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c-Myc is an important direct target of Notch1 in T-cell acute lymphoblastic leukemia/lymphoma

c-Myc is an important direct target of Notch1 in T-cell acute lymphoblastic leukemia/lymphoma Downloaded from Downloaded from Downloaded from genesdev.cshlp.org genesdev.cshlp.org genesdev.cshlp.org on November 4, 2021 - Published by on November 4, 2021 - Published by on November 4, 2021 - Published by Cold Spring Harbor Laboratory Press Cold Spring Harbor Laboratory Press Cold Spring Harbor Laboratory Press c-Myc is an important direct target of Notch1 in T-cell acute lymphoblastic leukemia/lymphoma 1,2,8 3,8 3 3 Andrew P. Weng, John M. Millholland, Yumi Yashiro-Ohtani, Marie Laure Arcangeli, 2 2 1 3 3 4 Arthur Lau, Carol Wai, Cristina del Bianco, Carlos G. Rodriguez, Hong Sai, John Tobias, 5 6 7 7 1 Yueming Li, Michael S. Wolfe, Cathy Shachaf, Dean Felsher, Stephen C. Blacklow, 3,10 1,9 Warren S. Pear, and Jon C. Aster Department of Pathology, Brigham and Women’s Hospital and Harvard Medical School, Boston, Massachusetts 02115, USA; Department of Pathology and Lab Medicine, Terry Fox Laboratory, British Columbia Cancer Agency, Vancouver, British Columbia V5Z 1L3, Canada; Department of Pathology and Lab Medicine, Abramson Family Cancer Research Institute, Institute for Medicine and Engineering, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA; University of Pennsylvania Bioinformatics Core, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA; Molecular Pharmacololgy and Chemistry Program, Memorial Sloan-Kettering Cancer Center, New York, New York 10021, USA; Center for Neurologic Diseases, Harvard Medical School and Brigham and Women’s Hospital, Boston, Massachusetts 02115, USA; Division of Oncology, Departments of Medicine and Pathology, Stanford University, Stanford, California 94305, USA Human acute T-cell lymphoblastic leukemias and lymphomas (T-ALL) are commonly associated with gain-of-function mutations in Notch1 that contribute to T-ALL induction and maintenance. Starting from an expression-profiling screen, we identified c-myc as a direct target of Notch1 in Notch-dependent T-ALL cell lines, in which Notch accounts for the majority of c-myc expression. In functional assays, inhibitors of c-myc interfere with the progrowth effects of activated Notch1, and enforced expression of c-myc rescues multiple Notch1-dependent T-ALL cell lines from Notch withdrawal. The existence of a Notch1–c-myc signaling axis was bolstered further by experiments using c-myc-dependent murine T-ALL cells, which are rescued from withdrawal of c-myc by retroviral transduction of activated Notch1. This Notch1-mediated rescue is associated with the up-regulation of endogenous murine c-myc and its downstream transcriptional targets, and the acquisition of sensitivity to Notch pathway inhibitors. Additionally, we show that primary murine thymocytes at the DN3 stage of development depend on ligand-induced Notch signaling to maintain c-myc expression. Together, these data implicate c-myc as a developmentally regulated direct downstream target of Notch1 that contributes to the growth of T-ALL cells. [Keywords: Notch; Myc; leukemia; T cell; transformation] Supplemental material is available at http://www.genesdev.org. Received February 17, 2006; revised version accepted June 6, 2006. Notch receptors participate in a conserved signaling cells (HSCs) (Varnum-Finney et al. 2000; Calvi et al. pathway that regulates the development of diverse cell 2003; Duncan et al. 2005), T-cell specification from a and tissue types in metazoans. Outcomes resulting from multipotent precursor (Pui et al. 1999; Radtke et al. Notch signals are highly pleiotropic, depending on dose 1999; Sambandam et al. 2005; Tan et al. 2005), matura- and context (Artavanis-Tsakonas et al. 1999). Within the tion of double-negative (DN) thymocytes, especially at hematolymphoid compartment, Notch signaling affects the -selection checkpoint (Wolfer et al. 2002; Tanigaki lineage commitment at multiple developmental stages et al. 2004; Ciofani and Zuniga-Pflucker 2005; Taghon et (for review, see Radtke et al. 2004; Maillard et al. 2005). al. 2006), and the differentiation of CD4 T cells along Notch influences the self-renewal of hematopoietic stem either T 1orT 2 pathways (Amsen et al. 2004; Tani- H H gaki et al. 2004; Minter et al. 2005; Tu et al. 2005). Since the discovery of Notch1 through the analysis of a rare (7;9) chromosomal translocation in human T-cell acute These authors contributed equally to this work. lymphoblastic leukemia/lymphoma (T-ALL) (Ellisen et Corresponding authors. E-MAIL jaster@rics.bwh.harvard.edu; FAX (617) 264-5169. al. 1991), abundant evidence has also accumulated im- E-MAIL wpear@mail.med.upenn.edu; FAX (215) 746-6725. plicating Notch1 in the pathogenesis of this aggressive Article published online ahead of print. Article and publication date are online at