Identifying active and inhibitor-resistant MGMT variants for gene therapy.

O6-methylguanine-DNA methyltransferase (MGMT) reverses alkylating-agent-induced methylation by 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) at the O⁶ position of guanine. MGMT is irreversibly inhibited by O⁶-benzylguanine (O6BG), while the Pro140Lys (P140K) variant is resistant. Combining the use of O6BG/BCNU with gene transfer of MGMT P140K into hematopoietic stem cells (HSCs) has enabled in vivo enrichment of gene-modified HSCs for therapeutic effect in preclinical studies. However, the P140K substitution cannot reliably be made using currently available gene-editing approaches. Identifying functional MGMT variants that are resistant to inhibitors and amenable to gene editing would enable in vivo enrichment of HSCs edited at both MGMT and a therapeutic locus. We used computational analyses to select putative variants and generated a library of MGMT variant-expressing plasmids (pMGMTs). For our functional screen, we treated MGMT-deficient U251 cells with O6BG and co-transfected them with pMGMT together with a plasmid cocktail including a fluorescent host cell reactivation reporter plasmid (mPlum_O⁶MeG) for MGMT activity. Flow cytometric analysis of MGMT activity identified active and O6BG-resistant MGMT variants. Treatment with a second MGMT inhibitor, PaTrin-2, confirmed these results. We also found MGMT variants that are detectable in the general population and tumors to be active and O6BG sensitive. Taken together, our findings establish a functional database for MGMT variants and a cell-based platform for screening DNA-repair proteins for unknown functional properties.