The advancement of CRISPR mediated gene engineering provides an opportunity to improve upon preclinical human cell line models of cancer predisposing syndromes. This review focuses on using CRISPR/Cas9 genome editing tools to model various human cancer predisposition syndromes. We examine the genetic mutations associated with neurofibromatosis type 1, Li‐Fraumeni syndrome, Gorlin syndrome, BRCA mutant breast and ovarian cancers, and APC mutant cancers. Furthermore, we discuss the possibilities of using next‐generation CRISPR‐derived precision gene editing tools to introduce a variety of genetic lesions into human cell lines. The goal is to improve the quality of preclinical models surrounding these cancer predisposition syndromes through dissecting the effects of these mutations on the development of cancer and to provide new insights into the underlying mechanisms of these cancer predisposition syndromes. These studies demonstrate the continued utility and improvement of CRISPR/Cas9‐induced human cell line models in studying the genetic basis of cancer.

The genetic tumor predisposition syndrome Neurofibromatosis type 1 (NF1) results from the inheritance of a mutant copy of NF1, a RAS-GAP tumor suppressor gene. Subsequent loss of the remaining wild-type NF1 allele in Schwann lineage cells of the peripheral nervous system leads to complete functional loss of the encoded protein, neurofibromin. Schwann cells lacking neurofibromin exhibit hyperactive RAS signaling, and contribute to the formation of benign plexiform neurofibromas (PNFs). Through additional mutations in tumor suppressor genes CDKN2A/CDKN2B, these PNFs may escape senescence and progress to atypical neurofibromatous neoplasms of uncertain biological potential (ANNUBP). Furthermore, loss of function mutations in Polycomb Repressive Complex 2 (PRC2) have been strongly implicated in the progression of ANNUBPs to lethal malignant peripheral nerve sheath tumors (MPNSTs). Although these alterations have been associated with MPNST formation, their temporal dependence during Schwann cell development and contribution to malignant transformation using a human cell model has not been studied. We hypothesize that sequential loss of NF1, CDKN2A, and finally SUZ12 in Schwann cells transforms them into MPNST, and this process is possible to study using a human induced pluripotent stem cell (iPSC) model system. To test our hypothesis, we generated Schwann lineage cells from commercially available iPSCs. Cells were subjected to targeted mutations using CRISPR/Cas9 ribonucleoproteins at different stages of development to mimic potential tumorigenesis. Our preliminary results show that multiple iPSC donor lines can reliably be differentiated to Neural Crest Cells (NCCs), and further to mature Schwann cells. We have edited multiple target loci at high levels (>70%) throughout Schwann cell development. NCCs lacking neurofibromin showed an increase in proliferation and migration. While iPSC-derived Schwann cells (iSCs) lacking NF1 and CDKN2A with or without SUZ12 loss became senescent after several passages. These mutant iSCs were unable to form tumors when subcutaneously injected into the flank of NRG mice, while commonly utilized immortalized human Schwann cell lines lacking NF1 were able to form large subcutaneous tumors. Further refinement of this induced-MPNST (iMPNST) model through targeting of CDKN2B, ATRX, or TP53 may allow for better understanding of the order and timing at which these transforming mutations are sufficient to result in MPNST formation. Additionally, the application of single-cell RNA sequencing technology to this system could uncover potential heterogeneity that could harbor a clone primed for malignant transformation. This model could aid in uncovering novel genetic mutations and pathways which contribute to transformation of benign PNFs to MPNSTs, providing additional targets for future therapeutic intervention. Citation Format: Garrett M. Draper, Daniel Panken, Mahathi Patchava, Wendy Hudson, Kyle B. Williams, David A. Largaespada. Developing a novel model of neurofibromatosis type 1 associated malignant peripheral nerve sheath tumors using induced pluripotent stem cells [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 1615.