myc drives group 3 medulloblastoma through …...medulloblastoma have distinct cell origins. wnt...

Tumor Biology and Immunology

MYC Drives Group 3 Medulloblastoma throughTransformation of Sox2þ Astrocyte ProgenitorCellsRan Tao1, Najiba Murad1, Zhenhua Xu1, Peng Zhang2, Konstantin Okonechnikov3,4,Marcel Kool3,4, Samuel Rivero-Hinojosa1, Christopher Lazarski1, Pan Zheng2, Yang Liu2,Charles G. Eberhart5, Brian R. Rood1, Roger Packer1, and Yanxin Pei1

Abstract

A subset of group 3 medulloblastoma frequently harborsamplification or overexpression of MYC lacking additionalfocal aberrations, yet it remains unclear whether MYC over-expression alone can induce tumorigenesis and which cellsgive rise to these tumors. Here, we showed that astrocyteprogenitors in the early postnatal cerebellum were susceptibleto transformation by MYC. The resulting tumors specificallyresembled human group 3 medulloblastoma based on his-tology and gene-expression profiling. Gene-expression analy-sis of MYC-driven medulloblastoma cells revealed alteredglucose metabolic pathways with marked overexpression oflactate dehydrogenase A (LDHA). LDHA abundance correlatedpositively withMYC expression and was associated with poor

prognosis in human group 3 medulloblastoma. Inhibition ofLDHA significantly reduced growth of bothmouse andhumanMYC-driven tumors but had little effect on normal cerebellarcells or SHH-associated medulloblastoma. By generating anew mouse model, we demonstrated for the first time thatastrocyte progenitors can be transformed byMYC and serve asthe cells of origin for group 3medulloblastoma. Moreover, weidentified LDHA as a novel, specific therapeutic target for thisdevastating disease.

Significance: Insights fromanewmodel identified LDHA asa novel target for group 3 medulloblastoma, paving the wayfor the development of effective therapies against this disease.

IntroductionMedulloblastoma is a highly malignant tumor of the cerebel-

lum that occurs most frequently in children (1). Medulloblasto-ma is widely recognized as comprising four molecular subgroupstermedWNT, SHH, group 3, and group 4 (2–5). Recently, group 3medulloblastoma has been further divided into three subtypes,distinguished by distinct gene copy-number aberrations, somaticmutations, potential oncogenic drivers, and clinical outcomes (6).Group 3a tumors frequently involve loss of MYC, group 3btumors exhibit activation of GFI1 and GFI1B, and group 3gtumors harbor MYC amplification or overexpression in theabsence of other focal aberrations (6). Another recent studyidentified four subtypes of tumors in group 3 medulloblastoma,in which subtype II is associated with MYC amplification (2).

Despite aggressive treatment, over 70% of patients with MYC-amplified medulloblastoma succumb to the disease (5). More-over, surviving patients often suffer severe treatment-related sideeffects, including permanent cognitive and motor disabilities.Thus, there is a critical need for novel targeted therapies that notonly improve life expectancy, but also limit damage to healthybrain tissue. The development of such therapies necessitates ananimal model that accurately recapitulates the molecular andcellular hallmarks of group 3 medulloblastoma subtypes.

In recent years, several animalmodels have been generated. Forexample, onemodel generated fromCD133þ cells overexpressingMyc and Gfi1 (7) may represent group 3b tumors. Other modelsinvolve Myc overexpression in combination with Trp53 inactiva-tion in eitherCD133þ cells orMath1þ granule neuronprogenitors(GNP; refs. 7–10). However, mutation or deletion of TP53 israrely detected in human group 3 medulloblastoma at diagno-sis (11, 12), indicating that loss of function of TP53 is not requiredfor human tumor initiation. Mouse models featuring Trp53mutation may thus be of limited relevance for understandinghuman tumor biology and therapy development.

Because group 3g tumors frequently harborMYC amplificationwithout additional focal mutations, it is of interest to determinewhetherMYC overexpression alone can initiate tumor formationin the developing cerebellum.MYC alone was thought incapableof inducing neoplastic transformation because high levels ofMYC drive apoptosis (13, 14). However, it is now clear thatMYC-induced apoptosis and tumor initiation depend uponcell type and developmental context (15–17). Medulloblastomasare thought to originate from immature cells at an early stageof cerebellar development, and different subgroups of

1Center for Cancer and Immunology, Brain Tumor Institute, Children's NationalHealth System, Washington, DC. 2Division of Immunotherapy, Institute ofHuman Virology, School of Medicine, University of Maryland, College Park,Maryland. 3Hopp Children's Cancer Center, NCT, Heidelberg, Germany. 4Divisionof Pediatric Neuro-oncology of the German Cancer Research Center andGermanCancer Consortium,Heidelberg, Germany. 5Division ofNeuropathology,Johns Hopkins University, Baltimore, Maryland.

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

Corresponding Author: Yanxin Pei, Children's National Health System, 111Michigan Avenue NW,Washington, DC 20010. Phone: 202-476-2558; Fax: 202-476-6994; E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-18-1787

�2019 American Association for Cancer Research.

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medulloblastoma have distinct cell origins. WNT subgrouptumors originate from progenitor cells in the lower rhombic lipof the brainstem (18), while SHH tumors arise from GNPs in theexternal granular layer (EGL; refs.19, 20). In contrast, the cell oforigin for group 3 medulloblastoma has not yet been wellcharacterized. Here, we showed that MYC overexpression wassufficient to drive tumorigenesis in astrocyte progenitors in theearly postnatal cerebellum in mice. The resulting tumors accu-rately resembled human group 3 medulloblastoma in terms ofhistology and gene expression, suggesting that astrocyte progeni-tors in the early postnatal cerebellum may represent the cell oforigin for group 3 medulloblastoma.

In the course of analyzing our new mouse model of MYC-driven medulloblastoma, we discovered that key genes involvedin glycolysis were significantly upregulated inMYC-driven tumorscompared with normal cerebellar tissue or SHH-associatedmedulloblastoma. Of these genes, LDHA (encoding lactate dehy-drogenase A) expression positively correlated with MYC and wasassociated with poor prognosis in group 3 medulloblastoma.Furthermore, inhibition of LDHA significantly suppressed growthof MYC-driven medulloblastoma, but had little effect on thegrowth of SHH-associated medulloblastoma or viability of nor-mal cerebellar cells. Thus, our studies identify LDHA as a novel,specific target for MYC-driven medulloblastoma treatment.

Materials and MethodsAnimals

Aldh1L1�GFP mice were provided by Dr. Jeffery Rothstein(John'sHopkinsUniversity School ofMedicine); Aldh1L1-CreERT2

mice were provided by Dr. Baljit Khakh (University of California,Los Angeles); Math1�GFP, Sox2�GFP, Sox2-CreERT2, Sox2�loxp,Rosa-CAG-LSL-tdTomato, NOD-SCID-IL2Rg null (NSG) andC57BL/6J mice were purchased from the Jackson Laboratory. Micewere maintained in the Animal Facility at Children's NationalHealth System (CNHS). All experiments were performed in accor-dance with national guidelines and regulations, and with theapproval of the animal care and use committee at CNHS.

