Inhibition of Mtor Pathway Sensitizes Acute Myeloid Leukemia Cells to Aurora Inhibitors by Suppression of Glycolytic Metabolism

Published OnlineFirst September 5, 2013; DOI: 10.1158/1541-7786.MCR-13-0172

Molecular Cancer Cell Death and Survival Research

Inhibition of mTOR Pathway Sensitizes Acute Myeloid Leukemia Cells to Aurora Inhibitors by Suppression of Glycolytic Metabolism

Ling-Ling Liu1,2, Zi-Jie Long1,3, Le-Xun Wang1,3, Fei-Meng Zheng2, Zhi-Gang Fang1, Min Yan2, Dong-Fan Xu1,3, Jia-Jie Chen1,3, Shao-Wu Wang4,5, Dong-Jun Lin1,3, and Quentin Liu1,2,3,4

Abstract Aurora kinases are overexpressed in large numbers of tumors and considered as potential therapeutic targets. In this study, we found that the Aurora kinases inhibitors MK-0457 (MK) and ZM447439 (ZM) induced polyploidization in acute myeloid leukemia (AML) cell lines. The level of glycolytic metabolism was significantly increased in the polyploidy cells, which were sensitive to glycolysis inhibitor 2-deoxy-D-glucose (2DG), suggesting that polyploidy cells might be eliminated by metabolism deprivation. Indeed, inhibition of mTOR pathway by mTOR inhibitors (rapamycin and PP242) or 2DG promoted not only apoptosis but also autophagy in the polyploidy cells induced by Aurora inhibitors. Mechanically, PP242 or2DGdecreased the level of glucose uptake and lactate production in polyploidy cells as well as the expression of p62/SQSTM1. Moreover, knockdown of p62/ SQSTM1 sensitized cells to the Aurora inhibitor whereas overexpression of p62/SQSTM1 reduced drug efficacy. Thus, our results revealed that inhibition of mTOR pathway decreased the glycolytic metabolism of the polyploidy cells, and increased the efficacy of Aurora kinases inhibitors, providing a novel approach of combination treatment in AML. Mol Cancer Res; 11(11); 1326–36. 2013 AACR.

Introduction apoptosis of acute myeloid leukemia (AML) cells (6). Our previous study showed that VX-680 (MK-0457), a rela- Anticancer drug that targets cell-cycle regulators has fi been widely applied in the treatment of the hematologic tively speci c Aurora A inhibitor, induced apoptotic cell malignancies (1–3). Aurora kinases A–C, which play death in AML cells, especially in the primary leukemic blasts with high Aurora A expression (4, 7). Although important roles in the process of mitosis, have been fi implicated in the tumorigenesis (1–3). Aurora A and B Aurora kinases inhibitors have signi cant therapeutic potential in AML, single-agent activity seems to be uni- proteins were overexpressed in a number of tumors, fi including leukemia cells, which were correlated with aneu- formly modest and the ef cacy needs improving (6). ploidy in tumor tissues and poor prognosis (4, 5). Previous Recently, we also showed that Aurora A inhibition trig- report showed that Aurora kinases inhibitors could sup- gered drug resistance could be reversed by autophagy press the growth of leukemia cells (1). ZM447439 (ZM), inhibitors in breast cancer cells (8). In addition, combi- the dual Aurora A and B inhibitor, inhibited the cell nation of Aurora kinases inhibitors with vincristine or proliferation, induced polyploidization, and promoted dasatinib presented synergistic effect to inhibit cell viability in human pre-B acute lymphoblastic leukemia (9). Thus, new strategy for drug combination is urgently required to Authors' Affiliations: 1Department of Hematology, the Third Affiliated overcome drug resistance induced by Aurora A inhibition. Hospital, Sun Yat-sen University, Guangzhou, China; 2State Key Labora- tory of Oncology in South China, Cancer Center, Sun Yat-sen University, Polyploidy is a prominent characteristic of human cancers Guangzhou, China; 3Sun Yat-sen Institute of Hematology, Guangzhou, and proposed as a driver of tumorigenesis (10). Study China; 4Institute of Cancer Stem Cell, Dalian Medical University, Dalian, showed that somatic polyploidy was associated with China; and 5Department of Radiology, the First Affiliated Hospital, Dalian Medical University, Dalian, China readjustment of main metabolic pathways. For example, mitochondrial process was depressed, and carbohydrate Note: Supplementary data for this article are available at Molecular Cancer Research Online (http://mcr.aacrjournals.org/). degradation and lipid biosynthesis were increased in the polyploidy cells (11). Warburg reported that tumors pre- L.-L. Liu and Z.-J. Long have contributed equally to this article. ferred aerobic glycolysis to mitochondrial oxidative phos- Corresponding Authors: Quentin Liu, Department of Hematology, the phorylation (12). Based on the reports, suppression of the Third Affiliated Hospital, Sun Yat-sen University, 600 Tianhe Road, Guangzhou 510630, China. Phone: 86-20-8525-3601; Fax: 86-20- aerobic glycolytic metabolism might help for restraining the 87343171; E-mail: [email protected]; and Dong-Jun Lin, proliferation of polyploidy cancer cells. Aurora kinases are [email protected] crucial for cytokinesis in a number of reports. Aurora kinases doi: 10.1158/1541-7786.MCR-13-0172 defection induced mitosis arrest and promoted polyploidy 2013 American Association for Cancer Research. formation (13). Therefore, readjustment of the metabolic

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mTOR Inhibition Enhances the Efﬁcacy of Aurora Inhibitors

