RESUMO
The PI3K/Akt pathway plays a crucial role in the pathogenesis of multiple myeloma (MM) in the bone marrow (BM) milieu. However, efficacy of selective and potent Akt inhibition has not yet been fully elucidated. In this study, we, therefore, examined the biologic impact of selective and potent Akt inhibition by a novel allosteric inhibitor TAS-117. TAS-117 induced significant growth inhibition, associated with downregulation of phosphorylated Akt (p-Akt), selectively in MM cell lines with high baseline p-Akt. Cytotoxicity of TAS-117 was also observed in patient MM cells, but not in normal peripheral blood mononuclear cells. Importantly, TAS-117 induced significant cytotoxicity in MM cells even in the presence of BM stromal cells, associated with inhibition of IL6 secretion. Oral administration of TAS-117 significantly inhibited human MM cell growth in murine xenograft models. TAS-117 triggered apoptosis and autophagy, as well as induction of endoplasmic reticulum (ER) stress response with minimal expression of C/EBP homologous protein (CHOP), a fatal ER stress marker. Importantly, TAS-117 enhanced bortezomib-induced cytotoxicity, associated with increased CHOP and PARP cleavage and blockade of bortezomib-induced p-Akt, suggesting that TAS-117 augments bortezomib-induced ER stress and apoptotic signaling. Carfilzomib-induced cytotoxicity was similarly enhanced by TAS-117. Importantly, TAS-117 enhanced bortezomib-induced cytotoxicity in vivo, associated with prolonged host survival. Our results show that selective and potent Akt inhibition by TAS-117 triggers anti-MM activities in vitro and in vivo, as well as enhances cytotoxicity of proteasome inhibition, providing the preclinical framework for clinical evaluation of selective Akt inhibitors, alone and in combination with proteasome inhibitors in MM.
Assuntos
Estresse do Retículo Endoplasmático/efeitos dos fármacos , Compostos Heterocíclicos com 3 Anéis/farmacologia , Mieloma Múltiplo/tratamento farmacológico , Inibidores de Proteassoma/farmacologia , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Animais , Apoptose/efeitos dos fármacos , Processos de Crescimento Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Modelos Animais de Doenças , Humanos , Imidazóis/farmacologia , Camundongos , Mieloma Múltiplo/enzimologia , Mieloma Múltiplo/patologia , Oxazinas/farmacologia , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Deregulation of cell-cycle control is a hallmark of cancer. Thus, cyclin-dependent kinases (Cdks) are an attractive target for the development of anti-cancer drugs. Here, we report the biological characterization of a highly potent pan-Cdk inhibitor with a macrocycle-quinoxalinone structure. Compound M inhibited Cdk1, 2, 4, 5, 6, and 9 with equal potency in the nM range and was selective against kinases other than Cdks. This compound inhibited multiple events in the cell cycle in vitro, including retinoblastoma protein (pRb) phosphorylation, E2F-dependent transcription, DNA replication (determined by bromodeoxyuridine incorporation), and mitosis completion (assayed by flow cytometry) in the 10 nM range. Moreover, this compound induced cell death, as determined by induction of the subG1 fraction, activated caspase-3, and anexin V. In vivo, Compound M showed anti-tumor efficacy at a tolerated dose. In a nude rat xenograft tumor model, an 8-h constant infusion of Compound M inhibited pRb phosphorylation and induced apoptosis in tumor cells at ~ 30 nM, which led to the inhibition of tumor growth. Immunosuppression was the only liability observed at this dose, but immune function returned to normal after 10 days. Suppression of pRb phosphorylation in tumor cells was clearly correlated with tumor cell growth inhibition and cell death in vitro and in vivo. In vivo, Compound M inhibited pRb phosphorylation in both tumor and gut crypt cells. Rb phosphorylation may be a suitable pharmacodynamic biomarker in both tumors and normal tissues for monitoring target engagement and predicting the efficacy of Compound M.
