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. 2019 Jun;14(6):1061-1076.
doi: 10.1016/j.jtho.2019.02.019. Epub 2019 Feb 27.

The Effect of LKB1 Activity on the Sensitivity to PI3K/mTOR Inhibition in Non-Small Cell Lung Cancer

Takehito Shukuya  1 , Tadaaki Yamada  2 , Michael J Koenig  1 , Jielin Xu  3 , Tamio Okimoto  1 , Fuhai Li  4 , Joseph M Amann  1 , David P Carbone  5
Affiliations

The Effect of LKB1 Activity on the Sensitivity to PI3K/mTOR Inhibition in Non-Small Cell Lung Cancer

Takehito Shukuya et al. J Thorac Oncol. 2019 Jun.

Abstract

Introduction: Liver kinase B1 (LKB1), also called serine/threonine kinase 11 (STK11), is a tumor suppressor that functions as master regulator of cell growth, metabolism, survival, and polarity. Approximately 30% to 35% of patients with NSCLC possess inactivated liver kinase B1 gene (LKB1), and these patients respond poorly to anti-programmed cell death 1 (PD-1)/programmed death ligand 1 (PD-L1) immunotherapy. Therefore, novel therapies targeting NSCLC with LKB1 loss are needed.

Methods: We used a new in silico signaling analysis method to identify the potential therapeutic targets and reposition drugs by integrating gene expression data with the Kyoto Encyclopedia of Genes and Genomes signaling pathways. LKB1 wild-type and LKB1-deficient NSCLC cell lines, including knockout clones generated by clustered regularly interspaced short pallindromic repeats-Cas9, were treated with inhibitors of mechanistic target of rapamycin kinase (mTOR) and phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) and a dual inhibitor.

Results: In silico experiments showed that inhibition of both mTOR and PI3K can be synergistically effective in LKB1-deficient NSCLC. In vitro and in vivo experiments showed the synergistic effect of mTOR inhibition and PI3K inhibition in LKB1-mutant NSCLC cell lines. The sensitivity to dual inhibition of mTOR and PI3K is higher in LKB1-mutant cell lines than in wild-type cell lines. A higher compensatory increase in Akt phosphorylation after rapamycin treatment of LKB1-deficient cells than after rapamycin treatment of LKB1 wild-type cells is responsible for the synergistic effect of mTOR and PI3K inhibition. Dual inhibition of mTOR and PI3K resulted in a greater decrease in protein expression of cell cycle-regulating proteins in LKB1 knockout cells than in LKB1 wild-type cells.

Conclusion: Dual molecular targeted therapy for mTOR and PI3K may be a promising therapeutic strategy in the specific population of patients with lung cancer with LKB1 loss.

Keywords: LKB1; Lung cancer; Molecular targeted agent; PI3K; mTOR.

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Conflict of interest statement

Conflict of interest statement:

TS reports personal fees from Eli Lilly and Company, Nichi-Iko Pharmaceutical Co., and Sanofi, outside the submitted work. DPC reports personal fees from Abbvie, Adaptimmune, Agenus, Amgen, Ariad, AstraZeneca, Biocept, Boehringer Ingelheim, Celgene, Clovis, EMD Serono, Foundation Medicine, Genentech/Roche, Gritstone, Guardant Health, Helsinn, Humana, Incyte, Inivata, Inovio, Janssen, Kyowa Kirin, Merck, Merck Sharp Dohme (MSD), Novartis, Palobiofarma, Pfizer, prIME Oncology, Stemcentrx, Takeda, Teva, and grants and personal fees from Bristol Myers-Squibb, outside the submitted work.

