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. Author manuscript; available in PMC: 2022 Aug 1.
Published in final edited form as: Mol Cancer Res. 2021 Oct 15;20(2):280–292. doi: 10.1158/1541-7786.MCR-21-0448

Enhanced vulnerability of LKB1-deficient NSCLC to disruption of ATP pools and redox homeostasis by 8-Cl-Ado

Ana Galan-Cobo 1,†, Christine M Stellrecht 1,2,3,†, Emrullah Yilmaz 1,4, Chao Yang 1,3, Yu Qian 1, Xiao Qu 1,5, Ishita Akhter 2, Mary L Ayres 2, Youhong Fan 1, Pan Tong 6, Lixia Diao 6, Jie Ding 2,7, Uma Giri 1, Jayanthi Gudikote 1, Monique Nilsson 1, William G Wierda 8, Jing Wang 6, Ferdinandos Skoulidis 1, John D Minna 9, Varsha Gandhi 2,3,8,*, John V Heymach 10,*
PMCID: PMC8816854  NIHMSID: NIHMS1750130  PMID: 34654720

Abstract

Loss of function somatic mutations of STK11, a tumor suppressor gene encoding LKB1 that contributes to the altered metabolic phenotype of cancer cells, is the second most common event in lung adenocarcinomas and often co-occurs with activating KRAS mutations. Tumor cells lacking LKB1 display an aggressive phenotype, with uncontrolled cell growth and higher energetic and redox stress due to its failure to balance ATP and NADPH levels in response to cellular stimulus. The identification of effective therapeutic regimens for LKB1-deficient non-small cell lung cancer (NSCLC) patients remains a major clinical need. Here, we report that LKB1-deficient NSCLC tumor cells displayed reduced basal levels of ATP and to a lesser extent other nucleotides, and markedly enhanced sensitivity to 8-Cl-adenosine (8-Cl-Ado), an energy-depleting nucleoside analogue. Treatment with 8-Cl-Ado depleted intracellular ATP levels, raised redox stress and induced cell death leading to a compensatory suppression of mTOR signaling in LKB1-intact, but not LKB1-deficient, cells. Proteomic analysis revealed that the MAPK/MEK/ERK and PI3K/AKT pathways were activated in response to 8-Cl-Ado treatment and targeting these pathways enhanced the anti-tumor efficacy of 8-Cl-Ado.

Implications:

Together, our findings demonstrate that LKB1-deficient tumor cells are selectively sensitive to 8-Cl-Ado and suggest that therapeutic approaches targeting vulnerable energy stores combined with signaling pathway inhibitors merit further investigation for this patient population.

Keywords: 8-Cl-Ado, LKB1, non–small cell lung cancer, mTOR, ERK, AKT

Introduction

Non-small lung cancer (NSCLC) is the leading cause of cancer-related mortality worldwide (1). Despite advances in targeted therapy, NSCLC patients have a 5-year survival rate of less than 20% (2). After TP53, STK11, which encodes serine/threonine kinase 11/liver kinase B1 (LKB1), is the most commonly mutated gene in NSCLC. STK11/LKB1 mutations occur in approximately 17% of lung adenocarcinomas (3) and poorly differentiated tumors have a higher frequency of LKB1-deficiency (4). In patients with stage III or IV NSCLC, LKB1 deficiency is associated with shorter cause-specific survival (5). STK11/LKB1 mutations or genomic loss frequently co-occur with KRAS alterations (6), resulting in a highly aggressive phenotype and reduced survival rates in both preclinical models (7) and NSCLC patients (6). In fact, many clinical laboratory improvement amendments (CLIA) based analyses of NSCLC tumor specimens, besides documenting KRAS alterations, also report back on tumors with and without STK11/LKB1 genetic abnormalities setting the stage of “precision medicine” selection of patients for STK11/LKB1 targeted therapy. Unfortunately, while numerous studies have focused on identifying vulnerabilities unique to LKB1-deficient tumors (8–11), to date there are no effective therapeutic regimens specific for this subset of patients. The urgency to discovery such regimens is additionally highlighted by the resistance of nearly all STK11/LKB1 altered NSCLC to immune checkpoint blockade (12). Thus, a crucial need for beneficial clinical translation is to identify a small molecule or targeted therapy that takes advantage of a NSCLC acquired “vulnerability” generated by STK11/LKB1 abnormalities.

LKB1, a master kinase that initiates the activation of numerous other kinases, plays a major role in the modulation of cell metabolism, polarity, differentiation and proliferation (13). The effects of LKB1 are largely mediated through its phosphorylation and activation of the energy sensor AMP-activated protein kinase (AMPK) (14). Under low cellular energy levels, AMPK restores intracellular homeostasis by balancing ATP and NADPH concentrations (15). Moreover, AMPK activation results in the inhibition of mammalian target of rapamycin (mTOR) signaling, which is a key protein in controlling cellular growth and proliferation (16). Likewise, AMPK activation has been shown to attenuate KRAS-mediated tumor cell proliferation (17). Therefore, LKB1-deficient tumors are unable to modulate cell growth and proliferation to maintain cellular ATP homeostasis in response to high energy stress. Consequently, LKB1-deficient tumors have an aberrant response to energy reduction (8,18) and a heightened sensitivity to energy-depleting agents (10,11,19), a vulnerability that can be exploited clinically.

8-chloro-adenosine (8-Cl-Ado), a nucleoside analogue that induces depletion of the endogenous ATP pool (20–24), has completed a phase I clinical trial in patients with chronic lymphocytic leukemia (CLL) (NCT00714103) (25) and is currently in a phase I/II clinical trial in patients with acute myeloid leukemia (NCT02509546). This analogue is rapidly metabolized to 8-Cl-ATP through the adenylate pathway. 8-Cl-ATP metabolite, 8-Cl-ADP, acts as a substrate for ATP synthase for conversion to 8-Cl-ATP, thereby blocking ATP synthase activity and preventing the catalysis of its natural substrate ADP to synthesize ATP (26). In addition to depleting ATP pools, 8-Cl-ATP incorporates into mRNA and prematurely terminates transcription (24), leading to a decline in transcription rates (20,22,23). This results in a loss of expression of genes with short lived transcripts which is detrimental for tumor cells which are dependent on these genes (27).

