Activated KRAS Cooperates with MLL-AF4 to Promote ... · and previous screenings of infants with MA4þ pro–B-ALL also 1Josep Carreras Leukemia Research Institute and Department

Tumor and Stem Cell Biology

Activated KRAS Cooperates with MLL-AF4 toPromote Extramedullary Engraftment andMigration of Cord Blood CD34þ HSPC But IsInsufficient to Initiate LeukemiaCristina Prieto1, Ronald W. Stam2, Antonio Agraz-Doblas1,3, Paola Ballerini4,Mireia Camos5, Julio Casta~no1, Rolf Marschalek6, Aldeheid Bursen6,Ignacio Varela3, Clara Bueno1, and Pablo Menendez1,7

Abstract

The MLL–AF4 (MA4) fusion gene is the genetic hallmark of anaggressive infant pro–B-acute lymphoblastic leukemia (B-ALL).Our understanding of MA4-mediated transformation is verylimited. Whole-genome sequencing studies revealed a silentmutational landscape, which contradicts the aggressive clinicaloutcome of this hematologic malignancy. Only RAS mutationswere recurrently detected in patients and found to be associatedwith poorer outcome. The absence of MA4-driven B-ALL modelsfurther questions whether MA4 acts as a single oncogenic driveror requires cooperating mutations to manifest a malignantphenotype. We explored whether KRAS activation cooperateswith MA4 to initiate leukemia in cord blood–derived CD34þ

hematopoietic stem/progenitor cells (HSPC). Clonogenic anddifferentiation/proliferation assays demonstrated that KRASactivation does not cooperate with MA4 to immortalize

CD34þ HSPCs. Intrabone marrow transplantation into immu-nodeficient mice further showed that MA4 and KRASG12V aloneor in combination enhanced hematopoietic repopulation with-out impairing myeloid–lymphoid differentiation, and thatmutated KRAS did not cooperate with MA4 to initiate leukemia.However, KRAS activation enhanced extramedullary hematopoi-esis of MA4-expressing cell lines and CD34þ HSPCs that wasassociated with leukocytosis and central nervous system infil-tration, both hallmarks of infant t(4;11)þ B-ALL. Transcriptionalprofiling of MA4-expressing patients supported a cell migrationgene signature underlying the mutant KRAS-mediated pheno-type. Collectively, our findings demonstrate that KRAS affects thehomeostasis of MA4-expressing HSPCs, suggesting that KRASactivation in MA4þ B-ALL is important for tumor maintenancerather than initiation. Cancer Res; 76(8); 2478–89. �2016 AACR.

IntroductionThe translocation t(4;11)(q21;q23) encodes the chimeric pro-

tein MLL–AF4 (MA4), the hallmark genetic abnormality associ-ated to ghastly infant pro–B-acute lymphoblastic leukemia (B-ALL) that arises prenatally during early hematopoiesis (1, 2).Newbornpro–B-ALL is highly aggressivewithhigh tumor burdensand white blood cell (WBC) counts, therapy resistance, and an

extremely short latency, raising the question of how it evolves soquickly (3). Our understanding ofMA4-mediated transformationis limited, and unlike other MLL fusions, MA4-induced leukemo-genesis has been difficult to model. Current murine-humandisease models do not faithfully recapitulate the pathogenesis/phenotype (4–6). It could be argued that the absence of a diseasemodelmay be because of (i) a cell in awrong developmental stagewas targeted or (ii) the impact of other secondary hits has not beenproperly addressed. We have previously reported that the expres-sion of MA4 on its own enhanced the hematopoietic engraftmentand clonogenic potential of human neonatal cord blood (CB)–CD34þ hematopoietic stem/progenitor cells (HSPC), but is notsufficient for leukemogenesis (7). MA4 also failed to transformCD34þ HSPCs in combination with FLT3 activation, which hasbeen proposed a candidate cooperating oncogenic insult (8).

Recent whole-genome sequencing (WGSeq) studies on MLL-rearranged (MLL-r) infants revealed that this infant leukemia hasone of the lowest frequencies of somatic mutations of anysequenced cancer, contradicting its aggressive clinical outcome(9–12). This, coupled to the absence of models reproducing thisleukemia, feeds the debate of whether MA4 acts as a single-drivermutation in a critical target cell or cooperating mutations need tobe accrued for a malignant phenotype. Despite the paucity ofmutations observed inWGSeq studies, activatingmutations in theRAS signaling pathway were found in half of the patients (9–12),and previous screenings of infants with MA4þ pro–B-ALL also

1Josep Carreras Leukemia Research Institute and Department of Bio-medicine, School of Medicine, University of Barcelona, Barcelona,Spain. 2Pediatric Oncology/Hematology, Erasmus MC-Sophia Chil-dren's Hospital, Rotterdam, theNetherlands. 3Institute of Biomedicineand Biotechnology of Cantabria (IBBTEC-CSIC-UNIVERSIDAD DECANTABRIA-SODERCAN), Santander, Spain. 4Pediatric HematologyDepartment, A. Trousseau Hospital, Paris, France. 5Hematology Lab-oratory, Hospital Sant Joan de Deu, Barcelona, Spain. 6Institute Phar-maceutical Biology, Goethe-University, Frankfurt/Main, Germany.7Instituci�o Catalana Recerca i Estudis Avancats (ICREA), Barcelona,Spain.

C. Bueno and P. Menendez contributed equally to this article.

Corresponding Authors: Pablo Menendez, Instituci�o Catalana Recerca i EstudisAvancats (ICREA), Carrer Casanova 143, 08036 Barcelona, Spain. Phone:34-935572809; Fax: 34-933231751; E-mail: [email protected];or Clara Bueno, E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-15-2769

�2016 American Association for Cancer Research.

CancerResearch

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found RAS mutations in approximately 25% of patients thatcorrelated with poor outcome. KRAS mutations have also beenassociated with higher WBC counts (13) and found subclonallypresent at birth in neonatal blood spots (14). Furthermore, in aMA4þ transgenic mouse model, activated KRAS accelerated leu-kemogenesis, although the latency/phenotype did not reproducethat seen in patients (15). Similarly, in a humanized MLL-AF10model, the coexpression of KRASG12Vwas required for a leukemicphenotype (16, 17).

