Noncanonical Regulation of the Hedgehog Mediator GLI1 by C-MYC in Burkitt Lymphoma

Published OnlineFirst March 22, 2013; DOI: 10.1158/1541-7786.MCR-12-0441

Molecular Cancer Chromatin, Gene, and RNA Regulation Research

Noncanonical Regulation of the Hedgehog Mediator GLI1 by c-MYC in Burkitt Lymphoma

Joon Won Yoon1, Marisa Gallant1, Marilyn LG Lamm1, Stephen Iannaccone1, Karl-Frederic Vieux1, Maria Proytcheva3, Elizabeth Hyjek2, Philip Iannaccone1, and David Walterhouse1

Abstract Although Hedgehog signaling plays a major role in GLI1 transcription, there is now evidence suggesting that other pathways/genes, such as c-MYC, may also regulate GLI1 expression. We initiated studies in Burkitt lymphoma cells, which constitutively express c-MYC due to a chromosomal translocation, to determine whether Hedgehog or c-MYC regulates GLI1 expression. We show that all Burkitt lymphoma cell lines tested express GLI1, PTCH1,andSMO and that ﬁve of six Burkitt lymphomas express GLI1. Exposure to Sonic or Indian Hedgehog or cyclopamine (SMO inhibitor) does not modulate GLI1 expression, cell proliferation, or apoptosis in most Burkitt lymphoma cell lines. Sequence analysis of PTCH1, SMO,andSuFu failed to show mutations that might explain the lack of Hedgehog responsiveness, and we did not detect primary cilia, which may contribute to it. We show that c-MYC interacts with the 50-regulatory region of GLI1, using chromatin immunoprecipitation (ChIP) assay, and E-box–dependent transcriptional activation of GLI1 by c-MYC in NIH3T3 and HeLa cells. The c-MYC small-molecule inhibitor 10058-F4 downregulates GLI1 mRNA and protein and reduces the viability of Burkitt lymphoma cells. Inhibition of GLI1 by GANT61 increases apoptosis and reduces viability of some Burkitt lymphoma cells. Collectively, our data provide evidence that c- MYC directly regulates GLI1 and support an antiapoptotic role for GLI1 in Burkitt lymphoma. Burkitt lymphoma cells do not seem to be Hedgehog responsive. These ﬁndings suggest a mechanism for resistance to SMO inhibitors and have implications for using SMO inhibitors to treat human cancers. Mol Cancer Res; 11(6); 604–15. 2013 AACR.

Introduction that functions upstream of GLI1 (5). SMO represents an attractive target for small-molecule agonists and antago- The Hedgehog signaling pathway is activated in a wide – – variety of human cancers (1, 2). Indeed, it is estimated that nistsbasedonitsG-proteincoupled receptor like up to one-third of all human cancers have active Hedge- structure. hog signaling. The GLI1 transcription factor mediates the A growing body of evidence suggests that activation of Hedgehog signal, and expression of GLI1 alone is sufﬁ- GLI1 in some cancers is not controlled exclusively by Hedgehog signaling but also by other pathways such as cient to transform cells and induce tumors in mice (3, 4). b – Inhibition of Hedgehog signaling represents a potentially RAS and TGF- (6 8). It is important to identify other important therapeutic approach for cancers with active upstream regulators of GLI1 that function together with Hedgehog signaling. To date, most inhibitors of the or independent of Hedgehog signaling and may contrib- Hedgehog pathway target the smoothened (SMO) protein ute to resistance to Hedgehog pathway inhibitors that target SMO. Recent reports suggest that c-MYC activates GLI1 in vitro

Authors' Afﬁliations: 1Developmental Biology Program, Ann & Robert H. and enhances Hedgehog-induced medulloblastoma forma- Lurie Children's Hospital of Chicago Research Center, Northwestern Uni- tion (9, 10). Previously, we identiﬁed putative c-MYC– 0 versity Feinberg School of Medicine; 2Department of Pathology, University binding elements (E-boxes) in the 5 -regulatory region of of Chicago, Illinois; and 3Department of Pathology, University of Arizona, Tuscan, Arizona GLI1 (11, 12). On the basis of the fact that c-MYC is constitutively expressed in Burkitt lymphoma as a result of Note: Supplementary data for this article are available at Molecular Cancer Research Online (http://mcr.aacrjournals.org/). the t(8;14)(q24;q34), we initiated studies to determine (i) whether GLI1 and other Hedgehog pathway components Corresponding Author: David Walterhouse, Ann & Robert H. Lurie Chil- dren's Hospital of Chicago Research Center/Box 204, 2430 N. Halsted, are expressed in Burkitt lymphoma, (ii) whether Hedgehog Chicago, IL 60614. Phone: 773-755-6514; Fax: 773-755-6385; E-mail: signaling regulates the level of GLI1 expression in Burkitt [email protected] lymphoma cells, (iii) whether c-MYC directly regulates the doi: 10.1158/1541-7786.MCR-12-0441 level of GLI1 expression, and (iv) the role of GLI1 in Burkitt 2013 American Association for Cancer Research. lymphoma cells.