In the present study, we performed an unbiased drug screen on a panel of NSCLC cell lines and identified 8-Cl-Ado as being highly effective in LKB1-deficient NSCLC cells, particularly in cell lines bearing KRAS co-mutations. Moreover, the heightened sensitivity of LKB1-deficient cells to 8-Cl-Ado was associated with an attenuated signaling response to ATP depletion due to inactivation of AMPK/mTOR signaling, which also lead to increased reactive oxygen species (ROS) production. Proteomic analyses revealed that MAPK/MEK/ERK and PI3K/AKT/mTOR pathways are upregulated as a compensatory mechanism in response to 8-Cl-Ado treatment. Hence, 8-Cl-Ado combinations with AMPK, MEK or PI3K inhibitors resulted in a synergistic anti-tumor effects in LKB1-deficient tumor cells. Collectively, our data indicate that LKB1-deficient tumors are highly sensitive to ATP depletion, suggesting that treatment with 8-Cl-Ado, alone or in combination with AMPK, MEK or PI3K inhibitors, is a promising treatment strategy for patients with NSCLC harboring this specific genetic background.

Materials and Methods

Drugs

8-Cl-Ado was obtained from Dr. V. Rao (Drug Development Branch, National Cancer Institute, Bethesda, MD). Metformin and 2-deoxyglucose were obtained from Sigma-Aldrich (St. Louis, MO). Trametinib, A66, PI-103 were purchased from Selleck Chemical (Houston, TX).

Cell culture, transfection, and transduction

NSCLC cell lines were established by Dr. J.D. Minna and Dr. A. Gazdar at The University of Texas Southwestern Medical Center, Dallas, TX, and the NCI or obtained from the ATCC and maintained in RPMI-1640 media (Sigma or Mediatech, Manassas, VA) supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin (Life Technologies, Carlsbad, CA) in the presence of 5% CO2 at 37°C. Cells were routinely tested for Mycoplasma infection and authenticated by short tandem repeat analysis by MD Anderson’s Characterized Cell Line Core Facility. LKB1 proficiency status was based on LKB1 genetic status, RNA, and protein levels (Table S1). LKB1 protein deficiency was assigned based on LKB1 levels in cell lines with known STK11 mutations (Fig. S1)

LKB1 isogenic A549, H460, H2030, and H23 cell lines (all NSCLC lines who developed in patients by virtue of endogenous STK11/LKB1 abnormalities) were generated with a pLenti-GIII-CMV-GFP-2A-Puro vector with or without a wild-type LKB1–encoding sequence (Applied Biological Materials Inc., Richmond, BC). LKB1-knockdown Calu6 cell line (initially STK11/LKB1 wild type) were generated with a scramble control vector or two independent shRNAs (V3LHS_348649 and V3LHS_639000, GE Healthcare Dharmacon Inc, Lafayette, CO).

Cell viability assay and IC50 estimation

An optimized number of viable cells for each cell line were seeded in 96-well plates (3,000 cells per well) and allowed to attach overnight. Cells were treated with the indicated doses (serial 3-fold dilutions) of 8-Cl-Ado for 72 hours. CellTiter-Glo® (Promega, Madison, WI) was added to each well as per the manufacturer’s protocol, and contents were briefly mixed and incubated for 15 minutes to determine cell viability. Bioluminescence was measured using a FLUOstar OPTIMA Multimode Microplate Reader (BMG Labtech). Average readings from triplicate wells were then expressed as a percentage of average bioluminescence measured from control DMSO wells treated with vehicle (DMSO), representing the highest DMSO concentration in drug-treated cells. A dose–response model was used to estimate IC50 values from cell viability data. Multiple models from the DoseFinding and drc packages were fitted and the best model was selected based on residual SE using the R software.

For combination studies, LKB1-deficient A549, EKVX, H1355, H1944, H2030, H2126, H460, H1573, H1993, H2122, and HCC15 cell lines, and LKB1-proficient Calu1, H1155, H1299, H1819, H1838, H2052, H508, H661, HCC4011, Hop62, Hop92, DFCI032, H2228 cell lines were treated with seven different concentrations (serial 3-fold dilutions) for the following agents either individually or in combination with 8-Cl-Ado for 96 hours. 8-Cl-Ado was combined at indicated fixed ratio with the following drugs: compound C at 100:30 ratio, PI-103 at 100:3 ratio, trametinib at 100:1 ratio, or A66 at 1:1 ratio. The concentration range for each drug was determined based on specific IC50 set as median dose. CalcuSyn software program (Biosoft, Cambridge, UK) was used to calculate combination indexes (CIs) to assess the efficacy of drug combinations.

Cell proliferation assay

For cell counting proliferation assay 1×105 cells were seeded in 6-wells plate. Twenty-four hours later, 8-Cl-Ado or DMSO were added at the indicated concentrations. After 72 hours of incubation cells were harvested and stained with Trypan Blue 0.4% and counted using Count II Automatic Cell Counter (Invitrogen).

Clonogenic assay

400 cells were plated in triplicate into 24-well plates containing 0.5 ml of medium. Cells were incubated for 24 hours in a humidified CO2 incubator at 37°C, and subsequently the medium was then replaced with medium containing indicated drug concentrations, allowing cells to continuously grow for colony formation for 10 to 14 days. To assess clonogenic survival, we stained cells with 1% crystal violet, wash with distillated water and dry out. Quantification of cell density was calculated by covered area using Image J software.

Intracellular NTPs and macromolecule synthesis quantification

Perchloric acid was used to extract nucleosides and nucleotides from LKB1 isogenic A549 and H460 cell lines treated with or without 8-Cl-Ado, and the neutralized extracts were analyzed by high-pressure liquid chromatography (HPLC) using a Waters 600E System Controller (Waters Corp., Milford, MA, USA) as described previously (21). Three independent experiments were performed for each dose assessed.

Macromolecule synthesis was measured by the addition of either 1 μCi/ml [3H]-thymidine, [3H]-uridine, or [3H]-leucine during the final 30 min of a drug treatment as described (22). Radioactivity incorporation into macromolecules was assessed in acid insoluble cellular materials retained on multiscreen-GV filters (Millipore Corp., Bedford, MA).

