Oncogenes and Tumor Suppressors

LIN28B Activation by PRL-3 PromotesLeukemogenesis and a Stem Cell–likeTranscriptional Program in AMLJianbiao Zhou1,2, Zit-Liang Chan1, Chonglei Bi1, Xiao Lu1, Phyllis S.Y. Chong1,Jing-Yuan Chooi2, Lip-Lee Cheong1, Shaw-Cheng Liu1, Ying Qing Ching1,Yafeng Zhou1, Motomi Osato1, Tuan Zea Tan1, Chin Hin Ng3, Siok-Bian Ng1,4,6,Shi Wang7, Qi Zeng5, and Wee-Joo Chng1,2,3

Abstract

PRL-3 (PTP4A3), a metastasis-associated phosphatase, is alsoupregulated in patients with acute myeloid leukemia (AML)and is associated with poor prognosis, but the underlyingmolecular mechanism is unknown. Here, constitutive expres-sion of PRL-3 in human AML cells sustains leukemogenesisin vitro and in vivo. Furthermore, PRL-3 phosphatase activitydependently upregulates LIN28B, a stem cell reprogrammingfactor, which in turn represses the let-7 mRNA family, inducinga stem cell–like transcriptional program. Notably, elevated

levels of LIN28B protein independently associate with worsesurvival in AML patients. Thus, these results establish a novelsignaling axis involving PRL-3/LIN28B/let-7, which confersstem cell–like properties to leukemia cells that is importantfor leukemogenesis.

Implications: The current study offers a rationale for targetingPRL-3 as a therapeutic approach for a subset of AML patients withpoor prognosis. Mol Cancer Res; 15(3); 294–303. �2016 AACR.

IntroductionThe 5-year overall survival of acute myeloid leukemia (AML)

remains about 30% to 40% and significantly poorer in patientsolder than 65 years (1, 2). Leukemia stem cells (LSC), also calledleukemia-initiating cells, a distinct subpopulation of AML cellswith self-renewal capacity, initiate and sustain thedevelopment ofthe bulk leukemic population (3–5). LSC has been considered asthe root source for the relapse and treatment failure of AML (6, 7).The identification of the molecular mechanisms underlying thetransformation of LSCs offers a potential opportunity to eradicateleukemia (8).

Protein tyrosine phosphatase of regenerating liver 3 (PRL-3,encoded by PTP4A3 gene) is a member of the VH1-like protein

tyrosine phosphatase (PTP) family with dual specificity (9).Elevated expression of PRL-3 was detected in a variety ofmetastatic and primary tumor tissues (10). PRL-3 activates thePI3K/AKT pathway (11) and Src-ERK1/2 pathways (12), thuspromoting epithelial–mesenchymal transition (EMT) andtumor angiogenesis in solid tumors.

PRL-3 protein is detected in approximately 50% and 90% ofbone marrow samples from patients with AML and multiplemyeloma, respectively (13–16). Importantly, several studies haveindependently confirmed that the high expression of PRL-3 isassociated with poor survival in AML (15–17). We previouslydemonstrated that LEO1, a component of RNA polymerase II–associated factor (PAF) complex, was induced by PRL-3 in AML(18). PAF complex plays an essential role in MLL-rearrangedleukemia (19). These findings collectively indicate that dysregu-lated PRL-3 is involved in the development of AML. However, themolecular mechanisms underlying the role of PRL-3 in AML arenot well understood.

Here, we report that LIN28B is a key oncogenic target in PRL-3–positive AML. Increased LIN28B in turn downregulates let-7mRNA family. The PRL-3/LIN28B/let-7 axis takes part in thetransformation of LSCs and leukemogenesis of AML. Antagoniz-ing PRL-3 may be of clinical benefit in the treatment of AML anderadication of LSCs.

Materials and MethodsCell lines and cell culture

AML cell linesMOLM-14, HL60, TF-1, and TF-1a were grown inRPMI1640 (Invitrogen) supplemented with 10% FBS (JRH Bios-ciences Inc.) at a density of 2–10 � 105 cells/ml in a humidincubator with 5% CO2 at 37�C. Additional human IL3 (Pepro-Tech) was added into growthmedium at 5 ng/mL to support TF-1

1Cancer Science Institute of Singapore, National University of Singapore, Centrefor Translational Medicine, Singapore, Republic of Singapore. 2Department ofMedicine, Yong Loo Lin School of Medicine, National University of Singapore,Singapore, Republic of Singapore. 3Department of Haematology-Oncology,National University Cancer Institute, NUHS, Singapore, Republic of Singapore.4Department of Pathology, National University Hospital, Singapore, Republic ofSingapore. 5Institute ofMolecular andCell Biology, A�STAR (Agency for Science,Technology and Research), Singapore, Republic of Singapore. 6Department ofPathology, Yong Loo Lin School of Medicine, National University of Singapore,Singapore. 7Department of Pathology, National University Hospital, NationalUniversity Health System, Singapore.

