JPO2/CDCA7L and LEDGF/p75 Are Novel Mediators of PI3K/AKT Signaling and Aggressive Phenotypes in Medulloblastoma

Medulloblastoma, a primitive neuroectodermal tumor of the cerebellum, is the most common malignant pediatric brain tumor (1). Despite recent advances in clinical risk stratification and intensified therapies, approximately one-third of children with medulloblastoma will succumb to their disease. Metastasis, which is found in 30% to 40% of patients at diagnosis, is one of the strongest predictors of survival. However, current approaches for controlling or preventing metastases are often ineffective and frequently leave survivors with severe long-term toxicity (2).

Recent genomic studies indicate four molecular subgroups of medulloblastoma (3) and confirmed early observations of the strong negative prognostic effect of MYCC amplification, which defines the most aggressive group 3 medulloblastoma (3–5). Our studies and those of others have shown that Myc can confer metastatic phenotypes in medulloblastoma (6–8). Although mechanisms of Myc-mediated transformation in medulloblastoma remain largely unknown, multiple lines of data suggest Myc-driven medulloblastoma are dependent on PI3K/AKT signaling (6–16) and point to the PI3K/AKT axis as a critical downstream effector of Myc in group 3 medulloblastoma (7, 16, 17). Activation of PI3K/AKT signaling has been shown to promote various oncogenic phenotypes in medulloblastoma including enhanced tumor growth, metastasis, and chemoresistance (14, 18–22). Thus, defining the scope of Myc regulatory network in medulloblastoma will clearly be important for informing more effective targeting of the Myc–PI3K/AKT axis.

In prior work, we discovered a Myc cotransforming protein partner—JPO2 (CDCA7L/RAM2/R1; ref. 23), which acts synergistically with Myc in transcriptional repression (6) and is upregulated in metastatic medulloblastoma (23, 24). Although these observations suggested important functional roles for JPO2 in medulloblastoma oncogenesis (25, 26), the specific role of JPO2 in medulloblastoma transformation remains unknown.

In this study, we demonstrate that JPO2 like Myc, potently and directly induces metastatic and infiltrative medulloblastoma in orthotopic models. We identify LEDGF/p75 as a binding partner of JPO2 in medulloblastoma, and show that JPO2 acts cooperatively with LEDGF/p75 to promote medulloblastoma cell migration via a novel feed-forward regulatory circuit that converges on AKT signaling. Our studies underscore AKT as a critical prometastatic signaling pathway in medulloblastoma and reveal JPO2 and LEDGF/p75 as novel therapeutic targets in AKT signaling.

Medulloblastoma cell lines D283, D341, D458 [gifts from Dr. Bigner (2003), Duke University (Durham, NC)] were maintained in zinc option medium (Life Technologies); UW228, UW426 [gifts from Dr. Silber (2003), University of Washington (Seattle, WA)), and DAOY (ATCC, 2005) were maintained in αMEM containing 10% FBS. 293T cells were maintained in DMEM (Life Technologies) with 10% FBS. For transient expression, 293TV cells were routinely transfected with 2 to 5 μg of plasmids using Fugene (Roche Applied Science) at a 3:1 ratio of Fugene/plasmid DNA and analyzed by RT-PCR and Western blotting 24 to 48 hours later.

To generate stable JPO2/LEDGF-expressing cell lines, cells were infected with pMN-GFP-JPO2/eGIPz-RFP-LEDGF or corresponding vector control. FACS-enriched cells were expanded and protein expression verified by Western blotting, as described previously (27). Stable expressions were tested each time when cells were grown from frozen stock prior to experiments.

For generating sh-JPO2–stable expressing cell lines, JPO2 RNAi construct as described previously: forward, 5′-CCGGAATGGTCGTGGAGTCAGATT TCAAGAGAATCTGACTCCACGACCATTTTTTTG-3′; and reverse, 5′-AATTGGAGTCAGATTCTCTTGAAATCTGACTCCACGACCATT-3′, were subcloned into psi-RNAi-plasmid with Zeocin selective marker. Cells were transfected with Fugene, selected with Zeocin, and tested for JPO2 expression by RT-PCR and Western blotting.

For RT-PCR, JPO2 primers (forward, 5′-GCGGAAGAGTTTTACAGC-3′; reverse, 5′-CTGACATCCTCCCCATAG-3′), LEDGF primers (forward, 5′-CACACAGAGATGATTACTACACTG-3′; reverse, 5′-CCATCTTGAGCATCAGATCCTC-3′), and previously described 36B4 primers were used (6).

