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Concurrent loss of LKB1 and KEAP1 enhances SHMT-mediated antioxidant defence in KRAS-mutant lung cancer

Nature Metabolism volume 6, pages 1310–1328 (2024)Cite this article

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Abstract

Non-small-cell lung cancer (NSCLC) with concurrent mutations in KRAS and the tumour suppressor LKB1 (KL NSCLC) is refractory to most therapies and has one of the worst predicted outcomes. Here we describe a KL-induced metabolic vulnerability associated with serine–glycine-one-carbon (SGOC) metabolism. Using RNA-seq and metabolomics data from human NSCLC, we uncovered that LKB1 loss enhanced SGOC metabolism via serine hydroxymethyltransferase (SHMT). LKB1 loss, in collaboration with KEAP1 loss, activated SHMT through inactivation of the salt-induced kinase (SIK)–NRF2 axis and satisfied the increased demand for one-carbon units necessary for antioxidant defence. Chemical and genetic SHMT suppression increased cellular sensitivity to oxidative stress and cell death. Further, the SHMT inhibitor enhanced the in vivo therapeutic efficacy of paclitaxel (first-line NSCLC therapy inducing oxidative stress) in KEAP1-mutant KL tumours. The data reveal how this highly aggressive molecular subtype of NSCLC fulfills their metabolic requirements and provides insight into therapeutic strategies.

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Fig. 1: LKB1 loss in the context of oncogenic KRAS mutations enhances serine and glycine biosynthesis in NSCLC.
Fig. 2: The LKB1–SIK axis inhibits SHMT expression in KRAS-mutant NSCLC.
Fig. 3: SHMT is required for KL NSCLC survival.
Fig. 4: KL cells require SHMT for redox homeostasis.
Fig. 5: SHMT suppression inhibits KL NSCLC tumour growth.
Fig. 6: SHIN2 and PTX combination treatment more effectively inhibits KL NSCLC tumour growth.
Fig. 7: Working model.

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Data availability

Data have been deposited in public databases. ATAC-seq data are available under the accession number GSE265860. Source data are provided with this paper. All materials used in this study are available either commercially or through collaboration, as indicated. GUI Natural Abundance Correction used for GC–MS data analysis are available on GitHub.(http://mzmine.github.io/, https://github.com/lparsons/accucor). Source data are provided with this paper.

Change history

  • 08 July 2024

    In the version of the article initially published, an earlier, incorrect version of Supplementary Table 9 was uploaded and has now been updated.

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Acknowledgements

We thank J. D. Rabinowitz (Princeton University) for providing SHIN2, and R. J. DeBerardinis (UTSW), P. M. Wehn (BridgeBio Pharma) and C.-H. Lee (YSM) for feedback. Jiyeon Kim (YSM) is supported by a Career Enhancement Program Grant from the Yale SPORE in Lung Cancer P50CA196530, the NCI 1K22CA226676-01A1, NCI 1R37CA285640-01A1, American Lung Association (IA-828202) and the American Cancer Society (RSG-21-153-01-CCB). C.C. was supported by NIH P01HL160469. G.S.D. was supported by NCI R00CA215307. K.L. is supported by the Fundamental Research Funds for the Central Universities (BMU2021YJ073). James Kim (UTSW) is supported by NCI 1R01CA258684, 1R01CA196851 and P50CA070907. D.B.A. is supported by P30CA177558. B.F. is supported by the NCI R00CA237724.

Author information

Authors and Affiliations

  1. Department of Urology, Yale School of Medicine, New Haven, CT, USA

    Hyun Min Lee, Isaac Y. Kim & Jiyeon Kim

  2. Department of Biochemistry and Molecular Genetics, College of Medicine, University of Illinois at Chicago, Chicago, IL, USA

    Nefertiti Muhammad, Elizabeth L. Lieu, Kenneth Joves, Constantinos Chronis & Hyosun Seo

  3. Children’s Medical Center Research Institute, UT Southwestern Medical Center, Dallas, TX, USA

    Feng Cai

  4. Department of Biochemistry and Biophysics, School of Basic Medical Sciences, Peking University Health Science Center, Beijing, China

