Cellular consequences of a germline <i>SMC5</i> variant enriched in medulloblastoma.

e14112 Background: Pediatric brain tumors are the second most common pediatric malignancy and are associated with a germline risk of approximately 15%. Medulloblastoma is a high-grade pediatric brain tumor with four subtypes, of which group 3 portends the worst outcomes and has the least molecular determinants identified. To this end, our work is the first to report the putative germline association of SMC5 variants in children with group 3 medulloblastoma in a large pediatric cancer predisposition study. SMC5 , encoding Structural Maintenance of Chromosome 5 (SMC5), is a core component of the SMC5/6 complex, essential for DNA replication, DNA damage repair, cell-cycle regulation, and chromosome maintenance. Tumor data show the occurrence of a second somatic hit in SMC5 with a tumor DNA mutational signature of late replication error, implicating the pathogenicity of SMC5 in medulloblastoma. Although germline variants in SMC5 have not been linked to cancer predisposition, biallelic alteration of SMC5 causes a neurodevelopmental disorder, Atelis Syndrome-2. In addition, somatic mutations in SMC5 have commonly been observed in adult cancers. These data suggest an important role of SMC5 in neurodevelopment and cancer biology, which is understudied and not well understood. Our study aims to elucidate the role of SMC5 in brain tumor biology by leveraging disease-relevant SMC5 mutations with the potential to shed light on the DNA repair vulnerability of these tumors. Methods: We assessed the function of SMC5 using the disease-relevant N940Y mutation in an iPSC and transiently overexpressed FLAG-tagged SMC5 wild-type and N940Y models. Baseline protein expression, cell cycle analysis, SMC5/6 complex integrity, and DNA damage signaling were assessed in wild-type and N940Y mutant models using molecular, immunofluorescence, and immunoprecipitation–mass spectrometry assays (IP-MS). Results: N940 SMC5 demonstrated unchanged protein expression compared to wild-type in the overexpression model. EdU-based flow cytometric analysis of cell cycle in isogenic and N940Y SMC5 iPSCs revealed no significant differences in cell-cycle distribution at baseline. To determine whether the N940Y mutant disrupts the formation of the SMC5/6 complex, which comprises eight proteins, we performed IP-MS and observed preserved complex formation in the N940Y mutant compared with wild-type. We next tested the effect of SMC5 dysfunction on DNA damage by immunofluorescence and demonstrated reduced 53BP1 foci in N940Y mutant iPSCs compared with wild-type iPSCs, suggesting that N940Y is unable to trigger DNA damage signaling. Conclusions: These data suggest that SMC5 N940Y preserves SMC5/6 complex integrity and cell cycle progression but impairs DNA damage signaling, warranting further investigation into the role of SMC5 in pediatric brain tumor biology.