Inactivation of Citron Kinase Inhibits Medulloblastoma Progression By
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Published OnlineFirst June 19, 2018; DOI: 10.1158/0008-5472.CAN-17-4060 Cancer Tumor Biology and Immunology Research Inactivation of Citron Kinase Inhibits Medulloblastoma Progression by Inducing Apoptosis and Cell Senescence Gianmarco Pallavicini1,2, Francesco Sgro1, 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 brain tumor in children. Current treatment for medulloblastoma consists of surgery followed by irradiation of the whole neuraxis and high-dose multiagent chemotherapy, a par- tially effective strategy associated with highly invalidating side effects. Therefore, identification and validation of novel target molecules capable of contrasting medulloblastoma growth without disturbing brain development is needed. Citron kinase protein (CITK), encoded by primary micro- cephaly gene MCPH17, is required for normal proliferation and survival of neural progenitors. Constitutive loss of CITK leads to cytokinesis failure, chromosome instability, and apoptosis in the developing brain, but has limited effects on other tissues. On this basis, we hypothesized that CITK could be an effective target for medulloblastoma treatment. In medulloblastoma cell lines DAOY and ONS-76, CITK knockdown increased both cytokinesis failure and DNA damage, impairing proliferation and inducing cell senes- cence and apoptosis via TP53 or TP73. Similar effects were obtained in the NeuroD-SmoA1 transgenic mouse model, in which CITK deletion increased apoptotic cells and senes- cence markers such as P21CIP1, P27KIP1, and P16INK4A. Most importantly, CITK deletion decreased tumor growth and increased overall survival in these mice, with no apparent side effects. These results suggest that CITK can be a useful molecular target for medulloblastoma treatment. Significance: In vitro and in vivo proof of concept identifies citron kinase protein as a suitable target for medulloblastoma treatment. Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/78/16/4599/F1.large.jpg. Cancer Res; 78(16); 4599–612. Ó2018 AACR. Introduction five subgroups based on histology and into four biological sub- Medulloblastoma (MB) is the most common malignant brain groups based on microarray and genomic sequencing technolo- tumor in children (1). medulloblastoma has been classified into gies: WNT, Sonic Hedgehog (SHH), group 3, and group 4 (2–6). 1Department of Molecular Biotechnology and Health Sciences, University of Turin, G. Pallavicini and F. Sgro contributed equally to this article. Torino, Italy. 2Neuroscience Institute Cavalieri Ottolenghi, University of Turin, Corresponding Author: Ferdinando Di Cunto, University of Turin, Neuro- Torino, Italy. 3Division of Stem Cells and Cancer, German Cancer Research Center science Institute Cavalieri Ottolenghi, Regione Gonzole 10, Torino, TO (DKFZ) and DKFZ-ZMBH Alliance, Heidelberg, Germany. 4Heidelberg Institute for 10126, Italy. Phone: 390116706616; Fax: 390116706621; E-mail: Stem Cell Technology and Experimental Medicine (HI-STEM gGmbH), Heidelberg, [email protected] Germany. 5Department of Neuroscience, University of Turin, Torino, Italy. doi: 10.1158/0008-5472.CAN-17-4060 Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). Ó2018 American Association for Cancer Research. www.aacrjournals.org 4599 Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 2018 American Association for Cancer Research. Published OnlineFirst June 19, 2018; DOI: 10.1158/0008-5472.CAN-17-4060 Pallavicini et al. WNT medulloblastoma are associated with relatively good prog- loblastoma express high levels of TUBB3 (49), which may increase nosis, whereas groups 3 and 4, which often show MYC amplifi- sensitivity to CITK loss (50). Finally, although CITK is essential for cation and TP53 mutation, are the most aggressive subtypes (5). normal brain development, it is not required in normal postnatal Current treatments for medulloblastoma include surgery fol- tissues (26, 34), and therefore its acute inactivation should be lowed by irradiation of the whole neuraxis and high-dose mul- associated with relatively mild side effects. tiagent chemotherapy (7). Five-year survival rate for children with In this report we analyzed the effects of deleting CITK in SHH average and high-risk disease, as defined by clinical criteria, is 80% medulloblastoma, using in vitro and in vivo models, including and 60–65%, respectively (8). Therefore, many of the high-risk conditional inactivation in primary tumors. Our results support patients die despite treatment (9). In addition, survivors have CITK as a novel drug target for this tumor