    Jiawei Mu & Kailong Li

  5. Department of Urology, School of Medicine, Kyungpook National University, Kyungpook National University Chilgok Hospital, Daegu, Korea

    Yun-Sok Ha

  6. Department of Biochemistry, University of Utah, Salt Lake City, UT, USA

    Guoshen Cao & Gregory S. Ducker

  7. Hamon Center for Therapeutic Oncology Research, UT Southwestern Medical Center, Dallas, TX, USA

    Chamey Suchors & James Kim

  8. Barer Institute, Philadelphia, PA, USA

    Kellen Olszewski & William R. Ewing

  9. Department of Population and Data Sciences, UT Southwestern Medical Center, Dallas, TX, USA

    Ling Cai

  10. Department of Pathology and Laboratory Medicine, University of Kentucky College of Medicine, Lexington, KY, USA

    Derek B. Allison & Sara E. Bachert

  11. Faculty of Pharmaceutical Sciences, University of British Columbia, Vancouver, BC, Canada

    Harvey Wong

  12. Department of Medicine-Hematology and Oncology, University of Chicago, Chicago, IL, USA

    Brandon Faubert

  13. Department of Cellular and Molecular Physiology, Yale School of Medicine, New Haven, CT, USA

    Jiyeon Kim

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  1. Hyun Min Lee
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Contributions

Jiyeon Kim and H.M.L. conceived the research project, designed experiments and wrote the paper. Jiyeon Kim, H.M.L., N.M., E.L.L. and H.S. performed research and/or contributed to analysis and discussions. B.F. ran GC–MS experiments and analysed data. L.C. analysed human NSCLC gene-expression data. K.J. and C.C. performed ATAC-seq and contributed to analysis and discussions. G.C. and G.S.D. designed, performed and analysed 2H labelling assays. J.M. and K.L. designed, performed and analysed ChIP–qPCR experiments. C.S. and James Kim performed orthotopic mouse lung experiments and analysed the data. D.B.A. and S.E.B. analysed TMA. F.C. developed LC–MS/MS methods to measure GSH biosynthesis rates and analysed the data. K.O. and W.R.E. provided SHIN2. K.O. and H.W. performed pharmacokinetic analysis and synergy calculation. Y.-S.H. and I.Y.K. calculated P value for the assays.

Corresponding author

Correspondence to Jiyeon Kim.

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Competing interests

W.R.E. and K.O. are employees of the Barer Institute, which is engaged in the clinical development of SHMT inhibitors. H.W. is a consultant for the following pharmaceutical companies: Genentech, Denali Therapeutics, Alector, Surrozen, Cleave Therapeutics, ORIC Pharmaceuticals, Barer Institute, Vincerx, Chinook and Cresenta. J.K. (UTSW) is on the scientific advisory board for Sanofi and is a consultant for Pulmatrix. The other authors declare no competing interests.

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Extended data

Extended Data Fig. 1 LKB1 loss in the context of oncogenic KRAS mutations enhances serine and glycine biosynthesis in NSCLC.

a, Metabolite set enrichment analysis using metabolome in an isogenic pair of A549 cells. SGOC metabolism-related pathways are in pink. Dots are colored by enrichment FDR values. b, Volcano plot presenting metabolites whose levels are significantly changed in A549-EV cells compared with A549-LKB1 cells. Blue dots represent metabolite depletion (log2 < -1) and red dots represent metabolite accumulation (log2 > 1) in A549-EV cells. non-SGOC metabolism/folate-methionine cycle intermediates are labeled in grey. c, serine and glycine pool size in H460-EV and -LKB1 cells used for [U-13C]glucose labeling in Fig. 1f. d and e, 13C labeling in glycine in the same set of samples in Fig. 1f (H460 and A549 (d) and H1373 (e) cells). f, Effect of LKB1 on serine m + 2/m + 1 from [U-13C]serine labeling in H460-EV and -LKB1 or H1373-shGFP and -shLKB1. g, Abundance of LKB1 in isogenic pairs of KL NSCLC cells. Vinculin was used as a loading control. h and i, 13C labeling in serine (h) and glycine (i) in a panel of NSCLC cells with different oncogenotypes (K, KRAS mutants; KL, KRAS/LKB1 co-mutants; L, LKB1 mutants)(n = 3 per cell line) cultured with [U-13C]glucose for 6 hours. j, Abundance of LKB1 in EV-, WT LKB1- and KD LKB1-H460 cells. CPS1 was used as a marker for LKB1 activity as reported previously22 and Actin was used as a loading control. k, 13C labeling in serine in cells used in Fig. 1i and Supplementary Fig. 1l. l, 13C labeling in glycine in three isogenic pairs of cells cultured with [U-13C]serine for 2 hours. Data are the mean ± s.d. of three independent cultures. Statistical significance was assessed using a two-tailed t-test (c to f, k and l) and a one-way ANOVA (h and i) with the data from each oncogenotype as a group. Metabolomics analysis was done once. All other experiments were repeated three times or more.

