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. Author manuscript; available in PMC: 2015 Sep 1.
Published in final edited form as: Mol Cancer Res. 2014 Jul 24;12(9):1205–1215. doi: 10.1158/1541-7786.MCR-14-0124

Targeting mTORC1-mediated Metabolic Addiction Overcomes Fludarabine Resistance in Malignant B Cells

Arishya Sharma 1,3, Allison J Janocha 4, Brian T Hill 2, Mitchell R Smith 2, Serpil C Erzurum 4, Alexandru Almasan 1
PMCID: PMC4163513  NIHMSID: NIHMS616425  PMID: 25061101

Abstract

mTORC1 activation occurs frequently in cancers, yet clinical efficacy of rapalogs is limited due to the associated activation of upstream survival pathways. An alternative approach is to inhibit downstream of mTORC1; therefore, acquired resistance to fludarabine (Flu), a purine analog and anti-metabolite chemotherapy, active agent for chronic lymphocytic leukemia (CLL) was investigated. Elevated phospho-p70S6K (T389), an mTORC1 activation marker, predicted Flu resistance in a panel of B-cell lines, isogenic Flu-resistant (FluR) derivatives, and primary human CLL cells. Consistent with the anabolic role of mTORC1, FluR cells had higher rates of glycolysis and oxidative phosphorylation than Flu-sensitive (FluS) cells. Rapalogs (everolimus, rapamycin), induced moderate cell death in FluR and primary CLL cells, and everolimus significantly inhibited glycolysis and oxidative phosphorylation in FluR cells. Strikingly, the higher oxidative phosphorylation in FluR cells was not coupled to higher ATP synthesis. Instead it contributed primarily to an essential, dihydroorotate dehydrogenase (DHODH) catalyzed, step in de novo pyrimidine biosynthesis. mTORC1 promotes pyrimidine biosynthesis by p70S6 kinase-mediated phosphorylation of CAD (Ser1859) and favors S-phase cell cycle progression. We found increased phospho-CAD (S1859) and higher S-phase population in FluR cells. Pharmacological inhibition of de novo pyrimidine biosynthesis using N-phosphonacetyl-L-aspartate (PALA) and leflunomide, RNAi-mediated knockdown of p70S6K, and inhibition of mitochondrial respiration were selectively cytotoxic to FluR, but not FluS cells. These results reveal a novel link between mTORC1-mediated metabolic reprogramming and Flu resistance identifying mitochondrial respiration and de novo pyrimidine biosynthesis as potential therapeutic targets.

Implications

This study provides the first evidence for mTORC1/p70S6K-dependent regulation of pyrimidine biosynthesis in a relevant disease setting.

Keywords: mTORC1, fludarabine, p70S6K, CAD, CLL

Introduction

Fludarabine (Flu; also known as F-ara-A) is a purine analog that is indicated for the treatment of hematological malignancies, including chronic lymphocytic leukemia (CLL) (1) and indolent non-Hodgkin’s lymphomas (2). Although Flu-based regimens have been successful in improving the outcome in patients, primary or acquired resistance limits the effectiveness of this therapy (1).

Recent research in B-cell malignancies, including CLL and non-Hodgkin lymphomas, suggests that constitutive activation of B-cell receptor-associated cellular signaling pathways and cues from the microenvironment are the key regulators for survival and maintenance of these cancers, as well as their response to chemotherapy (3). A critical downstream component of the B-cell receptor signaling pathway is the mammalian target of rapamycin (mTOR) kinase that is regulated by the phosphatidylinositol 3-kinase (PI3K)/acutely transforming retrovirus (Akt) pathway (4, 5). The mTOR kinase occurs in two distinct complexes: mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2) (6). Akt activates mTORC1, which in turn phophorylates p70S6 kinase (p70S6K) and the eukaryotic-initiation-factor 4E-binding protein-1 (4EBP1), whereas mTORC2 phosphorylates and activates Akt (5, 6).

Aberrant activation of mTORC1 occurs in the most common human cancers, suggesting that mTORC1 signaling confers survival and proliferative advantages to cancer cells (7). Therefore, allosteric inhibitors of mTORC1, rapamycin and its analogs (rapalogs), represent an attractive therapy for various tumors, including hematological malignancies (8, 9). However, these drugs failed to induce significant apoptosis of either cycling or quiescent cells and showed modest clinical responses that were also associated with toxicity (9). The mechanism of resistance to rapalogs is attributed to their ability to inhibit only one of several downstream targets of PI3K, leaving Akt unaffected. Moreover, they also disrupt a feedback mechanism that dampens PI3K activity, leading to a compensatory upregulation of Akt activity, causing counterproductive prosurvival effects. On the contrary, the ATP-competitive dual PI3K/mTORC1/2 and mTORC1/2 inhibitors display potent anticancer properties both in vitro and in vivo in a wide range of malignancies, including leukemia (9, 10). Several of these compounds are being tested in preclinical models and they show a consistently robust effect against tumors driven by PI3K/Akt signaling, while they are ineffective against tumors driven by mutations of Ras, which can signal through multiple pathways, such as those for MEK and ERK (11)..