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

Corresponding Author: Wee-Joo Chng, Department of Haematology-Oncolo-gy, National University Cancer Institute, NUHS, 1E, Kent Ridge Road, NUHSTower Block, Level 10, Singapore 119228. Phone: 656-772-4612; Fax: 656-777-5545; E-mail: mdccwj@nus.edu.sg

doi: 10.1158/1541-7786.MCR-16-0275-T

�2016 American Association for Cancer Research.

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cell growth. Bone marrow blast cells (>90%) from newly diag-nosed AML patients were obtained at National University Hos-pital in Singapore with informed consent. This study wasapproved by the Institutional Review Board of National Univer-sity of Singapore.

Cell viability assaysLeukemic cells were seeded in 96-well culture plates at a density

of 2 � 104 viable cells/100 mL/well in triplicates. CellTiter-GloLuminescent Cell Viability Assay (CTG assay, Promega) was usedto determine the cell growth and viability as described previously(20). Each experiment was in triplicate.

Affymetrix microarray and gene set enrichment analysesTF1-pEGFP and TF1-hPRL-3 cells were harvested, and total

RNA was extracted using the RNeasy Mini Kit, according tothe manufacturer's instructions (Qiagen). Gene expressionprofiling was performed using Affymetrix U133 Plus 2.0 genechip (Affymetrix) according to the manufacturer's protocol.The scanned data were processed using MicroArray Suite ver-sion 5.0 (Affymetrix). The detailed microarray analysis andsubsequent gene set enrichment analysis (GSEA) are describedin Supplementary Materials and Methods. Expression data(GSE64872) were deposited in the Gene Expression Omnibus.

Western blot analysisPreparation of the cell lysate and immunoblotting were per-

formed using standard techniques as described previously (21),except for PRL-3 analysis, and 16% gel was used. Anti-PRL3antibody was clone 318 (1:1,000) as reported previously (22).Anti-LIN28B antibody was purchased from Santa Cruz Biotech-nology (Cat. #sc-130802). Anti-b-actin (1:3,000, Sigma-Aldrich)was used as loading control.

Lentivirus LIN28B-shRNA infectionScramble shRNA control and two human LIN28B specifically

pLKO.1 lentiviral shRNAswere purchased fromOpenBiosystems.TF1-hPRL-3 or TF-1a cells (3�106)weremixedwith concentratedviral supernatant and 8 mg/mL of polybrene (Millipore) andcentrifuged at 2,500 rpm for 90minutes at 30�C. After additionalincubation at 37�C for 4 hours, themediumwas changed to freshcomplete medium. Two days later, cells were submitted forprotein extraction, followed by Western blot analysis of LIN28Band PRL-3.

mRNA mimics and inhibitorsPredesigned miRCURY LNA microRNA Mimics to let-7a

(#470408), let-7b (#470775), mRNA mimic negative control(#479903), specific miRCURY LNA microRNA Inhibitors to let-7a (#4101777), let-7b (#4100945), and control inhibitor(#199006) were purchased from Exiqon. Briefly, cells weretransfected with 30 nmol/L mRNA mimics or mRNA inhibitorsor their negative controls by electroporation with Neon Trans-fection System (Thermo Fisher Scientific) and were maintainedin a humidified incubator at 37�C in 5%CO2. Cell proliferationassays were carried out at days 1, 2, 4, and 6 after transfection.For colony-forming assays, colony numbers were counted atday 7.

Colony formation assay and serial replating assayTF1-pEGFP and TF1-hPRL-3 cells were washed twice with 1�

PBS, and cell viability was determined by Typan blue exclusionmethod. After that, about 20,000 vial cells from each cell linewere plated in MethoCult medium without cytokines (H4230,STEMCELL Technologies) in 6-well plates and cultured for 7 daysat 37�C in a 5%CO2 incubator. Colonies consisting of more than50 cells were counted under an invertedmicroscope. A total offiverandom 4 � 10 magnification fields were selected, and averagenumber of colonies of each sample was calculated. The experi-ments were duplicated. In serial replating assay, the colonynumberwas counted, and then subjected to replating every 7days.

Limited serial dilution and bone marrow transplantationThe protocol was reviewed and approved by Institutional

Animal Care and Use Committee in compliance with the Guide-lines on the Care and Use of Animals for Scientific Purpose.Detailed information is provided in Supplementary Materialsand Methods.

