Osalmid inhibits lung adenocarcinoma progression and enhances EGFR-TKI responses: a preclinical and translational study

Introduction

Globally, lung cancer ranks highest in both incidence and mortality among all cancers (1). Lung adenocarcinoma (LUAD) has a wide variety of driver mutations, including epidermal growth factor receptor (EGFR) and Kirsten rat sarcoma viral oncogene (KRAS) mutations (2-4). The prevalence of EGFR mutations of LUAD in the East Asian population is approximately 40–68%, making it the most common mutation type (5). The third-generation tyrosine kinase inhibitors (TKIs) have been approved as first-line options for LUAD patients with advanced or relapsed diseases of EGFR mutations (exon 19 deletions or exon 21 L858R mutation). However, in a phase II trial (NCT01407822), neoadjuvant EGFR-TKI monotherapy did not further reduce tumor regression to increase the complete resection rate or reduce the extent of resection (6). Meanwhile, a recent clinical trial (UMIN000023761) reported that the combination of third-generation TKI and bevacizumab showed promising results in EGFR-mutated patients of lung cancer (7), representing a promising strategy for the treatment to achieve better survival benefits. Notably, it is not uncommon when treatment-related adverse events occur during the application of TKIs (8), and previous studies have described severe interstitial pneumonitis and leukopenia in patients dosed with the third-generation TKI (8-10). These complications lead to decreased patient compliance, reducing survival benefits. Accordingly, there is an urgent need to expedite the development of novel pharmaceuticals for the clinical management of LUAD, improve the antitumor effect, and simultaneously reduce the side effects associated with third-generation TKIs.

Ribonucleotide reductase subunit-M2 (RRM2) is a small subunit of ribonucleotide reductase, which is involved in nucleotide metabolism, catalyzes the conversion of nucleotides to deoxynucleotides, and maintains the deoxyribonucleotide pool for DNA replication, repair, and synthesis (11). RRM2 is essential for cell proliferation and cancer progression and is related to chemoresistance, ferroptosis, and tumor immunity (12-15). Notably, the high RRM2 expression has been confirmed to be related to immune suppression in the tumor microenvironment, which promotes tumor immune escape, and the inhibition of RRM2 can effectively promote M1 macrophage polarization and suppress M2 macrophage polarization (15). RRM2 inhibitors, therefore, are expected to play a role in treating patients with LUAD. Osalmid, a drug that has received clinical approval for the management of cholecystitis, biliary tract inflammation, and postcholecystectomy syndrome, is an RRM2 inhibitor with tenfold greater activity than hydroxyurea (16). As an inhibitor of RRM2, osalmid can potentially augment the radiosensitivity of esophageal cancer (17). Furthermore, osalmid may promote apoptosis and induce cell cycle arrest in diffuse large B-cell lymphoma by targeting the JAK2/STAT3 pathway (18). In another study, osalmid and its analogues were shown to inhibit hepatocellular carcinoma activity, demonstrating significant anti-tumor effects (19). In addition, a clinical trial (NCT03670173) demonstrated the antitumor effect of osalmid in multiple myeloma (MM) (12). The above studies demonstrate that osalmid possesses antitumor activity, providing a foundation for its potential application in LUAD treatment.

In 2012, Brent R. Stockwell identified ferroptosis as an iron-dependent regulated cell death caused by lipid peroxidation and plasma membrane rupture (20). Ferroptosis involves iron overload, reduced glutathione (GSH) levels, glutathione peroxidase 4 (GPX4) suppression, and lipid peroxidation (20). RRM2 has been implicated as a protein involved in ferroptosis-related metabolism (21). RRM2 contributes to nucleotide synthesis through GSH dependence, with its upregulation promoting GSH biosynthesis and influencing ferroptosis metabolism. RRM2 knockdown induces ferroptosis in liver and LUAD cells (14,22), and its expression correlates with ferroptosis in prostate cancer (23). A previous review indicated that the MEK-ERK signaling pathway could regulate the expression of RRM2 (11), and EGFR is the upstream protein for activation of the MEK-ERK signaling pathway (24). Besides, another report uncovered that the expression of RRM2 and EGFR was regulated by the long non-coding RNA AFAP1-AS1 simultaneously (25). These findings suggest a potential biological connection between EGFR signaling and RRM2-dependent tumor vulnerabilities. Although EGFR-TKIs have substantially improved outcomes in patients with EGFR-mutant LUAD, primary or acquired resistance remains a major clinical challenge. Metabolic and cell-cycle-related vulnerabilities may provide complementary therapeutic opportunities to enhance EGFR-TKI efficacy. Therefore, we investigated whether pharmacological inhibition of RRM2 by osalmid could suppress LUAD progression and enhance EGFR-TKI responses, with particular attention to EGFR-mutant LUAD models. We present this article in accordance with the MDAR and ARRIVE reporting checklists (available at https://tlcr.amegroups.com/article/view/10.21037/tlcr-2026-0257/rc).

Methods

Patient and ferroptosis-related data obtained via CRISPR screens

Gene expression data (FPKM) and clinical information for LUAD patients were retrieved from TCGA. Data covered 572 cases, with clinical variables including survival status and time. Ferroptosis-related data obtained via CRISPR screens were obtained from an open access dataset. Dr. Zou and his colleagues performed CRISPR screening in renal cell carcinoma (RCC) 786-O cells treated with ML-210 (an inhibitor of GPX4, 5 µM) for four, six, or eight days (26). The names used for the screening datasets were 1-PMID30962421, 2-PMID30962421, and 3-PMID30962421 (https://orcs.thebiogrid.org/Download?type=screen&id=1256). The study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments.