Apoptosis assays

We assessed the viability of cells treated for 4 days with the indicated doses of 8-Cl-Ado. Cells were stained with annexin V-Cy5 (BD Biosciences, San Jose, CA) and propidium iodide (PI) according to the manufacturer’s instructions and analyzed with a FACSCalibur flow cytometer (BD Biosciences, San Jose, CA, USA) using CellQuest software (BD Biosciences, San Jose, CA, USA). Cells staining positive for either annexin V or PI were considered non-viable. A minimum of three independent experiments were performed for each dose assessed.

ROS level quantification

A549, H460, H2030, H23 and Calu-6 LKB1 isogenic pairs were treated for 72 hours with 5μM of 8-Cl-Ado. After treatment cell were incubated with CellRox DeepRed (Life Technologies, Carlsbad, CA, USA) at a final concentration of 5μM for 30 minutes at 37 ºC. After incubation cells were trypsinized, washed and re-suspended in FACS buffer for fluorescence emission quantification. ROS levels were assessed by flow cytometry using FACSCanto and FACSDiva software (BD Biosciences, San Jose, CA, USA).

Reverse phase protein array analysis

Reverse phase protein array (RPPA) analysis was performed and analyzed as described previously (28). Protein lysates were prepared from untreated and treated cells at the indicated times, and serial dilutions of the lysates were printed on nitrocellulose-coated glass FAST Slides (Schleicher & Schuell BioScience Inc., Dassel, Germany). Each array was incubated with primary antibodies (Table S2) extensively validated by immunoblot analysis.

Immunoblot analysis

Protein lysates were collected using RIPA lysis buffer (1% Triton X-100, 50 mM HEPES, 150 mM NaCl, 1.5 mM MgCl2, 1 mM EGTA, 100 mM NaF, 10 mM Na pyrophosphate, 1 mM Na3VO4, 10% glycerol) or 1x Cell Signaling Lysis Buffer with 1 mM PMSF, supplemented with protease (mini-Complete) and phosphatase (phosStop) inhibitors (Roche Diagnostics Corporation, Indianapolis, IN). Primary antibodies were rabbit polyclonal antibodies against phospho-p70S6K (Thr389), LKB1 clone 27D10, phospho-ACC Ser79 clone D7D11, phospho-AMPKα (Thr172) clone D79.5E, phospho-ribS6 clone D57.2.2E, p70 S6 clone 49D7, phospho-Erk1/2 clone 197G2, Erk1/2 #9102, Akt #9272, phosphor-Akt (Ser473), phosphor-GSK-3α/β (Ser21/9) #9331 (Cell Signaling Technology, Danvers, MA); and mouse monoclonal antibodies against, GSK-3α/β (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), GAPDH clone 6C6 (Novus Biologicals, Inc., Littleton, CO), vinculin clone H-10 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), and ribS6 clone 54D2 (Cell Signaling Technology).

Statistical analysis

All graphing, statistical, and regression analyses were performed using either Prism software (GraphPad Software, San Diego, CA) or the R system for statistical computing. The paired t-test, non-parametric Mann-Whitney t-test, and Holm-Sidak unpaired t-test were used as indicated. Box-and-whisker plots indicate medians with interquartile ranges. Synergy was assessed using CompuSyn software.

Results

LKB1-deficient NSCLC cells are associated with sensitivity to 8-Cl-Ado

To identify therapeutic agents with activity against LKB1-deficient NSCLC, we screened twenty-seven commonly used chemotherapeutic agents against a panel of fifty-seven NSCLC cell lines. LKB1 status was defined based on STK11 genetic status, STK11 mRNA, and LKB1 protein levels (Table S1), and deficiency was assigned based on LKB1 levels in cell lines with known STK11 mutations (Fig. S1). NSCLC cell lines harboring STK11/LKB1 mutations were significantly more sensitive to 8-amino-adenosine (8-NH2-Ado) and 8-Cl-adenosine (8-Cl-Ado), two 8-substituted-adenosine analogues, when compared with STK11/LKB1 wild type (wt) NSCLC cell lines. Likewise, 8-Cl-Ado IC50 values were significantly lower for STK11/LKB1 mutant cells than STK11 wild type (wt) NSCLC cells (P=0.003; Fig. 1A). Based on these initial findings, we next expanded our drug screen to a panel of 104 NSCLC cell lines for sensitivity to 8-Cl-Ado. Consistent with our initial observations, LKB1-deficient cells were significantly more sensitive to 8-Cl-Ado than LKB1-proficient cells (P=0.0080; Fig. 1B). Since STK11/LKB1 is frequently co-mutated with KRAS, we also assessed sensitivity to 8-Cl-Ado in regard to KRAS and STK11/LKB1 mutation status. There was not a significant difference in 8-Cl-Ado IC50 values (Fig. 1C) between KRAS wt and mutant cells. Overall, these results identify STK11/LKB1 alterations as a potential molecular subgroup with enhanced 8-Cl-Ado sensitivity.

Figure 1.

Figure 1.

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LKB1-deficient NSCLC cells display increased sensitivity to 8-Cl-Ado. (A) Comparison of 8-Cl-Ado growth/survival inhibitory IC50 values of individual NSCLC cell lines grouped by STK11 mutation status as indicated for 57 cell lines panel. Comparison of the growth/survival inhibitory IC50 values of 8-Cl-Ado from a panel of 104 NSCLC cell lines screen delineated by (B) LKB1 deficiency or (C) LKB1 deficiency and/or KRAS mutation status. Statistical analysis was performed using a two-tailed non-parametric Mann-Whitney t-test. (D-J) Scatter plots showing the correlation between 8-Cl-Ado sensitivity and basal expression levels for indicated proteins for LKB1-deficient and proficient NSCLC cell lines tested. P values were calculated by Spearman correlation.

