Induction of ectopic Myc target gene JAG2 augments hypoxic growth and tumorigenesis in a human B-cell model Jason T. Yusteina,1,2, Yen-Chun Liub, Ping Gaoc, Chunfa Jied, Anne Lec, Milena Vuica-Rossb, Wee Joo Chnge,f, Charles G. Eberhartb, P. Leif Bergsagele, and Chi V. Dangb,c,g,2 Departments of aPediatrics, bPathology, and cMedicine, dMicroarray Facility, and gSidney Kimmel Cancer Center, Johns Hopkins University School of Medicine, Baltimore, MD 21205; eComprehensive Cancer Center, Mayo Clinic, Scottsdale, AZ 85259; and fDepartment of Medicine, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 545523 Edited* by Robert N. Eisenman, Fred Hutchinson Cancer Research Center, Seattle, WA, and approved December 22, 2009 (received for review February 5, 2009) Ectopic Myc expression plays a key role in human tumorigenesis, provides an experimentally tractable system to identify putative and Myc dose-dependent tumorigenesis has been well established endogenous and ectopic Myc target genes (6). in transgenic mice, but the Myc target genes that are dependent on The P493-6 cells were derived from human peripheral blood B Myc levels have not been well characterized. In this regard, we used cells immortalized by an Epstein–Barr viral (EBV) genome that is the human P493-6 B cells, which have a preneoplastic state depend- complemented with an EBV nuclear antigen-estrogen receptor ent on the Epstein–Barr viral EBNA2 protein and a neoplastic state (EBNA2-ER) fusion protein and a tetracycline-repressible Myc with ectopic inducible Myc, to identify putative ectopic Myc target transgene (6). We selected P493-6 cells because they exist in at genes. Among the ectopic targets, JAG2 that encodes a Notch recep- least three states. With tetracycline, the P-493-6 cells withdraw tor ligand Jagged2, was directly induced by Myc. Inhibition of Notch from the cell cycle in a state with very low Myc levels, arbitrarily signaling through RNAi targeting JAG2 or the γ-secretase Notch termed the “NO Myc” state. In the presence of tetracycline and inhibitor N-[N-(3,5-difluorophenacetyl)-L-alanyl]-(S)-phenylglycine estradiol, which activates EBNA2-ER, the cells proliferate with t-butyl ester (DAPT) preferentially inhibited the neoplastic state in induction of endogenous Myc by EBNA2, achieving a “LOW Myc” CELL BIOLOGY vitro. Furthermore, P493-6 tumorigenesis was inhibited by DAPT in nontumorigenic state that is equivalent to EBV-immortalized vivo. Ectopic expression of JAG2 did not enhance aerobic cell pro- peripheral B lymphocytes (7). In the absence of tetracycline and liferation, but increased proliferation of hypoxic cells in vitro and estradiol, ectopic Myc is induced in a “HIGH Myc” tumorigenic significantly increased in vivo tumorigenesis. Furthermore, the state that resembles human Burkitt lymphoma. This system has expression of Jagged2 in P493-6 tumors often overlapped with allowed us to delineate a subset of genes that specifically respon- regions of hypoxia. These observations suggest that Notch signal- ded to ectopic Myc expression, potentially representing target ing downstream of Myc enables cells to adapt in the tumor genes that contribute to lymphomagenesis. hypoxic microenvironment. Here, we report JAG2, which encodes the Notch ligand Jag- ged2, as one of the genes most highly up-regulated upon ectopic neoplasia | Notch | target genes | transcription | hypoxia Myc expression in the P493-6 cells. Although the role of Notch signaling has not been thoroughly dissected in B-cell lymphomas, he Myc proto-oncogene encodes a transcription factor, c-Myc Jagged2 overexpression is prevalent in the B-cell malignancy T(herein termed Myc), that is implicated in genesis of many multiple myeloma (8). Recent evidence suggests that Notch may human malignancies (1, 2). Deregulated Myc expression induces synergize with the B-cell receptor to enhance B-cell activation tumorigenesis, presumably via its downstream targets comprising (9). In this report, we document that JAG2 is a direct Myc target about 10–15% of the human genome (2). It is therefore essential and that Jagged2 and Notch signaling participate in P493-6 lym- to determine whether the Myc target gene network is both phomagenesis. quantitatively and qualitatively different between nontumorigenic Results and Discussion and tumorigenic states. The chromosomal translocations of Burkitt lymphoma dereg- Human P493-6 B-Cell Neoplasm Model. The P493-6 cells were ulate the expression of the Myc transcription factor. Ectopic Myc engineered with a tetracycline-regulated Myc and an EBNA2- dosage effects on tumorigenesis are best exemplified by many ER fusion protein to achieve NO, LOW, and HIGH Myc states A–C transgenic mouse models, in which the targeted tissues succumb to as described previously (10). As shown in Fig. 1 , P493-6 tumor formation (3). The spectrum of hematologic neoplasms cells in the NO Myc state grew minimally with more compact depends on the dosage of Myc in bone marrow cells (4). In fact, Myc-induced hematologic transgenic tumors regressed when Myc Author contributions: J.T.Y., M.V.