Here, we explored whether activation of KRAS cooperates withMA4 to transform/immortalize CB-derived CD34þ HSPCs. Ourresults indicate that KRASG12V alone or in combination withMA4does not initiate leukemia. However, enforced KRASG12V expres-sion enhanced extramedullary hematopoiesis and invasion ofMA4-expressing CD34þHSPCs and MA4þ cell lines associated toelevated WBC counts and central nervous system (CNS) infiltra-tion, hallmarks ofMA4þ pro–B-ALL infants. These functional andclinical data indicate that activated KRAS does not cooperate withMA4 to initiate leukemia but influences the homeostasis ofMA4-expressing CD34þ HSPCs, suggesting that KRAS activation inMA4þ infant B-ALL may be important in tumor maintenancerather than initiation, thus supporting WGSeq studies showingthat RAS mutations in infant MLL-r leukemia are subclonal andlost at relapse. We conclude that MA4-mediated transformationmight depend on alternative (epi)-genetic cooperating lesionsand on a critical developmentally earlier window of stem cellvulnerability.

Materials and MethodsCB collection and CD34þ HSPCs isolation

Umbilical CB units (n ¼ 100) from healthy newborns wereobtained from the Catalonia Blood Tissue Bank following theinstitutional guidelines approved by our local InstitutionalReview Board. CBs were pooled to reduce variability amongindividual CB units, and mononuclear cells were isolatedby density gradient centrifugation using Ficoll–Hypaque. Afterlysing red cells, CD34þ cells were purified by magnetic beadseparation using the human CD34 MicroBead Kit and the Auto-MACS Pro-separator (Miltenyi Biotec) as per the manufacturer'sinstructions (Fig. 1A; refs.18, 19). Purity of the CD34þ fractionswas always assessed by flow cytometry using anti–CD34-PE(BD Biosciences), and only CD34þ fractions with purity >95%were used. The CD34� fraction was irradiated and used as acces-sory cells for cotransplantation with CD34þ HSPCs.

Plasmids and lentiviral transductionThe MA4 cDNA (7, 8) and the KRASG12V cDNA (kindly pro-

vided by professor Naoto Ishii, Tohoku University, Japan) weresubcloned into the pRRL-EF1a-PGK-GFP/dTo vector. The follow-ing lentivectors were used: pRRL-EF1a-PGK-GFP/dTo (emptyvector, EV), pRRL-EF1a-MA4-PGK-GFP (MA4), and pRRL-EF1a-KRASG12V-PGK-dTo (KRAS; Fig. 1B). Vesicular stomatitisvirus-G (VSV-G)–pseudotyped viral particles were generated on293T cells by calciumphosphate transfection and concentrated byultracentrifugation (7, 20). Human CD34þHSPCs (2� 106 cells)were infected overnight with concentrated viruses and polybrene(1 mg/mL; Sigma-Aldrich) and the hematopoietic cytokines StemCell Factor (100 ng/mL), FMS-like tyrosine kinase 3 ligand (100ng/mL), and IL3 (10 ng/mL, all from PeproTech). Viral superna-tant was removed 14 hours later, and transduced cells werewashed and maintained in culture in Stemspan medium (Stem

Cell Technologies; refs. 7, 21). For in vitro and in vivo experiments,transduced CD34þ cells were FACS-sorted (FACSAria) 36 hoursafter infection based on reporter expression: GFPþ for MA4-transduced CD34þ, dToþ for KRAS-transduced cells, and mergedyellow (GFPþdToþ) for MA4þKRAS cotransduced cells. The SEM(MA4þ) cell line was transduced with KRASG12V, FACS-purified,and xenografted as described below. SEM cells were obtainedfrom the DSMZ-German Collection Cell Bank (www.dsmz.de)and were routinely authenticated/characterized by PCR for MA4and FISH for MLL break-apart.

Mice transplantationNon-obese diabetic/LtSz-scid IL-2Rg�/� mice (NSG; n ¼ 50)

were used for in vivo assays (22, 23). Animals were housed underpathogen-free conditions, and the procedures were approved bythe Animal CareCommittee of Barcelona Parc Recerca Biomedica.Seven to 12-week-old mice were sublethally irradiated (2.25 Gy)6 to 16 hours before transplantation. They were anesthetized withisoflurane 3% inhalation, and intrabone marrow transplantation(IBMT) was performed (23, 24). A total of 3 � 104 transduced/sorted CD34þ HSPCs (along with 5 � 104 irradiated accessorycells) were transplanted in a 25-mL volume. For pain relief,buprenorphine:meloxicam was subcutaneously administeredduring the transplantation and 24 hours later. Mice were mon-itored throughout the entire experiment and killed at any sign ofdisease or 16 weeks after transplantation. For SEM assays, 2� 105

cells were IBM-transplanted.

Analysis of peripheral blood hematologic counts andmacroscopic parameters

Hematologic parameters, including absolute WBC, platelets,and hemoglobin levels, were determined in mice peripheralblood (PB) at sacrifice using the hematologic analyzer SysmexSX-800i (7). Spleen and liver were visualized macroscopically,measured, and weighted.

Analysis of engraftmentMice were killed 16 weeks after transplantation. Bonemarrow

(BM) from injected tibia (IT), contralateral tibia and femur(CL), liver, spleen, and PB were collected and analyzed forhuman chimerism. Cells were stained with anti-HLA.ABC-PEand CD45-APC.Cy7, and human chimerism was analyzedby FACS. All engrafted mice were assessed for multilineageanalysis using anti–CD33-APC for myeloid cells, anti–CD19-V450 for lymphoid cells, and anti–CD34-PE.Cy7 for immaturecells. Within the CD19þ cell subset, the percentage of CD34þ

and CD10þ cells was analyzed with anti–CD10-PerCP.Cy5.5to distinguish between pro-B and pre-B cells. In addition,the following CD34þ subsets were analyzed: immature pro-genitors (CD34þCD19�CD33�CD38�), myeloid progenitors(CD34þCD33þ), and B-cell progenitors (CD34þCD19þ).