Cell lines and cell culture

The LUAD cell lines A549, NCI-H1975, NCI-H322, NCI-H1299, NCI-H460, PC-9, NCI-H358, and SW-1573 were obtained from the American Type Culture Collection. All cell lines were maintained in RPMI-1640 medium supplemented with FBS and antibiotics under standard conditions (37 ℃, 5% CO₂). NCI-H1975 and PC-9 cells harbor EGFR mutations, whereas A549, NCI-H322, NCI-H1299, NCI-H460, and SW-1573 cells exhibit KRAS, TP53, or NRAS mutations.

Drug treatment

For in vitro drug-treatment experiments, three independent biological replicates were performed for each treatment group unless otherwise indicated. Osalmid was obtained from the Drug Discovery and Design Center, Shanghai Institute of Materia Medica, Chinese Academy of Sciences. AZD9291 (Osimertinib, a third-generation EGFR-TKI, TargetMol Co. Ltd., Boston, USA), Fer-1 (a ferroptosis inhibitor, TargetMol Co. Ltd., Boston, USA), RSL3 (a ferroptosis inducer, TargetMol Co. Ltd., Boston, USA), dimethyl sulfoxide (DMSO, TargetMol Co. Ltd., Boston, USA), and castor oil were purchased from MedChemExpress (State of New Jersey, USA), and AST2818 (Alflutinib, a third-generation EGFR-TKI) was obtained from Shanghai Allist Pharmaceuticals Co., Ltd. Cell viability assays were conducted to measure the half-maximal inhibitory concentration (IC₅₀) values of the compounds. Normal saline (NS; 0.9%) was obtained from Shanghai Pulmonary Hospital.

Cell viability assay

Cell viability was measured with CCK8 (TargetMol Co. Ltd., Boston, USA) after osalmid treatment at 0–4 days. Following incubation with 10% CCK8 reagent at 37 ℃ for 2 h in the dark, absorbance at 450 nm was recorded using an Infinite M Plex reader (Tecan Trading Co., Ltd., Shanghai, China) and Tecan.At.XFluor software.

IC₅₀ determination assay.

To calculate the IC₅₀ values of osalmid, AZD9291, RSL3, and AST2818 in LUAD cell lines, we evaluated cell viability with a CCK8 after 3 days of drug treatment. In this experiment, 2,000 cells were seeded in 96-well plates and treated for 72 h in complete media containing increasing concentrations of osalmid (0–1,000 µM), AZD9291 (0–10 µM), RSL3 (0–10 µM), and AST2818 (0–10 µM) as single agents or as constant ratio combinations. Fer-1 (1 µM) was used to block ferroptosis. After 72 h of treatment, old medium was removed, fresh medium with 10% CCK8 was added, and plates were incubated at 37 ℃ for 2 h. Notably, the plates were kept in the dark throughout this process. An Infinite M Plex microplate reader (Tecan Trading Co., Ltd., Shanghai, China) and Tecan.At.XFluor software were used for signal acquisition at a detection wavelength of 450 nm. Dose-response curves were generated, and IC₅₀ values were calculated with GraphPad Prism software version 9.0 (https://www.graphpad.com/) and analyzed by two-way ANOVA with the Bonferroni correction. All cell viability and IC₅₀ determination assays were performed in three independent biological replicates unless otherwise indicated.

Colony formation assay

Cells (1,000–2,000/well) were seeded into 6-well plates for colony formation assays. Medium was replaced every 48 h. After 14 days, colonies were fixed with 4% paraformaldehyde, stained with 0.5% crystal violet, and counted using ImageJ (https://imagej.net/ij/). Colony formation assays were performed in three independent biological replicates.

Lentivirus packaging

To construct RRM2-knockdown cell lines, human embryonic kidney 293 packaging cells were used for lentivirus production. 293T cells were transiently transfected with vector DNA, psPAX2 (#12260, Addgene, Cambridge, USA), pMD2.G (#12,259, Addgene, Cambridge, USA), and the target plasmid using Lipofectamine 3000 (Thermo Fisher Scientific, Waltham, USA). After 48 h, viral supernatants were collected and filtered (0.45 µm).

Cell transfection

Cells (NCI-H1975 and PC-9) were infected with viral supernatant, incubated for 24 h at 37 ℃, and selected with 2 µg/mL puromycin for 7 days.

Immunoblotting

Cells were seeded into 6-well plates at a density of 15,000 cells per well and treated with varying concentrations of Osalmid or AST2818 for 72 h. For immunoblotting, cells were washed once with ice-cold PBS and lysed in lysis buffer (50 mM Tris-HCl, pH 7.4; 150 mM NaCl; 1 mM EDTA; 1% Triton X-100; 5% glycerol) for 15 min at 4 ℃. The supernatant was separated via centrifugation for 15 min at 12,000 rpm and 4 ℃. Protein concentrations were measured using the Bradford assay. Cell lysates (30 µL) were combined with 30 µL of 2× SDS loading buffer and boiled for 10 min. Subsequently, 10 µL of protein samples were resolved on 4–10% gradient SDS-PAGE gels and transferred onto PVDF membranes (Millipore, MO, USA). Membranes were blocked with 5% nonfat milk for 1 h and incubated overnight at 4 ℃ with primary antibodies diluted in 1% BSA/PBS. After washing with PBS containing 0.05% Tween, membranes were incubated with secondary antibodies at room temperature for 1 h. The primary antibodies used included GPX4 (1:1,000; Abcam, UK), ACSL4 (1:3,000; Proteintech, USA), SLC7A11 (1:1,000; Cell Signaling Technology, USA), and GAPDH (1:3,000; Proteintech, USA). HRP-conjugated goat anti-rabbit IgG (H+L) (1:10,000; Biodragon Immunotech) and HRP-conjugated goat anti-mouse IgG (H+L) (1:10,000; Biodragon Immunotech) served as secondary antibodies. Protein bands were visualized using an electrochemiluminescence (ECL) detection system (Tanon-5200, Shanghai, China). Representative images from three independent experiments are shown.