status in polyploidy cells induced by Aurora kinases inhibi- Peripheral blood mononuclear cells (PBMC) were enriched tion could also be a promise approach to cancer therapy. by Ficoll–Hypaque density gradient centrifugation. The mTOR, a well-conserved serine/threonine kinase with 2 complexes, mTOR complex 1 (mTORC1) and Cell-proliferation assay mTOR complex 2 (mTORC2), is essential for cell growth, Cell viability was assessed by Cell counting kit-8 protein translation, autophagy, and metabolism (14). To (CCK8; Dojindo Biotechnology) according to the man- adapt the condition of hypermetabolic demand and thrive ufacturer's instructions. The optical density at 450 nm was rapidly, oncogenes in growth and nutrient-sensing signaling measured using a multiwell plate reader (Micro-plate pathways was activated (15). In numerous cancer types, Reader; Bio-Rad). mTOR signaling pathway was activated by deregulation of its upstream factors, such as Akt activation or deficiency of Cell-cycle analysis tuberous sclerosis complex 2 (TSC2; refs. 16 and 17). Cell cycle was analyzed by flow cytometer. Cells were Activation of mTORC1, a metabolic hallmark of cancer, treated with drugs for 24 hours. Then, cells were harvested increased glycolysis, and lipid biosynthesis (15). Although and stained with mixture of 50 mg/mL propidium iodide mTORC2 was not thoroughly studied in the metabolism of (PI), 0.2% Triton X-100, and 100 mg/mL RNAase inhib- tumor, RNAi against one of mTORC2 components also itor. Cell-cycle analysis was conducted using a FACS flow impaired the glucose and lipid metabolism in muscle and cytometer equipped with Modfit LT for Mac V2.0 software fat cells (18, 19). Furthermore, mTOR was associated with (BD Biosciences). the development of polyploidization. Ma and colleagues showed that S6K1, a downstream of mTOR, was involved Microscopic analysis of cells and nuclear morphology in polyploidization through its phosphorylation at Thr421/ Cells were treated with Aurora kinases inhibitors for 24 Ser424 (20). In addition, activation of mTOR pathway was hours, followed by PP242 or 2DG for another 24 hours. þ required for TSC2 /EK [EKer rat model carrying a germline After 48 hours, the cells were photographed under an insertional deletion in one copy of the Tsc2 (tuberous inverted microscopy (Olympus). Nuclear fragmentation was sclerosis 2) gene] smooth muscle cells of pulmonary lym- stained by Hoechst 33342 (Sigma) with 10 mg/mL for 15 phangioleiomyomatosis to undergo tetraploidization (21). minutes at 37C. Slides were viewed using a fluorescence Here, we found that mTOR signaling pathway was critical microscope. for maintaining the glycolytic metabolism state of polyploidy cancer cells induced by inhibition of Aurora kinases. More- Monodansylcadaverine staining over, we provided evidences that combination of mTOR Cells were incubated with the monodansylcadaverine inhibitors and Aurora kinases inhibitors effectively elimi- (MDC; Sigma) with a final concentration of 0.05 mmol/L nated leukemia cells by inducing apoptosis and autophagy. for 10 minutes at 37C. Then, cells were viewed on slides These results suggested a new approach of targeted thera- under a laser scanning confocal microscope (ZEISS, peutic drugs combination for AML treatment. Germany), and tested by flow cytermetry. MDC dye was applied to stain the late autophagic vacuoles.

Materials and Methods Western blotting analysis Reagents and cell culture Total cellular proteins were isolated with RIPA buffer. Reagents included MK-0457 (Selleck Chemicals, South Lysates were subjected to SDS-PAGE gel electrophoresis, Loop West, Houston), ZM447439 (AstraZeneca, Canada), and were transferred to nitrocellulose membranes. The PP242, 2-deoxy-D-glucose (2DG), and rapamycin (Sigma). membranes were blocked with 5% bovine serum albumin U937 cell line was obtained from the American Type and then incubated overnight with LC3 (Novus), p62 (Santa Culture Collection (ATCC). 293FT cell line was obtained Cruz), Aurora A (Upstate), p-mTOR (Ser2448, Ser2481), from Invitrogen. NB4 and HL-60 cell lines were provided by mTOR, p-Aurora A (Thr288), cleaved caspase 3 (Asp175), Shanghai Institute of Hematology, Ruijin Hospital. U937, cleaved PARP (Asp214), and b-actin antibodies (Cell Sig- NB4, and HL-60 cell lines were cultured in RPMI 1640 naling Technology). p-4EBP1/2/3 (Thr45) and 4EBP1/2/3 (Gibco; Invitrogen) supplemented with 10% (v/v) FBS antibodies were purchased from Epitomics, Inc. Subse- (Hyclone) at 37 Cin5%CO2 incubator. 293FT cell line quently, the membranes were incubated with an HRP- was cultured in DMEM supplemented with 10% (v/v) FBS conjugated secondary antibody (Protein Tech Group) at at 37 Cin5%CO2 incubator. KG1a cell line was obtained room temperature for 1 hour. The protein bands were from Deutsche Sammlung von Mikroorganismen und detected with an enhanced chemiluminescence reagent Zellkulturen GmbH (DSMZ; Braunschweig) and cultured (Sigma), according to the manufacturer's instructions. in RPMI 1640 supplemented with 20% (v/v) FBS at 37C in 5% CO2 incubator. The RPMI 1640 medium without Measurement of glucose uptake and lactate production glucose and DMEM were also purchased from Gibco of cells in the media (Invitrogen). Cell lines were authenticated by comparing Cells were counted using trypan blue staining before the short tandem repeat (STR) site and sex genes of the measurement of the glucose uptake and lactate production. extracted DNA with that of cell lines in ATCC in 2012. The media were collected, and the glucose and lactate levels

www.aacrjournals.org Mol Cancer Res; 11(11) November 2013 1327

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were immediately measured using Automatic Biochemical Statistics Analyzer (Olympus AU5400). The glucose consumption Data were expressed as mean SD of 3 determinations and lactate production were normalized to the number of with GraphPad Prism software. Statistical analysis was cells and results were presented as pmol/cell (22). analyzed using the Statistical Package for Social Sciences (SPSS) software (version 16.0). P-value < 0.05 was consid- Plasmid construction and retrovirus packaging ered statistically signiﬁcant. The coding sequence of p62/SQSTM1 was ampliﬁed from human leukemia cells. The primers were as followed: Results 50 Bgl II: TTAGATCTATGGCGTCGCTCACCGTGA- Aurora kinases inhibitors induce polyploidization of AGG; 30 Sal I: GTGTCGACTGGGCAAAAGTGGTCA- AML cell lines CAACG. The cDNA was cloned into MSCV-IRES-GFP To study the effect of Aurora kinases inhibitors on AML cell plasmid. 293FT cells were transfected with packaging lines, the proliferation of cells was tested. U937, NB4, HL-60, vectors and MSCV-p62-IRES-GFP or MSCV-IRES-GFP and KG1a cell lines treated with Aurora kinases inhibitors using Lipofectamine 2000 (Invitrogen). Media containing (MK or ZM) for 24 and 48 hours were subjected to CCK8 retrovirus were collected 48 hours after transfection for assay (Fig. 1A and Supplementary S1A). The 2 drugs had U937 cells infection. The short hairpin RNA (shRNA) slight effects on leukemia cell lines viability. For example, MK targeting p62 (NM_003900), Aurora A (NM_003600), 1 mmol/L suppressed the proliferation of U937 cells by 17.3% and the nontarget shRNA (SHC002) were obtained from 5.4% and 22% 6.3% at 24 and 48 hours respectively, Sigma. 293FT cells were used for packaging lentivirus. After and inhibited the proliferation of NB4 cells by 18.7% 1.8% infection with the lentivirus, infected U937 cells were and 20.4% 1.8% at 24 and 48 hours, respectively (Fig. 1A). selected with puromycin. As Aurora kinases were critical for the regulation of mitosis

