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Embryonic lethality after combined inactivation of Fancd2 and Mlh1 in mice
Henri J van de Vrugt1, Laura Eaton, Amy Hanlon Newell
1Oregon Stem Cell Center, Oregon Health and Science University, Portland, Oregon, USA. henrivandevrugt@gmail.com
Abstract:
DNA repair defects are frequently encountered in human cancers. These defects are utilized by traditional therapeutics but also offer novel cancer treatment strategies based on synthetic lethality. To determine the consequences of combined Fanconi anemia (FA) and mismatch repair pathway inactivation, defects in Fancd2 and Mlh1 were combined in one mouse model. Fancd2/Mlh1 double-mutant embryos displayed growth retardation resulting in embryonic lethality and significant underrepresentation among progeny. Additional inactivation of Trp53 failed to improve the survival of Fancd2/Mlh1-deficient embryos. Mouse fibroblasts were obtained and challenged with cross-linking agents. Fancd2-deficient cells displayed the FA-characteristic growth inhibition after mitomycin C (MMC) exposure. In primary fibroblasts, the absence of Mlh1 did not greatly affect the MMC sensitivity of Fancd2-deficient and Fancd2-proficient cells. However, in Trp53 mutant immortalized fibroblasts, Mlh1 deficiency reduced the growth-inhibiting effect of MMC in Fancd2 mutant and complemented cells. Similar data were obtained using psoralen/UVA, signifying that MLH1 influences the cellular sensitivity to DNA interstrand cross-links. Next, the effect of MLH1 deficiency on the formation of chromosomal aberrations in response to cross-linking agents was determined. Surprisingly, Mlh1 mutant fibroblasts displayed a modest but noticeable decrease in induced chromosomal breakage and interchange frequencies, suggesting that MLH1 promotes interstrand cross-link repair catastrophe. In conclusion, the combined inactivation of Fancd2 and Mlh1 did not result in synthetic lethality at the cellular level. Although the absence of Fancd2 sensitized Mlh1/Trp53 mutant fibroblasts to MMC, the differential survival of primary and immortalized fibroblasts advocates against systemic inactivation of FANCD2 to enhance treatment of MLH1-deficient tumors.
Insights
Combining Fanconi anemia (FA) and mismatch repair (MMR) gene defects in mice did not cause synthetic lethality. MLH1 deficiency surprisingly reduced DNA damage-induced errors, suggesting FANCD2 inactivation is not a viable strategy for MMR-deficient tumors.
Area of Science:
- Genetics
- Molecular Biology
- Cancer Research
Background:
- DNA repair defects are common in human cancers and are targets for therapy.
- Synthetic lethality offers novel cancer treatment strategies by exploiting specific gene inactivation combinations.
- The interplay between Fanconi anemia (FA) and mismatch repair (MMR) pathways in cancer remains incompletely understood.
Purpose of the Study:
- To investigate the consequences of combined Fanconi anemia (FA) and mismatch repair (MMR) pathway inactivation.
- To determine if combined defects in FANCD2 and MLH1 lead to synthetic lethality.
- To assess the impact of MLH1 deficiency on cellular sensitivity to DNA cross-linking agents in the context of FANCD2 deficiency.
Main Methods:
- Generated a mouse model combining defects in Fancd2 and Mlh1 genes.
- Analyzed embryonic lethality and progeny underrepresentation in double-mutant mice.
- Cultured mouse fibroblasts (primary and immortalized) and challenged them with DNA cross-linking agents like mitomycin C (MMC) and psoralen/UVA.
- Assessed cell viability, growth inhibition, and chromosomal aberration frequencies.
Main Results:
- Combined Fancd2 and Mlh1 inactivation resulted in embryonic lethality, but not synthetic lethality at the cellular level.
- Fancd2-deficient cells showed characteristic growth inhibition upon MMC exposure.
- MLH1 deficiency modulated MMC sensitivity in immortalized fibroblasts but not significantly in primary cells.
- MLH1 deficiency led to a decrease in chromosomal aberrations, suggesting a role in promoting DNA interstrand cross-link repair catastrophe.
- Inactivating Trp53 did not rescue the embryonic lethality of Fancd2/Mlh1 double mutants.
Conclusions:
- Combined inactivation of FANCD2 and MLH1 does not induce synthetic lethality at the cellular level.
- MLH1 influences cellular sensitivity to DNA interstrand cross-links, and its deficiency may prevent catastrophic DNA repair responses.
- The differential effects in primary versus immortalized cells suggest that systemic inactivation of FANCD2 is not a recommended strategy to enhance treatment for MLH1-deficient tumors.
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