Inactivation of Citron Kinase Inhibits Medulloblastoma ... · Medulloblastoma (MB) is the most common malignant brain tumor in children (1). medulloblastoma has been classiﬁed into

Tumor Biology and Immunology

Inactivation of Citron Kinase InhibitsMedulloblastoma Progression by InducingApoptosis and Cell SenescenceGianmarco Pallavicini1,2, Francesco Sgr�o1, Francesca Garello1, Mattia Falcone1,3,4,Valeria Bitonto1, Gaia E. Berto1,2, Federico T. Bianchi1,2, Marta Gai1,Alessandra M.A. Chiotto1,2, Miriam Filippi1, Juan C. Cutrin1, Ugo Ala1, Enzo Terreno1,Emilia Turco1, and Ferdinando Di Cunto1,2,5

Abstract

Medulloblastoma is the most common malignant braintumor in children. Current treatment for medulloblastomaconsists of surgery followed by irradiation of the wholeneuraxis and high-dose multiagent chemotherapy, a par-tially effective strategy associated with highly invalidatingside effects. Therefore, identification and validation of noveltarget molecules capable of contrasting medulloblastomagrowth without disturbing brain development is needed.Citron kinase protein (CITK), encoded by primary micro-cephaly geneMCPH17, is required for normal proliferationand survival of neural progenitors. Constitutive loss of CITKleads to cytokinesis failure, chromosome instability, andapoptosis in the developing brain, but has limited effects onother tissues. On this basis, we hypothesized that CITKcould be an effective target for medulloblastoma treatment.In medulloblastoma cell lines DAOY and ONS-76, CITKknockdown increased both cytokinesis failure and DNAdamage, impairing proliferation and inducing cell senes-cence and apoptosis via TP53 or TP73. Similar effects wereobtained in theNeuroD-SmoA1 transgenicmousemodel, inwhich CITK deletion increased apoptotic cells and senes-cence markers such as P21CIP1, P27KIP1, and P16INK4A. Mostimportantly, CITK deletion decreased tumor growth andincreased overall survival in these mice, with no apparentside effects. These results suggest that CITK can be a usefulmolecular target for medulloblastoma treatment.

Significance: In vitro and in vivo proof of concept identifies citron kinase protein as a suitable target for medulloblastomatreatment.

Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/78/16/4599/F1.large.jpg. Cancer Res; 78(16); 4599–612.�2018 AACR.

IntroductionMedulloblastoma (MB) is the most common malignant brain

tumor in children (1). medulloblastoma has been classified into

five subgroups based on histology and into four biological sub-groups based on microarray and genomic sequencing technolo-gies: WNT, Sonic Hedgehog (SHH), group 3, and group 4 (2–6).

1Department of Molecular Biotechnology and Health Sciences, University of Turin,Torino, Italy. 2Neuroscience Institute Cavalieri Ottolenghi, University of Turin,Torino, Italy. 3Division of Stem Cells and Cancer, German Cancer Research Center(DKFZ) and DKFZ-ZMBH Alliance, Heidelberg, Germany. 4Heidelberg Institute forStem Cell Technology and Experimental Medicine (HI-STEM gGmbH), Heidelberg,Germany. 5Department of Neuroscience, University of Turin, Torino, Italy.

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

G. Pallavicini and F. Sgr�o contributed equally to this article.

Corresponding Author: Ferdinando Di Cunto, University of Turin, Neuro-science Institute Cavalieri Ottolenghi, Regione Gonzole 10, Torino, TO10126, Italy. Phone: 390116706616; Fax: 390116706621; E-mail:[email protected]

doi: 10.1158/0008-5472.CAN-17-4060

�2018 American Association for Cancer Research.

CancerResearch

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WNT medulloblastoma are associated with relatively good prog-nosis, whereas groups 3 and 4, which often show MYC amplifi-cation and TP53 mutation, are the most aggressive subtypes (5).Current treatments for medulloblastoma include surgery fol-lowed by irradiation of the whole neuraxis and high-dose mul-tiagent chemotherapy (7). Five-year survival rate for childrenwithaverage andhigh-risk disease, as definedby clinical criteria, is 80%and 60–65%, respectively (8). Therefore, many of the high-riskpatients die despite treatment (9). In addition, survivors havesignificant neurologic and developmental consequences, such assevere cognitive deficits and endocrine disorders (10–15). Thus,novel and more specific therapies are urgently needed. A possiblestrategy to develop new selective therapies is to directly targetmolecular pathways altered by driver mutations. SHH subgroup,representing approximately 25%ofmedulloblastoma cases, is thebetter understood medulloblastoma subtype: it is characterizedby mutations of SHH signaling pathway downstream compo-nents (PTCH1, SMO, SUFU, and GLI2) and it is derived fromgranule neuron precursors (GNP) of the cerebellum (16–18).Small-molecule inhibitors of the SHH pathway have been devel-oped and tested in clinical trials (19). However, only a subgroupof these patients respond to treatment and even in these casesresistance rapidly develops (20–22). An alternative strategy fordrug development is to target molecules that, despite not beingmutated in cancer, are nevertheless required for tumor growth andprogression (23). Cancer cellsmaybecome specifically dependenton particular nonmutant proteins in consequence of peculiarfeatures of the cells from which they originate and/or of themodifications induced by oncogenic transformation (23).

CITK is a conserved multidomain protein, containing anAGC-type amino-terminal serine/threonine kinase domain(24, 25). CITK is ubiquitously expressed (26) and cell-cycleregulated, with highest levels in G2–M phase of the cell cycle(27). During mitosis, it is first localized to the nucleus (27), it isenriched at spindle poles before anaphase (28), it is concentratedat cleavage furrow and midbody from anaphase to cytokinesis(25). CITK is a specific regulator of abscission at the end ofcytokinesis (25, 29–32). In addition, it regulates mitotic spindleorientation (33) and it is involved in genomic stability indepen-dently of its role in cytokinesis (34). Despite ubiquitous expres-sion, CITK is functionally required in vivo only in few cell types,such as neural progenitors (26, 35) andmale germcells (36). CITKloss leads to severemicrocephaly in rodents (26, 37) and humans(38–41). Cells in the affected tissues display cytokinesis failure(26, 40), apoptosis (26, 36, 40), and accumulation of DNAdamage (34). Notably, the human CITKmicrocephaly syndrome,known as MCPH17, can be caused by homozygous kinase-inactivating mutations (40), indicating that catalytic activity isessential for function.

