Abstract 1568: Mechanistic studies of KL-50, a novel imidazotetrazine for the treatment of MGMT-/MMR- gliomas and glioblastoma
Authors: Huseman Eric D. , Lo Anna , Fedorova Olga , Lin Kingson , Gueble Susan , Sundaram Ranjini , Pyle Anna M. , Bindra Ranjit S. , Herzon Seth 
Published: April 2023

Abstract
The DNA damage repair protein O6-methylguanine methyl transferase (MGMT) reverses O6-alkylguanine lesions via SN2 transfer of the alkyl lesion to an active site cysteine which restores DNA to its native state. MGMT is ubiquitously expressed in healthy tissue but is silenced by promoter hypermethylation in approximately half of glioblastomas and up to 75% of lower grade gliomas. As such, patients with MGMT deficient (MGMT-) tumors benefit from DNA alkylation agents such as temozolomide (TMZ), an imidazotetrazine that deposits methyl lesions at O6-guanine. However, the cytotoxicity of TMZ requires an intact DNA mismatch repair (MMR) pathway and acquired resistance to TMZ via MMR silencing negates the efficacy of the drug. We recently reported the synthesis, discovery, and in vivo evaluation of KL-50, an imidazotetrazine derivative that overcomes this resistance mechanism while maintaining high selectivity for MGMT- cells in vitro and in vivo. Our preliminary mechanistic data suggest that KL-50 induces cell death independent of MMR status via the deposition of 2-fluoroethyl lesions at O6-guanine to give an O6-(2-fluoroethyl)guanine (O6FEtG) lesion that evolves to a deadly DNA interstrand cross-link (ICL). We hypothesize that this occurs via a two-step pathway involving slow cyclization of the initial O6FEtG lesion to an electrophilic N1,O6-ethanoguanine intermediate and subsequent nucleophilic ring-opening by the base-paired cytidine residue to yield a guanine(N1)-cytidine(N3) ethyl ICL. Our data suggest the high therapeutic index of KL-50 derives from the relative rates of MGMT reversal of O6FEtG and its conversion to an ICL, specifically that MGMT proficient healthy cells can repair the O6FEtG before it evolves to the ICL. However, direct MGMT reversal of O6FEtG has not been reported, and the rates of ICL formation are not known. Additionally, the formation of the putative ICL has not been demonstrated. Herein, we demonstrate that MGMT can repair the O6FEtG lesion. Kinetics studies using well-defined, photocaged oligonucleotides containing a single O6FEtG lesion show that ICL formation occurs on the order of hours. Furthermore, addition of MGMT following photo-deprotection prevents the formation of ICLs via rapid reversal of O6FEtG. Finally, we have detected a mass corresponding to the putative ICL in pUC19 DNA treated with KL-50 via LCMS . These findings bolster our mechanistic model and suggest strategies that exploit the relative rates of DNA damage and repair may be extensible to other tumor types harboring specific DNA repair defects.
Citation Format: Eric D. Huseman, Anna Lo, Olga Fedorova, Kingson Lin, Susan Gueble, Ranjini Sundaram, Anna M. Pyle, Ranjit S. Bindra, Seth Herzon. Mechanistic studies of KL-50, a novel imidazotetrazine for the treatment of MGMT-/MMR- gliomas and glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1568.