Tumor generationTo generate tumors, we isolated total cerebellar cells from P5

mice of C57BL/6J, Sox2-CreERT2/Rosa-CAG-LSL-tdTomato,Sox2-CreERT2 or Sox2-CreERT2/Sox2�loxp; or we FACS-sortedcells from P5 mice of C57BL/6J, Sox2�GFP, or Sox2-CreERT2/Aldh1L1�GFP/Rosa-CAG-LSL-tdTomato. These cells wereinfected with the indicated viruses and cultured overnight inNeuroCult medium (STEMCELL Technologies) supplementedwith Proliferation Supplement (STEMCELL Technologies), basicfibroblast growth factor (bFGF), and epidermal growth factor(EGF; PeproTech). Cells were then injected into the cerebella ofNSGmice (6–8weeks old) using a stereotaxic framewith amouseadaptor (DavidKopf Instruments), as describedpreviously (8). Topermanently label Sox2þ cells with tdTomato, total cerebellarcells from P5 Sox2-CreERT2/Rosa-CAG-LSL-tdTomato mice werecultured with 100 nmol/L 4-hydroxytamoxifen (Sigma) over-night. To knockout Sox2 in Sox2þ cerebellar cells, total cerebellarcells from P5 Sox2-CreERT2/Sox2�loxp mice were cultured with100 nmol/L 4-hydroxytamoxifen overnight. After transplanta-tion, animals were treated with tamoxifen for an additional 6days to ensure complete Sox2 deletion. Mice receiving mock-treated Sox2-CreERT2/Sox2�loxp cells or 4-hydroxytamoxifen–

treated Sox2-CreERT2 cells were used as controls. The mice receiv-ing 4-hydroxytamoxifen–treated Sox2-CreERT2 cells were alsotreatedwith tamoxifen for additional 6 days after transplantation.

Glycolysis pathway inhibition assaysTo assess the effects of small-molecule inhibitors of glucose

metabolism on cell growth, tumor cells were freshly isolated fromtumor-bearingmice and treatedwith the indicated concentrationsofGSK2837808a (Tocris Bioscience), FX11 (Calbiochem), PKI-III(Calbiochem), or DCA (Tocris Bioscience). Cells were cultured in384-well Greiner plates for 7 days in stem cell medium (Neuro-basal Media-Vitamin A þ DMEM/F12 þ Non-Essential AminoAcids þ Sodium pyruvate þ Hepes þ GlutaMAX þ Pen-Strep þB27 þ EGF þ bFGF þ Lif þ Heparin). Cell viability was thenassessed using the CellTiter-Glo assay (Promega). To determinethe effects of GSK 2837808a on cell viability of normal cells,mouse GNPs were cultured for 7 days on poly D-lysine-coatedplates with NeuroBasal medium (Life Technologies) supplemen-tedwith B27 (Gibco), SHH (PeproTech) and 2%FBS and contain-ing the indicated concentration of GSK 2837808a. Cell viabilitywas then assessed using the CellTiter-Glo assay.

LDHA knockdownTo assess the effects of LDHA knockdown on cell growth of

human group 3 or SHH group medulloblastoma in vitro, freshlyisolated patient-derived xenograft (PDX) tumor cells wereinfected with retrovirus encoding LDHA shRNA or correspondingcontrol shRNA overnight. Cells were then cultured in stem cellmedium for an additional 2 or 6 days. Cell viability was assessedusing the CellTiter-Glo assay.

To test the effects of LDHA knockdownon the growthof humangroup 3medulloblastoma in vivo, freshly isolated tumor cells wereinfected with retrovirus encoding LDHA shRNA or correspondingcontrol shRNA overnight. Cells were then injected into the cer-ebella of NSGmice (50,000 cells permouse). Mice were sacrificedonce they exhibited symptoms. Animal survival was assessed bythe Kaplan–Meier curve.

Mouse cells and PDXsAllmouse tumor cells or normal cellswere freshly isolated from

the indicated mice. PDX lines used for this study include MB002(G3) generated by the Cho lab (21); ICb-984 (SHH) generated bythe Li lab (22); Med-411-FH (G3) and Med-211-FH (G3) gener-ated by theOlson lab (23, 24); andRCMB20 (G3), RCMB40 (G3),and RCMB28 (G3) generated by theWechsler-Reya lab (25). PDXlines were generated by implanting patient cells directly into thecerebella of immune-compromised mice, and propagating themfrommouse tomouse without in vitro passaging. The identity andsubgroup of each line was validated by gene expression and/ormethylation analysis. We did not perform testing forMycoplasma.

Accession numbersRNA-seq data have been deposited in the GEO public database

(http://www.ncbi.nlm.nih.gov/geo/), with accession numberGSE114760.

ResultsOverexpression of MYC alone is sufficient to initiatetumorigenesis in the cerebellum

To investigate whether overexpression of the MYC oncogenealone is sufficient to initiate tumorigenesis, we isolated total

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cerebellar cells fromC57BL/6Jmice atpostnatal days2–7 (P2-7), asopposed to isolating specific cell populations as was done previ-ously (8, 9). In addition, we infected the cells withMYC lentivirusrather than retrovirus, allowing both quiescent and proliferatingcells tobe transduced.We culturedMYC-infected cerebellar cells for16 hours and then transplanted the cells into the cerebella ofNSG mice. Tumor growth was monitored in vivo using biolumi-nescence imaging to detect the expression of luciferase proteinencoded by the lentivirus vector. Most animals exhibited strongluciferase signal in the cerebellumanddeveloped tumorswithin60days after transplantation (Fig. 1A and B). Staining of tumor tissuewith hematoxylin and eosin showed large cell/anaplastic (LCA)histology, with nuclear molding and wrapping, focally prominentnucleoli, abundant mitotic and apoptotic cells as well as necroticfoci (Fig. 1C; Supplementary Fig. S1A), typical features of group3 medulloblastoma. IHC staining showed that these tumorswere highly proliferative (Ki67þ) and apoptotic (cleaved cas-pase-3, CC-3þ). Some tumor cells expressed neuronal markerSynaptophysin, and rare tumor cells expressed glial marker glialfibrillary acidic protein (GFAP; Supplementary Fig. S1B–S1E).These findings suggest thatMYC overexpression alone is sufficientto induce group 3-like medulloblastoma in the postnatal cerebel-lum, but the identity of the cell population(s) susceptible toMYC-driven transformation had yet to be defined.

Sox2þ cells in the postnatal cerebellum are susceptible totransformation by MYC

Sox2þ cells represent stem cell/progenitors in the earlypostnatal cerebellum, but their ability to form MYC-driven

medulloblastoma has never been evaluated. We thus examinedwhether these cells could be transformed by MYC. We firstevaluated Sox2 expression in the cerebella of P5 C57BL/6J andSox2�GFP mice (26) and found abundant Sox2þ cells located inthe Purkinje cell layer (PCL), white matter folium, and deepwhite matter (DWM) but not in the EGL (Supplementary Fig.S1F–S1H). We then examined the proliferation and apoptosis ofSox2þ cells after transduction withMYC. FACS-sorted GFPþ cellsfrom the cerebella of P5 Sox2�GFP mice (Fig. 1D) were infectedwith lentivirus encoding doxycycline (Dox) inducibleMYC.MYCoverexpression (Doxþ) resulted in a significant increase in pro-liferation, but little change in apoptosis compared with cellswithoutMYC overexpression (Dox�; Supplementary Fig. S1I andS1J). When the MYC-transduced Sox2þ cells were transplantedinto the cerebella of NSG mice, they developed highly aggressivetumors. In contrast,MYC-transduced Sox2� cells rarely developedtumors (Fig. 1E), suggesting that Sox2þ cells in the postnatalcerebellum are susceptible to MYC-induced transformation.

Most Sox2þ cells were observed to be quiescent, but a smallpopulation of Sox2þ cells was observed to be proliferative in thepostnatal cerebellum (Supplementary Fig. S1K). Lentivirus caninfect both quiescent and proliferating cells, but retrovirus onlyinfects proliferating cells. To evaluate whether MYC-driventumors arise from quiescent or proliferative Sox2þ cells, wetransplanted Sox2þ cells infected with MYC retrovirus into thecerebella of NSG mice. Our results showed that almost all micereceiving Sox2þ cells infected with MYC lentivirus developedtumors, but Sox2þ cells infected with MYC retrovirus did notgive rise to tumors (Supplementary Fig. S1L), indicating that the

Figure 1.

