antitumoreffectsofblockingproteinneddylation in t315i-bcr ... · tein neddylation, a type of...

Translational Science

AntitumorEffectsofBlockingProteinNeddylationin T315I-BCR-ABL Leukemia Cells and LeukemiaStem CellsChang Liu1, Danian Nie2, Juan Li3, Xin Du4, Yuhong Lu5, Yangqiu Li5, Jingfeng Zhou1,Yanli Jin1, and Jingxuan Pan1

Abstract

Imatinib revolutionized the treatment of chronic myeloid leu-kemia (CML), but drug resistance and disease recurrence remain achallenge. In this study, we suggest a novel strategy based onblocking protein neddylation to address BCR-ABL point muta-tions and leukemia stem cells (LSC) that lie at the root ofimatinib-resistant recurrences. On the basis of the finding thatthe NEDD8-activating enzyme subunit NAE1 is overexpressed inCML cells, we hypothesized that the function of certain neddyla-tion-dependent protein substrates might be targeted to therapeuticends in imatinib-resistant CML cells and LSCs. In support of thishypothesis, we demonstrated that the NAE1 inhibitor MLN4924induced G2–M-phase arrest and apoptosis in bulk CML cells withwild-type p53, regardless of their T315I mutation status in BCR-

ABL. Moreover, MLN4924 inhibited the survival and self-renewalof primary human CML CD34þ cells and LSCs in CML-bearingmice via accumulation of p27kip1 in the nucleus. Notably, p27kip1

silencing attenuated the suppressive effect of MLN4924 on themaintenance of LSCs in CML-bearing mice. Taken together, ourfindings offer a preclinical proof of concept for targeting proteinneddylation as a novel therapeutic strategy to override mutationaland LSC-derived imatinib resistance in CML.

Significance: These findings highlight a mediator of pro-tein neddylation, a type of protein turnover mechanism, as aviable therapeutic target against imatinib-resistant forms ofchronic myelogenous leukemia. Cancer Res; 78(6); 1522–36.�2018 AACR.

IntroductionChronic myeloid leukemia (CML) is a myeloproliferative dis-

ease, which is characterized by Philadelphia chromosome (Phþ),the genetic translocation t (9; 22) (q34; q11.2), involving the fusionof the Abelson oncogene (ABL) with the breakpoint cluster region(BCR) gene encoding the BCR-ABL fusion oncoprotein with con-stitutive tyrosine kinase activity (1). BCR-ABL is the single drivingforce in pathogenesis of CML. Tyrosine kinase inhibitors (TKI)including imatinib, dasatinib, and nilotinib are current first-linetherapy to treat CML (2). Approximately 80% of patients withchronic phase (CP)-CML achieve a complete cytogenetic remissionwithin 1 year of therapy with imatinib (3). Even though imatinib

can effectively control CML development, drug resistance mayoccur. Approximately 50% of imatinib-resistant patients are dueto emerging CML clones harboring BCR-ABL kinase mutants (4).T315I, the most vicious mutant to imatinib, accounts for at least15% of relapse cases of CML (5). The application of the third-generation TKI ponatinib against T315I-BCR-ABL may be limitedbecause of its severe toxic side effects (e.g., arterial thrombosis,myocardial infarction, and cerebrovascular events; ref. 6). There-fore, resistance to imatinib remains a therapeutic challenge inCML.

Reasons of the rest 50% of imatinib-resistant patients arevarious. CML, defined as a stem cell disease, has characteristicfeatures of leukemia stem cells (LSC; ref. 7). The maintenance ofLSCs is independent of BCR-ABL kinase activity (8), which isconsidered as roots of TKI resistance and CML relapse. Thus, it isurgent to seek novel agents to eliminate LSCs for CML cure.

Protein neddylation is a posttranslational modification thatadds an ubiquitin-like NEDD8 (neural precursor cell expressed,developmentally downregulated 8) to target proteins. Neddyla-tion cascade is catalyzed by three NEDD8-specific enzymes, an E1NEDD8-activating enzyme (NAE1), an E2-conjugating enzyme(UBC12), and one of the several E3 ligases (9). It is implicated inthe regulation of stability and subcellular localization of proteins,gene transcription, and DNA damage response (DDR), whicheventually controls diverse physiologic and pathologic cellularbehaviors such as survival, differentiation, senescence, and apo-ptosis (10). Overactivation of neddylation pathway is involved intumorigenesis and tumor progression (11). Furthermore, aber-rant neddylation confers drug resistance in ovarian cancer andmultiple myeloma (12, 13), which makes NAE1 inhibition anovel strategy to overcome drug resistance in these cancers.

MLN4924 (Pevonedistat), the first-in-class NAE1 inhibitor,blocks the entire neddylation modification of proteins including

1Jinan University Institute of Tumor Pharmacology, College of Pharmacy, JinanUniversity; State Key Laboratory of Ophthalmology, Zhongshan OphthalmicCenter, Sun Yat-sen University, Guangzhou, China. 2Department of Hematology,Sun Yat-sen Memorial Hospital, Sun Yat-sen University, Guangzhou, China.3Department of Hematology, The First Affiliated Hospital, Sun Yat-sen Univer-sity, Guangzhou, China. 4Department of Hematology, Guangdong GeneralHospital/Guangdong Academy of Medical Sciences, Guangzhou, China.5Department of Hematology, The First Affiliated Hospital, Jinan University,Guangzhou, China.

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

Corresponding Authors: Jingxuan Pan, Jinan University Institute of TumorPharmacology, College of Pharmacy, Jinan University; State Key Laboratoryof Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, 54South Xianlie Road, Guangzhou 510060, China. Phone: 8620-3762-8262; Fax:8620-8733-4279; E-mail: [email protected]; and Yanli Jin,[email protected]

doi: 10.1158/0008-5472.CAN-17-1733

�2018 American Association for Cancer Research.

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cullins, which results in accumulation of many cullin-RINGligase (CRL) substrates including phospho-IkBa, p27kip1, andCDT1. MLN4924 treatment induces apoptosis, DNA damage,and cell-cycle arrest in multiple types of solid and hematopoieticmalignant cells (14). Pevonedistat is currently under phase Iclinical trials (15, 16). Whether neddylation inhibition byMLN4924 induces apoptosis in T315I-BCR-ABLþ cells is elusive.