An alternative approach for inhibiting mTORC1 is to target its downstream effectors. A previous study, using unbiased genomic and metabolomic approaches, reported that gene sets related to specific metabolic pathways, including the pentose phosphate pathway, fatty acid biosynthesis, glycolysis, and cholesterol biosynthesis, comprised the top 20 mTORC1-induced genes (12). mTORC1 stimulates protein synthesis by regulating mRNA translation and ribosome biogenesis (13). Additional recent reports suggest regulation of glutamine (14) and pyrimidine metabolism by mTORC1 (15–17). Consistently, targeting the enzymes comprising metabolic pathways has been evaluated in various mTORC1-dependent cancer settings (18, 19). Targeting downstream metabolic pathways is unlikely to elicit the same unwanted feedback signaling events that appear to limit the usefulness of rapamycin and its analogues in the clinic. Additionally, it is possible that such metabolic inhibitors would elicit selective cytotoxic responses in the tumor, rather than the cytostatic effects routinely seen with rapamycin.

As mTORC1 is associated with poor treatment outcomes in B-cell malignancies (20), we examined the significance of mTORC1 pathway activation in Flu-resistant (FluR) cells that were generated by chronic exposure to Flu (21). Moreover, we investigated the metabolic consequences of mTORC1 activation in FluR cells, aiming to identify their selective vulnerability to interference with specific metabolic pathways. Our study reveals mTORC1-dependent increase in glycolysis and mitochondrial respiration in FluR cells. In addition, there was an increase in de novo pyrimidine biosynthesis, which contributed to addiction to mitochondrial respiration in FluR cells. We propose targeting de novo pyrimidine biosynthesis and mitochondrial respiration as potential strategies to overcome Flu resistance.

Materials and Methods

Reagents

Fludarabine (9-β-D-arabinofuranosyl-2-fluoroadenine 5′-phosphate) was purchased from Sigma Aldrich (St. Louis, MO), everolimus from Selleck (Houston, TX), and rapamycin from Calbiochem (Billerica, MA). N-phosphonacetyl-L-aspartate (PALA, NSC224131) was acquired from the NCI/DTP Open Chemical Repository (http://dtp.cancer.gov) for a study in Dr. Christine McDonald’s laboratory (Cleveland Clinic). Cells were treated with 10 μM fludarabine (Flu) and 200 nM everolimus, unless otherwise stated.

Cell lines and patient samples

Human pre-B acute lymphocytic leukemic Nalm-6, Reh, multiple myeloma RPMI-8226, histiocytic lymphoma U937, and acute T lymphocytic leukemic Molt-4 cell lines were obtained from the ATCC (Manassas, VA). Fludarabine-resistant (FluR) cells were generated by initially culturing cells with a lower concentration (1 μM) of Flu for short periods of time followed by 48 h of recovery time. The drug concentration was increased gradually until the desired resistance of twice the IC50 value was achieved. The resistant cells were intermittently treated with verapamil (Sigma Aldrich, St. Louis, MO) to eliminate the possibility of acquired resistance due to increased expression of efflux pumps. In addition to the derivattive FluR cells, we used Mec-1 and Mec-2 cells (a gift from Dr. Y. Saunthararajah, Cleveland Clinic), which are CLL-derived cell lines known to be inherently resistant to Flu (22, 23). Cells were maintained in RPMI-1640 medium supplemented with 10% fetal bovine serum (Atlanta Biologicals, Lawrenceville, GA), L-glutamine (Gibco BRL, Gaithersburg, MD), and antibiotic-antimycotic (Invitrogen, Carlsbad, CA). Cell lines were verified periodically for morphological characteristics, growth rates, and response to stimuli using Annexin V/Propidium iodide staining or Trypan blue exclusion. Passage number was not allowed to exceed 15–20, and cell lines were routinely tested to be mycoplasma free.

Peripheral blood samples were obtained from patients with CLL after patients gave informed consent according to protocols approved by the Cleveland Clinic Institutional Review Board, according to the Declaration of Helsinki. Briefly, lymphocytes from blood samples were purified by Ficoll-Paque PLUS (Amersham Biosciences, Piscataway, NJ) gradient centrifugation. Primary cells were cultured and cell death was assayed as previously described (21, 24).

Immunoblotting

Cell lysates for immunoblotting and immunoprecipitation were prepared, as described previously (21). The primary antibodies were used against p-p70S6K (T389), p70S6K, pCAD (S1859), cytochrome c, cleaved caspase-3 (Cell Signaling Technologies, Danvers, MA), and β-actin (Sigma, St. Louis, MO). Secondary antibodies were anti-mouse HRP (Millipore, Danvers, MA) and anti-rabbit HRP (Fisher Scientific, Pittsburg, PA).

Cell viability and apoptosis assays

Apoptosis was measured using annexin V–fluorescein isothiocyanate and propidium iodide staining (BD Biosciences, San Jose, CA), as described previously.(21) Cell death data were acquired on a BD FACSCalibur flow cytometer (BD Biosciences, San Jose, CA) and analyzed using CellQuest software. 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H tetrazolium inner salt (MTS) assay (25) (Promega, Madison, WI) was used to assess cell proliferation. Data are expressed as % reduction in metabolic activity i.e. 100- {[(O.D.490nm Untreated) − (O.D.490nm Treated)] / (O.D.490nm Untreated) × 100} versus the indicated concentrations of the drug.