MYC overexpression alone drives tumorigenesisfrom Sox2þ cells in the postnatal cerebellum.A–C, Tumors generated from total cerebellar cells.A, Bioluminescent imaging. B, Survival curve.C, Hematoxylin and eosin staining of tumorsections. Asterisks show an area of necrosis and alarge tumor cell with marked nuclear atypia(anaplasia). Arrow shows prominent nuclearmolding. Arrowhead showsmitotic figures. Normalindicates normal tissue. Scale bars, 25 mm. D, FACS-sorting of Sox2þ cells. E, Survival curve of tumorsgenerated from Sox2þ or Sox2� cells. F, Schema foridentifying tumor cells of origin. G, tdT expressionin tumors generated from Sox2-CreERT2/Rosa-CAG-LSL-tdTomato cells treated with or without4-OHT. Nuclei were counterstained with DAPI(blue). Scale bars, 1 mm. H, Flow cytometry analysisof tdT expression in tumors from G and summary ofthe percentage of tdTþ tumors.

MYC-Driven MB Derived from Astrocyte Progenitor Cells

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quiescent Sox2þ cells are the target of MYC-induced tumorigen-esis in this study.

We then validated Sox2þ cells as the major cell populationsusceptible to MYC-induced transformation by an alternativeapproach. Sox2-CreERT2 mice (26) were crossed with Rosa-CAG-LSL-tdTomato mice (27) and total cerebellar cells wereisolated from the P5 progeny. Cells were infected with MYClentivirus and treated with 4-hydroxytamoxifen (4-OHT) to acti-vate CreERT2 and permanently label Sox2þ cells with tdTomato(tdT).Cellswere then transplanted into the cerebella ofNSGmice.Under these conditions, we hypothesized that tumors arisingspecifically from Sox2þ cells will exhibit universal tdT expression.If tumors arise fromboth Sox2þ and Sox2� cells, only a fraction ofcells within a given tumor will express tdT; or if tumors developfrom Sox2� cells, the tumor will be tdT� (Fig. 1F). We firstexamined whether tdT expression specifically labeled Sox2þ cellsand their progeny after tamoxifen treatment.We treatedmicewithtamoxifen at P5 and found that tdT expression was specificallyobserved in Sox2þ cells at P6 (Supplementary Fig. S2A) and tdTexpression remained stable even after Sox2 expression ceasedduring cerebellar development at P10 (Supplementary Fig.S2B). Further studies revealed that mice receiving cells without4-OHT treatment all developed tdT� tumors and all animalsreceiving cells treated with 4-OHT developed tumors with nearlyuniversal tdT expression (Fig. 1G and H), indicating that MYC-driven tumors originate specifically from Sox2þ cells in thissystem. tdTþ and tdT� tumors were otherwise indistinguishableon the basis of IHC staining for markers as described above(Supplementary Fig. S2C).

Tumors generated from Sox2þ cells resemble human group 3medulloblastoma

We characterized the tumors arising from Sox2þ cells (hereafterreferred to as SOX2 tumors) at the cellular and molecular levels.SOX2 tumors exhibited LCA histology, high proliferation index,abundant apoptosis, and strong MYC expression (Fig. 2A–D),suggesting theymay represent group 3medulloblastoma. A smallnumber of tumor cells were positive for Synaptophysin or GFAP(Fig. 2E and F). Few tumor cells expressed Sox2, and a largenumber of cells were Olig2þ (Fig. 2G and H). Olig2 expressionwas also observed in a subset of human group 3medulloblastomaPDX (Fig. 2I–L).

To determine whether these tumors resemble group 3 medul-loblastoma at the molecular level, we performed RNA-seq anal-ysis on mouse MYC-driven tumors and compared the gene-expression profiles with those of 167 human tumor samples froma recent global medulloblastoma study (2). The comparativeanalysis was based on strict selection of 17,039 orthologous genesand precise batch-effect adjustment to address species differences.Principal component analysis (PCA) and/or hierarchical cluster-ing showed that mouseMYC-driven tumors generated from totalcerebellar cells (TC tumor) or Sox2þ cells (SOX2 tumor) weresimilar and indistinguishable from human group 3medulloblas-toma at the level of gene expression (Fig. 2M and N; Supplemen-tary Fig S3A; Supplementary Table S1–S3). They also demonstrat-ed strong similarity to previous models of Myc þ DNp53 tumor(MP tumor) and Myc þ Gfi1/Gfi1b tumor (MG/MGb tumor;Supplementary Fig. S3B; Supplementary Table S4). Specifically,mouse SOX2 tumors statistically resembled human MYC-ampli-fied group 3 medulloblastoma (Fig. 2M; SupplementaryFig. S3C). Further analysis revealed correspondence of SOX2

tumorsmainly to group 3 subtype II medulloblastoma associatedwith MYC amplification (Fig. 2O; ref. 2).

We also analyzed marker genes characterizing medulloblasto-ma molecular subgroups including DKK1 (WNT group), SFRP1(SHH group), NPR3 (group 3), and KCNA1, CDK6, and MYCN(group 4). DKK1 was not detected in SOX2 tumors. SFRP1 wassignificantly decreased in SOX2 tumors compared with normalcerebellar cells, suggesting that these are non-SHH subgrouptumors. Very low or almost no detectable expression of KCNA1,CDK6, andMYCN in SOX2 tumors suggests that these tumors donot model group 4 medulloblastoma. In contrast, NPR3 wassignificantly upregulated in SOX2 tumors compared with normalcerebellar cells (Supplementary Table S5). In addition, no Trp53mutation or Gfi1 overexpression was observed in SOX2 tumors(Supplementary Fig. S3D and E).

MYC-driven tumors arise from astrocyte progenitorsThe observation of Sox2 expression in distinct areas of the

cerebellum prompted the question of whether Sox2 is expressedin distinct cell lineages, one ormore of which is able to give rise toMYC-driven tumors. We found that Aldh1L1, which is a glialmarker, can be used to distinguish different populations of Sox2þ

cells. In the cerebellum of P5 Aldh1L1�GFP mice (28), Sox2þ cellsin the PCL expressed strong GFP (Fig. 3A and B), while mostSox2þ cells in the DWM expressed weak GFP and a fractionof Sox2þ cells in this area were GFP� (Fig. 3C and D). Furtheranalysis showed that the Sox2þ/Aldh1L1neg cells in theDWM expressed Olig2 (Fig. 3E–H). Using Aldh1L1�GFP/Sox2-CreERT2/Rosa-CAG-LSL-tdTomato mice, we could divide Sox2þ

cells into three populations: tdTþ/GFPhigh (Sox2þ/Aldh1L1high),tdTþ/GFPlow (Sox2þ/Aldh1L1low), and tdTþ/GFPneg (Sox2þ/Aldh1L1neg) cells (Fig. 3I).

To characterize Sox2þ/Aldh1L1high, Sox2þ/Aldh1L1low, andSox2þ/Aldh1L1neg cells, we FACS-sorted these cells (Fig. 3I; Sup-plementary Fig. S4A) and evaluated their self-renewal and differ-entiation potential in vitro. Sox2þ/Aldh1L1high cells exhibited thehighest frequency of neurosphere formation, while very fewneurospheres arose from Sox2þ/Aldh1L1low and almost no neu-rospheres were observed from Sox2þ/Aldh1L1neg cells (Fig. 3J;Supplementary Fig. S4B). Sox2þ/Aldh1L1high cells formed sec-ondary spheres but could not generate tertiary spheres (Fig. 3K).Instead, they formed an adherent cell layer with astrocytic mor-phology (Supplementary Fig. S4C), indicating they were incapa-ble of long-term self-renewal. When primary neurospheres werecultured in differentiation medium, they differentiated exclusive-ly into astrocytes (GFAPþ). No neurons (NeuNþ), interneurons(Pax2þ), or oligodendrocytes (O4þ) were observed (Fig. 3L).Furthermore, freshly isolated Sox2þ/Aldh1L1high, Sox2þ/Aldh1L1low, and Sox2þ/Aldh1L1neg cells cultured in differentia-tionmedium gave rise predominantly to astrocytes, interneurons,and oligodendrocytes, respectively (Supplementary Fig. S4D andS4E). These results suggest that Sox2þ cells are composed of threedistinct lineages, and Sox2þ/Aldh1L1high cells are unipotentastrocyte progenitors capable of short-term self-renewal. In vivolineage-tracing studies revealed that Aldh1L1þ cells differentiatedinto astrocytes and interneurons in the early postnatal cerebellum(Supplementary Fig. S4F and S4G), consistent with our in vitroobservations. We then examined the potential of each cell pop-ulation to generate tumors and found that tumors primarily arosefrom Sox2þ/Aldh1L1high cells. Rare tumors arose from Sox2þ/Aldh1L1low cells, with lower penetrance and longer latency.