We next performed an extensive protein expression analysis to validate the correlation of LKB1 protein expression and sensitivity to 8-Cl-Ado. Sixty-six LKB1-proficient and deficient NSCLC cell lines were proteomic profiled by reverse phase protein array (RPPA) and correlations between IC50 and levels of protein expression was assessed. Consistently, LKB1 and p-AMPK protein levels strongly correlated with the IC50 values for 8-Cl-Ado (Fig. 1D–F). A similar correlation was observed for p-AMPKT172 and AMPKα levels when only LKB1-deficient cell lines were analyzed (Fig. S2A&B), suggesting that inactivation of AMPK axis may play a role in the sensitivity to 8-Cl-Ado. Likewise, 8-Cl-Ado IC50 values negatively correlated with levels of proteins that are down-regulated by the LKB1/AMPK signaling pathway, such as MYC, S6, FOXO3 and CD98 (Fig. 1G–J) (13,29–32).

We next sought to determine whether LKB1 expression has a causal role in mediating sensitivity to 8-Cl-Ado. We generated human NSCLC LKB1 isogenic pairs using A549, H460 (LKB1-deficient), and Calu-6 (LKB1-proficient) cell lines (Fig. 2A). Consistent with our previous finding, re-expression of LKB1 significantly reduced sensitivity to 8-Cl-Ado in A549 and H460 LKB1-deficient cell lines assessed by IC50 estimation (Fig. 2B). Similar data were obtained using two additional LKB1-deficient cell lines, H23 and H2030 (Fig. S3A&B). Likewise, cell proliferation assessed by clonogenic and cell counting assays revealed a significantly reduced sensitivity to 8-Cl-Ado in A549, H460 and H2030 LKB1-proficient compared with LKB1-deficient cells (Fig. S3C&D). These methods displayed differences for a more limited range of concentrations when compared with cell viability by Cell-Titer Glo, which may be due to differences in technical precision as well as end time points and experimental design used for each method. Conversely, shRNA-mediated knock down of LKB1 in Calu-6 cells demonstrated a trend towards increased sensitivity (Fig. 2B). To determine whether 8-Cl-Ado treatment causes cytostatic or cytotoxic effects we assessed the apoptotic rate based on annexin-V/PI staining in A549, H460 and Calu-6 isogenic pairs treated with 0, 2 and 20μM of 8-Cl-Ado for four days (Fig. 2C–F). The fraction of late apoptotic (annexin V/PI positive) and necrotic (PI positive) cells was significantly higher in A549 LKB1-deficient cells following treatment with 8-Cl-Ado. The early apoptotic population (annexin V positive) was also significantly increased in LKB1-deficient cells compared to LKB1-proficient cells when treated with 20μM of 8-Cl-Ado (Fig. 2C). Likewise, we quantified the percentage of annexin V/PI positive cells after treating A549, H460 and Calu-6 LKB1 isogenic pairs with 2–50μM 8-Cl-Ado. Although the dose needed to induce substantial levels of cell death exceed IC50, the fraction of apoptotic cells was significantly increased after 8-Cl-Ado treatment in LKB1-deficient cells (Fig. 2D–F). These data suggest that the greater sensitivity in LKB1-deficient cells to lower doses may be associated with induction of cytostatic rather than cytotoxic effects. Taken together, these results indicate that LKB1 loss is associated with increased sensitivity to 8-Cl-Ado.

Figure 2.

Figure 2.

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LKB1 expression status determines 8-Cl-Ado–mediated cytotoxicity. (A) Immunoblot analysis for LKB1 protein expression in A549, H460 and Calu-6 isogenic pairs. (B) IC50 values of 8-Cl-Ado for H460, A549 and Calu-6 isogenic pairs determined by Cell Titer Glo assay after 72 hours of treatment. Statistical analysis performed using a two-tailed unpaired t-test. (C) Flow cytometry analysis of annexin V/PI staining in LKB1 isogenic A549 cells treated with 8-Cl-Ado for 4 days at indicated doses. (D-F) Quantitation of percentage of late apoptotic population represented by annexin V and PI positive cells for triplicate experiments performed in (D) A549, (E) H460 and (F) Calu-6 LKB1 isogenic pairs. Statistical analysis performed using an ANOVA Sidak’s multiple comparisons test. Bars represent the Mean ± SEM.

Impaired DNA, RNA or protein synthesis does not mediate the selective vulnerability of LKB1-deficient cells to 8-Cl-Ado

We previously demonstrated that 8-Cl-Ado inhibits RNA and DNA synthesis in hematologic malignancies which at least in part mediate its anti-tumor activity (33). To evaluate if the differential sensitivity of LKB1-deficient and proficient cell lines is associated with decreased DNA, RNA or protein synthesis impairment, we quantified incorporation of 3[H] thymidine, 3[H] uridine and 3[H] leucine respectively after treatments with 0–20μM of 8-Cl-Ado (Fig. 3). Consistent with the earlier finding in other tumor types, 8-Cl-Ado treatments resulted in an overall reduction of RNA and proteins synthesis in both LKB1-deficient and LKB1-proficient cell lines. These effects, however, were overall not greater in LKB1-deficient as compared with LKB1-intact cells. We observed a modest but significant difference in the effects of 8-Cl-Ado on RNA synthesis in H460-deficient compared with proficient cells after treatment with 2 and 20μM of 8-Cl-Ado, similar differences were not observed in the A549 model. Likewise, there was no significant difference in the impact of 8-Cl-Ado on proteins synthesis between LKB1-proficent and deficient models. (Fig. 3). It is interesting to note that the 8-Cl-Ado-induced inhibition of DNA synthesis was greater in LKB1-proficient A549 cells at 2 and 20μM than in LKB1-deficient cells. Although we did not specifically address this issue experimentally, this phenomenon may be due, at least in part, to the lack of mTOR pathway suppression observed under conditions of ATP depletion in LKB1-deficient cells, discussed in more detail below. These results indicate that while 8-Cl-Ado significantly reduced DNA, RNA or protein synthesis, this reduction is not the primary mechanism of the selective vulnerability of LKB1-deficient cells to 8-Cl-Ado.

Figure 3.

Figure 3.

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8-Cl-Ado–mediated DNA, RNA and protein synthesis impairment does not determine LKB1-dependent 8-Cl-Ado sensitivity. Macromolecules synthesis in NSCLC cells as measured by [3H]-thymidine, [3H]-uridine and [3H]-leucine incorporation after 72 hours of 8-Cl-Ado treatment at indicated concentration in H460 and A549 LKB1 isogenic pairs. Statistical analysis was performed using a two-tailed paired t-test. Data are presented as mean ± SEM. Bars represent the mean ± SEM.