For clonogenic assays, 20 cells/ml were seeded using 30% FBS containing RPMI media in poly-lysine coated plates, and treated as indicated. After 8–12 days, cells were stained with crystal violet and colonies were scored by an alpha image analyzer (Alpha Innotech Corp.). The % surviving fraction was then calculated according to the equation = (number of counts in treated sample/number of counts in NT sample) × 100. (26). The interaction between PALA and Flu in clonogenic assays was determined using the isobolographic method of Chou and Talalay (27). The combination index was calculated using the Compusyn software (www.combosyn.com), combination index < 1 indicates synergism, fraction affected = (100-% surviving fraction)/100.

Extracellular flux (XF) analysis

A Seahorse Bioscience XF-24 Flux Analyzer (Seahorse Bioscience, North Billerica, MA) was used to measure the OCR and ECAR. Cell density titrations were first performed to define the optimal seeding density for Nalm-6 and Nalm-6-FluR cells. Suspension cells were seeded in Seahorse Cell-Tak-coated XF 24-well cell culture microplates in 150 μL Seahorse assay medium [unbuffered DMEM (Sigma D5030), supplemented with 2 mM glutamine, 1 mM pyruvate and 11 mM glucose] pre-warmed to 37°C. In the subsequent experiments, Nalm-6 and Nalm-6-FluR cells were seeded in growth media in plates, as described above, with 95,000 Nalm-6 cells or 50,000 Nalm-6-FluR cells per well to ensure about 90% surface coverage at the time of the experiment. The cells were incubated for 30 min at 37°C to allow media temperature and pH to reach equilibrium. During this time, selective metabolic inhibitors were pre-loaded into injection ports of the cartridge to achieve final concentrations of 2-DG (100 mM), FCCP (1.5 μM), oligomycin (1.5 μM), rotenone (0.75 μM), and antimycin A (0.75 μM). Oligomycin and FCCP titrations were performed for each cell line. Before the first rate measurement, total volume was adjusted to 500 μL for mito-stress using the Seahorse media and incubated for an additional 15 min. At the end of incubation, the plate was placed in the Seahorse XF24 analyzer. During the assay, baseline rates were measured 3 times. OCR was reported in nmol/min and ECAR in milli-pH (mpH)/min and further normalized for each cell type. Substrates and selective inhibitors were injected during the measurements and mixed for 3 to 5 min. OCR and ECAR were then measured 3 times each.

Cytochrome c release

Cells were washed in 1 x PBS and resuspended in the lysis buffer (20 mM Hepes, 10 mM KCl, 1.5 mM MgCl2, 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol, and 250 mM sucrose). To ensure complete cell lysis, cells were drawn into a 28 1/2 or 30 1/2 -gauge needle using a syringe and then expelled a minimum of 20 times. Unbroken cells were removed by spinning at 5,000 rpm for 5 min. The supernatant was again centrifuged at 14,000 rpm for 30 min at 4°C to separate the mitochondrial fraction (pellet) from the cytoplasm (supernatant). The protein was quantified using Bradford’s method, 5 × SDS sample buffer was added to the supernatants, and analyzed on 15% SDS-PAGE gels to probe for cytochrome c release.

ATP quantification

The quantity of ATP was measured using the Mitochondrial ToxGlo luminescent cell viability assay (Promega, Madison, WI) according to the manufacturer’s protocol. Briefly, cells were seeded in white 96-well microplates at 1.0 × 104 cells per well in 100 μL growth media, and treated as indicated in Figure 3 for 2 h at 37ºC, and 5% CO2. Then, 100 μL luminogenic ATP detection reagent was added and luminescence intensity from each well was measured using a multi-label plate reader (Wallac Victor 1420; Perkin Elmer, Waltham, MA).

Figure 3. Constitutive mTORC1 activation leads to metabolic re-programming in FluR cells.

Figure 3

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Untreated Nalm-6 parental (95,000 cells/well), untreated or 16 h Ev-treated Nalm-6-FluR (50,000 cells/well) cells were seeded in V7 Seahorse tissue culture plates. (a) The basal extracellular acidification rate (ECAR) was calculated for each well for 45 min. In the case of Nalm-6-FluR-untreated and everolimus (Ev)-treated cells, ECAR was subsequently measured for another 45 min following 100 mM 2-deoxyglucose (2-DG) injection as a control to validate ECAR as a specific measure of glycolysis. (b) A series of basal oxygen-consumption rates (OCR) were measured for untreated or Ev treated Nalm-6 parental and derivative Nalm-6-FluR cells for the first 45 min and then following sequential injection of 1.5 μM oligomycin, 1.5 μM trifluorocarbonyl-cyanidephenylhydrazone (FCCP), and 0.75 μM rotenone + antimycin A. (c) Nalm-6 FluR cells were treated as indicated for 16 h. Mitochondria-free cytosol was then prepared and cytochrome c release was analyzed by western blotting. (d) Nalm-6 and Nalm-6-FluR cells were plated at 10,000 cells /well in 96-well plates and treated as indicated for 2 h and ATP was assayed using the mitochondrial ToxGlo assay from Promega. Data are presented as counts per second (cps) of luminescence intensity per 10,000 cells. Nalm-6, Nalm-6-FluR, and Mec-2 cells were: (e) treated with 200 mM 2-DG or cultured in glucose-free media for 72 h, or (f) treated with 0.75 μM rotenone and antimycin A for the indicated times and cell death was determined by annexin V/PI staining. Data represent mean ± SD (n=3), *p<0.05,**p<0.01, ***p<0.001, ****p<0.0001.