Figure 1. The effect of Aurora kinases inhibitors on AML cell lines. A, U937 and NB4 cells treated with different concentrations of MK or ZM for 24 and 48 hours were subjected to CCK8 assay. Data were mean SD (n ¼ 3). P < 0.05, P < 0.01. B, U937 and NB4 cells were treated with different concentrations of MK or ZM for 24 hours. Flow cytometry analysis was conducted for the cell-cycle distribution and the percentage of polyploidy cells was displayed. Western blotting was also conducted for the phosphorylated and total Aurora A protein expression (C).

1328 Mol Cancer Res; 11(11) November 2013 Molecular Cancer Research

process, the cell-cycle distribution of these cell lines was Polyploidy cells induced by Aurora inhibition have high analyzed. Flow cytometry analysis showed that treatment of level of glycolytic metabolism MK 1 mmol/L or ZM 2 mmol/L could induce polyploidiza- After 24 hours incubating with MK or ZM, U937 and NB4 tion of both U937 and NB4 cells, as well as HL-60 and KG1a cells converted to polyploidy cells. Study showed that metab- cells (Fig. 1B and Supplementary Fig. S1B). Western blotting olism was altered in polyploidy cells (23). So next, glucose conﬁrmed the effect of Aurora kinases inhibition by testing uptake and lactate production in culture media were measured the level of T288 phosphorylated Aurora A protein, and the to evaluate the level of glycolytic metabolism. Compared with results showed that the phosphorylated Aurora A decreased in that of control cells, the level of glucose uptake and lactate a dose-dependent manner (Fig. 1C). Moreover, knockdown production of U937 polyploidy cells was increased [Fig. 2A(a, of Aurora A in U937 cells reduced the cell viability (Supple- b); glucose uptake, 8.3 0.8 pmol/cell for control vs. 14.0 mentary Fig. S2), indicating that the antiproliferation effect of 0.4 pmol/cell for MK, 13.3 2.8 pmol/cell for ZM and MK and ZM was largely due to Aurora A inhibition. lactate production, 15.1 2.2 pmol/cell for control vs. 25.4

Figure 2. Polyploidy cells induced by Aurora inhibition have higher metabolism level than control cells. A, U937 and NB4 cells were treated with MK 1 mmol/L or ZM 2 mmol/L for 24 hours, and the level of glucose uptake (a, c) and lactate production (b, d) in the culture media was examined. The results were normalized to the cells number, and conducted as pmol/cell. B, U937 and NB4 cells were treated with MK 1 mmol/L or ZM 2 mmol/L for 24 hours, followed by 2DG for another 24 hours, and CCK8 assay was conducted. Data were mean SD (n ¼ 3). P < 0.05, P < 0.01.

Liu et al.

0.1 pmol/cell for MK, 24.4 1.7 pmol/cell for ZM]. induced by Aurora kinases inhibitors required higher level of Similar results were observed in NB4 cells [Fig. 2A(c, d)]. glycolytic metabolism than control cells. 2DG, a glucose analog, was used to confirm whether the polyploidy cells were sensitive to energy deprivation. 2DG Inhibition of mTOR pathway decreases the level of transiently inhibited glycolysis, which was considered as the glycolytic metabolism in AML cell lines mainly energy source of tumor cells (24). Figure 2B(left) Rapamycin is the specific inhibitor of mTORC1, and showed that 2DG inhibited the proliferation of U937 poly- PP242 inhibits mTORC1 and mTORC2. U937 and NB4 ploidy cells by 30% at 2 mmol/L and 40% at 5 mmol/L. cells were treated with different concentrations of rapamycin In Fig. 2B(right), the inhibition rate of NB4 polyploidy cells or PP242 for 24 hours, respectively. The activity of mTOR by 2DG was 30% at 2 mmol/L and 50% at 5 mmol/L. and its substrate 4EBP1 was detected by Western blotting. Two drugs combination test was also conducted in normal As shown in Fig. 3A, rapamycin inhibited the mTOR PBMCs and we found minimal cell death in PBMCs with activation, as well as phosphorylated 4EBP1. The same either single drug or 2 drugs combination treatment, indi- results were also detected in PP242 treated cells. Rapamycin cating the observed phenotypes were tumor specific(Supple- 0.05 mmol/L or PP242 1 mmol/L could inhibit mTOR mentary Fig. S3). Our data suggested that the polyploidy cells activation, and the 2 concentrations were chosen for further

Figure 3. Inhibition of mTOR pathway decreases the metabolic level in AML cell lines. A, different concentrations of rapamycin (Rapa) or PP242 were added to U937 and NB4 cells and the cell lysates were subjected to Western blotting analysis with indicated antibodies. Three independent experiments were conducted, and representative data were shown. B, U937 and NB4 cells were treated with rapa 0.05 or PP242 1 mmol/L for 24 hours, and cells were counted and the level of glucose uptake and lactate production in the culture media was immediately tested. The results were normalized to the cells number. Data were mean SD (n ¼ 3). P < 0.05. C, U937 and NB4 cells were treated with different concentrations of MK or ZM. Cell lysates were subjected to Western blotting analysis with indicated antibodies.