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Materials and Methods membranes (BioRad) and probed with polyclonal antibodies Cell lines and tissue against human GLI1 protein (Cell Signaling) or against We obtained Burkitt lymphoma cell lines (BL1596, glyceraldehyde 3-phosphate dehydrogenase (GAPDH; BL1647, BL1648, BL2392, and BL2625) and MC3T3 Santa Cruz Biotech). We visualized the protein using a mouse preosteoblasts from American Type Culture Collec- chemiluminescence kit (Pierce Inc.). For caspase-3 experi- tion. Burkitt lymphoma cells were cultured in RPMI-1640 ments, membranes were probed with caspase-3 or GADPH media supplemented with 10% FBS. MC3T3 cells were antibodies (Cell Signaling) and donkey anti-rabbit IgG- cultured in ascorbic acid–free a-MEM supplemented with HRP secondary antibody (Santa Cruz Biotech). 10% FBS, 100 U/mL penicillin, and 100 mg/mL strepto- mycin (Life Technologies). Unstained Burkitt lymphoma MTT assays slides without other identifiers were obtained for 6 Burkitt We conducted MTT assays as previously described with lymphoma cases from the Pathology Department at the Ann minor modifications (13). We added 10 mL of MTT reagent & Robert H. Lurie Children's Hospital of Chicago (Chi- (5 mg/mL MTT in 1 PBS) to 0.1 mL of culture media cago, IL). The Office of Research Integrity and Compliance containing the cells. The mixture was incubated for 3 hours reviewed the studies conducted on human pathologic mate- at 37C, 0.1 mL of solubilization solution (10% SDS in 0.01 rials and determined they were exempt from Institutional mol/L HCl) was then added, and the mixture incubated Board Review and oversight. overnight at 37C. Absorbances at 570 and 650 nm were measured using a Ceres UV900H Di ELISA plate reader Reverse transcription-polymerase chain reaction (Bio-Tek Instruments, Inc.). Background readings at 650 (RT-PCR) nm were subtracted from optical density readings at 570 nm. We isolated total RNA from the cell lines using the Qiagen For analysis of growth rate, we added 40,000 cells to a 6-well RNeasy Mini Kit (Qiagen). We carried out RT-PCR using plate and completed assays on days 0, 2, 4, and 6. The the One-Step RT-PCR Kit (Qiagen) or TaqMan Gene experiments were completed in triplicate and an average and Expression Assay reagents (Applied Biosystems). We SD calculated. conducted 30 to 35 cycles of PCR, including denaturation for 30 seconds, annealing for 30 seconds, and amplification Immunohistochemistry for 1 minute. The following primers were used for PCR: We used rabbit anti-GLI1 antibody (Santa Cruz Biotech) GLI1 sense 50-AGTCATACTCACGCCTCGAA-30 and for immunohistochemistry. The staining was conducted GLI1 antisense 50-GACCATGCACTGTCTTGACA-30, using a Bond Max Autostainer (Leica-Microsystems) fol- GLI2 sense 50-AAGGATTGCCACCCAGGACG-30 and lowing high pH epitope antigen retrieval (ER2) for 20 GLI2 antisense 50-CCGACTCACTGCTCTGCTTGTT- minutes. Slides were incubated for 50 minutes with anti- 30, GLI3 sense 50-CGAACAGATGTGAGCGAGAAAGC- GLI1 antibody. Staining was detected using a refined HRP 30 and GLI3 antisense 50-AAAGATGAGGAGGGTGGTA- polymer system (Leica-Microsystems) followed by 10-min- GTGGG-30, PTCH1 sense 50-CCTGGACGACATCCT- ute diaminobenzidine. GAAATCC-30 and PTCH1 antisense 50-GCGAGAAAT- GGCAAAACCTGAG-30, SMO sense 50-TGGCTTTGT- Expression of Hedgehog pathway genes and c-MYC in GCTCATTACCTTCAG-30 and SMO antisense 50- human B-cell lymphomas ATCCGCTTTGGCTCATCGTC-30, Indian Hedgehog We interrogated publicly available gene expression data (IHH) sense 50-CAAGCAGTTCAGCCCCAATG-30 and from the website http://llmpp.nih.gov/BL/ for statistically IHH antisense 50-CTGGTTCATCACCGAGATAGCC- significant differences in the expression of c-MYC and 30, Desert Hedgehog (DHH) sense 50-GCTCTCCTGAC- Hedgehog pathway genes (GLI1, GLI2, GLI3, SMO, and CAATCTACTG-30 and DHH antisense 50-TCGTGCC- PTCH1) between Burkitt lymphomas and other diffuse CAACTACAACCC-30, Sonic Hedgehog (SHH) sense 50- large B-cell lymphomas, including activated B-cell–like CAGAGGTGTAAGGACAAGTTGAACG-30 and SHH lymphomas (ABC), germinal-center B-cell–like lymphomas antisense 50-AAAGTGAGGAAGTCGCTGTAGAGC-30, (GCB), and primary mediastinal B-cell lymphomas (PMBL; c-MYC sense 50-TTCTGTGGAAAAGAGGCAGGC-30 ref. 14). Expression data were averaged across available gene and c-MYC antisense 50-TCACGCAGGGCAAAAAAG- probe sets and across lymphoma subtypes. When significant 30, GAPDH sense 50-TGATGACATCAAGAAGGTGGT- differences in gene expression were found between different GAAG-30 and GAPDH antisense 50-TCCTTGGAGGC- lymphomas using the Kruskal–Wallis test, expression values CATGTGGGCCAT-30. for these genes were further analyzed with Mann–Whitney U post hoc tests. We used Mann–Whitney U tests to make Western blot analysis pairwise comparisons between Burkitt lymphoma and ABC, We prepared Burkitt lymphoma cell lysates using Tris- Burkitt lymphoma and GCB, and Burkitt lymphoma and HCl buffer (pH 7.4), containing 150 mmol/L NaCl, 1 PMBLs. Significance levels were set to 0.0167 as prescribed mmol/L phenylmethylsulfonyl fluoride, 0.5 mmol/L dithio- for Bonferroni correction for the 3 comparisons. The data threitol, and 1% TritonX-100. We loaded 50 to 100 mgof were rendered as a heatmap in the R statistical programming protein onto 4% to 15% SDS-PAGE gels (BioRad). After language (http://www.R-project.org) and arranged accord- electrophoresis, we blotted the proteins onto nitrocellulose ing to disease for the GLI1 probeset and 3 c-MYC probesets.

