RESUMEN
Antisense oligonucleotides (ASOs) are a novel therapeutic approach to target difficult-to-drug protein classes by targeting their corresponding mRNAs. Significantly enhanced ASO activity has been achieved by the targeted delivery of ASOs to selected tissues. One example is the targeted delivery of ASOs to hepatocytes, achieved with N-acetylgalactosamine (GalNAc) conjugation to ASO, which results in selective uptake by asialoglycoprotein receptor (ASGR). Here we have evaluated the potential of GalNAc-conjugated ASOs as a therapeutic approach to targeting difficult-to-drug pathways in hepatocellular carcinoma (HCC). The activity of GalNAc-conjugated ASOs was superior to that of the unconjugated parental ASO in ASGR (+) human HCC cells in vitro, but not in ASGR (-) cells. Both human- and mouse-derived HCC displayed reduced levels of ASGR, however, despite this, GalNAc-conjugated ASOs showed a 5- to 10-fold increase in potency in tumors. Systemically administered GalNAc-conjugated ASOs demonstrated both enhanced antisense activity and antitumor activity in the diethylnitrosamine-induced HCC tumor model. Finally, GalNAc conjugation enhanced ASO activity in human circulating tumor cells from HCC patients, demonstrating the potential of this approach in primary human HCC tumor cells. Taken together, these results provide a strong rationale for a potential therapeutic use of GalNAc-conjugated ASOs for the treatment of HCC.
Asunto(s)
Acetilgalactosamina/química , Técnicas de Transferencia de Gen , Oligonucleótidos Antisentido/administración & dosificación , Oligonucleótidos Antisentido/química , Animales , Receptor de Asialoglicoproteína/genética , Receptor de Asialoglicoproteína/metabolismo , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patología , Línea Celular , Células Cultivadas , Expresión Génica , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patología , RatonesRESUMEN
The sonic hedgehog (Shh) pathway contributes to the initiation and progression of tumors with various origins when aberrantly activated. In this study, we investigated if the Shh pathway is important for the proliferation of hepatocellular carcinoma (HCC) cells and also began to identify which components of the pathway play a pivotal role in the biology of HCC. Expression levels of components in the pathway were measured, and glioma-associated oncogene (Gli) 2 levels were found to be considerably higher in human HCC lines compared with normal liver. Gli2 levels were also higher in tumor tissue from HCC patients compared with normal liver. Antisense oligonucleotides (ASO) were used to specifically down-regulate Gli2, and this led to decreased proliferation of various HCC cell lines. However, inhibition of Gli1 and Gli3 with ASOs did not decrease proliferation in most HCC cell lines and inhibitors targeting the upstream components of the pathway, including smoothened (Smo), displayed antiproliferative effects in only a subset of HCC cell lines. Moreover, in cancer cells harboring Smo mutations or unresponsive to the Smo inhibitor 3-keto-N-aminoethylaminoethylcaproyldihydrocinnamoyl cyclopamine, the Gli2 ASO was still able to inhibit proliferation. The importance of Gli2 in HCC proliferation was further confirmed by the changes in expression levels of genes, such as Bcl-2, c-Myc, and p27, following suppression of Gli2 expression. Taken together, these results suggest that, among the Gli transcription factors, Gli2 plays a predominant role in the proliferation of HCC cells and the suppression of Gli2 expression may provide a useful therapeutic option for the treatment of HCC.
Asunto(s)
Carcinoma Hepatocelular/genética , Factores de Transcripción de Tipo Kruppel/biosíntesis , Factores de Transcripción de Tipo Kruppel/genética , Neoplasias Hepáticas/genética , Proteínas Nucleares/biosíntesis , Proteínas Nucleares/genética , Animales , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patología , Procesos de Crecimiento Celular/fisiología , Regulación hacia Abajo , Regulación Neoplásica de la Expresión Génica , Proteínas Hedgehog/metabolismo , Humanos , Factores de Transcripción de Tipo Kruppel/metabolismo , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Proteínas Nucleares/metabolismo , Oligonucleótidos Antisentido/genética , Oligonucleótidos Antisentido/farmacología , Alcaloides de Veratrum/farmacología , Proteína Gli2 con Dedos de ZincRESUMEN
A library of 2'-methoxyethyl-modified antisense oligonucleotides (2'MOE ASO) targeting 1,510 different genes has been developed, validated, and used to identify cell cycle regulatory genes. The most effective molecular target identified was Eg5 (kinesin-like-1), which when inhibited gave the largest increase in 4N DNA in various tumor cells. The Eg5 ASO reduced Eg5 levels, inhibited proliferation, increased apoptosis, and altered the expression of other cell cycle proteins, including survivin and Aurora-A. To examine the therapeutic utility of the Eg5 ASO, the compound was also evaluated in xenograft models. Treatment with Eg5 ASO produced a statistically significant reduction of tumor growth, reduction in Eg5 expression in the tumors, and changes in histone phosphorylation, consistent with a loss of Eg5 protein expression. These data show, for the first time, the utility of a 2'MOE ASO library for high-throughput cell culture-based functional assays and suggest that an Eg5 ASO also has potential in a therapeutic strategy.