CITK is expressed at high levels in tumors (42–44) and isrequired for normal growth in tumor cell lines of different origin(45–47). Moreover, CITK knockdown reduces tumor growth inhepatocellular carcinoma cells in xenograft assays (45). Theseresults suggest that CITK could be an interesting target for anti-cancer drug development. However, the effects of CITK inactiva-tion have never been tested in primary tumors. Based on currentknowledge (30, 48), SHH medulloblastoma appears to be anideal tumor type for testing the potential of CITK as a candidatedrug target. Indeed, cerebellar GNPs, from which SHH medullo-blastoma develops (16–18), are among the neural progenitorsshowing highest sensitivity to CITK loss (26). Moreover, medul-

loblastoma express high levels of TUBB3 (49),whichmay increasesensitivity toCITK loss (50). Finally, althoughCITK is essential fornormal brain development, it is not required in normal postnataltissues (26, 34), and therefore its acute inactivation should beassociated with relatively mild side effects.

In this report we analyzed the effects of deleting CITK in SHHmedulloblastoma, using in vitro and in vivo models, includingconditional inactivation in primary tumors. Our results supportCITK as a novel drug target for this tumor type.

Materials and MethodsCell culture

DAOY cells were obtained from ATCC and were culturedaccording to the ATCC protocol.

ONS-76 cells were kindly provided by Luigi Varesio (GasliniHospital, Genova) and were cultured in RPMI medium supple-mented with 10% FBS and 1% penicillin/streptomycin (LifeTechnologies). No further authenticationwas performed on thesecells. For both cell lines, the number of in vitro passages fromthawing of the original aliquots to experiments was comprisedbetween 5 and 8. Cells were routinely analyzed formorphologicalfeatures and tested by PCR for mycoplasma contamination withthe following oligonucleotides sequences: MYCO1 50-ACTCC-TACGGGAGGCAGCAGTA-30; MYCO2: 50-TGCACCATCTGTCA-CTCTGTTAACCTC-30.

Transfection and RNAiPreviously published CITK double-stranded RNAs (31) and

nontargeting siRNA were used (Dharmacon). For knockdown ofTP53 and TP73, ONTARGETplus Dharmacon SMART pools wereused. D-001810-10 nontargeting pool was used as a negativecontrol. ONS-76 and DAOY cells plated on six-well plates weretransfected using 6.25 mL of the required siRNA (20 mmol/L)together with 1.5 mL Lipofectamine 2000 (Invitrogen), accordingto the manufacturer's instructions. Efficient knockdown wasobtained after 48 hours.

Lentiviral productionHEK293T cells were cultured in DMEM supplemented with

10% FCS and 1% penicillin/streptomycin (Life Technologies,ThermoFisher). Lentiviral vector stocks were produced inHEK293T cells by calcium phosphate–mediated transfection, asdescribed in ref. 51.

Generation of inducible shRNA cell lines and rescue of CITKexpression

Human CITK-specific shRNA sequences (31) were cloned intopLVTHM vector from Didier Trono's lab (Addgene plasmid#12247; Supplementary Fig. S1A). For inducible RNAi, cells weretransduced with pLV-DsRed-tTRKRAB from Didier Trono lab(Addgene plasmid #12247; Supplementary Fig. S1A), expanded,and used for transduction with pLVTH-GFP-shRNA lentiviralparticles. Next, cells were treated with doxycycline (2 mmol/L)for 12 hours; double GFPþDsRedþ cells were sorted by FACSAria(BD biosciences) and expanded. Cells expressing GFP in theabsence of doxycycline were considered "leaky" and removed bya second flow of cytometry sorting. Purified inducible cellsshowed efficient knockdown after 24 hours, as determined byWestern blot analysis. For rescue experiments, Myc-cherry-taggedCITK overexpression constructs encoding kinase-active or kinase-inactive (S222A) proteins previously described (31) were

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transfected at the same timeof doxycycline inductionwith 10.5mLof Lipofectamine 2000 (ThermoFisher, Invitrogen), accordingto the manufacturer's instructions. Live cells, as assessed bymethylene blue staining, were counted after 24 hours.

Analysis of cell death and proliferationFor apoptosis detection, 5 � 105 cells were collected, centri-

fuged at 2,000 rpm for 5 minutes, and resuspended in 300 mL ofbinding buffer. AnnexinV FITC (3mL)was added andmixed. Afterthe addition of 5 mL of propidium iodide (PI) staining solution,the cells were incubated for 15 to 30 minutes in the dark at roomtemperature and analyzed by flow cytometry to detect cell apo-ptosis. For cell proliferation analysis, 5� 103 ONS-76 and DAOYinducible cells were seeded into 24-well plates and activated usingdoxycycline (2 mmol/L). Live cells were counted in replica wellseach day for 6 days.

Experimental animal workExperiments involving samples from mutant and control mice

have been performed conforming to the Italian laws on animalexperimentation, under permission number 343/2015-PR,released on 08/05/2015 from Italian Ministry of Health, Depart-ment of Public Veterinary Health. Adherence to internationallyaccepted animal well-being standards was further supervised bythe veterinary service of the Molecular Biotechnology Centreanimal facility.

Xenograft assaysSubcutaneous medulloblastoma xenografts were obtained by

transplanting in mice DAOY cells expressing control or CITK-targeted shRNAs under doxycycline-inducible control. Four- toeight-week-old female NOD-SCID mice (Jackson Laboratory)were injected in the flank with 2 � 106 cells. After 6 weeks,doxycycline (10 mg/mL) was added to drinking water for other4 weeks. Xenograft tumors were measured every week and tumorvolume was estimated as 4pr3/3.

Mouse strainsThe mouse strain containing the "knockout first" allele of

CitK Cittm1a(KOMP)Wtsi, in a C57BL/6 background, was obtainedfrom UC Davis KOMP repository. The citron kinase conditionalknockout allele Citflox was obtained by crossing Cittm1a(KOMP)Wtsi

mice with the FLP-balancer line Gt(ROSA)26Sortm2(FLP�)Sor in aC57BL/6J congenic background (The Jackson Laboratory, BarHarbor, Main). The mouse strains Gt(ROSA)26Sortm1(cre/ERT2)Tyj

(ubiquitously-expressing tamoxifen-inducible ERT2-CRE) andND2:SmoA1 (expressing the constitutively active point mutantSmoA1 under the Neurod2 promoter in cerebellar granule cells),both in congenic C57BL/6 background, were obtained from TheJackson Laboratory.