Assuntos
Antineoplásicos/farmacologia , Quinases Ciclina-Dependentes/antagonistas & inibidores , Compostos Macrocíclicos/farmacologia , Quinoxalinas/farmacologia , Animais , Antineoplásicos/efeitos adversos , Antineoplásicos/química , Bromodesoxiuridina/metabolismo , Ciclo Celular/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Quinases Ciclina-Dependentes/metabolismo , Relação Dose-Resposta a Droga , Feminino , Células HCT116 , Humanos , Contagem de Leucócitos , Compostos Macrocíclicos/efeitos adversos , Compostos Macrocíclicos/química , Quinoxalinas/efeitos adversos , Quinoxalinas/química , Ratos , Ratos Nus , Especificidade por Substrato/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Immunosuppression is one of the common side effects of many anti-tumor agents targeting proliferating cells. We previously reported the development of a new class of pan-cyclin-dependent kinase (Cdk) inhibitor compounds that induce immunosuppression in rodents. Here, we demonstrated that a pan-Cdk inhibitor, Compound 1 very rapidly reduced white blood cells in mice, only 8 h after administration. Compound 1 induced death of peripheral blood cells or purified resting (non-stimulated) lymphocytes ex vivo. Cell death was induced very rapidly, after 4 h of incubation, suggesting that acute immunosuppression observed in rodents might be, at least in part, due to direct cytotoxic effects of Compound 1 on resting lymphocytes. While cell cycle-related Cdks were not activated, the carboxyl terminal domain (CTD) of the largest subunit of RNA polymerase II was phosphorylated, indicating activation of Cdk7 or Cdk9, which phosphorylates this domain, in resting lymphocytes. Indeed, the pan-Cdk inhibitor suppressed CTD phosphorylation in resting cells at the dose required for cell death induction. Inhibition of Cdk7 or Cdk9 by Compound 1 was also confirmed by suppression of nuclear factor-kappa B (NF-κB)-dependent transcription activity in the human cancer cell line U2OS. Interestingly, a Cdk4/6 inhibitor with selectivity against Cdk7 and Cdk9 did not induce cell death in resting lymphocytes. These results suggest that CTD phosphorylation possibly by Cdk7 or Cdk9 might be important for survival of resting lymphocytes and that Cdk inhibitors without inhibitory activity on these kinases might be an attractive agent for cancer chemotherapy.
Assuntos
Quinases Ciclina-Dependentes/antagonistas & inibidores , Linfócitos/citologia , Linfócitos/enzimologia , Inibidores de Proteínas Quinases/farmacologia , Animais , Morte Celular/efeitos dos fármacos , Quinases Ciclina-Dependentes/metabolismo , Células HEK293 , Humanos , Terapia de Imunossupressão , Concentração Inibidora 50 , Leucócitos/citologia , Leucócitos/efeitos dos fármacos , Linfócitos/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos ICR , NF-kappa B/metabolismo , Fosforilação/efeitos dos fármacos , Inibidores de Proteínas Quinases/química , Estrutura Terciária de Proteína , RNA Polimerase II/química , RNA Polimerase II/metabolismo , Ratos , Transdução de Sinais/efeitos dos fármacos , Fatores de Tempo , Transcrição Gênica/efeitos dos fármacosRESUMO
Abnormalities in the p16INK4a/ cyclin-dependent kinase (Cdk)4, 6/ Retinoblastoma (Rb) pathway frequently occur in various human cancers. Thus, Cdk4/6 is an attractive target for cancer therapy. Here we report the biological characterization of a 2-aminothiazole-derived Cdk4/6 selective inhibitor, named Compound A in vitro and in vivo. Compound A potently inhibits Cdk4 and Cdk6 with high selectivity (more than 57-fold) against other Cdks and 45 serine/threonine and tyrosine kinases. Compound A inhibits Rb protein (pRb) phosphorylation at Ser780, inhibits E2F-dependent transcription, and induces cell-cycle arrest at G1 in the T98G human glioma cell line. Among 82 human cells derived from various tissues, cell lines derived from hematological cancers (leukemia/lymphoma) tended to be more sensitive to Compound A in cell proliferation assay. Rb-negative cells tended to be insensitive to Compound A, as we had expected. In a nude rat xenograft model, Compound A inhibited pRb phosphorylation and bromodeoxyuridine (BrdU) incorporation in Eol-1 xenograft tumor at plasma concentration of 510 nM. Interestingly Compound A only moderately inhibited those pharmacodynamic and cell cycle parameters of normal crypt cells in small intestine even at 5 times higher plasma concentration. In F344 rats, Compound A did not cause immunosuppression even at 17 times higher plasma conc. These results suggest that Cdk4/6 selective inhibitors only moderately affects on the cell cycle of normal proliferating tissues and has a safer profile than pan-Cdk inhibitor in vivo.