After obtaining theobservation thatNAE1was overexpressed inthe primaryCD34þCD38� cell subpopulation fromCMLpatientswhen comparedwith the counterparts fromnormal bonemarrow(NBM),we hypothesized that the function of certain neddylation-dependent protein substrates might be targeted to therapeuticends in imatinib-resistant CML cells and LSCs. In support of thisplausibility, neddylation is reported to regulatemouse embryonicstem cells and self-renewal property of cancer stem-like cells(CSC) in nasopharyngeal carcinoma (17, 18). The purpose ofthis study was to investigate whether blocking neddylation path-way was active against BCR-ABL mutational imatinib-resistantcells, self-renewal capacity of LSCs in CML and the underlyingmechanisms. Our results showed that MLN4924 possessed aninhibitory activity against wild-type (WT) - and T315I-BCR-ABLþ

cells with WT-p53. MLN4924 treatment significantly eliminatedthe LSCs from CML patients and CML mice transformed byretrovirus BCR-ABL transduction. Overall, our findings providea rationale for targeting T315I-BCR-ABLþ bulk tumor cells andLSCs to overcome resistance to imatinib by neddylation inhibi-tion with MLN4924.

Materials and MethodsCell culture

KBM5 and KBM5-T315I cells were kindly provided byDr. Sai-Ching J. Yeung (University of Texas MD Anderson CancerCenter, Houston, TX) and cultured in Iscove's modified Dulbec-co's medium (Thermo Fisher Scientific) with 10% FBS (BiologicalIndustries; ref. 19). K562, KU812,M2-10B4, andHL-60 cells werepurchased from the ATCC and cultured in RPMI1640 medium(Thermo Fisher Scientific) supplemented with 10% FBS. 293T,Plat-E, BT-549,MCF-7, andU2OS cells were obtained fromATCCand cultured in DMEM (Thermo Fisher Scientific) supplementedwith 10% FBS. BV173 cells were obtained from DSMZ andcultured in RPMI1640 with 10% FBS. The parental BaF3 cellswere cultured in RPMI1640 with IL3. The BaF3 cells stablyexpressing either 210-kDa WT-BCR-ABL (BaF3-BCR-ABL) orT315I-BCR-ABL (BaF3-T315I) and KCL-22 cells were generouslyprovided by Dr. Jia Fei (Medical College of Jinan University,Guangzhou, China) andmaintained in RPMI1640 with 10% FBS(20). Cells were incubated at 37�C in a humidified incubatorcontaining 5% CO2. All the cell lines were authenticated by usingshort tandem repeat (STR) matching analysis last month. Nomycoplasma contamination was detected.

Plasmids and siRNA duplexesMSCV-BCR-ABL-IRES-EGFP constructwasdescribed previously

(21). HA-tagged p53 in pcDNA3.1 was a gift from Dr. TiebangKang (Sun Yat-Sen University Cancer Center, Guangzhou, China;ref. 22). Human p53 shRNA and mouse p27kip1 shRNA were fromSigma-Aldrich. siRNAduplexes against p27kip1, NAE1, and control(Mock)were purchased fromTransheep.Detailed information fortransfection of plasmids and siRNA duplexes is described inSupplementary Methods.

Western blot analysisWhole-cell lysates were prepared in RIPA buffer (23). Cyto-

solic fraction for detection of cytochrome c was prepared withdigitonin extraction buffer. Cytoplasmic and nuclear fractionswere obtained as described previously (24), with details shownin Supplementary Methods and Supplementary Table S1.

Measurement of apoptosis by flow cytometryAfter CML cells were treated with MLN4924, apoptosis was

measured by Annexin V-fluoresceinisothiocyanate (FITC)/propi-dium iodide (PI) apoptosis detection kit (Sigma-Aldrich), andanalyzed with a FACS C6 flow cytometry.

Primary humanCMLCD34þ cells were treatedwithMLN4924,imatinib alone or in combination for 24 hours, then collected andwashed. Cells were stainedwith Annexin V-FITC andCD38-PE for30 minutes, apoptotic cells (CD34þCD38�Annexin Vþ) weredetected by flow cytometry (BD LSRFortessa; ref. 21).

Real-time quantitative RT-PCRThe real-time PCRwas performed as described previously (24),

with details provided in Supplementary Methods.

Measurement of mitochondrial transmembrane potentialMitochondrial transmembrane potential (Dym) was detected

as described previously (25), with details provided in Supple-mentary Methods.

Primary cellsPeripheral blood or bonemarrow samples were obtained from

patients with CML (Supplementary Table S2) and healthy adultdonors in Sun Yat-sen Memorial Hospital of Sun Yat-sen Uni-versity, The First Affiliated Hospital of Sun Yat-sen University,Guangdong General Hospital/Guangdong Academy of MedicalSciences, and The First AffiliatedHospital of Jinan University afterwritten informed consent according to the institutional guidelinesand the Declaration of Helsinki principles. Primary humanCD34þ cells were isolated by using CD34 Microbeads Kit (Milte-nyi Biotec) according to the instructions of the manufacturer andincubated in Iscove's modified Dulbecco's medium supplemen-ted with 10% FBS (21, 24). The studies were approved byInstitutional Review Board, Sun Yat-sen University (Guangzhou,China).

Colony-forming cell/replating assayPrimary human CML CD34þ cells were pretreated with

MLN4924 for 24 hours, then washed with PBS, and 5,000 cellswere seeded in the methylcellulose medium (MethoCult H4434,STEMCELL Technologies). After incubation for 14 days, colonieswere counted. The cells were harvested and replated (5,000 cellsper well) for the secondary and tertiary rounds, respectively.Colonies were counted on day 14 (21, 24).

Long-term culture-initiating cell assayPrimary humanCML-nucleated cells (2� 106)were cocultured

with preestablished and irradiated (80 Gy)M2-10B4 cells in LTC-ICmedium (H5100, STEMCELL Technologies) in the presence ofMLN4924 (500 nmol/L) in combination with or without imati-nib (2,500 nmol/L) for 1 week. Cultures weremaintained in LTC-IC medium for 5 weeks with weekly half-medium replacement.The cellswereharvested, counted, andplated intomethylcellulose

Neddylation Blockade Eliminates LSCs in CML

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medium (MethoCult H4435, STEMCELL Technologies) for col-ony-forming assay. After incubation for 14 days, LTC-IC–derivedcolonies were counted (21).