AntibodiesThe following antibodies were used: mouse monoclonal anti-

citron (#611377; Transduction Laboratories, BD Biosciences),mouse monoclonal anti-vinculin (#V9131; Sigma-Aldrich), rab-bit polyclonal anti-gH2AX (S139; 20E3; #2577; Cell SignalingTechnology), mouse monoclonal anti-P27 (#610242; Transduc-tion Laboratories, BD Biosciences), rabbit polyclonal anti-cleavedcaspase-3 (#9661S; Cell Signaling Technology), goat polyclonalanti-cleaved caspase-7 (#sc-6138; Santacruz), rabbit polyclonalanti-P21 (#sc-756; Santacruz), rabbit polyclonal anti-P16(#sc-1207, Santacruz), mouse monoclonal anti-Ki67 (#ab15580,

Abcam), rabbit polyclonal anti-TP53 (#sc-6243; Santacruz),rabbit polyclonal anti-TP73 (#ab14430; Abcam), mouse mono-clonal anti-a-tubulin (#T5168; Sigma-Aldrich), rabbit polyclonalanti-b-actin (#A2066; Sigma-Aldrich).

Western blottingCell lines and tissues were lysed in RIPA buffer (1% NP40,

150 mmol/L NaCl, 50 mmol/L Tris-HCl, pH 8.5 mmol/L EDTA,0.01% SDS, 0.005% sodium deoxycholate, Roche proteaseinhibitors, PMSF) for 10 minutes at 4�C. Mouse tissues werepreviously homogenized with T 25 digital ULTRA-TURRAX.Samples were clarified 10 minutes at 13,000 rpm at 4�C. Forimmunoblots, equal amounts of proteins from both whole-celllysates were resolved by SDS–PAGE and blotted to nitrocellu-lose membranes.

ImmunofluorescenceCultured cells were fixed 5 minutes at RT using PFA 2% then

treated 10 minutes at RT using CSK buffer [100 mmol/L NaCl,300 mmol/L sucrose, 3 mmol/L MgCl2, 10 mmol/L PIPES(pH 6.8)], 0.7% Triton, and finally fixed again 5 minutes at RTusing PFA 2%. Mice were anesthetized by using a mixture ofRompun/Zoletil and trans-cardially perfused with fixative solu-tion (4% paraformaldehyde in PBS). Tumors were post-fixedovernight at 4�C, slowly dehydrated with increasing concentra-tions of sucrose, embedded with Tissue-TEK (O.C.T., SakuraFinetek, Alphen aan den Rijn, The Netherlands), and frozen byusing isopentane. Twenty-micrometers of cryo-sections wererehydrated in PBS before further processing. Cells and sectionswere permeabilized in 0.1% TritonX 100 in PBS for 10 minutes,saturated in 5% BSA in PBS for 30 minutes, and incubated with aprimary antibody for 1 hour at RT. Primary antibodies weredetected with anti-rabbit Alexa Fluor 488 or 568 (MolecularProbes, Invitrogen), anti-mouse Alexa Fluor 488 or 568 (Molec-ular Probes, Invitrogen) used at 1:500 dilution for 30 minutes.Cells were counterstained with 0.5 mg/mL DAPI for 10 minutesand washed with PBS. TUNEL assay was performed 48 hoursposttransfection using the TMR red In Situ Cell Death DetectionKit (Roche), according to manufacturer protocol. Images wereacquired using an ApoTome system (Zeiss) or using a Leica TCSSP5 confocal system (Leica Microsystems) equipped with a405nm diode, an argon ion, and a 561nm DPSS laser.

Detection of senescent cellsSenescent cells in cultures or in xenograft tumors were detected

using in situ b-galactosidase staining, following the protocolreported described in (52).

ImmunohistochemistryPeriodate-lysine-paraformaldehyde (PLP)-fixed and paraffin-

embedded tissue sections were stained with hematoxylin & eosin.For immunohistochemistry assays, de-waxed 5 mm sections weresubmitted to wet heat-induced antigen retrieval in a mixture of0.1M Tris and 0.01M EDTA solution at pH 9.0. Endogenousperoxidase was blocked in 0.6% hydrogen peroxide solution in0.05MTBS (pH7.6) for 20minutes at room temperature. Sectionswere then treated with 5% normal goat serum and reactedovernight with antibodies. Subsequently, sections were incubatedwith goat anti-rabbit (ab97051; Abcam)or anti-mouse (ab97240;Abcam) IgG conjugated to HRP for 1 hour at room temperature.Finally, sections were treated with diaminobenzidine enhancedliquid substrate chromogen system (D3939; Sigma-Aldrich) and

CITK Inactivation Inhibits Medulloblastoma Progression

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counterstained with haematoxylin. Apoptosis was assessed usingIn Situ Cell Death Detection Kit POD (Roche) according tomanufacturer's instructions.

Tamoxifen treatmentIn all in vivo experiments, littermatemice, homozygous for the

Citflox allele, homozygous for the ND2:SmoA1 and possessing(iCREþ) or nonpossessing (iCRE�) the ERT2 allele were treated for5 days with 0.100mg/g of tamoxifen by intraperitoneal injection.

Survival analysisKaplan–Meier method was used to estimate survival. For

tamoxifen-treated mice, 2 months-old littermates, divided iniCREþ and iCRE� cohorts, were treated and monitored forhealth status and movement abnormalities. All mice were eutha-nized at theonset of symptoms, including10%weight loss, ataxia,or lethargy.

To analyze human patients with medulloblastoma survival infunction of CITK expression, raw data of Gene Expression Omni-bus (GEO) dataset GSE85217 (33), generated on AffymetrixHuman Gene 1.1 ST Array platform, were downloaded andnormalized through RMA algorithm as in Bioconductor oligopackage (53). Mapping of Affy IDs to transcripts was based onNetAffx annotation (release na36, hg19). To specifically focus theanalysis on CITK and exclude CITN isoform expression, weselected kinase domain probe-sets (7966920, 7966921,7966922, 7966923, 7966924, 7966925, 7966926, 7966927) andaveraged the corresponding expression values in each sample.Overall Survival data were obtained from (33) and maximalsurvival time was cut at 15 years. Patients were divided in twogroups usingmedian expression as thediscriminant value. Inbothcases, statistical significance was set to P ¼ 0.05. Log-rank (Man-tel–Cox) test was used to compare survival between experimentalgroups.

