RESUMEN
Despite the high response rates of individuals with myelodysplastic syndrome (MDS) with deletion of chromosome 5q (del(5q)) to treatment with lenalidomide (LEN) and the recent identification of cereblon (CRBN) as the molecular target of LEN, the cellular mechanism by which LEN eliminates MDS clones remains elusive. Here we performed an RNA interference screen to delineate gene regulatory networks that mediate LEN responsiveness in an MDS cell line, MDSL. We identified GPR68, which encodes a G-protein-coupled receptor that has been implicated in calcium metabolism, as the top candidate gene for modulating sensitivity to LEN. LEN induced GPR68 expression via IKAROS family zinc finger 1 (IKZF1), resulting in increased cytosolic calcium levels and activation of a calcium-dependent calpain, CAPN1, which were requisite steps for induction of apoptosis in MDS cells and in acute myeloid leukemia (AML) cells. In contrast, deletion of GPR68 or inhibition of calcium and calpain activation suppressed LEN-induced cytotoxicity. Moreover, expression of calpastatin (CAST), an endogenous CAPN1 inhibitor that is encoded by a gene (CAST) deleted in del(5q) MDS, correlated with LEN responsiveness in patients with del(5q) MDS. Depletion of CAST restored responsiveness of LEN-resistant non-del(5q) MDS cells and AML cells, providing an explanation for the superior responses of patients with del(5q) MDS to LEN treatment. Our study describes a cellular mechanism by which LEN, acting through CRBN and IKZF1, has cytotoxic effects in MDS and AML that depend on a calcium- and calpain-dependent pathway.
Asunto(s)
Apoptosis/efectos de los fármacos , Calcio/metabolismo , Calpaína/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Factores Inmunológicos/farmacología , Síndromes Mielodisplásicos/tratamiento farmacológico , Receptores Acoplados a Proteínas G/efectos de los fármacos , Talidomida/análogos & derivados , Proteínas Adaptadoras Transductoras de Señales , Apoptosis/genética , Proteínas de Unión al Calcio/genética , Calpaína/genética , Calpaína/metabolismo , Línea Celular Tumoral , Redes Reguladoras de Genes , Humanos , Factor de Transcripción Ikaros/efectos de los fármacos , Factor de Transcripción Ikaros/genética , Factor de Transcripción Ikaros/metabolismo , Lenalidomida , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Síndromes Mielodisplásicos/genética , Síndromes Mielodisplásicos/metabolismo , Péptido Hidrolasas/metabolismo , Interferencia de ARN , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Talidomida/farmacología , Ubiquitina-Proteína LigasasRESUMEN
Myelodysplastic syndromes (MDSs) arise from a defective hematopoietic stem/progenitor cell. Consequently, there is an urgent need to develop targeted therapies capable of eliminating the MDS-initiating clones. We identified that IRAK1, an immune-modulating kinase, is overexpressed and hyperactivated in MDSs. MDS clones treated with a small molecule IRAK1 inhibitor (IRAK1/4-Inh) exhibited impaired expansion and increased apoptosis, which coincided with TRAF6/NF-κB inhibition. Suppression of IRAK1, either by RNAi or with IRAK1/4-Inh, is detrimental to MDS cells, while sparing normal CD34(+) cells. Based on an integrative gene expression analysis, we combined IRAK1 and BCL2 inhibitors and found that cotreatment more effectively eliminated MDS clones. In summary, these findings implicate IRAK1 as a drugable target in MDSs.
