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Translational Science

Targeting CDK6 and BCL2 Exploits the "MYBAddiction" of Phþ Acute Lymphoblastic LeukemiaMarco De Dominici1, Patrizia Porazzi1, Angela Rachele Soliera2,Samanta A. Mariani1, Sankar Addya1, Paolo Fortina1, Luke F. Peterson3,Orietta Spinelli4, Alessandro Rambaldi4, Giovanni Martinelli5, Anna Ferrari5,Ilaria Iacobucci5, and Bruno Calabretta1

Abstract

Philadelphia chromosome–positive acute lymphoblastic leu-kemia (Phþ ALL) is currently treated with BCR-ABL1 tyrosinekinase inhibitors (TKI) in combinationwith chemotherapy.How-ever, most patients develop resistance to TKI through BCR-ABL1–dependent and –independentmechanisms. Newly developed TKIcan target Phþ ALL cells with BCR-ABL1–dependent resistance;however, overcoming BCR-ABL1–independent mechanisms ofresistance remains challenging because transcription factors,which are difficult to inhibit, are often involved. We show herethat (i) the growth of PhþALL cell lines and primary cells is highlydependent on MYB-mediated transcriptional upregulation ofCDK6, cyclin D3, and BCL2, and (ii) restoring their expressioninMYB-silenced PhþALL cells rescues their impairedproliferationand survival. Levels of MYB and CDK6 were highly correlated in

adult PhþALL (P¼ 0.00008).Moreover, PhþALL cells exhibited aspecific requirement for CDK6 but not CDK4 expression, mostlikely because, in these cells, CDK6 was predominantly localizedin the nucleus,whereasCDK4was almost exclusively cytoplasmic.Consistent with their essential role in Phþ ALL, pharmacologicinhibition of CDK6 and BCL2markedly suppressed proliferation,colony formation, and survival of PhþALL cells ex vivo and inmice.In summary, these findings provide a proof-of-principle, rationalstrategy to target the MYB "addiction" of Phþ ALL.

Significance: MYB blockade can suppress Philadelphia chro-mosome-positive leukemia in mice, suggesting that this ther-apeutic strategy may be useful in patients who develop resis-tance to imatinib and other TKIs used to treat this disease.Cancer Res; 78(4); 1097–109. �2017 AACR.

IntroductionB-cell acute lymphoblastic leukemia (ALL) is a molecularly

heterogeneous malignancy with

and celastrol is a potent proteasome inhibitor that has pleiotropiceffects including the inhibition of NF-kB (28, 29).

To overcome these limitations, we sought to identify MYB-dependent pathways thatmaybe targeted therapeutically.We showhere that cyclin D3, CDK6, and BCL2 are relevant MYB targets withessential roles for in vitro growth and leukemogenesis of Phþ ALLcells. These findings provide a "proof of concept" demonstration ofhow to exploit the TF "addiction" of leukemic cells.

Materials and MethodsCell culture

BV173 (CML-lymphoid blast crisis cell line) were kindly pro-vided by Dr. N. Donato (NIH), SUP-B15 (Phþ ALL cell line) werepurchased from the ATCC, and Z181 (Phþ ALL cell line) werekindly provided by Dr. Z. Estrov (M.D. Anderson Cancer Center,Houston, TX). TKI-resistant BV173 cells were generated bystep-wise selection in the presence of increasing concentrationsof imatinib, which induced the outgrowth of cells with theBCR-ABL1 T315I mutation. Experiments were performed on celllines cultured for less than 30 passages. Mycoplasma was testedmonthly following an established procedure (30). Cell lines wereroutinely authenticated by monitoring B-cell markers and BCR-ABL1 isoform expression. Cell lines were cultured in Iscove'sMedium (Gibco) supplemented with 10% FBS, 100 U/mL pen-icillin–streptomycin, and 2mmol/L L-glutamine at 37�C. Primaryhuman Phþ ALL cells were maintained in SFEM (Stem CellTechnology) supplemented with SCF (40 ng/mL), Flt3L (30ng/mL), IL3 (10 ng/mL), IL6 (10 ng/mL), and IL7 (10 ng/mL;PeproTech). Information on primary Phþ ALL samples used inthis study is shown in Supplementary Table S1.

Cell proliferation, cell-cycle analysis, and colony formationassay

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro-mide (MTT) assay was performed in 96-multiwell plates. Cellswere incubated with 0.5 mg/mLMTT (Sigma Aldrich) at 37�C for2 hours; then, formazan crystals were dissolved with 0.1 mol/LHCl in 2-propanol and absorbance was measured at 570 nm.Cell-cycle analyses were performed by propidium iodide staining(50 mg/mL) of cells permeabilized with 0.1% Triton and 0.1%sodium citrate followed by flow cytometry determination ofDNAcontent. For clonogenic assays, cells were pretreatedwith 1 mg/mLdoxycycline (Research Product International) for 24 hours ortreatedwith drugs and immediately seeded in 1%methylcellulosemedium (Stem Cell Technology) at 2,500 to 5,000 cells/mL.Colonies were counted after 7 to 10 days.

ImmunoblotCells were counted and lysed at a density of 10,000/mL in

Laemmli Buffer. Lysateswhere run onpolyacrylamide gels (Biorad),transferred onto nitrocellulose membranes, and incubated withprimary antibodies (described in Supplementary Methods) andhorseradish peroxidase–conjugated secondary antibodies (ThermoFisher Scientific). Images were obtained by chemiluminescent reac-tion and acquisition on autoradiography films (Denville Scientific).Different antibodies were probed on the same nitrocellulose mem-brane; if necessary, previous signals were removed by incubation instripping buffer (62 mmol/L Tris-HCl, pH 6.8, 2% SDS, b-mercap-toethanol 0.7%) for 20minutes at 50�Corby incubationwith0.5%sodium azide for 10 minutes at room temperature.

Quantitative reverse-transcription PCRRNA was isolated with RNeasy Plus Mini Kit (Qiagen) and

reverse-transcribed with the High-Capacity cDNA Reverse Tran-scription Kit (Thermo Fisher Scientific). A total of 10 ng of cDNAwas used as template and amplified with Power SYBR-Green PCRMaster Mix (Thermo Fisher Scientific). When possible, primerswere designed to span exon–exon junctions and are listed in theSupplementary Methods section.

Lentiviral/retroviral vectorsFor MYB silencing, we used the MYB shRNA kindly provided