CML mouse modelHigh-titer helper-free retroviruses were produced by transient

transfection of Plat-E cells with the retroviral construct MSCV-BCR-ABL-IRES-EGFP as described previously (26). Donor maleC57BL/6 mice (Guangdong Medical Laboratory Animal Center)were pretreated with 5-fluorouracil (5-FU, 200 mg/kg). Five dayslater, bone marrow cells were harvested and transduced tworounds with MSCV-BCR-ABL-IRES-EGFP retrovirus in the pres-ence of cytokines (SCF, IL3, and IL6). The bonemarrow cells werethen transplanted into sublethally irradiated (550 cGy) recipientfemale C57BL/6 mice (21). Following transplantation, the micewere treated with vehicle, MLN4924 (60 mg/kg/day, i.p.), ima-tinib (100 mg/kg/day, gavage) or their combination for 14 days.

To examine the in vivo effect of p27kip1 knockdown, splenic cellsfrom CML mice transduced with scramble or murine p27kip1

shRNA lentivirus were transplanted into sublethally irradiatedrecipient C57BL/6mice followed by administration of MLN4924for 14 days (24). The detailed information for the detection ofLSK, LT-HSC, and ST-HSC cells is documented in SupplementaryMethods.

Limiting dilution assay of mouse LT-HSCsBone marrow cells from CML mice treated with or without

MLN4924 were harvested and transplanted via tail vein intosublethally irradiated recipient C57BL/6 mice at a serial concen-trations of cells (2 � 106, 1 � 106, 5 � 105 cells/mouse) inconjunction with normal bone marrow cells (2 � 105 cells/mouse). GFPþ cells in peripheral blood were monitored for 16weeks by flow cytometry once a week, andGFPþ cells > 0.5%wereconsidered as a CML mouse. At week 16, LSC frequency wasdetermined using Poisson statistics online at the Bioinformaticsfacility of The Walter & Eliza Hall Institute of Medical Research(21, 27). All animal studies were conducted with the approval ofthe Sun Yat-sen University Institutional Animal Care and UseCommittee.

Statistical analysisStatistical analyses were performed using GraphPad Prism 5.0

Software (GraphPad). All experiments were carried out at leastthree times, and data are presented as mean � SEM unlessotherwise specified. Paired analyses were calculated using Studentt test, and comparison of multiple groups by one-way ANOVA,post hoc intergroup comparisons, Tukey test. P < 0.05 was con-

sidered statistically significant. Kaplan–Meier survival curves wereanalyzed by log-rank test.

ResultsMLN4924 inhibits proliferation of CML cells with NAE1overexpressed and intact p53 regardless of T315I BCR-ABL

We first compared NAE1 levels in CML cells and other cancer-ous cells with normal humanperipheral bloodmononuclear cells(PBMC). Immunoblotting analysis showed that NAE1 levels werehigher in the tested 6 lines of CML cells and some Ph� tumor cells(HL-60, BT-549, and MCF-7 but not U2OS) than normal PBMCs(Fig. 1A). Consistently,NAE1was increased inBaF3-BCR-ABL andBaF3-T315I cells relative to their parent BaF3 cells (Fig. 1A). Wenext evaluated the specificity of MLN4924 inhibitory effect onneddylation pathway in CML cells harboring either WT-BCR-ABLor T315I-BCR-ABL. The results showed that MLN4924, but notMG132 and bortezomib (two proteasome inhibitors), inhibitedglobal protein neddylation (Fig. 1B). MLN4924 dramaticallysuppressed cullin1 neddylation and provoked stabilization ofkey CRL substrates (e.g., phospho-IkBa and p27kip1) in a dose-dependent manner (Fig. 1C).

Next, we examined the effect of MLN4924 on the proliferationof CML cells. CML cells and normal PBMCs were treated withMLN4924 at incremental concentrations for 72 hours. MTS assayshowed that MLN4924 markedly inhibited the cell viability ofKU812, BV173, and KBM5 cells all harboring WT-BCR-ABL withIC50 values of 204 nmol/L, 233 nmol/L, and 354 nmol/L, respec-tively (Fig. 1D). In contrast, MLN4924 did not reduce the cellviability at even up to 10, 000 nmol/L in PBMCs, which expressedextremely low NAE1 (Fig. 1D and A). MLN4924 also suppressedthe cell viability of KBM5-T315I to the similar extent as KBM5(Fig. 1D). Similarly, MLN4924 potently decreased the cell viabil-ity of BaF3-T315I as well as BaF3-BCR-ABL but not their parentalBaF3 cells (Fig. 1D).

To our surprise, MLN4924 did not counteract the cell viabilityof K562 and KCL-22 cells that are p53-null CML cells (IC50 valuesof 20,000 nmol/L and 16,000 nmol/L, respectively, Supplemen-tary Fig. S1A; ref. 28). Given that p53 stabilization inducesapoptosis in CML cells (29), we speculated that MLN4924 mightexert its antitumor activity in CML cells in a p53-dependentmanner. To test this hypothesis, K562 and KCL-22 cells trans-fected with full-length WT-p53 cDNA construct were exposed toMLN4924, and subjected to MTS assay. The results showed thatectopically restoring p53-sensitized K562 and KCL-22 cells toMLN4924 as demonstrated by approximately 3-fold decrease ofIC50 values relative to the corresponding CML cells transfected