-R., P.L.B., and C.V.D. designed research; J.T.Y., Y.-C.L., levels are diminished (5); however, the Myc dose-dependent target P.G., A.L., M.V.-R., and C.G.E. performed research; P.G. contributed new reagents/analytic genes have not been elucidated. Though these animal models tools; .J.T.Y., Y.-C.L., P.G., C.J., M.V.-R., W.J.C., C.G.E., P.L.B., and C.V.D. analyzed data; and show the importance of Myc dosage on tumorigenesis, they are not J.T.Y., C.J., A.L., W.J.C., and C.V.D. wrote the paper. easily amenable to molecular analysis to identify the putative Myc The authors declare no conflict of interest. * target genes that are induced in the tumorigenic state. This Direct Submission article had a prearranged editor. The critical Myc target genes that are necessary for cellular Data deposition: The data reported in this paper have been deposited in the Gene Ex- transformation and tumorigenesis are beginning to emerge, but pression Omnibus (GEO) database, www.ncbi.nlm.nih.gov/geo/ (accession no. GSE19703). 1 the effects of cell type and cellular context on target gene Present address: Division of Pediatric Hematology-Oncology, Texas Children’sCancer Center, Baylor College of Medicine, Houston, TX 77030. response to Myc remain poorly understood. The technical limi- 2To whom correspondence may be addressed. E-mail: [email protected] or yustein@bcm. tation has been the dearth of models of Myc-mediated tumori- edu. genesis that provide both nontumorigenic and tumorigenic states This article contains supporting information online at www.pnas.org/cgi/content/full/ in the same system. In this regard, the human B-cell line P-493-6 0901230107/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.0901230107 PNAS Early Edition | 1of6 Downloaded by guest on September 25, 2021 Fig. 1. c-Myc levels influence cell morphology, proliferation, and gene expression in P493-6 human B cells. (A) Immunoblot of Myc in NO (0.1 μg/mL tet- racycline), LOW (0.1 μg/mL tetracycline and 1 μM estradiol), or HIGH (untreated) Myc states of P493-6 cells. Tubulin serves as loading control. Relative expression levels to LOW Myc state are shown (numbers below the panel) as determined by densitometry of Western blot bands. (B) Histological appearance of P493-6 cells in NO, LOW, or HIGH Myc states. Note several mitotic cells, as well as deep basophilic cytoplasm with vacuoles in the ectopic Myc-expressing cells. (C) Proliferation of P493-6 cells with NO Myc, LOW Myc, or HIGH Myc. Experiment was repeated thrice, and a representative graph is shown (D). Unsupervised hierarchical cluster analysis of P493-6 and human B-cell malignancies using 445 gene probes with >5-fold expression changes that discriminate HIGH Myc from LOW Myc states. Analysis was performed with normal and malignant B-cell samples together with P493-6 in NO, LOW, or HIGH Myc states. Expression of the genes in the different samples is represented by the heat map where every row is a gene and every column is a sample. Red represents genes that are overexpressed, blue underexpressed, and white median expression. The diagnoses for the samples are indicated below the heat map. chromatin and pale cytoplasm, whereas those with LOW Myc conditions revealed that signals of >10,000 probe sets are dif- grew with the appearance of lymphoblastoid cells. Untreated ferent between the HIGH and NO Myc states, and >8,000 are cells expressing HIGH Myc grew even better, with the appear- different between the LOW and NO Myc states, whereas just ance of Burkitt cells characterized by perinuclear lipid vacuoles over 3,100 are different between EBNA-2 driven LOW Myc and and frequent mitotic figures. Because P-493-6 cells were derived ectopic HIGH Myc states. The intersection of all three sets yields from mature circulating B cells, they do not express Bcl-6, which 959 probe sets whose signals are altered in common. We focused is usually found in germinal center-derived Burkitt lymphoma on genes whose profiles are different between the HIGH and cells. In this regard, we sought to molecularly characterize the LOW Myc states and different between the HIGH and NO Myc P493-6 cell line by arbitrarily selecting 445 gene probes (Table states, but not between the LOW and NO Myc states. We sur- S1) whose expression is >5-fold different between the NO and mise that this set would contain genes that could be pathologic, HIGH Myc states for clustering analysis using expression profiles because they are significantly induced in the HIGH Myc but not of 169 primary human mature B-cell malignancies and 21 normal the LOW Myc state. The 874 probe sets, as shown in the dia- B-cell samples (11).