In vitro liquid culture of transduced CD34þ cells, apoptosis,cell-cycle, and senescence assays

For liquid culture, transduced CD34þ HSPCs were FACS-sorted based on reporter expression. Purified GFPþ (MA4-expressing), dToþ (KRAS-expressing), or merged yellow (MA4-and KRAS-coexpressing) CD34þ HSPCs were allowed toexpand for a period of 40 to 50 days. To determine the growthkinetics, cells were counted every 5 days and replated at adensity of 1 � 105 cells/cm2 (7, 25). Furthermore, the expres-sion of CD34 antigen was analyzed every 3 to 4 days. To

KRAS Mutation and MLL–AF4 in Cord Blood CD34þ Cells

www.aacrjournals.org Cancer Res; 76(8) April 15, 2016 2479




analyze cell-cycle distribution, transduced CD34þ HSPCs werefixed in 70% ice-cold ethanol and stored at �20�C. Subse-quently, cells were suspended in propidium iodide (PI) buffercontaining 5 mg PI and 100 mg/mL RNAase. Cell-cycle distri-bution was analyzed on a FACSCanto-II cytometer using theFACSDiva software to discriminate among resting cells (G0–

G1), cycling cells (S phase), and G2–M cells (25). The apoptoticstatus of transduced CD34þ HSPCs was assessed using AnnexinV as previously detailed (25). Senescence was measured onfixed CD34þ HSPCs by b-galactosidase staining as previouslydescribed (26).

Clonogenic progenitor assayHuman clonogenic progenitor assays were performed by plat-

ing 1,000 sorted EV-, MA4-, KRAS-, or MA4þKRAS-expressingCD34þHSPCs inmethylcellulose (n¼ 7). Colonies were countedand scored at days 10 to 12 of the assay using standard morpho-logic criteria (7, 27, 28). For secondary replating, all the colonyforming units (CFU) from each experimental condition wereharvested, and a single-cell suspension was achieved and replatedas above. The resultingday 12CFUcultureswereharvested, single-cell dissociated, washed, and immunophenotyped using anti–CD33-APC and anti–CD15-BV510 antibodies (BD Biosciences).

Cell migration assaysMouse serum was harvested from healthy mice by blood

centrifugation. A total volume of 600 mL of RPMI supplementedwith 10%murine serumwas added to the lower chamber of a 24-well transwell (Corning). CD34þ or SEM (1 � 105) in 100 mL ofmedium were loaded to the upper chamber (pore size 8 mm) andwere allowed to migrate for 4 hours at 37�C. Migrating cells werecollected from the lower chamber, counted, and characterizedimmunophenotypically.

RNA extraction, cDNA synthesis, PCR, and RT-PCRRNA was isolated from in vitro cultures and BM of transplanted

mice with an RNeasy kit (QIAGEN) and treated with DNaseI.cDNA was synthesized using the High-Capacity cDNA ReverseTranscription Kit (Applied Biosystems). cDNA was used for con-ventional (MA4, KRAS, and GAPDH) and quantitative (HOXA9,PROM1, CXCR4, MET, NELIN, and DCL1) PCR. PCR for MA4,KRAS, and GAPDH was done using the following primers: MA4,fwd 50-CAGAGCAAACAGAAAAAAGTG-30 and rev 50-TCCACAG-TCCCTTCCAGAAC-30; GAPDH, fwd 50-GAAAGCCTGCCGGT-GACTAA-30 and rev 50-CTCCGGGTGATGCTTTTCCT-30; andKRAS, fwd 50-TGGACTACAAAGACGATGACG-30 and rev 50-CCCTCCCCAGTCCTCATGTA-30. PCR conditions were 95�C

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Figure 1.Lentiviral-mediated expression and activation of MA4 and KRAS in CD34þ HSPCs. A, outline of the experimental design. B, schematic representation of thelentivectors used. MA4 and KRASG12V vectors express GFP and dTomato as reporter, respectively. C, representative flow cytometry plots showing howtransduced cellswere FACSpurified: GFPþ cells (green) inMA4-transduced, dToþ cells (red) in KRASG12V-transduced, andGFPþdToþ cells (yellow) in KRASG12V- andMA4-double transduced CD34þ HSPCs. Mock-transduced cells (left) were used as a reference control (n ¼ 12). D, RT-PCR confirming ectopic expressionofMA4 and KRASG12V in CB-CD34þ cells. E, RT-qPCR confirming approximately 2- to 10-fold upregulated expression of the MA4 downstream effectors HOXA9 andPROM1 in MA4-expressing CD34þ HSPCs. F, phosphoflow assay demonstrating activation of KRAS downstream effectors pERK1/2 and pAKT. G, KRASG12V

expression renders immortalization/IL3-independent growth of the pro–B-cell line BaF3 (n ¼ 2).

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Cancer Res; 76(8) April 15, 2016 Cancer Research2480




(2minutes) followed by 40 cycles of 95�C (20 seconds), 60�C (20seconds), and 72�C (30 seconds) and a final extension of 72�C(10 minutes). The resulted amplicons (KRAS, 253 bp; MA4, 382bp; GAPDH, 120 bp) were resolved on agarose gels. For compar-ative expression of HOXA9, PROM1, CXCR4, MET, NELIN, andDCL1 genes, the following primers were used: HOXA9, fwd 50-AAGACCGAGCAAAAGACGAG-30 and rev 50-GGGTGAGAGAA-GGGAGAAGG-30; PROM1, fwd 50-CTTTCTCCTGCCTCCCGC-30

and rev 50-TTTATGACCCGGCTTCTGGG-30; CXCR4, fwd 50-ATAGTCCCCTGAGCCCATTT-30 and rev 50-AGCAGGTAGCAAA-GTGACG-30; MET, fwd 50-CCATCCAGTGTCTCCAGAAGTG-30

and rev 50-TTCCCAGTGATAACCAGTGTGTAG-30, NELIN, fwd50-CGACGAACAGAGGAGGAAC-30 and rev 50-TTCTCTGCTC-AATCCAAAGGT-30; and DLC1, fwd 50-GGACAGAGATGCCATT-GAGGCT-30 and rev 50-CACAAGGCTCATCCTCGTCTGA-30. qRT-PCR conditions were 95�C (10 minutes) followed by 40 cycles of95�C (30 seconds), 60�C (60 seconds), and 72�C (60 seconds).