Figure 1.MLN4924 inhibits proliferation of CML cells with NAE1 overexpressed and intact p53 regardless of T315I BCR-ABL. A, NAE1 was increased in CML cells. Western blotanalysis of NAE1 in normal human PBMCs, CML cells, and a panel of Ph� cells (e.g., HL-60, BT-549, MCF-7, and U2OS). B, Cellular protein neddylationmodification was specifically blocked by the NAE1 inhibitor MLN4924 but not proteasome inhibitors MG132 and bortezomib. KBM5 and KBM5-T315I cells weretreated with MLN4924 (500 nmol/L), MG132 (20,000 nmol/L), and bortezomib (1,000 nmol/L) for 1 hour, followed by Western blot analysis with antibody againstglobal neddylation. C, Cullin-RING E3 ligase substrates were accumulated after MLN4924 treatment. The indicated CML cells were treated with incrementalconcentrations of MLN4924 for 36 hours. The protein levels of cullin1, p27kip1, phospho-IkBa, and IkBa were examined by Western blot analysis. D, MLN4924inhibited the cell viability of both imatinib-sensitive and -resistant cells harboring WT-p53. Normal PBMCs and the indicated cancerous cells were exposed toescalating concentrations of MLN4924 for 72 hours, and the cell viability was then determined by MTS assay. E, Clonogenicity of CML cells, BaF3-BCR-ABL, andBaF3-T315I cells was inhibited by MLN4924 in a concentration-dependent manner. Cells were pretreated with MLN4924 for 36 hours, then washed withPBS, and seeded in drug-free soft agar culture system. After incubation for 14 days, colonies composed of > 50 cells were counted. F and G, MLN4924 inducedG2–M-phase arrest in CML cells. KBM5 and KBM5-T315I cells were treated with MLN4924 (200 nmol/L) for the indicated time points, and subjected to flowcytometry analysis after PI staining. Representative histograms of the cell-cycle distribution (F) and results from three independent experiments in the graph(G) are shown. � , P < 0.05; �� , P < 0.01; �� , P < 0.001, one-way ANOVA, post hoc intergroup comparisons, Tukey test. H, MLN4924 induced DDR. KBM5 andKBM5-T315I cells were treated with MLN4924 for 36 hours, DNA damage–related proteins were examined by Western blot analysis.






Figure 2.

MLN4924 induces apoptosis in imatinib-sensitive and -resistant CML cells harboringWT-p53.A and B,MLN4924 induced apoptosis in a dose-dependent manner. Afterexposure to MLN4924 at the indicated concentrations for 36 hours, the indicated CML cells were subjected to either flow cytometry analysis after Annexin V-FITC/PIdouble staining (A) or Western blot analysis with the indicated antibodies (B). A, Representative histograms for each treatment (left) and results from threeindependent experiments (right) are shown. � , P < 0.05; �� , P < 0.01; �� , P < 0.001, one-way ANOVA, post hoc intergroup comparisons, Tukey test. B, Western blotanalysis of phospho-BCR-ABL, BCR-ABL, PARP, caspase-3, and -9 is shown. C, The protein levels of apoptosis-related proteins were detected by Western blotanalysis after treatment with MLN4924 for 36 hours in KBM5 and KBM5-T315I cells. D, MLN4924 treatment resulted in cytochrome c release in KBM5 and KBM5-T315Icells. Western blot analysis of cytochrome c in the cytosolic fractions prepared with digitonin buffer. Cox II is an indicator of mitochondrial fractionations. E andF, MLN4924 induced mitochondrial membrane depolarization in KBM5 and KBM5-T315I cells. The indicated CML cells were treated with MLN4924 (500 nmol/L) forthe indicated durations; mitochondrial potential (Dy) was analyzed by flow cytometry after staining with CMXRos and MTGreen. Representative histograms (E)and results from three independent experiments (F) are shown. � , P < 0.05; �� , P < 0.01; �� , P < 0.001, one-way ANOVA, post hoc intergroup comparisons, Tukey test.Gand H, Silencing NAE1 by siRNA induced DDR and decreased proliferation. KU812 cells transfected with siRNA control (Mock) or siRNA duplexes against NAE1 for48 hours were harvested for either immunoblotting analysis (G) or clonogenicity evaluation by being seeded in soft agar culture medium (H). �� , P < 0.01, Student t test.

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Figure 3.

MLN4924 reduces survival and self-renewal of primary CD34þ cells from patients with CML. A, qRT-PCR analysis of NAE1 mRNA levels in the purified CD34þ cellsfrom patients with CML (n ¼ 7) versus from healthy individuals (n ¼ 5). �� , P < 0.001, Student t test. B, The mRNA levels of NAE1 were significantly higherin the primary CML CD34þCD38� cells than those in the normal counterparts (GSE47927). �� , P < 0.01, Student t test. C, MLN4924 induced apoptosis in primaryhuman CML CD34þ cells. The purified primary CD34þ cells from CML patients (n ¼ 4) were exposed to imatinib (2,500 nmol/L), MLN4924 (500 nmol/L), ortheir combination for 24 hours. Annexin V–positive CD34þCD38� cells were analyzed by flow cytometry analysis after staining with Annexin V-FITC and CD38-PE.Representative flow cytometry histograms of apoptosis (left) and quantitative analysis of apoptotic CML CD34þCD38� cells (right) are shown. �� , P < 0.01;�� , P < 0.001, one-way ANOVA, post hoc intergroup comparisons, Tukey test. D, MLN4924 decreased CFC/replating capacity of human CML LSCs. The purifiedprimary CD34þ cells from CML patients were treated with MLN4924 (200 nmol/L or 500 nmol/L) for 24 hours, and 5,000 cells were seeded in drug-freemethylcellulose medium (MethoCult H4434). After 14 days of incubation, colonies were counted. The cells were harvested, and 5,000 cells were replated forthe secondary and tertiary rounds of colony formation assay. �, P < 0.05; �� , P < 0.01; �� , P < 0.001, one-way ANOVA, post hoc intergroup comparisons,Tukey test.E, LTC-ICs derived fromprimary humanCMLcellswere decreased afterMLN4924 treatment. A total of 2� 106CML-nucleated cells (n¼4)was overlaid onthe M2-10B4 cells in the LTC-IC medium and treated with MLN4924 (500 nmol/L) or in combination with imatinib (2,500 nmol/L) for 1 week. After beingmaintained for 5 weekswithweekly half-medium replaced, the cells were harvested, counted, and plated intomethylcellulosemedium (MethoCult H4435). Colonieswere counted at day 14. � , P < 0.05; �� , P < 0.01, one-way ANOVA, post-hoc intergroup comparisons, Tukey test.






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with empty vector (Supplementary Fig. S1B and S1C). In analternative set of experiments, KBM5-T315I cells transfected withshRNA against p53 were treated with MLN4924. As illustrated inSupplementary Fig. S1D and S1E, silencing p53 obviously atten-uated the apoptosis-inducing ability ofMLN4924 in KBM5-T315Icells, suggesting a requirement of p53 for the antitumor effect ofMLN4924 in CML cells.