by Dr. Tom Gonda (31). For silencing of p21 (the proteinproduct of the CDKN1A gene), CDK4, and CDK6, the pLKO.1plasmids constitutively expressing the shRNAs and conferringpuromycin resistance were purchased from GE Dharmacon[pLKO.1-Scramble: Addgene #1864; p21 (CDKN1A) shRNA:GE Dharmacon #TRCN0000040125; CDK4 shRNA: GE Dhar-macon #TRCN0000000363; CDK6 shRNA: GE Dharmacon#TRCN0000010081]. For exogenous expression of CDK6, theRNA extracted from BV173 cells was reverse transcribed, and thefull-length cDNA corresponding to transcript variant 1 (NCBI:NM_001259.6) was PCR-amplified with a forward primer intro-ducing the XbaI restriction site and a reverse primer introducingthe BamHI site. Then the product was digested and inserted in theXbaI-BamHI sites of the lentiviral vector pUltra-hot developed byDr.MalcolmMoore (Addgene plasmid # 24130), which expressesthe cDNA of interest and the mCherry protein as a bi-cistronictranscript under the control of the ubiquitin C promoter. ThecyclinD3 cDNA(NCBI:NM_001760.4)was similarly obtainedbytotal RNA purified from BV173 cells and inserted in the XbaI-BamHI sites of the pUltra-chili lentiviral vector (Dr. MalcolmMoore; Addgene plasmid #48687), which expresses dTomato as areporter protein. To obtain a nucleus-localized CDK4 protein,CDK4 (NM_000075.3) was PCR amplified from BV173 cDNA byusing a forward primer introducing an XbaI site and a reverseprimer introducing a BamHI site following a sequence encodingthe nuclear localization signal from the SV40 large T antigen(CCAAAGAAGAAGCGTAAGGTA). The CDK4-NLS product wasthen inserted in the XbaI-BamHI sites of the pUltra-Hot vector.MYB cDNA was PCR amplified from the MigR1-MYB plasmid(32) and inserted into theXbaI-NheI sites of pUltra-hot. To obtainthe shRNA-resistantMYB cDNA, the entire plasmidwas amplifiedwith primers harboring point mutations in the MYB sequencetargeted by the shRNA, which were designed to preserve theintegrity of the amino acid sequence of MYB. Then, the linearplasmid was self-ligated and sequenced to confirm that theexpected mutations were introduced. The E308G mutation wasintroduced by a similar method. The pLXSP-BCL2 retrovirus waspreviously described (33). Lentiviral or retroviral VSV-G pseudo-typed particles were produced by calcium phosphate transfectionof plasmid vectors into HEK-293T cells in combination withhelper plasmids. Twenty-four hours later, the supernatant wascollected, 0.45 mm filtered, and used to transduce BV173 or SUP-B15 cells by spinoculation in the presence of 8 mg/mL polybrene(Sigma-Aldrich). Transduced cells were either FACS-purifiedon the basis of the fluorescent reporter protein or selected with3 mg/mL puromycin (Sigma-Aldrich).

Microarray analysisMYB-shRNA SUP-B15 and BV173 cells were treated for 24

hours with doxycycline or left untreated. RNA was isolated by

De Dominici et al.

Cancer Res; 78(4) February 15, 2018 Cancer Research1098

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Published OnlineFirst December 12, 2017; DOI: 10.1158/0008-5472.CAN-17-2644

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using the RNeasy Plus Mini Kit (Qiagen). RNA was quantifiedon a Nanodrop ND-100 spectrophotometer, followed by RNAquality assessment by analysis on an Agilent 2200 TapeStation(Agilent Technologies). Fragmented, biotin-labeled cDNA wassynthesized using the Affymetrix WT Plus Kit (Thermo FisherScientific) for BV173 cells or the Ovation PICOWTA-system V2and cDNA biotin module (NuGen Technologies) for SUP-B15cells. cDNA from BV173 cells was hybridized onto Affymetrixgene chips, and Human Clariom S assays (Thermo FisherScientific) and cDNA from SUP-B15 cells were hybridized ontoHuman Gene 1.0 ST Array (Thermo Fisher Scientific) followingthe manufacturer's instructions. Arrays were scanned on anAffymetrix Gene Chip Scanner 3000, using Command ConsoleSoftware. Quality Control of the experiment was performed byExpression Console Software v1.4.1. Genes were considereddifferentially expressed when cDNA fold change was >1.5.Differentially expressed genes common to both datasets wereused for pathway analysis using IPA software. Microarray dataobtained in this study have been deposited in NCBI GeneExpression Omnibus under GEO Series accession numberGSE105826.

ImmunofluorescenceCells were cytospun on polylysine-coated glass slides, fixed

with 3.7% formaldehyde, permeabilized with Triton 0.1%, andincubated overnight at 4�Cwith primary antibodies anti-CDK4oranti-CDK6 followedbyAlexa Fluor 594–conjugated anti-rabbit oranti-mouse secondary antibodies (Thermo Fisher Scientific, 1hour at room temperature) and then mounted with DAPI-Fluor-omount G (Southern Biotech #0100-20). Imaging was acquiredwith a Nikon Eclipse Ti C2 laser confocal microscope withobjective Plan Apo 60X/1.40 oil and processedwithNIS ElementsAR 4.5 software.

Chromatin immunoprecipitationBV173 and SUP-B15 cells were processed with the SimpleChIP

Enzymatic Chromatin IP Kit (Cell Signaling Technology; #9002)following the manufacturer's instruction. Chromatin from 4 �106 cells was used for each immunoprecipitation with specificprimary antibodies or equal amounts of normal rabbit immu-noglobulins. The purified DNA segments were quantified byqPCR and normalized to the amount of input material.

AnimalsPhþ ALL cells (2� 106 cells/mouse) were injected intravenous-

ly in 6- to 8-week-old NOD/SCID-IL-2Rg–null mice (JacksonLaboratory). Doxycycline was administered at 2 g/L in the drink-ingwater starting 7days after injection. Peripheral blood leukemiccells were monitored by flow cytometry detection of GFP, humanCD10, and/or CD19 (based on cell type–specific expression). Forimmunoblot analysis, leukemic cells were purified from murinecells by FACS or with the EasySep Mouse/Human ChimeraIsolation Kit (StemCell Technologies). Palbociclib (obtained byPfizer) was dissolved at 15 mg/mL in 50 mmol/L sodium lactate,pH¼ 4.0, and given by oral gavage for 10 consecutive days at 150mg/kg. Sabutoclax (SelleckChem) was dissolved at 0.5 mg/mL in10:10:80 Kolliphor-EL (Sigma-Aldrich)-ethanol-PBS and admin-istered intraperitoneally every other day at 5 mg/kg for a total offivedoses. Animal experimentswere approvedby the InstitutionalAnimal Care and Use Committee of Thomas Jefferson Universityunder protocol number 00012.

Statistical analysisResults are expressed as mean � SEM. Statistical significance

was determined by unpaired two-tailed Student t test. Bonferronicorrection was applied in cases of multiple comparisons. Corre-lation studies were analyzed by the Pearson test, and significancewas calculated by the Student t distribution. Significance insurvival experiment was assessed by the log-rank test. For drugcombination studies, the combination index (CI) was calculatedby the Chou–Talalay method (34), and synergism was defined asCI < 1.

ResultsMYB silencing suppresses Phþ ALL cell growth

We showed previously that loss of aMyb allele impairs colonyformation of p190-BCR-ABL1–transformed B-cell progenitorsand suppresses B-cell leukemia in mice, but has no effect onnormal B-cell development (15).

To investigate if MYB is similarly required in Phþ ALL, weassessed the effects of MYB silencing in human cell lines BV173,SUP-B15, and Z181 transduced with the doxycycline (DOX)-inducible, GFP-expressing pLVTSH-MYB shRNA lentiviral vec-tor (31). DOX treatment abolished MYB expression in the threecell lines (Fig. 1A; Supplementary Fig. S1A and S1B). Comparedwith controls, DOX-treated BV173 cells exhibited (i) cell-cyclearrest in the G0–G1 phase (Fig. 1B); (ii) growth inhibitionrevealed by MTT assays (Fig. 1C); and (iii) reduced colonyformation (Fig. 1D). These effects were also observed in SUP-B15 and Z181 cells (Supplementary Fig. S1C–S1F). Thesefindings were not due to off-target effects, because expressionof an MYB cDNA carrying synonymous point mutations on theshRNA target sequence (Supplementary Fig. S2A–S2C) rescuedthe impaired proliferation and colony formation of MYB-silenced BV173 cells (Supplementary Fig. S2D and S2E). Theeffects of MYB silencing were also tested in TKI-resistant T315IBV173 cells; as shown in Fig. 1E–H, growth suppressioninduced by DOX treatment was undistinguishable from thatin parental BV173 cells. As expected, treatment with imatinib ordasatinib markedly suppressed colony formation of parentalBV173 cells but had no effect on the TKI-resistant derivative line(compare Fig. 1D and H).

