Abstract B024: Using CRISPR-Cas9 to understand the role of SNAI2 in malignant peripheral nerve sheath tumors (MPNST) cell lines


 Background: MPNST are highly aggressive soft tissue sarcomas. Various recurrent genetic mutations have been linked to the malignant transformation from neoplastic Schwann cells to MPNST, including the loss of SUZ12 and/or EED, which are core components of the polycomb repressive complex 2 (PRC2). This results in a heavily altered transcription landscape characterized by global loss of methylation on histone H3 lysine 27 (H3K27me3) and increased super enhancer (SE) -associated histone acetylation. We have previously identified SNAI2 as a core transcription factor (TF) driven by H3K27ac-decorated SE and is essential for the growth of MPNST. Studies by other have shown that SNAI2 is critical for biological processes including epithelial to mesenchymal transition, metastasis, radioprotection, dedifferentiation, and cancer stem cell maintenance in other cancers. Its function in maintaining MPNST survival through potential transcriptomic and epigenetic regulation has not been explored. Methods: Using CRISPR/Cas9-mediated genetic knockout and CRISPR/dead-Cas9-mediated epigenetic silencing (CRISPRi), we targeted either the SNAI2 DNA binding domain in exon 2 or the enhancer regions upstream of the gene. Knockout of SNAI2 was then confirmed using western blots, followed by phenotypic characterization through in vitro studies, such as 2-dimensional growth assays, anchorage independent colony formation assay, and scratch assay. Finally, an integrative analysis of SNAI2 genomic distribution and the transcriptomic effect of its knockout was performed using data from chromatin immunoprecipitation coupled with DNA sequencing (ChIP-seq) and RNAseq. The data was analyzed using packages in R to understand the deviation of the transcriptomic profile after SNAI2 knockout. Results: Western blots showed that SNAI2 was successfully knocked out using the CRISPR/Cas9 system. In contrast, epigenetic targeting through CRISPRi did not efficaciously repress the gene. Loss of SNAI2 slowed the growth of MPNST cells most significantly after passaging the cells in comparison to control. In vitro tumorigenic assays revealed that SNAI2 mostly regulated the cells’ capacity in invasion, but it was not significantly involved in the anchorage-independent cell growth. Finally, preliminary analysis of the RNAseq and ChIP-seq data demonstrated that SNAI2 binds to various promoter regions, and the knockout of SNAI2 resulted in both the downregulation and upregulation of its target genes. One example is the downregulation of CRELD1, a gene involved in cell adhesion, due to the loss of SNAI2 binding to its promoter (p-value <0.001. Conclusion: SNAI2 knockout using CRISPR/Cas9 slows the growth and invasion of MPNST cells; however, it does not affect the cells’ ability of colony formation. These phenotypic effects are accompanied by an altered transcriptomic profile through the loss of SNAI2 binding to the genome. Future efforts will focus on designing methods to employ targeted degradation of this essential TF and test preclinical efficacy in treating MPNST.
 Citation Format: Hilda M. Jafarah, Xiyuan Zhang, Bega Murray, Jack Shern. Using CRISPR-Cas9 to understand the role of SNAI2 in malignant peripheral nerve sheath tumors (MPNST) cell lines. [abstract]. In: Proceedings of the AACR Special Conference: Cancer Epigenomics; 2022 Oct 6-8; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_2):Abstract nr B024.