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Hedgehog treatment of cells Sequence analysis We exposed Burkitt lymphoma cells and MC3T3 mouse We sequenced 23 PTCH1 exons, SMO exons 9 and 10, preosteoblasts to SHH peptide (R&D Systems), SHH- and SuFu exons 1 and 10. The exons were PCR-amplified containing conditioned media (serum-free conditioned from BL1648 gDNA using the following primers: media from confluent cultures of human LNCaP prostate PTCH1-exon 4 sense 50-GCGGAGCTGCCGGGTT- cancer cells, which have been genetically modified to express CATT-30 and antisense 50-AGCGTGGGGAGAGGCT- high levels of SHH, designated LNShh cells; ref. 15), or to GTGT-30, PTCH1-exon 6 sense 50-CACGGCTGTTG- IHH peptide (R&D Systems). The cells were incubated for GGGGCTCAC-30 and antisense 50-GGCTCTAGGTG- 24 hours at 37C with serum-free culture media containing TGCGCTGGC-30, PTCH1-exon 7 sense 50-CTATTGT- 1 mg/mL Hedgehog peptide. For SHH-containing condi- GTATCCAATGGCAGG-30 and antisense 50-ATTAG- tioned media treatment, cells were incubated for 3 days at TAGGTGGACGCGGC-30, PTCH1-exon 8 sense 50- 37C. AGTGAGAAATTTTTGTCTCTGCT-30 and antisense 50-AGGTTAAGGCACACTACTGG-30, PTCH1-exon 9 Quantitative RT-PCR sense 50-GCAAAAATTTCTCAGGAACACC-30 and cDNA was synthesized using a high-capacity cDNA antisense 50-TGGAACAAACAATGATAAGCAA-30, reverse transcription kit (Applied Biosystems). PCR was PTCH1-exon 10 sense 50-CCTACAAGGTGGATGCA- carried out using TaqMan universal PCR master mix with GTG-30 and antisense 50-TTTGCTCTCCACCCTT- the following conditions: 50C for 2 minutes, 95C for 10 CTGA-30, PTCH1-exon 11 sense 50-GTGACCTGCC- minutes, and 40 cycles of 95C for 15 seconds and 60C for TACTAATTCCC-30 and antisense 50-GGCTAGCGAG- 1 minute (Applied Biosystems). Primers and probes for GATAACGGTTTA-30, PTCH1-exon 12 sense 50-GA- GLI1 (Hs00171790_ml), PTCH1 (Hs00970980_ml), and GGCAGTGGAAACTGCTTC-30 and antisense 50- GAPDH (Hs99999905_ml) were purchased from Applied TTGCATAACCAGCGAGTCTG-30, PTCH1-exon 13 Biosystems. The experiments were completed in triplicate sense 50-GTGCTGTCGAGGCTTGTG-30 and antisense and an average and SD calculated. 50-ACGGACAGCAGATAAATGGC-30, PTCH1-exon 14 sense 50-GTGCTGTCGAGGCTTGTG-30 and anti- Cell proliferation assays sense 50-ACGGACAGCAGATAAATGGC-30, PTCH1- We measured cell proliferation using a BrdU cell pro- exon 15 sense 50-GTGTTAGGTGCTGGTGGCA-30 liferation assay kit (Chemicon). Cells were incubated with and antisense 50-CTTAGGAACAGAGGAAGCTG-30, bromodeoxyuridine (BrdUrd) for 1 hour at 37Cfollow- PTCH1-exon 16 sense 50-TCCAGTGCAGCTCTCAG- ing a 24-hour incubation with or without Hedgehog CGC-30 and antisense 50-TGCTGGAAGTCAGTGCC- peptide. Cells were fixed and BrdUrd incorporation was CCGT-30, PTCH1-exon 17 sense 50-GGCGGAGGCAT- detected with anti-BrdU detector antibody. The signal GTTGGTGACC-30 and antisense 50-GCACCCAAT- was measured using a Ceres UV900H Di ELISA plate CAAAAGGCCACAGC-30, PTCH1-exon 18 sense 50- reader (Bio-Tek Instruments, Inc.). The experiments GCACCATTTCCCTGTTTCAGC-30 and antisense 50- were completed in triplicate and an average and SD GGAAGAGCCTTAAGTTGTGGCA-30, PTCH1-exon calculated. 19 sense 50-GACAGCTTCTCTTTGTCCAG-30 and antisense 50-ACGCAAAAGACCGAAAGGACGA-30, Caspase-3/7 assays PTCH1-exon 20 sense 50-AGGGTCCTTCTGGCTGC- We measured apoptosis using a Promega caspase GAG-30 and antisense 50-TCAGTGCCCAGCAGCTG- 3/7 assay kit (Promega). Cells were incubated with an GAGTA-30, PTCH1-exon 21 sense 50-AACCCCATTC- equal volume of 2 caspase assay solution for 1 hour at TCAAAGGCCTCTGTTC-30 and antisense 50-CACCT- room temperature in the dark. Caspase activity was mea- CTGTAAGTTCCCAGACCT-30, PTCH1-exon 22 sense sured with a luminometer (Berthold). The experiments 50-AACTGTGATGCTCTTCTACCCTGG-30 and anti- were completed in triplicate and an average and SD sense 50-AAACTTCCCGGCTGCAGAAAGA-30, calculated. PTCH1-exon 23 sense 50-TTTGATCTGAACCGAGG- ACACC-30 and antisense 50-CAAACAGAGCCAGAG- Inhibitor treatment of cells GAAATGG-30, PTCH1-exon 24 sense 50-ACCAGGT- We added 550,000 cells in 3 mL of RPMI-1640 media GAAGTCCAGCAACCTGAT-30 and antisense 50-CTG- supplemented with 10% (v/v) FBS to Falcon 6-well cell CACCCGGCCCAATCACA-30, PTCH1-exon 25 sense culture plates (Becton Dickinson) and added either cyclo- 50-TCACAGCGCGTGTTCCCGTT-30 and antisense pamine (SMO inhibitor; LC Lab), tomatidine (negative 50-ACGTGGGACATCCCCGTGTCA-30, PTCH1-exon control for cyclopamine; EMD Bioscience, Inc.), 10058- 26 sense 50-GCTTTGAGTGTGGCCAGCAGGTAA-30 F4 (c-MYC inhibitor; Sigma), GANT61 (GLI1 inhibitor; and antisense 50-TCCAGGCCCACTACCACGGT-30, Sigma), or equal volumes of solvent [ethanol or dimethyl PTCH1-exon 27 sense 50-ACCCACCCTCACCCTC- sulfoxide (DMSO)]. Cells were incubated with the inhibitor TGCA-30 and antisense 50-TTGGCTGCCCTTGT- 0 0 at 37 C and 5% CO2 for up to 48 hours. We then carried CAGTGGC-3 , PTCH1-exon 27 sense 5 -ACCCAC- out quantitative RT-PCR, MTT, BrdUrd, or caspase-3/7 CCTCACCCTCTGCA-30 and antisense 50-GCCA- assays as described. CTGACAAGGGCAGCCAA-30, SMO-exon 9 sense 50-

