Distinct initiating events underpin the immune and metabolic heterogeneity of KRAS-mutant lung adenocarcinoma

doi:10.1038/s41467-019-12164-y

. 2019 Sep 13;10(1):4190.

doi: 10.1038/s41467-019-12164-y.

Distinct initiating events underpin the immune and metabolic heterogeneity of KRAS-mutant lung adenocarcinoma

Sarah A Best^{1

2}, Sheryl Ding^{1

2}, Ariena Kersbergen¹, Xueyi Dong^{2

3}, Ji-Ying Song⁴, Yi Xie^{3

5}, Boris Reljic⁶, Kaiming Li^{1

7}, James E Vince^{2

8}, Vivek Rathi⁹, Gavin M Wright¹⁰, Matthew E Ritchie^{3

11}, Kate D Sutherland^{12

13}

Affiliations

¹ Cancer Biology and Stem Cells Division, The Walter and Eliza Hall Institute of Medical Research, Parkville, VIC, 3052, Australia.
² Department of Medical Biology, The University of Melbourne, Parkville, VIC, 3052, Australia.
³ Epigenetics and Development Division, The Walter and Eliza Hall Institute of Medical Research, Parkville, VIC, 3052, Australia.
⁴ Division of Experimental Animal Pathology, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX, Amsterdam, The Netherlands.
⁵ School of Life Sciences, Fudan University, Shanghai, China.
⁶ Department of Biochemistry and Molecular Biology and The Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, Parkville, VIC, 3052, Australia.
⁷ School of Life Sciences, Nanjing University, Nanjing, 210023, China.
⁸ Inflammation Division, The Walter and Eliza Hall Institute of Medical Research, Parkville, VIC, 3052, Australia.
⁹ Department of Anatomical Pathology, St Vincent's Hospital, The University of Melbourne, Fitzroy, VIC, 3065, Australia.
¹⁰ Department of Surgery, St Vincent's Hospital, The University of Melbourne, Fitzroy, VIC, 3065, Australia.
¹¹ School of Mathematics and Statistics, The University of Melbourne, Parkville, VIC, 3052, Australia.
¹² Cancer Biology and Stem Cells Division, The Walter and Eliza Hall Institute of Medical Research, Parkville, VIC, 3052, Australia. sutherland.k@wehi.edu.au.
¹³ Department of Medical Biology, The University of Melbourne, Parkville, VIC, 3052, Australia. sutherland.k@wehi.edu.au.

PMID: 31519898
PMCID: PMC6744438
DOI: 10.1038/s41467-019-12164-y

Distinct initiating events underpin the immune and metabolic heterogeneity of KRAS-mutant lung adenocarcinoma

Sarah A Best et al. Nat Commun. 2019.

. 2019 Sep 13;10(1):4190.

doi: 10.1038/s41467-019-12164-y.

Authors

Affiliations

¹ Cancer Biology and Stem Cells Division, The Walter and Eliza Hall Institute of Medical Research, Parkville, VIC, 3052, Australia.
² Department of Medical Biology, The University of Melbourne, Parkville, VIC, 3052, Australia.
³ Epigenetics and Development Division, The Walter and Eliza Hall Institute of Medical Research, Parkville, VIC, 3052, Australia.
⁴ Division of Experimental Animal Pathology, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX, Amsterdam, The Netherlands.
⁵ School of Life Sciences, Fudan University, Shanghai, China.
⁶ Department of Biochemistry and Molecular Biology and The Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, Parkville, VIC, 3052, Australia.
⁷ School of Life Sciences, Nanjing University, Nanjing, 210023, China.
⁸ Inflammation Division, The Walter and Eliza Hall Institute of Medical Research, Parkville, VIC, 3052, Australia.
⁹ Department of Anatomical Pathology, St Vincent's Hospital, The University of Melbourne, Fitzroy, VIC, 3065, Australia.
¹⁰ Department of Surgery, St Vincent's Hospital, The University of Melbourne, Fitzroy, VIC, 3065, Australia.
¹¹ School of Mathematics and Statistics, The University of Melbourne, Parkville, VIC, 3052, Australia.
¹² Cancer Biology and Stem Cells Division, The Walter and Eliza Hall Institute of Medical Research, Parkville, VIC, 3052, Australia. sutherland.k@wehi.edu.au.
¹³ Department of Medical Biology, The University of Melbourne, Parkville, VIC, 3052, Australia. sutherland.k@wehi.edu.au.