siRNA transfection

Transfections were performed with control-GFP or S6K1 siRNA Qiagen (Valencia, CA, USA) using the Amaxa Nucleofector Kit V (Lonza), according to the manufacturer's protocol. In brief, 3.0 × 106 cells were transfected with 500 nM siRNA using program D023.

Statistical analysis

Statistical comparisons between two groups were conducted by using the Student’s t test and between multiple groups using 2-way ANOVA with the Prism software (version 5.01). Error bars indicate standard deviation, which was calculated from three independent experiments performed in triplicates.

Results

Fludarabine resistance is associated with hyper mTORC1 activation

Deregulated mTORC1 activity is frequently associated with a variety of human cancers,(7) including leukemia (20, 28) and negatively influences the response to chemotherapy (20). To determine how mTORC1 regulates Flu-resistance, we derived Flu-resistant (FluR) cells from initially sensitive Nalm-6 and Reh cells (21). Examination of phospho-p70S6K at Thr-389 (p-p70S6K T389) using immunoblotting as an assay of mTORC1 activation status revealed higher mTORC1 activation in FluR-Nalm-6, -Reh, and CLL derived Mec-2 cell lines compared to parental Flu-sensitive (FluS) Nalm-6 and Reh cells (Figure 1A). Extending our findings to a panel of malignant B-cell lineage lines by comparing Flu-sensitivity (Figure 1C), determined by the dose-dependent effect of Flu on MTS reduction and mTORC1 activation (Figure 1B), we found a remarkably strong correspondence between hyperphosphorylation of p70S6K and Flu-resistance. In addition, we identified a similar relationship between p-p70S6K T389 (Figure 1D) and Flu resistance (Figure 1E) in primary CLL cells. Thus, Flu resistance is associated with hyper-mTORC1 activation in B cell leukemia and lymphoma cell lines and primary cells.

Figure 1. Fludarabine resistance is associated with mTORC1 activation.

Figure 1

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(a,b) Protein expression analysis of p-p70S6K T389, as a marker of mTORC1 activation in the indicated cell lines by immunoblot. Total p70S6K was used as a loading control. IC50, as determined in (c), is indicated at the bottom. (c) Dose response for the effect of 24 h fludarabine (Flu) treatment on cell growth in the indicated cell lines, as determined by the MTS assay. Data are expressed as mean ± SD (n=3). (d) Protein expression analysis of p-p70S6K T389 and p70S6K in the indicated primary CLL samples. Numbers indicate CLL patient numbers. FluR, fludarabine resistant; FluS, fludarabine sensitive (e) Effect of 48 h fludarabine (Flu) treatment on apoptosis in the indicated primary CLL samples, as determined by annexin V/PI staining and flow cytometry. % cell death following Flu treatment was normalized to % cell death in control cells using the formula: (Livecontrol- LiveFlu/ Livecontrol)*100.

mTORC1 activation is critical for survival of FluR cells

Next, we studied the effect of mTORC1 inhibition on cell death using two different rapalogs, rapamycin (Rap) and everolimus (Ev), alone or in combination with Flu, in FluS versus FluR cells. In Nalm-6, a FluS cell line, 100 nM Rap alone did not induce apoptosis and, in fact, may have led to reduced cleaved-caspase-3 in the presence of Flu (Figure 2A). In contrast, in Nalm-6-FluR cells, mTORC1 inhibition alone did induce cleaved-caspase-3 (Figure 2A). However, Rap (Figure 2A) did not sensitize FluR cells to Flu. Rap (100 nM) inhibited mTORC1 as measured by decreased phosphorylation of p70S6K in FluR cells (Figure 2B). Annexin V/PI staining further confirmed that Rap induced apoptosis in Nalm-6-FluR, but not in parental FluS Nalm-6 cells (Figure 2C). Similar data were obtained with Ev in Nalm-6 and Nalm-6-FluR cells (Figure 2D). Importantly, we found similar results in primary CLL cells cultured ex vivo, indicating that Rap (Figure 2E) or Ev (Figure 2F) alone induce significant cell death (p<0.05), but do not enhance sensitivity to Flu, as measured by annexin V/PI-staining. These findings suggest that even though constitutive mTORC1 activation is critical for survival of FluR cells, mTORC1 inhibition does not overcome Flu resistance.