To determine the antitumor activity specificity of MLN4924 inthe cancerous cells harboring BCR-ABL relative to Ph� cancerouscells, we included a panel of Ph� cancerous cells (e.g., HL-60, BT-549, MCF-7, and U2OS). The MTS results showed that MLN4924remarkably inhibited the cell viability of those cells (e.g., HL-60,BT-549, and MCF-7) overexpressing NAE1 but not U2OS weak-expressing NAE1 (Fig. 1D). The sensitivity of cancer cells toMLN4924 appeared to be associated with their NAE1 levels,which was consistent with previous reports (15–17).

In accordwith the results in PBMCs, the cell viability of parentalBaF3 cells was not diminished when MLN4924 was tested at theconcentrations even up to 20,000 nmol/L (Fig. 1D). These resultsimply existence of a therapeutic window of MLN4924.

Colony-forming assay revealed that MLN4924 selectivelyinhibited the clonogenicity in the BCR-ABLþ cells (KBM5,KBM5-T315I, BV173, and KU812) as well as BaF3-BCR-ABLand BaF3-T315I cells with NAE1 overexpressed and WT-p53,but not in parental BaF3 cells (Fig. 1E). Collectively, our resultssuggest that single treatment of MLN4924 exhibits equal effi-ciency against imatinib-resistant as well as -sensitive BCR-ABLþ

cells with NAE1 overexpressed and WT-p53 while sparingnormal PBMCs.

MLN4924 induces DNA damage response and G2–M-phasearrest in CML cells

Given that MLN4924 leads to abnormal accumulation ofthe CRL substrates, which subsequently induces DDR and cell-cycle arrest to inhibit cancer cell growth (12, 30), we examined theeffect of MLN4924 on cell-cycle distribution. Flow cytometryanalysis revealed that MLN4924 elicited G2–M-phase arrest inKBM5 and KBM5-T315I cells (Fig. 1F and G). MLN4924 alsoinduced DDR in KBM5 and KBM5-T315I cells, as reflected by theincrease in CDT1, gH2AX, and phospho-Chk2 (Fig. 1H). Besides,MLN4924 induced accumulation of endogenous inhibitor WEE1of G2–M-phase transition and downregulation of M-phase indi-cator phospho-H3 in CML cells (Fig. 1H).

MLN4924 induces apoptosis in imatinib-resistant CML cellsexpressing T315I-BCR-ABL with intact p53

Because of their sensitivity toMLN4924,we chose theCML cells(e.g., KBM5, KBM5-T315I, KU812, and BV173) with WT-p53 inthe subsequent experiments. We explored apoptosis-inducingability of MLN4924 in CML cells. Analysis of flow cytometryusing Annexin V/PI double staining showed that MLN4924

induced apoptosis in a dose- and time-dependent manner inthese 4 lines ofCML cells bearingWT-BCR-ABLor T315I-BCR-ABL(Fig. 2A; Supplementary Fig. S2A). Furthermore, MLN4924induced specific cleavage of PARP and caspase-3, and decreasein the proform of caspase-9 (Fig. 2B; Supplementary Fig. S2B).MLN4924, whereas, had minimal effect on BCR-ABL and phos-pho-BCR-ABL in the CML cells (Fig. 2B).

We also detected the apoptosis-related proteins by immuno-blotting analysis. The results showed that downregulation ofMcl-1, XIAP, Bid, and upregulation of Bim, and tBid, but no alterationin Bcl-2, Bcl-XL, and Survivin in the MLN4924-treated CML cells(Fig. 2C). The mRNA levels of Noxa were also increased in theMLN4924-treated CML cells (Supplementary Fig. S2C). We con-clude that disturbance of balance between the pro- and antia-poptotic proteins caused byMLN4924 treatmentmay favor onsetof apoptosis in CML cells.

Because the activation of caspase-9, -3 (Fig. 2B), and cyto-plasmic translocation of cytochrome c (Fig. 2D) were observedin MLN4924-treated cells, the effect of MLN4924 on mitochon-drial transmembrane potential (Dym)was further investigated inKBM5 and KBM5-T315I cells. The results showed that MLN4924significantly increased the proportions of CML cells with Dymloss (Fig. 2E and F). These data suggest that MLN4924 triggersintrinsic apoptosis pathway in CML cells.

Because the third-generation TKI ponatinib is active againstT315I-BCR-ABL (6), we examined whether MLN4924 was syner-gistic with ponatinib in T315I-BCR-ABLþ CML cells. The resultsindicated that the combination between MLN4924 and pona-tinib synergistically retarded the KBM5-T315I cell growth asmeasured by MTS assay (combinational index, CI

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of NAE1 were remarkably increased in CML CD34þCD38� cellsrelative to normal counterparts (Fig. 3B).

We next determined the effect of MLN4924 on the survivalof CML CD34þ cells. Flow cytometry analysis showed thatsingle treatment with MLN4924 significantly induced apoptosisof CML CD34þ cells, while imatinib alone was ineffective(Fig. 3C). Remarkably, inhibitory effect of MLN4924 on CFC/replating ability of human CML CD34þ cells was observed(Fig. 3D). Similarly, treatment with MLN4924 alone or in com-bination with imatinib but not imatinib alone led to a reducedLTC-IC derived from primary human CML CD34þ cells (Fig. 3E).Taken together, our results suggest that pharmacologic inhibitionof NAE1 by MLN4924 reduces survival and self-renewal of pri-mary human CML CD34þ cells.

MLN4924 prolongs survival of the BCR-ABL–driven CML miceand impairs maintenance of LSCs in vivo

We employed a human BCR-ABL gene–driven CML mousemodel to evaluate the in vivo effect of MLN4924 on CML (21).CMLmicewere randomized into 4 groups to be treatedwith eithervehicle, MLN4924, imatinib or their combination for 14 days(Fig. 4A). MLN4924 alone or in combination with imatinibinhibited the splenomegaly of CML mice (Supplementary Fig.S3A). Correspondingly, MLN4924 or imatinib sufficiently pro-longed the survival of CML mice, while the combinational treat-ment ofMLN4924 and imatinib elicited an enhanced extensionofsurvival in CML mice (Fig. 4B). Consistently, the populations ofGFPþ leukemia cells and myeloid leukemic cells in bone marrowand spleen were significantly reduced in the CML mice treatedwith MLN4924, and further reduced in the group treated withMLN4924 þ imatinib (Fig. 4C; Supplementary Fig. S3B).