PMID: 31519898
PMCID: PMC6744438
DOI: 10.1038/s41467-019-12164-y

Abstract

The KRAS oncoprotein, a critical driver in 33% of lung adenocarcinoma (LUAD), has remained an elusive clinical target due to its perceived undruggable nature. The identification of dependencies borne through common co-occurring mutations are sought to more effectively target KRAS-mutant lung cancer. Approximately 20% of KRAS-mutant LUAD carry loss-of-function mutations in KEAP1, a negative regulator of the antioxidant response transcription factor NFE2L2/NRF2. We demonstrate that Keap1-deficient Kras^G12D lung tumors arise from a bronchiolar cell-of-origin, lacking pro-tumorigenic macrophages observed in tumors originating from alveolar cells. Keap1 loss activates the pentose phosphate pathway, inhibition of which, using 6-AN, abrogated tumor growth. These studies highlight alternative therapeutic approaches to specifically target this unique subset of KRAS-mutant LUAD cancers.

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Conflict of interest statement

The authors declare no competing interests.

Figures

**Fig. 1**
*KEAP1* mutation is enriched in *KRAS*-mutant lung adenocarcinoma. a Frequency of *KRAS* mutation in lung adenocarcinoma (LUAD) obtained from the Broad Institute (n = 182), The Cancer Genome Atlas (n = 313) and the Memorial Sloan Kettering Cancer Center (MSKCC) (n = 864). b Frequency of *KEAP1* mutation status in *KRAS* WT (wild type; n = 921) or MUT (mutant; n = 627) LUAD samples. c Venn Diagram of co-mutation/mutual exclusivity of *KEAP1*, *TP53,* and *STK11* mutations in the subset of *KRAS*-mutant LUAD (n = 627). d *KEAP1*, *TP53,* and *STK11* mutation status in *KRAS*-mutant LUAD patient samples from the Clinical Lung Cancer Genome Project (CLCGP) cohort (n = 155). e Frequency of clinical stage in subsets of *KRAS*-mutant LUAD from (d). f Aggressive score assessed by clinical stage in each *KRAS*-mutant subgroup. g NQO1 immunostaining on *KRAS*^MUTKEAP1^WTTP53^MUT and *KRAS*^MUTKEAP1^MUTTP53^WT patient samples. Scale, 200 μm

**Fig. 2**
*Keap1* inactivation accelerates *Kras*^G12D-induced lung adenocarcinoma. a Survival analysis of K (n = 10) and KK (n = 19) mice following Ad5-CMV-Cre administration. Mantel-Cox test: ****p < 0.0001. b Hematoxylin and eosin (H&E) stained lungs of representative K and KK mice at ethical endpoint. Scale, 1 mm; inset, 100 μm. c Weight of left lung lobe of uninfected littermates (U, n = 8), K (n = 5) and KK (n = 14) mice 3 months following Ad5-CMV-Cre infection. Ordinary one-way ANOVA/Holm-Sidak’s multiple comparisons test: U v K: **p = 0.006; U v KK: ****p < 0.0001; K v KK: *p = 0.0327. Mean ± SEM. d Number of lesions in the lungs of K (n = 4) and KK (n = 7) mice 3 months post Ad5-CMV-Cre. Ordinary one-way ANOVA/Tukey’s multiple comparisons test K v KK *p = 0.0397. Mean ± SEM. e Lesion classification of K (n = 4) and KK (n = 7) lungs 3 months post Ad5-CMV-Cre infection. Two-way ANOVA/Tukey’s multiple comparisons test: alveolar hyperplasia K v KK *p = 0.05, adenoma K v KK *p = 0.05. Mean ± SEM. f Immunofluorescence of EYFP and Nrf2 expression in *EYFP*^T/+;*Kras*^G12D/+;*Keap1*^Δ/Δ three months following Ad5-CMV-Cre infection. Boxes represent EYFP positive and negative regions. Scale, 10 μm. g Quantification of 2,7-dichlorofluorescin diacetate (DCFDA) alone or in combination with 110 μM tert-butyl hydrogen peroxide (TBHP) to stimulate reactive oxygen species (ROS) in primary cell lines (n = 4 KP and KK). Mann–Whitney test DCFDA + TBHP KP v KK *p = 0.0286. Mean ± SEM