Asunto(s)
Cinesinas/antagonistas & inhibidores , Cinesinas/genética , Oligonucleótidos Antisentido/genética , Oligonucleótidos Antisentido/farmacología , Animales , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Apoptosis/genética , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Procesos de Crecimiento Celular/efectos de los fármacos , Procesos de Crecimiento Celular/genética , Línea Celular Tumoral , ADN de Neoplasias/genética , Biblioteca de Genes , Glioma/tratamiento farmacológico , Glioma/genética , Glioma/patología , Células HeLa , Humanos , Cinesinas/biosíntesis , Ratones , Ratones Desnudos , ARN Mensajero/antagonistas & inhibidores , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
BACKGROUND: The Janus kinase (JAK) and signal transduction and activation of transcription (STAT) signaling pathway is an attractive target in multiple cancers. Activation of the JAK-STAT pathway is important in both tumorigenesis and activation of immune responses. In diffuse large B-cell lymphoma (DLBCL), the transcription factor STAT3 has been associated with aggressive disease phenotype and worse overall survival. While multiple therapies inhibit upstream signaling, there has been limited success in selectively targeting STAT3 in patients. Antisense oligonucleotides (ASOs) represent a compelling therapeutic approach to target difficult to drug proteins such as STAT3 through of mRNA targeting. We report the evaluation of a next generation STAT3 ASO (AZD9150) in a non-Hodgkin's lymphoma population, primarily consisting of patients with DLBCL. METHODS: Patients with relapsed or treatment refractory lymphoma were enrolled in this expansion cohort. AZD9150 was administered at 2 mg/kg and the 3 mg/kg (MTD determined by escalation cohort) dose levels with initial loading doses in the first week on days 1, 3, and 5 followed by weekly dosing. Patients were eligible to remain on therapy until unacceptable toxicity or progression. Blood was collected pre- and post-treatment for analysis of peripheral immune cells. RESULTS: Thirty patients were enrolled, 10 at 2 mg/kg and 20 at 3 mg/kg dose levels. Twenty-seven patients had DLBCL. AZD9150 was safe and well tolerated at both doses. Common drug-related adverse events included transaminitis, fatigue, and thrombocytopenia. The 3 mg/kg dose level is the recommended phase 2 dose. All responses were seen among DLBCL patients, including 2 complete responses with median duration of response 10.7 months and 2 partial responses. Peripheral blood cell analysis of three patients without a clinical response to therapy revealed a relative increase in proportion of macrophages, CD4+, and CD8+ T cells; this trend did not reach statistical significance. CONCLUSIONS: AZD9150 was well tolerated and demonstrated efficacy in a subset of heavily pretreated patients with DLBCL. Studies in combination with checkpoint immunotherapies are ongoing. TRIAL REGISTRATION: Registered at ClinicalTrials.gov: NCT01563302 . First submitted 2/13/2012.
Asunto(s)
Linfoma/tratamiento farmacológico , Oligonucleótidos Antisentido/uso terapéutico , Oligonucleótidos/uso terapéutico , Adulto , Anciano , Anciano de 80 o más Años , Femenino , Humanos , Linfoma/patología , Masculino , Persona de Mediana Edad , Oligonucleótidos/farmacología , Oligonucleótidos Antisentido/farmacología , Factor de Transcripción STAT3 , Adulto JovenRESUMEN
Antisense oligonucleotides (ASOs) are widely accepted therapeutic agents that suppress RNA transcription. While the majority of ASOs are well tolerated in vivo, few sequences trigger inflammatory responses in absence of conventional CpG motifs. In this study, we identified non-CpG oligodeoxy-nucleotide (ODN) capable of triggering an inflammatory response resulting in B cell and macrophage activation in a MyD88- and TLR9-dependent manner. In addition, we found the receptor for advance glycation end product (RAGE) receptor to be involved in the initiation of inflammatory response to suboptimal concentrations of both CpG- and non-CpG-containing ODNs. In contrast, dosing RAGE KO mice with high doses of CpG or non-CpG ODNs lead to a stronger inflammatory response than observed in wild-type mice. Together, our data provide a previously uncharacterized in vivo mechanism contingent on ODN-administered dose, where TLR9 governs the primary response and RAGE plays a distinct and cooperative function in providing a pivotal role in balancing the immune response.