Source data

Extended Data Fig. 2 LKB1 loss increases gene expression in serine and glycine biosynthesis pathways.

a and d, Gene expression of serine/glycine biosynthesis enzymes in EV- and LKB1- expressing KL cells (H460 and H2122)(a) and shGFP- and shLKB1-expressing K cells (Calu-6, H1373, and Calu-1)(d) (n = 3). b and c, Abundance of serine/glycine biosynthesis proteins in EV- and LKB1-expressing KL cells (H460 and H2122)(b) and shGFP- and shLKB1- expressing K cells (Calu-1 and H1373)(c). CPS1 was used as a marker of LKB1 expression22 and Vinculin was used as a loading control. e and f, Gene expression (e) and protein abundance (f) of serine/glycine biosynthesis enzymes in K cells with AMPK silencing. g and h, Gene expression (g) and protein abundance (h) of serine/glycine biosynthesis proteins in EV- and LKB1-expressing KL cells with AMPK silencing. i and j, Gene expression (i) and protein abundance (j) of serine/glycine biosynthesis enzymes in EV- and LKB1- expressing KL cells with Torin treatment (100 nM). k and l, Gene expression (k) and protein abundance (l) of serine/glycine biosynthesis enzymes in EV- and LKB1-expressing KL cells with SIK1 and 3 silencing. Due to lack of good antibodies against SIK1, CRTC2 was used as a surrogate marker for SIK1 silencing. m and n, Gene expression (m) and protein abundance (n) of serine/glycine biosynthesis enzymes in K cells with SIK1 and 3 silencing. CRTC2 was used as a surrogate marker for SIK silencing. o, Gene expression of serine/glycine biosynthesis enzymes in EV- and LKB1-expressing H460 with SIK1 and 3 co-deletion. PEPCK1 and 2 were used as a surrogate marker for SIK1/3 deletion. p, Gene expression of serine/glycine biosynthesis enzymes in EV- and LKB1-expressing H1355 (left) and H2122 (right) with SIK1 deletion. q, Gene expression of SHMT1 and 2 in SIK1-deleted, EV- and LKB1-expressing H460 with or without SIK1 re-expression. Data are the mean ± s.d. of three independent cultures. Statistical significance was assessed using a two-tailed t-test (a, d, e, i and m) and a one-way ANOVA (g, k, o, p and q). All experiments were repeated three times or more.

Source data

Extended Data Fig. 3 SIK1 inhibits SHMT expression through NRF2-MAFK binding to the SHMT promoter region.

