Lack of mismatch repair enhances resistance to methylating agents for cells deficient in oxidative demethylation.

The human alkylation B (AlkB) homologs, ALKBH2 and ALKBH3, respond to methylation damage to maintain genomic integrity and cellular viability. Both ALKBH2 and ALKBH3 are direct reversal repair enzymes that remove 1-methyladenine (1meA) and 3-methylcytosine (3meC) lesions commonly generated by alkylating chemotherapeutic agents. Thus, the existence of deficiencies in ALKBH proteins can be exploited in synergy with chemotherapy. In this study, we investigated possible interactions between ALKBH2 and ALKBH3 with other proteins that could alter damage response and discovered an interaction with the mismatch repair (MMR) system. To test whether the lack of active MMR impacts ALKBH2 and/or ALKBH3 response to methylating agents, we generated cells deficient in ALKBH2, ALKBH3, or both in addition to Mlh homolog 1 (MLH1), another MMR protein. We found that MLH1ALKBH3 cells showed enhanced resistance toward S1- and S2-type methylating agents, whereas MLH1ALKBH2 cells were only resistant to S1-type methylating agents. Concomitant loss of ALKBH2 and ALKBH3 (ALKBH23) rendered cells sensitive to S1- and S2-agents, but the additional loss of MLH1 enhanced resistance to both types of damage. We also showed that ALKBH23 cells have an ATR-dependent arrest at the G/M checkpoint, increased apoptotic signaling, and replication fork stress in response to methylation. However, these responses were not observed with the loss of functional MLH1 in MLH1ALKBH23 cells. Finally, in MLH1ALKBH23 cells, we observed elevated mutant frequency in untreated and temozolomide treated cells. These results suggest that obtaining a more accurate prognosis of chemotherapeutic outcome requires information on the functionality of ALKBH2, ALKBH3, and MLH1.