Cell proliferation was assessed using MTS assays (Roche Colorimetric Cell Proliferation, Roche Pharmaceuticals) at regular intervals as described previously (6) and results were verified by direct Trypan blue cell count.

Bromodeoxyuridine (BrdUrd) assay was performed using the Cell Proliferation BrdUrd Chemiluminescent ELISA (Roche Pharmaceuticals). Briefly, cells were fixed and stained with anti-5-bromodeoxyuridine-POD antibody. Immune complexes and reactions were quantified using a multiwell luminometer.

Cell migration assays were performed as described previously (6) using Transwell Boyden chambers according to the manufacturer's instructions (BD Biosciences). In brief, 3.5 × 10⁴ cells were seeded in chambers at 37°C for 18 to 40 hours. To quantify migrated cells, membranes were stained with 1% toluidine, migrated cell counts were determined on the basis of 10 random microscopic fields, counted by two individuals in a blinded fashion.

Nu-Nu mice (Charles River Laboratories) were maintained in accordance with the Hospital for Sick Children Institutional Animal Care Committee–approved protocols. Briefly, cerebella of 4- to 6-week-old anesthetized male mice were injected stereotactically (guided coordinates X = −2.0, Y = 2.0, Z = 2.0) with 1 × 10⁵ UW228/DAOY cells stably expressing JPO2. Specifically, a total volume of 1.5 μL was injected over 3 to 4 minutes. All animals were monitored for posterior fossa tumor-related symptoms such as hunched posture, doomed head, ataxia, weight loss and euthanized as per standard tumor endpoint monitoring guidelines. Dr. Cynthia Hawkins performed histopathologic analyses of whole brain and spine from all mice.

IHC was performed on formalin-fixed paraffin-embedded tissue using standard procedures. Xenograft specimen tissues were subjected to antigen retrieval by pressure cooking (citrate buffer, pH 6, 20 minutes) and 0.3% H₂O₂ endogenous peroxidase blocking. Primary antibody: JPO2 antibody [1:500, rabbit, in-house (6, 22)], Ki-67 (1:150; Dako, Agilent Technologies), were incubated overnight at 4°C and detected using biotinylated secondary IgG antibodies for 30 minutes using ABC Reagent Kit and DAB Chromagen (Vector Laboratories). A final counterstain was performed in hematoxylin followed by dehydration in 70%, 80%, and 100% ethanol series to xylene and mounted in Permount (Thermo Fisher Scientific). Hematoxylin and eosin (H&E) stains were performed using standard protocols.

Cell lines were lysed with standard EBC whole cell lysis buffer (50 mM Tris-HCl, 120 mM NaCl, 0.5% NP-40, and 5 mM EDTA, pH 8.0) as described previously (23), and analyzed by Western blotting with anti-JPO2 (1:500, rabbit, in-house; refs. 6, 23), Myc (1:500, in-house 9E10 mouse monoclonal; refs. 6, 23), α-tubulin (1:5,000; mouse, T9026; Sigma-Aldrich), LEDGF (1:1,000, mouse, MABN674; EMD Millipore), PARP (1:1,000, rabbit, 9542; Cell Signaling Technology), p-AKT308 (1:1,000, rabbit, 2965; Cell Signaling Technology), pan-AKT (1:1,000, rabbit, 2920; Cell Signaling Technology), pRAC/CDC424 (1:1,000, rabbit, 2461; Cell Signaling Technology), RAC1-3 (1:1,000, rabbit, 2456; Cell Signaling Technology), CDC42 (1:1,000, rabbit, 2464; Cell Signaling Technology), pThr202/Tyr204ERK1/2 (1:1,000, rabbit, 9101; Cell Signaling Technology), and ERK1/2 (1:1,000, rabbit 9102; Cell Signaling Technology). Detection was performed using antispecies horseradish peroxidase–conjugated antibodies (Bio-Rad Laboratories) and Chemiluminescence Reagent Plus (PerkinElmer).

RNA and cDNA were generated using standard methods for quantitative PCR analysis (SYBR Green PCR Kit, Invitrogen, Life Technologies; ABI Prism 7900HT Sequence Detection Systems, Applied Biosystems). Specific mRNAs were quantified relative to 36B4 using the ΔΔC_t method.