a, Chromatin signatures at the SHMT1 and 2 loci in A549 cells. Promoter regions are shaded. b, Chromatin occupancy of NRF2 (left) and MAFK (right) on SHMT2 promoter in EV- and LKB1-expressing H460 cells treated with HG-9-91-01 (n = 5). c, Abundance of MAFK in A549 and H460 cells with MAFK silencing used in Fig. 2h. Vinculin was used as a loading control. d, Promoter design for luciferase assay with wild type (SHMT1 and 2 WT) and ARE-mutated (SHMT1 and 2 mt) promoters of SHMT. ChIP–seq data (H3K27ac ChIP: GSM1003578; MAFK ChIP: GSE127353; H3K4me3 ChIP: GSM2421528) of SHMT1 and 2 loci in A549 cells were used to design the promoter. e, Luciferase assay with ARE-WT and ARE-mt promoters of SHMT1 and 2 in 293 T cells with SIK1 silencing. f, Gene expression of PEPCK1 and SIK1 in SIK1-silenced 293 T cells. PEPCK1 was used as surrogate markers for SIK1 silencing (n = 3). g, Luciferase assay with ARE-WT and ARE-mt promoters of SHMT1 and 2 in 293 T cells with SIK1 overexpression. h, Abundance of FLAG-SIK1 protein in 293 T cells used in g. Vinculin was used as a loading control. i, Gene expression of PEPCK1 and SIK1 with SIK1 deletion in Calu-6. PEPCK1 was used as a surrogate marker for SIK1 deletion (n = 3). j, 13C labeling in glycine in KL cells with A769662 treatment (250 µM) cultured with [U-13C]serine for 2 hours (n = 3). k, Abundance of phospho-ACC in cells used in j. Actin was used as a loading control. l, Left, 13C labeling in glycine in an isogenic pair of H460 cells with AMPKα1/2 deletion cultured with [U-13C]serine for 2 hours (n = 3). Right, Abundance of LKB1 and AMPK in the same set of cells. Vinculin was used as a loading control. m, 13C labeling in glycine in K cells with HG-9-91-01 cultured with [U-13C]serine for 2 hours (n = 3). n, Effect of SIK suppression on m2/m1 serine ratio in H1373 cells. o, 13C labeling in glycine in Calu-6 cells with SIK1 and 3 silencing cultured with [U-13C]serine for 2 hours (n = 3). p, Effect of SIK1 and 3 silencing on m2/m1 serine ratio in Calu-6 cells. Data are the mean ± s.d. of independent cultures indicated in each panel. Statistical significance was assessed using a two-tailed t-test (f, i, j, m to p) and a one-way ANOVA (b, e, g, and l). j and luciferase assay in e were performed twice and all other experiments were repeated three times or more.

Extended Data Fig. 4 KL NSCLC requires SHMT for survival.

a and b, Abundance of SHMT1 (a) and 2 (b) protein in K (black) and KL (pink) cell lines transfected with a control siRNA or siRNA directed against SHMT1 or 2. Actin and Vinculin were used as a loading control. c, Abundance of SHMT1 (top) and 2 (bottom) protein in EV- and LKB1-expressing H460 cells with SHMT1/2 silencing. Vinculin was used as a loading control. d, Abundance of SHMT1 and SHMT2 protein used in Fig. 3c. Vinculin was used as a loading control. e, Abundance of SHMT2 protein in cells used in Supplementary Fig. 4k. Vinculin was used as a loading control. f and g, Effect of SHMT silencing on KL cell death. Representative dot plots of FACS results/cell lines (f) and quantified data (g) (n = 3). h, Abundance of SHMT1 (top) and 2 (bottom) protein used in Fig. 3d. Vinculin was used as a loading control. i, Left, Effect of SHMT1 (left) and SHMT2 (right) KO on anchorage-independent growth of KL cells (n = 3). Right, Abundance of SHMT1 and 2 in the cells. Vinculin was used as a loading control. j, Effect of siSHMT on an isogenic pair of H460 cell growth (n = 3). k, Effect of SHMT2 re-introduction on anchorage-independent growth of SHMT2 KO H460 cells (n = 6 for control, n = 3 for other conditions). l, Effect of SHIN1 treatment on cell viability in KL cells (n = 5). m, Effect of SHIN1 and 2 on anchorage-independent growth of H460 and A549 (n = 3). n, Effect of SHIN1 and 2 on cell death (n = 3). KL cells are in red-orange whereas K cells are in blue-green. o and p, 13C labeling in glycine in isogenic KL cells with SHIN1 (o) and SHIN2 (p) treatment cultured with [U-13C]serine for 2 hours (n = 3). q, Dose-response curves for two KL PDAC cells with KEAP1 deletion after 72 hours exposure to SHIN1 (n = 6). r, Effect of LKB1 silencing and KEAP1 deletion on siSHMT-induced viability loss in Calu-1 cells (n = 5). s, Effect of AMPK deletion on siSHMT-induced viability loss in two KL cells (n = 6). t, Effect of SHIN2 treatment on cell viability in SIK1 CA-expressing H460 cells (n = 3). u, Abundance of SHMT1, 2 and FLAG protein in the cells used in Fig. 3m. Vinculin was used as a loading control. Data are the mean ± s.d. of independent cultures indicated in each panel. Statistical significance was assessed using a two-tailed t-test (i, j, l and m) and a one-way ANOVA (g, k, n, o, p, and r to t). In t, *p < 0.05 compared to H460-EV with vehicle; #p < 0.05 compared to H460-EV with SHIN2; $p < 0.05 compared to H460-SIK1 CA with vehicle. All experiments were repeated three times or more.