Mouse experiments

We generated NCI-H1975 cell-derived xenografts (CDXs) to explore the antitumor effects of osalmid, AST2818, and the combination of osalmid with AST2818 in BALB/c nude mice (Shanghai SLAC Laboratory Animal Co., Ltd., Shanghai, China). NCI-H1975 cells (5×10⁶ cells/100 µL) were injected subcutaneously after one week of feeding. First, twenty-one mice were randomly divided into three groups (control, 100 mg/kg osalmid injection dose, and 150 mg/kg osalmid injection dose, 7 mice per group) when the tumor volume was about 50–100 mm³. Each mouse in the control and treatment groups was intraperitoneally administered 200 µL of a mixture of 5% DMSO + 4% castor oil + NS (control group) or osalmid (treatment groups) daily. Second, fifteen mice were randomly classified into three groups (control, AST2818, and combination of AST2818 and osalmid, 5 mice/group) when the tumor volume was approximately 50–100 mm³. The mice in the control group did not receive any drug; the mice in the AST2818 group were administered 20 mg/kg AST2818 by gavage daily (100 µL suspension); and the mice in the combination group were administered 100 mg/kg osalmid by intraperitoneal injection and 20 mg/kg AST2818 by gavage (100 µL suspension) every day. Sodium carboxymethylcellulose (0.5%, TargetMol Co. Ltd., Boston, USA) was used as the resuspension medium for AST2818 to obtain a stable homogeneous mixture. From day 0 to day 21, the longest and shortest diameters of the tumor were measured with vernier calipers every three days. Mice were monitored by regular weighing. After 28 days, tumors were collected, weighed, and fixed in 10% formalin following euthanasia by cervical dislocation. Experiments were performed under a project license (No. K22-366) granted by the Ethical Review Committee and Laboratory Animal Welfare Committee of Shanghai Pulmonary Hospital, in compliance with the European Community Council Directive of 24 November 1986 for the care and use of animals. A protocol was prepared before the study without registration.

Hematoxylin-eosin (HE)

CDX tumor tissues were fixed in 4% paraformaldehyde, embedded in paraffin, sectioned at 4 µm thickness, and subjected to HE staining. The stained sections were examined microscopically. Immunohistochemical staining of paraffin-embedded CDX tumor tissues was carried out using the EnVision system (Dako, Glostrup, Copenhagen, Denmark) following the manufacturer’s protocol.

Immunohistochemical staining

CDX tumor tissues were fixed with 4% paraformaldehyde, embedded in paraffin, cut into sections (4 µm), and subjected to immunohistochemical staining. A rabbit anti-4-hydroxynonenal (4-HNE) antibody (1:500; bs-6313R; BIOSS Co. Ltd., Beijing, China), a rabbit polyclonal anti-RRM2 antibody (1:500; DF7248; Affinity, Liyang, China), and a recombinant anti-Ki67 antibody (1:1,000; ab279653; Abcam, Cambridgeshire, UK) were used as the primary antibodies. Antibodies in the Dako REAL EnVision Detection System were used as secondary antibodies (1:100 dilution).

RNA extraction and qRT-PCR

RNA was extracted from NCI-H1975 and PC-9 cells using TRIzol reagent (Invitrogen, Carlsbad, USA), and 1 µg of mRNA was used to synthesize cDNA using HiScript III RT SuperMix for qPCR (R323-01, Vazyme, Nanjing, China). qRT-PCR was performed using ChamQ Universal SYBR qPCR Master Mix (Q711-02, Vazyme, Nanjing, China) according to a standard protocol (18). For each group, three technical replicates were collected. The sequences of the primers used for amplification of RRM2 were as follows: 5’-CACGGAGCCGAAAACTAAAGC-3’ and 3’-TCTGCCTTCTTATACATCTGCCA-5’. The relative expression of RRM2 was quantified using the 2^−ΔΔCT method. ACTIN, owing to its greater stability and consistency, was selected as the internal reference to normalize RNA input.

mRNA sequence

Total RNA was isolated from NCI-H1975 cells using TRIzol reagent (Invitrogen, Carlsbad, USA) following the manufacturer’s protocol. RNA purity and concentration were determined with a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, USA), and RNA integrity was evaluated using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, USA). Library preparation was performed with the VAHTS Universal V6 RNA-seq Library Prep Kit according to the manufacturer’s instructions (27,28). Sequencing was conducted on the Illumina NovaSeq 6000 platform, generating 150 bp paired-end reads (29). Each sample produced approximately 50 million raw reads in fastq format, which were processed using fastp to remove low-quality sequences. Roughly 40 million clean reads per sample were retained for downstream analyses. Clean reads were subsequently aligned to the reference genome using HISAT2 (30).

