Elucidation of molecular mechanisms of cytotoxicity resistance in nucleoside analog "alovudine"

A research group led by Professor Kouji Hirota, Graduate Student Md Bayejid Hosen, and Assistant Professor Ryotaro Kawasumi from the Graduate School of Science at Tokyo Metropolitan University announced that they have elucidated the molecular mechanisms by which human cells prevent the cytotoxicity of "alovudine," one of the nucleoside analogs. They found that the nuclease "FEN1," which contributes to DNA replication, removes incorporated alovudine, thereby reducing cytotoxicity. This discovery is expected to lead to applications in anticancer drug development through new mechanisms. The results were published in Nucleic Acids Research on July 18.

Nucleoside analogs are a general term for chemical substances that closely resemble nucleosides, which are the building blocks of DNA. They have been used since the 1980s for treating viral infections such as HIV and cancers such as leukemia. Because they closely resemble DNA building blocks, they are incorporated into newly synthesized genomic DNA during DNA replication, inhibiting the replication reaction. Nucleoside analogs more strongly suppress replication reactions of viruses and cancer cells with higher replication frequencies and lower reaction accuracy, making them useful for these treatments. However, their detailed molecular mechanisms were not understood.

Hirota and his team have been researching molecular mechanisms for preventing the cytotoxicity of various nucleoside analogs. Through their research, they discovered that different nucleoside analogs each exhibit completely different cytotoxicity, and that the DNA repair factors required to reduce this toxicity differ. They are aiming for next-generation therapeutic applications that specifically "target" DNA repair factor mutations found in cancers.

In this study, the research group used mutants of 24 genes involved in DNA repair to investigate how much alovudine suppresses cell proliferation.

As a result, they found that mutant cells of the enzyme gene "FEN1" that cleaves DNA in DNA replication reactions (- i.e., an enzyme that cuts flap structures at the ends of each newly synthesized strand during lagging strand synthesis, promoting efficient ligation) show high cytotoxicity to alovudine. Furthermore, in cells lacking the FEN1 gene, replication speed was significantly reduced after alovudine treatment.

However, this replication speed delay was almost restored to normal cell levels by simultaneous deficiency of the 53BP1 gene, which codes for a DNA repair enzyme. It was found that in FEN1-deficient cells, alovudine abnormally accumulates in the genome during replication, causing abnormal accumulation of 53BP1, which leads to DNA damage. Last year, the research group clarified that cellular resistance to alovudine toxicity involves the BRCA gene, which is essential for homologous recombination reactions.

To clarify this relationship, they created cells with simultaneously impaired FEN1 and homologous recombination and examined their relationship.

As a result, cells with deficiencies in both showed significantly reduced cellular resistance to alovudine compared with cells with only one impairment. It was found that FEN1 prevents alovudine cytotoxicity independently of homologous recombination.

Since FEN1 gene mutations and expression abnormalities are found in various cancers, it is expected to be applied to the treatment of cancers with FEN1 gene mutations.

Hirota commented: "We discovered that nucleoside analogs used for treating viral infections require different DNA repair factors for each drug. This can be called cancer's Achilles' heel, and we expect it to lead to new cancer treatments that exploit weaknesses in DNA repair. This time, we showed that the drug alovudine is particularly effective against cancers with abnormalities in the repair factor FEN1. We want to investigate the effects of more drugs and popularize nucleoside analogs as the main players in cancer treatment."

Journal Information
Publication: Nucleic Acids Research
Title: The flap endonuclease-1 promotes cellular tolerance to a chain-terminating nucleoside analog, alovudine, by counteracting the toxic effect of 53BP1
DOI: 10.1093/nar/gkaf617