Phosphoflow assays for AKT and ERK1/2For phosphosignaling studies, cells were resuspended in stain-

ing buffer, fixed with Cytofix buffer (BD Biosciences), and per-meabilized in prechilled Phosphoflow PermBuffer-III for 30minutes before staining with anti–phospho-ERK1/2 (pT302/pY204)-PE and anti–phospho-AKT (pS473)-PE. Data were ana-lyzed in a FACSCanto-II cytometer using FACSDiva. The meanfluorescence intensity was compared for KRASG12V-transducedversus EV-transduced CD34þ HSPCs.

BaF3 assayIL3-dependent pro–B-cell line BaF3 was used to confirm

KRASG12V activation. BaF3 cells (2� 106) were infected overnightwith concentrated EV- and KRASG12V-expressing lentivirus. Afterwashing viral particles, transduced BaF3 cells were recovered inRPMI þ 10% FBS þ 10 ng/mL mIL3 for 2 days. Then, mIL3 wasremoved and EV- and KRASG12V-BaF3 cells (2.5 � 104) wereplated without IL3 and counted every 5 days to determine IL3-independent growth.

CNS infiltrationMice skulls were retrieved at sacrifice and fixed in 10% neutral

buffered formalin (pH 6.8) for 2 weeks and then decalcified inEDTA (14%; pH 7.4) for 2 more weeks. Skulls were then embed-ded in paraffin, cut into 4 mm sections, rehydrated, and multiplesections were stained with hematoxylin and eosin (H&E) aspreviously described (29).

Gene expression profilingPB leukemic cells fromMA4-expressing pro–B-ALL infants (n¼

12) were purified for gene expression profiling (GEP). Five andseven patients harbored and lacked RAS mutations, respectively.Total RNA was extracted using TRIzol, and quantified on aspectrophotometer. The integrity of the RNA was assessed on anAgilent-2100 Bioanalyzer. High-quality RNA was reverse tran-scribed using T7-linked oligo-dT primers, and the obtained cDNAwas used as a template to synthesize biotinylated cDNA. LabeledcDNA was then fragmented and hybridized as duplicates toHU133plus2.0 GeneChips (Affymetrix) according to the manu-facturer's guidelines (30). Microarray data are deposited in NCBI(GSE19475). Hierarchical clustering of genes and samples wasperformed with the one minus correlation metric and theunweighted average distance. Because RAS mutations are sub-

clonal, a gene was considered differentially expressed when it was20% deregulated (up/down; P value < 0.05) as compared withthe control. Gene ontology (GO) term analysis was performedusing Gorilla (31–33) publicly available at http://cbl-gorilla.cs.technion.ac.il.

Statistical analysisData are expressed as mean � SEM of independent experi-

ments. Paired or impaired Student t tests were used as corre-sponding to perform statistical comparisons between condi-tions. Statistical significance was defined as P < 0.05.

ResultsSuccessful enforced coexpression of MA4 and KRASG12V in CB-derived CD34þ HSPCs

To determine whether KRASG12V cooperates with MA4 inregulating HSPCs, CD34þ cells were isolated from CB and trans-duced with a lentivector expressing (i) GFP reporter (EV), (ii)MA4-GFP (MA4), (iii) KRASG12V-dTomato (dTo) (KRAS), and(iv) MA4-GFP and KRAS-dTo, simultaneously (Fig. 1A and B).Transduced cellswere FACS-purified (purity>98%)48hours laterbased on GFP/dTo reporter and used for in vitro and in vivo assays(Fig. 1A and C). Proper transgene expression in HSPCs was alsoconfirmed by RT-PCR (Fig. 1D). The ectopic expression ofMA4 inCB-CD34þ HSPCs was comparable with that found in patientprimary blasts (data not shown), and induced the expression oftheMA4 target genesHOXA9 and PROM1 (Fig. 1E). Similarly, theectopic expression of KRASG12V resulted in robust RAS activationas demonstrated by phosphorylation of the downstream effectorsERK1/2 and AKT (Fig. 1F). Functionally, the immortalizationactivity of KRASG12V was confirmed by its capacity to conferIL3-independent growth to BaF3 cells (Fig. 1G).

KRASG12V does not cooperate with MA4 to immortalize in vitroCB-CD34þ cells.

MA4 was reported to enhance the in vitro clonogenic potentialwithout immortalizing CD34þ HSPCs (7, 8). We here examinedwhether KRAS cooperates with MA4 to immortalize CD34þ

HSPCs. The hematopoietic progenitor function of CD34þ

HSPCs was examined by quantitative and qualitative analysisof the CFU capacity of EV-, MA4-, KRAS-, and double-transducedcells (n ¼ 7). Scoring of primary CFUs revealed that progenitorsexpressing MA4 or KRAS alone displayed a higher clonogenicpotential (173 and 232 CFU, respectively, per 1,000 plated cells)compared with EV-transduced HSPCs (108 CFU/1,000 platedcells). However, KRAS did not synergize with MA4 to augmentthe clonogenic potential (Fig. 2A, left). Scoring of primary CFUsrevealed no significant differences in CFU types among condi-tions. Importantly, CFU replating assays revealed a significantdecrease in the secondary CFUs, indicating that KRAS does notcooperate with MA4 in progenitor immortalization (Fig. 2A,left). MA4-GFP and KRAS-dTo expressions were confirmedin CFUs by fluorescence microscopy and RT-PCR, (Fig. 2A, right).In addition, KRAS did not influence the MA4-mediated myeloidterminal differentiation, as demonstrated by similar proportionof CD33þCD15þ (45%–58%) and CD33þCD15� (42%–

55%; Fig. 2B), and identical kinetics of CD34 expression lossin liquid cultures (Fig. 2C), regardless of the genotype. Further-more, KRAS and MA4, either alone or combined, did notblock in vitro B-cell differentiation from CD34þ HSPCs on MS5






stromal cells using B-lineage growth conditions (data not shown;refs. 34, 35). Long-term liquid culture proliferation assaysrevealed that EV-, MA4-, KRAS-, and KRASþMA4-transducedCD34þ cultures grew similarly; cell growth gradually dropped

off from days 15 to 20, and by day 40, cultures were exhaustedregardless of the genotype (Fig. 2D). In line with the CFU assays,KRAS induced an enhanced transient expansion when activatedalone but not in cooperation with MA4, confirming that KRAS