MYB silencing suppresses leukemia development in NOD/SCID-IL-2Rg–null mice

Then, we assessed the requirement of MYB for leukemiadevelopment in NOD/SCID-IL-2Rg–null (NSG) mice (35)injected with BV173 cells and treated with DOX. Six weeksafter cell injection, DOX treatment induced a dramatic decreaseof leukemia burden revealed by flow cytometry and cytokine-independent colony formation of bone marrow BV173 cells(Fig. 2A and B).

Mice injected with shMYB BV173 cells were also monitored forsurvival. Untreated mice died of leukemia (bone marrow heavilyinfiltrated by leukemic cells and splenomegaly) within 8 weeks(average survival ¼ 42 days); mice given DOX to induce MYBsilencing survived up to 200 days with no signs of disease, exceptfor one animal whose cause of death could not be determined(Fig. 2C). All mice were sacrificed at the 200th-day endpoint, andflow cytometry of bone marrow cells revealed no leukemic cells(Supplementary Fig. S3A), indicating that MYB silencing marked-ly suppressed or eradicated the disease. As expected, ectopic

CDK6 and BCL2 Mediate the MYB Addiction of Phþ ALL

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expression of shRNA-resistant MYB rescued the leukemogenesisof shMYB BV173 cells (Fig. 2C).

DOX treatment also induced a marked increase in the survivalof NSG mice injected with shMYB SUP-B15 cells. Control micesurvived 53.0 � 2.2 days; by contrast, one DOX-treated mousedied 115 days after cell injection with no obvious signs of disease(50% (SupplementaryFig. S3C, lane 6).

We also investigated the requirement of MYB in NSG miceinjected with shMYB-transduced patient-derived Phþ ALL cells(ALL-674). For this experiment, transduced cells (approximately10% GFP-positive) were expanded in a recipient NSG mouse,

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MYB silencing reduces viability andproliferation of Phþ ALL BV173 cellline and of its TKI-resistant (T315I)derivative. A–C, immunoblot (A),cell-cycle analysis at 48 hours oftreatment (B), and cell growth at theindicated times (C) of untreated orDOX-treated BV173 shMYB cells. D,methylcellulose colony formationassay of BV173 shMYB cellsuntreated, treated with DOX toinduce MYB silencing (shMYB),imatinib (IM) 1 mmol/L, or dasatinib(DAS) 2 nmol/L. E–H, Experimentsperformed as in A–D on theTKI-resistant cell line T315I BV173shMYB. N.S., nonsignificant.

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GFP-sorted from the bone marrow and injected in female NSGrecipient mice to obtain a more efficient engraftment (33). Leu-kemia progression was monitored by periodic analysis of totalleukemic (CD19þ) or shMYB-transduced (GFPþ) cells. At 6weeks, DOX-treated mice showed reduced numbers of CD19þ

or GFPþ cells compared with untreated mice; however, by 10weeks, we observed an outgrowth of leukemic cells, a fraction ofwhichwasGFP-negative (Fig. 2D). In addition, amongGFPþ cells,average GFP intensity was reduced (Fig. 2E) and MYB expressionwas not downregulated (Fig. 2F), suggesting selection of leukemiccells expressing low levels of the MYB shRNA.

MYB modulates the expression of cell-cycle–regulatory genesTo investigate mechanisms responsible for the "MYB depen-

dence" of Phþ ALL cells, we performed microarray analysis ofMYB-silenced SUP-B15 and BV173 cells. Seventy-nine genes weredifferentially expressed in both cell lines (Fig. 3A). Ingenuitypathway analysis revealed that cell-cycle progression, DNA rep-

lication, and cell-cycle checkpoints were the pathways mostsignificantly affected by MYB silencing. In addition, BCL2 wasalso significantly downregulated (Fig. 3A). qPCR analysis con-firmed the downregulation of CDK6, cyclin D3 (CCND3), CDK2,cyclin E2 (CCNE2), and FOXM1 and the upregulation of the CDKinhibitor p21 (CDKN1A; Fig. 3B; Supplementary Fig. S4A). Immu-noblots of MYB-silenced BV173 and SUP-B15 cells confirmed theincrease in p21 expression and the downregulation of CDK6 andcyclin D3 (Fig. 3C and D); these changes were associated withmarkedly reduced CDK4/6-dependent RB phosphorylation (36,37) and expression of FOXM1 (Fig. 3C and D), a CDK4/6substrate stabilized through phosphorylation (38). ExpressionofRBwas partially reduced inMYB-silenced cells; instead, levels ofp130-RBL2were increased and the proteinmigrated faster than inuntreated cells consistent with loss of phosphorylation (Fig. 3Cand D). These changes were confirmed in MYB-silenced ALL-674cells purified from the bonemarrow of two DOX-treated (4 days)mice (Fig. 3E). By chromatin immunoprecipitation (ChIP) in

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MYB silencing suppresses leukemogenesis of Phþ ALL cells in immunodeficient mice. A and B, Leukemia burden in untreated or DOX-treated NSG mice (n ¼ 4)injected with BV173 shMYB cells assessed by flow cytometry analysis of GFP-positive bone marrow cells (A) or methylcellulose colony formation assay (B).C, Kaplan–Meier survival plot of untreated or DOX-treated NSG mice injected with BV173 shMYB cells or with BV173 shMYB cells expressing a shMYB-resistantform of MYB. D, Peripheral blood leukemia burden (percentage of GFPþ or CD19þ cells) in untreated (n ¼ 6) or DOX-treated (n ¼ 5) NSG mice injected withprimary Phþ ALL (#674) cells transduced with the shMYB lentivirus. E, Flow cytometry analysis of GFP positivity in the bone marrow of terminally ill untreatedor DOX-treated NSG mice injected with ALL-674 shMYB cells (gated to exclude GFP-negative cells, plots display results from one representative untreatedand one DOX-treated mouse). F, Immunoblot of MYB expression in GFP-sorted bone marrow cells from NSG mice injected with ALL-674 shMYB cells andcontinuously treated with DOX for 11 weeks (lanes 2–4) or after a four-day (4D) treatment initiated when peripheral blood GFP-positive cells were >50% (10 weeksafter injection). N.S., nonsignificant.






BV173 (Fig. 4A–C) and SUP-B15 cells (Supplementary Fig. S4B),we detected binding of MYB to the promoter of the CDKN1Aand CCND3 genes and to the promoter and intron 5 enhancerof the CDK6 gene (39). These regions contain putative MYB-binding sites, suggesting that CDK6, cyclin D3, and p21 expres-sion is directly regulated by MYB. As a positive control, MYBwas detected at the promoter of the BCL2 gene in BV173 andSUP-B15 cells (Supplementary Fig. S4C). To further investigatethe significance of these findings, mRNA levels of MYB and itsputative targets were analyzed in 24 primary human Phþ ALLsamples. We observed a strikingly positive correlation betweenMYB and CDK6 expressions (P ¼ 0.00008), whereas thatbetween MYB and cyclin D3 or p21 was not significant afterBonferroni correction (Fig. 4D–F). A positive correlationbetween MYB and CDK6 expressions was also noted in 226samples of adult B-cell ALL (Fig. 4G; ref. 40), in 207 samples ofPh-negative, high-risk childhood ALL (Fig. 4H; ref. 41), and in174 AML samples from The Cancer Genome Atlas (Fig. 4I).