Statistical analysisStudent t test (two-tailed paired) was used for examining the

statistical difference for in vitro cell line experiments, such as cellviability assay and colony-forming assay; P values of <0.05 wereconsidered to be significant. Data were presented as mean � SD.Multivariate survival analyses were conducted using R version3.1.2 and survival package version 2.37-7. Protein expressionof LIN28B and clinicopathologic parameters were first discretizedinto binary categories prior to Cox regression analysis. Kaplan–Meier analyses were conducted using GraphPad Prism version5.04 (GraphPad Software). Statistical significance of the Kaplan–Meier analysis was calculated by log-rank test (P < 0.05).

ResultsPRL-3 promotes AML maintenance and progression in vivo

As reported inprevious studies (15, 18),we established apair ofstable, isogenic cell lines, TF1-pEGFP and TF1-hPRL3, by trans-fecting pEGFP (vector control) and pEGFP-hPRL-3 vectors intoTF-1 cells, respectively, and showed that PRL-3 promoted cyto-kine-independent growth of AML cells. Here, we further deter-mined the in vivo function of PRL-3 on leukemogenesis by usingthis pair of TF1 cells in a bone marrow transplantation xenograftassay. NOD/SCID mice inoculated with TF1-hPRL3 cells via tailvein injection developed leukemia-like symptoms with enlargedspleen and liver and succumbed to the diseasewithin 3months. Incontrast, mice implanted with TF1-pEGFP did not display anysign of sickness, and all survived till the end of the experimentalperiod (Fig. 1A). We next performed serial bone marrow trans-plantation experiments. Bone marrow cells harvested from theprimary recipients were transplanted into secondary recipients.Compared with primary leukemia, there was a significant shortersurvival in the secondary recipients (Fig. 1A, P < 0.01). We thenperformed tertiary transplantation. Tertiary recipients succumbedto the disease even earlier than the secondary recipients (Fig. 1A,P < 0.001). Histologic analysis revealed that leukemic cells exten-sively infiltrated into the liver and spleen in diseased mice(Fig. 1B). Moreover, the expressions of PRL-3 and LIN28B weresignificantly elevated in engrafted mouse bone marrow cellsharvested from secondary and tertiary recipients, while their

Activation of LIN28B by PRL-3 Promotes Leukemogenesis

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expression of let-7 was significantly lower as compared with theprimary generation of mouse bone marrow cells (Fig. 1C). Thesedata bespeak a good correlation between the expressions of thesethree genes and aggressiveness of the disease.

Together, these studies demonstrate that PRL-3 promotes AMLmaintenance and progress in an animal model.

PRL-3 induces a "stemness" transcriptional programin AML cells

To gain insight into the role of PRL-3 in leukemogenesis,we conducted an mRNA expression microarray analysis of TF1-pEGFP and TF1-hPRL3 cell lines. Using 2-fold as the cut-off level,89 genes showed increased expression and24genes haddecreased

expression in TF1-hPRL3 cells compared with TF1-pEGFP cells(Supplementary Table S1). The top 10 up- and downregulatedgenes were shown in Supplementary Table S2. It is noteworthythat PRL-3 induced expression of many LSC or cancer stem cell–related genes, including LIN28B, KIT, IGF2BP1 (IGF2 mRNAbinding protein 1), LAPTM4B, FYN, EIF4E3, Caveolin 1, amongothers, while reducing the expression of cell surface markers ofmore differentiated cells, such as CD36 and CD38 (Supplemen-tary Table S1). LIN28B ranked as the third most elevated geneafter PTP4A3 (PRL-3) and MT1G in TF-hPRL3 cells (Supplemen-tary Table S2). It has been well established that LIN28B is a stemcell reprogramming factor and a marker for cancer stem cell (23).qRT-PCR analysis validated the changes of selected genes, includ-ing LIN28B, LAPTM4B, IGF2BP1, FYN, RAB27B, KIT, CD36, andCD38 (Supplementary Fig. S1), and increased LIN28B in TF1-hPRL3 cells was confirmed by additional RT-PCR and Westernblot assays (Fig. 2A). GSEA of this microarray result revealed asignificant enrichment of genes identified in a previously definedgene expression signature of leukemia stem cell (LSC SIGNA-TURE_SAITO, P < 0.001) and hematopoietic stem cell (HSCSIGNATURE_EPPERT, P < 0.001; Fig. 2B). This differentiallyexpressed gene list was uploaded to the MetaCore algorithm forgene cluster analysis, and the result showed LIN28B to act as afocal point in the regulation of many genes such as miRNA let-7family (Supplementary Fig. S2).