MRIMRIs were acquired at 7.1 T using a Bruker Avance 300 spec-

trometer. The animals were scannedweekly, starting from8weeksof age, in order to check the presence of tumors throughT2-weighted (T2w) images acquired with the following para-meters: repetition time (TR) ¼ 4,000 milliseconds; echo time(TE) ¼ 59 milliseconds; rare factor (RF) ¼ 24; slice thickness ¼1 mm; 15 slices; field of view ¼ 3.00 cm; matrix ¼ 256 � 256;number of averages (NAV) ¼ 4; total imaging time ¼ 2 minutes40 seconds). Animals bearing a tumormass (size 5–20mm3)werethen imaged with a high-resolution turbo-rare sequence (TR ¼2,500 milliseconds; TE ¼ 36 milliseconds; RF ¼ 8; slice thickness¼ 0.5mm; 20 slices; field of view¼ 3.50 cm;matrix¼ 384� 384;NAV¼ 10; total imaging time¼ 20minutes) before and 7,14, 21,and 28 days after tamoxifen administration, to monitor tumorprogression. Before undergoing MRI animals were anesthetizedby intramuscular injection of 5 mg/kg of xylazine (Rompun;Bayer) and 20 mg/kg of tiletamine/zolazepam (Zoletil 100;Virbac). Tumor volume was assessed in each animal through MRdata analysis, carried out with Bruker Paravision 5.1 Software.More in details, the tumor areawas delineated in each slice of T2whigh-resolution images by manually drawing regions of interestalong tumor borders. The tumor volume in each slice was esti-mated multiplying each tumor area for the slice thickness(0.5 mm). Finally, the total tumor volume was estimated byadding up all the single slice volumes.

Statistical analysisStatistical analyseswere performed usingMicrosoftOffice Excel

or Graphpad. Unpaired two-tails Student t test was used if nototherwise specified. Themean values shown represent the averageof at least three independent experiments and error bars arestandard errors of the mean.

ResultsSHH medulloblastoma cells are sensitive to CITK knockdown

To investigate the dependence of SHH medulloblastoma onCITK expression, we resorted to DAOY and ONS-76 cells, twoestablished human medulloblastoma lines showing a transcrip-tion profile typical of SHH activation (1), and carryingmutated orwild-type TP53, respectively (54). We induced transient CITKknockdown in these cells (Fig. 1A) using two validated siRNAsequences (31) andanalyzedCITK-associated cellular phenotypes48 hours after transfection. CITK loss induced apoptosis in bothcell lines, as shown by high levels of cleaved caspase-3 andcaspase-7 (Fig. 1A and B), as well as by increased rate of AnnexinV-positive cells (Fig. 1C) and of TUNEL-positive cells (Fig. 1D). Inaddition, after CITK knockdown, both lines showed increased rateof binucleated cells, indicative of cytokinesis failure (Fig. 1E andref. 34). We previously demonstrated that RNAi of CITK in ONS-76 cells leads to accumulation of DNA double-strand breaks(DSB), as shown by higher frequency of gH2AX- and TP53BP1-positive nuclear foci and, most importantly, by gain of chromo-some breaks (34). Accordingly, we found that also DAOY cellshave increased frequency of gH2AX nuclear foci after CITK knock-down (Fig. 2A). Interestingly, in both cell lines, transfection of thetwo CITK-specific siRNA sequences significantly enhanced theproportion of cells in G0–G1 (Fig. 2B and C). These result suggestthat, besides inducing cytokinesis failure, CITK loss reduces thenumber of medulloblastoma cells that progress into S-phase andmitosis. Consistent with this observation, CITK-interfered ONS-76 and DAOY cells showed high levels of the cyclin-kinaseinhibitors CDKN1A (P21) and CDKN1B (P27; Fig. 2D and E).

Previous work has shown that CITK loss may elicit both TP53-dependent and TP53-independent effects (34, 46). Accordingly,TP53 knockdown reverted to a great extent to the apoptoticphenotype resulting from CITK RNAi in ONS-76 cells but hadlittle effect in CITK-interfered DAOY cells (Fig. 3A and B). Becausewe previously showed that CITK loss increases TP73 RNA levelsindependently of the TP53 status (34), we assessed whether TP73is involved in phenotypes produced by CITK loss in DAOY cells.As expected, TP73 protein levels are significantly increased inDAOY cells after CITK RNAi (Fig. 3C and D). Moreover, when theinduction of TP73 was prevented by RNAi (Fig. 3C and D),apoptosis levels were significantly reduced (Fig. 3E) and theinduction of growth arrest markers was not observed (Fig. 3Cand F). Interestingly, TP53 or TP73 knockdown induced by itself amoderate increase of apoptosis levels. Taken together, theseobservations suggest that SHH medulloblastoma cells are highlysensitive to CITK loss, which increases the frequency of DNAdamage and cytokinesis failure and leads to apoptosis and cell-cycle progression block via TP53 or TP73.

Stable CITK knockdown impairs the expansion of SHHmedulloblastoma cells in vitro and in xenograft assays

To better evaluate the impact of CITK loss on SHH medullo-blastoma cells' growth, we generated DAOY and ONS-76 cell

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Figure 1.