Statistical analysis

Statistical analyses were conducted using GraphPad Prism 9. Gene expression levels were quantified as FPKM values, and read counts were obtained with HTSeq-count. Differential expression analysis was performed with DESeq2, applying a threshold of P<0.05 and fold change >2 or <0.5 to identify significantly differentially expressed genes (DEGs). Differences in tumor weight between groups were assessed using the Mann-Whitney U test. The standard deviation (SD) was calculated to evaluate the variation between experimental replicates. The combination index (CI) was used to assess the synergy effect of two drugs (CI =1: additive effect, CI <1: synergy effect, CI >1: antagonistic effect, https://synergyfinder.org/). A two-sided p value <0.05 was considered to indicate statistical significance. *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001; ns, not significant.

Results

Oncological effect of RRM2

Analysis of TCGA public data revealed that RRM2 transcriptional expression was significantly higher in LUAD tissues compared with normal lung tissues (P<0.001, Figure 1A). Moreover, within the LUAD cohort, patients exhibiting elevated RRM2 expression had markedly poorer survival outcomes than those with low RRM2 expression (P<0.001, Figure 1B). Thus, RRM2 expression may affect the progression of LUAD. We designed two shRNAs targeting RRM2 (sh1: 5’-GCTCAAGAAACGAGGACTGAT-3’; sh2: 5’-GCAGACAGACTTATGCTGGAA-3’). The qPCR results indicated that sh2 had a better knockdown effect than sh1 (Figure 1C). Therefore, we used the negative control shRNA (shNC), sh1, and sh2 to construct negative control and knockdown cell lines, respectively. Cell proliferation was decreased by RRM2 knockdown (Figure 1D,1E). In addition, colony formation assays showed similar results (Figure 1F,1G).

Figure 1 Oncological effect of RRM2. (A) Differential analysis of RRM2 expression in normal lung and lung tumor tissues. (B) Survival analysis of lung adenocarcinoma patients according to the expression of RRM2. (C) Effect of RRM2 knockdown by qPCR (n=3). Cell viability of PC-9 (D) and NCI-H1975 (E) cells after knockdown of RRM2 with different sh at 0, 1, 2, 3, and 4 days (cell viability reflects the status of cell proliferation, and a high fold value indicates a large number of alive cells, n=3). Clonal formation of PC-9 (F) and NCI-H1975 (G) cells treated with shNC, RRM2 sh-1, and RRM2 sh-2 (n=3). Data are presented as mean ± SD from three independent biological replicates unless otherwise indicated. Student’s t-test was performed in Figure (A): ****, P<0.0001. The log-rank test was performed in Figure (B). One-way analysis of variance (ANOVA) was performed in Figure (C-G): ***, P<0.001; **, P<0.01; *, P<0.05. SD, standard deviation; shNC, short hairpin negative control; TCGA, The Cancer Genome Atlas.

Osalmid inhibits LUAD progression

Previous research has shown that osalmid could selectively suppress the activity of RRM2 (12,16). In the present study, we used eight LUAD cell lines to evaluate the antitumor effect of osalmid. The results of Figure 2A revealed that the proliferation of these eight cell lines, especially the PC-9 and NCI-H1975 cell lines, was inhibited by osalmid. To further evaluate whether osalmid sensitivity differed according to EGFR mutation status, we compared the IC₅₀ values of osalmid across multiple non-small cell lung cancer (NSCLC) cell lines. EGFR-mutant cell lines, including NCI-H1975 and PC-9, exhibited lower IC₅₀ values than most non-EGFR-mutant NSCLC cell lines, suggesting relatively higher sensitivity to osalmid treatment in EGFR-mutant models (Table S1). The colony formation assay of these two cell lines also revealed the antitumor effect of osalmid on LUAD (Figure 2B). Western blot revealed that the RRM2 expression was inhibited by osalmid at these concentrations in the NCI-H1975 cell lines (Figure 2C). We further conducted animal experiments to investigate the therapeutic utility of osalmid in LUAD. Three groups were established: the control (n=7), 100 mg/kg osalmid (n=7), and 150 mg/kg osalmid (n=7) groups. NCI-H1975 cells were used to establish CDXs. The mean tumor volume in the control group (1,382.9±344.5 mm³) was the largest among the three groups (100 mg/kg group: 542.6±253.2 mm³, 150 mg/kg group: 292.8±151.9 mm³, all P<0.05; Figure 2D). The real-time photographs of the CDX tumors are presented in Figure 2E. The growth rate of the CDX tumors was evidently decreased in two treatment groups (Figure 2F). Additionally, the mean weight of the CDX tumors was lower in the 150 mg/kg group (0.26 g) than in the other groups (control group: 1.11 g, 100 mg/kg group: 0.43 g, all P<0.05; Figure 2G). To observe the side effects of osalmid, we measured the body weights of the mice at regular intervals and found that the mice in the treatment groups did not experience weight loss (Figure 2H). In addition, we harvested the liver and spleen from one mouse in each group and found no macroscopic differences in those tissues among the three groups (Figure 2I). HE staining of these tissues revealed similar results (Figure 2J). We also performed HE staining (Figure S1A) and immunohistochemical staining (Figure S1B) and found that the expression of Ki-67 was higher in the control group than in the treatment groups.