**Fig. 3**
Alveolar macrophages contribute to *Kras*^G12D-induced tumorigenesis. a Quantification of alveolar macrophages (CD11c⁺CD11b⁻CD103⁻) in CD45⁺ immune lung infiltrate in uninfected (U; n = 9) and K (n = 4), KP (n = 6), KK (n = 4) mice 3 months post Ad5-CMV-Cre infection. Kruskal–Wallis test/Dunn’s multiple comparisons test U v K *p = 0.0127; U v KP **p = 0.0042. Mean ± SEM. b F4/80 immunostaining on KP and KK lung tissue 3 months post Ad5-CMV-Cre infection. Scale, 50 μm. c Frequency of carcinomatous lesions in KP (n = 6) and KK (n = 7) lungs 3 months post Ad5-CMV-Cre infection. Unpaired t test ***p = 0.001. Mean ± SEM. d Schematic of treatment plan. Briefly, KP mice were administered weekly PBS-liposomes or Clodronate-liposomes four weeks following Ad5-CMV-Cre infection. Lungs were harvested and analyzed for alveolar macrophages and tumor burden following 8 weeks of liposome treatment. e Representative flow cytometry plot of alveolar macrophage population (CD45⁺CD11c⁺CD103⁻) in the lungs of PBS- or Clodronate-liposome (L) treated mice. f Quantification of alveolar macrophages as a proportion of CD45⁺ cells in the lungs of KP mice treated with PBS (P; n = 3) or Clodronate (C; n = 3) liposomes. Dotted line represents mean value of alveolar macrophages in KP and U from (a), above. Unpaired t test *p = 0.0443. Mean ± SD. g Representative flow cytometry plot of epithelial cells (EpCAM⁺) in the lungs of PBS or Clodronate liposome treated mice. h Quantification of EpCAM⁺ population as a proportion of lineage negative (CD31⁻CD45⁻) cells. Unpaired t test **p = 0.0025. Mean ± SD. i Quantification of lesion size in PBS- and Clodronate-liposome (L) treated mice. j Representative H&E of PBS- and Clodronate-liposome (L) treated mice. Scale, 200 μm

**Fig. 4**
*Keap1* loss preferentially transforms bronchiolar cells. a Expression of *Cx43* in KP (n = 5) and KK (n = 7) tumor pieces, relative to KP. Mann–Whitney test **p = 0.0025. Mean ± SEM. b Expression of *Connexin-43* (*Cx43*) in alveolar macrophages isolated from KP (n = 4) and KK (n = 5) tumor-bearing lungs, relative to *Gapdh* control. Unpaired student t test ****p < 0.0001. Mean ± SEM. c Quantification of alveolar (EpCAM⁺CD104⁻) compartment in uninfected (U; n = 9) and K (n = 4), KP (n = 4) and KK (n = 5) 3 months post Ad5-CMV-Cre administration. Kruskal–Wallis test/Dunn’s multiple comparisons test, U v K **p = 0.0034, U v KP **p = 0.0095. Mean ± SEM. d Quantification of bronchiolar (EpCAM⁺CD104⁺) compartment in uninfected (U; n = 9) and K (n = 3), KP (n = 4) and KK (n = 6) 3 months post Ad5-CMV-Cre administration. Kruskal–Wallis test/Dunn’s multiple comparisons test, U v KK **p = 0.0014, U v KP *p = 0.0308. Mean ± SEM. e Representative H&E stained KP and KK lungs 3 weeks post Ad5-CMV-Cre infection. Scale, 200 μm; inset, 50 μm. f Expression of *Nqo1* in alveolar (A) and bronchiolar (B) cells (n = 3). ****FDR p < 0.0001. Mean ± SEM. g Expression of *Cx43* mRNA in alveolar (A) and bronchiolar (B) cells (n = 3). **FDR p = 0.0055. Mean ± SEM