Cytotoxic effects of 8-Cl-Ado in LKB1-deficient NSCLC cells is mediated by cellular energetic stress.

To further investigate potential mechanisms underlying the enhanced sensitivity of LKB1-deficient cells to 8-Cl-Ado, we assessed the effect of 8-Cl-Ado on ribonucleoside triphosphates (ATP, GTP, CTP, and UTP) levels. Baseline ATP levels were increased by 20% when LKB1 was expressed in H460 and A549 cells (Fig. 4A). 8-Cl-Ado treatments reduced ATP levels to a greater extent in LKB1-deficient cells than in LKB1-proficient cells, showing an ATP depletion of 60% in H460-deficient cells and only 25% in LKB1-proficient cells compared with control LKB1-deficient non-treated cells (Fig. 4B). This greater ATP depletion found in LKB1-deficient cells was not due to a greater accumulation of the drug, since accumulation of active triphosphate form of 8-Cl-Ado (8-Cl-ATP) was comparable or lower in LKB1-deficient cells compared with their LKB1-proficient control (Fig. 4C). Evaluation of all four ribonucleoside triphosphates revealed a significant depletion of purine ribonucleotides, with similar levels of ATP and GTP depletion. By contrast, the effects of 8-Cl-Ado on pyrimidine ribonucleoside triphosphates (CTP and UTP) levels did not show an overall significant reduction either in LKB1-deficient or in proficient cells (Fig. 4D and Fig. S4). For comparison, H460 and A549 LKB1-proficient and LKB1-deficient isogenic pairs were next treated with two other ATP-depleting agents which have undergone clinical testing, metformin and 2-deoxyglucose (2-DG), at concentrations comparable to or greater than those typically achieved in the clinic (10,11,19). Consistent with the earlier findings, we observed that LKB1-deficient cells were more sensitive to ATP depletion than LKB1-proficient cells for all three drugs (Fig. 4E). The levels of ATP depletion by 8-Cl-Ado were comparable to, or greater than, those of metformin and 2-DG.

Figure 4.

Figure 4.

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LKB1 expression status attenuates 8-Cl-Ado–mediated depletion of ATP and induction of ROS production. (A) Relative ATP levels in H460 and A549 LKB1-proficient vs LKB1-deficient A549 and H460 cell lines by HPLC analysis. (B) Relative ATP levels in LKB1 isogenic H460 and A549 LKB1 isogenic pairs treated with increasing concentrations of 8-Cl-Ado assessed by HPLC analysis. Values are percent of the average nucleotide levels compared to H460 or A549 control LKB1-proficient 0 hours untreated cells. (C) Intracellular quantification of 8-Cl-Ado’s cytotoxic metabolite, 8-Cl-ATP, in LKB1 isogenic H460 and A549 cells treated with increasing concentrations of 8-Cl-Ado. (D) Relative NTPs levels in H460 and A549 LKB1 isogenic pairs treated with 2μM 8-Cl-Ado for 6 hours assessed by HPLC analysis. Values are percent of the average nucleotide levels compared to 0 hours untreated cells. (E) HPLC ATP analysis in LKB1 isogenic H460 and A549 cells treated with 2μM 8-Cl-Ado, 10 mM metformin (Metf) or 20 mM 2-deoxyglucose (2DG). (F) Flow cytometry analysis of cellular ROS levels in LKB1 isogenic A549, H23, H2030, H460, and Calu6 cells after treatment with 5μM 8-Cl-Ado for 72 hours. ROS levels are graphed as fold change to average of all LKB1-deficient untreated cells. Statistical analysis was performed using a two-tailed paired t-test. Data are presented as mean ± SEM. Bars represent the mean ± SEM.

Cytotoxic effects of 8-Cl-Ado in LKB1-deficient NSCLC cells is mediated by increased ROS stress

LKB1 loss affects not only ATP levels, but also redox homeostasis (8). Therefore, we next investigated whether the cytotoxic effect of 8-Cl-Ado in LKB1-deficient cells is mediated by an increase in intracellular ROS. We measured intracellular ROS levels in A549, H460, H23, H2030 and Calu-6 LKB1 isogenic pairs with and without 5μM of 8-Cl-Ado treatment for 72 hours. As expected, basal ROS levels were significantly higher in LKB1-deficient than LKB1-proficient cells (Fig. S5A). Furthermore, in LKB1-deficient cells 8-Cl-Ado significantly increased ROS levels, which is indicative of higher redox stress (Fig. 4F and Fig. S5B). In contrast, 8-Cl-Ado did not significantly increase ROS levels in the LKB1-proficient cells. Thus, ROS levels were significantly lower in 8-Cl-Ado–treated LKB1-proficient cells when compared with 8-Cl-Ado–treated LKB1-deficient cells, suggesting that LKB1 plays an important role in protecting cells from 8-Cl-Ado–mediated ROS accumulation.

Altogether, these data suggest that 8-Cl-Ado cytotoxicity in LKB1-defient tumor cells may be mediated by ATP depletions as well as an accumulation of intracellular ROS.

AMPK/mTOR axis deregulation enhances sensitivity to 8-Cl-Ado in LKB1-deficient NSCLC

Alterations in ATP or ROS levels can lead to activation of the cellular energy sensor AMPK through both LKB1-dependent and -independent mechanisms (14,34). Previously, we showed that 8-Cl-Ado induces AMPK activation in breast cancer and CLL cells (25,35). Therefore, we next evaluated the effects of 8-Cl-Ado on AMPK activation in LKB1-deficient NSCLC cells. H460, A549 and Calu-6 isogenic pairs were treated with 5 or 10 μM of 8-Cl-Ado and p-AMPK T172 levels were analyzed by Western blotting. 8-Cl-Ado increased p-AMPKT172 in LKB1-proficient but not LKB1-deficient cells (Fig. 5A). Likewise, 8-Cl-Ado increased the phosphorylation of the AMPK downstream target protein acetyl CoA carboxylase (ACC) in LKB1-proficient but not LKB1-deficient cells, demonstrating that activation of AMPK after 8-Cl-Ado treatment is LKB1-dependent (Fig. 5B&C).

Figure 5.

Figure 5.

