Investigating the role of BRCA2 in the development of pediatric rhabdomyosarcoma

Introduction: In a large-scale exome sequencing study of pediatric rhabdomyosarcoma (RMS) patients (n=615), we identified a statistical enrichment of patients with germline pathogenic variants in BRCA2 (Li et al., JNCI, 2020) . However, paired tumor-normal investigations of pediatric cancers have demonstrated that pediatric tumors do not typically exhibit somatic loss of BRCA2 , a departure from what is typical of adult cancers which result in profound homologous recombination (HR) deficiency. These findings prompted our multi-pronged study to assess the role of heterozygous BRCA2 variants in the development of pediatric RMS. Methods: All patient samples were obtained from ongoing Children's Oncology Group protocols. As part of the Gabriella Miller Kids First Program, paired tumor-normal whole genome sequencing and somatic RNA sequencing of RMS patients was conducted at HudsonAlpha. This included a subset of the initially detected six patients with germline BRCA2 variants. As a potential precursor model for RMS, we engineered BRCA2 loss-of-function (LOF) variants in immortalized mesenchymal stem cell (MSC) lines. We used CRISPR to induce frameshift alleles in exons 10 and 11 (c.1345dup and c.5680del) and prime editing to create two RMS patient-specific LOF variants in exons 5 and 17 (c.7857G>A and c.462_463del), followed by DNA repair and genomic stability assays of the recombinant cell lines. Results: Evaluation of three RMS tumors from patients with germline BRCA2 variants was negative for loss-of-heterozygosity at BRCA2 , additional somatic BRCA2 mutations, or decrease in BRCA2 expression. However, these tumors showed evidence of modest decrease in homologous recombination (HR) relative to other age-matched RMS tumors (n=21), measured via the DirectHRD pipeline. The BRCA2 heterozygous MSC cell lines demonstrate equivalent overall DNA double-strand break repair capacity but significantly diminished HR activity when assayed by integrating larger (700bp) repair cassettes, and increased micronucleus formation after treatment with ionizing radiation. Despite these functional impairments, we found no change in response to targeted therapies, including the PARP inhibitor olaparib and newer generation polymerase theta inhibitors. Conclusions: Through RMS tumor analysis and in vitro assays, we observed modest HR impairment resulting from heterozygous BRCA2 mutations, but to a lesser extent than seen in adult cancers with complete loss of BRCA2 and PARP sensitivity. These findings support a model where heterozygous germline variants in BRCA2 modestly increase genomic instability, leading to an increased risk of pediatric RMS and may explain other pediatric tumors where enrichment of BRCA2 germline variants has been described.