Lysates of UW228-FLAG-JPO2 and UW228-FLAG-vector cells extracted in lysis buffer (50 mmol/L Tris pH 7.4, 150 mmol/L NaCl, 1 mmol/L EDTA, and 0.5% Triton X-100) were subjected to immunoprecipitation using anti-FLAG M2 beads (A2220, Sigma-Aldrich) for 3 hours, followed by washes in lysis buffer (1% NP-40 and 0.1% Triton X-100) and wash buffer. Subsequently, beads were washed with 20 mmol/L Tris buffer (pH 8.0), and eluted proteins were analyzed by SDS–PAGE and silver staining. Protein bands excised from gels were digested with trypsin, and resulting peptides were identified by LC/MS-MS sequencing. Specific JPO2-interacting proteins were identified on the basis of (i) peptides originating from UW228-JPO2 samples that were absent in UW228-vector control; (ii) proteins yielding more than two unambiguous peptides and appearing in two or more independent experiments.

Publicly available gene expression data for a series of human primary medulloblastoma samples (GSE10327; ref. 28) and mouse medulloblastoma samples (GSE33201; ref. 11) performed on Affymetrix U133 platforms were downloaded from the Gene Expression Omnibus (GEO) repository, and CEL files were integrated and analyzed using the Partek Genomics Suite (Partek). Transcript levels of CDCA7L/JPO2 and PSIP1/LEDGF were investigated for subgroup-specific correlation using ANOVA and data presented in box plots. Log-rank analysis was performed using the Kaplan–Meier method to determine statistical difference of survival significance in the tumor xenograft studies. The number of samples used and the respective P values are listed in the figure legends. Significance for in vitro cell growth/death data was determined by Student two-tailed t test (N.S., not significant; *, P < 0.05; **, P < 0.01).

We previously observed that JPO2 promotes medulloblastoma transformation in vitro; however, the in vivo oncogenic potential of JPO2 has not yet been investigated. We therefore examined the effect of ectopic JPO2 expression on UW228 and DAOY medulloblastoma cells using orthotopic xenograft assays in nude mice. Strikingly, while mice injected with parental UW228-control cell lines exhibited no tumor growth even up to 60 days postinjection, 80% (8/10) of the mice injected with UW228-JPO2 cells developed rapidly growing, infiltrative cerebellar tumors. All mice (10/10) injected with stable DAOY-JPO2 cell lines also developed highly infiltrative cerebellar tumors whereas only 50% (6/12) of the mice injected with DAOY-control lines exhibited limited localized tumor growth (Fig. 1A). Xenografts from both UW228-JPO2 and DAOY-JPO2 cells exhibited anaplastic cellular features resembling the large cell/anaplastic histology of aggressive group 3 human medulloblastoma. Ki-67 stains confirmed that both UW228-JPO2 and DAOY-JPO2 xenografts infiltrated adjacent normal cerebellum with a particularly invasive growth pattern noted in DAOY-JPO2 tumors (Fig. 1B). Mice injected with UW228-JPO2 and DAOY-JPO2 exhibited significantly shortened survival (mean survival of 42 days; P < 0.001), as compared with mice with control cell injections (Fig. 1C). Notably, xenografts with JPO2 expression exhibited a higher frequency of metastases, spinal metastases were detected in 62.5% (5/8) of UW228-JPO2 and 70% (7/10) of DAOY-JPO2 tumor-bearing mice, as compared with only 8.3% (1/12) of mice with control DAOY xenografts (P = 0.03, Fig. 1D). These data indicate that like Myc, JPO2 is a potent oncogenic driver that can also directly induce aggressive and metastatic phenotypes in medulloblastoma.

To investigate mechanisms of JPO2 oncogenic signaling, we used mass spectrometry analyses to identify other binding partners of JPO2. These experiments revealed a spectrum of candidate JPO2 interactors with tumor-promoting and cell survival functions including LEDGF/p75, an HIV-integrase and known oncogenic protein (29–31), which has not been implicated in medulloblastoma to date. Transient transfection and coimmunoprecipitation assays (Fig. 2A) corroborated prior reported observations of JPO2 and LEDGF/p75 interaction in 293TV cells (32, 33). Significantly, coimmunoprecipitation experiments also revealed JPO2 binds to endogenous LEDGF/p75 in UW228-JPO2 medulloblastoma cells (Fig. 2B). These observations were further confirmed by immunofluorescence and confocal microscopy studies, which indicated nuclear colocalization of JPO2 and LEDGF/p75 in UW228-JPO2 as well as ONS76 medulloblastoma cells, which have high endogenous JPO2 expression (Fig. 2C).

