the mechanism was unknown. Here we studied a potent and specific Btk inhibitor, CC-292, in multiple myeloma (MM). In this report, we demonstrate that, although CC-292 increased OC differentiation, it inhibited OC function via inhibition of c-Src, Pyk2 and cortactin, all involved in OC-sealing zone formation. As CC-292 did not show potent in vitro anti-MM activity, we next evaluated it in combination with the proteasome inhibitor, carfilzomib. We first studied the effect of carfilzomib on OC. Carfilzomib did not have an impact on OC-sealing zone formation but significantly inhibited OC differentiation. CC-292 combined with carfilzomib inhibited both sealing zone formation and OC differentiation, resulting in more profound inhibition of OC function than carfilzomib alone. Moreover, the combination treatment in an in vivo MM mouse model inhibited tumor burden compared with CC-292 alone; it also increased bone volume compared with carfilzomib alone. These results suggest that CC-292 combined with carfilzomib augments the inhibitory effects against OC within the bone microenvironment and has promising therapeutic potential for the treatment of MM and related bone disease.
Asunto(s)
Acrilamidas/administración & dosificación , Mieloma Múltiple/tratamiento farmacológico , Oligopéptidos/administración & dosificación , Osteoclastos/efectos de los fármacos , Inhibidores de Proteasoma/administración & dosificación , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Pirimidinas/administración & dosificación , Acrilamidas/farmacología , Actinas/antagonistas & inhibidores , Agammaglobulinemia Tirosina Quinasa , Animales , Resorción Ósea/prevención & control , Diferenciación Celular , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Humanos , Ratones , Ratones SCID , Mieloma Múltiple/patología , Pirimidinas/farmacologíaRESUMEN
Histone deacetylases (HDACs) represent novel molecular targets for the treatment of various types of cancers, including multiple myeloma (MM). Many HDAC inhibitors have already shown remarkable antitumor activities in the preclinical setting; however, their clinical utility is limited because of unfavorable toxicities associated with their broad range HDAC inhibitory effects. Isoform-selective HDAC inhibition may allow for MM cytotoxicity without attendant side effects. In this study, we demonstrated that HDAC3 knockdown and a small-molecule HDAC3 inhibitor BG45 trigger significant MM cell growth inhibition via apoptosis, evidenced by caspase and poly (ADP-ribose) polymerase cleavage. Importantly, HDAC3 inhibition downregulates phosphorylation (tyrosine 705 and serine 727) of signal transducers and activators of transcription 3 (STAT3). Neither interleukin-6 nor bone marrow stromal cells overcome this inhibitory effect of HDAC3 inhibition on phospho-STAT3 and MM cell growth. Moreover, HDAC3 inhibition also triggers hyperacetylation of STAT3, suggesting crosstalk signaling between phosphorylation and acetylation of STAT3. Importantly, inhibition of HDAC3, but not HDAC1 or 2, significantly enhances bortezomib-induced cytotoxicity. Finally, we confirm that BG45 alone and in combination with bortezomib trigger significant tumor growth inhibition in vivo in a murine xenograft model of human MM. Our results indicate that HDAC3 represents a promising therapeutic target, and validate a prototype novel HDAC3 inhibitor BG45 in MM.
Asunto(s)
Inhibidores de Histona Desacetilasas/farmacología , Histona Desacetilasas/efectos de los fármacos , Mieloma Múltiple/enzimología , División Celular , Línea Celular Tumoral , Técnicas de Silenciamiento del Gen , Histona Desacetilasas/genética , Humanos , Mieloma Múltiple/patologíaRESUMEN
Small-molecule multi-targeted cyclin-dependent kinase (CDK) inhibitors (CDKIs) are of particular interest due to their potent antitumor activity independent of p53 gene alterations. P53 deletion is associated with a very poor prognosis in multiple myeloma (MM). In this regard, we tested the anti-MM activity of RGB-286638, an indenopyrazole-derived CDKI with Ki-nanomolar activity against transcriptional CDKs. We examined RGB-286638's mode-of-action in MM cell lines with wild-type (wt)-p53 and those expressing mutant p53. RGB-286638 treatment resulted in MM cytotoxicity in vitro associated with inhibition of MM tumor growth and prolonged survival in vivo. RGB-286638 displayed caspase-dependent apoptosis in both wt-p53 and mutant-p53 cells that was closely associated with the downregulation of RNA polymerase II phosphorylation and inhibition of transcription. RGB-286638 triggered p53 accumulation via nucleolar stress and loss of Mdm2, accompanied by induction of p53 DNA-binding activity. In addition, RGB-286638 mediated p53-independent activity, which was confirmed by cytotoxicity in p53-knockdown and p53-mutant cells. We also demonstrated downregulation of oncogenic miR-19, miR-92a-1 and miR-21. Our data provide the rationale for the development of transcriptional CDKIs as therapeutic agents, which activate p53 in competent cells, while circumventing p53 deficiency through alternative p53-independent cell death mechanisms in p53-mutant/deleted cells.