study. In Fig. 3B, rapamycin decreased the level of glucose cells. Under the microscopy, the polyploidy cells exposed uptake, whereas PP242 significantly downregulated the to PP242 broke into pieces [Fig. 4B(a)]. These morpho- glucose uptake and lactate production in U937 and NB4 logical changes were confirmed by flow cytometry. For cells. In addition, we also tested the inhibition of mTOR U937 cells treated with PP242, the sub-G1 rates of pathway in U937 and NB4 cells with increasing dose of polyploidy cells induced by MK or ZM were 33.5 Aurora kinases inhibitors. However, there was no change for 3.0% or 43.4 2.5% versus 2.15 0.55% of control the expression of phosphorylated mTOR and 4EBP1, sug- cells. For NB4 cells treated with PP242, the sub-G1 rates gesting that the mTOR pathway could not be suppressed by of polyploidy cells induced by MK or ZM were 24.3 Aurora inhibitors (Fig. 3C). 1.1% or 22.5 1.6% versus 2.0 0.3% of control cells [Fig. 4B(b)]. As mentioned in Fig. 3B, inhibition of Polyploidy cells induced by Aurora inhibition are mTOR decreased the metabolism level of the control sensitive to the mTOR inhibitors cells. Moreover, the metabolism level significantly Then we examined the effect of mTOR inhibitor on declined after the polyploidy cells were treated with PP242 polyploidy cells. After exposed to MK 1 mmol/L or ZM 2 (Fig. 4C). In U937 cells, the glucose uptake decreased mmol/L for 24 hours, U937 and NB4 cells were treated from 23.1 2.5 to 14.7 1.1 pmol/cell, and lactate with PP242 for another 24 hours. As shown in Fig. 4A, production decreased from 33.9 0.8 to 18.6 2.8 PP242 significantly suppressed the viability of polyploidy pmol/cell. In NB4 cells, the glucose uptake decreased

Figure 4. Polyploidy cells induced by Aurora inhibition are sensitive to the mTOR inhibitors. U937 and NB4 cells were treated with MK 1 mmol/L or ZM 2 mmol/L for 24 hours, followed by different concentrations of PP242 for another 24 hours. CCK8 assay was conducted for the proliferation detection (A). Data were mean SD (n ¼ 3). P < 0.05, P < 0.01. Cells were photographed under microscopy (B-a) and ﬂow cytometry analysis for the sub-G1 percentage was shown (B-b). , P < 0.001. C, the level of glucose uptake and lactate production in the culture media was tested in polyploidy cells treated with or without PP242 for 24 hours. Data were mean SD (n ¼ 3). P < 0.05, P < 0.01.

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from 14.8 2.0 to 8.3 1.1 pmol/cell, and lactate mTOR inhibitors or the glycolysis inhibitor induce not production decreased from 31.2 5to21.8 4.4 pmol/ only apoptosis but also autophagy in polyploidy cells cell. To further investigate the combinational effect, U937 Next, control cells and polyploidy U937 cells induced and NB4 cells were treated with Aurora kinases inhibitors by Aurora inhibitors were treated with PP242 or 2DG, (MKorZM)andmTORinhibitors(rapamycinorPP242) respectively. After 24 hours, the cells were stained with simultaneously for 48 hours. The metabolism examina- Hoechst 33342 dye and observed under ﬂuorescence micro- tion also showed the same outcomes (Supplementary Fig. scopy. Figure 5A showed that much more apoptotic bodies S4). Of note, as shown in Supplementary Figs. S5 and S6, in polyploidy cells than that in control cells, and the the combination had synergistic effect on the proliferation percentage of apoptotic bodies was presented (MK, inhibition of AML cells, but showed little effect on the 14.0% 5.1% for control, 56.3% 5.7% for PP242, normal PBMCs. These results indicated that, compared 47.3% 11.0% for 2DG; ZM, 15.7% 5.5% for control, with control cells, the polyploidy cells had higher metab- 56.0% 15.6% for PP242, 46.7% 10.7% for 2DG). olism level and were more sensitive to mTOR inhibitors. MDC dye was applied to stain the late autophagic vacuoles.

Figure 5. PP242 or 2DG induces not only apoptosis but also autophagy in polyploidy cells. U937 cells were treated with MK 1 mmol/L or ZM 2 mmol/L for 24 hours, followed by PP242 1 mmol/L or 2DG 5 mmol/L for another 24 hours. The apoptotic bodies in the cells were stained by Hoechst 33342 dye and the percentage of apoptosis cells was tested by flow cytometry (A). Autophagic vacuoles in the cells were detected by MDC dye and the percentage of MDC staining in cells was quantified by flow cytometry (B). The apoptosis and autophagy-related proteins in U937 cells were determined by Western blotting (C). , P < 0.05; , P < 0.01; , P < 0.001.

Figure 6. P62/SQSTM1 acts as a regulator in the metabolism of polyploidy cells induced by Aurora kinases inhibitors. A, U937 cells were treated with MK 1 mmol/L or ZM 2 mmol/L for 24 hours. After washed with PBS, cells were reseeded in culture media with or without glucose. The expression of cleaved caspase 3 and PARP was conducted. B, P62 was knocked down (shp62-1#) or overexpressed (Oep62) in U937 cells, and the cells were treated with MK 1 mmol/L for 24 hours. Cell lysates were subjected to Western blotting analysis with indicated antibodies.

