Suppression of the SLC7A11/glutathione axis causes synthetic lethality in KRAS-mutant lung adenocarcinoma

Abstract

Oncogenic KRAS is a major driver in lung adenocarcinoma (LUAD) that has yet to be therapeutically conquered. Here we report that the SLC7A11/glutathione axis displays metabolic synthetic lethality with oncogenic KRAS. Through metabolomics approaches, we found that mutationally activated KRAS strikingly increased intracellular cystine levels and glutathione biosynthesis. SLC7A11, a cystine/glutamate antiporter conferring specificity for cystine uptake, was overexpressed in patients with KRAS-mutant LUAD and showed positive association with tumor progression. Furthermore, SLC7A11 inhibition by either genetic depletion or pharmacological inhibition with sulfasalazine resulted in selective killing across a panel of KRAS-mutant cancer cells in vitro and tumor growth inhibition in vivo, suggesting the functionality and specificity of SLC7A11 as a therapeutic target. Importantly, we further identified a potent SLC7A11 inhibitor, HG106, that markedly decreased cystine uptake and intracellular glutathione biosynthesis. Furthermore, HG106 exhibited selective cytotoxicity toward KRAS-mutant cells by increasing oxidative stress- and ER stress-mediated cell apoptosis. Of note, treatment of KRAS-mutant LUAD with HG106 in several preclinical lung cancer mouse models led to marked tumor suppression and prolonged survival. Overall, our findings reveal that KRAS-mutant LUAD cells are vulnerable to SLC7A11 inhibition, offering potential therapeutic approaches for this currently incurable disease.

Keywords: Cancer; Drug therapy; Oncology.

Figure 1. Mutant KRAS drives glutathione metabolism reprogramming.

(A) Heatmap showing significantly differently expressed metabolites (P < 0.05) between HPNE and HPNE/KRAS groups. Values are scaled as indicated (2 to −2) (n = 5). (B) The top 10 enriched pathways from integrated pathway analysis of significantly changed metabolites. The P value cutoff was 0.05 and represents the significance of enrichment of the pathway. (C) Illustration of the GSH metabolism pathway (left) and the relative levels of cystine, glutamate, and glutathione (GSH; right) (n = 5). ASC, alanine-serine-cysteine transporter; DP, dipeptidase; GGT, γ-glutamyl transpeptidase; GSSG, glutathione disulfide; MRP, multidrug resistance–associated protein; TRR1, thioredoxin reductase 1; GCL, glutamate-cysteine ligase; GS, glutamine synthetase; γ-GC, γ-glutamylcysteine. (D) Cystine, GSH, and ROS levels quantified in KRAS isogenic cell lines. Na⁺-independent [¹⁴C]-cystine uptake was analyzed by a scintillation counter. The intracellular GSH content was measured using a GSH/GSSG-Glo assay kit. For the determination of ROS production, the cells were loaded with DCFH-DA, and fluorescence intensity was measured by flow cytometry. The levels in KRAS WT cells were defined as 100%. Results shown are representative of 3 independent experiments. Data are represented as mean ± SD of biological triplicates. **P < 0.01, ***P < 0.001 by unpaired, 2-tailed Student’s t tests.

Figure 2. SLC7A11 is overexpressed in KRAS-mutant LUAD.

(A) mRNA levels of SLC7A11 and NRF2. (B) Protein levels of SLC7A11 and Nrf2. For each cell pair, SLC7A11 and Nrf2 are contemporaneous immunoblots that run in parallel from the same biological replicate. (C) SLC7A11 and Nrf2 expression in normal lung tissues (N) and tumors (T) from LSL-Kras^G12D mice (n = 4). p-ERK was stripped, and the same membrane was then immunoblotted for total ERK. (D) Nrf2 and SLC7A11 expression in mouse embryonic fibroblasts (MEFs). MEFs were generated from LSL-Kras^G12D mice and induced by adenovirus-Cre (Ad-cre) for 48 hours (n = 3). p-ERK and total ERK are contemporaneous immunoblots that run in parallel from the same biological replicate. (E) Nrf2 and SLC7A11 expression upon KRAS silencing. For individual cell lines, KRAS, SLC7A11, and Nrf2 are contemporaneous immunoblots that run in parallel from the same biological replicate. (F) SLC7A11 suppression upon genetic depletion of Nrf2. Nrf2-sg1 and Nrf2-sg2 represent 2 individual small guide RNAs targeting Nrf2 for editing. (G and H) SLC7A11 and Nrf2 expression upon blockade of KRAS signaling at the mRNA (G) and protein levels (H). A549 cells were treated with LY294002 (1 μM, PI3K inhibitor), afuresertib (5 μM, Akt inhibitor), dabrafenib (20 μM, Raf inhibitor), AZD6244 (20 nM, MEK inhibitor), and RBC8 (10 μM, Ral GTPase inhibitor) for 48 hours, respectively. Results are shown as mean ± SD of biological triplicates. **P < 0.01 by unpaired, 2-tailed Student’s t tests. (I) SLC7A11 expression in clinical samples. Scale bars: 50 μm. (J) Box plots showing SLC7A11 IHC scores. The horizontal lines represent the median; the bottom and top of the boxes represent the 25th and 75th percentiles, respectively; and the vertical bars represent the range of the data. (K) Box plot of SLC7A11 expression in clinical tumors. Stage classification of LUAD refers to the TNM classification. *P < 0.05, **P < 0.01, ***P < 0.001 by 1-way ANOVA with Tukey’s multiple-comparisons test.