Notably, the let-7 miRNA family members play a "gatekeeper"role of embryonic stem cell pluripotency and act as tumorsuppressors in a wide range of human malignancies (24). LIN28was identified to selectively repress the expressionof let-7miRNAsor directly bindmRNA (23, 25, 26). Therefore, we quantitated thelevel of let-7 miRNAs in TF1-pEGFP and TF1-hPRL3 cells. Withthe exception of let-7c, the other let-7miRNAmembers, includinglet-7a, let-7b, let-7d, let-7f, let-7g, let-7i, and miR-98, were allsignificantly repressed in TF1-hPRL3 cells relative to TF1-pEGFPcells (Fig. 2C). To rule out the effect of cell type specificity, weoverexpressed PRL-3 into other AML cell line, HL60, and mea-sured the expression of LIN28B and let-7a miRNA. Similarly, weobserved that HL60-hPRL3 cells had significant higher expressionof PRL-3 and LIN28B, but lower let-7a as compared with HL60-pEGFP cells (Fig. 2D). In summary, our data indicate that PRL-3enforces a stem-like transcription program in AML cells, driven bychanges of LIN28B and let-7 miRNAs.

PRL-3–expressing AML cells have leukemia-initiatingcapabilities

It has been well established that leukemia-initiating cells areenriched in CD34þCD38� subpopulation of AML cells. Lineageanalysis demonstrated that the percentage of CD34þCD38� cellswas 8-fold higher in TF1-hPRL3 cell lines than in TF1-pEGFP celllines (Fig. 3A; Supplementary Fig. S3A).We also found that PRL-3was more highly expressed on CD34þCD38� AML thanCD34þCD38þ AML cells, as well as CD34þCD38� cells fromhealthy controls, suggesting PRL-3 was upregulated in AML-ini-tiating cells (Fig. 3B; Supplementary Fig. S3B and S3C). To assessthe functional involvement of PRL-3 in leukemia-initiatingcells, we used serial replating assay to assess another intrinsiccharacteristic of leukemia-initiating cells, self-renewal capacity, inTF1-hPRL3 cells. As expected, TF1-hPRL3 cells could be seriallyreplated in basic methylcellulose without additional cytokines(Fig. 3C), whereas no colonies could be observed in TF1-pEGFPcells. To exclude possible artefacts accompanied by clone

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PRL-3 promotes AML maintenance and progression in vivo. A, Survival analysisof immunodeficient mice transplanted with TF1-pEGFP, TF1-hPRL3 cells andsecondary and tertiary transplantation from primary recipient mice ofTF1-hPRL3. Each group consisted of 10 mice. Survival curves were constructedaccording to the Kaplan–Meier method. B, Representative images of H&Estaining of spleen and liver tissues harvested from TF1-pEGFP mice andTF1-hPRL3 mice. C, qRT-PCR analysis of PRL-3, LIN28B, and let-7a expression inthe bone marrow cells of recipient mice. Error bars indicate the mean � SDof 3 mice per group. � , P < 0.05; �� , P < 0.01.

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selection, we established another pair of isogenic TF1-FUGW andTF1-FUGW-PRL3 after transduced TF-1 cells with either FUGWcontrol lentivirus or FUGW-PRL3 lentivirus and selection. Inaccordance with the previous report, TF1-FUGW-PRL3 cellsgained cytokine-independent growth advantage, whereas TF1-FUGW cells failed to do (Supplementary Fig. S4). We nextassessed the replating capacity of this pair of isogenic cells.In agreement with the results from TF1-pEGFP and TF1-hPRL3,TF1-FUGW-PRL3 cells could be serially replated, whereas TF1-FUGW control cells could not form colonies (Supplementary Fig.S4). To address the question of whether TF1-hPRL3 cellsmaintainleukemia-initiating cell properties in vivo, we conducted limitingdilution transplantation and serial BMT assays. The ability of TF1-hPRL3 cells to propagate leukemia in immunodeficient mice wasevaluated using a 5-time cell dilution (50,000, 10,000, 2,000, and400 cells). Leukemic symptoms development characterized byhepatosplenomegaly was observed in all mice injected with50,000 and 10,000 cells (5mice/group), and 4 of 5mice receiving2,000 cells (Supplementary Table S3; Supplementary Fig. S5A).