As shown in Fig. 5B, inhibition of mTOR or the glycolysis cells (Fig. 6A). Expression of cleaved caspase 3 and PARP was in polyploidy cells increased the appearance of autophagic significantly increased when compared with cells cultured vacuoles, which seemed as distinct dot-like structures dis- with glucose-containing media (Fig. 6A). Furthermore, we tributed within the cytoplasm or localized in the perinuclear chose one plasmid which showed great efficiency to knock- regions in cells. Moreover, the percentage of autophagic down p62 expression for virus packaging and U937 infection vacuoles in cells tested by flow cytometry also showed the (Supplementary Fig. S8). P62 knockdown-U937 cells were same results (MK, 36.5% 17.3% for control, 79.2% treated with MK and subjected to Western blotting. The 10.0% for PP242, 77.7% 18.7% for 2DG; ZM, 64.8% result showed that MK increased PARP cleavage and 6.0% for control, 80.4% 9.6% for PP242, 85.3% decreased more p62 expression in the p62 knockdown- 6.9% for 2DG; Fig. 5B). U937 cells (Fig. 6B). On contrast, overexpression of p62 in The expression of apoptosis-associated proteins and U937 cells with treatment of MK partly reversed the MK- autophagy markers was further determined. As shown induced apoptosis with decreased expression of cleaved PARP in Fig. 5C, PP242 or 2DG inhibited mTOR activation at (Fig. 6B). These results implied that p62 might act as a serine 2448 and 2481 phosphorylated sites in polyploidy regulator involving in polyploidy cells metabolism pathway. cells. Either PP242 or 2DG increased the expression of cleaved caspase 3 and PARP (poly ADP-ribose polymerase) in polyploidy cells. P62/SQSTM1 was downregulated and Discussion LC3II were upregulated in polyploidy cells with PP242 or In this study, we found that Aurora kinases inhibitors 2DG treatment, indicating autophagy occurred (Fig. 5C). could induce polyploidization in various AML cell lines. The Moreover, in Supplementary Fig. S7, the expression of p62 induced polyploidy cells had higher level of glycolytic metab- decreased and cleaved caspase 3 expression increased in cells olism than control cells. Importantly, mTOR inhibitors with drug combination simultaneous for 48 hours. could target p62 to promote apoptotic and autophagic death in Aurora kinases inhibitors induced polyploidy cells. Our P62/SQSTM1 acts as a regulator in the metabolism of data suggested that suppression the metabolism of polyploidy polyploidy cells induced by Aurora kinase inhibitors cells by mTOR inhibition was a novel approach to improve P62 is a key regulator of nutrient sensing in the mTOR the therapeutic effect of Aurora kinases inhibitors in AML. pathway (25). The glucose deprivation was applied to further Aurora kinases were participated in a wide range of study the function of p62 in polyploidy cells. We found that cell-cycle events and overexpressed in a numerous of tu- the expression of p62 in polyploidy cells was significantly mors. Aurora kinases inhibitors have been considered as the decreased when cultured in glucose-free media, suggesting attractive anticancer regents (4, 6, 26). In our study, MK that p62 was correlated with the metabolism of polyploidy and ZM inhibited Aurora A phosphorylation in a dose-

Liu et al.

dependent manner and showed modest inhibition on the The mTOR signaling pathway integrates various meta- proliferation of AML cell lines (Fig. 1A and C and Supple- bolic pathways in cells. mTORC1 positively regulates the mentary Fig. S1A), which was consistent with the result in genes encompassed glycolysis, pentose phosphate pathway, Aurora A knockdown cells (Supplementary Fig. S2). These lipid/sterol biosynthesis, and mitochondrial function (37, data indicated the antitumor effect of Aurora kinases inhi- 38). For glycolysis, mTORC1 signaling stimulates glucose bitors was largely related to Aurora A inhibition. Further- uptake, promotes glycolysis flus, and increases lactate secre- more, the cells treated with MK and ZM showed polyploi- tion (37, 38). mTORC2 modulates cells metabolism through dization (Fig 1B and Supplementary Fig. S1B). P53 played a phosphorylating Akt/PKB and activating the downstream vital role during mitosis and loss of p53 supported the such as FoxO3a (19, 39). Furthermore, knockout of rictor survival of polyploidy cells (27, 28). However, our data (one of the mTORC2 components) in mice liver (40), suggested that Aurora kinases inhibitors induced poly- adipose (19), or muscle (20) cells impaired glycolysis and ploidy might be independent of p53 pathways (data not lipogenesis. These studies suggested that mTOR pathway show). The study of ZM on leukemia cells also indicated that played an important role in activating cellular bio-energetic polyploidy induced by Aurora kinases inhibitors did not processes contributed to cancer. In this study, mTOR inhi- inevitably result in apoptosis (29). Thus, polyploidization bitors decreased the glucose uptake and lactate production of induced by Aurora kinases inhibitors seemed to be the cause AML cell lines (Fig. 3B). Numerous studies reported that of modest effect on proliferation. Eliminating polyploidy rapamycin activated Akt signaling pathway via a negative cells might enhance the effect of Aurora kinase inhibitors in feedback loop by inhibiting mTORC1 activation (41, 42). AML treatment. Low concentrations of rapamycin increased Akt and Erk Metabolic pathways are activated in tumor cells, as well as phosphorylation through an mTORC1-dependent mecha- polyploidy tumor cells (30). In the study of comparison in nism, but high doses of rapamycin inhibited Akt and Erk the whole genomic transcripts of polyploidy and nonpoly- phosphorylation mainly via mTORC2 signaling pathway ploidy decidual cells in mouse, genes involved in metabolic (43). Compared with mTORC1 inhibitor rapamycin, the process were upregulated (23). In addition, immortalized dual mTORC1/2 inhibitor PP242 showed more effective to nontumorigenic human prostate and mammary epithelial reduce cellular metabolic level in cancer cells (Fig. 3B). cells which overexpressed pim-1, could gradually convert to Furthermore, PP242 significantly decreased the proliferation polyploidy cells, with increase of reactive oxygen species of polyploidy cells, as well as the glucose uptake and lactate (ROS) level (31). In aneuploid cells, the metabolic properties production, compared with that of control cells (Fig. 4), were also altered with increased glucose and glutamine providing evidence that mTOR pathway regulated the consumption (32). Anatskaia and colleagues reported that metabolism of polyploidy cells. Thus, mTORC1/2 inhibi- the rearrangement of the mainly metabolic pathways such as tion might be a potential approach to eliminate the polyploidy depression of mitochondrial processes and increase of car- cells. Indeed, the improved anticancer effect of mTOR bohydrate degradation and lipid biosynthesis in polyploidy inhibitors and Aurora kinases inhibitors combination was cells, triggered pentose–phosphate pathway and depressed reported in HCT116 cells recently (44). In our study, we oxidative stress (33). Furthermore, these metabolic changes elucidated a novel mechanism of the synergistic combination could increase metabolic plasticity and protect replicating by suppression of cell metabolism. DNA from oxidative damage (33), suggesting that polyploi- Previous study reported T-cell lymphomas that overex- dy cells might be sensitive to metabolic inhibitors. Polyploi- pressed Myc were sensitive to VX-680 (MK), and the dy induced by Aurora kinases inhibitors had been showed synthetic lethal interaction was executed by sequential apo- with more than 4N chromosomes (Fig. 1B and Supplemen- ptosis and autophagy. Exposed to VX-680 (MK) for 3 days, tary Fig. S1B). The most obvious phenomenon of polyploi- 30% of the cells were in apoptosis, and the survived cells dy-related changes was the decrease of nuclear surface-to- entered a new killing phase induced by autophagy after 3 volume ratio, which modified chromatin architecture and days (45). In our study, apoptosis slightly increased and exerted spectacular effects on genes activity (33, 34). A great autophagy elevated in AML cells incubating with Aurora of ATPase and energy were required for stemming from the kinases inhibitors for 2 days. Significantly, PP242 and 2DG metabolic shift from tricarboxylic acid cycle to glycolysis increased the apoptosis and autophagy of the polyploidy cells (35). Therefore, polyploidy induced by Aurora kinases (Fig. 5). The results implied that inhibition the metabolism inhibitors might display elevated glycolysis. Consistent with of polyploidy cells provoked not only apoptotic cell death, the previous reports, we showed that polyploidy cells but also significant autophagy. induced by Aurora kinases inhibitors had higher level of Autophagy, an evolutionary highly conserved process, is glycolytic metabolism than control cells (Fig. 2A). 2DG, involved in degrading proteins and organelles in response to glucose analog, had been reported to inhibit hexokinase, cellular stress (46). However, the function of autophagy in decrease intracellular ATP, and induce autophagy (36). In tumorgenesis is complex and paradoxical. On the one hand, our study, 2DG could inhibit the proliferation of the autophagy protects tumor cells from stress including nutrient polyploidy cells (Fig. 2B), suppress the mTOR pathway, starvation, radiotherapy, and certain cytotoxic drugs (47). On and result in apoptotic and autophagic death (Fig. 5), the other hand, autophagy plays a vital role in suppressing indicating that the polyploidy cells with high metabolism tumorgenesis. Overexpression Beclin-1 gene exhibited were susceptible to 2DG. increased susceptibility to cancer (48). Prolonged stress and