CITK knockdown leads to apoptosis and cytokinesis failure in medulloblastoma cell lines. A, Western blot analysis of total cell lysates obtained fromDAOY and ONS-76 cells, treated for 48 hours with nontargeting (siCtrl) or CITK-specific (siCITK1, siCITK2) siRNA sequences. Levels of CITK and ofcleaved forms of caspase-3 (cC3) and 7 (cC7) were analyzed. Internal loading control was vinculin (VINC). B, Quantification of the relative density ofcleaved caspase-3 and cleaved caspase-7 in siCITK1 cells, compared with cells treated with siCtrl (N ¼ 5). C, Quantification of Annexin V–positive DAOY andONS-76 cells, 48 hours after transfection with siCtrl or with two different CITK-specific siRNA sequences (N ¼ 15). D, DAOY and ONS-76 cells weretreated for 48 hours with siCtrl or siCITK1 and apoptotic cells were quantified. Left, representative images of DAOY and ONS-76 analyzed with TUNEL(red) and counterstained with DAPI. Right, quantification of TUNEL positive cells. E, DAOY cells, seeded at low density, were processed forimmunofluorescence 48 hours after transfection with nontargeting or CITK-specific siRNA sequences and stained with DAPI and anti-a-tubulin antibodies.The percentage of binucleated cells was then assessed. All quantifications were based on three independent biological replicates, unless notedotherwise. Error bars, SEM. � , P < 0.05; ��, P < 0.01; �� , P < 0.001, two-tailed Student T test. Scale bars, 20 mm. A.U., arbitrary unit.






lines conditionally expressing control and CITK-specific shRNAs.To this aim, we cloned previously validated CITK sequences intoa tightly regulated lentiviral-based system, in which shRNAexpression can be induced by doxycycline administration (Sup-plementary Fig. S1A; ref. 51). Polyclonal populations obtainedfrom both medulloblastoma cell lines showed efficient CITKknockdown 48 hours after doxycycline administration (Supple-mentary Fig. S1B). Stable knockdown of CITK recapitulated allthe phenotypes produced by transient one (Supplementary Fig.

S1C and S1D). Furthermore, in long-term colony forming assay,both DAOY and ONS-76 cells showed reduced growth ratescompared with controls, after CITK shRNA induction by con-tinuous exposure to doxycycline (Fig. 4A and B). In addition,CITK knocked-down cells had a senescent morphology (Fig. 4C).Accordingly, CITK shRNA induction led to strong increase ofcells positive for b-galactosidase cytochemical reaction (Fig. 4D),which is typical of senescent cells (52). To assess whetherantiproliferative effects of CITK loss are dependent on kinase

Figure 2.

medulloblastoma cell linesaccumulate DNA damage and arresttheir cell cycle after CITK knockdown.A, High magnification of DAOY cellstreated for 48 hours with siCtrl orsiCITK1, stainedwith the DNA-damagemarker gH2AX, and counterstainedwith DAPI. Right, quantification ofgH2AX foci per nucleus. B, Flow-cytometric analysis of DNA content inDAOY and ONS-76 cells, treated as inA. C, Quantification of diploid (G0–G1),S-phase, and tetraploid (G2–M) cellsdistribution in experiments performedas in B. All the differences betweenCITK-specific siRNAs and controlswere significant (P < 10�7, Chi-squaretest performed assuming the nullhypothesis that cell-cycle distributionis independent on siRNAs condition).D, Western blot analysis of total celllysates obtained fromDAOYandONS-76 cells, treated as in A. Levels of P27and P21 were analyzed. Internalloading control was vinculin (VINC). E,Quantification of the relative intensityof P27 and P21 inWestern blot analysisperformed as in D. All quantificationswere based on four independentbiological replicates. Error bars, SEM.� , P < 0.05; �� , P < 0.01; ��, P < 0.001(Mann–Withney U test for A;two-tailed Student T test for blots).Scale bars, 20 mm. A.U., arbitrary unit.

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activity, we used ONS-76 cells expressing CITK1 shRNA andperformed rescue assays with overexpression vectors, coding formouse CITK proteins. Interestingly, in CITK knocked down cells,transfection of RNAi-resistant wild-type CITK (Supplementary

Fig. S1E) restored cell growth at control levels, whereas kinase-dead mutant did not revert the phenotype (Fig. 4E). These resultindicate that CITK catalytic activity is required for SHH medul-loblastoma cells proliferation.

Figure 3.

CITK loss in medulloblastoma cellsinduces TP53-dependent andTP53-independent response. A,Western blot analysis of total celllysates obtained from DAOY andONS-76 cells, treated for 48 hourswiththe indicated siRNA sequences (þ).Samples in which a specific targetingsequencewasmissing (-)were treatedwith same amount of nontargetingcontrol sequence. Internal loadingcontrol was vinculin (VINC). B,Quantification of Annexin V-positiveDAOY and ONS-76 cells, 48 hoursafter transfection with the indicatedsiRNA sequences (N ¼ at least to 8).C, Western blot analysis of total celllysates obtained from DAOY cells,treated for 48 hourswith the indicatedsiRNA sequences. D, Quantification ofthe relative intensity of TP73 inWestern blot analyses performed as inC (N¼ 4). E,Quantification of AnnexinV-positive DAOY, 48 hours aftertransfection with the indicated siRNAsequences (N ¼ at least 7).F, Quantification of the relativeintensity of P27 and P21 in Westernblot analyses performed as in C(N ¼ 4). Error bars, SEM. � , P < 0.05;�� , P < 0.01; �� , P < 0.001, two-tailedStudent T test. n.s., nonsignificant;A.U., arbitrary unit.






Figure 4.

CITK loss decreases proliferation and induces senescence in medulloblastoma cell lines. A, DAOY and ONS-76 cells, stably transfected with inducible nontargeting(shCtrl) or CITK-specific shRNA sequences (CITK1 and CITK2), were plated (5,000 cells) in control medium (Vehicle) or in doxycycline-containing (Doxy) medium (2mmol/L). Growth curves were then obtained by assessing cell number in dishes at the indicated times. B, DAOY and ONS-76 cells, expressing nontargeting or CITK-specific shRNA sequences under doxycycline-inducible control, were plated in 35 mm dishes (500 cells/dish) 48 hours after shRNA induction with 2 mmol/Ldoxycycline. After additional 5 days of continuous exposure to doxycycline, the obtained colonieswerefixed and stainedwith crystal violet.C,Phase contrast imagesof representative fields, in cultures of DAOY and ONS-76 cells, expressing shCtrl or shCITK1, 5 days after doxycycline induction. D, Quantification of thepercentage of b-galactosidase–positive colonies in cultures, treated as in B, 5 days after doxycycline administration. E, ONS-76 cells, obtained as in B, wereinduced with doxycycline for 24 hours and then transfected with control vector (OE Myc) or with vectors overexpressing mouse wild type (OE CitK) or Citronkinase inactive form (OE CitKd). Cell number in dishes was then quantified 24 hours later. All quantifications were based on five independent biological replicates.Error bars, SEM. � , P < 0.05; �� , P < 0.01; �� , P < 0.001; n.s., nonsignificant; two-tailed Student T test. Scale bars, 20 mm.

