Ari M. Vanderwalde

RRM1 and RRM2 inhibition reduces cell viability in oncogene-driven lung adenocarcinomas. Bar graphs representing the IC50 values of (A) triapine and (B) gemcitabine in different lung adenocarcinoma lines. C and D, Comparison of proliferation after genetic knockdown (KD) and overexpression (OE) of RRM1 or non-targeting control (NTC) in H358 cells. Bar graphs represent cell-cycle phases upon triapine treatment (1.5 μmol/L, 72 hours) in different lung adenocarcinoma cell lines, namely (E) H358, (F) PC9, and (G) A925L. H, Representative pathway enrichment plot from the MSigDB database of genes associated with RRM1 and RRM2. For comparisons among multiple groups, one-way analysis of variance (ANOVA) was performed; between two groups, a Student t test followed by Dunnett post hoc test was performed. ***, P < 0.001 were considered significant [P > 0.05 was considered nonsignificant (ns)]. The error bar represents the ± standard error of the mean (SEM), and the results are a representation of n = either 2 or 3 based on the experiments.

Non–small cell lung cancer (NSCLC) is responsible for the majority of cancer-related mortality worldwide. Lung adenocarcinoma is the most common NSCLC subtype. Despite advances in targeted therapies, treatment resistance remains a critical challenge. Ribonucleotide reductase (RNR), a crucial enzyme in deoxyribonucleotide triphosphate biosynthesis, is frequently upregulated in cancer, contributing to genomic instability and poor prognosis in multiple malignancies. However, the role of the RNR complex in driving tumorigenesis is not fully understood in oncogene-driven lung adenocarcinoma. Transcriptomic analysis of more than 27,000 real-world samples of patients with NSCLC revealed that RNR subunits (RRM1 and RRM2) are significantly upregulated in TP53-mutated NSCLC and are correlated with significantly poor prognosis in multiple oncogene-driven lung adenocarcinoma. Using pharmacologic and genetic approaches to inhibit RNR in lung adenocarcinoma models, we assessed functional consequences through molecular, biochemical, and imaging techniques. RNR inhibition induced appreciable replication stress and triggered DNA damage, leading to cell death in lung adenocarcinoma cells. Notably, we uncovered that RNR suppression preferentially induced ferroptosis, an iron-dependent cell death driven by lipid peroxidation. This represents a previously unrecognized mechanism of RNR-mediated cell death by which mutant lung adenocarcinoma cells can be selectively targeted. Our study establishes RNR inhibition as a potent strategy to selectively induce ferroptosis in oncogene-addicted lung adenocarcinoma, offering a new therapeutic avenue for genetically defined patient subgroups. Targeting nucleotide metabolism could serve as an effective approach to overcome treatment resistance and improve clinical outcomes for patients with high-risk lung adenocarcinoma. Significance:Our findings highlight RNR as a promising therapeutic target in oncogene-driven lung adenocarcinoma. By demonstrating that RNR inhibition induces ferroptosis, our study opens up new possibilities for developing targeted therapies that selectively eliminate cancer cells in lung adenocarcinoma, paving the way for personalized treatment strategies and potentially overcoming resistance to current therapies.

Higher levels of RRM1 and RRM2 expression are linked to worse prognosis in oncogene-driven NSCLC. A, Box plot representing the expression of RRM1 and (B) RRM2 in the clinical dataset of real-world NSCLC cases (n = 27,280; Caris data) according to the TP53 mutational status (TP53WT and TP53MUT). C, Box plot representing the expression of RRM1 and (D) RRM2 in oncogene-addicted NSCLC, namely KRASMUT, EGFRMUT, and ALKFUS according to the TP53 mutational status. E, Kaplan–Meier plot according to the RRM1 expression in the overall cohort. F,KRASMUT, (G) EGFRMUT, and (H) ALKFUS populations. I, Kaplan–Meier plot according to the RRM2 expression in the overall cohort; (J) KRASMUT, (K) EGFRMUT, and (L) ALKFUS populations. χ2, Fisher exact, Mann–Whitney U, and t tests (with Welch correction) were used and adjusted for multiple hypothesis testing using the Benjamini–Hochberg method where appropriate. Statistical significance was considered when q < 0.05. *, P < 0.05; ***, P < 0.001; ****, P < 0.0001 were considered as significant [P > 0.05 was considered as non-significant (ns)]. LUAD, lung adenocarcinoma; med OS, Median overall survival.