Figure 2. mTORC1 inhibition causes moderate cell death in FluR cells and does not enhance the cytotoxic efficacy of Flu.

Figure 2

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(a) Western blot analysis for cleaved caspase-3 and β-actin, as a loading control, in Nalm-6 and Nalm-6-Flu-resistant (Nalm-6-FluR) cells following inhibition of mTORC1 using rapamycin (Rap) in combination with Flu. (b) Nalm-6-FluR cells were treated with the indicated concentrations of Rap and cell lysates analyzed by western blotting for p-p70S6K T389 and p70S6K. (c) Nalm-6 and Nalm-6-FluR cells were treated with the indicated concentrations of Rap for 48 h and cell death was determined by annexin V/PI staining. (d) Nalm-6 and FluR cells were treated with everolimus (Ev) and analyzed by western blotting for the levels of indicated proteins. β-actin was used as a loading control. Primary CLL cells were treated with (e) Flu ± Rap or Rap alone and (f) Flu ± Ev or Ev alone for 48 h and cell death was determined by annexin V/PI staining. Data represent mean ± SD (n=7), *p<0.05.

High basal mTORC1 activation leads to higher aerobic glycolysis and oxygen consumption rates in FluR cells

The efficacy of mTORC1 inhibition is limited by compensatory activation of oncogenic pathways due to loss of negative feedback on the upstream PI3K/Akt pathway and by regulation of mTORC1 by other signaling pathways (9, 11, 29). Therefore, we intended to investigate whether targeting downstream functions of mTORC1 activation was an effective alternative to overcome Flu resistance. As recent studies suggest that activation of oncogenic pathways, including mTORC1, must induce metabolic reprogramming in order to provide ATP and substrates for biosynthesis to support tumor growth (30), we next investigated whether FluR cells had different metabolic requirements than FluS cells.

We measured two metabolic parameters: the extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) using a label-free system with the Seahorse XF-24 Metabolic Flux Analyzer. ECAR correlates with the rate of glycolysis because lactic acid is produced from pyruvate generated through glycolysis, in order to replenish the NAD+ needed for glycolysis. OCR represents mainly the mitochondrial respiration rate. We found that Nalm-6-FluR cells had a significantly higher basal rates of glycolysis (p<0.001; Figure 3A) and mitochondrial respiration (p<0.0001; Figure 3B) compared with Nalm-6 cells. Ev treatment significantly inhibited both ECAR (p<0.02) (Figure 3A) and OCR (p<0.001) (Figure 3B) in FluR cells, suggesting that mTORC1 regulates both glycolysis as well as mitochondrial respiration in FluR cells. As 2-deoxyglucose (2-DG) is an inhibitor of hexokinase, the first enzyme required for glycolysis, it also inhibits glycolysis and thus, glucose utilization. Addition of 2-DG blocked ECAR in both untreated as well as Ev-treated Nalm-6-FluR cells (Figure 3A), confirming that ECAR was a specific measure of glycolysis. Moreover, Ev treatment for 16 h had no effect, whereas bendamustine (Bd), which is known to induce apoptosis in FluR cells (21), led to cytochrome c release in FluR cells (Figure 3C), indicating that the decrease in OCR following Ev treatment was not an outcome of mitochondrial membrane permeabilization (31).

We next defined the metabolic profile of Nalm-6 and Nalm-6-FluR cells using a series of mitochondrial chemical probes (32). Oligomycin blocks ATP synthesis (and degradation) by the F0 /F1 ATPase, therefore, reducing the OCR in cells in which oxygen consumption is coupled to ATP synthesis. A decrease in basal OCR on addition of oligomycin thus provides an estimation of mitochondrial ATP synthesis. Trifluorocarbonylcyanide phenylhydrazone (FCCP) disrupts the proton gradient across the inner mitochondrial membrane and therefore uncouples the electron transport chain from oxidative phosphorylation. As a result, the electrons continue to pass through the chain and reduce oxygen to water, but with no ATP synthesis taking place. As a consequence, mitochondrial oxygen consumption abruptly increases when FCCP is added to coupled cells. Moreover, the response to the combination of rotenone and antimycin A, which blocks the respiratory chain at complexes 1 and 3, respectively, provides a measure of non-mitochondrial oxygen consumption. The nearly complete inhibition of OCR with rotenone and antimycin A confirmed that OCR is, indeed, a measure of mitochondrial oxygen consumption (Figure 3B). Oligomycin treatment reduced the OCR, which then abruptly rose when FCCP was added (Figure 3B). These data indicate that mitochondrial function is not compromised in either Nalm-6 or Nalm-6-FluR cells. Nevertheless, there was only a low decrease in basal OCR on addition of oligomycin (<70%) (Figure 3B), whereas the maximal OCR achieved using optimal concentrations of FCCP was close to the basal OCR in both cell lines (Figure 3B). These results suggest that the higher basal OCR in FluR cells was not coupled to higher ATP synthesis. Consistently, we found no significant difference in the basal ATP levels between Nalm-6 and Nalm-6-FluR cells (Figure 3D). 2-DG inhibition of glycolysis significantly decreased ATP levels in Nalm-6 as well as Nalm-6-FluR cells (p<0.001) (Figure 3D), however, the fold decrease in ATP due to 2-DG treatment, as compared to respective controls, was almost twice as much in the resistant cells as it was observed in the FluS cells. These results suggest that glycolysis contributes to ATP synthesis in both cell types. however, FluR cells depend more than FluS cells on glycolysis. Moreover, both cell types showed a much greater decrease in ATP levels with inhibition of glycolysis than with inhibition of the electron transport chain using rotenone and antimycin A (Figure 3D), further confirming that oxidative phosphorylation is inefficiently coupled to ATP synthesis in these cell lines. Thus, mTORC1 activation led to higher rates of glycolysis and mitochondrial respiration in FluR cells. Moreover, higher OCR was not related to higher ATP synthesis in FluR cells.