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Sox2þ/Aldh1L1neg cells did not develop tumors (Fig. 3M). Thesefindings strongly suggest thatMYC-driven tumors originate fromastrocyte progenitors.

A previous study showed that CD133þ cells in the postnatalcerebellum consist of glial progenitors (29). We thus examined

whether Sox2andCD133were expressed in the samecells. Amongtotal cerebellar cells, approximately 4% were CD133þ and 20%were Sox2þ. However, only 10% of Sox2þ cells were CD133þ,indicating that while CD133 and Sox2 markers indeed overlap tosome degree, Sox2 expression identifies a distinct population(s)

Figure 2.

SOX2 tumors resemble group 3 medulloblastoma at the cellular and molecular levels. A, Hematoxylin and eosin (H&E) staining of tumor sections. Asterisks showan area of necrosis and a large tumor cell with marked nuclear atypia (anaplasia). Arrow shows prominent nuclear molding. Arrowhead shows mitotic figures.B–H, IHC staining of SOX2 tumor tissue. I–L,Olig2 expression in human group 3 medulloblastoma PDX. In all IHC staining, scale bars, 25 mm.M, PCA for batch-effect–adjusted gene expression (RPKM) quantifies data from humanmedulloblastoma and SOX2 tumors for the top 500most variable genes across thedataset. N, Unsupervised hierarchical clustering for four groups of humanmedulloblastoma and SOX2 tumor applied on the ortholog subset from significantmedulloblastoma group-specific differentially expressed genes.O, Unsupervised hierarchical clustering of batch-effect–adjusted SOX2 tumors and group3 medulloblastoma samples assigned with subtypes I, II, III, and IV.






of cells (Fig. 4A andB).We then characterized the Sox2þ/CD133þ,Sox2þ/CD133�, and Sox2�/CD133þ populations by evaluatingself-renewal, differentiation, expression of lineage markers andpotential for tumorigenesis. Neurospheres were generated from

Sox2þ/CD133þ cells and Sox2þ/CD133� cells, but not fromSox2�/CD133þ cells (Fig. 4C). When neurospheres were culturedin differentiation medium, they differentiated exclusively intoastrocytes, suggesting that the cells capable of self-renewal are

Figure 3.

MYC-driven tumors arise from astrocyte progenitors.A–H, Sox2þ cells are composed of Aldh1L1high, Aldh1L1low, and Aldh1L1neg cells. Scale bars, 100 mm (A, C, E,and G). B, D, F, and H, High magnification images corresponding to the inset boxes inA, C, E, and G. Scale bars, 25 mm. I, Isolation of Sox2þ/Aldh1L1high, Sox2þ/Aldh1L1low, and Sox2þ/Aldh1L1neg cells by flow cytometry. J, Number of primary neurospheres from 5,000 cells. K,Number of primary, secondary, or tertiaryneurospheres from 5,000 Sox2þ/Aldh1L1high cells. Error bars, mean� SD. L, Differentiation of primary neurospheres from Sox2þ/Aldh1L1high cells. Nuclei werecounterstained with DAPI (blue). Scale bars, 100 mm.M, Survival curve (n¼ 6 each).

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astrocyte progenitors (Fig. 4D). Flow cytometry analysis showedthat neuron progenitors (PSA-NCAMþ) and oligodendrocyteprogenitors (O4þ; ref. 29) were enriched in Sox2�/CD133þ cells,but not in Sox2þ/CD133þ or Sox2þ/CD133� cells (Supplemen-tary Fig. S5A). Sox2þ/CD133þ and Sox2þ/CD133� cells infectedwith MYC lentivirus developed tumors, while Sox2�/CD133þ

cells did not (Fig. 4E). Although a subpopulation of CD133þ cellsexpress Sox2, CD133þ cells infected with Myc retrovirus failed toform tumors in previous studies (8, 9). To uncover the reasons forthis, we examined cell fate of Sox2þ cells infected with MYClentivirus or CD133þ cells infected with Myc retrovirus shortly

after transplantation. Two days after transplantation, we foundthat allMYC lentivirus–infected cells were Sox2þ, while only rareMyc retrovirus–infected cells were Sox2þ (Fig. 4F), suggesting thatMyc retrovirus did not infect the Sox2þ subpopulation in CD133þ

cells. FewCC-3þ cells were detectable in eitherMYC virus infectedCD133þ cells or Sox2þ cells at day 2 or day 9. At day 14, however,CC-3 staining was abundant in the transplanted CD133þ cellswhereas few CC-3þ cells were observed in the transplanted Sox2þ

cells (Fig. 4F). Sox2þ cells infected with MYC lentivirus furthergrew tumors while CD133þ cells infected withMyc retrovirus didnot, even though the MYC levels achieved by these viruses were

Figure 4.

Characterization of Sox2þ versus CD133þ cells in the early postnatal cerebellum. A and B, Expression of CD133 and Sox2 in the early postnatal cerebellum. Totalcerebellar cells from P5 wild-type (A) or Sox2�GFP (B) mice were stained with APC-conjugated isotype or anti-CD133 antibody and then analyzed for GFP orCD133 expression by flow cytometry. C, Number of primary neurospheres from 5,000 cells. D, Differentiation of primary neurospheres. E, Survival curve. F, IHCstaining for Sox2 or CC-3 in preneoplasms. GFP indicatesMYC virus–infected cells shown as green. Normal, normal tissue. In all IHC staining, scale bars, 50 mm,and nuclei were counterstained with DAPI (blue).






similar (Supplementary Fig. S5B and S5C). These observationssuggest that similar level of MYC induce different levels of apo-ptosis in Sox2þ cells versus CD133þ cells, whichmay render thesecells differentially susceptible to MYC-induced transformation.

Sox2 promotes MYC-induced tumorigenesisThe observation that MYC-driven tumors predominantly arise

from Sox2þ cells, without requiring other mutations, promptedthe question whether Sox2 itself plays a critical role in MYC-induced tumorigenesis. We first determined whether knockout ofSox2 affected MYC-induced apoptosis in Sox2þ cells. P5 Sox2-CreERT2/Sox2�loxp mice were treated with tamoxifen to knockout

Sox2 in Sox2þ cells, and brain tissue was collected at P10. In theabsence of tamoxifen treatment, Sox2 expression was colocalizedwith Sox9. With tamoxifen treatment, Sox2 was completely delet-ed, but Sox9 expression was not affected (Fig. 5A and B). Thus,Sox9 traced the presence of Sox2þ cells when Sox2 is knocked out.Moreover, no CC-3 staining was observed in Sox2 knockout cells(Fig. 5C and D). These data suggest that deletion of Sox2 did notaffect survival of Sox2þ cells. We then examined CC-3 expressionin Sox2þ cells or Sox2 knockout cells isolated from tamoxifen-treated Sox2-CreERT2/tdTomato or Sox2-CreERT2/Sox2�loxp/tdTomato mice and infected with control or MYC lentivirus invitro. The result showed that MYC overexpression markedly

Figure 5.