http://www.genesdev.org/cgi/doi/10.1101/gad.1450406. cancer (Pear et al. 1996; Aster et al. 2000; Bellavia et al. 2096 GENES & DEVELOPMENT 20:2096–2109 © 2006 by Cold Spring Harbor Laboratory Press ISSN 0890-9369/06; www.genesdev.org Downloaded from genesdev.cshlp.org on November 4, 2021 - Published by Cold Spring Harbor Laboratory Press Notch1 induces c-Myc in T-ALL 2000). Recently, activating mutations in Notch1 were Results discovered in 55%–60% of human T-ALLs (Weng et al. Identification of c-myc as a putative Notch1 target 2004), and emerging data indicate that similar types of gene Notch1 mutations occur frequently in many different murine T-ALL models as secondary events (Dumortier et To identify potential Notch1 target genes, we performed al. 2006; Lin et al. 2006; O’Neil et al. 2006). expression profiling on a set of RNAs obtained from T6E Normal Notch signaling is initiated by the binding of T-ALL cells in which Notch was turned “off” (-secre- ligands of the Delta/Serrate/Lag-2 (DSL) family to the tase inhibitor [GSI]-treated or DN-MAML1 transduced) Notch ectodomain, which result in cleavage at a site just or left “on” (untreated and mock GSI-treated cells, external to the transmembrane domain by ADAM me- sorted GFP cells transduced with empty MigRI virus, talloproteases (Brou et al. 2000; Mumm et al. 2000). This and sorted GFP cells from MigRI-DN-MAML1 cul- TM event creates a short-lived Notch intermediate (N *) tures). RNAs were hybridized to Affymetrix U74Av2 that is recognized by nicastrin (Shah et al. 2005), a com- GeneChip arrays (∼12,000 genes). Raw data were ana- TM ponent of -secretase, which in turn, cleaves N * lyzed with dChip software (Li and Hung Wong 2001) by within its transmembrane domain (Schroeter et al. 1998; applying standard normalization and modeling routines, De Strooper et al. 1999; Kimberly et al. 2003). This final and by filtering gene lists based on “presence” call, ex- cleavage releases the intracellular domain of Notch pression level, and variation criteria. Hierarchical clus- (ICN) from the membrane, allowing it to translocate to tering performed using filtered gene sets resulted in un- the nucleus and form a transcriptional activation com- supervised discovery of the Notch “on” and “off” sample plex with a DNA-binding protein termed CSL (for CBF1, groups. Supervised analysis was then applied to a filtered Suppressor of Hairless, Lag-1) (Jarriault et al. 1995; Kopan list of ∼600 genes to identify genes for which the expres- et al. 1996; Struhl and Greenwald 1999; Ye et al. 1999) sion levels were most highly correlated with Notch ac- and transcriptional coactivators of the Mastermind-like tivation status. (MAML) family (Petcherski and Kimble 2000a,b; Wu et A total of 83 genes demonstrated multiple-compari- al. 2000). son-adjusted p-values of <0.05 (Fig. 1A; see Supplemen- Although signals mediated through Notch receptors tary Table S1 for the full list of genes, fold changes, and have diverse outcomes, only a fairly limited set of Notch p-values). Genes down-regulated in the Notch “off” target genes have been identified in various cellular/de- group included the majority of known/previously de- velopmental contexts. The hairy/enhancer of split (Hes) scribed Notch targets in immature T cells (hes1, hey1, genes are highly conserved target genes that are regu- pre-T, deltex1, and several interferon-induced genes), lated by Notch in multiple cell types (Preiss et al. 1988; and c-myc. We also performed an independent set of ex- Jarriault et al. 1995). On the other hand, investigators pression profiling experiments in which we sought to studying Notch1 function in immature T cells identified identify pathways that were perturbed by GSI treatment several likely T-cell-specific target genes, including of T6E. When analyzed with Ingenuity software, the CD25 and pre-T (Deftos et al. 2000; Reizis and Leder pathway most highly down-regulated by Notch inhibi- 2002). Other putative context-specific target genes that tion was that involving c-myc (data not shown). may promote cell growth include cyclinD1, which was The identification of c-myc as a potential Notch1 tar- identified as a transcriptional target in RKE cells get in studies using different inhibitors and several inde- (Ronchini and Capobianco 2001), and c-myc (Satoh et al. pendent analytical tools provided the impetus for more 2004), which was identified as a possible Notch target in focused studies. As an initial test, we assessed the effects hematopoietic stem cells. In this latter study, Notch re- of -secretase blockade on c-myc mRNA levels in five sponsiveness was linked to an ∼200-base-pair (bp) ele- human T-ALL cell lines that require Notch signals for ment lying immediately 5 of the c-myc transcriptional growth (Weng et al. 2004). Notch pathway blockade with start site, but neither direct association of Notch with a GSI led to the down-regulation of c-myc in all five of this