Consistently, even though 2-DG induced apoptosis in FluS as well as FluR cells, the Nalm-6-FluR (p<0.05) and Mec-2 FluR (p<0.01) cells were significantly more sensitive to 2-DG than the FluS Nalm-6 cells (Figure 3E). Glucose deprivation, however, had no effect on survival of Nalm-6-FluR and Mec-2 FluR cells, but was significantly more toxic to the FluS Nalm-6 cells, p<0.001 (Figure 3E). The opposite effects on cell death due to 2-DG and glucose deprivation in FluS and FluR cells suggest that FluS, but not FluR cells, depended more on exogenously added glucose for glycolysis.

Interestingly, treatment with antimycin A and rotenone to inhibit mitochondrial respiration induced significant cell death in a time-dependent manner in Nalm-6-FluR [48 h (p<0.01), 72 h (p<0.0001)] and Mec-2 [(48 h (p<0.001), 72 h (p<0.0001)] cells (Figure 3F). In contrast, cell death was negligible in Nalm-6 cells up to 72 h (Figure 3F).

Overall, these results indicate that mTORC1 activation leads to higher rates of glycolysis and mitochondrial respiration in FluR cells, which translated into higher sensitivity of FluR cells to pharmacological inhibition of glycolysis as well as mitochondrial respiration. However, the effect of inhibition of mitochondrial respiration was much more profound in FluR cells, whereas FluS cell survival depended on glycolysis, and not mitochondrial respiration. Thus, these data indicate that mitochondrial respiration is a potential target to overcome Flu resistance.

FluR cells have higher de novo pyrimidine biosynthesis

Given our findings that inhibition of mitochondrial respiration was selectively toxic to FluR compared to FluS cells, and that the higher OCR was not coupled to higher ATP synthesis in FluR cells, we next investigated potential downstream effects. The mitochondrial respiratory chain has been shown to be involved in de novo pyrimidine synthesis via the activity of the enzyme dihydroorotate dehydrogenase (DHODH).(33) mTORC1 promotes glutamine flux through pyrimidine synthesis via p70S6K, which directly phosphorylates CAD (carbamoyl-phosphate synthetase 2, aspartate transcarbamoylase, and dihydroorotase) on Ser-1859, leading to its oligomerization and increased activity (15, 16). We found higher levels of phosphorylated Ser1859-CAD in FluR than in FluS cell lines (Figure 4A), which corresponded to greater phosphorylated p70S6K levels (Figure 4A). Inhibiting mTORC1 with Ev diminished phosphorylation of both CAD and p70S6K (Figure 4B). Therefore, higher activation of CAD in FluR cells was indeed an outcome of higher mTORC1 activation. Moreover, PALA [N-(phosphonacetyl)-L-aspartate], a pharmacological inhibitor of CAD (34) synergized with Flu to induce cell death in FluR cells (Figure 4C). And, inhibition of de novo pyrimidine biosynthesis using PALA and leflunomide, an inhibitor of DHODH, decreased the clonogenic survival of FluR cells (Figure 4D). To unambiguously define the role of mTORC1 activation on CAD signaling through p70S6K, we next modulated p70S6K expression levels. Remarkably, p70S6K knockdown by siRNA in FluR cells caused massive cell death (Figure 4E), however it did not affect viability of FluS cells (Supplementary Figure S1a,b). Increased de novo biosynthesis of pyrimidines, as a result of CAD activation, favors progression through S phase of the cell cycle because of the increased DNA synthesis (35). Indeed, we found a significantly higher S-phase population in Nalm-6-FluR (p<0.05), Mec-1, and Mec-2 cells compared to Nalm-6 cells (Figure 4F).

Figure 4. Constitutive mTORC1 activates CAD in FluR cells.

