Heightening Energetic Stress Selectively Targets LKB1-Deficient Non-Small Cell Lung Cancers

Abstract

Inactivation of the LKB1 tumor suppressor is a frequent event in non-small cell lung carcinoma (NSCLC) leading to the activation of mTOR complex 1 (mTORC1) and sensitivity to the metabolic stress inducer phenformin. In this study, we explored the combinatorial use of phenformin with the mTOR catalytic kinase inhibitor MLN0128 as a treatment strategy for NSCLC bearing comutations in the LKB1 and KRAS genes. NSCLC is a genetically and pathologically heterogeneous disease, giving rise to lung tumors of varying histologies that include adenocarcinomas and squamous cell carcinomas (SCC). We demonstrate that phenformin in combination with MLN0128 induced a significant therapeutic response in KRAS/LKB1-mutant human cell lines and genetically engineered mouse models of NSCLC that develop both adenocarcinomas and SCCs. Specifically, we found that KRAS/LKB1-mutant lung adenocarcinomas responded strongly to phenformin + MLN0128 treatment, but the response of SCCs to single or combined treatment with MLN0128 was more attenuated due to acquired resistance to mTOR inhibition through modulation of the AKT-GSK signaling axis. Combinatorial use of the mTOR inhibitor and AKT inhibitor MK2206 robustly inhibited the growth and viability of squamous lung tumors, thus providing an effective strategy to overcome resistance. Taken together, our findings define new personalized therapeutic strategies that may be rapidly translated into clinical use for the treatment of KRAS/LKB1-mutant adenocarcinomas and squamous cell tumors.

Figure 1. Phenformin and the mTOR kinase inhibitor MLN0128 compound energy stress to induce apoptosis in KRAS/LKB1 mutant NSCLC cells

(A) A549 NSCLC cells were treated with increasing doses of MLN0128 or rapamycin. Immunoblots were probed with the indicated antibodies. (B and C) Cellular ATP measurement using cell titer and caspase 3/7 glo assays. LKB1 and KRAS/LKB1 mutant tumor lines (A549, H157, H460, A427, H1568, H23, H838, and RH2) were treated with DMSO (NT), phenformin (2mM), MLN0128 (2μM) or phenformin + MLN0128 for 24 hours. (D) Flow cytometry analysis of A549 cells expressing pBABE vector (B), wildtype LKB1 (WT) or kinase dead LKB1 (KD) that were treated with DMSO (NT), phenformin (2mM), MLN0128 (2μM) or phenformin + MLN0128 for 24 hours. Annexin V staining of A549-B, WT and KD cells following 24 hours of treatment. Statistical significance (p-values: * < 0.05; ** < 0.01; *** < 0.001) calculated using a non-parametric one-way ANOVA (Tukey test). The data are represented as the mean ± SEM. Error bars represent the ± S.E.M.

Figure 2. 4E-BP1 is a regulator of cell survival and energetics in KRAS/LKB1 mutant NSCLC tumor cells

(A) Trypan blue cell viability assay of A549 and H460 cells expressing 4E-BP1 4Ala (A549-4A; H460-4A) or vector (A549-V; H460-V) that were either not treated (NT) or treated with 1μg/ml doxycycline (Dox) for 3 days. (B-D) H460-4A cells were either untreated (NT) or treated with 1μg/ml doxycycline for two days (Dox). NT and Dox treated cells were then treated with 2mM phenformin for 24 hours (Phen) and (Dox+Phen). Cell viability, cell lysates and cellular ATP were analyzed. (B) Cell viability by trypan blue staining of NT, Dox, Phen and Dox+Phen H460-4A cells. (C) Immunoblots of H460-4A lysates probed with antibodies recognizing cleaved PARP (Clv'd PARP), cleaved caspase 3 (Clv'd Casp3) and actin. (D) Cellular ATP measurement by cell titer glo in H460-4A cells. Statistical significance (p-values: * < 0.05; ** < 0.01; *** < 0.001) calculated using a non-parametric one-way ANOVA (Tukey test). The data are represented as the mean ± SEM. Error bars represent the ± S.E.M.

Figure 3. MLN0128 inhibited glucose metabolism in KRAS/LKB1 mutant NSCLC in vitro and in vivo

(A) Lung tumor lysates from tumors isolated from KL_luc mice (M1, M2, M3 or M4) that were treated with vehicle (Veh) or MLN0128 (MLN) for five days were probed with indicated antibodies. (B) IHC staining of P-4E-BP1 in KL_luc lung tumors following five days MLN0128 treatment. Scale bars (black) = 50μM. (C) H460 cell line was treated with vehicle (NT) or 2μM MLN128 (MLN) for 24hr or 48hr. Lysates were probed with indicated antibodies. (D) Lung tumor lysates from tumors isolated from KL_luc mice (M1, M2, M3 or M4) that were treated with vehicle (Veh) or MLN0128 (MLN) for 5 days were probed with indicated antibodies. (E and F) Relative levels of glucose consumption and lactate production in H460 cells (E) or lung tumor cell line derived from a Kras^G12D;p53^−/−;Lkb1^−/− (KPL) mouse that were treated with vehicle (NT) or 2μM MLN0128 (MLN) for 24 hours. Statistical significance (p-values: * < 0.05; ** < 0.01; *** < 0.001;) calculated using a non-parametric one-way ANOVA (Tukey test). The data are represented as the mean ± SEM. Error bars represent the ± S.E.M.