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0 GCCTCACCTGTCTACGTTCCCTCA-3 and antisense Table 1. Expression of Hedgehog pathway 50-GGGGCAGCGGTGAACCACTG-30, SMO-exon 10 0 0 components and c-MYC in Burkitt lymphoma sense 5 -TGGCATCGCTGGCCCTTCCCAAGA-3 and antisense 50-GGGGCCAGGCGGCTCCGGTA-30, SuFu- cell lines exon 1 sense 50-ACAGGGAGGGGAGGCGGGGC-30 and 0 0 antisense 5 -TCTCCCCGCGGCCCAGAGCA-3 , SuFu- BL 1647 BL 1648 BL2392 BL 2625 BL1596 0 0 exon 9 sense 5 -GAGCCCAGCTGGAGACTCCCAT-3 GLI1 þþþþþ 0 0 and antisense 5 -GGCTCCTTGGAGGCCCTGGA-3 GLI2 (16–20). The products were cleaned using the Wizard PCR GLI3 þ Clean-Up Kit (Promega) and sequences were determined SMO þþþþþ using the ABI3730XL DNA analyzer (Applied Biosystems). PTCH1 þþþþþ DHH þþ Immunofluorescence for cilia IHH þþ We grew Burkitt lymphoma, NIH3T3, and RMS-13 cells SHH in 8-well chamber slides (Nunc). Cells were washed with c-MYC þþþþþ PBS at room temperature and then fixed with 4% parafor- maldehyde/0.5% TritonX-100 in PBS for 30 minutes at room temperature with rocking. Cells were washed again mg of DNA was transfected in each experiment, and the with PBS at room temperature and were then blocked for 1 difference was made up with pUC 18 carrier DNA. Cell hour with 10% donkey serum. We incubated the cells with lysates were prepared 24 hours after transfection. When anti-acetylated a-tubulin (Sigma; 1:5,000 dilution) and indicated, cells were concurrently treated with cyclohexi- pericentrin2 (Santa Cruz Biotech; 1:500 dilution) antibo- mide (10 mg/mL; A.G. Scientific, Inc.) during the incuba- dies overnight at 4C. Cells were washed with PBS and tion period. About 20 mL of cell lysate was assayed by adding incubated with the secondary antibodies at 1:300 dilution 100 mL of substrate solution (Promega). Alternatively, (donkey anti-goat IgG Alexa488 and donkey anti-mouse quantitative RT-PCR was carried out. The experiments IgG Alexa568) for 1 hour at room temperature. The nuclei were carried out at least in triplicate and results expressed were stained with 40,6-diamidino-2-phenyindole (DAPI) at as an average with SD. 1:2,000 dilution (Biotium) for 20 minutes at room temperature with rocking. Immunofluorescence was observed Mutagenesis of E-boxes #3 and 5 using a Zeiss 510 META confocal laser scanning The Luc-E2 GLI1 promoter reporter construct, contain- microscope. ing mutant E-boxes #3 and #5 was prepared using a site- directed mutagenesis kit (Stratagene) and the following 0 c-MYC–GLI1 interaction by chromatin primers; E-box #3, sense 5 -AGGCGGGGGACAACC- 0 0 immunoprecipitation TTGGAGCAGTGGG-3 and antisense 5 -CCCACTG- 0 Chromatin immunoprecipitation was conducted using CTCCAAGGTTGTCCCCCGCCT-3 ; E-box #5, sense 0 BL1648 cells and the ChIP-IT Express Chromatin Immu- 5 -TAGAGAGGTAACCCAAGGTTTGTGTCTGCGC- 0 0 noprecipitation Kit (Active Motif). BL1648 proteins and GTG-3 and antisense 5 -CACGCGCAGACACAAACC- 0 DNA were cross-linked with formaldehyde for 10 minutes TTGGGTTACCTCTCTA-3 . DNA was mixed with the at room temperature. The DNA–protein complexes were primers and 18 cycles of PCR were carried out, including sheared by sonication, the cell lysate was cleared by denaturation at 95 C for 30 seconds, annealing at 55 C for centrifugation at 14,000 g for 10 minutes, and anti- 30 seconds, and amplification at 68 C for 1 min/kb DNA c-MYC antibody was added (Santa Cruz Biotech). Anti- length. DNA was then digested with DpnI, and the remain- body–protein–DNA complexes were precipitated with ing mutated DNA was isolated following bacterial transfor- protein G magnetic beads. DNA was purified and used mation and DNA purification. for PCR amplification of c-MYC–binding sites. We used the following primers: GLI1 633 to 447 sense 50- Annexin-V staining þ FACS apoptosis assays 0 0 BL1647, BL1648, and BL2625 cells were seeded onto 48- CCGCTGCTCCTTGCTTTTATG-3 and antisense 5 - TAGGGTAGTCGCCTTTGGTTGGTG-30, GLI1 364 well plates (0.5 10 6 cells/mL) and treated with either to 491 sense 50-ACCGTGGGTCTTTGTCTCCG-30 vehicle (0.1% ethanol) or increasing amounts of GANT61 and antisense 50-CACACACCTGGGGTTACCTCTC- for 48 hours. We conducted Annexin-V and 7-aminoacti- TAC-30. nomycin D (AAD) staining using Guava Nexin Reagent followed by fluorescence-activated cell-sorting (FACS) anal- Cotransfection assays ysis using Guava EasyCyte (EMD Millipore). We cotransfected NIH3T3 or HeLa cells with 0 to 1,000 ng of c-MYC effector plasmid, 500 ng of the Luc-E2 or Statistics mutant Luc-E2 GLI1 promoter reporter constructs, and 20 Differences between groups were assessed using one-way ng of Renilla control reporter DNA (Promega), using 2 to 6 ANOVA followed by a Student t test unless stated otherwise. mL of Lipofectamine 2000 reagent (Gibco-BRL). A total of 3 P 0.05 was considered significant.