Figure 4

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(a) Western blot analysis of Ser1859-CAD and p-P70S6K protein expression in the indicated cell lines. (b) Nalm-6 and Nalm-6-FluR cells were treated with Ev for 24 h, and cell lysates were analyzed by western blotting for p-p70S6K (T389) and pCAD (S1859). (c) Combination Index-fraction affected plot of the effect of combination of fludarabine (Flu) and N-(phosphonacetyl)-l-aspartate (PALA) on clonogenic cell survival in Nalm-6-FluR cells. Combination index < 1 indicates synergism, fraction affected = (100-%surviving fraction)/100. (d) Effect of PALA and leflunomide treatment on clonogenic cell survival in Nalm-6-FluR cells. (e) Cell death analysis in siControl and si-p70S6K-expressing Nalm-6-FluR cells in response to 24 h Flu treatment as determined by annexin V/PI staining. (f) Cell cycle distribution in the indicated cell lines was determined by BrdU and 7-AAD double staining and FACS analysis. Data represent mean ± SD (n = 3 independent experiments), **p<0.01.

Thus, we establish that high mTORC1 activation leads to CAD phosphorylation, which provides a survival advantage to FluR cells. The selective dependence of FluR cells on mTORC1-dependent mitochondrial respiration was not related to ATP synthesis, and may be attributed to multiple functions, including de novo pyrimidine biosynthesis (Figure 5). Importantly, we show that this specific metabolic dependence can be effectively exploited by pharmacological inhibition of mitochondrial respiration, and of de novo pyrimidine biosyntheisis to induce cell death in FluR cells.

Figure 5. Model for targeting metabolic vulnerability of FluR cells.

Figure 5

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1. Hyperactive mTORC1 is associated with Flu-resistance. 2. mTORC1 causes higher rates of glycolysis, as measured by extracellular acidification rate (ECAR) and mitochondrial respiration, as measured by oxygen consumption rate (OCR), both of which are essential for FluR cell survival. Inhibition of mitochondrial respiration using rotenone and antimycin induces a more dramatic cell death than inhibition of glycolysis using 2-deoxyglucose, However, the increase in OCR is not related to ATP synthesis. 3. In fact, constitutive mTORC1 activation causes CAD S1859 phosphorylation in FluR cells, which leads to de novo pyrimidine biosynthesis and promotes survival in these cells. As such, FluR cells are also highly susceptible to inhibition of de novo pyrimidine biosynthesis using PALA and leflunomide, 4. Dihydroorotate dehydrogenase (DHODH), an essential enzyme in de novo pyrimidine biosynthesis, requires mitochondrial respiratory chain electron acceptors to oxidize dihydroorotate (DHO) to orotate, Thus, high mitochondrial respiration contributes to increase in de novo pyrimidine biosynthesis, in addition to other functions in FluR cells.

Discussion

In this study we establish that mTORC1 activation, as measured by p-p70S6K T389, defines malignant B-cell response to Flu. This study reveals that mTORC1 activation in FluR cells is associated with specific metabolic adaptation, which renders these cells highly vulnerable to the inhibition of mitochondrial respiration and de novo pyrimidine biosynthesis. Aberrant activation of mTORC1 signaling is a common feature of human cancers, including hematological malignancies (5, 36). In addition, inhibition of B-cell receptor-associated signaling pathways, including mTORC1, is a potential treatment target in B cell malignancies, including CLL (28). p-p70S6K T389 activation status per se has not been previously studied in the context of CLL or Flu-responsiveness. We show that mTORC1 activation correlates with Flu resistance in a panel of leukemic cell lines and in primary CLL cells. Despite high mTORC1 activity in FluR cells, mTORC1 inhibition by rapalogs had limited effect on cell death, likely due to the previously identified feedback activation of other oncogenic pathways (37).

To address these limitations, we evaluated an alternative approach by targeting downstream metabolic reprogramming associated with mTORC1 activation in FluR cells (12). Consistent with the well-established role of mTORC1 in regulation of cellular metabolism, our study highlights three important aspects of metabolic reprogramming in FluR compared to parental Nalm-6 cells. FluR cells exhibited: (i) accelerated rates of glycolysis and mitochondrial respiration, (ii) higher de novo pyrimidine biosynthesis, as suggested by hyper-phosphorylation of CAD, and (iii) cell death in response to inhibition of mitochondrial respiration and de novo pyrimidine biosynthesis.

An increased rate of glycolysis in the presence of sustained OCR has been previously reported in leukemic cells, using electrons from non-glucose carbon sources (38). Glutamine-dependent, glucose-independent Krebs cycle activity has been also reported in glioblastoma and melanoma cells (38). Our data suggest that both FluS and FluR cells utilize glycolysis for ATP synthesis, therefore, cell death occurs in both cases in response to 2-DG. However, FluR cells are sensitive to a greater extent to 2-DG, which indicates that FluR cells are more dependent on glycolysis for ATP synthesis and overall survival, which, in turn, can be explained by an overall increase in biosynthetic pathways, such as pyrimidine biosynthesis. Nevertheless, the higher cell death in Nalm-6 cells than FluR cells in response to glucose starvation seems contradictory. However, it suggests that the resistant cells have adapted to survive without exogenous glucose. Thus, Flu-sensitive cells require exogeneous glucose, and hence they die in response to glucose starvation. In contrast, FluR cells make their own glucose by activation of endogeneous glucose-deprivation response pathways, such as autophagy (39) which, therefore, do not die in response to lack of glucose in the cell culture media. High glycolysis and intracellular utilization of glucose coexisting with lower dependence on exogenous glucose due to Increased expression of the glucose deprivation response network, including unfolded protein response, autophagy, glucagon signaling, and gluconeogenesis, genes, has been described before in the context of acquired resistance to lapatinib in breast cancer cell lines (40). Selective targeting of these pathways associated with glucose-deprivation could overcome resistance (40). Similarly, we recently reported that FluR cells could be selectively targeted by inhibition of autophagy (21). Thus, glucose deprivation response pathways could potentially be targeted to overcome Flu resistance.