excessive cellular damage might lead to cell death by over- apoptosis, indicating that p62 might act as a potential target stimulating autophagy (49). In this study, AML cell lines were for cancer treatment (Fig. 6B). showed increased autophagy level with polyploidization after In summary, we found that polyploidy cells induced by treating with Aurora kinases inhibitors (Fig. 5B). Further- Aurora inhibitors required more energy than that of the more, either mTOR inhibitor PP242 or glycolysis inhibitor control cells. Suppression of metabolism by mTOR pathway 2DG promoted the polyploidy cells death by significantly inhibition increased the apoptosis and autophagy of the inducing apoptosis and autophagy (Fig. 5). We previously polyploidy cells. Moreover, p62 expression was decreased by showed that Aurora inhibition induced autophagy and mTOR inhibitor or glucose deprivation in polyploidy cells triggered drug resistance, which could be reversed by and might act as an attractive target. Thus, inhibition of autophagy inhibitors (8). These paradoxical data suggested mTOR pathway could increase the potential efficiency of that cell death or survival promoted by autophagy depended Aurora kinases inhibitors in AML treatment. on the physiologic state of the cell (47). In our present fl system, mTOR inhibition might overstimulate autophagy Disclosure of Potential Con icts of Interest No potential conflicts of interest were disclosed. in polyploidy cells and eventually induce the polyploidy cells death. Authors' Contributions The adaptor p62 is initially identified as a partner of the Conception and design: L.-L. Liu, Z.-J. Long, D.-J. Lin, Q. Liu Development of methodology: L.-L. Liu, F.-M. Zheng, D.-F. Xu, J.-J. Chen, Q. Liu atypical PKC (protein kinase C), interacting with various Acquisition of data (provided animals, acquired and managed patients, provided signaling proteins to regulate multiple cellular functions, facilities, etc.): L.-L. Liu, Z.-J. Long, L.-X. Wang, Q. Liu including apoptosis and autophagy (50–52). UBA and LIR Analysis and interpretation of data (e.g., statistical analysis, biostatistics, compu- tational analysis): L.-L. Liu, L.-X. Wang, F.-M. Zheng, Z.-G. Fang, D.-F. Xu, domains of p62 confer the protein to function as an adaptor J.-J. Chen, Q. Liu between autophagy and ubiquitinated proteins (53). As an Writing, review, and/or revision of the manuscript: L.-L. Liu, Z.-J. Long, autophagy substrate, p62 is recruited to the autophagosomal F.-M. Zheng, D.-J. Lin, Q. Liu Administrative, technical, or material support (i.e., reporting or organizing data, membrane dependent or independent of LC3 for degrada- constructing databases): L.-L. Liu, Z.-J. Long, F.-M. Zheng, M. Yan, S.-W. Wang, tion (54, 55). Upregulation of p62 by defective autophagy D.-J. Lin, Q. Liu was a key factor in tumorigenesis, and elimination of p62 Study supervision: D.-J. Lin, Q. Liu might benefit to cancer therapy (56, 57). Deficiency of p62 Acknowledgments increased ROS levels, enhanced cell death, and reduced The authors thank the members of LiuQ lab and Medical Research Center of the Third Affiliated Hospital of Sun Yat-sen University for their critical comments and tumorigenicity of Ras (58). mTORC1 activation inhibited technical support. autophagy by abolishing phosphorylated ATG13 protein binding to ATG1, promoting the accumulation of p62 Grant Support protein via reducing its degradation (59). Indeed, decrease This work was supported by the National Natural Science Foundation of China (No. 81130040 to Q. Liu; No. 81000217 to Z.-J. Long), the Science and Technology the p62 expression by mTOR inhibitor was showed in some Foundation of Guangzhou (No. 2012J2200077 to Z.-J. Long), the Fundamental reports (8, 60). Similarly, we found that expression of p62 Research Funds for the Central Universities (No. 11ykpy37 to Z.-J. Long), and National decreased in mTOR inhibitors treated polyploidy cells (Fig. Basic Research Program of China (973 Program; No. 2012CB967000 to Q. Liu). The costs of publication of this article were defrayed in part by the payment of page 5C), implying that p62 might be a target for oncotherapy. charges. This article must therefore be hereby marked advertisement in accordance with In addition, our data showed that p62 knockdown U937 18 U.S.C. Section 1734 solely to indicate this fact. cells treated with MK were observed with more apoptosis, Received April 9, 2013; revised July 29, 2013; accepted August 1, 2013; whereas p62 overexpressed U937 cells reversed MK induced published OnlineFirst September 5, 2013.