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8-Cl-Ado–mediated AMPK activation is LKB1 dependent. Immunoblot analysis of (A) phospho-AMPK and LKB1 in indicated LKB1 isogenic pairs; (B) phospho-ACC, LKB1 and phospho-p70S6K for H460 and A549 LKB1 isogenic pairs; and (C) phospho-ACC, LKB1, phospho-ACC, phospho-p70S6K and phospho- and total-S6 ribosomal protein levels in Calu-6 cells. Cells were treated with the indicated concentrations of 8-Cl-Ado for 24 h. Data shown in panel B and C for A549 and Calu-6 pairs respectively were generated by re-probing blots used in panel A. For presentation purpose loading controls from panel A have been repurposed in panel B and C for A549 and Calu-6 pairs, respectively. (D) IC50 values and (E) combination indexes for 8-Cl-Ado and the AMPK inhibitor compound C (CmpC) at ED25, ED50, and ED75. Statistical analysis was performed using an ANOVA Sidak’s multiple comparisons test. Bars represent the median with interquartile range.

AMPK regulates cellular energy in part by inhibiting the mTOR pathway to restore energy balance through the reduction of cell proliferation and therefore ATP-consuming processes. 8-Cl-Ado has been shown to inhibit mTOR signaling in breast cancer cells (35). In LKB1-proficient cells, but not LKB1-deficient cells, 8-Cl-Ado treatment resulted in inhibition of mTOR activity, as evidenced by the phosphorylation of its downstream targets p70S6K and S6 (Fig. 5B&C). Consistent with these findings, 8-Cl-Ado suppressed DNA synthesis to a greater extent in LKB1-proficient compared with LKB1-deficient cells (Fig. 3). These data suggest that LKB1 loss impairs the ability of the cell to suppress DNA synthesis through mTOR signaling under conditions of ATP depletion induced by 8-Cl-Ado, which may lead to an inability to maintain ATP and redox homeostasis, and contribute to the enhanced sensitivity to 8-Cl-Ado in LKB1-deficient tumor cells.

To determine the role of AMPK in 8-Cl-Ado sensitivity we then evaluated the synergistic effects of compound C, an AMPK inhibitor (36–38) with 8-Cl-Ado in a panel of 14 LKB1-proficient and 10 LKB1-deficient NSCLC cell lines (Fig. 5D&E). IC50 values of compound C were significantly reduced in LKB1-deficient and proficient cells when combined with 8-Cl-Ado (Fig. 5D). To further determine synergistic effect between these two drugs we calculated the combination index (CI) for each cell line at 25%, 50% and 75% the effective dose (ED25, ED50 and ED75, respectively); CIs <1.0 were considered to indicate synergy (39). CI values indicated synergistic effects of compound C when combined with 8-Cl-Ado in both, LKB1-proficient and deficient cells (Fig. 5E), although the synergistic effect was more pronounced in LKB1-deficient cell lines. These results indicate that LKB1-deficient cells are more sensitive to energy-depleting agents and that dysregulation of AMPK/mTOR axis may be partially responsible of the higher sensitivity associated with LKB1 loss.

8-Cl-Ado treatment activates MEK/ERK and PI3K/AKT pathways

We next sought to identify other proteins or signaling pathways associated with sensitivity to energy depletion. Proteomic profiling revealed that basal levels of ERK1/2 and MEK1/2 proteins correlated with 8-Cl-Ado sensitivity (Fig. 6A–E). Among LKB1-deficient cell lines, ERK2 as well as MEK levels, were specifically associated with 8-Cl-Ado sensitivity (Fig. S6A&B). Likewise, AKT significantly correlated with 8-Cl-Ado sensitivity (Fig. 6F) and we observed same trend in LKB1-deficient cells analysis (Fig. S6C). These data suggest that the MEK/ERK and PI3K/AKT pathways may play a role in the response to 8-Cl-Ado.

Figure 6.

Figure 6.

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Basal protein expression patterns correlated with 8-Cl-Ado sensitivity. Protein markers as assessed by RPPA that significantly correlate with 8-Cl-Ado sensitivity/resistant in all NSCLC cell lines tested. Scatter plots showing the correlation between 8-Cl-Ado sensitivity and basal expression levels of (A-F) indicated proteins for LKB1-proficient and deficient NSCLC cell lines tested. P values were calculated by Spearman correlation.

To further investigate the role of MEK/ERK and PI3K/AKT pathways on 8-Cl-Ado sensitivity, we assessed 8-Cl-Ado–mediated changes in the proteome of LKB1-deficient A549, H1355, H1944, H2030, and H2126 cell lines and LKB1-proficient H1155, HOP-62, H1299, H1819, H1838, HCC15, and H2052 cell lines. Cells were treated with 10μM 8-Cl-Ado for 16, 24, or 48 hours and then evaluated by RPPA. p-ERK1/2T202Y204 (p-MAPKT202Y204), p-AKTS473 and the AKT target p-GSK3βS21/9 showed the greatest significant increase after 8-Cl-Ado treatment in LKB1-deficient and proficient cells (Fig. 7A and Fig. S7A&B). Furthermore, 8-Cl-Ado treatment resulted in activation of these signaling molecules regardless of the LKB1 status (Fig. S7C&D). To confirm whether 8-Cl-Ado induces phosphorylation of these proteins and evaluate the impact of LKB1 expression on this modulation, we then analyzed total and phosphorylated levels of ERK 1/2, AKT and GSK3β by Western blotting in three LKB1 isogenic pairs after treatment with 10 μM 8-Cl-Ado for 24 h (Fig. 7B). 8-Cl-Ado induced ERK1/2(T202/204), AKTS473, and GSK3βS9 phosphorylation in both LKB1-deficient and proficient cells.

Figure 7.

Figure 7.