We next measured the effect of MLN4924 on the proportionsof stem/progenitor cells in CML mice. MLN4924 alone or incombination with imatinib dramatically reduced the propor-tions of LSK, LT-HSC, and ST-HSC cells, as well as GMP andCMP in bone marrow and spleen (Fig. 4D–G; SupplementaryFig. S3C–S3J). Consistent with previous studies (21, 32), ima-tinib alone did not eliminate LSCs (Fig. 4E). We also examinedthe effect of MLN4924 on the proportions of quiescent LSKcells. A significant decrease of the subset of quiescent LSK cellsas defined by Ki67lowHoechst3342low G0-phase populationwas observed in the mice treated with MLN4924 (Supplemen-tary Fig. S3K). Meanwhile, MLN4924 treatment inhibited theglobal neddylation in splenic nucleated cells from CML mice(Supplementary Fig. S3L).

We further investigated the exact in vivo impact ofMLN4924 onthe frequency of CML LSCs in the secondary transplantation bylimiting dilution assay (Fig. 4H). The secondary recipient micereceived transplantation of the bone marrow cells isolated fromthe primary CML mice that were treated with MLN4924 alone orin combinationwith imatinib showed significantly lower engraft-ment of GFPþ cells and the frequency of CML LSCs at week 16posttransplantation (Fig. 4I and J; Supplementary Table S3).

MLN4924 induces nucleus p27kip1 accumulation in bulkleukemia cells as well as stem/progenitor cells in CML

Given that cell-cycle inhibitor p27kip1 is a substrate of CRL, andthat p27kip1 deficiency increases LSK cell population and accel-erates leukemogenesis in CML mice (33), we examined the effectof MLN4924 on the expression of p27kip1 and its E3 ligase Skp2.The results revealed thatMLN4924 treatment caused a decrease inSkp2, while an accumulation in p27kip1 protein levels, in CMLcells (Fig. 5A). Further immunoblotting analysis with nuclear andcytoplasm extractions revealed that increased accumulation ofp27kip1 protein caused by MLN4924 treatment predominantlyoccurred in nucleus but not in cytoplasm (Fig. 5B). To examinewhether downregulation of Skp2 protein by MLN4924 is due toproteasome-dependent degradation, the in vivo ubiquitinationassay was performed. MLN4924 treatment in KBM5-T315I cellssignificantly increased Skp2 ubiquitination (Fig. 5C). Takentogether, our results suggest that MLN4924 induces Skp2 degra-dation in a proteasome-dependent manner with concurrentp27kip1 accumulation in nucleus.

Flow cytometry analysis further confirmed that MLN4924treatment significantly increased protein levels of intracellularp27kip1 in the gated LSK cells from CMLmice (Fig. 5D). Increasedprotein levels of intracellular p27kip1 detected by flow cytometryanalysis were also identified in the primary human CML CD34þ

from2 of 3 patients (Fig. 5E). Furthermore, the nucleus relocationof p27kip1 was also exceptional in the MLN4924-treated KBM5and KBM5-T315I cells (Fig. 5F) and primary human CML CD34þ

cells as observed with immunofluorescence staining (Fig. 5G).To define the role of p27kip1 in MLN4924-induced apoptosis,

we knocked down p27kip1 by siRNA duplexes in KU812 cells, andthen treated these cells with MLN4924. The results showed thatsilencing p27kip1 at least partially attenuated the MLN4924-induced apoptosis in CML cells (Fig. 5H).

Collectively, these data suggest that MLN4924 leads to Skp2degradation and p27kip1 accumulation in nucleus in CML bulkleukemia cells as well as LSCs.

Figure 5.MLN4924 induces nucleus p27kip1 accumulation in bulk leukemia cells as well as LSK cells in CML. A and B, MLN4924 treatment resulted in Skp2 degradation andp27kip1 accumulation. A, CML cells were exposed to MLN4924 for 36 hours. Western blot analysis of Skp2 and p27kip1 was then performed. B, CML cells weretreated with MLN4924 (500 nmol/L) for 36 hours. The protein levels of p27kip1 in the cytoplasmic and nuclear fractions were detected by Western blot analysis.a-Tubulin andPCNA servedasmarkers of cytoplasmic andnuclear extractions, respectively.C,MLN4924 increasedSkp2ubiquitination inCML cells. KBM5-T315I cellspretreated with or without MLN4924 (MLN, 500 nmol/L) for 18 hours were exposed to MG132 (20,000 nmol/L) for another 6 hours. The cell lysate pelletsimmunoprecipitated by anti-Skp2were subjected to immunoblottingwith anti-ubiquitin.D, The CMLmicewere administratedwith or without MLN4924 for 2weeks,and the gated LSK cells (n¼ 5 each group) frombonemarrow–nucleated cellswere subjected to flowcytometry analysiswith anti-p27kip1. Representative histograms(left) and quantitative analysis (right) of median fluorescence intensity (MFI) of intracellular of p27kip1 protein levels are shown. �� , P < 0.001, Student t test.E, The primary purified CD34þ cells from CML patients (n¼ 3) were treated with MLN4924 (500 nmol/L) for 24 hours, and the intracellular of p27kip1 protein levelswere measured by flow cytometry. F and G, Subcellular localization analysis of p27kip1 by immunofluorescence staining assay. Twenty-four hours after treatingwith MLN4924 (500 nmol/L), KBM5, KBM5-T315I (F), and primary human CML CD34þ cells (G) were stained with anti-p27kip1 and corresponding Alexa Fluor488–conjugated secondary goat-anti-rabbit antibody, followed by recording under fluorescence confocal microscopy. Scale bar, 20 mm. H, Knockdown ofp27kip1weakened the effect of MLN4924-induced apoptosis in CML cells. KU812 cells were transfected with siRNA control (Mock) or siRNA duplexes against p27kip1.Forty-eight hours later, the cellswere subjected to eitherWestern blot analysis (top) or further treatmentwithMLN4924 (500nmol/L) for another 24 hours, followedby flow cytometry analysis after Annexin V-FITC/PI double staining (bottom). Results from three independent experiments are shown. � ,P

