Pilot: DNA Mismatch Repair-Dependent Metabolic Adaptation After Chemotherapy

Abstract DNA mismatch repair (MMR) is the cellular spellcheck mechanism for preventing the accumulation of replication errors. However, MMR proteins also prevent mutagenesis from exogenous agents that disrupt DNA base structure resulting in the incorporation of the incorrect base opposite the damaged base. These damage-induced mispairs occur during common chemotherapy treatments including those causing DNA base alkylation (Temodar), platination (cisplatin), or oxidation. Interestingly, DNA mismatch repair cannot repair damage induced mispairs, rather the pathway recognizes the mispair and results in ATR/CHK1 DNA damage response activation, G2/M cell cycle arrest, and apoptotic cell death. This MMR- mediated recognition of damage induced mispairs significantly contributes to the cytotoxicity of these chemotherapeutics. Loss of MMR results in increased resistance to these therapeutics. The chain of cellular events linking MMR mediated recognition of the mispair to eventual cell death is not well defined. To further study MMR-mediated cell death we generated CRISPR/Cas9 knock-out cell lines of two critical MMR proteins, MSH2 and MLH1 in Hela-S3 cells. MSH2 and MLH1 are required for MMR-mediated cell death. We treated parental and knockout cells with the laboratory alkylating agent, Methyl- nitronitrosoguanidine (MNNG), which causes the O6-methylguanine lesion that causes the MMR- mediated cell death seen with clinical alkylating agent, Temodar. Using the Seahorse metabolic flux analyzer we found that both basal respiration and spare respiratory capacity and ATP production increased with MNNG treatment. This adaptive metabolic response was lost in both MSH2 and MLH1 knock out cells. Up to 15% of colorectal cancers and 30% of endometrial cancers have mutated MMR genes or promoter methylation of MLH1 and are treated with cisplatin. Around 12% of primary or reoccurring glioblastoma tumors, for which Temodar is the first line treatment, have at least partial immunohistochemical loss of a MMR protein. However, immunotherapy works poorly for these patients and there is a desperate need for additional treatments. Understanding the link between MMR processing of damage-induced mispairs and cellular metabolism will provide functional insights into the events linking mispair recognition with eventual cell death. It will also aid in formulating metabolic interventions that could be combined with DNA damaging chemotherapy to enhance response in MMR-defective tumors that are resistant to chemotherapy. We hypothesize that MMR processing of damage-induced mispairs results in repair intermediates that trigger consumption of metabolic precursors, AMPK activation and result in upregulation of oxidative phosphorylation in an attempt to promote cell survival. In the absence of functional MMR these repair intermediates are not generated and there is no resulting metabolic stress.