To evaluate the significance of JPO2:LEDGF/p75 interaction in medulloblastoma, we examined gene expression patterns of both loci in publicly available human and mouse medulloblastoma expression datasets. Interestingly, these analyses showed JPO2/jpo2 and LEDGF/p75/ledgf/p75 were concordantly expressed at high levels across all medulloblastoma subtypes and in a spectrum of murine medulloblastoma models (Fig. 3A and B). Western blot analyses also showed frequent high coexpression of LEDGF/p75 and JPO2 in a majority of human medulloblastoma cell lines examined (Fig. 3C).

To determine how JPO2 and LEDGF/p75 may act together to promote medulloblastoma transformation, we examined the cellular effects of JPO2 and LEDGF/p75 expression in medulloblastoma cells. In contrast to Myc, BrdUrd, MTS, and cell count assays revealed that neither ectopic expression of JPO2 (Supplementary Fig. S1A–S1C) nor JPO2 knockdown (Supplementary Fig. S1D and S1E) altered cell growth or proliferation across several different medulloblastoma cell lines. JPO2 expression also had little effect on medulloblastoma cell survival when cells were grown under conditions of serum starvation or exposed to radiation and various chemotherapeutic agents (Supplementary Fig. S2A–S2C). Consistent with its known function as a prosurvival protein, LEDGF/p75 expression enhanced survival of medulloblastoma cells exposed to serum starvation or chemotherapeutic drugs (Supplementary Fig. S3A) but had insignificant effects on medulloblastoma cell growth under normal proliferative conditions (Supplementary Fig. S3B).

As Myc and JPO2 are associated with metastatic medulloblastoma (5, 11), we examined whether JPO2 or LEDGF/p75 expression altered migratory and invasive phenotypes in medulloblastoma. Expression of JPO2 and LEDGF/p75 individually significantly enhanced medulloblastoma cell migration and invasion in Boyden chamber assays. Specifically, UW228, UW426, and DAOY cell lines with stable JPO2 or LEDGF/p75 expression showed 2- to 5-fold greater cell migration/invasion as compared with control cell lines (P < 0.01; Fig. 4A and B). Conversely, siRNA-mediated knockdown of JPO2 or LEDGF/p75 in medulloblastoma cell lines ONS76, D341, and D458, which express high endogenous levels of both proteins, diminished medulloblastoma cell migration and invasion by more than 50% (Fig. 4C and D).

To further investigate whether medulloblastoma cell migration was functionally linked to JPO2 and LEDGF/p75 interaction, we performed siRNA-mediated knockdown of JPO2 and LEDGF/p75, respectively, in medulloblastoma cells with LEDGF/p75 or JPO2 overexpression. Significantly, migration of UW228-JPO2 cells with LEDGF/p75 knockdown and UW228-LEDGF cells with JPO2 knockdown were, respectively, diminished by 35% and 50% relative to controls (Fig. 4E). Interestingly, corresponding qRT-PCR and Western blot analyses showed that JPO2 mRNA and protein expression were also diminished with LEDGF/p75 knockdown in UW228-JPO2 (Fig. 4E and F) and ONS76 cells (Supplementary Fig. S4A) and conversely, JPO2 mRNA was upregulated in medulloblastoma cells with stable LEDGF/p75 expression (Supplementary Fig. S4B). In contrast, LEDGF/p75 mRNA and protein expression remained unchanged with JPO2 overexpression or knockdown (Figs. 4E and 5 and Supplementary Fig. S4C). These findings suggest LEDGF/p75 and JPO2 may act via a feed-forward regulatory loop to modulate a common signaling axis and promote medulloblastoma cell migration/invasion.

To further elucidate mechanisms underlying the promigratory effects of JPO2 and LEDGF/p75 in medulloblastoma cells, we investigated effects of ectopic JPO2 and LEDGF/p75 expression on signaling pathways that have been implicated in medulloblastoma migration/metastases including PI3K/AKT and mitogen-associated protein kinase ERK1/2 pathways (13, 34–36). Interestingly, Western blot analyses of medulloblastoma cell lines with ectopic or endogenous LEDGF/p75 expression showed that LEDGF/p75 expression was accompanied by increased phospho-AKT-T308 as well as JPO2 expression (Fig. 5 and Supplementary Fig. S5). Similarly, ectopic JPO2 expression correlated with increased phospho-AKT-T308 levels. In contrast, we did not observe a consistent correlation of JPO2 expression with phospho-ERK1/2 signaling in different medulloblastoma cell lines, whereas LEDGF/p75 expression was associated with increased phospho-ERK1/2 level. These findings indicate that medulloblastoma cell migration and invasion is mediated by convergence of LEDGF/p75 and JPO2 on AKT.