Treatment with mitochondrial toxins induced robust cell death in FluR cells. Although we observed higher OCR in FluR than in parental Nalm-6 cells, with carefully titrated concentrations of FCCP both cell lines demonstrated basal OCR close to their maximal capacities. Yet, coupling efficiency was low in both cell lines. Moreover, the two cell lines showed no significant difference in ATP levels. Overall, these results suggest that the higher OCR in FluR cells was not coupled to higher ATP synthesis. Mitochondrial respiration in hematopoietic and various other cell types is known to be affected by de novo pyrimidine synthesis in a Krebs cycle- and glucose-independent manner (38). Moreover, mTORC1 activation was recently shown to enhance glutamine flux through pyrimidine biosynthesis (1, 16) and leflunomide was reported to overcome Flu resistance in CLL (41). Consistent with those data, we found higher pCAD (S1859) levels in FluR cells. Moreover, inhibition of pyrimidine biosynthesis using two different inhibitors, PALA and leflunomide, reduced clonogenic survival of FluR cells. Importantly, PALA acted synergistically with Flu in inducing cell death in FluR cells. These findings conclusively establish that constitutive mTORC1 activation promotes de novo pyrimidine synthesis in FluR cells, to which they are addicted.

Notably, p70S6K knockdown induced remarkable cell death in FluR cells compared to FluS cells. This further supports the importance of mTORC1/p70S6K/CAD axis in regulating pyrimidine biosynthesis and, therefore, survival in FluR cells. The fact that rapalog treatment, despite reducing active p70S6K levels more effectively than S6K knockdown, was less effective in inducing cell death, seems intriguing. However, rapalog treatment will also affect other targets of mTORC1 which, in turn, may be associated with pro-survival pathways (29). For example, mTORC1 inhibition activates the ULK-1 (ATG1) complex which, in turn, will activate autophagy, which is indeed a well established pro-survival pathway (42). Consistently, we have previously reported that FluR cells depend on autophagy for their survival (21). Therefore, these findings further underscore the importance of targeting downstream pathways in mTORC1-dependent cancers.

We recognize that OxPhos inhibition may cause cell death due to multiple reasons, e.g. inhibition of recycling of NAD+ (43), inhibition of de novo pyrimidine biosynthesis (44), ROS(45) and, disruption of MMP leading to Bax/Bak oligomerisation (38). Indeed, uridine supplementation could only modestly rescue the cell death caused by rotenone and antimycin treatment in these cells (data not shown). Nevertheless, our data suggest that one of the reasons should be de novo pyrimidine biosynthesis given high proportion of S-phase cells and high pCAD S1859. The DHODH enzyme, a critical component of this pathway, is located in the inner mitochondrial membrane and must use mitochondrial electron transfer chain (ETC) components, i.e. ubiqinone as the proximal acceptor and coenzyme q as the ultimate electron acceptor, in order to carry out oxidation of DHO to orotate.

In summary, we established mTORC1 activation, as measured by p-p70S6K T389, and downstream pCAD S1859 as potential biomarkers of Flu-resistance in leukemic cells (Figure 5). FluR cells depend on mTORC1-dependent de novo pyrimidine biosynthesis and mitochondrial respiration for survival. Thus, directly targeting de novo pyrimidine biosynthesis pathway enzymes using PALA and leflunomide, or targeting mitochondrial respiration represent effective strategies to overcome Flu resistance.

Supplementary Material

1
2

Acknowledgments

We would like to thank Drs. C. McDonald, Y. Sountharajah, and N. Gupta (Cleveland Clinic), R. Dalla Favera (Columbia University Medical Center) for critical reagents. Dr. C. Talerico (Cleveland Clinic) provided substantive editing and comments. This work was supported by a research grant from National Institutes of Health CA127264 (awarded to AA) HL103453 and HL60917 (awarded to SCE), and a fellowship from Cleveland State University (Molecular Medicine Program to AS).

Footnotes

Disclosure of Potential Conflicts of Interest: No potential conflicts of interest were disclosed

Note: Supplementary data for this article are available at Molecular Cancer Research Online (http://mcr.aacrjournals.org/).

Authors' Contributions: Conception and design: A. Sharma, A. Almasan

Development of methodology: A. Sharma, A.J. Janocha, A.

Acquisition of data and provided samples: A. Sharma, B.T. Hill, M.R. Smith

Analysis and interpretation of data: A. Sharma, A. Almasan, Writing, review, and/or revision of the manuscript: A. Aharma, A. Almasan, B.T. Hill, M.R. Smith

Study supervision: A. Almasan, S.C. Erzurum

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Supplementary Materials

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