**Fig. 5**
Cell-of-origin determines the alveolar macrophage composition in LUAD. a Schematic of cell type-specific Ad5-Cre experimental design. b Expression of *Sftpc* and *Scgb1a1* in KK lung tumor pieces 3 months following administration of Ad5-CC10-Cre (n = 3) or Ad5-SPC-Cre (n = 4). Unpaired t test, *Sftpc* *p = 0.0168; *Scgb1a1* *p = 0.0107. Mean ± SEM. c F4/80 immunostaining on lung tissue from KK and KP mice infected with Ad5-CC10-Cre or Ad5-SPC-Cre. Scale, 50 μm. d Quantification of alveolar macrophages in CD45⁺ immune lung infiltrate of Ad5-CMV-Cre infected KK (n = 4) and KP (n = 4), Ad5-CC10-Cre infected KK (n = 4) and KP (n = 2) and Ad5-SPC-Cre infected KK (n = 4) and KP (n = 3) mice. KK, ordinary one-way ANOVA/Holm-Sidak’s multiple comparisons test CMV-Cre v SPC-Cre ***p = 0.0002, SPC-Cre v CC10-Cre **p = 0.0016. KP, unpaired t test CMV-Cre v CC10-Cre ***p = 0.0006. Mean ± SEM. e Schematic of cell-of-origin hypothesis and enhanced macrophage number in lung tumors arising from alveolar epithelial cells. Lightning bolt depicts genetic alterations. f Immunostaining of CD68 on *KRAS*^MUTKEAP1^WT and *KRAS*^MUTKEAP1^MUT LUAD. Scale, 200 μm. g Analysis of *CD68* expression in *NQO1*^low (n = 7) and *NQO1*^high (n = 10) *KRAS*-mutant LUAD TCGA patient samples. **FDR p = 0.0012. Mean ± SEM. h Analysis of *SFTPC* expression in *NQO1*^low (n = 7) and *NQO1*^high (n = 10) *KRAS*-mutant LUAD TCGA patient samples. **FDR p = 0.0108. Mean ± SEM

**Fig. 6**
Enhanced glucose metabolism in *Keap1*-mutant *Kras*^G12D LUAD. a Volcano plot of top KEGG metabolic pathways differentially expressed in *NQO1*^low (n = 10) and *NQO1*^high (n = 10) *KRAS*-mutant LUAD from the TCGA dataset. Pathways involved in drug metabolism, pentose phosphate pathway, glutathione metabolism, and glycolysis have been highlighted. Dotted line represents p = 0.05. b Heatmap of KEGG pentose phosphate pathway in *NQO1*^low (n = 10) and *NQO1*^high (n = 10) *KRAS*-mutant LUAD from the TCGA dataset. c Analysis of *TALDO1* expression in *NQO1*^low (n = 7) and *NQO1*^high (n = 10) *KRAS*-mutant LUAD TCGA patient samples. ***FDR p = 0.0006. Mean ± SEM. d Correlation of TALDO1 and NQO1 immunostaining in *KRAS*-mutant LUAD (n = 46). e TALDO1 immunostaining on *KRAS*^MUTKEAP1^WT and *KRAS*^MUTKEAP1^MUT patient samples. Scale, 200 μm; inset, 50 μm. f Expression of glycolytic enzyme (*G6PD*), pentose phosphate pathway enzymes (*Tkt, Pgd, Taldo1*) and malic enzyme (*Me1*) in tumor pieces from KP (n = 7) and KK (n = 7) lungs. Expression relative to *Gapdh* housekeeper control and quantified relative to KP expression. Two-way ANOVA/Sidak’s multiple comparisons test *Taldo1* KP v KK ****p < 0.0001. Mean ± SEM. g Rate of glycolysis measured by extracellular acidification rate (ECAR) of uninfected lung epithelium (U; n = 2 experiments, n = 6 mice per experiment) and KP (n = 4) as well as KK (n = 4) flow cytometry isolated tumor cells 3 months following Ad5-CMV-Cre infection. Ordinary one-way ANOVA/Holm-Sidak’s multiple comparisons test, KP v KK *p = 0.0198. Mean ± SEM

