Functional Characterization of POLE1 Variant Fibroblasts Reveals Replication Stress and Increased Sensitivity to Genotoxic Stress

TL;DR

This study shows that fibroblasts with POLE1 variants are more sensitive to DNA-damaging stress and experience replication issues.

Contribution

First functional analysis of POLE1-variant fibroblasts from a compound-heterozygous patient, revealing replication stress and genotoxic sensitivity.

Findings

POLE1-variant fibroblasts show reduced clonogenic survival after ionizing radiation compared to healthy controls.

Increased chromosomal breakage and γH2AX foci suggest impaired DNA damage processing in these cells.

Cells exhibit delayed proliferation, S phase accumulation, and heightened senescence under genotoxic stress.

Abstract

Background/Objectives: DNA polymerase ε (Pol ε), encoded by POLE1, plays a pivotal role in high-fidelity DNA replication and in coordinating DNA repair. While pathogenic exonuclease-domain variants are well established in cancer, biallelic POLE1 variants remain largely unexplored in non-malignant human cells. Methods: Here, we analyzed primary fibroblasts derived from a skin biopsy of a compound-heterozygous patient carrying two POLE1 variants. Western blot analysis confirmed detectable Pol ε protein levels, indicating preserved protein expression despite the underlying variants. Results: Nevertheless, functional alterations were observed across multiple independent assays. Compared with healthy control fibroblasts, this patient-derived Pol ε fibroblast line exhibited reduced clonogenic survival following ionizing radiation. Surviving fractions were consistently lower across radiation doses from 2 to 4 Gy, with an approximately twofold reduction at 2 Gy and progressively greater differences at higher doses. The isoeffect dose corresponding to 10% survival was reduced relative to pooled control fibroblasts. In addition, chromosomal breakage was increased, supporting altered processing of radiation-induced DNA damage in this cellular model. Live-cell imaging and senescence assays revealed delayed proliferation and an increased proportion of senescent or senescence-like cells under baseline and genotoxic stress conditions, including enhanced senescence-associated β-galactosidase activity. Flow-cytometric analysis demonstrated S phase accumulation and G2/M arrest, consistent with replication stress and cell-cycle perturbation. Immunofluorescence staining revealed increased γH2AX foci, consistent with persistent DNA double strand breaks. RAD51 foci formation was not reduced; instead, increased RAD51 recruitment was observed under combined cisplatin and irradiation treatment, arguing against a primary defect in RAD51-mediated homologous recombination. POLE1-variant fibroblasts also showed impaired proliferative recovery, reduced wound closure, increased γH2AX accumulation following cisplatin exposure, suggesting heightened susceptibility to DNA crosslinking stress. Conclusions: Collectively, these findings provide the first functional characterization of a patient-derived POLE1-variant fibroblast cell line and indicate that altered Pol ε function may influence cellular responses to genotoxic stress. While based on primary fibroblasts from a single compound-heterozygous patient, validation in additional patient-derived or isogenic models will be required to determine the broader relevance of these findings.

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