CDK6 but not CDK4 expression is necessary for Phþ ALL cellproliferation

InMYB-silenced BV173 and SUP-B15 cells, expression of CDK6is markedly downregulated while levels of CDK4 are not affected(Fig. 5A and B). Because CDK4 and CDK6 should have redundant

roles in G1–S phase transition and CDK4 is expressed in mostcases of Phþ ALL (Fig. 5C), we asked whether decreased CDK6levels can explain the cell-cycle arrest of MYB-silenced cells. Thus,BV173 and SUP-B15 cells were transduced with CDK4 or CDK6shRNAs. Surprisingly, CDK4 silencing had negligible effects,whereas CDK6 silencing suppressed cell-cycle progression, RBphosphorylation, and FOXM1 expression in both lines (Fig.5D–G). These data suggest that in Phþ ALL cells, CDK6 exerts afunction that is not shared by CDK4. Interestingly, confocalmicroscopy analysis revealed that CDK6 is predominantly local-ized in the nucleus of Phþ ALL cells, whereas CDK4 is almostexclusively cytoplasmic (Fig. 5H). To investigate whether thecytoplasmic localization of CDK4 may explain its inability torescue the growth suppression induced by CDK6 silencing, anucleus-localized form of CDK4 (CDK4-NLS) was expressed inBV173 shMYB cells transduced with a lentiviral vector expressingcyclin D3 (because the latter is also downregulated in MYB-silenced cells). In cells expressing cyclin D3 alone, CDK4 waspredominantly cytoplasmic; by contrast, CDK4wasmostly nucle-ar in cells expressing CDK4-NLS (Supplementary Fig. S5A). UponDOX treatment to silence MYB expression, coexpression of cyclinD3 and CDK4-NLS partially restored RB phosphorylation andFOXM1 expression (Supplementary Fig. S5B) and resulted in asignificant increase in the percentage of proliferating cells

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MYB silencing alters the expression of cell-cycle–regulatory genes. A, Heatmap of 79 genes significantly modulated in BV173 shMYB and SUP-B15 shMYBcell lines (C1, C2, and C3 denote untreated controls; D1, D2, and D3 denote samples treated with DOX for 24 hours). B, qPCR analysis of CDK6, cyclin D3 (CCND3),p21 (CDKN1A), CDK2, cyclin E2 (CCNE2), and p21 (CDKN1A) expression in BV173 shMYB cells untreated or DOX-treated for the indicated time points (normalized toGAPDH). C–E, Immunoblot analysis of cell-cycle–regulatory genes in MYB-silenced Phþ cell lines (C and D) or ALL-674 shMYB cells DOX-treated for 4 daysin vivo and GFP-sorted (E).

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(Supplementary Fig. S5C) compared with empty vector (EV)–transduced cells.

Restoring expression of CDK6, cyclin D3, and BCL2 rescuescell-cycle arrest and apoptosis induced by MYB silencing

Next, we asked whether restoring the expression of cyclin D3and CDK6 would be sufficient to rescue the proliferative arrest ofMYB-silenced cells.

Expression of CDK6 alone partially restored RB phosphor-ylation and rescued FOXM1 expression, whereas expression ofcyclin D3 alone did not (Supplementary Fig. S6A). CDK6expression partially rescued the reduced growth of MYB-silenced cells, whereas cyclin D3 expression had a modest effect(Supplementary Fig. S6B).

Coexpression of CDK6 and cyclin D3 (K6/D3) restored RBphosphorylation and FOXM1, CDK2, and cyclin E2 (CCNE2)expression (Fig. 6A–C), and rescued the cell-cycle arrest of cellstreatedwithDOXfor2days (Fig. 6DandE).However,MYB-silencedK6/D3 cells grew less vigorously after 4 days of DOX treatment(Fig. 6F) and did not form colonies in methylcellulose (Fig. 6G).

Based on these findings, we asked whether induction of apo-ptosis could explain the reduced growth of MYB-silenced K6/D3BV173 cells. Indeed, although sub-G1 apoptotic cells were notdetected by cell-cycle analysis of shMYB BV173 or SUP-B15 cellsperformed after 2 days of DOX treatment (Fig. 1B and F, Fig. 6Dand E; Supplementary Fig. S1B), a 4-dayDOX treatment increasedthe percentage of Annexin Vþ cells and induced caspase 3 cleavagein EV and K6/D3 cells (Fig. 6H and I).

0.750.9

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MYB regulates the expression of cell-cycle–regulatory genes and is coexpressed with CDK6 in different types of leukemia. A–C, MYB binding by ChIP toregulatory regions of the CDK6 (A), CCND3 (B), or CDKN1A (C) genes in BV173 cells (numbers refer to the position of the forward primer relative to the TSS).As negative controls, ChIPs were performed with a nontargeting antibody (white bars) or with an anti-MYB antibody on lysates from MYB-silenced BV173cells (gray bars).D–F,Plot of the correlation between themRNA levels (by qPCR) ofMYB andCDK6 (D),MYB andCCND3 (E), orMYB andCDKN1A (F) in 24 samples ofprimary human Phþ ALL samples. G, Plot of the correlation between the expression (by microarray) of MYB and CDK6 in a panel of 226 B-cell ALL of mixedcytogenetics (GSE79533). H, Plot of the correlation between the expression (by microarray) of MYB and CDK6 in a panel of 207 Ph-negative childhood, high-riskALL (GSE11877). I, Plot of the correlation between the expression of MYB and CDK6 based on The Cancer Genome Atlas data on 174 samples of AML ofmixed cytogenetics (RNA-seq, median expression).






BCL2 was previously reported to be a MYB target (42–45),and thus, its decreased expression may contribute to the apo-ptosis of MYB-silenced cells (Fig. 6J and K). To test thishypothesis, we generated a derivative shMYB BV173 K6/D3cell line named KDB in which levels of ectopically expressedBCL2 were not affected by MYB silencing (Fig. 6L). In thesecells, BCL2 expression inhibited apoptosis, restored cellgrowth, and partially rescued the loss of colony formationinduced by MYB silencing (Fig. 6M–O).

The defective colony formation potential ofMYB-silenced KDBcellswasnot causedby increasedp21 expressionbecause silencingp21 did not further increase colony formation of DOX-treatedKDB cells (Supplementary Fig. S6C–S6F).

Finally, we tested whether these derivative lines induce leuke-mia in NSG mice. Mice injected with shMYB BV173 cells expres-sing K6/D3 or BCL2 alone and given DOX did not developleukemia. However, 10 of 12 DOX-treated mice injected withKDB or KDBp cells developed leukemia, and 9 of them suc-cumbed to the disease by 200 days (Fig. 6P).

The recruitment of p300/CBP is required for MYB-dependentcontrol of proliferation but is dispensable for its antiapoptoticeffects

The interactionbetweenMYBand thehistone acetyltransferasesp300/CBP is critically important for MYB function in hemato-poietic cells (46–48). To assess the importance of p300/CBP

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Expression of CDK6 but not CDK4 is required for the proliferation of Phþ ALL cells. A–C, Immunoblot of CDK6 and CDK4 expression in untreated or DOX-treated BV173 shMYB cells (A) or SUP-B15 shMYB cells (B) and in untreated primary Phþ ALL cells (C). D and E, Representative experiment showing thecell-cycle analysis of scramble, CDK4, or CDK6 constitutive shRNA-transduced BV173 (D) or SUP-B15 (E) cells. Compared with scramble-transduced cells, thedecrease in the percentage of S phase cells is significant in BV173 shCDK6 cells (P ¼ 10�5) and in SUP-B15 shCDK6 cells (P ¼ 0.001), but not in BV173 shCDK4or SUP-B15 shCDK4 cells (results are from three independent experiments). F and G, Immunoblot analysis of CDK4, CDK6, RB phosphorylation, and FOXM1expression in scramble, CDK4, or CDK6 shRNA–transduced BV173 (F) or SUP-B15 (G) cells. H, Subcellular localization of CDK6 (left panels) or CDK4 (right panels)in BV173, SUP-B15, and primary human Phþ ALL cells (ALL-1222 and ALL-3934) by immunofluorescence-confocal microscopy (scale bar, 20 mm).

