Reduction of Tumor Growth with RNA-Targeting Treatment of the NAB2–STAT6 Fusion Transcript in Solitary Fibrous Tumor Models

Abstract

Solitary fibrous tumor (SFT) is a rare soft-tissue sarcoma. This nonhereditary cancer is the result of an environmental intrachromosomal gene fusion between NAB2 and STAT6 on chromosome 12, which fuses the activation domain of STAT6 with the repression domain of NAB2. Currently there is not an approved chemotherapy regimen for SFTs. The best response on available pharmaceuticals is a partial response or stable disease for several months. The purpose of this study is to investigate the potential of RNA-based therapies for the treatment of SFTs. Specifically, in vitro SFT cell models were engineered to harbor the characteristic NAB2–STAT6 fusion using the CRISPR/SpCas9 system. Cell migration as well as multiple cancer-related signaling pathways were increased in the engineered cells as compared to the fusion-absent parent cells. The SFT cell models were then used for evaluating the targeting efficacies of NAB2–STAT6 fusion-specific antisense oligonucleotides (ASOs) and CRISPR/CasRx systems. Our results showed that fusion specific ASO treatments caused a 58% reduction in expression of fusion transcripts and a 22% reduction in cell proliferation after 72 h in vitro. Similarly, the AAV2-mediated CRISPR/CasRx system led to a 59% reduction in fusion transcript expressions in vitro, and a 55% reduction in xenograft growth after 29 days ex vivo.

STAT6-targeting antisense oligonucleotides against solitary fibrous tumor

“…We previously established an SFT cell line (NS-poly) by editing the NAB2exon6-STAT6exon17 fusion into human colon cancer cells (HCT116) 30 using the CRISPR-SpCas9 system. 31 , 32 , 33 , 34 , 35 , 36 However, SFTs are believed to originate from mesenchymal stem cells with fibroblastic differentiation.…”

“…To address this concern, for each variant of the NAB2-STAT6 fusion, an alternative approach could involve the design of ASOs specifically aimed at the fusion junctions. For example, in our recent publication, 30 we developed an ASO tailored to target specifically the NAB2exon6-STAT6exon17 fusion junction that, when delivered using RNAiMAX, achieved a 58% suppression of expression of NAB2-STAT6 transcript at 1 μM within 48 h. Nevertheless, it should be noted that STAT6-targeting ASO 993523 showed even greater targeting efficiency, particularly when a gymnotic delivery method was adopted. Taken together, these results shed light on the delicate balance between two competing aspects: (a) specificity for the fusion transcript to prevent potential off-target effects and (b) efficacy to ensure a therapeutic benefit.…”

NAB2-STAT6 drives an EGR1-dependent neuroendocrine program in Solitary Fibrous Tumors

“…Detection of NAB2-STAT6 protein products has become the primary diagnostic tool for this rare tumor type, however the role of the fusion protein in the etiology of SFTs has remained elusive (8). A variety of mechanisms have been proposed for NAB2-STAT6's function, including activation of STAT6 targets and conversion of NAB2 from a repressor to an activator (6,34,39,40,50). Here we show that NAB2-STAT6 activates an EGR1-driven neuroendocrine gene expression signature by translocating EGR1, NAB1, and wild type NAB2 to the nucleus (Fig.…”

“…SFTs are presumed to be mesenchymal in origin, therefore we used the osteosarcoma-derived U2OS cell line to generate an inducible model of NAB2-STAT6 expression and study the early transcriptional events driven by the fusion protein. Previous work on NAB2-STAT6 relied on overexpression in bulk cell populations that were primarily of non-mesenchymal origin, potentially limiting their utility (6,34,39,40). We generated a single cell derived clone expressing the most common isoform of NAB2-STAT6 (exons 1-4 of NAB2 and exons 2-22 of STAT6) under the control of a doxycycline inducible promoter (tet-ON).…”

“…The determinants of NAB2-STAT6 occupancy genome-wide have not been previously investigated, nor have the epigenetic and transcriptional consequences of the fusion protein been identified (6,34,39,40). Here, we employ functional genomics and proteomics strategies to demonstrate that NAB2-STAT6 drives a EGR1dependent neuroendocrine transcriptional program via epigenetic activation of a network of distal enhancers and proximal promoters that is normally operating in neuronal cells.…”