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Extended Data Fig. 5 KL NSCLC requires SHMT for survival.

Abundance of proteins in Fig. 3i (a), 3j (b, right), Supplementary Fig. 4r (b, left), Supplementary Fig. 4n (c, Tubulin was used as a loading control), Supplementary Fig. 4s (d), Fig. 3l (e), Fig. 3m (f). g, 13C labeling in glycine in EV-, LKB1- and KEAP1-expressing H460 cells cultured with [U-13C]serine for 2 and 6 hours (n = 3). h, Effect of SHMT suppression on NADP+/NADPH ratio in EV-, LKB1- and KEAP1-expressing H460 cells (n = 3). i, Abundance of KEAP1 and LKB1 in the cells used in Supplementary Fig. 5g. Vinculin was used as a loading control. Statistical significance was assessed using a two-way (g) and a one-way (h) ANOVA. All experiments were repeated twice or more.

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Extended Data Fig. 6 KL NSCLC requires SHMT for redox balance.

a, Effect of metabolic intermediates in SGOC metabolism on SHMT silencing-induced viability loss (n = 6). b, Effect of antioxidants on SHIN1-induced viability loss (n = 5). c, Effect of glycine and formate on SHMT silencing-induced viability loss (n = 5). d, Effect of SHMT silencing on cellular ROS (n = 3). e, Effect of SHIN1 treatment on cellular ROS (n = 3). f, Effect of LKB1 on SHIN1-induced ROS accumulation in two isogenic pair cell lines (n = 3). g, Effect of LKB1 on SHIN1-induced alteration of NADP + /NADPH ratio in two isogenic pair cell lines (n = 3). h, Effect of LKB1 on SHIN1-induced alteration of GSSG/GSH ratio in two isogenic pair cell lines (n = 3). i and j, Relative NADP+ and NADPH levels in SHMT1 (i) and 2 (j) KO KL cells used in Fig. 4g. k and n, Relative NADP+ and NADPH levels in H460 cells (k) and H1373 cells (n) used in Supplementary Fig. 6g. l and m, Abundance of SHMT1 (l) and 2 (m) protein in cells used in Fig. 4f. Vinculin was used as a loading control. o and p, Relative GSSG and GSH levels in H460 cells (o) and H1373 cells (p) used in Supplementary Fig. 4h. q, 13C labeling in GSH in an isogenic pair of H460 cells with SHMT silencing cultured with [U-13C]serine for 6 hours (n = 3). r, Effect of MTHFD1 and 2 silencing on cell viability in various K and KL cells (n = 6). s, Abundance of MTHFD1 and 2 protein used in Supplementary Fig. 6r. Vinculin was used as a loading control. t, Time course 13C labeling (left) and 2H labeling (right) in proline in an isogenic pair of H460 cells cultured with [U-13C]glutamine (left) and [2,3,3-2H]serine (right) (n = 3). u, Effect of NADK2 silencing on cell viability in H1355 cells (n = 6). v, Abundance of NADK2 and LKB1 in H460 cells used in Fig. 4k and H1355 used in Supplementary Fig. 6u. Vinculin was used as a loading control. Data are the mean ± s.d. of independent cultures indicated in each panel. Statistical significance was assessed using a one-way ANOVA (a to k, n to q, and u), and a two-way ANOVA (t). In a, * compared to each siCtrl condition. In b, *p < 0.05 compared to no treatment; #p < 0.05 compared to NAC treatment. In c, *p < 0.05 compared to no treatment; #p < 0.05 compared to glycine treatment; $p < 0.05 compared to formate treatment. In q, *p < 0.05 compared to EV-siCtrl; #p < 0.05 compared to EV-siSHMT1/2; $p < 0.05 compared to LKB1-siCtrl. In u, *p < 0.05 compared to EV-siCtrl; #p < 0.05 compared to LKB1-siCtrl. 13C and 2H labeling (q, t) was performed twice. All other experiments were repeated at least twice or more.