References 1. Farag SS. The potential role of Aurora kinase inhibitors in haemato- death in all-trans retinoid acid-resistant acute promyelocytic leukemia logical malignancies. Br J Haematol 2011;155:561–79. cells. J Transl Med 2011;9:74. 2. Marumoto T, Zhang D, Saya H. Aurora A—a guardian of poles. Nat Rev 8. Zou Z, Yuan Z, Zhang Q, Long Z, Chen J, Tang Z, et al. Aurora kinase Cancer 2005;5:42–50. A inhibition-induced autophagy triggers drug resistance in breast 3. Meraldi P, Honda R, Nigg EA. Aurora kinases link chromosome cancer cells. Autophagy 2012;8:1798–810. segregation and cell division to cancer susceptibility. Curr Opin Genet 9. Fei F, Lim M, Schmidhuber S, Moll J, Groffen J, Heisterkamp N. Dev 2004;14:29–36. Treatment of human pre-B acute lymphoblastic leukemia with the 4. Huang XF, Luo SK, Xu J, Li J, Xu DR, Wang LH, et al. Aurora kinase Aurora kinase inhibitor PHA-739358 (Danusertib). Mol Cancer 2012; inhibitory VX-680 increases Bax/Bcl-2 ratio and induces apoptosis in 11:42. Aurora-A-high acute myeloid leukemia. Blood 2008;111:2854–65. 10. Duesberg P. Does aneuploidy or mutation start cancer? Science 5. Lucena-Araujo AR, de Oliveira FM, Leite-Cueva SD, Dos SG, Falcao RP, 2005;307:41. Rego EM. High expression of AURKA and AURKB is associated with 11. Anatskaia OV, Vinogradov AE. [Somatic polyploidy associated meta- unfavorable cytogenetic abnormalities and high white blood cell count bolic changes revealed by modular biology]. Tsitologiia 2010;52:52–62. in patients with acute myeloid leukemia. Leuk Res 2011;35:260–4. 12. Warburg O. On the origin of cancer cells. Science 1956;123:309–14. 6. Hauf S, Cole RW, LaTerra S, Zimmer C, Schnapp G, Walter R, et al. The 13. Eggert US, Mitchison TJ, Field CM. Animal cytokinesis: from parts list small molecule Hesperadin reveals a role for Aurora B in correcting to mechanisms. Annu Rev Biochem 2006;75:543–66. kinetochore-microtubule attachment and in maintaining the spindle 14. Meric-Bernstam F, Gonzalez-Angulo AM. Targeting the mTOR signal- assembly checkpoint. J Cell Biol 2003;161:281–94. ing network for cancer therapy. J Clin Oncol 2009;27:2278–87. 7. Xu DR, Huang S, Long ZJ, Chen JJ, Zou ZZ, et al. Inhibition of mitotic 15. Yecies JL, Manning BD. mTOR links oncogenic signaling to tumor cell kinase Aurora suppresses Akt-1 activation and induces apoptotic cell metabolism. J Mol Med (Berl) 2011;89:221–8.

Liu et al.