Assuntos
Quinase 4 Dependente de Ciclina/antagonistas & inibidores , Quinase 6 Dependente de Ciclina/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Pirimidinas/farmacologia , Tiazóis/farmacologia , Animais , Linhagem Celular Tumoral , Quinase 2 Dependente de Ciclina/antagonistas & inibidores , Quinase 2 Dependente de Ciclina/genética , Quinase 2 Dependente de Ciclina/metabolismo , Quinase 4 Dependente de Ciclina/metabolismo , Quinase 6 Dependente de Ciclina/metabolismo , Fatores de Transcrição E2F/antagonistas & inibidores , Fatores de Transcrição E2F/metabolismo , Fase G1 , Humanos , Masculino , Fosforilação , Inibidores de Proteínas Quinases/química , Pirimidinas/química , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Ratos , Ratos Nus , Proteína do Retinoblastoma/antagonistas & inibidores , Proteína do Retinoblastoma/metabolismo , Tiazóis/química , Transplante HeterólogoRESUMO
Aurora-A kinase is a one of the key regulators during mitosis progression. Aurora-A kinase is a potential target for anticancer therapies because overexpression of Aurora-A, which is frequently observed in some human cancers, results in aberrant mitosis leading to chromosomal instability and possibly tumorigenesis. MK-5108 is a novel small molecule with potent inhibitory activity against Aurora-A kinase. Although most of the Aurora-kinase inhibitors target both Aurora-A and Aurora-B, MK-5108 specifically inhibited Aurora-A kinase in a panel of protein kinase assays. Inhibition of Aurora-A by MK-5108 in cultured cells induced cell cycle arrest at the G(2)-M phase in flow cytometry analysis. The effect was confirmed by the accumulation of cells with expression of phosphorylated Histone H3 and inhibition of Aurora-A autophosphorylation by immunostaining assays. MK-5108 also induced phosphorylated Histone H3 in skin and xenograft tumor tissues in a nude rat xenograft model. MK-5108 inhibited growth of human tumor cell lines in culture and in different xenograft models. Furthermore, the combination of MK-5108 and docetaxel showed enhanced antitumor activities compared with control and docetaxel alone-treated animals without exacerbating the adverse effects of docetaxel. MK-5108 is currently tested in clinical trials and offers a new therapeutic approach to combat human cancers as a single agent or in combination with existing taxane therapies.
Assuntos
Antineoplásicos/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Ácidos Cicloexanocarboxílicos/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Taxoides/farmacologia , Tiazóis/farmacologia , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/química , Aurora Quinase A , Aurora Quinase B , Aurora Quinases , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Ácidos Cicloexanocarboxílicos/administração & dosagem , Ácidos Cicloexanocarboxílicos/química , Docetaxel , Humanos , Concentração Inibidora 50 , Camundongos , Mitose/efeitos dos fármacos , Inibidores de Proteínas Quinases/administração & dosagem , Inibidores de Proteínas Quinases/química , Ratos , Taxoides/toxicidade , Tiazóis/administração & dosagem , Tiazóis/química , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Because cyclin-dependent kinases (CDK) play a pivotal role in cancer progression, the development of CDK inhibitors has attracted attention in antitumor therapy. However, despite significant preclinical and clinical developments, CDK inhibition biomarkers for predicting efficacy against certain cancers in individual patients have not been identified. Here, we characterized a macrocyclic quinoxalin-2-one CDK inhibitor, compound A, and identified a gene biomarker for predicting its efficacy. Compound A showed 100-fold selectivity for CDK family proteins over other kinases and inhibited both E2F transcriptional activity and RNA polymerase II phosphorylation. Compound A treatment resulted in decreased proliferation in various tumor cell lines; however, the apoptosis induction rate differed significantly among the cell lines examined, which was consistent with roscovitine. By comparing the mRNA expression profiles of sensitive and resistant cell lines, we found that expression levels of an endogenous CDK inhibitor, p18(INK4C), showed a strong negative correlation to the sensitivity. In fact, p18 status was correlated with the response to CDK inhibitor in an independent data set of multiple myeloma cell lines and silencing p18 expression increased the susceptibility of resistant cells to CDK inhibitors. The analysis of molecular mechanisms revealed that cells with lowered p18 had aberrant CDK6 and E2F activities, which resulted in a transcriptional down-regulation of Mcl-1, a key molecule associated with flavopiridol-induced apoptosis, thereby leading to susceptibility to therapeutic intervention with CDK inhibitors. These results identified a molecular basis for CDK inhibitors to exert an antitumor effect in p18-deficient cancers and support the clinical use of CDK inhibitors.