Asunto(s)
Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Mieloma Múltiple/patología , Pirazoles/farmacología , Transcripción Genética/efectos de los fármacos , Proteína p53 Supresora de Tumor/fisiología , Urea/análogos & derivados , Animales , Apoptosis/efectos de los fármacos , Humanos , Masculino , Ratones , Fosforilación , Inhibidores de Proteínas Quinasas/farmacología , Reacción en Cadena en Tiempo Real de la Polimerasa , Urea/farmacología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Given the prevalence of osteolytic bone disease in multiple myeloma (MM), novel therapies targeting bone microenvironment are essential. Previous studies have identified activin A to be of critical importance in MM-induced osteolysis. Lenalidomide is a known and approved treatment strategy for relapsed MM. Our findings demonstrate that lenalidomide acts directly on bone marrow stromal cells via an Akt-mediated increase in Jun N-terminal kinase-dependent signaling resulting in activin A secretion, with consequent inhibition of osteoblastogenesis. Here, we attempted to augment the antitumor benefits of lenalidomide while overcoming its effects on osteoblastogenesis by combining it with a neutralizing antibody to activin A. Increased activin A secretion induced by lenalidomide was abrogated by the addition of activin A-neutralizing antibody, which effectively restored osteoblast function and inhibited MM-induced osteolysis without negating the cytotoxic effects of lenalidomide on malignant cells. This provides the rationale for an ongoing clinical trial (NCT01562405) combining lenalidomide with an anti-activin A strategy.
Asunto(s)
Activinas/antagonistas & inhibidores , Inhibidores de la Angiogénesis/farmacología , Anticuerpos Neutralizantes/farmacología , Antineoplásicos/farmacología , Mieloma Múltiple/metabolismo , Talidomida/análogos & derivados , Activinas/metabolismo , Diferenciación Celular/efectos de los fármacos , Línea Celular Tumoral , Humanos , Lenalidomida , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Mieloma Múltiple/genética , Osteoblastos/citología , Osteoblastos/efectos de los fármacos , Osteoblastos/metabolismo , Células del Estroma/efectos de los fármacos , Células del Estroma/metabolismo , Talidomida/farmacologíaRESUMEN
Upregulation of cytokines and chemokines is a frequent finding in multiple myeloma (MM). CCL3 (also known as MIP-1α) is a pro-inflammatory chemokine, levels of which in the MM microenvironment correlate with osteolytic lesions and tumor burden. CCL3 and its receptors, CCR1 and CCR5, contribute to the development of bone disease in MM by supporting tumor growth and regulating osteoclast (OC) differentiation. In this study, we identify inhibition of osteoblast (OB) function as an additional pathogenic mechanism in CCL3-induced bone disease. MM-derived and exogenous CCL3 represses mineralization and osteocalcin production by primary human bone marrow stromal cells and HS27A cells. Our results suggest that CCL3 effects on OBs are mediated by ERK activation and subsequent downregulation of the osteogenic transcription factor osterix. CCR1 inhibition reduced ERK phosphorylation and restored both osterix and osteocalcin expression in the presence of CCL3. Finally, treating SCID-hu mice with a small molecule CCR1 inhibitor suggests an upregulation of osteocalcin expression along with OC downregulation. Our results show that CCL3, in addition to its known catabolic activity, reduces bone formation by inhibiting OB function, and therefore contributes to OB/OC uncoupling in MM.
Asunto(s)
Remodelación Ósea/fisiología , Calcificación Fisiológica/fisiología , Quimiocina CCL3/fisiología , Regulación Neoplásica de la Expresión Génica/fisiología , Mieloma Múltiple/complicaciones , Proteínas de Neoplasias/fisiología , Osteoblastos/fisiología , Osteocalcina/biosíntesis , Osteogénesis/fisiología , Osteólisis/etiología , Animales , Células de la Médula Ósea/metabolismo , Línea Celular Tumoral/metabolismo , Regulación hacia Abajo , Quinasas MAP Reguladas por Señal Extracelular/biosíntesis , Quinasas MAP Reguladas por Señal Extracelular/genética , Humanos , Células Madre Mesenquimatosas/metabolismo , Ratones , Ratones SCID , Mieloma Múltiple/metabolismo , Mieloma Múltiple/patología , Proteínas de Neoplasias/biosíntesis , Proteínas de Neoplasias/genética , Trasplante de Neoplasias , Osteocalcina/genética , Osteoclastos/fisiología , Osteólisis/metabolismo , Osteólisis/patología , Receptores CCR1/biosíntesis , Receptores CCR1/genética , Receptores CCR5/biosíntesis , Receptores CCR5/genética , Factor de Transcripción Sp7 , Células del Estroma/metabolismo , Factores de Transcripción/biosíntesis , Factores de Transcripción/genéticaRESUMEN
Dysregulated cell cycling is a universal hallmark of cancer and is often mediated by abnormal activation of cyclin-dependent kinases (CDKs) and their cyclin partners. Overexpression of individual complexes are reported in multiple myeloma (MM), making them attractive therapeutic targets. In this study, we investigate the preclinical activity of a novel small-molecule multi-CDK inhibitor, AT7519, in MM. We show the anti-MM activity of AT7519 displaying potent cytotoxicity and apoptosis; associated with in vivo tumor growth inhibition and prolonged survival. At the molecular level, AT7519 inhibited RNA polymerase II (RNA pol II) phosphorylation, a CDK9, 7 substrate, associated with decreased RNA synthesis confirmed by [(3)H] Uridine incorporation. In addition, AT7519 inhibited glycogen synthase kinase 3beta (GSK-3beta) phosphorylation; conversely pretreatment with a selective GSK-3 inhibitor and shRNA GSK-3beta knockdown restored MM survival, suggesting the involvement of GSK-3beta in AT7519-induced apoptosis. GSK-3beta activation was independent of RNA pol II dephosphorylation confirmed by alpha-amanitin, a specific RNA pol II inihibitor, showing potent inhibition of RNA pol II phosphorylation without corresponding effects on GSK-3beta phosphorylation. These results offer new insights into the crucial, yet controversial role of GSK-3beta in MM and show significant anti-MM activity of AT7519, providing the rationale for its clinical evaluation in MM.
