Metabolic rewiring renders KEAP1 -mutant lung cancers sensitive to AKR1C3-activated prodrugs

The AKR1C1-3 family (aldo-keto reductase family 1, members C1-C3) comprises cytosolic NADPH-dependent oxidoreductases that catalyze the reduction of carbonyl groups on steroids, prostaglandins, and xenobiotics. Upregulation of AKR1C enzymes in certain cancers can contribute to resistance to chemotherapeutic agents and oxidative stress, making them key effectors in redox homeostasis and drug resistance. AKR1C3 expression has been exploited to convert inert prodrugs to their active form with tumor selectivity. ACHM-025 is a second-generation AKR1C3-activated prodrug that is reduced in a single enzymatic step to ACHM-025H, a nitrogen mustard that bis-alkylates DNA to form intra- and interstrand cross-links (ICLs) leading to stalled DNA replication fork progression and cell death. ACHM-025 was shown to have potent activity in preclinical models of T-ALL, a cancer that can express high levels of AKR1C3. Loss-of-function alterations in KEAP1 occur frequently in lung cancer, often co-occurring with alterations in KRAS , STK11 , and TP53 , forming distinct molecular subgroups that influence therapeutic response. KEAP1 -mutant tumors are typically resistant to immune checkpoint blockade and platinum doublet chemotherapy, leading to significantly worse clinical outcomes. Previously, we showed that AKR1C1/2/3 are among the most significantly up-regulated genes in KEAP1- mutant lung cancers, which supports preclinical testing ACHM-025 as novel therapeutic agent in this subset of tumors.Using a series of isogenic lung adenocarcinoma cell lines, we found that lines deficient in KEAP1 express high levels of AKR1C3 and are sensitive to ACHM-025. Colony formation and cell viability assays demonstrated dose-dependent sensitivity to ACHM-025, which was positively correlated with the amount of AKR1C3 protein expressed. The KEAP1 -mutant isogenic clones of H292 and H358 cell lines exhibited IC₅₀ values of 171 nM (95% CI: 124-240 nM) and 466 nM (95% CI: 329-689 nM), respectively. Among the tested cell lines, H460 cells showed the greatest sensitivity to ACHM-025, with an IC₅₀ of 4 nM (95% CI: 2-10 nM). Co-treatment with SN34037, a potent and selective AKR1C3 inhibitor, abolished ACHM-025 activity, which demonstrated selective activation. Clonal competition assays admixing KEAP1-mutant and wild-type human adenocarcinoma cell lines demonstrated rapid depletion of KEAP1 -mutant cells with minimal bystander effect. Finally, we confirmed that the cytotoxic effect of ACHM-025 led to increased phosphorylated H2AX (gammaH2AX) which is consistent with a mechanism of action involving DNA damage.Taken together, our data support the rationale that metabolic rewiring in KEAP1-mutant tumors leads to enhanced sensitivity to ACHM-025 and, therefore, supports AKR1C3 mediated prodrug activation as a potential therapeutic strategy for this subset of lung cancer patients.