Abstract 5610: Investigating the role of BRCA2 in the development of pediatric rhabdomyosarcoma
Authors: Weinstein P. Adam , Zabriskie Ryan , Ozdemir Cem , Miller Kyle M. , Medina-Martinez Olga , Heaney Jason D. , Sabo Aniko , Lupo Philip J. , Plon Sharon E. 
Published: March 2024

Introduction: In a large-scale exome sequencing study of pediatric rhabdomyosarcoma (RMS) patients, we identified a statistical enrichment of patients with germline variants in BRCA2 (Li, et al. JNCI, 2020), which prompted this study to assess the role of heterozygous BRCA2 variants in the development of pediatric RMS in both precursor cell and mouse models. Methods: We engineered BRCA2 loss of function (LoF) variants in lines of immortalized mesenchymal stem cells, a potential precursor cell to RMS, using CRISPR and Prime Editing (PE). CRISPR was used to induce frameshift alleles in Exons 10 and 11 and with PE, we created two RMS patient specific LOF variants in Exons 5 and 17. We generated a genetically engineered mouse model (GEMM) with heterozygous excision of Exon 11 of Brca2. This allele is now being crossed into two different previously established GEMMs of RMS: a myoblast-specific (MyoD1-Cre) conditional loss of p53, and a gain-of-function mutant p53, LSL-p53R172H (Nakahata, et al. Dis. Model. Mech., 2022), to assess changes in the pace of tumorigenesis and metastatic potential. Through an X01 agreement with the Gabriella Miller Kids First Program, we have submitted paired tumor-normal samples for additional whole-genome sequencing and RNA sequencing (tumor only) on &gt;500 patients with RMS, including those with germline variants in BRCA2. Results: Using CRISPR, we generated cell lines heterozygous for the frameshift variants c.1345dup, c.5680del, and c.5680dup. With Prime Editing, we created heterozygous lines mimicking two specific variants detected in the RMS cohort, c.462_463del and c.7857G&gt;A. Generation of a C57BL6 GEMM with a heterozygous removal of Exon 11 in Brca2 has been successful. No homozygous mice were obtained following 7 heterozygous crosses (50 pups), a departure from Mendelian ratios (p=0.0001) consistent with embryonic lethality. Heterozygous Brca2 mice derived from crosses into two RMS models are being monitored for tumor development. We have completed tumor-normal whole-genome and tumor RNA sequencing for 609 RMS patients, including 228 subjects not previously analyzed. We will directly assess whether there is evidence for mutation or loss of expression of the remaining BRCA2 allele in tumors and whether these tumor genomes exhibit signatures of DNA repair deficiency. Conclusions: The goal of this project is to determine the molecular basis for the enrichment in germline BRCA2 LOF variants in pediatric malignancies, including RMS. Ongoing experiments using the engineered heterozygous BRCA2 in vitro cell line models will assess genomic stability, homologous recombination proficiency, and DNA replication fork stability. Assessment of the effect of heterozygous Brca2 variants in GEMMs of RMS, and subsequent analysis of additional sequencing data, will further elucidate what role this gene may play in increasing risk or progression of pediatric cancers.

Citation Format: P. Adam Weinstein, Ryan Zabriskie, Cem Ozdemir, Kyle M. Miller, Olga Medina-Martinez, Jason D. Heaney, Aniko Sabo, Philip J. Lupo, Sharon E. Plon. Investigating the role of BRCA2 in the development of pediatric rhabdomyosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5610.