To confirm the specific role of JPO2:AKT signaling in medulloblastoma cell migration, we examined the effect of PI3K inhibitor, LY294002, which blocks AKT phosphorylation, on migration of medulloblastoma cell lines with high endogenous (D458) and exogenous JPO2 expression (UW228-JPO2; DAOY-JPO2). Treatment with LY294002 inhibited D458 cell migration and reversed the promigratory effects of exogenous JPO2 expression in UW228 and DAOY cell lines (Fig. 6A). However in contrast to UW228-Myc cells, survival of UW228-JPO2 cells was not altered by LY294002 treatment (Supplementary Fig. S6A and S6B). Importantly, the inhibitory effect of LY294002 treatment on JPO2-induced cell migration was highly specific as treatment with MEK1/2 inhibitor, U0126, which blocks ERK1/2 phosphorylation, as well as a control inactive analog, U0124, had no effects UW228-JPO2 and DAOY-JPO2 cell migration (Fig. 6B). In keeping with the specific association of JPO2-induced cell migration with the AKT pathway, we observed a decrease in phospho-AKT expression in all 3 medulloblastoma cell lines with inhibition of cell migration. Interestingly, this was also accompanied by a decrease in JPO2 protein (Fig. 6A) but not mRNA expression (Supplementary Fig. S7), thus indicating posttranslational regulation of JPO2 by AKT.

Our collective experimental studies support a model in which the prometastatic functions of AKT signaling are dependent and promoted by two novel and synergistic feed-forward regulatory circuits between LEDGF/p75 and JPO2 and between JPO2 and AKT (Fig. 7). These findings highlight JPO2 and LEDGF/p75 as new and important avenues for targeting the prometastatic AKT signaling cascade in medulloblastoma.

Substantial data implicate Myc and PI3K activation in aggressive phenotypes and treatment resistance in medulloblastoma (5, 7, 15, 19, 20, 37–39). To date, multiple studies have demonstrated that pharmacologic inhibitors of PI3K signaling have potent antineoplastic effects in medulloblastoma (7, 13, 14, 16, 17, 19, 36), and underscore the importance of PI3K signaling particularly in Myc-driven medulloblastoma. To effectively exploit PI3K/AKT signaling for medulloblastoma treatment, it would be important to delineate the extent of the Myc–PI3K/AKT signaling axis through identification of novel targetable effector and mediators of this pathway. In this study, we identified JPO2 and LEDGF/p75 as novel components of PI3K/AKT signaling, and have uncovered complex new signaling circuits driven by JPO2 and LEDGF/p75, which promote migratory and invasive phenotypes in medulloblastoma via upregulation of PI3K/AKT signaling.

Our in vitro and in vivo studies indicate JPO2 specifically promotes medulloblastoma cell migration/invasion and metastases without effects on cell proliferation or survival. Although our observations strongly support a role for the JPO2:AKT axis on medulloblastoma cell migration, gross morphologic changes were not evident in medulloblastoma cells with JPO2 overexpression. Interestingly, we observed in preliminary studies that N-cadherin expression is diminished in medulloblastoma cells with ectopic JPO2 expression, thus suggesting JPO2 may mediate medulloblastoma migratory phenotypes in part by promoting epithelial–mesenchymal transition (EMT). Future studies to determine how JPO2 and the PI3K/AKT pathway act convergently or independently to mediate EMT and migratory/metastatic medulloblastoma phenotypes would clearly be of therapeutic interest.