Figures

Figure 1.
Figure 1.. Combined inhibition of mTOR and PI3K as a promising target for LKB1-deficient tumors in numerical experiments.
A, Yellow nodes are proteins from PI3K-Akt-mTOR signaling pathways, the red and green edges are the ones exclusively exist in mTOR and PI3K-Akt signaling pathways, respectively, blue edges are common edges with respect to both pathways. B, When adding mTOR inhibitors alone, there exists local paths (in red) in the loop structure which could finally activate PI3K and Akt.
Figure 1.
Figure 1.. Combined inhibition of mTOR and PI3K as a promising target for LKB1-deficient tumors in numerical experiments.
A, Yellow nodes are proteins from PI3K-Akt-mTOR signaling pathways, the red and green edges are the ones exclusively exist in mTOR and PI3K-Akt signaling pathways, respectively, blue edges are common edges with respect to both pathways. B, When adding mTOR inhibitors alone, there exists local paths (in red) in the loop structure which could finally activate PI3K and Akt.
Figure 2.
Figure 2.. Synergistic effect between mTOR inhibition and PI3K inhibition in LKB1-deficient non-small cell lung cancer cells in vitro.
The MTT assay was used to assess sensitivity to the drugs for metabolic activity. Cells were seeded at 4 × 103 per well of a 96-well plate and incubated for 48 hours with the drugs. Data are representative of three independent experiments. A, Non-small cell lung cancer cell lines without LKB1 mutation (Calu1, H358, H520, H522, H1299, and H2087) and with LKB1 mutation (HCC15, H23, H157, and A549) were incubated with rapamycin alone (100 nmol/L), BKM-120 alone (100 nmol/L), or in combination. When compared LKB1 mutant cells and LKB1 wild type cells, p values were 0.1356, 0.8312, and 0.0190 in rapamycin alone, BKM-120 alone, and their combination, respectively. B, Non-small cell lung cancer cell lines without LKB1 mutation (Calu1, H358, H520, H522, H1299, and H2087) and with LKB1 mutation (HCC15, H23, H157, and A549) were incubated with GSK2126458 alone (100 nmol/L). When compared LKB1 mutant cells and LKB1 wild type cells, p value was 0.0105. C, LKB1 knock out clones and wild-type clones of H358 and H1299 cells were incubated with GSK2126458 alone. LKB1 knock out clones were more sensitive than wild type clones. D, LKB1 mutated lung cancer cells (HCC15, H23, H157, and A549) and LKB1 knock out clones of H358 and H1299 were treated with various doses of rapamycin and BKM-120. Combination index values were analyzed according to the Chou and Talalay equation using the CompuSyn software. Abbreviations; WT; wild type, MT; mutant, KO; knock out.
Figure 2.
Figure 2.. Synergistic effect between mTOR inhibition and PI3K inhibition in LKB1-deficient non-small cell lung cancer cells in vitro.
The MTT assay was used to assess sensitivity to the drugs for metabolic activity. Cells were seeded at 4 × 103 per well of a 96-well plate and incubated for 48 hours with the drugs. Data are representative of three independent experiments. A, Non-small cell lung cancer cell lines without LKB1 mutation (Calu1, H358, H520, H522, H1299, and H2087) and with LKB1 mutation (HCC15, H23, H157, and A549) were incubated with rapamycin alone (100 nmol/L), BKM-120 alone (100 nmol/L), or in combination. When compared LKB1 mutant cells and LKB1 wild type cells, p values were 0.1356, 0.8312, and 0.0190 in rapamycin alone, BKM-120 alone, and their combination, respectively. B, Non-small cell lung cancer cell lines without LKB1 mutation (Calu1, H358, H520, H522, H1299, and H2087) and with LKB1 mutation (HCC15, H23, H157, and A549) were incubated with GSK2126458 alone (100 nmol/L). When compared LKB1 mutant cells and LKB1 wild type cells, p value was 0.0105. C, LKB1 knock out clones and wild-type clones of H358 and H1299 cells were incubated with GSK2126458 alone. LKB1 knock out clones were more sensitive than wild type clones. D, LKB1 mutated lung cancer cells (HCC15, H23, H157, and A549) and LKB1 knock out clones of H358 and H1299 were treated with various doses of rapamycin and BKM-120. Combination index values were analyzed according to the Chou and Talalay equation using the CompuSyn software. Abbreviations; WT; wild type, MT; mutant, KO; knock out.
