Research Assistant The University of Texas at Dallas PLANO, Texas, United States
Introduction:: Solitary fibrous tumors (SFT) are rare, soft tissue tumors of mesenchymal origin that account for less than 2% of all soft tissue sarcomas. These tumors traditionally present unique diagnostic challenges because of the common gross and histologic features they express. In 2013, a defining discovery was made that all SFTs have a version of a hallmark intrachromosomal gene fusion between NAB2 and STAT6 on chromosome 12. Despite advances in the classification and understanding of the molecular mechanisms of SFTs, no systemic therapeutic options are yet available. New treatment regimens are needed, but a critical bottleneck in progress is the lack of clinically relevant models of SFTs. To address the bottleneck in progress in the field of SFTs, we used CRISPR-mediated knock-in technology and successfully established stable SFT cell models harboring the NAB2-STAT6 fusion types and provided results of the initial characterization assays.
Materials and Methods:: To generate our SFT cell models, two guide RNAs (gRNAs) were designed to target NAB2 and STAT6. Next, a homologous directed repair (HDR) template, containing the fusion mutation, was designed. Human colorectal carcinoma (HCT116) cells were transiently transfected with the CRISPR/Cas9 system, the gRNAs, and the HDR template. The resulting stable cell lines were then subjected to characterization assays. Long-range genomic PCR and RT-PCR were performed to validate the acquisition of the fusion mutation at both the DNA and RNA levels. The PCR products were subjected to gel electrophoresis and the bands of interest were purified and subjected to direct Sanger sequencing. Western blot was performed on whole cell lysates to confirm the expression of NAB2-STAT6 fusion proteins in the stable SFT cell lines.
Results, Conclusions, and Discussions:: Using CRISPR-mediated knock-in technology, we have successfully established stable SFT cell models harboring the NAB2-STAT6 fusion types while preserving all original NAB2-STAT6 gene fusion information, including endogenous NAB2 promoters, 5’-UTRs of NAB2, and 3’-UTRs of STAT6. We have validated the acquisition of the NAB2-STAT6 fusion mutation at the DNA, RNA, and protein level with long-range genomic PCR, RT-PCR, and western blot, respectively. We have also developed CasRx-based constructs packaged into AAV viral vectors that exhibit dose-dependent suppression of specific NAB2-STAT6 fusion transcripts in our SFT cell model. With our SFT model, the CasRx-based constructs efficiently and specifically suppressed the NAB2-STAT6 fusion transcript in vitro and suppressed SFT tumor growth ex vivo. Our work highlights the use of CRISPR/Cas systems to generate SFT cell models, provides evidence of the efficiency of RNA-based therapy in vitro and ex vivo, and aims to accelerate solitary fibrous tumor research. Further investigations are needed to evaluate their in vivo efficacies and safety before being translated into clinical practice. This work could also be applied to other gene fusion-related diseases.
Acknowledgements (Optional): : We thank the laboratory members in the Bleris and Hayenga labs for their support and discussions.