Silencing p27kip1 attenuates MLN4924-mediated decrease inLSK cells in CML mice

The fact that MLN4924 induces nuclear accumulation ofp27kip1 in CML LSCs prompted us to assess whether the repressiveeffect of MLN4924 on CML LSCs in vivowas mediated by p27kip1.We knocked down p27kip1 by lentiviral shRNA in splenic GFPþ

cells collected from the first generation of CML mice, and thentransplanted such cells into the secondary recipient C57BL/6mice sublethally irradiated. The mice were then treated withMLN4924 or vehicle for 14 days (Fig. 6A and B). As anticipated,the secondary recipient mice received with MLN4924 did notdisplay splenomegaly (Supplementary Fig. S4A). However,p27kip1-knockdown alone or p27kip1-knockdown combined withMLN4924 exhibited splenomegaly, suggesting that p27kip1-knock-down attenuated the efficacy of MLN4924 in CML mice (Supple-mentary Fig. S4A). This was confirmed by the observation thatMLN4924 significantly reduced the percentages of GFPþ andmyeloid (Mac-1þGr-1þ) cells in bone marrow, while p27kip1-knockdown efficiently rescued such effects of MLN4924 (Fig.6C and D). Of note, flow cytometry analysis revealed that thepercentages of LSKs, LT-HSCs, and ST-HSCs, as well as GMP andCMP cells in bone marrow were also decreased by MLN4924treatment, which was obviously reversed by p27kip1-knockdown(Fig. 6E–I). Similar results were obtained in the splenic cells ofCML mice (Supplementary Fig. S4B–S4H). Of importance, therecipient mice received injection of p27kip1-knockdown CML cellsin combination with MLN4924 treatment showed a significantlydecreased survival when compared with the CML mice treatedwith MLN4924 alone (Fig. 6J), further confirming that p27kip1-knockdown reversed the in vivo suppressive effect of MLN4924 inCML. Collectively, these data suggest that MLN4924 inhibits themaintenance of CML LSK cells at least partially through p27kip1

protein accumulation in nucleus.

MLN4924 inhibits the engraftment capacity of human CMLCD34þ cells in NSI mice

To evaluate the impact of MLN4924 on the long-term in vivorepopulating potential, human CML CD34þ cells were trans-planted into sublethally irradiated NOD-scid-IL2Rg�/� (NSI)mice (Fig. 7A). MLN4924 treatment reduced the percentages ofhuman CD45þ cells in bone marrow and spleen (Fig. 7B–D). Inaddition, the proportions of human myeloid cells (CD33þ andCD14þ) and lymphocytes (B- and T-cells) were also reduced(Fig. 7E–G). These results indicated that MLN4924 suppressedthe long-term engraftment capacity of human CML CD34þ cellsin vivo.

DiscussionResistance to imatinib due to T315I mutation and LSCs

remains a challenge in patients with CML. In this study, wediscovered that targeting neddylation pathway by MLN4924led to cell-cycle arrest at G2–M-phase, and apoptosis of CMLcells harboring either WT-BCR-ABL or T315I-BCR-ABL in a WT-p53–dependent manner. Furthermore, MLN4924 inhibited thesurvival and self-renewal of primary human CML CD34þ cells.MLN4924 significantly prolonged the survival of CMLmice andreduced LSK cells from CML mice at least partially throughp27kip1 protein accumulation in nucleus. In addition,MLN4924 inhibited the engraftment capacity of human CMLCD34þ cells in NSI mice.

In our study, we found that MLN4924 efficiently blockedcullin1 neddylation, which confers stabilization of variousproteins including phospho-IkBa, p27kip1, and activation ofthe DDR effector Chk2 in WT- and T315I-BCR-ABLþ cells. Ourresults showed that MLN4924 treatment in CML cells inducedG2–M-phase arrest, which is consistent with the observations inpancreatic cancer, gastric cancer cells, as well as acute leukemiacells (30, 34). Distinctly, MLN4924 elicits S-phase arrest andDNA rereplication in other type of tumor cells (e.g., colorectalcarcinoma cells), which may be explained by accumulatedCDT1 that is required for loading of the DNA replicationhelicase complex (MCM2-7) onto chromatin at G1-phase ofthe cell cycle (35). Given the wide-range of substrates of CRLand genetic backgrounds of tumors, it is likely that the eventualoutcome is dependent on context comprised of DNA damage,DDR extent, susceptibility, and cell type etc. The MLN4924-treated WT- and T315I-BCR-ABLþ cells died by apoptosis, asreflected by cleavage of PARP and caspase-3. This can beexplained by downregulation of Mcl-1, XIAP, Bid, and upre-gulation of Bim and tBid, which may favor onset of apoptosisin these MLN4924-treated CML cells.

Previous studies demonstrated that MLN4924 might induceapoptosis through p53-dependent or p53-independent mannerin different cancer cells (35–37). The absence of p53 or p21sensitized the cells to the MLN4924-mediated cellular senescencein HCT-116 cells (35). In contrast, our data reveal that p53-nullK562 and KCL-22 cells are insensitive to MLN4924 when com-pared with KBM5, KBM5-T315I, KU812, and BV173 cells, whichharbor WT-p53. Ectopic restoration of WT-p53 sensitized K562and KCL-22 cells to MLN4924. Conversely, knockdown of p53attenuated the effect of MLN4924-induced apoptosis in the p53-intact CML cells (e.g., KBM5-T315I). Given the instrumentalfunctions of p53 in apoptosis, cell-cycle arrest, and DDR (38),it is plausible that MLN4924 induces apoptosis in CML cells in ap53-dependent manner. This effect of MLN4924 may have par-ticular advantage because rare mutations in p53 are observed inpatients with CML (39).