Figure 4. FDG-PET guided treatment of KL_luc GEMMs revealed MLN0128 as a single agent or in combination with phenformin induced a significant metabolic and therapeutic response in lung tumors

(A) A schematic showing the treatment regimen for KL_luc mice. Mice were fed phenformin ad lib in drinking water (1.8mg/mL) and given an i.p. injection of vehicle (PEG/NMP) or MLN128 1.0mg/kg q.d. (B) 18F-FDG-PET and CT images of KL_luc mice at 6 weeks and 8 weeks post treatment with vehicle, phenformin, MLN0128 or phenformin + MLN0128. (C) Quantitative measure of tumor volume by CT of KL_luc mice at 6 and 8 weeks post treatment. (D) Quantitative measure of SUVmax by 18F-FDG PET of KL_luc mice at 6 and 8 weeks post treatment. Statistical significance (p-values: * < 0.05; ** < 0.01; *** < 0.001) calculated using a non-parametric one-way ANOVA (Tukey test). The data are represented as the mean ± SEM. Error bars represent the ± S.E.M.

Figure 5. Phenformin and MLN0128 therapy induced a differential therapeutic response between KL_luc lung adenocarcinomas and squamous cell carcinomas

(A) Representative H&E images of KL_luc whole lungs following 8 weeks treatment with vehicle, phenformin, MLN0128 or phenformin + MLN0128 combination therapy (as described in Figure 4A). Scale bars (black) = 4mm. (B) Distribution of KL_luc lung tumor subtypes: atypical adenomatous hyperplasia (AAH), adenocarcinoma in situ (AIS), mucinous adenocarcinoma in situ (mAIS), adenocarcinoma (ADC), squamous cell carcinoma SCC following treatment. (C) IHC staining of therapy resistant lung tumors with antibodies against p63 and TTF1. Scale bars (white) = 50μM. (D) Quantitative histology analysis of lung ADC tumor area from H&E stained whole lung sections of KL_luc mice using morphometric analysis software. (E) Total lesion counts in whole lung sections of KL_luc mice. Statistical significance (p-values: * < 0.05; ** < 0.01; *** < 0.001;) calculated using a non-parametric one-way ANOVA (Tukey test). The data are represented as the mean ± SEM. Error bars represent the ± S.E.M.

Figure 6. Phenformin + MLN0128 combination therapy inhibit tumor cell proliferation and induce apoptosis in KL_luc lung ADCs

(A) Immunohistochemical analysis of lung ADCs from KL_lucfollowing 8 weeks of treatment. Whole lung sections were stained with H&E or the indicated antibodies: Ki67, cleaved caspase 3 (Clv'd Casp3), P-S6 or HkII. Scale bars (white) = 50μM. (B-E) Quantitative IHC analysis of whole lung sections from KL_luc mice stained Ki67, cleaved caspase 3, PS6 and HkII. Statistical significance (p-values: * < 0.05; ** < 0.01; *** < 0.001) calculated using a non-parametric one-way ANOVA (Tukey test). The data are represented as the mean ± SEM. Error bars represent the ± S.E.M.

Figure 7. Co-targeting SCC with MLN0128 and MK2206

(A) Analysis of lung tumor lysates from KL_luc SCC following 8 weeks of MLN0128 treatment. Immunoblots were stained with the indicated antibodies. (B) Immunoblots from whole cell lysates from RH2 cells treated with DMSO, MLN0128 (20nM and 100nM), MK2206 (1μM), 20nM MLN0128 + 1μM MK2206 and 100nM MLN0128 + 1μM MK2206 for 24hours. Immunoblots were stained with the indicated antibodies. (C) Representative immunohistochemistry images from therapy resistant SCC tumors stained with phospho-GSKα/β (Ser21/9) antibody. Scale bars (black) = 50 μM and inset = 2mm. (D) Cell viability measured by trypan blue staining of SCC cell lines (RH2, H157, H226, H460, H520, H596, H1703, and SW900) treated with DMSO, low dose MLN0128 (20nM), MK2206 (1μM) or MLN0128 (20nM) + MK2206 (1μM) for 3 days. Statistical significance (p-values: * < 0.05; ** < 0.01; *** < 0.001;) calculated using a non-parametric one-way ANOVA (Tukey test). The data are represented as the mean ± SEM. Error bars represent the ± S.E.M.