Figure 2 Osalmid inhibits lung adenocarcinoma progression. Cell viability of eight lung adenocarcinoma cell lines (A) after treating with different concentrations of osalmid at 72 h. Clonal formation of PC-9 and NCI-H1975 (B) cells treated with PBS, 200 μM, and 300 μM osalmid (n=3). (C) Western blot analysis of the expression of RRM2 in NCI-H1975 cells treated with PBS, 200 μM, and 300 μM osalmid (n=3). (D) Tumor volume growth curves during the 21-day therapeutic period. (E) Photographs of tumors after treatments with NS, 100 mg/kg, and 150 mg/kg osalmid (n=7). (F) The fold change of tumor volume over time. (G) The weight of excised tumors on Day 21. (H) Body weight of BALB/c nude mice bearing NCI-H1975 tumors during 21-day treatments (n=7). Macroscopic view (I) and microscopic view (J) of the liver and spleen. One-way analysis of variance (ANOVA) was performed in Figure (A,B). Data are presented as mean ± SD from three independent biological replicates unless otherwise indicated. Tumor-volume and body-weight curves are presented as mean ± SEM, and endpoint tumor weights are presented as mean ± SD. Student’s t-test was performed in Figure (D,F-H). ****, P<0.0001; ***, P<0.001; **, P<0.01; *, P<0.05. NS, normal saline; PBS, phosphate-buffered saline; SD, standard deviation; SEM, standard error of the mean.

Relationship between RRM2 and ferroptosis

Previous studies have indicated that RRM2 plays a role in ferroptosis-associated metabolism, and that silencing RRM2 can trigger ferroptosis in both liver cancer and LUAD cells (14,21,22). Thus, we analyzed ferroptosis-related data from CRISPR screens obtained from an open access dataset (26). Upon ferroptosis induction with ML-210, the expression of antiferroptotic genes, including GPX4 and apoptosis-inducing factor mitochondria-associated 2 (AIFM2), was reduced, whereas the expression of proferroptotic genes, such as acyl-CoA synthetase long-chain family member 4 (ACSL4), was elevated. The RRM2 level exhibited a trend similar to the levels of GPX4 and AIFM2 (Figure 3A-3D). In addition, previous studies revealed that RRM2 knockdown increased ferroptosis (14,22). Therefore, we performed RSL3 treatment experiments to explore the role played by RRM2 in EGFR-mutated cell lines. To functionally validate the involvement of ferroptosis, we treated RRM2-knockdown NCI-H1975 and PC-9 cells with the ferroptosis inducer RSL3, with or without the ferroptosis inhibitor ferrostatin-1 (Fer-1). RRM2 knockdown increased the sensitivity of both cell lines to RSL3-induced cell viability loss, whereas Fer-1 partially rescued this effect, supporting a functional link between RRM2 suppression and ferroptosis susceptibility (Figure 3E). Consistently, RRM2 knockdown decreased the expression of the ferroptosis suppressors GPX4 and SLC7A11 and increased ACSL4 expression (Figure 3F). In CDX tissues, osalmid treatment was accompanied by increased 4-HNE staining, suggesting enhanced lipid peroxidation in vivo (Figure 3G). To further determine whether ferroptosis contributes to osalmid-induced growth inhibition, we performed additional rescue experiments using Fer-1. In both NCI-H1975 and PC-9 cells, Fer-1 increased the IC₅₀ value of osalmid, from 149.9 to 323.3 µM in NCI-H1975 cells and from 164.0 to 471.4 µM in PC-9 cells. These results indicate that inhibition of ferroptosis partially attenuates osalmid-induced cell viability loss, supporting a functional contribution of ferroptosis to the antitumor activity of osalmid (Figure S2).

Figure 3 Relationship between RRM2 and ferroptosis. (A-D) Analysis of CRISPR screening data. Cell viability of shNC, shRRM2, and shRRM2 + Fer-1 in NCI-H1975 and PC-9 cell lines (E) after treating with different concentrations of RSL3 at 72 h (n=3). (F) Western blot analysis of the expression of ACSL4, SLC7A11, and GPX4 in NCI-H1975-shNC, NCI-H1975-shRRM2#1, NCI-H1975-shRRM2#2, PC-9-shNC, PC-9-shRRM2#1, and PC-9-shRRM2#2 cells (n=3). (G). Immunohistochemical staining of 4-HNE in CDX tissues treated with osalmid or vehicle control. Scale bars indicate 100 and 20 μm. Data are presented as mean ± SD from three independent biological replicates unless otherwise indicated. One-way analysis of variance (ANOVA) was performed: **, P<0.01; *, P<0.05; ns, no significance. FC, fold change; SD, standard deviation; shNC, short hairpin negative control.