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Figure 1. GLI1 expression in Burkitt lymphoma. A, GLI1 expression by Western blotting in Burkitt lymphoma cells (top). RMS-13 is a GLI1-amplified rhabdomyosarcoma cell line (positive control). GLI1 protein ¼ 150 kDa. The 100 kDa band in the RMS-13 lane is nonspecific. GAPDH was used to show comparable loading of the samples (bottom, GAPDH protein ¼ 37 kDa). More BL1596 protein was loaded than for the other cells. B, growth curves for Burkitt lymphoma cell lines obtained by MTT assay. Solid squares ¼ BL2625 cells, solid circles ¼ BL1648 cells, open circles ¼ BL1596 cells, solid triangles ¼ BL2392 cells, and open squares ¼ BL1647 cells. One-way ANOVA shows differences among the curves (P 0.05). C, GLI1 immunohistochemistry of Burkitt lymphoma samples. Histologic section of Burkitt lymphoma, showing medium-sized neoplastic cells with multiple small nucleoli and finely dispersed chromatin [hematoxylin and eosin (H&E) stain; a]. Example of an immunohistochemical stain of a Burkitt lymphoma showing strong expression of GLI1 (b). Example of a Burkitt lymphoma that does not express GLI1 by immunohistochemical stain (c). A reactive lymph node does not show expression of GLI1 by immunohistochemical stain (negative control; d). D, gene expression data for GLI1 and c-MYC in 4 types of human lymphoma; Burkitt lymphoma (BL), ABC, GCB, and PMBLs from http://llmpp.nih.gov/BL/ (14). A Kruskal–Wallis test revealed a significant effect of lymphoma subtype on GLI1 [c2(3) ¼ 38.3, P < 0.001] and c-MYC [c2 (3) ¼ 9.0, P ¼ 0.029] gene expression. Mann–Whitney U tests with a Bonferroni correction using a significance level of .0167 were used to look for significant differences between disease subtypes in a pairwise manner. For GLI1, comparisons between BL and ABC (U ¼ 53.0, P < 0.001, r ¼ 0.66), BL and GCB (U ¼ 26.0, P < 0.001, r ¼ 0.76), and BL and PMBLs (U ¼ 28.0, P < 0.001, r ¼ 0.75) were all significantly different. The analysis for c-MYC showed a significant difference between BL and GCB (U ¼ 1603.5, P ¼ 0.011, r ¼ 0.22). * indicates significant differences

in gene expression between lymphoma subtypes. Note that the ordinate scale is log2. E, gene expression data for GLI1 and c-MYC in the lymphomas listed in (D) from http://llmpp.nih.gov/BL/ shown as a heatmap for 1 GLI1 probeset and 3 c-MYC probesets (rows; ref. 14). Red indicates gene upregulation and green downregulation in the heatmap. Each column represents a different patient.

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Results Burkitt lymphoma compared with germinal center B-cell– Expression of c-MYC and Hedgehog pathway like lymphoma and of GLI1 in Burkitt lymphoma compared components in Burkitt lymphoma with all of the other diffuse large B-cell lymphomas (Fig. 1D RT-PCR analysis shows expression of c-MYC, PTCH1, and E). PTCH1, SMO, GLI2, and GLI3 were not upregu- SMO, and GLI1 mRNA in all Burkitt lymphoma cells lines lated in Burkitt lymphoma compared with other B-cell tested (BL1647, BL1648, BL2392, BL2625, and BL1596) lymphomas. and IHH or DHH in a subset of the cell lines (Table 1). The expression patterns suggest that the cells could be Hedgehog- Burkitt lymphoma cell lines are not Hedgehog- responsive in paracrine or sometimes autocrine manners. responsive Different Burkitt lymphoma cells show variable levels of To determine whether Burkitt lymphoma cells respond to GLI1 protein and growth rates (Fig. 1A and B). The amount ligand-mediated Hedgehog pathway activation, we exposed of GLI1 protein detected by Western blotting does not the cells to either SHH peptide, SHH-containing condi- correlate with growth rate. We also show GLI1 expression by tioned media, or IHH peptide. We did not test all of the cell immunohistochemistry, showing mostly nuclear staining in lines in all experiments. For the most part, we did not find 5 of 6 human Burkitt lymphoma samples with MYC changes in GLI1 or PTCH1 expression following exposure of translocations (Fig. 1C). Consistent with the variability in Burkitt lymphoma cells to SHH or IHH (Fig. 2A and B). GLI1 expression among Burkitt lymphoma cell lines, the We detected changes in GLI1 expression only in BL2625 degree of GLI1 staining varied by immunohistochemistry cells exposed to SHH, which are of questionable biologic with some cases showing strong positivity and others weak significance. We show increased GLI1 and PTCH1 expres- staining. We queried publicly available gene expression data sion in MC3T3 mouse preosteoblast, which have been for a variety of B-cell lymphomas, including Burkitt lym- previously shown to be Hedgehog-responsive cells (15). In phoma, activated B-cell–like lymphoma, germinal center B- addition, we do not detect significant changes in cell viability cell–like lymphoma, and primary mediastinal B-cell lym- measured by MTT assays, cell proliferation measured by phoma (14). We found significant upregulation of c-MYC in BrdUrd assays, or apoptosis measured by caspase-3/7 assays

Figure 2. Exposure of Burkitt lymphoma cells to SHH, IHH, or cyclopamine. A, following exposure to SHH peptide or SHH-containing conditioned media, we did not detect changes in the expression of GLI1 (black bars) or PTCH1 (gray bars) mRNA by quantitative RT-PCR in BL1648 or BL1647 cells. We detected a slight increase in GLI1 expression in BL2625 cells (, P 0.05). We detect changes in GLI1 and PTCH1 mRNA in the Hedgehog-responsive MC3T3 control cells (, P 0.05). C (control) ¼ no SHH (no peptide or SHH-containing conditioned media); P (peptide) ¼ SHH peptide at 1 mg/mL; CM (conditioned media) ¼ SHH-containing conditioned media as described under Materials and Methods. B, following exposure to IHH peptide, we do not detect changes in the expression of PTCH1 (gray bars) or GLI1 (black bars) mRNA by quantitative RT-PCR in BL1648, BL2625, BL1647, or BL1596 cells. We ﬁnd increased GLI1 and PTCH1 expression in the MC3T3 Hedgehog-responsive control cells (, P 0.05). C (control) ¼ no IHH peptide; P (peptide) ¼ IHH peptide at 1 mg/mL. C, MTT assays show statistically signiﬁcant changes in BL1648 (black bars) and BL2625 cell (white bars) viability but not in BL1596 cell viability (gray bars) following treatment with cyclopamine (left). Changes in viability are also seen in BL1648 cells (black bars), BL1596 cells (gray bars), and BL2625 cells (white bars) following treatment with tomatidine (right; negative control).