Source data

Extended Data Fig. 7 KL cells require SHMT for antioxidant defenses.

a, Abundance of HA-TPNOX and HA-mitoTPNOX in cells used in Fig. 4m and n. Vinculin was used as a loading control. b and c, Effect of G6PD (left), ME1 (middle), IDH1 (right) silencing on SHMT silencing-induced loss of viability in H2122 cells (b) and H1373 cells (c)(n = 3). d, Abundance of G6PD, ME1, IDH1, SHMT1 and 2 in cells used in Fig. 4o, and p. Vinculin was used as a loading control. e and f, Abundance of G6PD, ME1, IDH1, SHMT1 and 2 in cells used in Supplementary Fig. 7b (e) and c (f). Vinculin was used as a loading control. g and h, Effect of G6PDi (G6PD inhibitor, left), ME1i (ME1 inhibitor, middle), GSK321 (IDH1 inhibitor, right) on SHMT inhibition-induced viability loss in H460 cells (g) and H1373 cells (h)(n = 3). i and j, Effect of G6PDi (left), ME1i (middle), GSK321 (right) on SHMT inhibition-induced alteration of NADP+/NADPH ratio in H460 cells (i) and H1373 cells (j)(n = 3). k, Abundance of NADK2 used in Fig. 4l. Vinculin was used as a loading control. Data are the mean ± s.d. of independent cultures indicated in each panel. Statistical significance was assessed using a one-way ANOVA (b, c, g to j). In b and c, *p < 0.05 compared to EV-siCtrl; #p < 0.05 compared to EV-siSHMT1 + 2; $p < 0.05 compared to EV-siG6PD, ME1 or IDH1. In g to j, *p < 0.05 compared to DMSO; #p < 0.05 compared to SHIN2; $p < 0.05 compared to G6PDi, ME1i or GSK321. All experiments were repeated at least twice or more.

Source data

Extended Data Fig. 8 KL NSCLC requires SHMT for tumor growth in vivo.

a, Growth of H2122 WT (sgCtrl) or SHMT1 KO pools (left) and SHMT2 KO pools (right) xenografts. Relative tumor growth and SEM are shown for each group (n = 5 per group). b, Abundance of SHMT1 and 2 from xenograft tumors with SHMT1 or 2 KO H460 (pools, tumor growth data are shown in Fig. 5a) and H2122 cells (pools, tumor growth data are shown in Supplementary Fig. 8a). Vinculin was used as a loading control. c, Tumor bearing mice in Fig. 5c. d, Abundance of SHMT1 and 2 from xenograft tumors with SHMT1 or 2 KO Calu-6 and H1373 cells (pools, tumor growth data are shown in Fig. 5b). Vinculin was used as a loading control. e, Growth of shGFP- and shLKB1-expressing H1373 xenografts with SHIN1 (100 mg/kg, every day for 16 days); the arrow indicates when SHIN1 was first injected. Relative tumor growth and SEM are shown for each group (n = 5 per group). Data were normalized to first measurement. f, 13C labeling in serine (left) and glycine (right) in mice used in Supplementary Fig. 8e. g, Representative TUNEL staining images of tumor tissues from Fig. 5e. DAPI was used to stain DNA. Scale bars, 100μm. h, TUNEL+ cells in Supplementary Fig. 8g and total cells/tumor were quantified. i, Left, Representative DCFDA staining images of A549 tumors in the presence and absence of SHIN2 treatment. Right, DCFDA+ cells and total cells/tumor were quantified. Data were normalized to first measurement. Statistical significance was assessed using a two-tailed t-test (h and i), a one-way ANOVA (f), and a two-way ANOVA (a and e). a and d were performed twice. All other experiments are performed once.