We observed that JPO2 binds to LEDGF/p75, a known oncoprotein highly expressed in multiple cancer types (31, 40, 41), that is also coordinately upregulated with JPO2 in medulloblastoma. Our data, which is the first to implicate LEDGF/p75 in medulloblastoma suggest LEDGF/p75 and JPO2 may act via a common transcriptional signaling complex to promote Myc:PI3K/AKT-associated medulloblastoma transformation. Our observations that LEDGF/p75 regulates and functions cooperatively with JPO2 to promote prometastatic AKT signaling in medulloblastoma cells are concordant with reported LEDGF/p75 and JPO2 interactions in HEK 293 cells (32). We observed changes in JPO2 mRNA and protein levels with LEDGF/p75 expression in medulloblastoma cells that suggest transcriptional regulation of JPO2 by LEDGF/p75. However, while we observed a significant overall upregulation of both genes in primary human and murine medulloblastoma, detailed in silico analyses of individual human medulloblastoma subtypes revealed a positive, but nonsignificant trend between JPO2 and LEDGF/p75 expression only in human SHH and group 3 medulloblastoma (Supplementary Fig. S8). Interestingly, studies by Bartholoomeeusen and colleagues (32) in HEK 293 cells demonstrate LEDGF/p75 also regulates JPO2 protein stability. These aggregate data suggest that LEDGF/p75 may regulate JPO2 via transcriptional as well as posttranscriptional mechanisms in a cell context–dependent manner. In addition to direct transcriptional functions, both LEDGF/p75 and JPO2 (42, 43) have been implicated in chromatin remodelling (44–46), thus JPO2:LEDGF/p75 transcriptional complexes may have more potent, global gene regulatory roles with impact on additional as yet undefined transforming pathways in medulloblastoma. Interestingly, recent studies also suggest potential E3 ubiquitin ligase functions for JPO2 (44, 47), thus JPO2 may also regulate signaling via posttranslational mechanisms. Future biochemical studies of JPO2 and JPO2:LEDGF/p75 interactions will be important for defining critical domains required for JPO2 and LEDGF/p75 cooperation in oncogenic signaling that may be exploited therapeutically.

LEDGF/p75 is a potent oncogene with known diverse functions in cell survival, chemotherapy resistance, tumor progression (41, 48, 49), and leukemic transformation via interactions with MLL oncoproteins (30). Although our studies indicated a tight convergence of LEDGF/p75 and JPO2 on promigratory AKT signaling, we observed that JPO2 and LEDGF/p75 did not always have overlapping cellular and signaling effects in UW228 and UW426 medulloblastoma cell lines. Specifically, while we observed inconsistent effects of JPO2 expression on p-ERK1/2 levels in UW228 and UW426 cells, ERK1/2 was consistently upregulated with LEDGF/p75 expression in the same cell lines (Fig. 5). Although effects of cell context remains to be examined with a wider panel of medulloblastoma cell lines, it is interesting to note that while pERK1/2 expression did not correlate with promigratory effects of LEDGF/p75 on medulloblastoma cells, each of the UW228, UW426, and DAOY cells examined exhibited enhanced survival with LEDGF/p75 expression (Supplementary Fig. S3A). Our collective observations support LEDGF/p75 as a novel and important driver of prometastatic phenotypes in medulloblastoma and indicate additional distinct oncogenic roles for LEDGF/p75 in medulloblastoma. Whether LEDGF/p75 can recapitulate the potent in vivo oncogenic and metastatic effects of Myc and JPO2 on medulloblastoma cells remains to be investigated. The relatively high expression of LEDGF/p75 and JPO2 in various human medulloblastoma subgroups and murine medulloblastoma models also suggest broader roles for LEDGF/p75 and/or JPO2 in medulloblastoma pathogenesis. Given the high expression of JPO2 expression in Myc as well as N-Myc–associated medulloblastoma, we anticipate JPO2 and LEDGF to also have roles in N-myc–driven PI3K signaling seen in the SHH subtype of medulloblastoma (50, 51).

In summary, we have discovered a new and complex signaling circuit that promotes prometastatic activity of the PI3K/AKT pathway in medulloblastoma. Our data implicates two oncoproteins JPO2 and LEDGF/p75 as novel modulators of PI3K/AKT signaling and suggest potential new avenues for targeting the potent Myc:PI3K/AKT signaling axis, which underlies the most lethal phenotypes in medulloblastoma.

No potential conflicts of interest were disclosed.

Conception and design: T.S.Y. Chan, C.J. McGlade, A. Huang

Development of methodology: T.S.Y. Chan, C.J. McGlade, A. Huang

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): T.S.Y. Chan, C. Hawkins, J.R. Krieger, A. Huang

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): T.S.Y. Chan, C. Hawkins, J.R. Krieger, A. Huang

Writing, review, and/or revision of the manuscript: T.S.Y. Chan, A. Huang

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): A. Huang

Study supervision: A. Huang

The authors thank L. Penn for Myc antibodies and D. Picard for technical help in generating JPO2 stably expressing cell lines.

This work was supported by Canadian Institute of Health Research grant #MOP-119591. T.S.Y. Chan received a University of Toronto Fellowship, Frank Fletch Memorial Research Fund, and Clayton Pediatric Research Fund.

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