type. significant neurologic and developmental consequences, such as severe cognitive deficits and endocrine disorders (10–15). Thus, Materials and Methods novel and more specific therapies are urgently needed. A possible Cell culture strategy to develop new selective therapies is to directly target DAOY cells were obtained from ATCC and were cultured molecular pathways altered by driver mutations. SHH subgroup, according to the ATCC protocol. representing approximately 25% of medulloblastoma cases, is the ONS-76 cells were kindly provided by Luigi Varesio (Gaslini better understood medulloblastoma subtype: it is characterized Hospital, Genova) and were cultured in RPMI medium supple- by mutations of SHH signaling pathway downstream compo- mented with 10% FBS and 1% penicillin/streptomycin (Life nents (PTCH1, SMO, SUFU, and GLI2) and it is derived from Technologies). No further authentication was performed on these granule neuron precursors (GNP) of the cerebellum (16–18). cells. For both cell lines, the number of in vitro passages from Small-molecule inhibitors of the SHH pathway have been devel- thawing of the original aliquots to experiments was comprised oped and tested in clinical trials (19). However, only a subgroup between 5 and 8. Cells were routinely analyzed for morphological of these patients respond to treatment and even in these cases features and tested by PCR for mycoplasma contamination with resistance rapidly develops (20–22). An alternative strategy for the following oligonucleotides sequences: MYCO1 50-ACTCC- drug development is to target molecules that, despite not being TACGGGAGGCAGCAGTA-30; MYCO2: 50-TGCACCATCTGTCA- mutated in cancer, are nevertheless required for tumor growth and CTCTGTTAACCTC-30. progression (23). Cancer cells may become specifically dependent on particular nonmutant proteins in consequence of peculiar Transfection and RNAi features of the cells from which they originate and/or of the Previously published CITK double-stranded RNAs (31) and modifications induced by oncogenic transformation (23). nontargeting siRNA were used (Dharmacon). For knockdown of CITK is a conserved multidomain protein, containing an TP53 and TP73, ONTARGETplus Dharmacon SMART pools were AGC-type amino-terminal serine/threonine kinase domain used. D-001810-10 nontargeting pool was used as a negative (24, 25). CITK is ubiquitously expressed (26) and cell-cycle control. ONS-76 and DAOY cells plated on six-well plates were regulated, with highest levels in G2–M phase of the cell cycle transfected using 6.25 mL of the required siRNA (20 mmol/L) (27). During mitosis, it is first localized to the nucleus (27), it is together with 1.5 mL Lipofectamine 2000 (Invitrogen), according enriched at spindle poles before anaphase (28), it is concentrated to the manufacturer's instructions. Efficient knockdown was at cleavage furrow and midbody from anaphase to cytokinesis obtained after 48 hours. (25). CITK is a specific regulator of abscission at the end of cytokinesis (25, 29–32). In addition, it regulates mitotic spindle Lentiviral production orientation (33) and it is involved in genomic stability indepen- HEK293T cells were cultured in DMEM supplemented with dently of its role in cytokinesis (34). Despite ubiquitous expres- 10% FCS and 1% penicillin/streptomycin (Life Technologies, sion, CITK is functionally required in vivo only in few cell types, ThermoFisher). Lentiviral vector stocks were produced in such as neural progenitors (26, 35) and male germ cells (36). CITK HEK293T cells by calcium phosphate–mediated transfection, as loss leads to severe microcephaly in rodents (26, 37) and humans described in ref. 51. (38–41). Cells in the affected tissues display cytokinesis failure (26, 40), apoptosis (26, 36, 40), and accumulation of DNA Generation of inducible shRNA cell lines and rescue of CITK damage (34). Notably, the human CITK microcephaly syndrome, expression known as MCPH17, can be caused by homozygous kinase- Human CITK-specific shRNA sequences (31) were cloned into inactivating mutations (40), indicating that catalytic activity is pLVTHM vector from Didier Trono's lab (Addgene plasmid essential for function. #12247; Supplementary Fig. S1A). For inducible RNAi, cells were CITK is expressed at high levels in tumors (42–44) and is transduced with pLV-DsRed-tTRKRAB from Didier Trono lab required for normal growth in tumor cell lines of different origin (Addgene plasmid #12247; Supplementary Fig. S1A), expanded, (45–47). Moreover, CITK knockdown reduces tumor growth in and used for transduction with pLVTH-GFP-shRNA lentiviral hepatocellular carcinoma cells in xenograft assays (45). These particles. Next,