De Dominici et al.





recruitment for MYB transcriptional effects, shMYB BV173cells were transduced with the EV, the shRNA-resistant wild-type(WT) MYB, or the shRNA-resistant MYB-E308G mutant thatdoes not interact with p300/CBP (48) and were treated withDOX to silence endogenous MYB.

Although expression of shRNA-resistant MYB WT complete-ly rescued the effects of MYB silencing, DOX-treated MYB-E308G–expressing cells displayed decreased S phase and col-ony formation but no increase in Annexin V positivity (Sup-plementary Fig. S7A–S7C). By immunoblot analysis, expres-sion of MYB-E308G rescued levels of BCL2 but not CDK6 and

FOXM1 or RB phosphorylation (Supplementary Fig. S7D). Toinvestigate mechanisms that may explain the differentialrequirement of p300/CBP in the regulation of CDK6 andBCL2, ChIP experiments were performed at the regulatoryregions of these genes.

DOX-treated EV-transduced cells showed reduced levels ofMYB and p300 binding and H3K27 acetylation at the promoterand enhancer of CDK6 and at the promoter of BCL2, comparedwith cells expressing MYB-WT. MYB-E308G–expressing cells alsoshowed reduced p300 binding and H3K27 acetylation at thepromoter and enhancer of CDK6, but H3K27 acetylation was

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Coexpression of CDK6/Cyclin D3 and BCL2 partially rescues the effects of MYB silencing. A, Immunoblot for MYB and its targets in untreated or DOX-treated(96 hours) BV173 shMYB EV or BV173 shMYB K6/D3 cells.B andC, qPCR forCDK2 (B) and cyclin E2 (C) in untreated or DOX-treated (48 hours) BV173 shMYB EV cellsor BV173 shMYB K6/D3 cells. D and E, Cell-cycle analysis of untreated or DOX-treated (48 hours) BV173 shMYB EV (D) or BV173 shMYB K6/D3 (E) cells. F,Cell growth by MTT assay of untreated or DOX-treated BV173 shMYB EV cells or BV173 shMYB K6/D3 cells (vertical axis is shown as a logarithmic scale). G, Colonyassay of untreated or DOX-treated BV173 shMYB K6/D3 cells. H and I, Annexin V positivity (top) and expression of cleaved caspase-3 by immunoblot (bottom) inuntreated or DOX-treated (2, 4, or 6 days) BV173 shMYB EV (H) and BV173 shMYBK6/D3 (I) cells. J andK, qPCR forBCL2 (top) and immunoblot for BCL2 (bottom) inuntreated or DOX-treated (2 or 4 days) BV173 shMYB EV (J) or BV173 shMYB K6/D3 (K) cells. L, Immunoblot of EV and KDB derivative BV173 shMYB cell linesuntreated or treated with DOX for 4 days. M–O, Annexin V positivity (M), cell growth by MTT assay (N; vertical axis is shown as a logarithmic scale), andmethylcellulose colony formation assay (O) of untreated or DOX-treated KDB cells. P, Kaplan–Meier survival plot of NSG mice injected with untreatedBV173 shMYB or DOX-treated BV173 shMYB derivative cell lines.






not reduced at the BCL2 promoter, in spite of lower p300binding (Supplementary Fig. S7E–S7G).

Cotreatment with theCDK4/6 inhibitor palbociclib and a BCL2antagonist inhibits growth of Phþ ALL cells ex vivo andsuppresses leukemia burden in NSG mice

Based on their essential role in cell growth, CDK6 and BCL2might serve as targets to exploit the MYB "addiction" of Phþ ALLcells. First, we investigated the effects of the CDK4/CDK6 inhib-itor palbociclib in primary Phþ ALL cells. Similar to its effects incell lines (49), palbociclib inhibited the S phase of Phþ ALL cells(Supplementary Fig. S8A). Moreover, it suppressed colony for-mation of primary Phþ ALL cells, whereas normal CD34þ cellswere less affected (Supplementary Fig. S8B). Then,we assessed theeffect of palbociclib inNSGmice injected with three different Phþ

ALL samples. Compared with vehicle-treated mice, drug-treatedanimals displayed lower numbers of peripheral blood CD19þ

leukemia cells 6 to 8weeks after cell injection (Supplementary Fig.S8C). However, the effect was transient, probably reflecting thecytostatic effects of palbociclib and/or the insufficient length ofthe treatment.

To mimic more faithfully the effects of MYB silencing, we usedpalbociclib in combination with the BCL2 antagonist venetoclax(50). The palbociclib/venetoclax combination had synergisticeffects in BV173 and additive effects in SUP-B15 cells (Supple-mentary Fig. S9A–S9D). SUP-B15 cells were markedly moresensitive to venetoclax than BV173 cells (Supplementary Fig.S9E and S9F), likely because of lower MCL1 and BCL-XL andhigher BIM expression (Supplementary Fig. S9G). Primary Phþ

ALL cells display low proliferation in vitro, preventing analysis ofthe effects induced by the palbociclib/venetoclax combination.However, treatment with venetoclax induced apoptosis in three

Phþ ALL samples, albeit at different drug concentrations (Sup-plementary Fig. S9H–S9J). In particular, sample #1006 wasresistant to treatment with venetoclax, most likely because ofmuch higher BCL-XL andMCL1 levels than in the other samples(Supplementary Fig. S9K).

Due to the sample-to-sample variability in expression of BCL2familymembers and sensitivity to venetoclax, we tested the effectsof the pan-BCL2 inhibitor sabutoclax (51). Treatment with thepalbociclib/sabutoclax combination synergistically reduced thenumber of BV173 and SUP-B15 cells at most doses (Supplemen-tary Fig. S9L–S9O, respectively).

Next, we evaluated the effect of the palbociclib/sabutoclaxcombination on leukemia progression in NSGmice injected withALL-674 or ALL-1222 cells.

Treatment with palbociclib induced a moderate reduction inperipheral blood leukemia burden (17% and 32% decrease inALL-674 and ALL-1222 samples, respectively), whereas treat-ment with sabutoclax had negligible effects. However, thecombined treatment was significantly more effective thaneither drug alone (77% reduction for ALL-674, Fig. 7A andB and 90% reduction for ALL-1222, Fig. 7C and D) in sup-pressing leukemia load.

DiscussionWe show here that MYB expression is critically important for

growth and leukemogenesis of PhþALL cells. Mechanistically, theeffects of MYB depend on transcriptional regulation of manytargets, among which, CDK6 and BCL2 are crucial mediators ofits proliferative and antiapoptotic functions.

In addition toMYB-dependent regulation, several mechanismsmay contribute to enhanced expression/activity of CDK6 in Phþ

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Sabutoclax and palbociclib cooperateto suppress Phþ ALL in NSG mice. Aand B, Percentage of CD19þ cells inthe peripheral blood (A) and foldchanges in leukemia burden (B) ofuntreated and drug-treated NSGmiceinjected with sample ALL-674 before(5 weeks after injection) and aftertreatment (7 weeks after injection).C and D, Percentage of CD19þCD10þ

cells in the peripheral blood (C) andfold changes in leukemia burden (D)of untreated and drug-treated NSGmice injected with sample ALL-1222before (5 weeks after injection) andafter treatment (7 weeks afterinjection); palbociclib was given byoral gavage for 10 consecutive days at150 mg/kg, whereas sabutoclax wasadministered intraperitoneally everyother day (five doses, 5 mg/kg).