Figure 3. Silencing SLC7A11 selectively kills KRAS-mutant LUAD cells.

(A) Effects of SLC7A11 depletion on cell survival. Isogenic cells were transfected with SLC7A11 siRNAs (siSLC7A11) or a scrambled siRNA (siControl). The knockdown efficiency of siSLC7A11 was examined by immunoblotting. Cell viability was measured 72 hours after transfection. Relative cell viability was calculated by setting the values of the siControl-alone group as 100%. (B) Effects of SLC7A11 depletion on ROS production. Relative ROS production was calculated by setting the values of the siControl-alone group as 100%. (C) SLC7A11 depletion led to selective toxicity toward KRAS-mutant cancer cell lines. Six KRAS-mutant and 9 WT cancer cell lines (see Supplemental Table 3) were transfected with siSLC7A11 or a scrambled siRNA. The percent cell viability is relative to the untreated controls. (D) Inhibitory effects of sulfasalazine (SAS) on isogenic cell lines. (E) SAS treatment led to selective toxicity toward KRAS-mutant cancer cells. Seven KRAS-mutant and 7 WT cancer cell lines were treated with SAS for 72 hours. Dots indicate IC₅₀ value of each cell line. (F) Colony formation of A549 cells after SAS treatment. A549 cells were plated in 6-well plates and treated with the indicated concentrations of SAS for 7 days. The relative number of colonies was calculated by normalization to untreated group as 100%. Scale bar: 0.5 cm. (G) Effect of SAS on A549 cell soft agar colony formation. A549 cells were uniformly dispersed in agar and treated with the indicated concentrations of SAS for 21 days. The medium containing SAS was changed twice a week. At the end of the experiment, the colonies were photographed. Scale bar: 2 mm. All data are representative of 3 independent experiments and shown as mean ± SD of biological triplicates. *P < 0.05, **P < 0.01, ***P < 0.001 by unpaired, 2-tailed Student’s t tests.

Figure 4. Sulfasalazine leads to tumor regression in vivo.

(A) Schematic illustration of the LSL-Kras^G12D allele and drug treatment protocol. LSL-Kras^G12D mice were induced with adenovirus-Cre. After a 12-week induction, mice were treated with vehicle, trametinib (1 mg/kg), or sulfasalazine (250 mg/kg) for an additional 4 weeks. P1 and P2, qPCR forward and reverse primers, respectively. (B) Representative images of tumors from LSL-Kras^G12D mice. Animals were scanned by microCT. Red arrows indicate lung tumors, and yellow asterisks indicate heart. (C) Box plots showing the tumor volumes at the endpoint of the indicated treatments based on microCT (n = 5). The horizontal lines representing the median; the bottom and top of the boxes represent the 25th and 75th percentiles, respectively; and the vertical bars represent the range of the data. All data are shown as mean ± SD. **P < 0.01 by 1-way ANOVA with Tukey’s multiple-comparisons test. (D) Kaplan-Meier survival curves for LSL-Kras^G12D mice after the indicated treatments (n = 8). Med., median. *P < 0.05 by log-rank tests.

Figure 5. Identification of HG106 as a potent SLC7A11 inhibitor.

(A) Chemical structure of HG106. (B and C) HG106 dose-dependently inhibited cystine uptake and GSH level. A549 and H441 cells were treated with HG106 and sulfasalazine (1 mM). Relative cystine uptake and GSH levels were calculated by setting the values of the vehicle control group as 100%. (D) Metabolic pathway enrichment in A549 cells after HG106 treatment. A549 cells were treated with 5 μM HG106 for 6 hours. Cell lysates were subjected to metabolomic profiling. For metabolite pathway enrichment analysis, subsets of significantly affected metabolites were chosen. The bar plot shows the top 10 enriched pathways (n = 4). (E) Significantly changed metabolites involved in GSH biosynthesis are shown in the heatmap (n = 4). Changes in cystine and glutathione between the vehicle control– and HG106-treated groups are shown according to the metabolomic data. (F) Effect of β-mercaptoethanol (β-ME, 100 μM) and l-cysteine (5 mM) on HG106-induced cell death in H441 cells. H441 cells were treated with an HG106 concentration gradient with or without β-ME and l-cysteine for 72 hours, and cell viability was measured. (G) Effect of SLC7A11 knockdown by RNA interference on HG106-induced cell death. H441 cells were treated with the indicated concentrations of HG106 72 hours after transfection with SLC7A11 siRNAs or a scrambled siRNA. Relative cell viability was calculated by setting the values of the siControl group as 100%. All data are representative of at least 2 independent experiments, and shown as mean ± SD of biological triplicates. *P < 0.05, **P < 0.01, ***P < 0.001 by 1-way ANOVA with Tukey’s multiple-comparisons test (B, C, and G) or by unpaired, 2-tailed Student’s t tests (E).