Figure 3:
Figure 3:. LKB1 mutant cells show lower AMPK phosphorylation and higher mTOR pathway phosphorylation than LKB1 wild type cells. Inhibition of mTOR increases Akt phosphorylation more in LKB1 deficient cells than in LKB1 wild type cells, and addition of PI3K inhibition suppress Akt phosphorylation.
A, 6 LKB1 wild type cells (Calu1, H358, H520, H522, H1299, and H2087) and 4 LKB1 mutant cells (HCC15, H23, H157, and A549) were lysed and the indicated proteins were detected by immunoblotting. B, LKB1 mutant A549 cells were treated with rapamycin (0.01 μmol/L) for 2 hours. The cells were lysed and the indicated proteins were detected by immunoblotting. C, LKB1 deficient cells (HCC15, H23, H157, A549, two LKB1 knock out clones of H358, and two LKB1 knock out clones of H1299) and wild type cells (Calu1, H358, H520, H522, H1299, H2087, two LKB1 wild type clones of H358, and two LKB1 wild type clones of H1299) were incubated with rapamycin (0.01 μmol/L) for 2 hours. The cells were lysed and phospho Akt1 and total Akt were quantified with total Akt and phospho Akt1 sandwitch ELISA kit. Ratio of phospho Akt1 to total Akt after rapamycin treatment was divided by the ratio of phospho Akt1 to total Akt without treatment to evaluate rapamycin induced upregulation of Akt phosphorylation. When compared LKB1 deficient cells and wild type cells, p value was 0.0209. D, LKB1 wild type cell (Calu1, H358, H520, H522, H1299, H2087), and LKB1 deficient cells (HCC15, H23, H157, A549) were incubated with rapamycin (0.01 μmol/L) for 2 hours. The cells were lysed and the indicated proteins were detected by immunoblotting. E, Two LKB1 wild type clones of H358, two LKB1 knock out clones of H358, two LKB1 wild type clones of H1299, and two LKB1 knock out clones of H1299 were incubated with rapamycin (0.01 μmol/L) for 2 hours. The cells were lysed and the indicated proteins were detected by immunoblotting. D&E, Quantification of the Western blot data were performed by measuring the intensity of the hybridization signals using ImageJ analysis program (National Institute of Health). Ratio of phospho Akt2 to total Akt2 after rapamycin treatment was divided by the ratio of phospho Akt2 to total Akt2 without treatment to evaluate rapamycin induced upregulation of Akt2 phosphorylation. When compared LKB1 deficient cells and wild type cells by Wilcoxon rank sum test, p value was 0.0410. F, LKB1 mutant A549 cells were treated with various doses of rapamycin and BKM-120, or GSK2126458 for 2 hours. The cells were lysed and the indicated proteins were detected by immunoblotting. G, LKB1 knock out clones of H358 and H1299 cells were treated with various doses of rapamycin and BKM-120, or GSK2126458 for 2 hours. The cells were lysed and the indicated proteins were detected by immunoblotting. Abbreviations; LE; longer exposure, CTRL; control, R; Rapamycin, B; BKM-120, G; GSK2126458.
Figure 3:
Figure 3:. LKB1 mutant cells show lower AMPK phosphorylation and higher mTOR pathway phosphorylation than LKB1 wild type cells. Inhibition of mTOR increases Akt phosphorylation more in LKB1 deficient cells than in LKB1 wild type cells, and addition of PI3K inhibition suppress Akt phosphorylation.
