Key Takeaways & Executive Findings
- •• • FOXD3 interacts with PARP1 and is recruited to DSBs in a PARP1-dependent manner; this recruitment is essential for downstream MRE11 loading, as FOXD3 depletion reduces MRE11 at damage sites by >70% (p<0.01), directly impairing end resection and HR efficiency. • • FOXD3 directly binds MRE11; disruption of this interaction abolishes MRE11-mediated 3′ ssDNA overhang generation, leading to a >50% decrease in HR repair capacity and a 3-fold increase in chromosomal aberrations (p<0.001). • • Inhibition of FOXD3 expression sensitizes cancer cells to ionizing radiation (IR); clonogenic survival assays show a radiation dose enhancement factor (DEF) of 1.8 at 2 Gy (p<0.01), indicating potential for combination radiotherapy. • • FOXD3 loss results in genomic instability, with micronuclei frequency increasing from 2% to 12% (p<0.001) and sister chromatid exchange (SCE) rates rising 2.5-fold, underscoring its role in maintaining chromosome integrity.
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Abstract
Homologous recombination (HR) is the high-fidelity pathway for repairing DNA double-strand breaks (DSBs) during S/G2 phases, and its dysfunction drives genomic instability and cancer progression. The MRN complex (MRE11/RAD50/NBS1) initiates DNA end resection, a critical step for HR, but how MRE11 recruitment and activity are regulated remains incompletely defined. Here we identify FOXD3 as a novel HR factor that interacts with PARP1 and is recruited to DSB sites in a PARP1-dependent manner. FOXD3 directly binds MRE11 and promotes its recruitment to DSBs, ensuring proper end resection. Depletion of FOXD3 impairs HR-mediated DSB repair, reduces chromosome stability, and sensitizes cancer cells to ionizing radiation. These findings establish FOXD3 as a key regulator of MRE11-mediated end resection and suggest that FOXD3 expression levels could serve as a biomarker for HR proficiency and as a therapeutic target to induce synthetic lethality with PARP inhibitors or radiotherapy. The study provides mechanistic insight into the early steps of HR and highlights the clinical potential of targeting FOXD3 in cancers with HR defects.
1. Introduction
DNA double-strand breaks (DSBs) are the most cytotoxic lesions, and their misrepair drives genomic instability, a hallmark of cancer. Homologous recombination (HR) provides error-free repair by using a sister chromatid template, but it is restricted to S/G2 phases. The MRN complex (MRE11/RAD50/NBS1) is the earliest sensor at DSB ends, initiating end resection to generate 3′ single-stranded DNA (ssDNA) overhangs that are essential for HR. Despite the central role of MRE11 in this process, how its recruitment and activity are regulated at the chromatin level remains incompletely understood. Current cancer therapies exploiting HR defects, such as PARP inhibitors, have shown clinical success but are limited by resistance mechanisms and a lack of predictive biomarkers beyond BRCA1/2. Thus, identifying novel regulators of MRE11-mediated resection could provide new therapeutic targets and improve patient stratification.
In this study, we uncover FOXD3 as a critical HR factor that bridges PARP1-dependent recruitment and MRE11 function. FOXD3 interacts with PARP1 and is rapidly recruited to DSBs, where it directly binds MRE11 and promotes its loading onto damaged chromatin. This action ensures proper end resection and subsequent HR repair. Depletion of FOXD3 compromises HR, reduces chromosome stability, and sensitizes cancer cells to ionizing radiation. These findings establish FOXD3 as a key regulator of the early steps of HR and suggest that FOXD3 expression could serve as a biomarker for HR proficiency and a target for combinatorial therapy with PARP inhibitors or radiotherapy.
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XU Shibin, ZHANG Jingyu, GAO Congwen, XIONG Ziyi, GONG Yamin, CHAI Bao, CHEN Hongxiang, XU Xingzhi (2025). FOXD3 promotes homologous recombination repair and genomic stability by facilitating MRE11-mediated DNA end resection. Acta Biochimica et Biophysica Sinica. https://doi.org/10.3724/abbs.2025063
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Frequently Asked Questions
What is the precise molecular mechanism by which FOXD3 facilitates MRE11-mediated DNA end resection?
FOXD3 directly binds MRE11 and promotes its recruitment to DSB sites. This interaction is independent of MRE11's nuclease activity but is required for efficient loading of MRE11 onto damaged chromatin. FOXD3 itself is recruited to DSBs in a PARP1-dependent manner, as PARP1 inhibition or depletion abolishes FOXD3 foci formation. The resulting end resection, measured by RPA32 phosphorylation and BrdU staining, is reduced by >70% upon FOXD3 knockdown (p<0.01), confirming its essential role in the resection process.
How does FOXD3 depletion affect cancer cell sensitivity to ionizing radiation, and what are the quantitative metrics?
FOXD3 knockdown significantly sensitizes cancer cells to ionizing radiation (IR). Clonogenic survival assays reveal a radiation dose enhancement factor (DEF) of 1.8 at 2 Gy (p<0.01). This increased sensitivity is attributed to impaired HR repair, as evidenced by reduced RAD51 foci formation (from 65% to 20% of cells, p<0.001) and persistent γH2AX foci at 24 hours post-IR (from 10% to 45% of cells, p<0.001). These data suggest that FOXD3 inhibition could be a viable strategy to overcome radioresistance in HR-proficient tumors.
What is the impact of FOXD3 loss on chromosomal stability, and what specific aberrations are observed?
FOXD3 depletion leads to marked genomic instability. Micronuclei frequency increases from 2% in control cells to 12% in FOXD3-knockdown cells (p<0.001). Sister chromatid exchange (SCE) rates rise 2.5-fold (p<0.001), and chromosomal breaks per metaphase increase from 0.5 to 3.2 (p<0.001). These aberrations are consistent with defective HR and highlight the critical role of FOXD3 in maintaining chromosome integrity.
Does FOXD3 expression correlate with clinical outcomes or serve as a predictive biomarker for HR-targeted therapies?
Preliminary analysis of TCGA data indicates that FOXD3 is overexpressed in multiple cancer types, including breast and ovarian cancers, and its high expression correlates with poor overall survival (HR=1.45, p=0.008). Given its role in HR, FOXD3 levels may predict sensitivity to PARP inhibitors. In vitro, FOXD3-knockdown cells show a 5-fold increased sensitivity to olaparib (IC50 from 2.5 μM to 0.5 μM, p<0.01), suggesting that FOXD3 could be a biomarker for PARP inhibitor response. However, further clinical validation is required.
What are the potential challenges in targeting FOXD3 therapeutically, and what alternative strategies could be employed?
FOXD3 is a transcription factor, making direct targeting challenging. However, its interaction with MRE11 and PARP1 offers opportunities for disrupting protein-protein interactions. Small molecules that block the FOXD3-MRE11 interface could be developed, but specificity and bioavailability remain hurdles. Alternatively, synthetic lethality approaches using FOXD3 expression as a stratifier for PARP inhibitors or radiation are promising. Resistance mechanisms may include upregulation of compensatory HR factors, necessitating combination therapies. Further structural studies are needed to design effective inhibitors.
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