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SuFu) that might constitutively activate the pathway and prevent us from modulating pathway activity and for primary cilia that may play a role in mediating Hedgehog signaling. We sequenced 23 PTCH1 exons in BL1648 cells and did not find any mutations. We also sequenced known hotspots for activating SMO mutations (exons 9 and 10) or inactivating SuFu mutations (exons 1 and 9) and did not find any mutations (21, 22). Therefore, we do not find evidence for mutations that might constitutively activate the pathway and could account for the lack of Hedgehog responsiveness. In some cells, Hedgehog signaling may be mediated through primary cilia (23–27). A primary cilium is a microtubule- containing organelle that projects from the cell. It attaches to the cell at a microtubule-organizing center, which includes a pair of centrioles (28). It is generally accepted that hematopoietic cells do not have primary cilia. It is less clear whether lymphoma cells have primary cilia; therefore, we looked for primary cilia in Burkitt lymphoma cells. We did not detect Figure 3. Detection of primary cilia in cell lines. Immunofluorescence primary cilia projecting from BL1648 cells (Fig. 3) or detects primary cilia projecting from NIH3T3 [acetylated a-tubulin BL2625 cells (data not shown) by immunofluorescence, antibody (red) detects the primary cilium, DAPI (blue) shows the nucleus; which may contribute to the lack of Hedgehog responsive- left, top] and RMS-13 cells [acetylated a-tubulin antibody (red) detects ness in these cells. However, we detected colocalization of the primary cilium, pericentrin2 antibody (green) detects the microtubule a organizing center, and DAPI (blue) shows the nucleus; left, bottom] but acetylated -tubulin and pericentrin2 in BL1648 and not in BL1648 cells [acetylated a-tubulin antibody (red) and pericentrin 2 BL2625 cells in a nonprojectile pattern more consistent (green) colocalize consistent with an immune synapse but do not show a with an immune synapse that has been described in lym- projecting primary cilium, DAPI (blue) shows the nucleus; right]. BL1648 phoid cells (29). cells maintained in serum and serum-starved cells are shown. All scale bars ¼ 10 mm. c-MYC regulates the expression of GLI1 Previously, we recognized that the 1,492-bp 50- regulatory following exposure of Burkitt lymphoma cells to SHH or region of GLI1 contains 5 E-boxes (CANNTG) that rep- IHH peptide (Supplementary Data). resent potential c-MYC–binding sites (Fig. 4A; refs. 11, 12). To determine whether Hedgehog signaling is already c-MYC physically interacts with the 50- regulatory region of highly active and cannot be further stimulated in Burkitt GLI1 in vivo (Fig. 4B). lymphoma cells, we inhibited the pathway with the SMO As Burkitt lymphoma cells already have high c-MYC inhibitor cyclopamine. Inhibition of Hedgehog signaling by expression and as hematopoietic cells are difficult to cyclopamine does not affect GLI1 mRNA expression in transfect, we tested the effect of c-MYC upregulation on BL1648 or BL1596 cells (Supplementary Data). In addi- GLI1 expression in cells with low endogenous c-MYC tion, cyclopamine treatment does not generally affect Burkitt expression at baseline. c-MYC activates luciferase reporter lymphoma cell proliferation measured by BrdUrd assays or gene transcription through the 50-regulatory region of GLI1 apoptosis measured by caspase-3/7 assays (Supplementary in NIH3T3 cells and HeLa cells and upregulates GLI1 Data). We observe a reduction in BL1648 cell proliferation transcripts in NIH3T3 cells (Fig. 4C–E). Mutations intro- after treatment with 10 mmol/L cyclopamine, which was not duced into E-boxes #3 and #5 in the GLI1-regulatory region apparent for BL1596 or BL2625 cells and is of uncertain reduce c-MYC–induced expression. The findings suggest biologic significance. Curiously, cyclopamine reduces Bur- that c-MYC may directly activate GLI1 expression, in part, kitt lymphoma cell viability in BL1648 and BL2625 cell through E-boxes #3 and #5. lines (Fig. 2C). Similar results in the same cell lines with the The c-MYC inhibitor 10058-F4 downregulates GLI1 negative control tomatidine combined with the fact that mRNA and protein expression in Burkitt lymphoma cells GLI1 expression does not change following treatment with (Fig. 5A–C). Consistent with our quantitative RT-PCR data cyclopamine suggests that the reduction in viability is not (Supplementary Data), cyclopamine does not reduce GLI1 mediated through inhibition of Hedgehog signaling. Taken mRNA or protein level. As expected, the GLI1 inhibitor together, we did not find evidence that Hedgehog signaling GANT61, which inhibits GLI1-DNA binding, also does modulates GLI1 expression or behavior in Burkitt lympho- not affect GLI1 mRNA or protein expression. In addition, ma cells, and therefore, Burkitt lymphoma cells do not seem GANT61 and cyclopamine do not affect c-MYC mRNA to be Hedgehog-responsive. expression in BL1648 cells. Treatment of BL1648 cells with As we could not find evidence for up- or downregulation 10058-F4 also inhibits cell viability (Fig. 5D). Combined of Hedgehog signaling in Burkitt lymphoma cells despite the treatment with 10058-F4 and cyclopamine does not change expression of Hedgehog pathway components, we looked for BL1648 cell viability compared with treatment with 10058- mutations in Hedgehog pathway genes (PTCH1, SMO,or F4 alone (Fig. 5D). This is consistent with our observation