Consistently, we also observed significantly higher expression ofPRL-3 and LIN28B and decreased expression of let-7a miRNA inbonemarrow cells harvested from diseasedmice (SupplementaryFig. S5B). In contrast, none of the mice (n¼ 5) transplanted with400 cells developed disease up to 6 months postinoculation(Supplementary Table S3; Supplementary Fig. S5A). So the esti-mated frequency of leukemia-initiating cell in TF1-hPRL3 cellscould be 1 in 1,771 [95% confidence interval (CI), 712–4,410)cells according to the analysis with ELDA software. In contrast,mice (n¼ 5) receiving 50,000 TF1-pEGFP cells were all healthy atthe end of experiments (Supplementary Table S3). Collectively,these results demonstrate a functional role for PRL-3 on trans-formation of leukemia-initiating cells in vitro and in vivo.

LIN28B is a key downstream target of PRL-3Through our microarray study, LIN28B is one of the most

significantly upregulated gene in TF1-hPRL3 compared withTF1-pEGFP. The C(X)5R motif within the P-loop of the PRL-3protein possess the PTP enzymatic activity. To examine the

Figure 2.

Gene expression changes driven by PRL-3 indicate acquisition of "stemness" properties in AML cells. A, Validation of increased expression of LIN28B on mRNAand protein levels by RT-PCR and Western blot (including densitometric quantification), respectively. M, 100 bp DNA ladder; lane 1, TF1-pEGFP; lane 2,TF1-hPRL3. B, GSEA analysis revealed that the TF1-hPRL3 cell expression profile significantly enriched previously reported LSC and HSC signatures. ES,enrichment score; NES, normalized enrichment score; FDR: False Discovery Rate. C, qRT-PCR analysis of the human let-7miRNA family in TF1-pEGFP and TF1-hPRL3cells. D, qRT-PCR analysis of PRL-3, LIN28B, and let-7a expression in HL60 AML cells transfected with either pEGFP or pEGFP-hPRL3 plasmid. Error barsindicate the mean � SD of three independent experiments. �� , P < 0.01.

Activation of LIN28B by PRL-3 Promotes Leukemogenesis

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importance of PRL-3 phosphatase activity in the regulation ofLIN28B, we transfected a catalytic domain mutant of PRL-3(C104S) into TF-1 cells and checked LIN28B protein level. West-ern blotting analysis showed that the substitution of C104 withserine effectively diminished thePRL-3–mediatedupregulationofLIN28, suggesting a phosphatase activity–dependent mechanismof regulation of LIN28B (Supplementary Fig. S6A). To verify therelevance of LIN28B overexpression in clinical samples, we exam-ined the expression of PRL-3 and LIN28B in CD34þCD38� cellsisolated from 28 primary AML samples using quantitative RT-PCR. As shown in Fig. 4A, there was a significant positive corre-lation between the expression levels of PRL-3 and LIN28B (Pear-son r¼ 0.51; two-tailed P¼ 0.007). Furthermore, as LIN28B playsan important role in repressing miRNAs and cellular reprogram-ming (27),we therefore focusedon the biological significance andregulatory mechanisms of LIN28B in the transformation of LSCsinduced by PRL-3. We used two independent LIN28B-shRNAs toselectively knockdown LIN28B expression in TF1-hPRL3 cells,and then performed serial replating assays. As shown in Fig. 4B,significant suppression of LIN28B protein was achieved by bothshRNAs. The knockdown efficacy of shRNA2 was higher thanshRNA1. Scrambled shRNA–treated TF1-hPRL3 cells could bereplated and the numbers of the colonies increased with eachround, whereas the colony number of LIN28B-shRNAs trans-duced cells was markedly diminished in each round of plating(Fig. 4C). Importantly, the significant reduction in the number ofcolonies correlated with the level of suppression of LIN28 expres-sion (Fig. 4B), indicating theon-target effect fromspecific shRNAs.We used lentiviral transduction method to specifically knock-down PRL-3 in MOLM-14 cells and then performed serial replat-

ing assays. Next, stable shRNA-mediated knockdown led to stablereductions in PRL-3 expression by 80%(Supplementary Fig. S6B).These cells and cells transduced with scramble shRNA were thenplated in methylcellulose for CFU in vitro assays. We observedsignificant reduced number of colonies in PRL-3 shRNA-expres-sing cells than scramble shRNA-expressing cells (SupplementaryFig. S6C). Collectively, these observations imply that inhibition ofPRL-3 and LIN28B effectively diminishes the replating capacity ofhuman AML cells.