Sox2 promotesMYC-induced tumorigenesis.A–D, IHC staining for Sox2, Sox9, or CC-3 in P10 cerebellum. Scale bars, 50 mm. Nuclei were counterstained withDAPI (blue). E, Expression of CC-3 assessed byWestern blot. F, Survival curve of mice transplanted with cells from Sox2-CreERT2/Sox2loxp mice or Sox2-CreERT2

mice treated with or without tamoxifen (n¼ 5 each). G, Survival curve of mice transplanted with Sox2þ cells infected withMYC lentivirus or Sox2� cells infectedwith viruses ofMYC orMYCþ DNp53 (n¼ 4 each). H, Survival curve of mice transplanted with Sox2þ cells infected withMYC lentivirus or Sox2� cells infectedwith viruses ofMYC orMYCþ Sox2 (n¼ 8 each). I, Survival curve of mice transplanted with Sox2� cells infected with viruses ofMYC orMYCþ Sox2 (n¼ 4 each).Exogenous Sox2 expression was shut off at 10 days after transplantation. J, Sox2 expression assessed by qRT-PCR. mRNA level in Sox2� cells was set up to 1.Error bars, mean� SD. � , P < 0.05; ��, P < 0.0001.

Tao et al.





induced CC-3 expression in Sox2 knockout cells but not in Sox2þ

cells (Fig. 5E), suggesting that Sox2 antagonizes MYC-inducedapoptosis.

Second, we tested whether Sox2 deletion in Sox2þ cellsaffected their susceptibility to MYC-induced transformation.Total cerebellar cells isolated from P5 mice of Sox2-CreERT2/Sox2loxp or Sox2-CreERT2 were infected with MYC lentivirus,cultured with or without 4-OHT for 16 hours and transplantedinto the cerebella of NSG. 4-OHT treatment itself (i.e., in theabsence of Sox2 deletion) did not affect tumor growth, whiledeletion of Sox2 was associated with significantly decreasedtumor penetrance and increased tumor latency (Fig. 5F). Sox2expression was not detected in the tumors generated from4-OHT–treated cells compared with control tumors (Supple-mentary Fig. S6A and B). Sox2 knockout delayed tumor growthbut did not completely prevent tumorigenesis. We reasonedthat high levels of Sox1, Sox3, Sox8, and Sox10 expression inSox2þ cells (Supplementary Fig. S6C–S6F) may partially com-pensate for loss of Sox2 function (30, 31).

Third, we determined whether overexpression of Sox2 inSox2� cells could promote MYC-induced tumorigenesis. Sox2�

cells overexpressing MYC alone did not give rise to tumors,while mice receiving Sox2� cells overexpressing MYC/Sox2 orMYC/DNp53 and Sox2þ cells overexpressing MYC developedtumors with LCA histology (Fig. 5G and H; Supplementary Fig.S6G). We further evaluated the role of Sox2 in tumor initiationand progression. Sox2� cells were infected with viruses encod-ing MYC and Dox-inducible Sox2 and transplanted into thecerebellum. Following transplantation, mice were fed Doxsupplemented diet to induce Sox2 expression for the first10 days. Subsequently, the mice were switched to a normaldiet to eliminate Sox2 expression. Eliminating Sox2 expressiondid not affect tumor development (Fig. 5I), indicating that Sox2promotes the onset of MYC-induced tumorigenesis but is notrequired for tumor progression.

Sox2þ/Aldh1L1low and Sox2þ/Aldh1L1neg cells express Sox2,but they failed to develop MYC-driven tumors. Sox2 levels inSox2þ/Aldh1L1low and Sox2þ/Aldh1L1neg cells were significantlylower than in Sox2þ/Aldh1L1high cells (Fig. 5J), which mayaccount for the nonsusceptibility of Sox2þ/Aldh1L1low andSox2þ/Aldh1L1neg cells to MYC-induced transformation.

Transcriptional profiling identifies pathways involved in thegrowth of murine MYC-driven medulloblastoma

To identify potential therapeutic targets for treatingMYC-driv-en medulloblastoma, we compared the gene-expression profilesof SOX2 tumors with those of freshly isolated Sox2þ andMath1þ

postnatal cerebellar cells. PCA showed that the tumor cells weredistinct from Sox2þ cells, from which they were derived, andalso distinct fromMath1þGNPs (Fig. 6A). Hierarchical clusteringconfirmed that the SOX2 tumors differed from their normalcell counterpart (Fig. 6B). Focusing on 2-fold and higherchanges in gene expression (P value with FDR correction <0.05), we identified 1,013 upregulated and 896 downregulatedgenes in SOX2 tumors compared with Sox2þ cerebellar cells(Supplementary Table S6).

To gain insight into the pathways controlling the growth ofmouse MYC-driven tumors, we studied genes differentiallyexpressed in tumors versus normal cerebellar cells using func-tional enrichment analysis (KEGG pathway annotation). Theanalysis showed that the genes upregulated inMYC-driven tumors

were associated with ribosome biogenesis, RNA polymerase, RNAtransport, metabolic pathways, and glycolysis/gluconeogenesis(Fig. 6C; Supplementary Table S7).

Targeting LDHA inhibits the growth of MYC-drivenmedulloblastoma

Upregulated expression of genes associated with glycolysisin mouse MYC-driven medulloblastoma suggested that therapid proliferation of tumor cells may rely on this pathway,and that targeting this pathway may inhibit tumor growth. ByRNA-seq analysis, we found upregulation of genes encodingkey enzymes controlling glucose metabolism including Ldha,Pkm2 (Pyruvate kinase muscle isozyme M2), Hk2 (Hexokinase2), and Pdk1 (pyruvate dehydrogenase kinase 1) in SOX2tumor cells compared with Sox2þ normal cerebellar cells(Fig. 6D; Supplementary Table S8). qRT-PCR and Western blotanalysis confirmed the upregulation of these genes at mRNAand protein levels in SOX2 tumors compared with normalcerebellar cells or mouse SHH medulloblastoma (Fig. 6Eand F). To determine whether growth of mouse MYC-driventumors was dependent on glucose metabolism, we tested theeffects of LDHA, PKM2, and PDK1 antagonists on tumor cellgrowth in vitro. GSK 2837808a and FX11, two antagonists ofLDHA, dramatically inhibited the growth of SOX2 tumor cellsbut exhibited minimal growth inhibition on mouse SHHtumor cells (Fig. 6G and H). In contrast, neither pyruvatekinase inhibitor III (PKI-III, PKM2 antagonist) nor dichloroa-cetate (DCA, PDK1 antagonist) showed inhibitory effects, evenat the highest concentration (Fig. 6I and J). LDHA inhibitorhad little effect on viability of normal cerebellar cells(GNP; Fig. 6K).

LDHA, PKM2, HK2, and PDK1 expressionwas also upregulatedin human group 3 medulloblastoma compared with normalcerebellar tissue or other subtypes of medulloblastoma (Fig. 7Aand B; Supplementary Table S9). LDHA antagonists dramaticallyinhibited the growth of group 3 PDX cells but showed minimalgrowth inhibition on SHH subgroup PDX cells (Fig. 7C and D).Neither PKM2antagonist nor PDK1antagonist showed inhibitoryeffects, even at the highest concentration (Fig. 7E and F). LDHAcatalyzes the conversion of pyruvate to lactate (32). We thusanalyzed lactate production in group 3 medulloblastoma PDXcells treated with LDHA antagonist. We found that inhibition ofLDHA significantly decreased lactate levels in group 3 PDX cellscompared with control (Fig. 7G).

Further studies showed that LDHA expression positively cor-related with MYC (Fig. 7H and I; Supplementary Table S9) andwas associated with poor prognosis in human group 3 medullo-blastoma (Fig. 7J; Supplementary S7A). Knockdown of LDHAsignificantly reduced viability of group 3 PDX cells in vitro (Fig. 7Kand L; Supplementary S7B and C) and tumor growth in vivo(Fig. 7M), indicating that group 3 medulloblastoma growth isdependent on LDHA. In contrast, knockdown of LDHA did notaffect the viability of SHH PDX cells in vitro (SupplementaryFig. S7D). In addition, LDHA knockdown did not affect MYCexpression in group 3 PDX cells (Supplementary Fig. S7E).