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Figure 2.KRASG12V does not immortalize CD34þ HSPCs but impairs the clonogenic potential ofMA4-expressing CD34þ HSPCs. A, representative experiment (n¼ 7) of CFUsshowing that MA4 and KRASG12V do not support replating clonogenic potential. In MA4/KRASG12V cotransduced CD34þ HSPCs, the clonogenic potentialis partially impaired. E, erythroid; M, monocytic; G, granulocytic; GM, granulomonocytic; GEMM, granulocyte–erythroid–monocytic-megakaryocitic. � , P < 0.05. Topright, representative fluorescence image of CFU colonies (10�) harvested after 12 to 14 days. Bottom right, RT-PCR showing stable expression of MA4 andKRASG12V in individual CFUs. B, flow cytometry analysis at day 14 of CFU assays shows very similar content of CD33þCD15þ (neutrophils) and CD33þCD15�

(monocytes) for the indicated genotypes. C, differentiation over time, measured as loss of CD34 antigen, of the indicated genotypes of HSPCs seeded inliquid cultures (n ¼ 6). D, representative experiment (n ¼ 7) of in vitro cell growth kinetics over a period of 40 to 50 days of FACS-purified, EV-, MA4-,KRAS-, and MA4þKRAS–expressing CD34þ HSPCs. Right plots are representative fluorescence images confirming reporter expression on day 15 cultures. E and F,apoptotic levels (AnnexinVþ; E) and cell-cycle distribution (F) of the indicated genotypes of CD34þHSPCs in liquid culture (n¼ 3). G, percentage ofb-galþ senescentcells in the indicated genotypes of growing CD34þ HSPCs (n ¼ 2). Representative b-gal staining is shown.

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activation does not cooperate with MA4 to immortalize CD34þ

HSPCs. Interestingly, KRASG12V slightly impaired the clonogenicpotential of MA4-expressing CD34þ HSPCs. Apoptosis levels(Fig. 2E) and cell-cycle distribution (Fig. 2F) were not affectedamong genotypes. However, KRAS induced in vitro senescence ofCD34þ cells (Fig. 2G) and cooperated in vivo with MA4 to shiftthe CD34þ population toward a more immature and B-cellprogenitor phenotypes while compromising the myeloid phe-notype (Fig. 3D, bottom). This may explain, at least in part, theKRAS-driven lower clonogenic potential of MA4-expressingCD34þ HSPCs because human clonogenic assays represent amyeloid read-out.

KRAS activation does not cooperate with MA4 to enhancemultilineage hematopoietic engraftment and fails to initiateMA4-mediated leukemogenesis in vivo

To determine whether KRAS activation cooperates withMA4 inregulating HSPCs in vivo, purified CD34þHSPCs were transducedwith EV, MA4, KRAS, and MA4þKRAS, sorted based on reporterexpression, and 30,000 cells were IBMT-transplanted into suble-thally irradiated NSG mice (n ¼ 50). Animals were monitoredthroughout the entire experiment, and none of the mice showedany sign of disease 16 weeks after transplantation. Enforcedexpression of KRAS, alone or combined with MA4, enhanced thehematopoietic engraftment 3-fold as compared with EV-trans-duced cells (36% and 38%vs. 12%; Fig. 3A), indicating that KRASand MA4 do not synergize to promote more robust/sustainedengraftment potential. The ectopic expression of MA4 and/orKRAS was confirmed by RT-PCR in BM cells derived fromengrafted animals (Fig. 3B).

Next, the composition of the human graft was characterized byFACS, and multilineage repopulation was consistently observedin all engraftedmice, regardless of the expression ofMA4 or KRASand the tissue analyzed (Fig. 3C). The graft was consistentlylymphoid-biased (CD45þCD19þ; 70% � 6.0%), followed byCD45þCD33þ myeloid cells (19% � 4.9%) and CD45þCD34þ

immature cells (11% � 3.6%; Fig. 3D, top). Because leukemicblasts in t(4;11)þ B-ALL are characterized by a CD34þ

CD19þCD10� pro-B phenotype, we further analyzed the pheno-type of the CD45þCD19þ B-cell graft (Fig. 3D, middle). Withinthe CD45þCD19þ B-cell graft, approximately 7% of B-cell pro-genitors (CD19þCD10þCD34þ) coexisted with a predominantpre–B-cell population (CD19þCD10þCD34�; �58%) and moremature B cells (CD19þCD10�CD34�; �5%), confirming a nor-mal B-cell development. Importantly, KRAS cooperates withMA4 to shift the CD34þ population toward a more immaturephenotype (CD34þCD19�CD33�CD38�; 18% MA4þKRAS vs.10% other genotypes) and a B-cell progenitor phenotype(CD34þCD19þ; 72% vs. 58%) while compromising the myeloidphenotype (CD34þCD33þ; 16% MA4þKRAS vs. 33% othergenotypes; Fig. 3D, bottom).

To further confirm that KRAS did not contribute to MA4-mediated leukemia initiation, all the animals lacking diseasesignals after 16 weeks were sacrificed, and neither splenomegalynor hepatomegaly was observed regardless the genotype (Fig.3E). Besides, hemoglobin levels and platelet counts were nor-mal and similar between conditions (Fig. 3F). These resultssupport that enforced coexpression ofMA4 and KRASG12V is notsufficient for leukemogenesis, and KRAS does not cooperatewith MA4 to enhance multilineage hematopoietic engraftmentin vivo.

Activated KRAS cooperates with MA4 to promoteextramedullary engraftment andmigration of CB-CD34þHSPC

As previously reported (36), in our cohort of MA4þ B-ALLinfants (n ¼ 24), those harboring KRAS mutations presented 2-fold higher WBC counts at diagnosis than those with germlineKRAS (Fig. 4A). Similarly, the human WBC counts in PB ofxenografts transplanted with MA4þKRAS-transduced CB-CD34þ

cells were 4- to 8-fold higher than inmice transplantedwith singleMA4- or KRAS-transduced CD34þ HSPCs (Fig. 4B). The PBleukocytosis observed in t(4;11) patients harboring KRAS muta-tions and reproduced inNSGmice xenografted withMA4þKRAS-expressing CD34þ HSPCs suggests that RAS activation may con-tribute to the maintenance rather than to the initiation of theleukemia by altering the aggressiveness and migration ability ofMA4-expressing blasts and primary CD34þ HSPCs, respectively.