De Dominici et al.





ALL. TheCDK4/6 inhibitor INK4A is structurally altered in 30% to50% of Phþ ALL (11, 52), the CDK6 promoter is hypomethylatedin BCR-ABL1–transformed cells (53), and miR-124 is epigenet-ically silenced in PhþALL, resulting in higher CDK6 levels (54). Inaddition to its kinase-dependent effects, CDK6 is also involved intranscriptional regulation (55, 56).

Silencing CDK6 (but not CDK4) alone suppressed S phaseentry of PhþALL cells; however, this effectmay not require kinase-independent mechanisms because it was undistinguishable fromthat induced by pharmacologic inhibition of CDK4/6. The essen-tial role of CDK6 was noted in MLL-rearranged leukemias(57, 58), but no explanation was provided for the differentialrequirement of CDK4 and CDK6. Here, we show that CDK6 isreadily detectable in thenucleus ofPhþALL cells, whereasCDK4 isalmost exclusively cytoplasmic. The exclusion of CDK4 from thenucleus likely explains its inability to compensate for the growthinhibition induced by CDK6 downregulation because expressionof a nucleus-localized form of CDK4, in combination with cyclinD3 expression, partially rescued the impaired proliferation ofMYB-silenced cells.

We do not know the reasons for the lack of nuclear import ofCDK4 in PhþALL cells. A possible explanationmight reside in theexpression levels of different cyclin D proteins because CDK4appears to interact preferentially with cyclin D1, whereas CDK6binds preferentially to cyclin D3 (59) and levels of cyclin D3mRNA are much higher than those of cyclin D1 in BV173 andSUP-B15 cells, based on microarray data. This may affect bothnuclear import and kinase activity of CDK4.

Interestingly,MYB expression is required for leukemogenesis inamodel ofMLL-AF9AML (22) andMLL-rearrangedALL, andAMLcells are also dependent on CDK6 but not on CDK4 expression(57, 58). Furthermore, MYB and CDK6 expression is highlycorrelated in ALL and in adult AML, suggesting that MYB mightlink different oncogenic pathways to the activation of CDK6expression.

Decreased susceptibility to apoptosis mediated by BCL2 is alsoimportant for MYB-dependent regulation of Phþ ALL cell growth.However, even in combination with CDK6/Cyclin D3, BCL2enabled MYB-deficient BV173 cells to develop leukemia onlyafter long latency, suggesting that other MYB-regulated pathwaysare important for a complete recovery of the leukemogenicpotential of these cells.

A criticalmechanismwherebyMYB regulates gene expression isthrough its interaction with p300/CBP, because mutations ofMYB in the p300/CBP-binding sites phenocopy the impairedhematopoiesis induced by MYB knockout and prevent leukemiaformation (46–48). The MYB–p300 interaction seems necessaryfor CDK6 expression and cell-cycle progression but not for reg-ulation of BCL2 expression and cell survival. Thus, drugs thatdisrupt this interaction might have limited efficacy in Phþ ALLunless MYB-regulated antiapoptotic pathway effects are alsotargeted.

Our approach of simultaneously inhibiting MYB-dependentproliferative andantiapoptotic programs recapitulatesmore faith-fully the effects of MYB silencing in Phþ ALL, as indicated by themore potent suppression of ex vivo and in vivo cell growth by

combining palbociclib with venetoclax or sabutoclax. Of interest,venetoclax treatment of patient-derived Phþ ALL cells revealedsample-to-sample variations in induction of apoptosis. Thesefindings likely depend on the expression profile of BCL2 familymembers and suggest that the choice of a particular BCL2 antag-onist in the clinic should be guided by the pattern of BCL2 familyexpression and the ex vivo drug sensitivity of Phþ leukemia cellsfrom individual patients.

On the other hand, given that CDK4 is dispensable in Phþ ALL,CDK6-selective inhibitors might prove as effective as dual CDK4/6 inhibitors in blocking the proliferation of Phþ ALL cells andmay cause fewer side effects in normal cells.

In conclusion, our data indicate that targeting CDK6 andBCL2 is an effective strategy to exploit therapeutically the MYBaddiction of Phþ ALL cells and might provide an alternativetreatment for patients who develop resistance to TKI-basedtherapies.

Disclosure of Potential Conflicts of InterestG. Martinelli is a consultant/advisory boardmember for Abbvie, Incyte, Jazz

Pharma, J&G, and Pfizer. No potential conflicts of interest were disclosed by theother authors.

Authors' ContributionsConception and design: M. De Dominici, B. CalabrettaDevelopment of methodology: M. De Dominici, S. AddyaAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): M. De Dominici, P. Porazzi, A.R. Soliera, P. Fortina,O. Spinelli, A. Rambaldi, A. FerrariAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): M. De Dominici, S.A. Mariani, S. Addya, P. Fortina,G. Martinelli, B. CalabrettaWriting, review, and/or revision of the manuscript: M. De Dominici,P. Porazzi, S. Addya, O. Spinelli, B. CalabrettaAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): M. De Dominici, P. Fortina, L.F. Peterson,A. FerrariStudy supervision: M. De Dominici, P. Fortina, B. CalabrettaOther (provided patient samples): I. Iacobucci

AcknowledgmentsThis work was supported by NCI grant RO1-CA167169 (B. Calabretta).We thankDrs. A.M.Mazo and C.M. Eischen for critically reviewing the article

and V. Minieri for edits to the text. We thank Drs. S.B. McMahon, T.L. Manser,and A.E. Aplin for helpful discussions and sharing of materials throughout theproject. We thank Dr. M. Caliguri from the Ohio State University and Dr. M.Carrol from the StemCell and Xenograft Core of the University of Pennsylvaniafor providing primary Phþ ALL samples. We thank Dr. N. Flomenberg and theBoneMarrowTransplantation unit at Thomas JeffersonUniversity for providingCD34þ human hematopoietic progenitor cells. We thank Pfizer for kindlyproviding palbociclib and Dr. T. Gonda for kindly providing the pLVTSH-MYBshRNA vector. We also thank C. Kugel, E. Greenawalt, and R. DeRita for theirhelp in obtaining preliminary results.

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received August 30, 2017; revised October 25, 2017; accepted December 8,2017; published OnlineFirst December 12, 2017.

References1. Rowe JM, Buck G, Burnett AK, Chopra R, Wiernik PH, Richards SM, et al.

Induction therapy for adults with acute lymphoblastic leukemia: results ofmore than 1500 patients from the international ALL trial: MRCUKALL XII/ECOG E2993. Blood 2005;106:3760–7.






2. Dores GM, Devesa SS, Curtis RE, Linet MS, Morton LM. Acute leukemiaincidence and patient survival among children and adults in the UnitedStates, 2001–2007. Blood 2012;119:34–43.

3. PulteD, Jansen L, Gondos A, Katalinic A, Barnes B, RessingM, et al. Survivalof adults with acute lymphoblastic leukemia in Germany and the UnitedStates. PloS One 2014;9:e85554.

4. Gleissner B, GokbugetN, BartramCR, JanssenB, RiederH, Janssen JW, et al.Leading prognostic relevance of the BCR-ABL translocation in adult acuteB-lineage lymphoblastic leukemia: a prospective study of the GermanMulticenter TrialGroup and confirmedpolymerase chain reaction analysis.Blood 2002;99:1536–43.