Asunto(s)
Quinasas Asociadas a Receptores de Interleucina-1/antagonistas & inhibidores , Síndromes Mielodisplásicos/tratamiento farmacológico , Animales , Apoptosis , Línea Celular , Perfilación de la Expresión Génica , Humanos , Quinasas Asociadas a Receptores de Interleucina-1/genética , Quinasas Asociadas a Receptores de Interleucina-1/fisiología , Ratones , Síndromes Mielodisplásicos/patología , FN-kappa B/metabolismo , Fosforilación , Proteínas Proto-Oncogénicas c-bcl-2/antagonistas & inhibidores , Factor 6 Asociado a Receptor de TNF/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
In vitro studies suggest that haploinsufficiency is involved in the pathogenesis of myelodysplastic syndromes (MDS). In patients with del5q cytogenetic abnormality, RPS-14 and microRNAs (miRNAs) play a major role. In a multicenter phase II single-arm trial with lenalidomide in anemic primary del5q MDS patients with low- or int-1 risk IPSS, biological changes from baseline were investigated. Gene expression profiling of selected genes was performed (TaqMan® Low Density Array Fluidic card, Applied Biosystems PRISM® 7900HT) and normalized against the expression of the 18S housekeeping gene from a pool of healthy subjects. Thirty-two patients were evaluated at baseline and after 3 and 6 months of treatment. RPS-14, miR-145, and miR-146 were downregulated at baseline and significantly increased during treatment. Nuclear factor kappa B, IL-6, interferon regulatory factor-1, IFNγ-R2, IL-2, and many genes in the apoptotic pathways (TNF, IL-1B, and IL-10) were upregulated at baseline and significantly downregulated during lenalidomide treatment, while forkhead box P3, FAS, IFNγ, IL-12A, and IL-12B were downregulated at baseline and progressively upregulated during treatment. The crucial role of aberrant immunological pathways and haploinsufficiency in the pathogenesis of del5q MDS is confirmed in the present patient setting. Our results indicate that lenalidomide may act through defined immunological pathways in this condition.
Asunto(s)
Anemia Macrocítica/genética , Perfilación de la Expresión Génica , Regulación de la Expresión Génica/efectos de los fármacos , Síndromes Mielodisplásicos/genética , Talidomida/análogos & derivados , Anciano , Anemia Macrocítica/tratamiento farmacológico , Anemia Macrocítica/inmunología , Apoptosis/genética , Apoptosis/inmunología , Células de la Médula Ósea/metabolismo , Células de la Médula Ósea/patología , Deleción Cromosómica , Cromosomas Humanos Par 5/genética , Cromosomas Humanos Par 5/inmunología , Femenino , Factores de Transcripción Forkhead/biosíntesis , Factores de Transcripción Forkhead/genética , Dosificación de Gen , Estudios de Asociación Genética , Humanos , Inmunidad Innata/genética , Lenalidomida , Masculino , MicroARNs/biosíntesis , MicroARNs/genética , Modelos Genéticos , Síndromes Mielodisplásicos/tratamiento farmacológico , Síndromes Mielodisplásicos/inmunología , Proteínas Ribosómicas/deficiencia , Proteínas Ribosómicas/genética , Transducción de Señal/genética , Transducción de Señal/inmunología , Talidomida/farmacología , Talidomida/uso terapéuticoRESUMEN
Bortezomib (Velcade) is used widely for the treatment of various human cancers; however, its mechanisms of action are not fully understood, particularly in myeloid malignancies. Bortezomib is a selective and reversible inhibitor of the proteasome. Paradoxically, we find that bortezomib induces proteasome-independent degradation of the TRAF6 protein, but not mRNA, in myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) cell lines and primary cells. The reduction in TRAF6 protein coincides with bortezomib-induced autophagy, and subsequently with apoptosis in MDS/AML cells. RNAi-mediated knockdown of TRAF6 sensitized bortezomib-sensitive and -resistant cell lines, underscoring the importance of TRAF6 in bortezomib-induced cytotoxicity. Bortezomib-resistant cells expressing an shRNA targeting TRAF6 were resensitized to the cytotoxic effects of bortezomib due to down-regulation of the proteasomal subunit α-1 (PSMA1). To determine the molecular consequences of loss of TRAF6 in MDS/AML cells, in the present study, we applied gene-expression profiling and identified an apoptosis gene signature. Knockdown of TRAF6 in MDS/AML cell lines or patient samples resulted in rapid apoptosis and impaired malignant hematopoietic stem/progenitor function. In summary, we describe herein novel mechanisms by which TRAF6 is regulated through bortezomib/autophagy-mediated degradation and by which it alters MDS/AML sensitivity to bortezomib by controlling PSMA1 expression.