A, 6 LKB1 wild type cells (Calu1, H358, H520, H522, H1299, and H2087) and 4 LKB1 mutant cells (HCC15, H23, H157, and A549) were lysed and the indicated proteins were detected by immunoblotting. B, LKB1 mutant A549 cells were treated with rapamycin (0.01 μmol/L) for 2 hours. The cells were lysed and the indicated proteins were detected by immunoblotting. C, LKB1 deficient cells (HCC15, H23, H157, A549, two LKB1 knock out clones of H358, and two LKB1 knock out clones of H1299) and wild type cells (Calu1, H358, H520, H522, H1299, H2087, two LKB1 wild type clones of H358, and two LKB1 wild type clones of H1299) were incubated with rapamycin (0.01 μmol/L) for 2 hours. The cells were lysed and phospho Akt1 and total Akt were quantified with total Akt and phospho Akt1 sandwitch ELISA kit. Ratio of phospho Akt1 to total Akt after rapamycin treatment was divided by the ratio of phospho Akt1 to total Akt without treatment to evaluate rapamycin induced upregulation of Akt phosphorylation. When compared LKB1 deficient cells and wild type cells, p value was 0.0209. D, LKB1 wild type cell (Calu1, H358, H520, H522, H1299, H2087), and LKB1 deficient cells (HCC15, H23, H157, A549) were incubated with rapamycin (0.01 μmol/L) for 2 hours. The cells were lysed and the indicated proteins were detected by immunoblotting. E, Two LKB1 wild type clones of H358, two LKB1 knock out clones of H358, two LKB1 wild type clones of H1299, and two LKB1 knock out clones of H1299 were incubated with rapamycin (0.01 μmol/L) for 2 hours. The cells were lysed and the indicated proteins were detected by immunoblotting. D&E, Quantification of the Western blot data were performed by measuring the intensity of the hybridization signals using ImageJ analysis program (National Institute of Health). Ratio of phospho Akt2 to total Akt2 after rapamycin treatment was divided by the ratio of phospho Akt2 to total Akt2 without treatment to evaluate rapamycin induced upregulation of Akt2 phosphorylation. When compared LKB1 deficient cells and wild type cells by Wilcoxon rank sum test, p value was 0.0410. F, LKB1 mutant A549 cells were treated with various doses of rapamycin and BKM-120, or GSK2126458 for 2 hours. The cells were lysed and the indicated proteins were detected by immunoblotting. G, LKB1 knock out clones of H358 and H1299 cells were treated with various doses of rapamycin and BKM-120, or GSK2126458 for 2 hours. The cells were lysed and the indicated proteins were detected by immunoblotting. Abbreviations; LE; longer exposure, CTRL; control, R; Rapamycin, B; BKM-120, G; GSK2126458.
Figure 4:
Figure 4:. Loss of LKB1 affects cell cycle regulating proteins and increase susceptibility to mTOR and PI3K dual inhibition
A, LKB1 knock out clones and wild type clones of H358 and H1299 cells were treated with GSK2126458 (0.1 μmol/L) for 48 hours. The cells were lysed, and the indicated proteins were detected by immunoblotting. Quantification of the Western blot data were performed by measuring the intensity of the hybridization signals using ImageJ analysis program (National Institute of Health). The indicated protein expression levels after GSK2126458 exposure for 48 hours were compared between LKB1 wild type and knock out clones. Expression levels were normalized by GAPDH, and Wilcoxon rank sum tests were performed (LKB1 WT vs KO; cyclin A p = 0.0209, phosphorylated-Rb p = 0.0209, CDK2 p = 0.1489, p27 p = 0.2482, cyclin D1 p = 0.0433, respectively). B, HCC15, H23, H157, and A549 were treated with GSK2126458 (0.1 μmol/L) for 48 hours. The cells were lysed, and the indicated proteins were detected by immunoblotting. C, LKB1 knock out clones and wild type clones of H358 cells were treated with GSK2126458 (0.01 μmol/L) for 6 hours and 24 hours. Cell-cycle histograms were generated after analysis of propidium iodide stained cells by FACS. Abbreviations; WT; wild type, KO; knock out.
Figure 4:
Figure 4:. Loss of LKB1 affects cell cycle regulating proteins and increase susceptibility to mTOR and PI3K dual inhibition