Previously, a subclone of TF-1 cell line, TF-1a, was generated.TF-1a can proliferate continuously without cytokines anddeveloptumor in nude mice, in contrast to its parental line (28). Immu-nophenotypically, TF-1a cells are more immature and primitive(CD34þ/CD38�) than the parental TF-1 cells (CD34þ/CD38þ;ref. 28). TF-1a cells and TF1-hPRL3 cells share similarities in thesefeatures, which are indicators of self-renewal and uncontrolledproliferative capacity. In accordance with results obtained fromTF1-hPRL3 cells, high expression of PRL-3 and LIN28B proteinwas found in TF-1a cells compared with TF-1 cells (Fig. 4D). It isworth pointing out that the expression of PRL-3 and LIN28B inTF1-hPRL3 cells was comparable with their endogenous levels inTF-1a cells (Fig. 4D), suggesting our observations were not arti-ficially produced. The different levels of PRL-3 and LIN28Bproteins between TF-1a and TF-1 might be the underlying causefor the abovementioned different characteristics between thesetwo cell lines. To exclude any possible artifacts caused by over-expression or nonspecific effect of the shRNA approach, we nextconducted a loss-of-function together with a rescue experiment inthe TF-1a cell line expressing endogenously high level of PRL-3and LIN28B. TF-1a cells transduced with PRL-3-shRNAs or

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PRL-3 is responsible for thetransformation of leukemia-initiatingcells. A, FACS analysis ofCD34þCD38� population in TF1-pEGFP and TF1-hPRL3 cell lines.B, qRT-PCR analysis of PRL-3expression in CD34þCD38�,CD34þCD38þ fractions isolated fromAML patient bone marrows (n ¼ 6,mean � SD) and healthy control bonemarrow cells (n ¼ 6, mean � SD). C,Serial replating assay using TF1-hPRL3cells. Bar figures represent threeseparate experiments (mean � SD).� , P < 0.05; �� , P < 0.01.

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LIN28B plays a pivotal role in PRL-3–enhanced LSC phenotype. A, The correlation of LIN28B and PRL-3 expression in CD34þCD28� primary bone marrowcells from 28 patients with AML at diagnosis. B, Immunoblot and densitometric analyses showed that LIN28B-specific shRNAs downregulated LIN28Bprotein expression in TF1-hPRL3 cells. C, Quantification of colonies of indicated cells over three rounds of replating in methylcellulose medium (n ¼ 3, mean �SD). � , P < 0.05; �� , P < 0.01. D, Comparison of PRL-3 and LIN28B proteins in TF-1, TF-1a, and TF1-hPRL3 cell lines. E, Western blot and densitometricanalyses of PRL-3 and LIN28B protein levels in TF1-a cells receiving different treatments as indicated. OE, overexpression. F, Quantification of colonies ofindicated cell lines over three rounds of replating (n ¼ 3, mean � SD). The colony number of LIN28B overexpression was significantly higher thanthose of LIN28B-shRNA and PRL-3 shRNA (P < 0.05).

Activation of LIN28B by PRL-3 Promotes Leukemogenesis

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LIN28B-shRNAs produced significantly less number of coloniesin the serial replating assay, while replating activity of PRL-3-shRNA transduced cells was restored to near normal level by theoverexpression of LIN28B (Fig. 4E and F). In conclusion, thesedata support that LIN28B is essential for PRL-3–mediated replat-ing capacity of leukemic cells.

Repression of let-7 miRNAs is required for the PRL-3/LIN28B-promoted leukemogenesis

The RNA-binding protein, LIN28B, functions to repress let-7miRNAs via blocking the microprocessor complex (29). As it iswell established that let-7 is an important tumor suppressor gene,which inhibits numerous oncogenes, such as RAS, c-MYC, NFkB,and HMGA (30, 31), we decided to examine whether the repres-sion of let-7 miRNAs is required for PRL-3/LIN28B-inducedleukemogenesis. Ectopic expression of let-7a and let-7b ledto decreased cell colony-forming capacity of TF1-hPRL3 cells(Fig. 5A, P < 0.05) and proliferation (Fig. 5B, P < 0.01). Concor-dantly, antagomiRs to let-7a and let-7b significantly increasedcolony-forming capacity of TF1-hPRL3 cells (Fig. 5C, P < 0.05)and cell proliferation (Fig. 5D, P < 0.01). To further validate theimportant role of let-7 miRNAs, we repeated the same experi-ments by replacing TF1-hPRL3 cell line with MOLM-14, expres-

sing high level of endogenous PRL-3 and LIN28B protein.Indeed, similar results were observed for MOLM-14 cells (Sup-plementary Fig. S7). Taken together, these evidences demonstratethat let-7 plays an essential role in the PRL-3/LIN28B-mediatedleukemogenesis.