Direct injection of cells within the BM cavity provides theopportunity to assess the migration of transplanted cells in vivo.Thus, the levels of extramedullary hematopoiesis of transducedCD34þ HSPCs were assessed by analyzing the chimerism in theBM, PB, spleen, and liver. Regardless of the expression ofMA4 orKRAS, CD34þHSPCswere capable ofmigrating to and colonizingPB, spleen, and liver in all analyzed animals. Surprisingly, how-ever, MA4þKRAS-transduced CD34þ cells consistently displayeda 3- to 15-fold higher level of extramedullary engraftment in PB,spleen, and liver (Fig. 4C). To gain further insights, the cell lineSEM [t(4;11)þ and germline RAS] was transduced with EV andKRASG12V, FACS-sorted, and IBM-transplanted in NSG mice (n ¼12). After 3 weeks, BM engraftment was comparable between EVand KRAS conditions, whereas PB colonization was 3-fold higherby KRASG12V-expressing SEM (Fig. 4D, left). KRAS expression inengrafted mice was confirmed by fluorescence microscopy andRT-PCR (Fig. 4D, right).

Infant t(4;11)þ B-ALL commonly associates with CNS involve-ment (37). We thus analyzed whether KRAS activation facilitatesCNS infiltration inMA4-expressing leukemia cells that were IBM-transplanted. Three weeks after IBMT, brain-skull sections frommice engrafted with SEM-EV (n ¼ 6) or SEM-KRAS (n ¼ 6) wereanalyzed by histopathology, andH&E staining showed that KRASactivation consistently (100% of mice) conferred t(4;11)þ cells arobust ability to cross blood–brain barrier, as revealed by thepresence of leukemic infiltration within leptomeningeal space,indicative of CNS involvement (Fig. 4E). In vivo data with CB-CD34þ HSPCs and SEM cell line indicate that KRAS activationpromotes extramedullary hematopoiesis and CNS infiltration,suggesting a migratory role for KRASG12V in MA4-expressing(pre)-leukemia cells. To explore this further, in vitro migratorytranswell assays were performed with SEM (Fig. 4F and G) andCD34þ HSPCs (Fig. 4F and H). KRAS-expressing SEM andMA4þKRAS-transduced CB-CD34þ HSPCs migrated toward agradient of mouse serum 4-fold and 2-fold, respectively, morethan the comparing genotypes (Fig. 4F–H). Together, our in vitroand in vivo functional data suggest that KRAS activation confers anextramedullary migratory phenotype to MA4-expressing CB-CD34þ and leukemic cells.

Global transcriptional analysis of t(4;11)þ B-ALL infantssupports the KRAS-mediated migratory role/extramedullaryhematopoiesis

To identify patterns of gene expression that could explainmolecularly the impact of KRAS activation on MA4-expressingcells, we harnessed existing GEP data generated from PB leukemic






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Figure 3.KRASG12V does not cooperate withMA4 to initiate leukemia in CB-CD34þHSPCs. A, long-term hematopoietic engraftment of CB-CD34þ expressingMA4, KRASG12V,or both MA4 and KRASG12V together (n ¼ 50 mice). B, RT-PCR confirming stable ectopic expression of MA4 and KRASG12V in xenografts killed 16 weeks aftertransplantation. C, representative flow cytometry analysis of engrafted mice. The human graft identified as CD45þ HLA-ABCþ comprises CD19þ B-lymphoid cells(CD10þ pre-B and CD10� pro-B), CD33þ myeloid cells, and CD34þ immature cells. D, top, graft composition confirming normal multilineage engraftment in micetransplanted with 30,000 sorted/transduced CD34þ HSPCs expressing the indicated genes. Middle, B-cell graft demonstrating normal, nonleukemic B-celldifferentiation. B-cell graft is comprised of a majority (�80%–90%) of pre-B cells (CD34�CD19þCD10þ) and a minority (�10%–20%) of the pro-B cells(CD34þCD19þCD10�). Bottom, engraftment composition of the CD34þ fraction, including the more immature CD34þ cells (CD34þCD19�CD33�CD38�),B-cell progenitors (CD34þCD19þ), and myeloid progenitors (CD34þCD33þ). KRASG12V cooperates with MA4 to half reduce the contribution of the myeloidengraftment (n ¼ 30 mice). E, weight (top) and macroscopic images (bottom) of spleen (left) and liver (right) showing lack of splenomegaly or hepatomegalyin transplanted mice (n ¼ 50). F, hemoglobin (left) and platelet (right) levels analyzed in the indicated mice groups (n ¼ 50) revealing no sign ofleukemia in reconstituted mice.

Prieto et al.





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Figure 4.Coexpression ofMA4 andKRASG12V promotes extramedullary engraftment and/ormigration of CB-CD34þHSPCs andMA4þ cell lines. A, KRASmutMA4þ infantswithB-ALL show 2-fold higher WBC counts at diagnosis than KRASWT group. B, approximately 4- to 8-fold higher human absolute WBC counts in PB of micexenografted with CB-CD34þ cells coexpressing both MA4 and KRASG12V. C, mice xenografted with CB-CD34þ cells coexpressing both MA4 and KRASG12V display amuch higher engraftment in extramedullary hematopoietic sites, including liver (top), spleen (middle), and PB (bottom). D, expression of KRASG12V in thecell line SEM derived from a t(4;11)(q21;q23) infant B-ALL renders an increased engraftment in (colonization of) PB but not in the BM (n ¼ 6, left). Representativefluorescence image and RT-PCR confirming KRAS expression in transduced SEM cell line (right). E, photomicrographs of brain-skull sections stained withH&E from mice engrafted with SEM-EV or SEM-KRASG12V cells showing that expression of KRASG12V confers a robust ability to infiltrate the CNS (n ¼ 6 mice).The asterisk identifies leukemia infiltrate in leptomeningeal space. White arrowhead, the skull. B, brain tissue. F, overview of the transwell assay with SEMand CB-CD34þ HSPCs. G, 4-fold enhanced migration of SEM-KRASG12V cells toward mouse PB serum as compared with SEM-EV. H, CB-CD34þ HSPCs coexpressingboth MA4 and KRASG12V migrate toward mouse PB serum twice than those expressing MA4 or KRASG12V alone.