Asunto(s)
Apoptosis/efectos de los fármacos , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Glucógeno Sintasa Quinasa 3/metabolismo , Mieloma Múltiple/tratamiento farmacológico , Piperidinas/farmacología , Pirazoles/farmacología , ARN Polimerasa II/antagonistas & inhibidores , Animales , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Activación Enzimática/efectos de los fármacos , Glucógeno Sintasa Quinasa 3 beta , Humanos , Masculino , Ratones , Ratones SCID , Mieloma Múltiple/patologíaRESUMEN
Earlier studies have shown that ascorbic acid (vitamin C) inhibits bortezomib-induced cytotoxicity against cancer cells in vitro. However, the clinical significance of vitamin C on bortezomib treatment is unclear. In this study, we examined whether daily oral intake of vitamin C inhibits antimultiple myeloma (MM) activities of bortezomib. Vitamin C, at orally achievable concentrations, inhibited in vitro MM cell cytotoxicity of bortezomib and blocked its inhibitory effect on 20S proteasome activity. Specifically, plasma collected from healthy volunteers taking 1 g/day vitamin C reduced bortezomib-induced MM cell death in vitro. This antagonistic effect of vitamin C against proteasome inhibitors is limited to the boronate class of inhibitors (bortezomib and MG262). In vivo activity of this combination treatment was then evaluated using our xenograft model of human MM in SCID (severe combined immune-deficient) mice. Bortezomib (0.1 mg/kg twice a week for 4 weeks) significantly inhibits in vivo MM cell growth, which was blocked by oral vitamin C (40 mg/kg/day). Therefore, our results for the first time show that vitamin C can significantly reduce the activity of bortezomib treatment in vivo; and importantly, suggest that patients receiving treatment with bortezomib should avoid taking vitamin C dietary supplements.
Asunto(s)
Antineoplásicos/antagonistas & inhibidores , Antioxidantes/farmacología , Ácido Ascórbico/farmacología , Ácidos Borónicos/antagonistas & inhibidores , Pirazinas/antagonistas & inhibidores , Animales , Bortezomib , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Humanos , Ratones , Mieloma Múltiple/tratamiento farmacológico , Inhibidores de ProteasomaRESUMEN
Cyclin D dysregulation and overexpression is noted in the majority of multiple myeloma (MM) patients, suggesting its critical role in MM pathogenesis. Here, we sought to identify the effects of targeting cyclin D in MM. We first confirmed cyclin D mRNA overexpression in 42 of 64 (65%) patient plasma cells. Silencing cyclin D1 resulted in >50% apoptotic cell death suggesting its validity as a potential therapeutic target. We next evaluated P276-00, a clinical-grade small-molecule cyclin-dependent kinase inhibitor as a way to target the cyclins. P276-00 resulted in dose-dependent cytotoxicity in MM cells. Cell-cycle analysis confirmed either growth arrest or caspase-dependent apoptosis; this was preceded by inhibition of Rb-1 phosphorylation with associated downregulation of a range of cyclins suggesting a regulatory role of P276-00 in cell-cycle progression through broad activity. Proliferative stimuli such as interleukin-6, insulin-like growth factor-1 and bone-marrow stromal cell adherence induced cyclins; P276-00 overcame these growth, survival and drug resistance signals. Because the cyclins are substrates of proteasome degradation, combination studies with bortezomib resulted in synergism. Finally, in vivo efficacy of P276-00 was confirmed in an MM xenograft model. These studies form the basis of an ongoing phase I study in the treatment of relapsed/refractory MM.