The role of RRM2/osalmid in treatment with third-generation TKIs

Expression correlation analysis in LUAD based on the TCGA database revealed that RRM2 was positively related to EGFR (R=0.16, P=0.00043; Figure 4A). RNA sequencing was performed in two groups of NCI-H1975 cells, i.e., a control group (n=3) and a third-generation TKI treatment group (cells were stimulated with a 5-fold 72h-IC50 concentration of the drug for 24 h, n=3). AZD9291 inhibited purine metabolism, pyrimidine metabolism, and nucleotide metabolism pathways, where RRM2 is situated (Figure S3). RRM2 expression was decreased significantly after AZD9291 treatment at the transcriptomic level (Figure 4B), suggesting that EGFR-TKI exposure suppresses RRM2-associated nucleotide metabolism. Consistently, AST2818 treatment markedly reduced RRM2 protein expression compared with the untreated control in NCI-H1975 cells (Figure 4C), and RRM2 staining was also decreased in AST2818-treated CDX tumors (Figure 4D). Notably, among AST2818-treated groups, RRM2 protein showed a partial rebound at certain concentrations after 72 h of exposure, although its expression remained lower than that in the control group. This pattern may reflect adaptive or feedback regulation of RRM2 under sustained EGFR-TKI pressure and provides a rationale for simultaneous inhibition of EGFR and RRM2. For combination assays, osalmid concentration was fixed at IC10, while TKIs were administered in escalating doses. The IC10 of osalmid was 24 µM in NCI-H1975 cells and 116 µM in PC-9 cells. After pretreatment with the IC10 of osalmid, the IC50 values of AST2818 and AZD9291 were decreased in the PC-9 and NCI-H1975 cell lines (Figure 4E). Overall, the combination of the two drug types resulted in CI values less than 1, indicating a synergistic effect of osalmid with AZD9291 or osalmid with AST2818 (Figure 4F,4G, Tables S2,S3). As shown in Figure 4H, under RRM2 knockdown conditions, AZD9291 had a lower IC50 value than it did in the control cell line. The colony formation assay results showed that RRM2 knockdown increased the sensitivity of NCI-H1975 and PC-9 cells to third-generation TKIs (Figure 4I). Furthermore, we performed animal experiments to validate the effect of combining these two types of drugs. The flow chart of this experiment is shown in Figure 5A. The weights of the CDX tissues were the lowest in the combination group (AST2818: 20 mg/kg; osalmid: 100 mg/kg) among the three groups (all P<0.05, Figure 5B). Photographs of tumors presented a similar trend in each group (Figure 5C). The mean tumor volume was the lowest in the combination group (AST2818: 20 mg/kg; osalmid: 100 mg/kg) among the three groups (all P<0.05, Figure 5D). In addition, the tumor suppression rate in the drug combination group was larger than that in the AST2818 monotherapy group (89% vs. 70%, P<0.001). In addition, there was no significant difference in the mouse body weight among the three groups (Figure 5E). Immunohistochemical staining for Ki-67 also showed that the combination had significant antitumor efficacy (Figure 5F). These results suggest that osalmid suppresses LUAD progression and enhances the antitumor effect of AST2818 in vivo. However, because a concurrent osalmid monotherapy group was not included in the combination experiment, the in vivo data should be interpreted as evidence of enhanced AST2818 efficacy rather than formal proof of in vivo synergy. In addition, RRM2 may be involved in regulating ferroptosis (Figure 6). However, further proof is needed.

Figure 4 The role of RRM2/osalmid in treatment with third-generation TKIs. (A) Correlation analysis between EGFR and RRM2 based on the TCGA database. (B) Differential analysis of RNA sequencing data (n=3). (C) Western blot analysis of RRM2 expression in NCI-H1975 cells treated with increasing concentrations of AST2818 for 72 h (n=3). RRM2 expression was reduced compared with the untreated control, with partial rebound observed at some AST2818 concentrations. (D) Immunohistochemical staining of RRM2 in CDX tissues (scale bars indicated 100 and 20 μm). Cell viability of AZD9291, AZD9291 + IC10 osalmid, AST2818, and AST2818 + IC10 osalmid in NCI-H1975 and PC-9 cell lines (E) after treating with different concentrations of AZD9291 or AST2818 at 72 h (n=3). Combination index analysis of osalmid plus AST2818 in the NCI-H1975 (F) and PC-9 (G) cells (n=3). CI <1 indicates synergistic interaction. Cell viability of shNC and shRRM2 in NCI-H1975 and PC-9 cell lines (H) after treating with different concentrations of AZD9291 at 72 h (n=3). Clonal formation (I) of PC-9 (shNC and shRRM2) and NCI-H1975 (shNC and shRRM2) cells treated with PBS, 0.01 μM, and 1 μM AST2818 (n=3). Data are presented as mean ± SD from three independent biological replicates unless otherwise indicated. One-way analysis of variance (ANOVA) was performed: **, P<0.01; *, P<0.05. CDX, cell-derived xenograft; CI, combination index; FC, fold change; SD, standard deviation; shNC, short hairpin negative control; TCGA, The Cancer Genome Atlas; TPM, transcripts per million.

Figure 5 Osalmid enhances the antitumor effect of AST2818 in NCI-H1975 xenografts. (A) In vivo therapeutic schedule of osalmid and AST2818 (n=5). (B) The weight of excised tumors on Day 21 (n=5). (C) Photographs of tumors after treatments with NS, 20 mg/kg AST2818, and 100 mg/kg osalmid plus 20 mg/kg AST2818 (n=5). (D) Tumor volume growth curves during the 21-day therapeutic period. (E) Body weight of BALB/c nude mice bearing NCI-H1975 tumors during 21-day treatments (n=5). (F) The results of immunohistochemical staining for Ki-67 (scale bars indicated 100 and 20 μm). Tumor-volume and body-weight curves are presented as mean ± SEM, and endpoint tumor weights are presented as mean ± SD. Student’s t test was performed in Figure (B): **, P<0.01. One-way analysis of variance (ANOVA) was performed in Figure (D,E): ****, P<0.0001. NS, normal saline; SD, standard deviation; SEM, standard error of the mean.

Figure 6 Diagram of the mechanism of the study.

