Small Molecule Inhibition of SMARCA2 Informs Pediatric Sarcoma Therapy
Introduction
Ewing’s sarcoma (EwS) and rhabdomyosarcoma (RMS) are the most common bone and soft tissue tumors in children and adolescents. These tumors are largely driven by gene fusion events that create abnormal oncogenic chimeric transcription factors. The EwS and RMS cells also possess molecular machines such as BAF (BRG1/BRM-associated factors) complexes that alter the architecture of certain parts of the genomic DNA and associated proteins. Genomic alterations in BAF complexes are observed in >20% of all cancers. A combination of defective BAFs and their interplay with chimeric oncofusion proteins is advantageous for the proliferation and survival of several types of pediatric sarcoma cells (EwS and RMS). These chimeras are considered highly important proteins for therapeutic targeting, but so far, such efforts have not been successful. Thus, the investigative focus has shifted toward finding new molecular pathways, such as BAF complexes, that may provide novel therapeutic windows of intervention by small molecules. SMARCA2, also known as Brahma (BRM), and its biochemical paralog SMARCA4 (Brahma-related gene 1 or BRG1) are the two mutually exclusive DNA-dependent ATPases of the BAF complex. Cancer cells that harbor alterations in SMARCA4 rely upon SMARCA2 activity for their growth and survival. SMARCA2 knockout mice are fully viable. Thus, targeting SMARCA2 by novel small molecules is an emerging focus of drug discovery.
Methods
We expressed and purified human SMARCA2 from insect cells in large quantities. Using a fluorescence-polarization-based assay, we identified a high-affinity DNA substrate and established a biochemical assay to measure high ATP-hydrolysis activity of SMARCA2 in the presence of DNA. Next, we performed a high-throughput drug screen of 40,000 compounds at our CPRIT-funded Center for Innovative Drug Discovery core (CIDD). We identified 15 top hits that could inhibit SMARCA2 ATPase activity >60%. We tested the cellular activity of our top hits in cell proliferation assays. Finally, using single-mouse testing (SMT) approach, we tested the activity of our lead compound (BAF-X-1) in a PDX model of Ewing’s sarcoma.
Results
We validated the top hits in secondary ATPase assays of SMARCA2. Our lead compound called BAF-X-1 was further characterized for its inhibitory effects on the ATPase activity of SMARCA2 on various substrates: short ds DNA (29mer), a longer (147bp) ds DNA, and a reconstituted nucleosome core particle (NCP). BAF-X-1 uniformly inhibits the ATPase activity of SMARCA2 assembled on different substrates. We show that SMARCA2 is essential for Ewing’s sarcoma (EwS) growth, and BAF-X-1 suppresses the proliferation of several pediatric cancer cells, including EwS and rhabdomyosarcoma (RMS) that rely upon SMARCA2 activity. We also conducted a single-mouse testing study to show that an oral administration of BAF-X-1 (50mg/kg, three times a week for four weeks) suppressed the tumor growth in a patient-derived xenograft (PDX) model of Ewing’s sarcoma. Mechanistically, BAFx1 perturbs the assembly of aberrant BAF complexes in EwS and RMS cells. We also filed a patent application on BAF-X-1 (PCT/US2023/010236), highlighting the potential of SMARCA2 inhibition as an effective strategy to treat tumors that harbor aberrant BAF assemblies.
Conclusion
Taken together, these data indicate that BAF-X-1 is a novel and excellent drug candidate with transformative potential for treating pediatric sarcomas. We are currently determining the impact of BAF-X-1 treatment on the integrity and activity of the BAF complexes to develop it as a novel drug for pediatric sarcoma therapy. Our ongoing structural, mechanistic, and animal studies will help develop BAF-X-1 as a more efficacious and less toxic therapy for treating pediatric sarcomas. Given the pro-tumorigenic role of BAF in other tumors, the outcomes of this study may have broader significance in cancer therapy.