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Extended Data Fig. 9 SHMT inhibition enhance therapeutic efficacy of PTX in KL NSCLC in vivo.

a, Growth of A549 xenografts with SHIN2 (100 mg/kg), BSO (20 μM), and SHIN2 plus BSO; the arrow indicates when SHIN2 was first injected. Relative tumor growth and SEM are shown for each group (n = 5 per group). b, Effect of SHIN2 and BSO on cellular ROS in vivo (tissues are from Supplementary Fig. 9a). c, The joint action of combination drug under each model (Response Additive results, Bliss Independence results, and Highest Agent results) is assessed using a combination index55. Index score above 0 is considered as a synergy. d, Time course of SHIN2 (left), and PTX (right) concentrations expressed in units of ng/mL of plasma. f, Growth of A549 xenografts treated with SHIN2 (100 mg/kg) plus PTX (10 mg/kg) in the presence and absence of NAC (1 g/L in drinking water); the arrows indicate when SHIN2 was first injected. Tumor volume and SEM are shown for each group (n = 5 per group). g, Left, Representative DCFDA staining images of tumor tissues from Supplementary Fig. 9f. DAPI was used to stain DNA. Scale bars, 100μm. Right, DCFDA+ cells and total cells/tumor were quantified. e, Growth of H460 (left) and Calu-6 (right) xenografts with SHIN2 (100 mg/kg), MTX (10 mg/kg), and SHIN2 plus MTX; the arrows indicate when SHIN2 was first injected. Relative tumor growth and SEM are shown for each group (n = 5 per group). Data were normalized to first measurement at day 6. h, Left, Dose-response curves for two murine KP and KPL NSCLC cells after 72 hours of exposure to SHIN1 (n = 6). Right, LKB1 status in these cells. Vinculin was used as a loading control. i, Left, Dose-response curves for murine KPL and KPLK NSCLC cells after 72 hours of exposure to SHIN2 (n = 6). Right, KEAP1 and NRF2 status in murine NSCLC cells. Vinculin was used as a loading control. j, Left, BLI signal intensity from H460 cells orthotopically grown with SHIN2 (100 mg/kg), PTX (10 mg/kg) or SHIN2 plus PTX; the arrow indicates when SHIN2 was first injected. Right, mouse body weight in each group. k, Left, Representative Ki67 staining images of orthotopic lung tumors. Scale bar, 2 mm. Right, Ki67+ tumor cells/lung was quantified. l, Left, Full-scan liver H&E images of lung orthotopic tumor xenografts. Scale bar, 2 mm. Right, Tumor area/liver was quantified. m, Left, Representative Ki67 staining images of liver metastases from orthotopic lung tumors. Scale bar, 2 mm. Right, Ki67+ tumor cells/liver was quantified. n, Left, Relative TUNEL staining images of orthotopic syngeneic lung tumors. Scale bar, 100μm. Right, TUNEL+ cells and total cells/tumor were quantified. Data were normalized to first measurement. Statistical significance was assessed using a one-way ANOVA (b, g, k to n) and a two-way ANOVA (a, e, f and j). In a and b, *p < 0.05 compared to vehicle; #p < 0.05 compared to BSO; $p < 0.05 compared to SHIN2. In g, *p < 0.05 compared to vehicle; #p < 0.05 compared to NAC alone; $p < 0.05 compared to SHIN2 plus PTX. j performed three times. All other experiments were performed once.

Extended Data Fig. 10 KL NSCLC requires SHMT for survival.

a and b, Kaplan−Meier plot associating SHMT1 (a) and SHMT2 (b) mRNA expression with survival. Dataset is from KM Plotter (http://kmplot.com/analysis/index.php?p=service&cancer=lung). c, Representative TMA staining for SHMT1 and 2 shown in Fig. 3h. Scale bars, 200μm. d, Scoring of SHMT1 and 2 expression in TMA samples. Scoring method is described in Methods.

