ABSTRACT
PURPOSE: The successful clinical translation of compounds that target specific oncogenic transcription factors will require an understanding of the mechanism of target suppression to optimize the dose and schedule of administration. We have previously shown trabectedin reverses the gene signature of the EWS-FLI1 transcription factor. In this report, we establish the mechanism of suppression and use it to justify the reevaluation of this drug in the clinic in patients with Ewing sarcoma.Experimental Design: We demonstrate a novel epigenetic mechanism of trabectedin using biochemical fractionation and chromatin immunoprecipitation sequencing. We link the effect to drug schedule and EWS-FLI1 downstream target expression using confocal microscopy, qPCR, Western blot analysis, and cell viability assays. Finally, we quantitate target suppression within the three-dimensional architecture of the tumor in vivo using 18F-FLT imaging. RESULTS: Trabectedin evicts the SWI/SNF chromatin-remodeling complex from chromatin and redistributes EWS-FLI1 in the nucleus leading to a marked increase in H3K27me3 and H3K9me3 at EWS-FLI1 target genes. These effects only occur at high concentrations of trabectedin leading to suppression of EWS-FLI1 target genes and a loss of cell viability. In vivo, low-dose irinotecan is required to improve the magnitude, penetrance, and duration of target suppression in the three-dimensional architecture of the tumor leading to differentiation of the Ewing sarcoma xenograft into benign mesenchymal tissue. CONCLUSIONS: These data provide the justification to evaluate trabectedin in the clinic on a short infusion schedule in combination with low-dose irinotecan with 18F-FLT PET imaging in patients with Ewing sarcoma.
Subject(s)
Antineoplastic Agents, Alkylating/pharmacology , Chromatin/genetics , Gene Expression Regulation, Neoplastic/drug effects , Oncogene Proteins, Fusion/antagonists & inhibitors , Proto-Oncogene Protein c-fli-1/antagonists & inhibitors , RNA-Binding Protein EWS/antagonists & inhibitors , Trabectedin/pharmacology , Transcription Factors/genetics , Active Transport, Cell Nucleus , Animals , Cell Line, Tumor , Disease Models, Animal , Dose-Response Relationship, Drug , Humans , Mice , Oncogene Proteins, Fusion/blood , Oncogene Proteins, Fusion/genetics , Protein Binding , Proto-Oncogene Protein c-fli-1/blood , Proto-Oncogene Protein c-fli-1/genetics , RNA-Binding Protein EWS/blood , RNA-Binding Protein EWS/genetics , Sarcoma, Ewing/drug therapy , Sarcoma, Ewing/genetics , Sarcoma, Ewing/metabolism , Sarcoma, Ewing/pathology , Xenograft Model Antitumor AssaysABSTRACT
Growth factors, such as insulin, can induce both acute and long-term glucose uptake into cells. Apart from the rapid, insulin-induced fusion of glucose transporter (GLUT)4 storage vesicles with the cell surface that occurs in muscle and adipose tissues, the mechanism behind acute induction has been unclear in other systems. Thioredoxin interacting protein (TXNIP) has been shown to be a negative regulator of cellular glucose uptake. TXNIP is transcriptionally induced by glucose and reduces glucose influx by promoting GLUT1 endocytosis. Here, we report that TXNIP is a direct substrate of protein kinase B (AKT) and is responsible for mediating AKT-dependent acute glucose influx after growth factor stimulation. Furthermore, TXNIP functions as an adaptor for the basal endocytosis of GLUT4 in vivo, its absence allows excess glucose uptake in muscle and adipose tissues, causing hypoglycemia during fasting. Altogether, TXNIP serves as a key node of signal regulation and response for modulating glucose influx through GLUT1 and GLUT4.
Subject(s)
Adipose Tissue/metabolism , Carrier Proteins/metabolism , Glucose/metabolism , Insulin/metabolism , Muscle, Skeletal/metabolism , Proto-Oncogene Proteins c-akt/metabolism , Signal Transduction , Thioredoxins/metabolism , 3T3-L1 Cells , Animals , Carrier Proteins/genetics , Endocytosis , Glucose Transporter Type 1/genetics , Glucose Transporter Type 1/metabolism , Glucose Transporter Type 4/genetics , Glucose Transporter Type 4/metabolism , Mice , Mice, Transgenic , Proto-Oncogene Proteins c-akt/genetics , Thioredoxins/geneticsABSTRACT
LIN28 has emerged as an oncogenic driver in a number of cancers, including neuroblastoma (NB). Overexpression of LIN28 correlates with poor outcome in NB, therefore drugs that impact the LIN28/Let-7 pathway could be beneficial in treating NB patients. The LIN28/Let-7 pathway affects many cellular processes including the regulation of cancer stem cells and glycolytic metabolism. Polyamines, regulated by ornithine decarboxylase (ODC) modulate eIF-5A which is a direct regulator of the LIN28/Let-7 axis. We propose that therapy inhibiting ODC will restore balance to the LIN28/Let-7 axis, suppress glycolytic metabolism, and decrease MYCN protein expression in NB. Difluoromethylornithine (DFMO) is an inhibitor of ODC in clinical trials for children with NB. In vitro experiments using NB cell lines, BE(2)-C, SMS-KCNR, and CHLA90 show that DFMO treatment reduced LIN28B and MYCN protein levels and increased Let-7 miRNA and decreased neurosphere formation. Glycolytic metabolic activity decreased with DFMO treatment in vivo. Additionally, sensitivity to DFMO treatment correlated with LIN28B overexpression (BE(2)-C>SMS-KCNR>CHLA90). This is the first study to demonstrate that DFMO treatment restores balance to the LIN28/Let-7 axis and inhibits glycolytic metabolism and neurosphere formation in NB and that PET scans may be a meaningful imaging tool to evaluate the therapeutic effects of DFMO treatment.