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Next, we investigated whether CITK might be required for SHHmedulloblastoma cell expansion also in vivo, by performing xeno-graft assays with shRNA-expressing cell lines. In particular, con-sidering the establishedmalignant potential ofDAOY cells and thehigher effectiveness of the CITK1 sequence, we subcutaneouslyinjected into immunodeficient mice DAOY cells, transduced withinducible control or with CITK1 shRNAs-containing lentiviruses.Inmice fed with doxycycline-free diet, palpable tumors developedwithin 6 weeks after injection and underwent rapid growth after-wards (Fig. 5A). Doxycycline administration had no significanteffects on growth of tumors expressing control shRNA (Fig. 5A). Incontrast, treatment reduced significantly the growth of CITK1shRNA-expressing tumors (Fig. 5A–C). Interestingly, tumorsexpressing CITK1 shRNA showed also higher positivity to b-galac-tosidase staining, if compared with controls (Fig. 5D). Takentogether, these results suggest that the inhibition of CITK impairsgrowth and induces cell senescence in SHHmedulloblastoma cellsindependently of TP53 status, both in vitro and in vivo.

CITK inactivation inhibits SHH-medulloblastoma progressionCITK is not frequently mutated in human cancers (46) but

is overexpressed in different tumor types (43–45), although it isexpressed at very low levels in normal brain (55, 56). Byre-analyzing gene expression data of a recently published medul-loblastoma cohort (33), we found that tumors expressing CITK atlevels higher than median have worse prognosis (SupplementaryFig. S2A). We therefore asked whether CITK loss-of-function mayelicit anti-neoplastic effects in patient-like medulloblastoma. Tothis aim, we set out to induce CITK inactivation in tumors arisingin the cerebellum of SmoA1 transgenic mice (57). This lineexpresses a constitutively active form of Smoothened gene (Smo)in cerebellar GNPs under the control of NeuroD2 promoter (57)and shows, especially when crossed to homozygosity, high inci-dence ofmedulloblastoma, closelymimicking the human disease(58). SmoA1 medulloblastoma show high expression of CITK, ifcompared with the levels observed during normal cerebellardevelopment (Supplementary Fig. S2B). To inactivate CITK inthese tumors in a temporally-controlled manner, we developed amouse conditional CitK allele, in which the third exon is flankedby LoxP sites (CitKflox; Supplementary Fig. S3A). We then crossedthis allele to homozygosity into transgenic mice constitutivelyexpressing a tamoxifen-inducible ERT2CRE enzyme from Rosa26locus (59). We assessed the effectiveness of conditional deletionby analyzing the ratio between recombined and non-recombinedalleles (Supplementary Fig. S3B), as well as CITK protein levels(Supplementary Fig. S3C), in tissues of mice homozygous forCitKflox (CitKflox/flox) and heterozygous for ERT2CRE, treated withtamoxifen at postnatal day (P)60. Recombination of the CitKflox

allele was efficiently induced by tamoxifen in lymphoid tissues(Supplementary Fig. S3B and S3C). No significant histologicdifferences were observed in thymus and spleen of CITK-deletedand control mice (Supplementary Fig. S3D). One-year follow updid not show increased mortality or gross behavioral differencesof treated ERT2CREþCitKflox/floxmice as comparedwith ERT2CRE-CitKflox/flox controls. CITK deletion was not detected in maturebrain (Supplementary Fig. S3B), which mostly expresses thekinase domain-lacking isoform CITN (Fig. 6A; ref. 56). However,because effectiveness of tamoxifen-induced recombination coulddepend on proliferative activity or differentiation-related phe-nomena, we crossed the above mice with the SmoA1 line, togenerate mice homozygous for SmoA1 and CitKflox alleles, and

Figure 5.

CITK knockdown decreases growth and induces senescence in DAOYxenograft tumors. A, DAOY cells stably transduced with the indicated shRNAvectors were injected subcutaneously in NOD-SCID mice. Tumor growth wasmonitored weekly. Doxycycline (Doxy) 10 mg/mL or ethanol (Vehicle) wasadded to the drinking water 6 weeks after cells injection, and tumor growthwasmonitored for three additional weeks. Analysis of xenograft tumors growth wasperformed by measuring subcutaneous tumor diameter. B, Quantification oftumor weight after explantation, performed at the end of the experimentsdescribed in A. C, Representative pictures of xenograft tumors, obtained afterexplantation at the end of the experiments described in A. D, Representativepictures of xenograft tumors obtained from doxycycline-treated mice andstained with b-galactosidase assay. All quantifications were based on sevenindependent biological replicates. Error bars, SEM. � , P < 0.05; �� , P < 0.01,two-tailed Student t test. Scale bars, 50 mm.






Figure 6.

Loss of CITK leads to senescence and apoptosis in primary medulloblastoma. A, Western blot analysis, performed with indicated antibodies, of total cell lysatesobtained from medulloblastoma explanted from mice homozygous for SmoA1 transgene and for conditional CitKflox allele, and either negative (iCRE�) orpositive (iCREþ) for ERT2Cre allele, whose activity was induced by tamoxifen treatment (5 days, 0.100mg/g). Explantswere obtained 5 days after the last tamoxifeninjection. Results of two independent tumors per each genotype are shown. Cleaved caspase-3, cC3; cleaved caspase-7, cC7. Internal loading control was vinculin(VINC). Note that the anti-CIT antibodies recognize both CITK and CITN isoforms. B, Quantification of the ratio between signals obtained in iCREþ and iCRE� micein experiments performed as described in A. C, Paraffin sections from tumors obtained as in A were analyzed by histology using hematoxylin and eosin(H&E) staining, as well as by IHC with antibodies anti-Ki67 and -P27 antigens. IHC samples were counterstained with hematoxylin. D, Frozen sections ofmedulloblastoma obtained as in A were processed for immunofluorescence, stained using anti-P27 antibodies, and counterstained with DAPI. Note the nuclearaccumulation of P27 in iCREþ tumors. E, TUNEL staining (brown nuclei) of paraffin sections obtained as in A, counterstained with hematoxylin and quantificationof TUNEL-positive cells. All Western blot analysis quantifications were based on five independent biological replicates. IHC analysis and TUNEL quantificationbased on three independent biological replicates. Error bars, SEM. � , P < 0.05; �� , P < 0.01; �� , P < 0.001, two tailed Student t test. Scale bars, 30 mm.