RNR inhibition causes DNA damage and replication stress in oncogene-driven lung adenocarcinomas. A, Representative IF staining images for H358 cells, stained with γ-H2AX and DAPI, −/+ triapine treatment (1.5 μmol/L, 72 hours). B, Representative IF staining images for PC9 cells, stained with γ-H2AX and DAPI, −/+ triapine treatment (1.5 μmol/L, 72 hours). C, Representative IF staining images for A925L cells, stained with γ-H2AX and DAPI, −/+ triapine treatment (1.5 μmol/L, 72 hours). D, Bar graph representation of the quantification of γ-H2AX and phospho-RPA32 mean fluorescence intensity (MFI) in different lung adenocarcinoma cell lines after triapine treatment. E, Representative IF staining images for H358 cells, stained with pRPA32 and DAPI, −/+ triapine treatment (1.5 μmol/L, 72 hours). F, Representative IF figure panel of PC9 cells stained with pRPA32, −/+ triapine treatment (1.5 μmol/L, 72 hours), and with phospho-RPA32 and DAPI. G, Representative IF staining images for A925L cells, stained with pRPA32 and DAPI, −/+ triapine treatment (1.5 μmol/L, 72 hours). H, Representative Western blot panel for γ-H2AX and phospho-RPA32 expression in PC9 and A925L cells upon triapine treatment (1.5 μmol/L, 72 hours). For comparisons among multiple groups, one-way analysis of variance (ANOVA) was performed; between two groups, a Student t test followed by Dunnett post hoc test was performed. *, P < 0.05; **, P < 0.01; ***, P < 0.001 were considered significant [P > 0.05 was considered nonsignificant (ns)]. The error bar represents the ± standard error of the mean (SEM), and the results are a representation of n = either 2 or 3 based on the experiments.

RNR inhibition induces ferroptosis in oncogene-driven lung adenocarcinoma cells by increasing lipid peroxidation. A, Scatter plot representation of correlation analysis of RRM1 and ferroptosis signature gene set or RRM1 and fatty acid metabolism (FAM) signature gene sets. B–E, Bar graph representation of flow cytometry–based BODIPY/C11 staining for lipid peroxidation readout after triapine treatment (1 μmol/L, 72 hours) and gemcitabine (0.5 μmol/L, 72 hours) treatment with and without ferroptosis inhibitor, Fer-1, treatment on different lung adenocarcinoma cells. F, Bar graph representation of thiobarbituric acid reactive substance assay for measuring cellular MDA levels upon triapine treatment (1.5 μmol/L, 72 hours) in PC9, H358, and A925L cells. G, Bar graph representation of GSH assay for measuring cellular GSH levels upon triapine treatment (1.5 μmol/L, 72 hours) in PC9, H358, and A925L cells. H, Graphical representation of the effect of RNR inhibition in lung adenocarcinoma cells. For comparisons among multiple groups, one-way analysis of variance (ANOVA) was performed; between two groups, a Student t test followed by Dunnett post hoc test was performed. *, P < 0.05; **, P < 0.01; ***, P < 0.001 were considered significant [P > 0.05 was considered nonsignificant (ns)]. The error bar represents the ± standard error of the mean (SEM), and the results are a representation of n = either 2 or 3 based on the experiments.

RNR inhibition modulates ferroptosis markers in NSCLC. A, Scatter plot representing the coexpression correlation between RRM1 and SLC7A11 and GPX4 in clinical samples with KRAS, EGFR, and ALK driver mutations. B, Representative Western blot panel for ferroptosis markers ACSL4, FSP1, GPX4, NRF2, SLC7A11, and FTH1 in PC9 and A925L cells upon triapine treatment (1.5 μmol/L, 72 hours). For comparisons among multiple groups, one-way analysis of variance (ANOVA) was performed; between two groups, a Student t test followed by Dunnett post hoc test was performed. P < 0.001 were considered significant [P > 0.05 was considered nonsignificant (ns)]. The error bar represents the ± standard error of the mean (SEM), and the results are a representation of n = either 2 or 3 based on the experiments.

RRM1 and RRM2 expression in NSCLC clinical samples

Stage progression is the primary driver of increased risk of death, independent of RRM1 expression or driver mutation status.

Genetic inhibition of RNR decreases cell proliferation, which is rescued by RNR re-expression

Stage progression is the primary driver of increased risk of death, independent of RRM2 expression or driver mutation status.