5. Wetzler M, Dodge RK, Mrozek K, Carroll AJ, Tantravahi R, Block AW,et al. Prospective karyotype analysis in adult acute lymphoblasticleukemia: the cancer and leukemia Group B experience. Blood 1999;93:3983–93.

6. Fielding AK, Rowe JM, Richards SM, BuckG,Moorman AV,Durrant IJ, et al.Prospective outcome data on 267 unselected adult patients with Philadel-phia chromosome-positive acute lymphoblastic leukemia confirms supe-riority of allogeneic transplantation over chemotherapy in the pre-imatinibera: results from the International ALL Trial MRC UKALLXII/ECOG2993.Blood 2009;113:4489–96.

7. YanadaM, Takeuchi J, Sugiura I, AkiyamaH, Usui N, Yagasaki F, et al. Highcomplete remission rate and promising outcome by combination ofimatinib and chemotherapy for newly diagnosed BCR-ABL-positive acutelymphoblastic leukemia: a phase II study by the Japan Adult LeukemiaStudy Group. J Clin Oncol 2006;24:460–6.

8. Soverini S, De Benedittis C, Papayannidis C, Paolini S, Venturi C, IacobucciI, et al. Drug resistance and BCR-ABL kinase domain mutations in Phila-delphia chromosome-positive acute lymphoblastic leukemia from theimatinib to the second-generation tyrosine kinase inhibitor era: The mainchanges are in the type of mutations, but not in the frequency of mutationinvolvement. Cancer 2014;120:1002–9.

9. Calabretta B, Perrotti D. The biology of CML blast crisis. Blood 2004;103:4010–22.

10. Williams RT, Sherr CJ. The INK4-ARF (CDKN2A/B) locus in hematopoiesisand BCR-ABL-induced leukemias. Cold Spring Harbor Symposia QuantitBiol 2008;73:461–7.

11. Mullighan CG, Miller CB, Radtke I, Phillips LA, Dalton J, Ma J, et al. BCR-ABL1 lymphoblastic leukaemia is characterized by the deletion of Ikaros.Nature 2008;453:110–4.

12. FeldhahnN,HenkeN,Melchior K,DuyC, SohBN,Klein F, et al. Activation-induced cytidine deaminase acts as a mutator in BCR-ABL1-transformedacute lymphoblastic leukemia cells. J Exp Med 2007;204:1157–66.

13. KlemmL, Duy C, Iacobucci I, Kuchen S, von LevetzowG, FeldhahnN, et al.The B cell mutator AID promotes B lymphoid blast crisis and drugresistance in chronic myeloid leukemia. Cancer cell 2009;16:232–45.

14. Perrotti D, Cesi V, Trotta R, Guerzoni C, Santilli G, Campbell K, et al. BCR-ABL suppresses C/EBPalpha expression through inhibitory action ofhnRNP E2. Nature Genet 2002;30:48–58.

15. WaldronT,DeDominiciM, SolieraAR, AudiaA, Iacobucci I, Lonetti A, et al.c-Myb and its target Bmi1 are required for p190BCR/ABL leukemogenesisin mouse and human cells. Leukemia 2012;26:644–53.

16. Chai SK,NicholsGL, RothmanP.Constitutive activation of JAKs and STATsin BCR-Abl-expressing cell lines and peripheral blood cells derived fromleukemic patients. J Immunol 1997;159:4720–8.

17. Soliera AR, Lidonnici MR, Ferrari-Amorotti G, Prisco M, Zhang Y, MartinezRV, et al. Transcriptional repression of c-Myb andGATA-2 is involved in thebiologic effects of C/EBPalpha in p210BCR/ABL-expressing cells. Blood2008;112:1942–50.

18. Lidonnici MR, Corradini F, Waldron T, Bender TP, Calabretta B. Require-ment of c-Myb for p210(BCR/ABL)-dependent transformation of hemato-poietic progenitors and leukemogenesis. Blood 2008;111:4771–9.

19. Ratajczak MZ, Hijiya N, Catani L, DeRiel K, Luger SM, McGlave P, et al.Acute- and chronic-phase chronic myelogenous leukemia colony-formingunits are highly sensitive to the growth inhibitory effects of c-myb antisenseoligodeoxynucleotides. Blood 1992;79:1956–61.

20. Calabretta B, Sims RB, Valtieri M, Caracciolo D, Szczylik C, Venturelli D,et al. Normal and leukemic hematopoietic cells manifest differentialsensitivity to inhibitory effects of c-myb antisense oligodeoxynucleotides:an in vitro study relevant to bone marrow purging. Proc Natl Acad Sci USA1991;88:2351–5.

21. Lahortiga I, De Keersmaecker K, Van Vlierberghe P, Graux C, Cauwelier B,Lambert F, et al. Duplication of the MYB oncogene in T cell acutelymphoblastic leukemia. Nature Genet 2007;39:593–5.

22. Zuber J, Rappaport AR, Luo W, Wang E, Chen C, Vaseva AV, et al. Anintegrated approach to dissecting oncogene addiction implicates a Myb-coordinated self-renewal program as essential for leukemia maintenance.Genes Dev 2011;25:1628–40.

23. Bujnicki T, Wilczek C, Schomburg C, Feldmann F, Schlenke P, Muller-Tidow C, et al. Inhibition of Myb-dependent gene expression by thesesquiterpene lactone mexicanin-I. Leukemia 2012;26:615–22.

24. GuzmanML, Rossi RM, Karnischky L, Li X, Peterson DR, Howard DS, et al.The sesquiterpene lactone parthenolide induces apoptosis of human acutemyelogenous leukemia stem and progenitor cells. Blood 2005;105:4163–9.

25. Kawasaki BT, Hurt EM, Kalathur M, DuhagonMA, Milner JA, Kim YS, et al.Effects of the sesquiterpene lactone parthenolide on prostate tumor-ini-tiating cells: An integrated molecular profiling approach. Prostate2009;69:827–37.

26. Uttarkar S, Dukare S, Bopp B, Goblirsch M, Jose J, Klempnauer KH.Naphthol AS-E phosphate inhibits the activity of the transcription factorMyb by blocking the interaction with the KIX domain of the coactivatorp300. Mol Cancer Ther 2015;14:1276–85.

27. Uttarkar S, Dasse E, Coulibaly A, Steinmann S, Jakobs A, Schomburg C,et al. Targeting acute myeloid leukemia with a small molecule inhibitor ofthe Myb/p300 interaction. Blood 2016;127:1173–82.

28. Yang H, ChenD, Cui QC, Yuan X, DouQP. Celastrol, a triterpene extractedfrom the Chinese "Thunder of God Vine," is a potent proteasome inhibitorand suppresses human prostate cancer growth in nude mice. Cancer Res2006;66:4758–65.

29. Lee JH, Koo TH, Yoon H, Jung HS, Jin HZ, Lee K, et al. Inhibition of NF-kappa B activation through targeting I kappa B kinase by celastrol, aquinone methide triterpenoid. Biochem Pharmacol 2006;72:1311–21.

30. Young L, Sung J, Stacey G, Masters JR. Detection of mycoplasma in cellcultures. Nat Protoc 2010;5:929–34.

31. Drabsch Y, Hugo H, Zhang R, Dowhan DH, Miao YR, Gewirtz AM, et al.Mechanism of and requirement for estrogen-regulated MYB expression inestrogen-receptor-positive breast cancer cells. Proc Natl Acad Sci USA2007;104:13762–7.

32. Corradini F, Cesi V, Bartella V, Pani E, Bussolari R, Candini O, et al.Enhanced proliferative potential of hematopoietic cells expressing degra-dation-resistant c-Myb mutants. J Biol Chem 2005;280:30254–62.