A, LKB1 knock out clones and wild type clones of H358 and H1299 cells were treated with GSK2126458 (0.1 μmol/L) for 48 hours. The cells were lysed, and the indicated proteins were detected by immunoblotting. Quantification of the Western blot data were performed by measuring the intensity of the hybridization signals using ImageJ analysis program (National Institute of Health). The indicated protein expression levels after GSK2126458 exposure for 48 hours were compared between LKB1 wild type and knock out clones. Expression levels were normalized by GAPDH, and Wilcoxon rank sum tests were performed (LKB1 WT vs KO; cyclin A p = 0.0209, phosphorylated-Rb p = 0.0209, CDK2 p = 0.1489, p27 p = 0.2482, cyclin D1 p = 0.0433, respectively). B, HCC15, H23, H157, and A549 were treated with GSK2126458 (0.1 μmol/L) for 48 hours. The cells were lysed, and the indicated proteins were detected by immunoblotting. C, LKB1 knock out clones and wild type clones of H358 cells were treated with GSK2126458 (0.01 μmol/L) for 6 hours and 24 hours. Cell-cycle histograms were generated after analysis of propidium iodide stained cells by FACS. Abbreviations; WT; wild type, KO; knock out.
Figure 5:
Figure 5:. Loss of LKB1 confers sensitivity to dual inhibition of mTOR and PI3K by GSK2126458 in vivo.
A, An LKB1 knock out clone and a wild type clone of H358 cells (5 × 106 cells) were inoculated into the flank of NSG mice, and when tumor volumes reached approximately 200 mm3, the mice were treated with GSK2126458 0.5 mg/kg/mouse or vehicle, p.o.. days 1-5, 8-12, 15-19, and 22-26. Data shown are mean ± SE for 7 to 9 mice in each group (7 mice injected LKB1 knock out clones and 8 mice injected LKB1 wild type clones were treated with GSK2126458, and 9 mice injected LKB1 knock out clones and 9 mice injected LKB1 wild type clones were treated with vehicle). Xenograft tumors of mice were evaluated as described in Materials and Method. Tumor size was analyzed by Wilcoxon rank sum test (KO veh vs. KO GSK day 0 p = 0.5251, day 3 p = 0.0018, day 7 p = 0.0502, day 10 p = 0.0036, day 14 p = 0.0050, day 17 p = 0.0528, day 21 p = 0.0389, day 24 p = 0.0117, day 28 p = 0.0163; WT GSK vs. KO GSK day 0 p = 1.0000, day 3 p = 0.1052, day 7 p = 0.2976, day 10 p = 0.0206, day 14 p = 0.0078, day 17 p = 0.0707, day 21 p = 0.0282, day 24 p = 0.0707, day 28 p = 0528). B, C, D Two LKB1 mutant cells (H157 and A549) and one LKB1 wild type cells (H522) were inoculated into the flank of NSG mice, and when tumor volumes reached approximately 200 mm3, the mice were treated with GSK2126458 0.5 mg/kg/mouse or vehicle, p.o.. days 1-5, 8-12, 15-19, and 22-26. Data shown are mean ± SE for 10 to 11 mice in each group (11 mice injected H157 were treated with vehicle, 11 injected H157 were treated with GSK2126458, 11 injected A549 were treated with vehicle, 11 injected A549 were treated with GSK2126458, 10 injected H522 were treated with vehicle, and 10 injected H522 were treated with GSK2126458). Xenograft tumors of mice were evaluated as described in Materials and Method. Tumor size was analyzed by Wilcoxon rank sum test (H157 veh vs. H157 GSK day 0 p = 0.4698, day 3 p = 0.0138, day 7 p = 0.0012; A549 veh vs. A549 GSK day 0 p = 0.7928, day 3 p = 0.0009, day 7 p = 0.0043, day 10 p = 0.0006, day 14 p = 0.0009; H522 veh vs. H522 GSK day 0 p = 0.1988, day 3 p = 0.1208, day 7 p = 0.8206, day 10 p = 0.6230, day 14 p = 0.9397, day 17 p = 0.4963, day 21 p = 0.5967, day 24 p = 0.4057, day 28 p = 0.5453). The H157 cells grew very rapidly and the majority of the vehicle treated mice (10 of 11) had to be euthanized because they met early removal criteria by day seven and the study was stopped. Although tumors in the treated mice grew slower, several of the mice (5 of 11) treated with GSK2126458 also had to be euthanized due to meeting early removal criteria at day seven. For the A549 cell line, at day 14, most of the vehicle treated mice (9 of 11) met early removal criteria while GSK2126458 treated mice (4 of 11) met early removal criteria at this time and the study was stopped. Abbreviations; WT; wild type, KO; knock out, Veh; vehicle, GSK; GSK2126458.
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