Discussion

In this study, we examined the antitumor activity of osalmid against LUAD both in vitro and in vivo. Our results demonstrated that either osalmid treatment or RRM2 knockdown suppressed the proliferation of LUAD cells. Moreover, Fer-1 rescue experiments suggested that ferroptosis contributes to osalmid-induced cell viability loss in EGFR-mutant LUAD cells. Western blot analysis indicated that RRM2 silencing reduced the expression of GPX4 and SLC7A11. Collectively, these findings suggest that osalmid may impede LUAD progression through RRM2-associated mechanisms, with ferroptosis acting as a contributing process rather than the sole mechanism. We further assessed the impact of osalmid on the sensitivity of EGFR-mutant LUAD to third-generation TKIs. Notably, the IC50 values of TKIs were reduced upon osalmid treatment in vitro. Additionally, combined administration of AST2818 and osalmid produced greater tumor growth inhibition than AST2818 monotherapy in vivo. Taken together, our data support the notion that osalmid suppresses LUAD progression and enhances responsiveness to EGFR-TKIs, potentially through inhibition of RRM2. In addition, RRM2 may be involved in regulating ferroptosis. However, further proof is needed.

RRM2 could be an indicator of prognosis in LUAD patients. As we described, LUAD patients of high RRM2 expression had poorer survival than those of low RRM2 expression. Previous studies also confirmed that the expression of RRM2 could be a prognostic indicator in other malignancies, such as MM, prostate cancer, and esophageal squamous cell carcinoma (12,31,32). I In addition, some recent in vitro and in vivo studies have shown, similar to our findings, that high RRM2 expression can promote NSCLC progression (14,33). Therefore, RRM2 was functionally validated oncologically as a prognostic indicator in LUAD. Moreover, previous studies have shown that RRM2 inhibitors, such as osalmid and its analogues, effectively inhibit tumors and induce apoptosis in tumor cells (18,33). Additionally, in a clinical trial for MM, sulfasamine, used as a late-line drug, halted disease progression in 77.8% of patients (12). In esophageal cancer, osalmid has also demonstrated the ability to enhance the efficacy of sensitizing radiotherapy (17). These findings suggest that osalmid and its analogues may have therapeutic potential, either alone or in combination with other drugs, warranting further validation in tumor-specific contexts.

Neoadjuvant targeted therapy is essential for the treatment of LUAD patients of EGFR mutations (exon 21 L858R mutation, exon 20 T790M mutation, and exon 19 deletion) with locally advanced diseases. Preoperative drug treatment provides a greater rate of R0 (complete) resection and greater survival benefits than surgery alone. In a recent report (NCT03433469), however, Osimertinib (AZD9219) therapy alone did not result in a satisfactory outcome (34). Moreover, the data indicated a major pathological response rate of only 16.7% and a pathological complete response rate of zero. Another clinical trial (ChiCTR1800016948) of neoadjuvant therapy revealed that only one patient achieved a pathological complete response (35). These clinical trials suggest that patients receiving targeted therapy alone may not achieve optimal survival outcomes. On the basis of these findings, a phase III clinical trial (NCT04351555) is currently underway to evaluate neoadjuvant osimertinib, with or without chemotherapy, compared with chemotherapy alone in patients with EGFR-mutated resectable LUAD (36). Accordingly, it is important to increase the rates of major pathological response and pathological complete response for patients with locally advanced disease. With respect to preclinical and translational research, the findings of this study may provide preliminary preclinical rationale for further evaluating osalmid-based combinations in EGFR-mutant or RRM2-dependent LUAD models. In our in vivo combination experiment, AST2818 plus osalmid produced greater tumor growth inhibition than AST2818 monotherapy, suggesting that osalmid may enhance the antitumor effect of third-generation EGFR-TKIs. This design was aligned with our translational objective of evaluating osalmid as an add-on strategy to EGFR-TKI treatment. However, because a concurrent osalmid monotherapy arm was not included in this combination experiment, the current data cannot formally determine the independent contribution of osalmid or establish in vivo synergy. Therefore, these findings should be interpreted as preliminary preclinical evidence supporting further evaluation of osalmid-based combinations, rather than as direct evidence sufficient for clinical implementation or EGFR-TKI dose reduction.

RRM2 expression affects ferroptosis. A previous report suggested that knockdown of RRM2 could increase ferroptosis in vivo (14). In the present study, we also found that RRM2 knockdown increased ferroptosis in the NCI-H1975 cell line and, moreover, the abundance of 4-HNE, an indicator of lipid peroxidation, increased with increasing concentration of osalmid. In addition, we revealed that the expression of GPX4 and SLC7A11 was lower in the shRRM2 cell line than in the shNC cell line. Thus, our data suggest that RRM2 suppression increases ferroptosis susceptibility, at least partly through regulation of GPX4 and SLC7A11. Importantly, the newly added Fer-1 rescue experiments showed that inhibition of ferroptosis increased the IC₅₀ value of osalmid in both NCI-H1975 and PC-9 cells, indicating that ferroptosis contributes to osalmid-induced growth inhibition. However, the precise regulatory mechanism linking RRM2 inhibition to ferroptosis remains to be further clarified. Direct lipid ROS assessment, such as C11-BODIPY staining, should be performed in future studies to further validate this mechanism.