Figure 6. HG106 preferentially decreases the viability of KRAS-mutant LUAD cells.

(A) 3D plot showing ROS production. A549 cells were treated with H₂O₂ for 1 hour or different concentrations of HG106 for 6 hours. (B) Effect of NAC (10 mM) on HG106-induced cell death. A549 cells were treated with HG106 alone or in combination with NAC for 72 hours, and cell viability was measured. Survival data for the HG106 group and the HG106+NAC group were normalized to the untreated control group and NAC-alone group, respectively. Cell survival was also determined by calcein-AM staining (right; green, viable cells). Scale bar: 50 μm. (C) Basal mitochondrial oxygen consumption rate (OCR) changes in HG106-treated A549 cells. OCR values were normalized to sulforhodamine staining. (D) MMP change in HG106-treated A549 cells. CCCP, carbonyl cyanide 3-chlorophenylhydrazone. (E) Mitochondria morphology. Red arrowheads indicate swelling of mitochondria. Scale bar: 0.5 μm. (F) HG106 activated ER stress–related markers. A549 cells were treated with HG106 and sulfasalazine (1 mM) for 24 hours. Immunoblots were contemporaneous and run in parallel from the same biological replicate. (G) Effect of HG106 on cell viability of KRAS isogenic cells. (H) HG106 selective kills KRAS-mutant cancer cells. A panel of KRAS mutant (n = 18) and WT KRAS (n = 8) cancer cell lines (see Supplemental Table 9) were treated with HG106 for 72 hours. Dots indicate IC₅₀ value of each cell line. (I) A549 cell apoptosis induced by HG106. (J) The effect of HG106 on A549 cell colony formation. Colony number was normalization to the control. Scale bar: 0.5 cm. All data are representative of at least 3 independent experiments and shown as mean ± SD of biological triplicates. **P < 0.01, ***P < 0.001 by 1-way ANOVA with Tukey’s multiple-comparisons test (C, D, I, and J) or by unpaired, 2-tailed Student’s t tests (H).

Figure 7. In vivo responses of KRAS-mutant LUAD to HG106.

(A) A549 xenograft growth curve (n = 8). Mean weights of tumors on day 26 are shown in the inset. (B) Growth curve of patient-derived xenografts (n = 8). Mean tumor weight on day 20 is shown in the inset. Values are expressed as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 by 1-way ANOVA with Tukey’s multiple-comparisons test. (C) Representative tumor images of LSL-Kras^G12D/+;Trp53^fl/fl (KP) mice. KP mice bearing established tumors were treated with HG106. Animals were scanned by microCT during treatment. (D) Tumor volumes in KP mice (n = 6~7). Tumor volumes at the endpoint of the indicated treatments are shown as box plots. Data are shown as mean ± SD. The horizontal lines represent the median; the bottom and top of the boxes represent the 25th and 75th percentiles, respectively; and the vertical bars represent the range of the data. ***P < 0.001 by unpaired, 2-tailed Student’s t tests. (E) Kaplan-Meier survival curves of KP mice (n = 6~7). **P < 0.01 by log-rank tests.

Figure 8. Schematic representation of KRAS-regulated glutathione metabolic reprogramming and the selective killing of KRAS-mutant cells by HG106.

(A) Activated KRAS induces SLC7A11 overexpression through activation of transcription factor Nrf2. As a consequence, SLC7A11 takes up a high level of cystine from the extracellular environment to generate more glutathione (GSH), which plays an important role in sustaining the oxidative balance in KRAS-mutant cells. Ultimately, KRAS-mutant cells are highly dependent on SLC7A11-mediated GSH biosynthesis. (B) When treated with HG106, a potent SLC7A11 inhibitor, cellular oxidant-antioxidant homeostasis is severely disrupted, coupled with significant mitochondrial dysfunction and ER stress, ultimately leading to massive cell death of KRAS-mutant cancers. Activated KRAS is indicated by an asterisk. Red arrows indicate pathway activation, and gray arrows indicate pathway inhibition.