16. Ma L, Chen Z, Erdjument-Bromage H, Tempst P, Pandolfi PP. Phos- 37. Duvel€ K, Yecies JL, Menon S, Raman P, Lipovsky AI, Souza AL, et al. phorylation and functional inactivation of TSC2 by Erk implications for Activation of a metabolic gene regulatory network downstream of tuberous sclerosis and cancer pathogenesis. Cell 2005;121:179–93. mTOR complex 1. Mol Cell 2010;39:171–83. 17. Inoki K, Li Y, Zhu T, Wu J, Guan KL. TSC2 is phosphorylated and 38. Cunningham JT, Rodgers JT, Arlow DH, Vazquez F, Mootha VK, inhibited by Akt and suppresses mTOR signalling. Nat Cell Biol 2002; Puigserver P. mTOR controls mitochondrial oxidative function through 4:648–57. a YY1-PGC-1a transcriptional complex. Nature 2007;450:736–40. 18. Kumar A, Harris TE, Keller SR, Choi KM, Magnuson MA, Lawrence JJ. 39. Ye X, Guo Y, Zhang Q, Chen W, Hua X, Liu W, et al. bKlotho suppresses Muscle-specific deletion of rictor impairs insulin-stimulated glucose tumor growth in hepatocellular carcinoma by regulating Akt/GSK-3b/ transport and enhances basal glycogen synthase activity. Mol Cell Biol Cyclin D1 signaling pathway. PLoS One 2013;8:e55615. 2008;28:61–70. 40. Hagiwara A, Cornu M, Cybulski N, Polak P, Betz C, Trapani F, et al. 19. Kumar A, Lawrence JC Jr, Jung DY, Ko HJ, Keller SR, Kim JK, et al. Fat Hepatic mTORC2 activates glycolysis and lipogenesis through Akt, cell-specific ablation of rictor in mice impairs insulin-regulated fat cell glucokinase, and SREBP1c. Cell Metab 2012;15:725–38. and whole-body glucose and lipid metabolism. Diabetes 2010;59: 41. Carracedo A, Pandolfi PP. The PTEN-PI3K pathway: of feedbacks and 1397–406. cross-talks. Oncogene 2008;27:5527–41. 20. Ma D, Yu H, Lin D, Sun Y, Liu L, Liu Y, et al. S6K1 is involved in 42. Carracedo A, Ma L, Teruya-Feldstein J, Rojo F, Salmena L, Alimonti A, polyploidization through its phosphorylation at Thr421/Ser424. J Cell et al. Inhibition of mTORC1 leads to MAPK pathway activation through Physiol 2009;219:31–44. a PI3K-dependent feedback loop in human cancer. J Clin Invest 2008; 21. Gui Y, He GH, Walsh MP, Zheng XL. Predisposition to tetraploidy in 118:3065–74. pulmonary vascular smooth muscle cells derived from the Eker rats. 43. Chen XG, Liu F, Song XF, Wang ZH, Dong ZQ, Hu ZQ, et al. Rapamycin Am J Physiol Lung Cell Mol Physiol 2007;293:L702–11. regulates Akt and ERK phosphorylation through mTORC1 and 22. Sun Q, Chen X, Ma J, Peng H, Wang F, Zha X, et al. Mammalian target of mTORC2 signaling pathways. Mol Carcinog 2010;49:603–10. rapamycin up-regulation of pyruvate kinase isoenzyme type M2 is 44. Shionome Y, Yan L, Liu S, Saeki T, Ouchi T. Integrity of p53 associated critical for aerobic glycolysis and tumor growth. Proc Natl Acad Sci pathways determines induction of apoptosis of tumor cells resistant to U S A 2011;108:4129–34. Aurora-A kinase inhibitors. PLoS One 2013;8:e55457. 23. Ma X, Gao F, Rusie A, Hemingway J, Ostmann AB, Sroga JM, et al. 45. Yang D, Liu H, Goga A, Kim S, Yuneva M, Bishop JM. Therapeutic Decidual cell polyploidization necessitates mitochondrial activity. potential of a synthetic lethal interaction between the MYC proto- PLoS One 2011;6:e26774. oncogene and inhibition of aurora-B kinase. Proc Natl Acad Sci U S A 24. Brown J. Effects of 2-deoxyglucose on carbohydrate metablism: 2010;107:13836–41. review of the literature and studies in the rat. Metabolism 1962;11: 46. Klionsky DJ, Emr SD. Autophagy as a regulated pathway of cellular 1098–112. degradation. Science 2000;290:1717–21. 25. Duran A, Amanchy R, Linares JF, Joshi J, Abu-Baker S, Porollo A, et al. 47. White E, DiPaola RS. The double-edged sword of autophagy modu- p62 is a key regulator of nutrient sensing in the mTORC1 pathway. Mol lation in cancer. Clin Cancer Res 2009;15:5308–16. Cell 2011;44:134–46. 48. Liang XH, Jackson S, Seaman M, Brown K, Kempkes B, Hibshoosh H, 26. Dar AA, Goff LW, Majid S, Berlin J, El-Rifai W. Aurora kinase inhibi- et al. Induction of autophagy and inhibition of tumorigenesis by beclin tors—rising stars in cancer therapeutics? Mol Cancer Ther 2010; 1. Nature 1999;402:672–6. 9:268–78. 49. Mathew R, Karantza-Wadsworth V, White E. Role of autophagy in 27. Ohshima S. Centrosome aberrations associated with cellular senes- cancer. Nat Rev Cancer 2007;7:961–7. cence and p53 localization at supernumerary centrosomes. Oxid Med 50. Moscat J, Diaz-Meco MT, Albert A, Campuzano S. Cell signaling and Cell Longev 2012;2012:217594. function organized by PB1 domain interactions. Mol Cell 2006;23: 28. Gogolin S, Batra R, Harder N, Ehemann V, Paffhausen T, Diessl N, et al. 631–40. MYCN-mediated overexpression of mitotic spindle regulatory genes 51. Sanchez P, De Carcer G, Sandoval IV, Moscat J, Diaz-Meco MT. and loss of p53-p21 function jointly support the survival of tetraploid Localization of atypical protein kinase C isoforms into lysosome- neuroblastoma cells. Cancer Lett 2013;331:35–45. targeted endosomes through interaction with p62. Mol Cell Biol 29. Walsby E, Walsh V, Pepper C, Burnett A, Mills K. Effects of the aurora 1998;18:3069–80. kinase inhibitors AZD1152-HQPA and ZM447439 on growth arrest and 52. Moscat J, Diaz-Meco MT. p62 at the crossroads of autophagy, polyploidy in acute myeloid leukemia cell lines and primary blasts. apoptosis, and cancer. Cell 2009;137:1001–4. Haematologica 2008;93:662–9. 53. Puissant A, Fenouille N, Auberger P. When autophagy meets cancer 30. Moreno-Sanchez R, Rodriguez-Enriquez S, Marin-Hernandez A, through p62/SQSTM1. Am J Cancer Res 2012;2:397–413. Saavedra E. Energy metabolism in tumor cells. FEBS J 2007;274: 54. Noda NN, Ohsumi Y, Inagaki F. Atg8-family interacting motif crucial for 1393–418. selective autophagy. FEBS Lett 2010;584:1379–85. 31. Roh M, van der Meer R, Abdulkadir SA. Tumorigenic polyploid cells 55. Itakura E, Mizushima N. p62 targeting to the autophagosome forma- contain elevated ROS and ARE selectively targeted by antioxidant tion site requires self-oligomerization but not LC3 binding. J Cell Biol treatment. J Cell Physiol 2012;227:801–12. 2011;192:17–27. 32. Williams BR, Prabhu VR, Hunter KE, Glazier CM, Whittaker CA, Hous- 56. Sanz L, Diaz-Meco MT, Nakano H, Moscat J. The atypical PKC- man DE, et al. Aneuploidy affects proliferation and spontaneous interacting protein p62 channels NF-kB activation by the IL-1-TRAF6 immortalization in mammalian cells. Science 2008;322:703–9. pathway. EMBO J 2000;19:1576–86. 33. Anatskaya OV, Vinogradov AE. Somatic polyploidy promotes cell 57. Mathew R, Karp CM, Beaudoin B, Vuong N, Chen G, Chen HY, et al. function under stress and energy depletion: evidence from tissue- Autophagy suppresses tumorigenesis through elimination of p62. Cell specific mammal transcriptome. Funct Integr Genomics 2010;10: 2009;137:1062–75. 433–46. 58. Duran A, Linares JF, Galvez AS, Wikenheiser K, Flores JM, Diaz-Meco 34. Comai L. The advantages and disadvantages of being polyploid. Nat MT, et al. The signaling adaptor p62 is an important NF-kB mediator in Rev Genet 2005;6:836–46. tumorigenesis. Cancer Cell 2008;13:343–54. 35. Tchernaenko V, Radlinska M, Lubkowska L, Halvorson HR, Kashlev M, 59. Díaz-Troya S, Perez-P erez ME, Florencio FJ, Crespo JL. The role of Lutter LC. DNA bending in transcription initiation. Biochemistry 2008; TOR in autophagy regulation from yeast to plants and mammals. 47:1885–95. Autophagy 2008;4:851–65. 36. Ben Sahra I, Laurent K, Giuliano S, Larbret F, Ponzio G, Gounon P, et al. 60. Huang S, Yang ZJ, Yu C, Sinicrope FA. Inhibition of mTOR kinase by Targeting cancer cell metabolism: the combination of metformin and 2- AZD8055 can antagonize chemotherapy-induced cell death through deoxyglucose induces p53-dependent apoptosis in prostate cancer autophagy induction and down-regulation of p62/sequestosome 1. cells. Cancer Res 2010;70:2465–75. J Biol Chem 2011;286:40002–12.

Inhibition of mTOR Pathway Sensitizes Acute Myeloid Leukemia Cells to Aurora Inhibitors by Suppression of Glycolytic Metabolism

Ling-Ling Liu, Zi-Jie Long, Le-Xun Wang, et al.

Mol Cancer Res 2013;11:1326-1336. Published OnlineFirst September 5, 2013.

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