33. Corradini F, Bussolari R, Cerioli D, Lidonnici MR, Calabretta B. A degra-dation-resistant c-Myb mutant cooperates with Bcl-2 in enhancing prolif-erative potential and survival of hematopoietic cells. Blood Cells Mol Dis2007;39:292–6.

34. Chou TC, Talalay P. Quantitative analysis of dose-effect relationships: thecombined effects of multiple drugs or enzyme inhibitors. Adv EnzymeRegulation 1984;22:27–55.

35. Ito M, Hiramatsu H, Kobayashi K, Suzue K, Kawahata M, Hioki K, et al.NOD/SCID/gamma(c)(null) mouse: an excellent recipient mouse modelfor engraftment of human cells. Blood 2002;100:3175–82.

36. Schmitz NM, Hirt A, Aebi M, Leibundgut K. Limited redundancy inphosphorylation of retinoblastoma tumor suppressor protein by cyclin-dependent kinases in acute lymphoblastic leukemia. Am J Pathol 2006;169:1074–9.

37. Takaki T, Fukasawa K, Suzuki-Takahashi I, Semba K, Kitagawa M, Taya Y,et al. Preferences for phosphorylation sites in the retinoblastomaprotein ofD-type cyclin-dependent kinases, Cdk4 and Cdk6, in vitro. J Biochem2005;137:381–6.

38. Anders L, Ke N, Hydbring P, Choi YJ, Widlund HR, Chick JM, et al. Asystematic screen for CDK4/6 substrates links FOXM1 phosphorylation tosenescence suppression in cancer cells. Cancer cell 2011;20:620–34.

39. Roe JS, Mercan F, Rivera K, Pappin DJ, Vakoc CR. BET bromodomaininhibition suppresses the function of hematopoietic transcription factorsin acute myeloid leukemia. Mol Cell 2015;58:1028–39.

40. Hirabayashi S, Ohki K, Nakabayashi K, Ichikawa H, Momozawa Y, Oka-mura K, et al. ZNF384-related fusion genes define a subgroup of childhoodB-cell precursor acute lymphoblastic leukemia with a characteristic immu-notype. Haematologica 2017;102:118–29.

41. Kang H, Chen IM, Wilson CS, Bedrick EJ, Harvey RC, Atlas SR, et al. Geneexpression classifiers for relapse-free survival andminimal residual disease

De Dominici et al.





improve risk classification and outcomeprediction in pediatric B-precursoracute lymphoblastic leukemia. Blood 2010;115:1394–405.

42. Taylor D, Badiani P, Weston K. A dominant interfering Mybmutant causesapoptosis in T cells. Genes Dev 1996;10:2732–44.

43. Frampton J, Ramqvist T, Graf T. v-Myb of E26 leukemia virus up-regulatesbcl-2 and suppresses apoptosis in myeloid cells. Genes Dev 1996;10:2720–31.

44. Salomoni P, Perrotti D, Martinez R, Franceschi C, Calabretta B. Resistanceto apoptosis in CTLL-2 cells constitutively expressing c-Myb is associatedwith induction of BCL-2 expression andMyb-dependent regulation of bcl-2 promoter activity. Proc Natl Acad Sci USA 1997;94:3296–301.

45. Jing D, Bhadri VA, Beck D, Thoms JA, YakobNA,Wong JW, et al. Opposingregulation of BIM and BCL2 controls glucocorticoid-induced apoptosis ofpediatric acute lymphoblastic leukemia cells. Blood 2015;125:273–83.

46. Sandberg ML, Sutton SE, Pletcher MT, Wiltshire T, Tarantino LM, Hogen-esch JB, et al. c-Myb and p300 regulate hematopoietic stem cell prolifer-ation and differentiation. Dev Cell 2005;8:153–66.

47. Papathanasiou P, Tunningley R, Pattabiraman DR, Ye P, Gonda TJ, WhittleB, et al. A recessive screen for genes regulating hematopoietic stem cells.Blood 2010;116:5849–58.

48. PattabiramanDR,McGirr C, Shakhbazov K, Barbier V, Krishnan K,Mukho-padhyay P, et al. Interaction of c-Myb with p300 is required for theinduction of acute myeloid leukemia (AML) by human AML oncogenes.Blood 2014;123:2682–90.

49. Nemoto A, Saida S, Kato I, Kikuchi J, Furukawa Y, Maeda Y, et al. Specificantileukemic activity of PD0332991, a CDK4/6 Inhibitor, against Phila-delphia chromosome-positive lymphoid leukemia. Mol Cancer Ther2016;15:94–105.

50. Souers AJ, Leverson JD, Boghaert ER, Ackler SL, Catron ND, Chen J, et al.ABT-199, a potent and selective BCL-2 inhibitor, achieves antitumoractivity while sparing platelets. Nature Med 2013;19:202–8.

51. Wei J, Stebbins JL, Kitada S, Dash R, Placzek W, Rega MF, et al. BI-97C1, anoptically pure Apogossypol derivative as pan-active inhibitor of antiapop-totic B-cell lymphoma/leukemia-2 (Bcl-2) family proteins. J Med Chem2010;53:4166–76.

52. Iacobucci I, Ferrari A, Lonetti A, Papayannidis C, Paoloni F, Trino S, et al.CDKN2A/B alterations impair prognosis in adult BCR-ABL1-positive acutelymphoblastic leukemia patients. Clin Cancer Res 2011;17:7413–23.

53. Kollmann K, Heller G, Ott RG, Scheicher R, Zebedin-Brandl E, Schneck-enleithner C, et al. c-JUN promotes BCR-ABL-induced lymphoid leukemiaby inhibiting methylation of the 5' region of Cdk6. Blood 2011;117:4065–75.

54. Agirre X, Vilas-Zornoza A, Jimenez-Velasco A, Martin-Subero JI, Cordeu L,Garate L, et al. Epigenetic silencing of the tumor suppressormicroRNAHsa-miR-124a regulates CDK6 expression and confers a poor prognosis in acutelymphoblastic leukemia. Cancer Res 2009;69:4443–53.

55. Kollmann K, Heller G, Schneckenleithner C, Warsch W, Scheicher R, OttRG, et al. A kinase-independent function of CDK6 links the cell cycle totumor angiogenesis. Cancer cell 2016;30:359–60.

56. Scheicher R, Hoelbl-Kovacic A, Bellutti F, Tigan AS, Prchal-Murphy M,HellerG, et al. CDK6as a key regulatorof hematopoietic and leukemic stemcell activation. Blood 2015;125:90–101.

57. van der Linden MH, Willekes M, van Roon E, Seslija L, Schneider P, PietersR, et al. MLL fusion-driven activation of CDK6 potentiates proliferation inMLL-rearranged infant ALL. Cell cycle (Georgetown, Tex) 2014;13:834–44.

58. Placke T, Faber K, Nonami A, Putwain SL, Salih HR, Heidel FH, et al.Requirement for CDK6 inMLL-rearranged acute myeloid leukemia. Blood2014;124:13–23.

59. Spofford LS, Abel EV, Boisvert-AdamoK, Aplin AE. CyclinD3 expression inmelanoma cells is regulated by adhesion-dependent phosphatidylinositol3-kinase signaling and contributes to G1-S progression. J Biol Chem2006;281:25644–51.






2018;78:1097-1109. Published OnlineFirst December 12, 2017.Cancer Res Marco De Dominici, Patrizia Porazzi, Angela Rachele Soliera, et al. Acute Lymphoblastic Leukemia

+Targeting CDK6 and BCL2 Exploits the ''MYB Addiction'' of Ph

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