**Fig. 7**
PPP blockade abrogates the growth of *KEAP1*-mutant LUAD. a Schematic of spontaneous tumor treatment study. Briefly, KK or KP mice were randomized into 20 mg/kg 6-AN or vehicle treated 40 days post Ad5-CMV-Cre intranasal infection. Lungs were harvested for analysis on day 64. b Representative H&E images of KK vehicle or 6-AN-treated lungs. Scale, 1 mm. c Quantification of KK superior lobe lung weight (milligrams, mg) following vehicle (n = 6) or 6-AN (n = 8) treatment. Unpaired t test *p = 0.0106. Mean ± SEM. d Ratio of KK hyperplasia relative to large airway size in vehicle (n = 6) and 6-AN (n = 8) treated lungs. Unpaired t test *p = 0.0116. Mean ± SEM. e Colony assay 72 h following treatment with 62.5 μM 6-AN vs vehicle in *KEAP1*^MUT and *KEAP1*^WT NSCLC cell lines. Scale, 5 mm. f Relative change in tumor size of xenograft models (n = 3/cell line) of *KEAP1*^MUT and *KEAP1*^WT 48 h following treatment with 20 mg/kg 6-AN or vehicle. Change in size relative to vehicle of each cell line. Two-way ANOVA/Sidak’s multiple comparisons test: A549 **p = 0.0012; H460 ***p = 0.001. Mean ± SD. g Tumor measurements of A549 cell line xenograft following treatment with 20 mg/kg 6-AN (n = 6) or vehicle (n = 6) every 10 days. Survival log-rank (Mantel-Cox) test **p = 0.0023. h Kaplan–Meier survival curve of A549 xenograft NSG mice treated with 20 mg/kg 6-AN (n = 6) or vehicle (n = 6) every 10 days. Mantel–Cox test **p = 0.0017. i Tumor measurements of H358 xenograft following treatment with 20 mg/kg 6-AN (n = 8) or vehicle (n = 4) every 10 days. j Kaplan–Meier survival curve of H358 xenograft NSG mice treated with 20 mg/kg 6-AN (n = 8) or vehicle (n = 4) every 10 days

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References

1. Prior IA, Lewis PD, Mattos C. A comprehensive survey of Ras mutations in cancer. Cancer Res. 2012;72:2457–2467. doi: 10.1158/0008-5472.CAN-11-2612. - DOI - PMC - PubMed
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[1] Prior IA, Lewis PD, Mattos C. A comprehensive survey of Ras mutations in cancer. Cancer Res. 2012;72:2457–2467. doi: 10.1158/0008-5472.CAN-11-2612. - DOI - PMC - PubMed

[2] Prior IA, Lewis PD, Mattos C. A comprehensive survey of Ras mutations in cancer. Cancer Res. 2012;72:2457–2467. doi: 10.1158/0008-5472.CAN-11-2612. - DOI - PMC - PubMed

[3] Quinlan MP, Settleman J. Isoform-specific ras functions in development and cancer. Future Oncol. 2009;5:105–116. doi: 10.2217/14796694.5.1.105. - DOI - PubMed

[4] Quinlan MP, Settleman J. Isoform-specific ras functions in development and cancer. Future Oncol. 2009;5:105–116. doi: 10.2217/14796694.5.1.105. - DOI - PubMed

[5] Papke B, Der CJ. Drugging RAS: know the enemy. Science. 2017;355:1158–1163. doi: 10.1126/science.aam7622. - DOI - PubMed

[6] Papke B, Der CJ. Drugging RAS: know the enemy. Science. 2017;355:1158–1163. doi: 10.1126/science.aam7622. - DOI - PubMed

[7] Cancer Genome Atlas Research, N. Comprehensive molecular profiling of lung adenocarcinoma. Nature. 2014;511:543–550. doi: 10.1038/nature13385. - DOI - PMC - PubMed

[8] Cancer Genome Atlas Research, N. Comprehensive molecular profiling of lung adenocarcinoma. Nature. 2014;511:543–550. doi: 10.1038/nature13385. - DOI - PMC - PubMed

[9] Jackson EL, et al. Analysis of lung tumor initiation and progression using conditional expression of oncogenic K-ras. Genes Dev. 2001;15:3243–3248. doi: 10.1101/gad.943001. - DOI - PMC - PubMed

[10] Jackson EL, et al. Analysis of lung tumor initiation and progression using conditional expression of oncogenic K-ras. Genes Dev. 2001;15:3243–3248. doi: 10.1101/gad.943001. - DOI - PMC - PubMed

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Distinct initiating events underpin the immune and metabolic heterogeneity of KRAS-mutant lung adenocarcinoma

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Distinct initiating events underpin the immune and metabolic heterogeneity of KRAS-mutant lung adenocarcinoma

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