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8-Cl-Ado treatment induces ERK1/2 and AKT activity and synergizes with MEK and PI3K inhibitors. (A) Heat map showing 8-Cl-Ado–mediated fold changes in the levels of proteins and phosphoproteins which showed significant differences at 24 hours of 8-Cl-Ado treatment measured by RPPA analysis only for LKB1-deficient cell lines assessed. (B) Immunoblot analysis of phospho-ERK1/2, total ERK1/2, phospho-AKT(S473), total AKT, phospho-GSK3β(S9), and total-GSK3β protein levels in LKB1 isogenic H460, A549, and Calu-6 cells treated with 8-Cl-Ado for 24 hours. (C) IC50 values and (D) combination index values for 8-Cl-Ado and MEK inhibitor (trametinib). (E) IC50 values and (F) combination index values for 8-Cl-Ado and PI3K inhibitor (A66) at ED25, ED50, and ED75 in the cell lines assessed. Statistical analysis was performed using an ANOVA Sidak’s multiple comparisons test. Bars represent the median with interquartile range.

Altogether, these finding suggest that the MAPK/ERK and PI3K/AKT pathways play a compensatory role in response to the energetic stress induced by 8-Cl-Ado. Therefore, higher expression of these proteins following 8-Cl-Ado treatment as seen in LKB1-proficient cells may mediate reduced sensitivity to 8-Cl-Ado.

MEK inhibition synergizes with 8-Cl-Ado in LKB1-deficient cells

Since cells with higher levels of ERK1/2 and MEK1/2 were less sensitive to 8-Cl-Ado, we sought to determine the efficacy of combined MEK inhibitor and 8-Cl-Ado treatment. We treated ten LKB1-deficient and fourteen LKB1-proficient cell lines with 8-Cl-Ado and the MEK inhibitor trametinib, alone or in combination. In LKB1-deficient cells, the IC50 values for 8-Cl-Ado in combination with trametinib were significantly lower than those for 8-Cl-Ado or trametinib alone, suggesting that the cells had enhanced sensitivity to 8-Cl-Ado when MEK was inhibited (Fig. 7C). Similarly, in LKB1-proficient cells, the IC50 values for 8-Cl-Ado in combination with trametinib were significantly lower than single agent treatments IC50, although to a lesser extent compared to LKB1-deficient cells (Fig. 7C). To discern whether 8-Cl-Ado synergizes with trametinib, we calculated the CI value for each cell line. Strong synergy was noted in the LKB1-deficient cell lines, where all but one cell line had a CI ≤0.5. Moreover, CI values of most LKB1-proficient cell lines indicated synergistic effects, however two cell lines indicated additive effects at ED75 and three cell lines even displayed antagonistic effects (Fig. 7D). These results indicate that inhibition of the MEK pathway could synergize with 8-Cl-Ado in LKB1-deficient and some LKB1-proficient cells.

PI3K inhibition synergizes with 8-Cl-Ado in LKB1-deficient cells

Because we found that 8-Cl-Ado also mediated induction of PI3K/AKT signaling, we next tested the effect of 8-Cl-Ado in combination with two PI3K inhibitors, PI-103 and A66, in the same set of cell lines. Combination of 8-Cl-Ado with the PI3K inhibitor A66 significantly reduced IC50 in LKB1-deficient and proficient cell lines, although increment in sensitivity was more pronounced in LKB1-deficient compared to LKB1-proficient cell lines (Fig. 7E). Strong synergy was found in all cell lines regardless LKB1 expression. CI values for these combinations yielded synergistic effect in a portion of cell lines and additive effects in others, including in LKB1-deficient cell lines (Fig. 7F). Similarly, the addition of PI3K inhibitor, PI-103, significantly enhanced sensitivity to 8-Cl-Ado in all cell lines (Fig. S8A), although synergy appeared more robust with the A66 compound. These findings provide evidence that inhibition of the PI3K pathway may enhance the cytotoxic effects and may compensate for ATP-depletion induced by 8-Cl-Ado in LKB1-deficient and, to a lesser extent, in LKB1-intact cells.

Discussion

STK11 (LKB1) is the second most altered tumor suppressor gene in NSCLC, and loss of LKB1 expression leads an aggressive tumor phenotype with worse prognosis and reduced responsiveness to PD1/PD-L1 inhibitors (6,12,40). There are currently no approved treatment strategies for this subset of patients. Here, we identified 8-Cl-Ado, an adenosine analogue which induces an imbalance in intracellular ATP/AMP levels, as active agent for LKB1-deficient NSCLC cells. Additionally, we show that 8-Cl-Ado treatment results in the activation of the MAPK/MEK/ERK and PI3K/AKT/mTOR pathways and targeting of these pathways synergistically enhances the anti-tumor activity of 8-Cl-Ado in LKB1 deficient tumor cells, identifying these regimens as a potential therapeutic strategy for NSCLC patients harboring LKB1-deficient tumors.

LKB1 is a master regulator of cellular growth and metabolism. Under cellular stress condition, LKB1/AMPK axis senses low levels of ATP and NADPH to restore intracellular energy homeostasis (15,41) at least in part by modulating cell proliferation through activation of the mTOR pathway (16). LKB1-deficient tumors display a disrupted balance between energy consumption and production, bypassing normal growth inhibitory signals, resulting in unrestricted cell growth which reduces cellular ATP levels, therefore increasing energetic and oxidative stress (8,42). Thus, LKB1-deficient tumors display increased vulnerability to energetic stress induced by ATP depletion.

The C8-modified adenosine analogues 8-Cl-Ado and 8-NH2-Ado are cytotoxic to a number of liquid and solid tumor cells (43,44) and currently, 8-Cl-Ado is under a phase I/II clinical trial for hematological malignancies (NCT02509546) (21). Compared with nucleoside analogues established for cancer therapeutics, these analogues are unique as they possess a ribose sugar. We previously identified that 8-Cl-Ado is metabolized to 8-Cl-AMP, 8-Cl-ADP, and 8-Cl-ATP (21). 8-Cl-ADP and 8-Cl-ATP act as mimetics to disrupt the metabolism of the endogenous adenylate pools (26), which results in diminished cellular ATP levels (20,22,45). Increased 8-Cl-ATP also inhibits mRNA transcript synthesis by incorporating into the RNA as a chain terminator or by inhibiting polyadenylation (24). Previous studies have shown that 8-Cl-Ado and 8-Cl-cAMP, a prodrug form of the analogue, inhibit the growth and/or survival of murine and human lung cancer cells. Although these studies did not assess the context in which LKB1 affects sensitivity and energy depletion, they did show that the mechanism of action of 8-Cl-Ado in NSCLC cells was associated with G2/M arrest and mitotic catastrophe (43,44). In agreement with these previous findings, our data indicate that basal ATP depletion in LKB1-deficient tumor cells selectively increases vulnerability to 8-Cl-Ado. Treatment with this compound significantly impairs cell proliferation by reducing intracellular ATP levels to greater extent in LKB1-deficient tumor cells. While other ATP-depleting agents (metformin and 2-DG) also show preferential ATP depletion in LKB1-deficient cells, the concentrations of these agents needed to achieve similar levels of ATP depletion have not been shown to be clinically achievable (10,11,19). Moreover, our data showed that 8-Cl-Ado activates AMPK signaling in a LKB1-dependent manner, indicating that LKB1-AMPK axis activation may play a key role in compensating for the ATP reduction induced by 8-Cl-Ado in LKB1-proficient tumors.