cells from t(4;11)/MA4þ pro–B-ALL infants (GSE19475; ref. 30).Twelve infants were analyzed, of which five harbored RAS muta-tions and seven were RAS germline. Because RAS mutations areoften subclonal, only patients with >40% RAS clonality wereconsidered for GEP. A heatmap representation of hierarchicalclustering of genes differentially expressed (20% dysregulated upor down; P value < 0.05) between t(4;11)þ patients with versuswithout KRASmutations is shown in Fig. 5A. A total of 764 geneswere differentially expressed between KRAS-mutated and KRAS-germline t(4;11)þpatients.Of them, 658 (86%)were upregulatedand 106 (14%)downregulated inKRAS-mutated t(4;11) patients,suggesting that KRAS functions as a global transcriptional activa-tor in t(4;11)/MA4þ infants (Fig. 5B). To get insight into thebiologic functions affected by differentially expressed genes, weusedGorilla software (38–40) to performGOanalysis comparingKRAS-mutated versus KRAS nonmutated t(4;11)þ patients.Among the top significant GO biologic processes enriched in theKRAS-mutated upregulated genes, we found "cell migration" and"cell motility" (Fig. 5C). In contrast, among the top most signif-icant GObiologic processes enriched in theKRAS-mutated down-regulated genes, "leukocyte cell adhesion" and "leukocyte aggre-gation" pathways as well as "mesenchymal–epithelial transition"pathway were observed (Fig. 5D).

To functionally support the t(4;11)þ patients-based GEPdata, we compared in MA4- versus MA4/KRAS-expressingCD34þ HSPCs harvested from xenograft extramedullary tissues(spleen and PB) the expression of CXCR4 and MET, mastergenes involved in HSPC retention to the BM and leukocyte celladhesion aggregation, respectively, and the expression ofNELIN (41) and DCL1 (42), master regulators of cell migrationand motility. A robust downregulation of CXCR4 and METcoupled to a significant upregulation of the migration-promot-ing genes NELIN and DCL1 was observed in CB-CD34þ HSPCscoexpressing KRAS and MA4. These data support the GOsignature found in PB t(4;11)þ patient blasts, suggesting aKRAS-mediated BM exit and extramedullary hematopoiesis ofMA4-expressing CB-CD34þ and SEM cells.

DiscussionDespite tremendous improvement in clinicalmanagement and

survival of childhood B-ALL, the outcome of infants with t(4;11)/MA4þ B-ALL remains dismal, with overall survival < 30% (43).Studies in monozygotic twins and archived blood spots haveprovided compelling evidence of a prenatal cellular origin astarget forMA4 fusion, explaining the brief latency of this leukemia(1, 2, 44, 45). Despite its aggressiveness/short latency, currentprogress about its etiology, pathogenesis, and cellular origin isvery limited as evidenced by the lack of models recapitulating thedisease phenotype/latency (4–6, 46). The lack of bona fideMA4þ

B-ALL disease models may be explained from a developmentalangle because developmental cues and the prenatal nature of thetarget cell remain elusive as they have not been properlyaddressed. To understand the biology underlying the initiation ofMA4þ B-ALL, two key questions must be addressed. First, it isunclear whether t(4;11)/MA4þ alone is sufficient for overt leu-kemia or secondary cooperating oncogenic insults are required(9–11). Second, the developmentally early nature of the cell oforigin of this leukemia remains unknown (34, 39).

In sharp contrast with its dismal clinical evolution, indepen-dent recent WGSeq studies have revealed a silent mutational

landscape in MLL-r infant B-ALL (9–12), supporting the possi-bility that MA4 functions as a single oncogenic driver that sufficesto spawn aggressive B-ALL. Despite the paucity of mutationsobserved in WGSeq studies, activation of FLT-3 and/or RAS wassubclonally found in 30% to 50% of patients (9–12) and corre-lated with poor outcome (36, 47). Using CB-CD34þ HSPCs, wehave previously reported that the expression of MA4 itself or incombination with activated FLT3 is not sufficient for leukemo-genesis (7). KRAS mutations have also been associated withhigher WBC counts and poor disease evolution (36) and havebeen found subclonally present at birth in matched neonatalblood spot diagnosis samples (14). Furthermore, in a MA4þ

transgenic mouse model, activated KRAS accelerated leukemo-genesis, although the phenotype did not reproduce that seen inpatients (15). Similarly, in a humanized MLL-AF10 model, coex-pression of KRASG12Vwas required for a leukemic phenotype (16,17). However, whether RAS activation cooperates with MA4 inleukemia initiation/maintenance has not been addressed in ahuman system. Here, we successfully developed a dual-reporter–based transduction strategy to stably overexpress both MA4 andKRASG12V in CB-CD34þ HSPCs.

Regardless of MA4 expression, expression of KRAS enhancedthe hematopoietic engraftment 3-fold, indicating that KRAS andMA4 do not synergize to promote more robust engraftmentpotential. Mice follow-up, characterization of the graft composi-tion, and hematopathology analyses revealed that enforced coex-pression of MA4 and KRASG12V is not sufficient to initiate leuke-mia in vivo. Similarly, in vitro assays showed that KRASG12V doesnot cooperate with MA4 to immortalize CB-CD34þ HSPCs, asdemonstrated by the inability of KRASG12V to confer CFU replat-ing potential or overgrowth to MA4-expressing CD34þ cells.Interestingly, KRASG12V slightly impairs the clonogenic potentialof MA4-expressing CD34þ HSPCs. Although no differences weredetectable in cell cycle and apoptosis, KRAS induced in vitrosenescence of CD34þ cells and cooperated with MA4 to shift theCD34þ population toward a nonmyeloid phenotype, explainingthe KRAS-driven lower clonogenic potential of MA4-expressingCD34þ HSPCs since human CFU assays only read-out myeloidclonogenic potential.

