2574 - Mutational Landscape of MLH1-Deficient Colorectal Tissues and Impact of Ionizing Radiation: 3D Organoid-Based Whole Genome Sequencing Analysis
Presenter(s)
J. B. Park1, J. Lee2, K. S. Kim3, M. J. Kim4, J. Youk5, T. G. Son6, S. J. Choi6, and E. K. Chie3; 1Department of Radiation Oncology, Seoul National University College of Medicine, Seoul, Korea, Republic of (South), 2Seoul National University Cancer Research Institute, Seoul, Korea, Republic of (South), 3Department of Radiation Oncology, Seoul National University Hospital, Seoul National University College of Medicine, Seoul, Korea, Republic of (South), 4Department of Radiation Oncology, Seoul National University Hospital, Seoul, Korea, Republic of (South), 5Department of Internal Medicine, Seoul National University Hospital, Seoul, Korea, Republic of (South), 6Research Center, Dongnam Institute of Radiological and Medical Science, Busan, Korea, Republic of (South)
Purpose/Objective(s):
Colorectal cancers in patients with Lynch syndrome (LS) are characterized by high microsatellite instability (MSI) due to mismatch repair (MMR) deficiency, most commonly caused by MLH1 mutations. Ionizing radiation (IR), which plays an important role in colorectal cancer treatment, induces DNA double-strand breaks but may also contribute to radiation-induced carcinogenesis in normal tissues. However, the mechanisms of tumorigenesis in the context of MLH1 mutations with IR exposure remain poorly understood. This study aimed to investigate the effects of MLH1 mutations and IR exposure on normal colorectal tissues using murine 3D organoid models and whole-genome sequencing (WGS).Materials/Methods:
Organoid cultures were clonally expanded from single cells derived from the lower gastrointestinal tracts of wild-type (Mlh1+/+; WT), heterozygous (Mlh1+/-; HE), and homozygous (Mlh1-/-; HM) knockout mice. WGS and mutational signature profiling were performed to analyze mutational burden and associated signatures. MSI testing was performed to evaluate MMR status. Organoids were exposed to gamma irradiation at doses of 0, 2, 4, and 8 Gy, and viability assays were conducted to assess radiation sensitivity and dose-response relationships. Differences in continuous variables were analyzed using the Wilcoxon rank-sum test, with p < 0.05 considered statistically significant.Results:
HM mice exhibited a marked increase in mutational burden, with 5.90-fold higher single-nucleotide variants (SNVs), 74.67-fold higher insertion–deletion mutations (indels), with exclusive enrichment of SBS15, an MMR deficiency-associated mutational signature. In contrast, neither WT nor HE mice demonstrated MMR-deficient mutational patterns or elevated MSI rates. Age-related accumulation of mutations and MSI was evident only in HM mice, with a 1.38-fold increase from 6-weeks- to 6-months-old samples. IR exposure did not significantly increase SNVs, MMR-related signatures, or MSI across all genotypes (all p > .05). Although cell viability decreased in a dose-dependent manner after IR exposure, radiation dose was not associated with increased mutational burden or altered profiles. HE mice showed no significant differences from WT in mutational patterns, MSI, or radiosensitivity, indicating that heterozygous MLH1 mutation alone maintains a wild-type phenotype under baseline and irradiated conditions.Conclusion:
Homozygous MLH1 loss induces distinct MMR-deficient mutational signatures and elevated MSI. In contrast, heterozygous MLH1 mutations do not result in MMR-related mutational features or radiosensitization in normal colorectal tissues. IR exposure did not significantly exacerbate mutational burden in any genotype. These findings suggest that MLH1 heterozygosity alone may not increase susceptibility to radiation-induced carcinogenesis in normal colorectal epithelium, with potential implications for risk assessment and treatment planning in LS patients.