Asunto(s)
Inmunidad Celular/inmunología , Inflamación/inmunología , Oligonucleótidos Antisentido/uso terapéutico , Receptor para Productos Finales de Glicación Avanzada/metabolismo , Receptor Toll-Like 9/metabolismo , Animales , Linfocitos B/inmunología , Citocinas/sangre , Humanos , Macrófagos/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Factor 88 de Diferenciación Mieloide/metabolismo , Cultivo Primario de Células , ARN/genética , ARN/metabolismo , Receptor para Productos Finales de Glicación Avanzada/genética , Análisis de la Célula Individual , Receptor Toll-Like 9/genética , Transcripción GenéticaRESUMEN
Next-generation sequencing technologies have greatly expanded our understanding of cancer genetics. Antisense technology is an attractive platform with the potential to translate these advances into improved cancer therapeutics, because antisense oligonucleotide (ASO) inhibitors can be designed on the basis of gene sequence information alone. Recent human clinical data have demonstrated the potent activity of systemically administered ASOs targeted to genes expressed in the liver. We describe the preclinical activity and initial clinical evaluation of a class of ASOs containing constrained ethyl modifications for targeting the gene encoding the transcription factor STAT3, a notoriously difficult protein to inhibit therapeutically. Systemic delivery of the unformulated ASO, AZD9150, decreased STAT3 expression in a broad range of preclinical cancer models and showed antitumor activity in lymphoma and lung cancer models. AZD9150 preclinical activity translated into single-agent antitumor activity in patients with highly treatment-refractory lymphoma and non-small cell lung cancer in a phase 1 dose-escalation study.
Asunto(s)
Antineoplásicos/uso terapéutico , Carcinoma de Pulmón de Células no Pequeñas/terapia , Regulación Neoplásica de la Expresión Génica , Técnicas de Silenciamiento del Gen , Neoplasias Pulmonares/terapia , Linfoma/terapia , Oligonucleótidos Antisentido/uso terapéutico , Oligonucleótidos/uso terapéutico , Factor de Transcripción STAT3/antagonistas & inhibidores , Adulto , Anciano , Anciano de 80 o más Años , Animales , Apoptosis , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/patología , Línea Celular Tumoral , Proliferación Celular , Regulación hacia Abajo , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Linfoma/genética , Linfoma/metabolismo , Linfoma/patología , Masculino , Ratones Endogámicos BALB C , Ratones Endogámicos NOD , Ratones Desnudos , Ratones SCID , Persona de Mediana Edad , Factor de Transcripción STAT3/genética , Factor de Transcripción STAT3/metabolismo , Factores de Tiempo , Resultado del Tratamiento , Carga Tumoral , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Advances in the medicinal chemistry of antisense oligonucleotide drugs have been instrumental in achieving and optimizing antisense activity in cell types other than hepatocytes, the cell type that is most sensitive to antisense effects following systemic treatment. To broadly characterize the effects of antisense drugs on target messenger RNA (mRNA) levels in different organs and cell types in animals, we have developed a sensitive RNA in situ hybridization technique using the noncoding RNA metastasis associated lung adenocarcinoma transcript 1 (MALAT1) as a surrogate target. We have used this technique to evaluate the effects of 2'-O-methoxy ethyl (MOE) and constrained ethyl bicyclic nucleic acid (cEt) gapmer antisense oligonucleotides (ASOs). ASO tissue distribution was also characterized using immunohistochemical techniques, and MALAT1 mRNA reductions were confirmed by quantitative real time-polymerase chain reaction. Our findings demonstrate that systemic antisense drug administration in both mice and non-human primates resulted in marked reductions in MALAT1 RNA in many tissues and cell types other than liver including kidney, muscle, lung, adipose, adrenal gland, and peripheral nerve tissue. As expected, ASOs with cEt chemistry were more efficacious than MOE ASO in all tissues examined.
Asunto(s)
Técnicas de Silenciamiento del Gen , Oligonucleótidos Antisentido/administración & dosificación , ARN Largo no Codificante/genética , ARN Mensajero/genética , Animales , Células Cultivadas , Hibridación in Situ , Inyecciones Subcutáneas , Macaca fascicularis , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Oligonucleótidos Antisentido/genética , Especificidad de Órganos , ARN Largo no Codificante/metabolismo , ARN Mensajero/metabolismo , Sensibilidad y EspecificidadRESUMEN
Genome-wide studies have identified thousands of long noncoding RNAs (lncRNAs) lacking protein-coding capacity. However, most lncRNAs are expressed at a very low level, and in most cases there is no genetic evidence to support their in vivo function. Malat1 (metastasis associated lung adenocarcinoma transcript 1) is among the most abundant and highly conserved lncRNAs, and it exhibits an uncommon 3'-end processing mechanism. In addition, its specific nuclear localization, developmental regulation, and dysregulation in cancer are suggestive of it having a critical biological function. We have characterized a Malat1 loss-of-function genetic model that indicates that Malat1 is not essential for mouse pre- and postnatal development. Furthermore, depletion of Malat1 does not affect global gene expression, splicing factor level and phosphorylation status, or alternative pre-mRNA splicing. However, among a small number of genes that were dysregulated in adult Malat1 knockout mice, many were Malat1 neighboring genes, thus indicating a potential cis-regulatory role of Malat1 gene transcription.