ABSTRACT
Rhabdoid tumor (RT) is a pediatric cancer characterized by the inactivation of SMARCB1, a subunit of the SWI/SNF chromatin remodeling complex. Although this deletion is the known oncogenic driver, there are limited effective therapeutic options for these patients. Here we use unbiased screening of cell line panels to identify a heightened sensitivity of rhabdoid tumor to mithramycin and the second-generation analogue EC8042. The sensitivity of MMA and EC8042 was superior to traditional DNA damaging agents and linked to the causative mutation of the tumor, SMARCB1 deletion. Mithramycin blocks SMARCB1-deficient SWI/SNF activity and displaces the complex from chromatin to cause an increase in H3K27me3. This triggers chromatin remodeling and enrichment of H3K27ac at chromHMM-defined promoters to restore cellular differentiation. These effects occurred at concentrations not associated with DNA damage and were not due to global chromatin remodeling or widespread gene expression changes. Importantly, a single 3-day infusion of EC8042 caused dramatic regressions of RT xenografts, recapitulated the increase in H3K27me3, and cellular differentiation described in vitro to completely cure three out of eight mice.
Subject(s)
Rhabdoid Tumor , Animals , Cell Differentiation , Chromosomal Proteins, Non-Histone , Humans , Mice , Plicamycin/pharmacology , Rhabdoid Tumor/drug therapy , Rhabdoid Tumor/genetics , Transcription Factors/geneticsABSTRACT
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
Melanin-concentrating hormone receptor antagonists containing thieno- and a benzopyridazinone cores were designed and tested as potential anorectic agents. These ligands showed high affinity for the receptor, potent functional activity in vitro, and good oral bioavailabilty in rats. The thiophene analogue exhibited low iv clearance, long half-life, and high brain penetration. In obese rats, the thienopyridazinone demonstrated a dose-dependent reduction in feeding and body weight with doses between 1 and 10 mg kg-1.