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either ERT2CRE-negative (control, iCRE�) or ERT2CRE-positive(iCREþ). Under basal conditions, both iCRE� and iCREþ micedevelopedmedulloblastomawithin 1 year, with similar frequency(87% and 84%, respectively) and in comparable times (118� 60days and 125 � 55 days, respectively). To evaluate whether CITKcould be inactivated in these tumors, as well as the effects of CITKacute loss, we injected tamoxifen for five consecutive days in miceshowing at MRI medulloblastoma of similar size (3–20 mm3

diameter) and analyzed them 5 days after the last injection.Western blot analysis confirmed that tamoxifen treatment iseffective in blunting CITK expression in iCREþ tumors, but notin iCRE� controls (Fig. 6A). DNA analysis confirmed the effec-tiveness of recombination, but also showed that a significantfraction of cells in the tumors did not undergo CRE-mediateddeletion (Supplementary Fig. S3E). Nevertheless, tamoxifen-trea-ted iCREþ tumors showed increased levels of gH2AX, P21, P27,andP16, aswell as cleaved caspase-3andcaspase-7 (Fig. 6AandB).

These results confirm that CITK loss leads to increased DNAdamage, apoptosis, and cell senescence in medulloblastoma.Histopathologic analysis revealed striking differences in cellularorganization between tamoxifen-treated iCRE� and iCREþ

tumors. In iCRE� tumors, cells showed a whorl-packed-likearrangement organization (Fig. 6C); it is interesting to note thehigh number of macrophagic cells phagocitying apoptotic celldebries giving a pattern of "starry sky," as it was reported in tumorswith high-proliferative index like Burkitt lymphoma (60). Incontrast, the morphologic structure of tamoxifen-treated iCREþ

tumors wasmore heterogeneous, characterized by areas of packedsmall cells interleaved by areas of low cellular density (Fig. 6C).Immunohistochemical analysis underlined that the latter cellsexpress low levels of the proliferationmarker Ki67 and high levelsof P27 (Fig. 6C). In addition, in P27-positive cells of CITK-deletedtumors, P27 was mostly localized in the nucleus, as it would beexpected in senescent cells (61), whereas in CITK-proficient

Figure 7.

CitK deletion reduces MBs progression andincreases SmoA1 mice survival. A, RepresentativeT2 weighted MRI-based follow up ofmedulloblastoma originating in iCRE� or iCREþ miceat different time points after tamoxifen treatment(time 0). Original images in grayscale and pseudo-colorized images are displayed; red and yellow linesoutline the tumor area in MRI sections. B,Quantitative analysis of tumor growth in follow upexperiment performed as described in A. Tumorvolumes were reconstructed, summing up thecorresponding voxels in each section (N ¼ 6). Errorbars, SEM. � , P < 0.05; �� , P < 0.01, two-tailed Studentt test. C, Kaplan–Meier survival curves of control(iCRE�) and CITK-deleted (iCREþ) mice treated for 5days with tamoxifen at 9 weeks of age (N ¼ at least30). Log-rank (Mantel–Cox) test was used tocompare survival between experimental groups. D,Western blot analysis of CITK levels in cell lysatesobtained from control (iCRE�) and CITK-deleted(iCREþ) tumors explanted 1 week or 28 weeks afterthe end of tamoxifen treatment. Internal loadingcontrol was b-actin (ACTB).






tumors itwas found in thecytoplasm(Fig. 6D).Conversely, cells indense areas weremostly Ki67-positive and P27-negative (Fig. 6C).Consistentwith biochemical data, we observed increased frequen-cy of TUNEL-positive cells in sections of iCREþ tumors (Fig. 6E).

To assess the overall antitumor effect of CitK deletion, wemonitored tumor growth by MRI, before and after tamoxifenadministration. This analysis showed that CITK loss impairsmedulloblastoma growth during the first 4 weeks after treatment(Fig. 7A and B). In the end, we evaluated whether CitK deletionmay significantly affect the survival of SmoA1 mice. To this aim,we treated with tamoxifen 2 months old iCRE� and iCREþ mice.At this stage more than 90% of SmoA1 homozygous mice areexpected to develop subclinical tumors (58). Kaplan–Meiercurves showed a significant increase in survival of the iCREþmice(Fig. 7C). Interestingly, Western blot analysis of advanced tumorsisolated from both groups revealed that all of them expressedsimilar levels of CITK, whereas the spleen of iCREþwas still CITK-deficient (Fig. 7D). Altogether, these results indicate that CITKdeletion inhibits SmoA1 medulloblastoma progression.

DiscussionIn this report we explored the potential suitability of CITK as a

target for medulloblastoma therapy, by producing transient orstable RNAi-mediated knockdown in human medulloblastomacell lines, as well as temporally-controlled genetic inactivation inmouse medulloblastoma of SmoA1 transgenic model. Dataobtained in human medulloblastoma cells confirmed that CITKis necessary for their in vitro expansion and that CITK loss leads tohigh frequency of cytokinesis failure and apoptosis, similarly towhat has previously been described in developing neural progeni-tors (26, 40) and in nonneural tumor cell lines (31, 45, 46, 62).Wealso confirmed that DNA damage is a prominent consequence ofCITK loss (34). Interestingly, the strongest effect produced by CITKknockdown on cell-cycle profile was an increase in the proportionofG0–G1 cells, suggestive of high frequency of cell-cycle arrest. Thisfinding was partially unexpected, because previous studies intumor cell lines (46, 62) and in CitK knockout mice (26) reportedan increase of G2–M or binucleated cells as the main alteration,correlating with high frequency of cytokinesis failure. Consideringthe change of cell morphology, as well as the increased positivityfor b-galactosidase and the strong induction of cyclin kinaseinhibitors P21 and P27, we conclude that human medulloblasto-ma cell lines respond to CITK loss mainly by undergoing growtharrest and senescence, although cytokinesis failure is also detect-able. A combination of apoptosis and senescencemay therefore beresponsible for the reduced expansion of cells after CITK knock-down, both in culture and in xenograft assays.