DiscussionThe present work demonstrates that MYC overexpression

alone, in the absence of other genetic alterations, can drivetumorigenesis from astrocyte progenitors in the postnatal






Figure 6.

Inhibition of LDHA suppresses growth of mouseMYC-driven medulloblastoma. A, PCA of SOX2 tumors and P5 Sox2þ and Math1þ normal cerebellar cells using allgenes exhibiting variation across the data set. B, Heat map showing dysregulated genes in SOX2 tumors compared with normal Sox2þ cells. C, Biologicalpathways enriched in SOX2 tumors. D, Heat map showing genes involved in glycolysis in SOX2 tumor and normal Sox2þ cells. E, Expression of Ldha, Pkm2, Hk2,and Pdk1 assessed by qRT-PCR. Mean mRNA value in normal cerebellar cells was set to 1. � , P < 0.05; �� , P < 0.01; �� , P < 0.001. F, Expression of Ldha, Pkm2, Hk2,and Pdk1 assessed byWestern blot. G–J, Effect of LDHA inhibitors (G and H), PKM2 inhibitor (I), or PDK1 inhibitor (J) on the growth of tumor cells in vitro.K, Effect of the LDHA inhibitor on the viability of GNPs in vitro. Error bars, mean� SEM.

Tao et al.





cerebellum. The tumors accurately model human group 3medul-loblastoma associated with amplification of MYC and thereforeprovide a valuable tool for the evaluation of novel therapies. By

studying this novelmousemodel,we identified LDHA as a specifictarget for the development of less toxic therapies for this highlymalignant disease.

Figure 7.

Inhibition of LDHA suppresses the growth of humanMYC-driven medulloblastoma. A, Expression of LDHA, PKM2, HK2, and PDK1 assessed by RNA-seq. B,Expression of LDHA, PKM2, HK2, and PDK1 in group 3 and SHH group medulloblastoma PDX or normal human cerebellum examined byWestern blot. C–F, Effectof LDHA inhibitors (C and D), PKM2 inhibitor (E), or PDK1 inhibitor (F) on the growth of PDX cells in vitro. Error bars inA and C–F indicate mean� SEM. G, Lactatelevels in group 3 PDX cells treated with an LDHA inhibitor in vitro. Value of untreated sample collected at 4 hours was set to 100. H and I, Positive correlation ofLDHA andMYC expression in human group 3 medulloblastoma. H, Linear regression of LDHA andMYCmRNA level, R-squared is indicated. I, Bar plot of LDHAandMYCmRNA levels. J, Kaplan–Meier analysis of group 3 medulloblastoma patients. K, Knockdown of LDHA in group 3 PDX cells examined byWestern blot.L, Knockdown of LDHA inhibits growth of group 3 PDX cells in vitro. Viability of cells without virus infection (Ctr) was set to 100%.M, Knockdown of LDHA inhibitstumor growth of group 3 PDX in vivo. n¼ 10 in each group. In G and L, error bars indicate mean� SD. Asterisk represents statistical significance betweenindicated treated groups and untreated groups. � , P < 0.05; �� , P < 0.01; �� , P < 0.001.






Sox2þ astrocyte progenitors are susceptible to MYCtransformation

MYC-induced tumorigenesis often requires a defined range ofMYC levels. Excessive activation of MYC is associated with DNAdamage and apoptosis, while less robustMYC activation inducesproliferative arrest and cellular senescence (33, 34). In previouslydescribed group 3 medulloblastoma mouse models, in uteroelectroporation of MYC plasmid into distinct populations ofembryonic cerebellar progenitor cells did not induce tumors (10),possibly because MYC levels achieved by this approach were toolow to overcome proliferation arrest. In other models (8, 9),inactivation of Trp53 was required for MYC-induced tumorigen-esis, suggesting that inactivation of Trp53 may be required toovercome the MYC-induced apoptosis in those cells.

MYC-induced transformation also depends on geneticand epigenetic cellular context (15, 35). Mycn overexpressionfailed to initiate tumor formation in several experimental mod-els (36, 37) but was shown to transform Glt1-positive glial cellsand certain distinct neural stem cell populations (38, 39), suggest-ing that distinct cell types are susceptible toMycn-induced tumor-igenesis. Our studies show that Sox2þ astrocyte progenitors in thepostnatal cerebellum are susceptible toMYC-induced tumorigen-esis and Sox2 itself plays a critical role in this process. Sox2 hasbeen reported to play important roles in cancer, where it promotestumor cell proliferation, colony formation and self-renewal ofcancer stem cells (CSC; refs. 40, 41). Here, we report that Sox2 iscritical forMYC-induced tumor initiation. We demonstrated thatSox2 antagonized MYC-induced apoptosis in Sox2þ cerebellarcells and knockout of Sox2 in these cells significantly delayed theformation of MYC-driven tumors. Moreover, overexpression ofSox2 promoted MYC-induced tumorigenesis from Sox2� cells,which otherwise cannot be transformed by MYC overexpressionalone. In this process, Sox2was required only for tumor initiation,but not for tumor progression. A subpopulation of CD133þ cellsexpressed Sox2 (CD133þ/Sox2þ), but CD133þ cells infected withMyc retrovirus failed to form tumors. The observation that theSox2þ subpopulation in CD133þ cells was not infected by Mycretrovirus and that Myc retrovirus infected cells underwent dra-matic apoptosis post-transplantation may explain the failure ofthese cells to form tumors. Sox2þ/Aldh1L1low and Sox2þ/Aldh1L1neg cells also expressed Sox2, but theywere not susceptibleto MYC-induced transformation. These cells had very limitedcapacity for self-renewal and expressed significantly lower levelsof Sox2 compared with Sox2þ/Aldh1L1high cells. Sox2 expressionlevels are linked to maintenance of progenitor cell identity andincreased capacity for self-renewal (42). We therefore reasonedthat the differential tumorigenesis potential of distinct Sox2þ

subpopulations may be related to Sox2 levels.SOX2 tumors included quiescent Sox2þ cells comprising less

than 5% of the tumor cell population. Rare and quiescent Sox2þ

cells have also been observed in SHH subgroup medulloblasto-ma, where theywere identified as CSCs driving tumor progressionand contributing to treatment resistance (43). It will be of greatinterest to determine in future studies whether Sox2þ cells inMYC-driven medulloblastoma are also CSCs and whether thesecells contribute to treatment resistance and therapeutic failure.

Identification of LDHA as a novel therapeutic target for MYC-driven medulloblastoma

In order to identify novel therapeutic targets for MYC-drivenmedulloblastoma treatment, we compared the gene-expression

profiles of mouse MYC-driven tumors to those of normalcerebellar cells from which they were derived. In addition tothe identification of pathways that are reportedly enriched inhuman group 3 medulloblastoma such as ribosome biogenesis,RNA polymerase, and RNA transport (5), our gene-expressionanalysis also revealed upregulation of genes involved in gly-colysis, which has not been previously well studied in MYC-driven medulloblastoma.

Cancer cells predominantly produce energy through a highrate of glycolysis with secretion of lactate even under conditionsof abundant oxygen (44), a process called aerobic glycolysis orthe Warburg effect. The key enzymes involved in aerobicglycolysis, including HK2, PKM2, PDK1, and LDHA, have beenidentified as potential targets for therapeutic intervention inmany cancers, including pediatric brain tumors. Deletion of thegene encoding HK2 in the developing brain was shown toinhibit SHH-induced aerobic glycolysis and reduce SHH-asso-ciated medulloblastoma growth (45), while PKM2 deletionaccelerated the growth of SHH-associated medulloblasto-ma (46). The roles of PDK1 and LDHA in medulloblastomahave not previously been evaluated.