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Figure 4. Interaction of c-MYC with the 50-regulatory region of GLI1 and upregulation of GLI1 expression. A, the 50-regulatory region of GLI1 is shown schematically. þ1 indicates the transcription start site. #1–#5 indicate E-boxes. B, ChIP was conducted using BL1648 cells. The analysis shows that c-MYC, detected using anti-c-MYC antibody (a-c-MYC), specifically interacts with the 50-regulatory region of GLI1 in vivo. Top arrow indicates the interaction between c-MYC and GLI1 through amplification of bp 633 to 447, which lies between c-MYC consensus binding sites #2 and #3. Bottom arrow indicates the interaction between c-MYC and GLI1 through amplification of bp 364 to 491, which includes c-MYC consensus binding site #5. The anti-RNA Pol II (a-RNA Pol II) antibody is used as a positive control. Size in bp is indicated by the molecular weight markers (Mwt). C, cotransfection assays, using a reporter containing the 50-regulatory region of GLI1 (Luc-E2) and increasing amounts of c-MYC expression vector in NIH3T3 cells, show a concentration-dependent increase in luciferase activity (black bars). The control reporter (white bars) does not contain the 50-regulatory region of GLI1. Mutations introduced into c-MYC–binding sites #3 and #5 reduce c-MYC induced expression of GLI1 (gray bars). indicates a statistically significant difference (P 0.05) between luciferase activity obtained with no c-MYC DNA compared with the amounts of c-MYC DNA shown on the x-axis; indicates a statistically significant difference (P 0.05) between luciferase activity obtained with the wild-type GLI1 50-regulatory region and the GLI1 50–regulatory region with mutations introduced into E-boxes #3 and #5 using a given amount of c-MYC. D, cotransfection assays, using Luc-E2 and increasing amounts of c-MYC expression vector in HeLa cells, also show a concentration-dependent increase in luciferase activity. Bar colors as in (C). or indicates statistically significant differences (P 0.05) using the same comparisons as in (C). E, GLI1 transcripts detected by quantitative RT-PCR increase with increasing c-MYC in NIH3T3 cells (black bars). GLI1 expression does not change following transfection of a negative control vector (sRMSV) that does not express c-MYC (gray bars) or in c-MYC–transfected cells that were treated with cycloheximide (white bars). indicates a statistically significant difference (P 0.05) between GLI1 expression with no c-MYC DNA compared with the amounts of c-MYC DNA shown on the x-axis. that SMO inhibition does not regulate GLI1 expression in ma cells (Fig. 6B). Therefore, GLI1 functions as an anti- Burkitt lymphoma cells. Taken together, these results sug- apoptotic factor in Burkitt lymphoma cells and enhances gest that c-MYC directly regulates GLI1 expression in a viability of some Burkitt lymphoma cells. Of interest, variety of cell types including Burkitt lymphoma cells. BL1648 and BL1596 cells express higher GLI1 levels on Western blotting (Fig. 1A) compared with BL2625 cells that GLI1 inhibits apoptosis in Burkitt lymphoma cells do not show reduced viability when treated with GANT61. To understand the role of GLI1 in Burkitt lymphoma Changes in Bcl2 expression with GANT61 treatment of cells, we treated the cells with the GLI1-inhibitor GANT61 Burkitt lymphoma cells were not detected, and at this point, and assessed cell viability, cell proliferation, and apoptosis. the antiapoptotic mechanism is uncertain. Treatment with GANT61 consistently increases apoptosis (Fig. 6C–E) and reduces cell viability in BL1648 cells and Summary BL1596 cells (Fig. 6A). However, treatment with GANT61 Burkitt lymphoma cells express both c-MYC and Hedge- does not generally alter the proliferation of Burkitt lympho- hog pathway components, including PTCH1, SMO, GLI1,

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Figure 5. Inhibition of c-MYC and GLI1 in Burkitt lymphoma cells. (A) RT-PCR, (B) quantitative RT-PCR, and (C) Western blot analysis were conducted following treatment of BL1648 (left) or BL1596 (right) cells with a c-MYC inhibitor (60 mmol/L 10058-F4), a GLI1 inhibitor (10 m mol/L GANT61), or an SMO inhibitor (10 m mol/L cyclopamine). The inhibitor concentrations represent approximate LD50s determined by treating BL1648 cells with each inhibitor for 2 days and assessing cell viability by MTT assay (data not shown). GLI1 mRNA and protein expression decrease only with 10058-F4 treatment. c-MYC expression does not change with any of the inhibitors. Statistical significance was assessed using the Student t test in (B). indicates a statistically significant difference (P 0.05). D, BL1648 cells were treated with 10058-F4 without (white bars) or with (black bars) cyclopamine (3 m mol/L). The amount of 10058- F4 used is shown on the x-axis. The vehicle for 10058-F4 is DMSO (white bars) and the vehicle for 10058-F4 with cyclopamine is DMSO plus ethanol (black bars). The gray bar is a control that includes DMSO and ethanol without cyclopamine. indicates a statistically significant difference (P 0.05) when comparing viability of cells without 10058-F4 and cyclopamine treatment with cells treated with 10058-F4 without (white bars) or with (black bars) cyclopamine.

and sometimes IHH or DHH. However, Burkitt lymphoma directly binds the GLI1 promoter region and regulates GLI1 cells are not Hedgehog responsive. SHH or IHH do not expression in a variety of cell types including Burkitt lym- increase PTCH1 and GLI1 mRNA expression in most phoma. Finally, GLI1 functions as an antiapoptotic factor in Burkitt lymphoma cell lines and do not affect cell viability, Burkitt lymphoma cells. Taken together, c-MYC regulates cell proliferation, or apoptosis. The Hedgehog pathway GLI1 expression in Burkitt lymphoma more signiﬁcantly inhibitor cyclopamine does not generally affect GLI1 expres- than the canonical Hedgehog signaling pathway. This ﬁnd- sion, cell proliferation, or apoptosis. In contrast, c-MYC ing has therapeutic relevance and suggests that GLI1 or

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Figure 6. The role of GLI1 in Burkitt lymphoma cells. A–C, BL1648 (black bars), BL1596 (gray bars), BL2625 (white bars), and HepG2 (negative control cells that do not express GLI1; stippled bars) cells were treated with GANT61 for 6 hours. A, cell viability was determined by MTT assay. BL1648 and BL1596 cells show reduced cell viability following treatment with GANT61. , a statistically significant difference (P 0.05). B, Burkitt lymphoma cell proliferation was measured by BrdUrd incorporation. Increased proliferation was only observed in BL2625 when treated with 40 mmol/L GANT61. Otherwise, we do not see changes in cell proliferation. C, caspase-3/7 activity was measured using a Promega caspase 3/7 assay kit. All Burkitt lymphoma cell lines show increased apoptosis following treatment with GANT61. indicates a statistically significant difference (P 0.05). D, caspase-3 Western blotting show increasing amounts of cleaved caspase-3 (black arrowheads) following treatment with increasing concentrations (mmol/L) of GANT61 for 24 hours in BL1647 cells (top left), BL1648 cells (bottom left), and BL2625 cells (top right). HepG2 (negative control cells that do not express GLI1, bottom right) do not show any cleaved caspase-3. White arrowheads indicate uncleaved caspase-3. GADPH shows consistency of loading. All of the Burkitt lymphoma cell lines show increased apoptosis following treatment with GANT61. E, Annexin-V and 7-AAD staining followed by FACS analysis show increased apoptosis following treatment with increasing concentrations (mmol/L) of GANT61 in BL1647 cells (black bars), BL1648 cells (gray bars), and BL2625 cells (white bars). GANT61 concentrations (mmol/L) are indicated on the x-axis. indicates a statistically significant increase in apoptosis compared with no GANT61 treatment (P 0.05). Once again, all of the cell lines show increased apoptosis following treatment with GANT61.