Our data support previous WGSeq studies in MLL-r B-ALLwhere KRASmutations are subclonal and lost at relapse, suggest-ing thatMA4-mediated transformation may rely on a critical fetaltarget cell or on epigenetic cooperating lesions (12). This stablemutational landscape is not unique to infant leukemia. It has alsobeen observed in other pediatric/infant tumors, such as rhabdoidtumors (48) andbrain/hindbrain ependymomas (49, 50), furthersupporting infant cancer as a developmental disease (51). Epige-netically enhanced promoter methylation is typically accompa-nied by global loss of methylation in nonpromoter regions of thegenome of cancer cells. However, MA4þ B-ALL cells display aglobal hypermethylated genomic state, both at promoter andnonpromoter regions (52). Global hypomethylation leads togenomic instability linked to cancer development, thus explain-ing the global genomic stability/silent mutational landscapefound in MA4þ infant B-ALL. Alternatively, AF4-MLL, the recip-rocal product of MA4, has been suggested to be an oncogenicdriver in t(4;11)þ B-ALL (6), even in the absence ofMA4.WhetherAF4-MLL is oncogenic in human CD34þ cells remains to beexplored.

Importantly, however, enforced KRASG12V expression in IBM-transplantedMA4-expressing CD34þ HSPCs and MA4þ cell lines

Prieto et al.





KRASwt t(4;11) pts

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KRASmut vs. KRAS germline t(4;11)+ ptsGene regulationDifferentially expressed genes

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Figure 5.Gene expression profiling comparing KRAS-mutated versus KRAS nonmutated MA4þ B-ALL infants. A, heatmap diagram depicting the genes differentiallyexpressed (20%up- or downregulated;P<0.01) in PBblasts from t(4:11) patientswith (n¼ 5) orwithout (n¼ 7)KRASmutations. The right color bar codifies the geneexpression level in a log2 scale. B, scheme representing the number of genes differentially expressed in KRAS-mutated versus nonmutated t(4;11)þ infants(left). A total of 764 geneswere differentially expressed, of which 658 (86%) were upregulated and 106 (14%) were downregulated in KRAS-mutated t(4;11) patients(right). C and D, statistically significant GO biologic functions identified using Gorilla software of the genes differentially upregulated (C) or downregulated(D) in KRAS-mutated versus KRAS-germline PB blasts from t(4;11)þ infant B-ALL, ranked by enrichment score. Enrichment score, black bars (right y-axis);�log P value, filled red circle with red line (left y-axis). E, qRT-PCR confirming that coexpression of both MA4 and KRASG12V induces a pronounceddownregulation of CXCR4 and MET and an upregulation of NELIN and DLC1 in CB-CD34þ HSPCs.






induced highWBC counts, robustly enhanced in vivo extramedul-lary hematopoiesis and BM exit. Similarly, KRASG12V expressionconferred t(4;11)þ cells a robust ability to cross blood–brainbarrier, inducing CNS infiltration. In vitro migratory transwellassays performed with SEM cells and CB-CD34þ HSPCs con-firmed the migratory role for KRASG12V in MA4-expressing (pre)-leukemia cells. Furthermore, global gene expression profiling andGO analysis in PB blasts from t(4;11)þ patients revealed anupregulation of a migratory-motility genes coupled to down-regulation of cell adhesion aggregation and anchoring genes,further supporting a KRAS-mediated BM exit and extramedullaryhematopoiesis of MA4-expressing CB-CD34þ and SEM cells.Elevated WBC counts and CNS involvement are hallmark phe-notypes observed in MA4þ pro–B-ALL infants (37).

Our study indicates that KRAS does not cooperate with MA4 toinitiate leukemia in CB-CD34þHSPCs but affect the homeostasisof MA4-expressing HSPCs, suggesting that KRAS activation inMA4þ B-ALL may be important in tumor maintenance/dissem-ination rather than initiation. Our data support previous WGSeqstudies inMLL-r B-ALL where KRASmutations are subclonal andlost at relapse. We propose that MA4-mediated transformationdepends on alternative epigenetic cooperating lesions and on acritical target cell in a developmentally earlier windowof stem cellvulnerability.

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

Authors' ContributionsConception and design: C. Prieto, C. BuenoDevelopment of methodology: C. Prieto, C. Bueno, P. MenendezAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.):C. Prieto, R.W. Stam, P. Ballerini,M. Camos, A. BursenAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): C. Prieto, A. Agraz-Doblas, J. Casta~no, R. Marschalek,I. Varela, C. Bueno, P. MenendezWriting, review, and/or revision of the manuscript: C. Prieto, C. Bueno,P. MenendezAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): C. Prieto, C. Bueno, P. MenendezStudy supervision: C. Bueno, P. Menendez

Grant SupportThis work was supported by the European Research Council (P. Menendez),

The Spanish Ministry of Economy (ISCIII/FEDER-PI14/01119 to C. Bueno;SAF2013-43065R to P. Menendez; and SAF2012-31627 to I. Varela), TheSpanish Cancer Association (P. Menendez and C. Bueno), Fundaci�on Inocente(P.Menendez), and theDeutsche Jos�eCarreras Leuk€amie Stiftung (P.Menendezand R. Marschalek). C. Bueno is supported by aMiguel Servet contract (CPII13/00011). I. Varela is supported by Ram�on Cajal Program. C. Prieto is supportedby PFIS scholarship (FI12/00468). P. Menendez also acknowledges supportfrom Obra Social La Caixa-Fundaci�o Josep Carreras and Generalitat de Cata-lunya (SGR330).

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 October 13, 2015; revised December 31, 2015; accepted January 8,2016; published OnlineFirst February 2, 2016.

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2016;76:2478-2489. Published OnlineFirst February 2, 2016.Cancer Res Cristina Prieto, Ronald W. Stam, Antonio Agraz-Doblas, et al. HSPC But Is Insufficient to Initiate Leukemia

+Extramedullary Engraftment and Migration of Cord Blood CD34 Cooperates with MLL-AF4 to PromoteKRASActivated

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