LIN28Boverexpression independently predicts poor survival ofpatients with AML

To examine the clinical importance of LIN28B in AML, weused IHC to determine LIN28B protein expression in bonemarrow samples from 159 patients with AML at diagnosis.LIN28B staining was calculated as the number of LIN28B-positive blast cells divided by the total number of blast cells inat least 10 fields and then expressed as a percentage. Scoring ofthe tissue microarray was completed independently by twoclinically qualified pathologists who were blinded to the clinicalinformation. We observed that LIN28B mainly localizes innuclear of leukemia blast cells. We classified the LIN28B stainingpattern as follows: negative, <20% leukemia cells with nuclearstaining, 20%–<50% leukemia cells with nuclear staining, 50%–

<75% leukemia cells with strong nuclear staining, and �75%leukemia cells with strong nuclear staining. The scoring resultswere shown in Supplementary Table S4, and representative

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Role of let-7 miRNAs in PRL-3/LIN28B-mediated leukemogenesis. A and B, Impact of let-7a and let-7b on colony forming (A) and cell proliferation (B) ofTF1-hPRL3 cells. Ambion Pre-miR let-7a, let-7b, and a negative control (NC) miRNA (30 nmol/L each) were transfected into TF1-hPRL3 cells by electroporationwith Neon Transfection System. C and D, Effect of inhibitors specific to let-7a and let-7b (30 nmol/L each) on colony forming (C) and cell proliferation (D)of TF1-hPRL3 cells. The results were shown as mean � SD of three independent experiments. � , P < 0.05; �� , P < 0.01.

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LIN28B staining images were presented in Fig. 6A. Notably, thefive normal bone marrow samples from healthy controls dem-onstrated very little LIN28B expression (Fig. 6A).

The survival data were available for 108 patients. We classifiedpatients with LIN28B scoring 50% and above as the "LIN28Bhigh" group and patients with negative or LIN28B scoring lessthan 50% as the "LIN28B low" group. The comparison of variousclinical features between LIN28B high and low groups was sum-marized in Supplementary Table S5.We then compared event-freesurvival between these two groups of patients. On the basis of theKaplan–Meier survival analysis, the group of patients who wereLIN28B high had amuch shorter event-free survival than patientswhowere LIN28B low (Fig. 6B, P¼ 0.0159). Multivariate analysisrevealed that LIN28B protein expression was an independentprognostic factor of FLT3 status, NPM mutation, and cytogeneticrisk. The HR of LIN28B expression was 3.96 (95% CI, 2.15–7.30;P < 0.001; Supplementary Table S6). Overall, high expression ofLIN28B protein was a novel, independent predictor for poorsurvival in our cohort.

DiscussionThe phosphatase of regenerating liver (PRL) family contains

three members, PRL-1, PRL-2, and PRL-3. Among the PRL family,PRL-3 has been most frequently studied in cancer, followed byPRL-2 and PRL-1. PRL-2 has been reported to increase EPO andIL3 response in hematopoietic cells (32) and play critical roles inregulating HSC self-renewal (33). A previous study reported thatgenetic disruption of PRL-3 reduces clonogenicity and growth ofCD133þ mouse colon cancer cells in an in vitro culture system(34). CD133 is also a specific marker for human hematopoieticstem/progenitor cells (35, 36). The observations from these

published studies indicate a possibility that aberrant expressionof PRL family in hematopoietic cells may promote the transfor-mation of LSC in AML. The association between PRL-3 expressionand poor prognosis has been widely confirmed in a number ofsolid tumors and AML (10, 13, 15–17), arguing for the role ofPRL-3 as a biomarker or a therapeutic target in stratifying patientsfor personalized therapy (9, 33, 37, 38). Thus, it is of clinicalimportance to further understand the molecular mechanism bywhich PRL-3 is regulated and contributes to leukemogenesis. Inthis study, we demonstrate that PRL-3 contributes to the leukemicphenotype of AML and plays an important role in the transfor-mation of LSCs via the LIN28B/let-7 regulatory axis (graphicsummary in Fig. 7).

Our global gene expression analysis showed the upregula-tion of several stemness factors, such as LIN28B, c-KIT, andIGF2BP1, and the downregulation of cell surface markers ofmore mature cells, such as CD36 and CD38, by PRL-3. c-KITfunctions as an important regulator of HSC proliferation andself-renewal through binding to soluble steel factor (39). Gain-of-function mutations in c-KIT have been characterized in AMLand other types of cancers (40). GSEA analysis further revealedthat our gene expression profile significantly enriched withpreviously reported LSC and HSC gene signatures (41, 42),suggesting PRL-3 induces a stem cell-like transcriptional pro-gram in leukemia cells.

