Biomarker-Targeted O6-Guanine Alkylation Potentiates ATR Inhibitor Response in De Novo and Relapsed/Refractory AML
Acute myeloid leukemia (AML) remains limited by high relapse rates and a scarcity of biomarker-directed therapies, underscoring the need to identify actionable vulnerabilities and mechanisms of therapeutic resistance. O6-methylguanine-DNA methyltransferase (MGMT), a DNA repair enzyme that directly reverses mutagenic O6-alkylguanine lesions, is epigenetically silenced in multiple cancers. Although MGMT silencing is predictive of temozolomide (TMZ) response in glioblastoma with significant survival advantage, prior clinical trials of TMZ in AML have shown only modest responses. Therefore, the prevalence and therapeutic relevance of MGMT silencing, as well as genetic factors that modify its therapeutic response in AML, remain to be comprehensively investigated. Here, we first profiled MGMT status across a diverse cohort of 23 de novo and 16 relapsed/refractory (R/R) primary AMLs using an integrated analysis of MGMT mRNA expression, promoter methylation, and protein expression. We found that approximately 25-30% of AMLs harbor MGMT silencing, in contrast to consistent MGMT expression in healthy CD34+ hematopoietic stem and progenitor cells. We further identified frequent loss of mismatch repair (MMR) proteins in these AML cohorts, a known resistance mechanism against TMZ in glioblastoma. Using CRISPR knockout screening and genetically engineered AML models, we found that MMR loss also drives both upfront and acquired TMZ resistance in MGMT-silenced AML. These findings identify MMR deficiency as an important and previously underappreciated genetic contributor to the modest responses observed in prior trials of TMZ in AML. To therapeutically exploit MGMT silencing while bypassing genetic determinants of resistance, including MMR deficiency, we evaluated clinically and preclinically characterized MGMT-dependent DNA-alkylating agents and identified a fluoroethylating analog of TMZ, N3-(2-fluoroethyl) imidazotetrazine (KL50), with pronounced and selective activity in MGMT-silenced AML. KL50 retained antileukemic activity irrespective of MMR status, significantly prolonging survival in humanized MISTRG6 mice harboring primary AML patient-derived xenografts. Mechanistically, KL50 induced DNA damage through the time-dependent formation of interstrand DNA crosslinks, bypassing MMR dependence and triggering a replication stress response dominated by ATR signaling. Pharmacologic ATR inhibition synergized with KL50, producing marked antileukemic activity and significantly extending survival across AML models without compromising hematologic safety. Together, these findings establish MGMT silencing as a prevalent and therapeutically actionable biomarker in AML, define MMR status as a key determinant of TMZ response in AML but not KL50 sensitivity, and provide a translational rationale for combining low-dose O6-fluoroethylating imidazotetrazines with ATR inhibitors to target both de novo and R/R AML across diverse genetic backgrounds.