ROS are a by-product of mitochondrial respiration. Low to moderate ROS levels stimulate cell proliferation and differentiation (46), by contrast excess ROS levels cause oxidative stress and subsequent cell death by damaging cellular components, such as DNA, proteins and lipids (47). As a consequence of increased energetic and metabolic stress, LKB1-deficient cells generate elevated levels of ROS (8,42), and the induction of ROS production has also been shown to promote AMPK phosphorylation and activation but may occur in an LKB1-independent manner (34). Glutamine-derived glutamate is the main precursor of glutathione (GSH), the major cellular antioxidant for redox homeostasis. We recently reported that LKB1 alterations drive metabolic adaptations resulting in a glutamine “addiction” which leads to reduced intracellular GSH stores and, consequently vulnerability to oxidative stress (8). Indeed, LKB1-deficient tumor cells used in the present work showed increased level of intracellular ROS. Furthermore, 8-Cl-Ado treatment raised ROS levels primarily in LKB1-deficient cells, but not in LKB1-proficient cells, suggesting that induction of cell death after 8-Cl-Ado treatment may be also driven by higher ROS stress.

Response to energy depletion involves shifting of cellular metabolism to restore the energy balance by stimulating catabolism and inhibiting ATP-consuming processes. Several pathways are involved in regulating cell proliferation and metabolism to guarantee the correct maintenance of cellular homeostasis. Mitogen-activated protein kinase (MAPK) and phosphatidylinositol-3 kinase (PI3K) modulate extracellular signals to control cell growth, differentiation and migration (48,49). Consequently, combination of mTOR and PIK3 inhibitors has shown therapeutic effects in LKB1-deficient NSCLC (50). Our proteomic analysis revealed differential baseline levels of MAPK and PI3K pathway signaling molecules associated with response to 8-Cl-Ado. Consequently, 8-Cl-Ado treatment activated these signaling in sensitive and resistant cell lines, and suggested that targeting these pathways may potentiate the effects of 8-Cl-Ado. In agreement with this, the combination of 8-Cl-Ado with the MEK inhibitor trametinib, displayed a strong synergistic effect particularly in LKB1-deficient cell lines. Phosphorylation of AKTS473 and its target, GSK3S21/9 was also induced by 8-Cl-Ado, and basal AKT levels correlated with 8-Cl-Ado sensitivity. Moreover, two different PI3K inhibitors (PI-103 and A66) showed increased effects when combined with 8-Cl-Ado in most NSCLC cell lines. These results demonstrate that combinations of 8-Cl-Ado with either MEK inhibitors or PI3K inhibitors merits further investigation as a potential therapeutic regimen for patients harboring LKB1-deficient tumors.

Taken together, our findings indicate that LKB1-deficient NSCLC cells are selectively vulnerable to energy depletion. Treatment with 8-Cl-Ado demonstrated specific anti-tumor activity, depleted ATP levels and raised redox stress in LKB1-deficient cells. Dysregulation of AMPK/mTOR axis associated with LKB1 loss may be responsible for greater sensitivity to 8-Cl-Ado by reducing ability to compensate energy and redox imbalance induced by this compound. Data provided herein support the future clinical testing of energy-depleting agents such as 8-Cl-Ado combined with signaling pathway inhibitors in patients with LKB1-deficient NSCLC and other tumor types.

Supplementary Material

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Financial support

This work was supported by The University of Texas Southwestern Medical Center and The University of Texas MD Anderson Cancer Center Lung UT; Lung SPORE P50CA07907; The LKB1 R01 CA205150; CPRIT CP160652; The Lung Cancer Moon Shot, including donations from; Kyte Family, Jeff Hepper, and Normal Godinho; Rexanna’s Foundation for Fighting Lung Cancer; Weaver Foundation; CCSG CA016672; a Stand Up To Cancer-American Cancer Society Lung Cancer Dream Team Translational Research Grant (Grant Number: SU2C-AACR-DT17-15), a Jane Ford Petrin donation, and Helen H. Laughery donation (V.G.). The indicated Stand Up To Cancer grant is administered by the American Association for Cancer Research, the scientific partner of SU2C.”

Disclosure statement

J.D. Minna reports receiving licensing fees for tumor cell lines from the NIH and University of Texas Southwestern Medical Center; J.V. Heymach reports receiving research support from AstraZeneca, Spectrum Pharmaceuticals, and Boehringer Ingelheim and has served on advisory committees for AstraZeneca, Boehringer Ingelheim, Bristol-Myers Squibb, Catalyst, EMD Serono, Foundation Medicine, Hengrui Therapeutics, Genentech, GSK, Guardant Health, Eli Lilly, Merck, Novartis, Pfizer, Roche, Sanofi, Seattle Genetics, Spectrum, Takeda, and receives royalties and licensing fees from Spectrum; M. Nilsson receives royalties and licensing fees from Spectrum Pharmaceuticals; V. Gandhi reports receiving sponsored research support from Pharmacyclics, Acerta, AstraZeneca, AbbVie, Infinity, Sunesis, and Loxo Oncology. No potential conflicts of interest were disclosed by the other authors.

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Associated Data

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Supplementary Materials

1
NIHMS1750130-supplement-1.pdf (503.3KB, pdf)
2
NIHMS1750130-supplement-2.xlsx (13.5KB, xlsx)
3
NIHMS1750130-supplement-3.xlsx (19.3KB, xlsx)

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