Abstract A36: Validation of candidate cancer genes using the CRISPR system

Abstract

Abstract We have used Sleeping Beauty (SB) transposon mutagenesis to study the development of several types of sarcomas including osteosarcoma (OS) and malignant peripheral nerve sheath tumors (MPNST). This work has revealed the power of SB mutagenesis to identify critical genes and genetic pathways that, when altered, can cooperate to cause these tumors. We have discovered specific novel oncogenes, such as FOXR2 in MPNST and SEMA4D in OS, recurrent alterations in certain pathways, such as the Wnt/β−catenin pathway in MPNST, and information about what genetic alterations can cooperate in sarcomagenesis, such as loss of NF1 and PTEN in MPNST. We have functionally validated these genetic alterations in several ways, including the use of genetically engineered mouse (GEM) models and TAL effector nucleases (TALEN) or CRISPR RNA-guided Cas9 nuclease modified human cell lines. However, these studies typically analyzed only single candidates of great interest due to the alteration observed in comparative genomics analyses or novelty in the cancer field. In an attempt to further study our candidate cancer genes (CCGs) we have implemented the CRISPR nuclease system to perform a medium throughput screen of all candidate tumor suppressor genes (TSGs). To this end, we are generating and validating CRISPR gRNAs to target all TSGs identified in our MPNST and OS SB screens (~300 genes). Using immortalized human cell lines we have targeted these candidate TSGs and screened for their ability to increase colony formation in soft agar, migration, and xenograft tumor formation. We have identified numerous genes that when mutated by CRISPR resulted in increased tumorigenic properties. These genes are currently being tested for their ability to induce xenograft formation in immunocompromised mice. This CRISPR screen for MPNST and OS CCGs is ongoing and new results will be reported. Citation Format: Natalie Wolf, Branden Smeester, Madison Weg, David A. Largaespada, Branden Scott Moriarity. Validation of candidate cancer genes using the CRISPR system. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Pediatric Cancer Research: From Mechanisms and Models to Treatment and Survivorship; 2015 Nov 9-12; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(5 Suppl):Abstract nr A36.