The activation status of EGFR affects the expression of RRM2. In the present study, we subjected NCI-H1975 cells to targeted inhibition and then performed RNA sequencing analysis of the control and treated cells. The RNA sequencing and western blot analysis results revealed that RRM2 expression was significantly downregulated. In addition, a previous review indicated that the MEK-ERK signaling pathway could regulate the expression of RRM2 (11). EGFR is the upstream protein for activation of the MEK-ERK signaling pathway. Therefore, EGFR may regulate the expression of RRM2 via the MEK-ERK pathway. The relationship between EGFR-TKI exposure and RRM2 expression appears to be context-dependent. In our RNA-sequencing analysis, acute AZD9291 treatment suppressed RRM2 expression and downregulated nucleotide metabolism-related pathways, consistent with inhibition of EGFR-driven proliferative signaling. In the AST2818 dose-response western blot assay, RRM2 protein levels were also lower than those in untreated controls; however, a partial rebound was observed at certain concentrations after 72 h of treatment. This may reflect adaptive feedback regulation or compensatory recovery of nucleotide metabolism under sustained EGFR-TKI pressure. Given that RRM2 supports deoxyribonucleotide synthesis and DNA replication stress adaptation, such compensatory recovery may contribute to incomplete EGFR-TKI response. These findings provide a biological rationale for combining EGFR-TKIs with RRM2 inhibition, although further time-course studies are needed to define the dynamic regulation of RRM2 after EGFR-TKI exposure.

This study has several limitations. First, our study was limited to functional experiments, and the underlying mechanisms were not further explored. Nevertheless, these functional validation experiments provide preliminary evidence for future preclinical and translational studies. Second, part of the clinical data analyzed in this study was derived from a public dataset with a relatively small sample size; therefore, expanding the cohort will be necessary to validate our conclusions. The ferroptosis-related CRISPR screening data used in this study were derived from RCC 786-O cells rather than LUAD models. Therefore, these screening data should be interpreted as exploratory evidence supporting a potential association between RRM2 and ferroptosis-related vulnerability, rather than as LUAD-specific mechanistic proof. Future studies using LUAD-specific CRISPR screening or genetic-dependency models are warranted to further validate this relationship. Third, the in vivo combination experiment did not include a concurrent osalmid monotherapy arm. Although we evaluated osalmid monotherapy in a separate CDX experiment and observed dose-dependent tumor growth inhibition, the absence of a concurrent osalmid-only group in the combination experiment precluded direct comparison among osalmid alone, AST2818 alone, and the combination. Therefore, the in vivo combination results should be interpreted as showing enhanced AST2818 efficacy with osalmid co-treatment, rather than as formal evidence of in vivo synergy. Although EGFR-mutant cell lines appeared more sensitive to osalmid in our cell-line panel, we acknowledge that the current data do not establish strict EGFR-mutant specificity. Future studies using larger panels of EGFR-mutant and EGFR-wild-type LUAD models, together with systematic analyses of RRM2 expression and drug-combination synergy, are warranted. Fifth, the clinical relevance of the osalmid concentrations used in vitro remains to be fully established. In this study, the osalmid concentration range was used to generate dose-response curves and determine IC50 values, while the combination experiments used IC10 concentrations to test whether low-dose osalmid could enhance EGFR-TKI responses. Although osalmid/4-hydroxysalicylanilide has been evaluated in a phase I clinical trial in MM with a favorable safety profile and preliminary clinical activity, whether comparable plasma or intratumoral exposures can be achieved in patients with LUAD remains uncertain. Therefore, future pharmacokinetic, pharmacodynamic, toxicity, and dose-optimization studies are needed before clinical translation. In the future, we still need to conduct further tests on the basis of in vitro experiments. Osalmid is not approved for the treatment of malignancies; thus, we could not obtain posttreatment samples from patients to measure RRM2 expression.

Conclusions

Osalmid may inhibit the progression of LUAD and enhance EGFR-TKI responses through RRM2-associated mechanisms. Functional rescue experiments using Fer-1 suggest that ferroptosis contributes to osalmid-induced growth inhibition, although the precise regulatory mechanism requires further validation. These findings support further investigation of osalmid-based combinations, particularly in EGFR-mutant or RRM2-dependent LUAD contexts. Further pharmacokinetic, pharmacodynamic, and biomarker-guided validation is warranted before clinical translation.

Acknowledgments

The abstract of this submission was accepted for poster presentation in IASLC 2024 World Conference on Lung Cancer, at San Diego (DOI: 10.1016/j.jtho.2024.09.564). We appreciate the help in bioinformatic analysis from the staff of OE Biotech Co., Ltd, Shanghai, China.

Footnote

Reporting Checklist: The authors have completed the MDAR and ARRIVE reporting checklists. Available at https://tlcr.amegroups.com/article/view/10.21037/tlcr-2026-0257/rc

Data Sharing Statement: Available at https://tlcr.amegroups.com/article/view/10.21037/tlcr-2026-0257/dss

Peer Review File: Available at https://tlcr.amegroups.com/article/view/10.21037/tlcr-2026-0257/prf

Funding: This work was supported by the National Natural Science Foundation of China (No. 82272943), Zhejiang Provincial Cancer Hospital Cultivation Fund (Nos. PY2024064; PY2024034), and Zhejiang Provincial Medical Health Science and Technology Project (No. 2025KY695).

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://tlcr.amegroups.com/article/view/10.21037/tlcr-2026-0257/coif). The authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. The study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. Animal experiments were performed under a project license (No. K22-366) granted by the Ethical Review Committee and Laboratory Animal Welfare Committee of Shanghai Pulmonary Hospital, in compliance with the European Community Council Directive of 24 November 1986 for the care and use of animals.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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