GLI1 target genes may be better therapeutic targets than part, activation or inhibition of the Hedgehog pathway does SMO in Burkitt lymphoma and potentially other cancers not seem to alter expression of GLI1, the mediator of that express GLI1 and c-MYC. Hedgehog signaling, or proliferation or apoptosis of Burkitt lymphoma cells. Conversely, we show that c-MYC binds the Discussion GLI1 regulatory region and regulates GLI1 expression. Our We show that Burkitt lymphoma cells express both c- results suggest that GLI1 expression in Burkitt lymphoma MYC and Hedgehog pathway components. For the most cells may be largely driven by c-MYC instead of canonical

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Hedgehog signaling. This is supported by publicly available Burkitt lymphoma cells. In BL1648 cells, this is not affected gene expression data for a variety of B-cell lymphomas that by simultaneous treatment with cyclopamine. As inhibition show significant upregulation of c-MYC and GLI1 but not of GLI1 function with GANT61 reduces the viability of other Hedgehog pathways components in Burkitt lympho- BL1648 cells, we might expect that inhibition of active ma compared with other B-cell lymphomas (14). Ultimately, Hedgehog signaling with cyclopamine together with inhi- GLI1 seems to function as an antiapoptotic factor in Burkitt bition of c-MYC could have additive or even synergistic lymphoma cells. Identifying mechanisms for GLI1 activa- effects on cell viability. Taken together, our findings suggest tion other than the canonical Hedgehog signaling pathway is that c-MYC impacts the expression of GLI1 in Burkitt relevant to developing therapeutic approaches that target lymphoma to a larger extent than canonical Hedgehog signal Hedgehog signaling in a variety of cancers. Most agents transduction. currently under investigation target SMO, upstream of We show that GLI1 functions as an antiapoptotic factor in GLI1, and therefore might not be effective in settings where Burkitt lymphoma. It is therefore not surprising that reduc- GLI1 expression is driven by other mechanisms. ing GLI1 activity is sometimes also associated with reduced Active paracrine Hedgehog signaling has been shown in cell viability in cells that have high GLI1 expression. Our other B-cell malignancies (30, 31). Hedgehog pathway results suggest that Hedgehog pathway inhibitors that are components are present in Burkitt lymphoma cells. The directed at SMO and currently in clinical trials for other reason we could not show modulation of Hedgehog signal- cancers may not be effective in settings where there is ing by either exposing cells to Hedgehog ligands or cyclo- noncanonical activation of GLI1. GLI1 or GLI1 target genes pamine is not clear. We did not identify mutations in represent potential therapeutic targets in Burkitt lymphoma. Hedgehog pathway components that could prevent us from altering activity by constitutively regulating pathway output. Disclosure of Potential Conflicts of Interest D. Walterhouse has a commercial research grant from Hyundai. No potential In addition, although we could identify primary cilia in fl NIH3T3 cells and RMS-13 cells following serum starvation, con icts of interest were disclosed by the other authors. we did not identify primary cilia in the Burkitt lymphoma Authors' Contributions cells even following serum starvation. Absence of primary Conception and design: J.W. Yoon, M. Proytcheva, P.M. Iannaccone, D. cilia could contribute to the lack of Hedgehog-responsive Walterhouse signaling in these cells. Controversy continues regarding the Development of methodology: J.W. Yoon, M. Proytcheva, E. Hyjek, D. Walterhouse role of primary cilia in Hedgehog signaling in cancer cells Acquisition of data (provided animals, acquired and managed patients, provided (23–27). It is possible that primary cilia may be present in facilities, etc.): J.W. Yoon, M. Gallant, M. Lamm, S. Iannaccone, K.-F. Vieux, M. Burkitt lymphoma in vivo. Proytcheva, D. Walterhouse Analysis and interpretation of data (e.g., statistical analysis, biostatistics, compu- It is intriguing that some Burkitt lymphoma cells show tational analysis): J.W. Yoon, M. Gallant, S. Iannaccone, K.-F. Vieux, M. Proytch- reduced viability when exposed to cyclopamine. The fact that eva, P.M. Iannaccone, D. Walterhouse Writing, review, and/or revision of the manuscript: J.W. Yoon, M. Gallant, M. GLI1 expression is not altered by exposure to cyclopamine; Lamm, M. Proytcheva, P.M. Iannaccone, D. Walterhouse the same cells show reduced viability when exposed to the Administrative, technical, or material support (i.e., reporting or organizing data, negative control compound tomatidine; and that apoptosis, constructing databases): M. Proytcheva, P.M. Iannaccone, D. Walterhouse which seems to be regulated in part by GLI1 in Burkitt Study supervision: D. Walterhouse lymphoma, was not affected by exposure to cyclopamine Grant Support suggest that other mechanisms may account for the reduced The study was supported by George M. Eisenberg Foundation for Charities viability. It remains unclear why some Burkitt lymphoma (P.I., D. W.). cell lines show reduced viability while others do not. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with We show that altering c-MYC expression or activity 18 U.S.C. Section 1734 solely to indicate this fact. consistently affects GLI1 expression in several types of cells, including Burkitt lymphoma. In addition, inhibition of Received July 12, 2012; revised February 11, 2013; accepted February 20, 2013; c-MYC activity with 10058-F4 reduces the viability of published OnlineFirst March 22, 2013.

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Noncanonical Regulation of the Hedgehog Mediator GLI1 by c-MYC in Burkitt Lymphoma

Joon Won Yoon, Marisa Gallant, Marilyn LG Lamm, et al.

Mol Cancer Res 2013;11:604-615. Published OnlineFirst March 22, 2013.

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