LSCs are believed a source of therapy failure in CML. In thisstudy, we demonstrated that NAE1 was overexpressed in CMLstem/progenitor cells. MLN4924 induced apoptosis and inhib-ited the self-renewal of primary CML CD34þ cells. MLN4924also effectively reduced the survival, frequency, and self-renew-al capacity of LSK cells in BCR-ABL–driven CML mice. Ourresults are in agreement with previous report that MLN4924 istoxic to AML and MDS stem/progenitor cells without toxicity tonormal stem/progenitor cells (31). Therefore, MLN4924 mightbe a promising strategy and the relevant clinical trial in CML iswarranted. Actually, ongoing phase I clinical trials of single-agent pevonedistat showed that the MTD can reach 83 mg/m2,with Cmax, maximum plasma concentration, of 2.0 mmol/L,which is much higher than the IC50 values in CML cells in ourstudy (40). Seventeen percent (4/23) of the overall completeresponse, partial response rate, and modest clinical activity inpatients with acute myeloid leukemia and myelodysplasticsyndromes at the MTD or below were observed (41). Theclinical efficacy apparently correlated with the accumulationof CRL substrates CDT1 and NRF2 in biopsies of these treatedpatients. Despite adverse effects such as hepatotoxicity, fever,and thrombocytopenia, pevonedistat was generally well toler-ated in these patients (42). Encouragingly, more clinical trialsof pevonedistat in combinational regimens with azacitidine,

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Figure 6.

Silencing p27kip1 attenuates the MLN4924-mediated elimination of LSK cells in CML mice. A, The schema of experimental procedure. B, Western blot analysis ofp27kip1 in the CML mouse spleen cells after lentiviral transduction with p27kip1 shRNA. C–I, After intravenously transplanting with the donor CML mouse splenicnucleated cells that were transduced with p27kip1 shRNA, the recipient mice were treated with MLN4924 or vehicle (Control) for 2 weeks. The subpopulationsof bulk tumor cells including GFPþ (C) and myeloid (D) leukemia cells, leukemia stem cells including LSK (E), LT-HSC (F), ST-HSC (G), and leukemia progenitor cells(H and I) in bonemarrowwere then analyzed by flow cytometry. Control (n¼ 10); shp27 (n¼ 10); MLN4924 (n¼ 8); shp27þMLN4924 (n¼ 9). � ,P

Figure 7.

MLN4924 inhibits the engraftment capacity of human CML CD34þ cells in NSI mice. A, The schema of experimental procedure. B–D,MLN4924 treatment decreasedthe percentage of human CD45þ cells. Representative flow cytometry histograms of engraftment human CD45þ cells in NSI mice (B), quantitative analysis ofengraftment human cells in bone marrow (C) and spleen (D) are shown. Each datum point represents one mouse. Control (n¼ 7); MLN4924 (n¼ 10). ��, P < 0.001,Student t test. E–G, MLN4924 inhibited diverse human CML myeloid cell lineages in NSI mice. Representative flow cytometry plots of human CD45 and CD33expression inmicewith cells from one of the twoCML patients (E). Control (n¼ 7); MLN4924 (n¼ 10). The percentages of engraftment of humanCD34, CD33, CD11B,CD14, CD19, and CD3 in bonemarrow (F) and spleen (G) were decreased after MLN4924 treatment. Control (n¼ 7); MLN4924 (n¼ 10). �� , P < 0.001, Student t test.


Liu et al.




doceraxel, gemcitabine, paclitaxel, and cisplatin in patientswith solid tumors are going on (40).

Little is known about the mechanism of NAE1 inhibitor killingCSCs. Our results revealed that MLN4924 induced a prominentaccumulation of p27kip1 in nucleus of primary human CMLCD34þ cells. The increased nucleus p27kip1 may in turn impairmaintenance and self-renewal of LSCs. Zhang and colleagues havedemonstrated that the mice of p27kip1 deficiency exhibits anaccelerated leukemogenesis and a marked increase in total LSK,LT-HSC, and ST-HSC cells when induced by retroviral BCR-ABLtransduction in comparison withWT-p27kip1 CMLmice (33). It isreported that BCR-ABL can elicit relocation of p27kip1marked by areduced nucleus p27kip1 by the activated AKT in the human CMLCD34þ cells, which facilitates cell cycling and expansion ofCD34þ pool (43). Consistently, reduced nucleus p27kip1 causedby CaMKIIg-dependent phosphorylation of p27kip1 at T187 canreactivate dormant LSCs in CML (44). In this sense, the increasednucleus p27kip1 by MLN4924 may favor restriction of LSCs.

Of note, neddylation inhibition by MLN4924 may disruptdegradation of multiple proteins (e.g., c-myc). A previous studyrevealed that MLN4924 inhibited cullin1 neddylation, therebystabilizing E3 ligase Fbxw7 and degradation of its substrate c-myc(45). Considering the critical role of c-myc in the maintenance ofstemness of LSCs (46), the possibility of MLN4924-mediated c-myc degradation in the elimination of LSCs cannot be excluded.

In conclusions, our findings offer a preclinical proof of conceptfor targeting protein neddylation as a novel therapeutic strategy tooverride mutational and LSC-derived imatinib resistance in CML.This study provides a rationale for clinical trials of pevonedistat asa single agent and in combination with imatinib in refractorypatients with CML. However, our results suggest that MLN4924may also cause reduced lineages of B- and T-cells in the long-term

engraftment experiments, implying that intensively monitoringthe immune systemmay be needed in patients received long-termadministration with MLN4924.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: C. Liu, Y. Jin, J. PanDevelopment of methodology: C. Liu, J. Zhou, Y. JinAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): C. Liu, D. Nie, J. Li, X. Du, Y. Lu, Y. Li, J. PanAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): C. Liu, J. Zhou, J. PanWriting, review, and/or revision of the manuscript: C. Liu, Y. Jin, J. PanAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases):Study supervision: Y. Jin, J. Pan

AcknowledgmentsThis study was supported by grants from National Natural Science Funds

(nos. U1301226 and 81373434 to J. Pan; nos. 81473247 and 81673451 toY. Jin). The Natural Science Funds of Guangdong Province for DistinguishedYoung Scholars (grant no. 2016A030306036 to Y. Jin); the Research Foun-dation of Education Bureau of Guangdong Province, China (Grant cxzd1103to J. Pan), and Natural Science Foundation of Guangdong province (grant2015A030312014 to J. Pan).

The costs of publication of this articlewere defrayed inpart by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received June 16, 2017; revised October 27, 2017; accepted January 5, 2018;published OnlineFirst January 10, 2018.

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Liu et al.




2018;78:1522-1536. Published OnlineFirst January 10, 2018.Cancer Res Chang Liu, Danian Nie, Juan Li, et al. T315I-BCR-ABL Leukemia Cells and Leukemia Stem CellsAntitumor Effects of Blocking Protein Neddylation in

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