Supplementary information

Supplementary Information

Supplementary Figs. 1 and 2: Gating strategy of flow cytometry analysis. Supplementary Fig. 1: Gating strategy of flow-cytometry analysis for PI/Annexin V-FITC staining (Extended Data Fig. 4f). Supplementary Fig. 2. Gating strategy of flow cytometry analysis for DCFDA (Extended Data Fig. 6f) and MitoSOX (Fig. 4n).

Reporting Summary

Supplementary Tables 1–12

Supplementary Table 1: Variable importance in the projection (VIP) analysis of metabolomic differences between K and KL NSCLC tissues. Primary data used in the analysis are from our previous report22. Data from TIC normalization (left) and tissue-weight normalization (right) are shown. Supplementary Table 2: Metabolomics in EV and LKB1-expressing A549 cells. A set of 113 metabolites was monitored in an isogenic pair of A549 cells (n = 4 per group). Data from TIC normalization (left) and protein-amount normalization (right) are both shown. Supplementary Table 3: VIP analysis of metabolomic differences between A549-EV and A549-LKB1 cells. Primary data used in the analysis are in Supplementary Table 2. Data from TIC normalization (left) and protein-amount normalization (right) are both shown. Supplementary Table 4: Metabolite pathway enrichment using human NSCLC metabolomics data. Key metabolic pathways altered in KL NSCLC compared with K NSCLC are shown. Under each metabolic pathway, metabolites detected in the metabolomics analysis are highlighted in red. Data from TIC normalization (top) and tissue-weight normalization (bottom) are both shown. Statistical significance of pathway enrichment analysis was assessed using a hypergeometric test26. The FDR was calculated using the Benjamini–Hochberg test. Supplementary Table 5: Metabolite pathway enrichment using A549 metabolomics data. Key metabolic pathways altered in A549-EV compared with A549-LKB1 are shown. Under each metabolic pathway, metabolites detected in the metabolomics analysis are highlighted in red. Data from TIC normalization (left) and protein-amount normalization (right) are both shown. Statistical significance of pathway enrichment analysis was assessed using a hypergeometric test26. The FDR was calculated using the Benjamini–Hochberg test. Supplementary Table 6: Statistical analysis of TCGA NSCLC data. Statistical significance was assessed using a one-way ANOVA followed by Tukey’s multiple-comparisons test. Supplementary Table 7: Expression score of LKB1, SHMT1 and SHMT2 in NSCLC TMA. Expression levels of SHMT1 and SHMT2 along with a comprehensive annotation of each sample are shown. The scoring system was based on intensity and percent staining; for SHMT1 and SHMT2, expression score was calculated by multiplying the percentage (0 = 0, 1 = 1–25%, 2 = 25–75%, 3 = >75%) by intensity (weak = 1, moderate = 2, strong = 3). 0 = negative; 1+ = score < 3, 2+ = 3 ≤ score <6, 3+ = score ≥ 6. Supplementary Table 8: Summary table of combination indices and global combination indices under each of the reference model in xenograft. Actual tumour volumes are shown in the left panel and combination indices from three different methods are shown in the right panel. The drug synergism was assessed using CombPDX55. Supplementary Table 9: Mutation status of LKB1, P53 and KEAP1 in cell lines used in this study. Supplementary Table 10: Mutation status of KRAS, STK11 (LKB1) and P53 in the TCGA cohort. Statistical significance for co-mutation frequency was assessed using a two-tailed Fisher’s exact test. Supplementary Table 11: Primer sequences for sgRNA, custom siRNA, ChIP–qPCR, RT–qPCR and overexpression and promoter sequences for luciferase assays used in this study. Supplementary Table 12: Information of antibodies and siRNAs (for example, catalogue numbers) used in this study.

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Lee, H.M., Muhammad, N., Lieu, E.L. et al. Concurrent loss of LKB1 and KEAP1 enhances SHMT-mediated antioxidant defence in KRAS-mutant lung cancer. Nat Metab 6, 1310–1328 (2024). https://doi.org/10.1038/s42255-024-01066-z

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