In the current study, we found that PKM2 and PDK1 inhibitiondid not affect the growth of MYC-driven tumors. In contrast,inhibition of LDHA significantly reduced the growth of MYC-driven medulloblastoma with reduced secretion of lactate, buthad minimal effects on the growth of SHH-associated medullo-blastoma or normal cerebellar cells. The LDHA gene is known tobe regulated by MYC (47, 48) and HIF1A (49). However, we didnot observe upregulation of HIF1A in either mouse or humanMYC-drivenmedulloblastoma. We therefore suggested that upre-gulation of LDHA is very likely a result ofMYC overexpression ingroup 3 medulloblastoma. This is supported by our observationthat LDHA expression positively correlated with MYC levels andwas associated with poor prognosis.

Our studies present the first evidence that targeting LDHAeffectively inhibits growth of MYC-driven medulloblastoma,but not MYC-independent tumors or normal cerebellar cells.Although the results presented here are promising, existingLDHA inhibitors are not amenable to immediate preclinicaltranslational studies due to low blood–brain barrier pene-trance (50). Nevertheless, our findings warrant further studiesand should act as a spur to efforts to develop LDHA antagonistsclinically suitable for the treatment of MYC-driven medullo-blastoma patients.

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

Authors' ContributionsConception and design: R. Tao, R. Packer, Y. PeiDevelopment of methodology: R. Tao, C. Lazarski, R. Packer, Y. PeiAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): R. Tao, N. Murad, Z. Xu, C. Lazarski, C.G. Eberhart,Y. PeiAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): R. Tao, N. Murad, P. Zhang, K. Okonechnikov,M. Kool, S. Rivero-Hinojosa, Y. Liu, B.R. Rood, R. Packer, Y. PeiWriting, review, and/or revisionof themanuscript:R. Tao,N.Murad, P. Zhang,K. Okonechnikov, M. Kool, Y. Liu, C.G. Eberhart, B.R. Rood, Y. PeiAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): R. Tao, N. Murad, C. Lazarski, P. Zheng,R. Packer, Y. PeiStudy supervision: R. Tao, Y. Pei

Tao et al.





AcknowledgmentsThe authors would like to acknowledge Jeffery Rothstein for Aldh1L1�GFP

mice and Baljit Khakh for Aldh1L1-CreERT2 mice; Daniel Treisman, Yuan Zhu,and Zeng-jie Yang for providing mouse SHH medulloblastoma cells or cellpellets; Mirna Lechpammer for normal childhood cerebellar tissue; HuadongPei for LDHAshRNA,WilliamHahn forGFP shRNA, Scott Lowe forMSCV-IRES-Luciferase, and Didier Trono for pWPI plasmids; Yoon-Jae Cho, Robert Wechs-ler-Reya, Xiao-Nan Li, and JamesOlson for the PDX lines; RobertWechsler-Reyafor helpful discussions; and Adam Richman and Naomi Luban for editing the

manuscript. Thisworkwas supported by funds fromChildren'sNational HealthSystems, NIH R21CA180063 (YPEI) and R21NS102776 (YPEI).

The costs of publication of this articlewere defrayed inpart by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received June 14, 2018; revised August 7, 2018; accepted February 28, 2019;published first March 12, 2019.

References1. Packer RJ, Macdonald T, Vezina G, Keating R, Santi M. Medulloblastoma

and primitive neuroectodermal tumors. Handb Clin Neurol 2012;105:529–48.

2. Northcott PA, Buchhalter I, Morrissy AS, Hovestadt V, Weischenfeldt J,Ehrenberger T, et al. The whole-genome landscape of medulloblastomasubtypes. Nature 2017;547:311–7.

3. Cho YJ, Tsherniak A, Tamayo P, Santagata S, Ligon A, Greulich H, et al.Integrative genomic analysis of medulloblastoma identifies a molecularsubgroup that drives poor clinical outcome. J Clin Oncol 2011;29:1424–30.

4. Kool M, Korshunov A, Remke M, Jones DT, Schlanstein M, Northcott PA,et al. Molecular subgroups of medulloblastoma: an international meta-analysis of transcriptome, genetic aberrations, and clinical data of WNT,SHH, Group 3, and Group 4 medulloblastomas. Acta Neuropathol 2012;123:473–84.

5. Taylor MD, Northcott PA, Korshunov A, Remke M, Cho YJ, Clifford SC,et al. Molecular subgroups of medulloblastoma: the current consensus.Acta Neuropathol 2012;123:465–72.

6. Cavalli FMG, Remke M, Rampasek L, Peacock J, Shih DJH, Luu B, et al.Intertumoral heterogeneity within medulloblastoma subgroups. CancerCell 2017;31:737–54.e6.

7. Northcott PA, Lee C, Zichner T, St€utz AM, Erkek S, Kawauchi D, et al.Enhancer hijacking activates GFI1 family oncogenes in medulloblastoma.Nature 2014;511:428–34.

8. Pei Y, Moore CE, Wang J, Tewari AK, Eroshkin A, Cho YJ, et al. Ananimal model of MYC-driven medulloblastoma. Cancer Cell 2012;21:155–67.

9. Kawauchi D, Robinson G, Uziel T, Gibson P, Rehg J, Gao C, et al. A mousemodel of the most aggressive subgroup of human medulloblastoma.Cancer Cell 2012;21:168–80.

10. KawauchiD,OggRJ, Liu L, ShihDJH, FinkelsteinD,MurphyBL, et al.NovelMYC-driven medulloblastoma models from multiple embryonic cerebel-lar cells. Oncogene 2017;36:5231–42.

11. Hill RM, Kuijper S, Lindsey JC, Petrie K, Schwalbe EC, Barker K, et al.Combined MYC and P53 defects emerge at medulloblastoma relapse anddefine rapidly progressive, therapeutically targetable disease. Cancer Cell2015;27:72–84.

12. Zhukova N, Ramaswamy V, Remke M, Pfaff E, Shih DJ, Martin DC, et al.Subgroup-specific prognostic implications of TP53 mutation in medullo-blastoma. J Clin Oncol 2013;31:2927–35.

13. Pelengaris S, Rudolph B, Littlewood T. Action of Myc in vivo - proliferationand apoptosis. Curr Opin Genet Dev 2000;10:100–5.

14. Murphy DJ, Junttila MR, Pouyet L, Karnezis A, Shchors K, Bui DA, et al.Distinct thresholds govern myc's biological output in vivo. Cancer Cell2008;14:447–57.

15. Beer S, Zetterberg A, Ihrie RA, McTaggart RA, Yang Q, Bradon N, et al.Developmental context determines latency of MYC-induced tumorigene-sis. PLoS Biol 2004;2:e332.

16. Gabay M, Li Y, Felsher DW. MYC activation is a hallmark of cancerinitiation and maintenance. Cold Spring Harb Perspect Med 2014;4. doi:10.1101/cshperspect.a014241.

17. Baudino TA, Maclean KH, Brennan J, Parganas E, Yang C, AslanianA, et al. Myc-mediated proliferation and lymphomagenesis, but notapoptosis, are compromised by E2f1 loss. Mol Cell 2003;11:905–14.

18. Gibson P, Tong Y, Robinson G, Thompson MC, Currle DS, Eden C, et al.Subtypes ofmedulloblastoma have distinct developmental origins. Nature2010;468:1095–9.

19. Yang ZJ, Ellis T, Markant SL, Read TA, Kessler JD, Bourboulas M, et al.Medulloblastoma can be initiated by deletion of Patched in lineage-restricted progenitors or stem cells. Cancer Cell 2008;14:135–45.

20. Schuller U,Heine VM,Mao J, KhoAT, Dillon AK,Han YG, et al. Acquisitionof granule neuron precursor identity is a critical determinant of progenitorcell competence to formShh-inducedmedulloblastoma.CancerCell 2008;14:123–34.

