Abstract LB060: Single-cell CRISPR screen of malignant peripheral nerve sheath tumors (MPNST)

Abstract

Abstract Background: Malignant peripheral nerve sheath tumors (MPNST) are aggressive sarcomas of the peripheral nervous system. Loss of function of the polycomb repressive complex 2 (PRC2) have been identified in up to 80% of MPNST cases, via recurrent genetic mutations in the core components, EED and/or SUZ12. However, the transcriptional mis-regulation incurred by the loss of transcriptional repression in MPNST, through loss of H3K27me3, has yet to be fully deciphered. We hypothesize that PRC2 loss leads to transcriptional regulatory imbalance between key epigenetic regulating complexes in this malignancy, which may contribute to the oncogenesis of this disease. Therefore, this project aims to elucidate the role of the SWI/SNF chromatin remodeling complexes in MPNST, epigenetic regulators known to act antagonistically to PRC2. Method: This project uses a CRISPR knock out screen combined with a single cell RNA sequencing readout to target 44 known components of the SWI/SNF complexes. This technology enables large scale phenotypic assays, which we leverage to decipher the complicated epigenetic regulatory network within these lesions. Resulting genes of interest were further studied via loss-of-function assays coupled with colony formation assay and chromatin immunoprecipitation DNA sequencing (ChIP-seq) experiments. Results: This CRISPR screen highlighted core SWI/SNF components, SMARCA4 and SMARCE1, as genes of interest in MPNST. Knockdown of these genes in MPNST cell lines reduced proliferation, indicating a role for these genes in MPNST growth and viability. Further, investigations via soft agar assay found both SMARCA4 and SMARCE1 knock out to decrease the tumorigenic potential of MPNST cells. The potential dependency of SMARCA4 function in MPNST cells on PRC2 mutation was investigated using a doxycycline-inducible PRC2-restoration MPNST cell line model. ChIP-seq results suggest that loss of PRC2 does not substantially affect the binding of SMARCA4-containing SWI/SNF complexes across the genome. DPF1, a member of the neuronal-specific SWI/SNF chromatin remodeling complex, was further highlighted as a unique regulator of MPNST transcription via the CRISPR single-cell screen. CRISPR knockdown of this gene in MPNST cell lines has been found to decrease proliferation, while soft agar assay experiments highlighted that DPF1 loss reduces cell tumorigenicity. The unique dependency of this gene observed in MPNST but not in any other cancer subtypes highlights the potential of DPF1 to be a novel and unique therapeutic target in MPNST. Conclusion: Thus far, our studies have shown that members of the SWI/SNF complex play key roles in the MPNST cell proliferation and tumorigenicity. The function of these complexes in MPNST, however, may be independent of the transcriptional imbalance caused by PRC2 mutation. Citation Format: Béga Murray, Xiyuan Zhang, Shahroze Abbas, Hilda Jafarah, Jack F. Shern. Single-cell CRISPR screen of malignant peripheral nerve sheath tumors (MPNST) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB060.