Serial transplantation in mouse model and serial replatingassay provided functional evidence that PRL-3 confers stemcell–like properties in AML cells and mediates their transforma-tion into leukemia-initiating cells. We further demonstrated thatincreased expression is dependent on the phosphatase activity ofPRL-3, and LIN28B is essential for this transformation process.LIN28B is a stemness factor and an miRNA regulator, whichinhibits the biogenesis of let-7 miRNA family (25, 26). A dysreg-uated LIN28B/let-7 circuit has been implicated in the develop-ment of many types of malignancies, including T-cell acutelymphoblastic leukemia (43), neuroblastoma (44), and livercancer (45). Interestingly, MUC1-C, a transmembrane oncopro-tein, induces activation of LIN28B and downregulation of let-7 innon–small cell lung cancer, thus stimulating EMT and stem cellself-renewal (46). Here, we demonstrate that the LIN28B/let-7axis plays a key role in PRL-3–induced leukemia. The colony-

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The clinical relevance of LIN28B expression in patients with AML. A,Representative IHC images of AML#1 (LIN28B: strong positive), AML#2 (LIN28B:negative), and 1 healthy control (LIN28B: weak positive). B, Impact of LIN28Bprotein expression on outcome of patients with AML. Survival curves wereconstructed according to the Kaplan–Meier method. LIN28B low: staining <50%(n ¼ 67); LIN28B high: staining �50% (n ¼ 41); P ¼ 0.0158.

Figure 7.

Schematic description of the role of PRL3/LIN28B/let-7 axis in the pathogenesis

of AML. ", increased expression; #, decreased expression; activate; ?, inhibit.

Activation of LIN28B by PRL-3 Promotes Leukemogenesis

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forming capacity of leukemic cells is alleviated after knockingdown LIN28B or upon ectopic expression of let-7 miRNAs, whileoverexpression of LIN28B partially rescues the decreased colonyforming capacity caused by viral-mediated silencing of PRL-3 inserial replating assays. By studying a cohort of clinical samples, weshowed the clinical significance of LIN28B expression in bonemarrow samples of AML patients. The shared gene signatures ofLSCs between our expression profile and others derived fromclinical AML samples further underscores the clinical relevance ofour findings (41, 42).

Leukemia-initiating or stem cells can arise from malignanttransformation of normal hematopoietic stem cells or the dereg-ulation of genes that induce stem cell–like properties in moremature progenitor cells. Our investigation has uncovered a noveland critical regulatory PRL-3/LIN28B/let-7 network, which playsan important role in transformation of LSCs and development ofAML and identifies AML patients with poor outcomes. We havepreviously shown that PRL-3 is deregulated in myeloid malig-nancies by STAT activation by upstream oncogenic kinases, suchas FLT3 (e.g., in FLT3-ITD AML; refs. 13, 21, 47). Our resultstherefore provide potential mechanistic insights to the highrelapse rates and poor outcome of FLT3-ITD AML. This novelregulatory network may represent the "Achilles' heel" of LSCs inAML, and its therapeutic disruption could lead to the eliminationof LSCs in a molecular subset of AML in which PRL-3/LIN28Bappears pivotal for the transformation.

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

Authors' ContributionsConception and design: J. Zhou, W.-J. ChngDevelopment of methodology: J. Zhou, J.-Y. Chooi, W.-J. ChngAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): J. Zhou, Z.-L. Chan, C. Bi, X. Lu, J.-Y. Chooi,S.-C. Liu, Y.Q. Ching, Y. Zhou, C.H. Ng, S.-B. Ng, S. WangAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): J. Zhou, Z.-L. Chan, C. Bi, J.-Y. Chooi, M. Osato,T.Z. Tan, W.-J. ChngWriting, review, and/or revision of the manuscript: J. Zhou, W.-J. ChngAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): C. Bi, P.S.Y. Chong, J.-Y. Chooi, L.-L. Cheong,Y.Q. Ching, Q. ZengStudy supervision: W.-J. Chng

AcknowledgmentsThe authors thank the Singapore National Research Foundation and the

Ministry of Education under the Research Center of Excellence Program andNational Medical Research Council (NMRC) for financial support.

Grant SupportThis work was supported by the Singapore National Research Founda-

tion and the Ministry of Education under the Research Center of Excel-lence Program (to W.-J. Chng) and NMRC Clinician-Scientist IRG grantCNIG11nov38 (to J. Zhou). W.-J. Chng was also supported by NMRCClinician Scientist Investigator Award.

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 August 11, 2016; revised October 29, 2016; accepted November 18,2016; published OnlineFirst December 23, 2016.

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like Transcriptional Program in AML−CellLIN28B Activation by PRL-3 Promotes Leukemogenesis and a Stem

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