Interestingly, we observed a similar response in TP53-proficientONS-76 cells and in DAOY cells, expressing nonfunctional TP53,supporting the notion that CITK loss may lead to bothTP53-dependent and TP53-independent antiproliferativeresponses (34, 46). Functional studies (Fig. 3) confirmed thatwild-type TP53 is a crucial player of the phenotypes elicited byCITK loss, and suggest that its role may be at least partiallycompensated by TP73. It is worth noting that TP53 knockdowninDAOY cells andboth TP53or TP73knockdown inONS-76 cellsmoderately increased per se apoptosis levels. In the first case, thephenomenon could be justified by the oncogenic activity of amutant TP53, as previously reported in other cancer cell lines(63). A possible explanation of the ONS-76 results could be

instead that wild-type TP53 or TP73 knockdown may increaseapoptosis in these cells by increasing the frequency of catastrophicmitoses. However, more detailed studies are necessary to betterunderstand this phenomenon.

Analysis of a large gene expression dataset of patients withmedulloblastoma revealed that CITK expression is associatedwith worse prognosis (Supplementary Fig. S2A), but we cannotexclude that this correlation may indirectly depend on mitoticactivity, because CITK expression is cell-cycle dependent (27).

To analyze the effects of CITK inactivation in tumors mim-icking patients' medulloblastoma, we resorted to the SmoA1transgenic model (57), in which we engineered temporally-controlled CitK deletion. With regard to these experiments, itmust be underscored that tamoxifen-induced CitK deletion wasonly partially effective, although it acutely abolished CITKexpression. Nevertheless, after tamoxifen treatment, CITK-delet-ed tumors showed increased apoptosis, as well as morpholog-ical, immunohistochemical, and biochemical changes highlysuggestive of senescence induction. Accordingly, CITK-deletedtumors showed reduced growth rate. Moreover, and mostimportantly, mice in which CITK was deleted at a preclinicalstage had significantly improved survival, compared with non-deleted littermates similarly treated with tamoxifen. At the endof the follow-up period, tumors that developed in tamoxifen-treated iCREþ mice showed CITK protein levels similar to thoseof iCRE� tumors, whereas CITK expression remained almostundetectable in spleen (Fig. 7D). This result indicates thatmedulloblastoma developing in treated iCREþ mice arise fromcells that do not undergo CITK deletion, further suggesting thatCITK-deleted medulloblastoma cells have a selective disadvan-tage if compared with nondeleted cells. These results indicatethat deletion ofCitKmay favorably affect the clinical outcome ofprimary SHH-medulloblastoma. It will be interesting to testwhether similar effects can be produced by inactivating CITK inmodels of type III and type IV medulloblastoma. In addition, itwill be important to assess whether combination of CITK inac-tivation and established anti-medulloblastoma treatmentscould act synergistically to increase tumor cells' death.

Based on the DAOY and ONS-76 data, it seems reasonable toassume that the antitumor effects elicited by in vivo CITK deletionare due to the above-proposedmechanisms, operating cell-auton-omously. However, because in this model CITK is also deleted innontumor cell types, we cannot exclude the possibility that non-cell-autonomous mechanisms may play a role. Investigatingwhether CITK inactivation in cells contributing to medulloblas-toma microenvironment may affect tumor growth will be animportant subject for future studies.

Reduced growth rate of medulloblastoma cells expressingshRNAs against CITK could be rescued by expression of wild-typeCITK, but not by the corresponding kinase-defective mutant,suggesting that CITK catalytic activity is permissive for medullo-blastoma growth. Little is known about the CITK substrates thatmay be important for this activity. Myosin light chain, initiallyproposed as potential substrate, was not supported by in vivoevidence (64). Presently, the only bona-fide CITK substrate is themitotic protein INCENP (65) but it is unknown whether CITK-dependent INCENP phosphorylation may affect genomic stabil-ity. CITK is capable of interacting with different proteins linked tothe accumulation of DNA damage such as P27 (66), ASPM (28),CK2a (50), andRAD51 (34), but it is still unknownwhether someof these partners are direct substrates.

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Nevertheless, based on the present results, as well as on findingthat patients with microcephaly with homozygous CITK nullmutations or kinase-inactivating mutations show overlappingclinical phenotypes (40), we expect that CITK-specific inhibitorsshould induce in medulloblastoma biological responses resem-bling thoseproducedbyCITK lossof function. Thedevelopment ofsuch molecules will therefore represent a crucial topic for futureresearch. As a final remark, our work adds further support to theemerging view thatmany genes implicated ingeneticmicrocephalymay represent promising targets for brain tumors treatment (67).

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

Authors' ContributionsConception and design: G. Pallavicini, F. Di CuntoDevelopment of methodology: M. Falcone, E. Turco, F. Di CuntoAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): G. Pallavicini, F. Sgr�o, F. Garello, V. Bitonto,G.E. Berto, F.T. Bianchi, M. Filippi, E. Turco

Analysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): G. Pallavicini, F. Sgr�o, F.T. Bianchi, M. Gai,A.M.A. Chiotto, U. Ala, E. Turco, F. Di CuntoWriting, review, and/or revision of themanuscript:G. Pallavicini, F. Di CuntoAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): F. Di CuntoStudy supervision: J.C. Cutrin, E. Terreno, E. Turco, F. Di Cunto

AcknowledgmentsWe thank Dr. Luigi Varesio (Gaslini Hospital, Genova) for providing

us with ONS-76 cells. We also thank Damiana Sattanino for technicalassistance. This work was mainly supported by Associazione Italiana perla Ricerca sul Cancro (AIRC) with grant IG17527 to F. Di Cunto.G. Pallavicini and V. Bitonto were supported by a PhD fellowshipfrom Italian Ministry of University and Research (MIUR). F. Garellowas supported by the Fondazione Umberto Veronesi fellowshipprogram. The contribution of EPIGEN flagship project from CNR and ofUniversity of Torino ex-60% fund to F. Di Cunto is also gratefullyacknowledged.

Received December 30, 2017; revised May 1, 2018; accepted June 7, 2018;published first June 19, 2018.

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Pallavicini et al.




2018;78:4599-4612. Published OnlineFirst June 19, 2018.Cancer Res Gianmarco Pallavicini, Francesco Sgrò, Francesca Garello, et al. by Inducing Apoptosis and Cell SenescenceInactivation of Citron Kinase Inhibits Medulloblastoma Progression

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Inactivation of Citron Kinase Inhibits Medulloblastoma ... · Medulloblastoma (MB) is the most common malignant brain tumor in children (1). medulloblastoma has been classiﬁed into