21. Bandopadhayay P, Bergthold G, Nguyen B, Schubert S, Gholamin S, TangY, et al. BET bromodomain inhibition of MYC-amplified medulloblasto-ma. Clin Cancer Res 2014;20:912–25.

22. Zhao X, Liu Z, Yu L, Zhang Y, Baxter P, Voicu H, et al. Global geneexpression profiling confirms the molecular fidelity of primary tumor-based orthotopic xenograft mouse models of medulloblastoma.Neuro Oncol 2012;14:574–83.

23. Girard E, Ditzler S, Lee D, Richards A, Yagle K, Park J, et al. Efficacy ofcabazitaxel inmousemodels of pediatric brain tumors. NeuroOncol 2015;17:107–15.

24. Morfouace M, Shelat A, Jacus M, Freeman BB 3rd, Turner D, Robinson S,et al. Pemetrexed and gemcitabine as combination therapy for the treat-ment of Group3 medulloblastoma. Cancer Cell 2014;25:516–29.

25. Brun SN, Markant SL, Esparza LA, Garcia G, Terry D, Huang JM, et al.Survivin as a therapeutic target in Sonic hedgehog-driven medulloblasto-ma. Oncogene 2015;34:3770–9.

26. Arnold K, Sarkar A, Yram MA, Polo JM, Bronson R, Sengupta S, et al. Sox2(þ) adult stem and progenitor cells are important for tissue regenerationand survival of mice. Cell Stem Cell 2011;9:317–29.

27. Madisen L, Zwingman TA, Sunkin SM, Oh SW, Zariwala HA, Gu H, et al. Arobust and high-throughput Cre reporting and characterization system forthe whole mouse brain. Nat Neurosci 2010;13:133–40.

28. Yang Y, Vidensky S, Jin L, Jie C, Lorenzini I, Frankl M, et al. Molecularcomparison of GLT1þ and ALDH1L1þ astrocytes in vivo in astroglialreporter mice. Glia 2011;59:200–7.

29. Lee A, Kessler JD, Read TA, Kaiser C, Corbeil D, HuttnerWB, et al. Isolationof neural stem cells from the postnatal cerebellum. Nat Neurosci 2005;8:723–9.

30. Graham V, Khudyakov J, Ellis P, Pevny L. SOX2 functions to maintainneural progenitor identity. Neuron 2003;39:749–65.

31. Collignon J, Sockanathan S, Hacker A, Cohen-Tannoudji M, Norris D,Rastan S, et al. A comparison of the properties of Sox-3 with Sry and tworelated genes, Sox-1 and Sox-2. Development 1996;122:509–20.

32. Le A, Cooper CR, Gouw AM, Dinavahi R, Maitra A, Deck LM, et al.Inhibition of lactate dehydrogenase A induces oxidative stress and inhibitstumor progression. Proc Natl Acad Sci U S A 2010;107:2037–42.

33. FelsherDW, Zetterberg A, Zhu J, Tlsty T, Bishop JM.Overexpression ofMYCcauses p53-dependentG2 arrest of normalfibroblasts. ProcNatl Acad Sci US A 2000;97:10544–8.

34. Grandori C, Wu KJ, Fernandez P, Ngouenet C, Grim J, Clurman BE, et al.Werner syndrome protein limits MYC-induced cellular senescence.Genes Dev 2003;17:1569–74.

35. Li Y, Casey SC, Felsher DW. Inactivation of MYC reverses tumorigenesis.J Intern Med 2014;276:52–60.

36. Browd SR, Kenney AM, Gottfried ON, Yoon JW, Walterhouse D, PedoneCA, et al. N-myc can substitute for insulin-like growth factor signaling in amouse model of sonic hedgehog-induced medulloblastoma. Cancer Res2006;66:2666–72.

37. McCall TD, Pedone CA, Fults DW. Apoptosis suppression by somatic celltransfer of Bcl-2 promotes Sonic hedgehog-dependent medulloblastomaformation in mice. Cancer Res 2007;67:5179–85.






38. Swartling FJ,GrimmerMR,Hackett CS,Northcott PA, FanQW,GoldenbergDD, et al. Pleiotropic role forMYCN inmedulloblastoma.GenesDev2010;24:1059–72.

39. Swartling FJ, Savov V, Persson AI, Chen J, Hackett CS, Northcott PA, et al.Distinct neural stem cell populations give rise to disparate brain tumors inresponse to N-MYC. Cancer Cell 2012;21:601–13.

40. Chou YT, Lee CC, Hsiao SH, Lin SE, Lin SC, Chung CH, et al. The emergingrole of SOX2 in cell proliferation and survival and its crosstalk withoncogenic signaling in lung cancer. Stem Cells 2013;31:2607–19.

41. Boumahdi S, Driessens G, Lapouge G, Rorive S, Nassar D, Le Mercier M,et al. SOX2 controls tumour initiation and cancer stem-cell functions insquamous-cell carcinoma. Nature 2014;511:246–50.

42. Hutton SR, Pevny LH. SOX2 expression levels distinguish between neuralprogenitor populations of the developing dorsal telencephalon. Dev Biol2011;352:40–7.

43. Vanner RJ, Remke M, Gallo M, Selvadurai HJ, Coutinho F, Lee L, et al.Quiescent sox2(þ) cells drive hierarchical growth and relapse in sonichedgehog subgroup medulloblastoma. Cancer Cell 2014;26:33–47.

44. Liberti MV, Locasale JW. The Warburg effect: how does it benefit cancercells? Trends Biochem Sci 2016;41:211–8.

45. Gershon TR, Crowther AJ, Tikunov A, Garcia I, Annis R, Yuan H, et al.Hexokinase-2-mediated aerobic glycolysis is integral to cerebellar neuro-genesis and pathogenesis of medulloblastoma. Cancer Metab 2013;1:2.

46. Tech K, Tikunov AP, Farooq H, Morrissy AS, Meidinger J, Fish T, et al.Pyruvate kinase inhibits proliferation during postnatal cerebellar neuro-genesis and suppresses medulloblastoma formation. Cancer Res 2017;77:3217–30.

47. Lewis BC, Shim H, Li Q, Wu CS, Lee LA, Maity A, et al. Identification ofputative c-Myc-responsive genes: characterization of rcl, a novel growth-related gene. Mol Cell Biol 1997;17:4967–78.

48. Shim H, Dolde C, Lewis BC, Wu CS, Dang G, Jungmann RA, et al. c-Myctransactivation of LDH-A: implications for tumormetabolism and growth.Proc Natl Acad Sci U S A 1997;94:6658–63.

49. Semenza GL, Jiang BH, Leung SW, Passantino R, Concordet JP, Maire P,et al. Hypoxia response elements in the aldolase A, enolase 1, and lactatedehydrogenase A gene promoters contain essential binding sites for hyp-oxia-inducible factor 1. J Biol Chem 1996;271:32529–37.

50. Valvona CJ, Fillmore HL, Nunn PB, Pilkington GJ. The regulation andfunction of lactate dehydrogenase a: therapeutic potential in brain tumor.Brain Pathol 2016;26:3–17.


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2019;79:1967-1980. Published OnlineFirst March 12, 2019.Cancer Res Ran Tao, Najiba Murad, Zhenhua Xu, et al.

Astrocyte Progenitor Cells+Sox2 Drives Group 3 Medulloblastoma through Transformation ofMYC

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http://cancerres.aacrjournals.org/lookup/doi/10.1158/0008-5472.CAN-18-1787http://cancerres.aacrjournals.org/content/suppl/2019/03/12/0008-5472.CAN-18-1787.DC1http://cancerres.aacrjournals.org/content/79/8/1967.full#ref-list-1http://cancerres.aacrjournals.org/content/79/8/1967.full#related-urlshttp://cancerres.aacrjournals.org/cgi/alertsmailto:[email protected]://cancerres.aacrjournals.org/content/79/8/1967http://cancerres.aacrjournals.org/

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myc drives group 3 medulloblastoma through …...medulloblastoma have distinct cell origins. wnt...

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