site nor its functional importance was demon- the human T-ALL cell lines that require Notch signals, strated. Thus, the identities of the genes downstream of as well as murine T6E cells (Fig. 1B). Notch1 that maintain the growth of T-ALL cells have yet to be determined. c-Myc is a direct target of Notch1 To address this uncertainty, we used expression pro- filing to identify genes that are down-regulated by Notch Forms of Notch1 bearing activating mutations within pathway inhibitors in T6E, a murine T-ALL cell line the extracellular domain are susceptible to ligand-inde- whose growth depends on a membrane-tethered form of pendent cleavage by metalloproteases at site S2 TM Notch1 resembling N (Weng et al. 2003). Among the (Sanchez-Irizarry et al. 2004). The product of this cleav- TM potential target genes identified was c-myc, which has age, N *, is normally rapidly recruited to the -secre- been shown to induce T-ALL in animal models when tase complex and further processed into ICN1 (Shah et TM overexpressed (Girard et al. 1996; Felsher and Bishop al. 2005). However, we noted previously that N *in 1999; Langenau et al. 2003). This insight led to a series of T-ALL cells is stabilized and accumulates in the pres- TM functional studies, which showed that c-myc is an im- ence of GSI (Weng et al. 2003), creating a pool of N * portant target of Notch not only in T-ALL cells, but also that can be rapidly converted to ICN1 upon GSI wash- at a critical stage of normal pre-T-cell development. out. GENES & DEVELOPMENT 2097 Downloaded from genesdev.cshlp.org on November 4, 2021 - Published by Cold Spring Harbor Laboratory Press Weng et al. Figure 1. Withdrawal of Notch1 signals down-regulates c-myc expression in T-ALL cells. (A) Identification of Notch1-sensitive genes in T6E cells. Columns represent experimental samples, while rows represent genes. Each colored box indicates relative expression level (normalized for each gene), where red indicates high and blue indicates low. The seven columns on the left are samples from T6E cells with active Notch signaling, and the six columns on the right are samples from T6E cells in which Notch signaling was inhibited. Expression data from 82 genes significantly correlated with the Notch “on” (left) versus “off” (right) distinction (p < 0.05) are depicted; expression of the 25 genes in the upper cluster increased on inhibition of Notch signaling, expression of the 57 in the lower cluster decreased. Genes implicated in the literature as related to Notch signaling as well as the novel gene c-myc are highlighted. Sample label key: (GSI [1 µM compound E] + ICN1) sorted ICN1 transduced cells treated with GSI; (GSI mock) DMSO vehicle-treated cells; (GSI 0 h) untreated cells; (GFPposA/B) sorted GFP-only cells; (GFPnegA/B) sorted untransduced cells from the same cultures as transduced cells; (GSI) cells treated with GSI for the indicated number of hours; (DN-MamA/B) sorted dominant-negative MAML1 transduced cells. (B) Northern blot analysis demonstrating the down-regulation of c-myc following treatment with GSI (1 µM com- pound E). Notch-dependent T6E cells and human T-ALL cell lines were treated with carrier (DMSO) or GSI for the indicated time periods. Blots were hybridized with probes indicated in the right margin. 2098 GENES & DEVELOPMENT Downloaded from genesdev.cshlp.org on November 4, 2021 - Published by Cold Spring Harbor Laboratory Press Notch1 induces c-Myc in T-ALL We used this maneuver to determine if the transcrip- To determine if Notch stimulates the synthesis of c- tional up-regulation of c-myc by Notch1 requires protein myc transcripts, nuclear runoff experiments were per- synthesis. These experiments used the human T-ALL formed with KOPT-K1 nuclei obtained from cells treated cell line KOPT-K1, which bears a mutation in the extra- with DMSO or GSI (Fig. 2B). GSI treatment diminished cellular heterodimerization domain of Notch1 that re- c-myc transcription, whereas washout of GSI for as little sults in ligand-independent S2 cleavage (Weng et al. as 2 h produced a rebound in c-myc transcription com- 2004; Malecki et al. 2006). Washout of GSI in KOPT-K1 parable to that observed in the experiments conducted cells led to the rapid up-regulation of c-myc transcripts with cycloheximide. Importantly, the increase in c-myc even in the presence of cycloheximide, an inhibitor of pro- transcription was largely abrogated when cells were tein synthesis (Fig. 2A). The up-regulation of c-myc upon refed fresh medium containing GSI (mock washout). GSI washout was observed over a wide range of cyclohexi- Thus, Notch1 increases c-myc RNA in T-ALL cells by mide doses (5–40 µg/mL), all of which were sufficient to stimulating transcription. suppress the growth of KOPT-K1 cells (data not shown). Scanning of c-myc genomic sequences revealed two Figure 2. c-Myc is a direct target of Notch1. (A) c-Myc up-regulation by Notch does not require de novo protein synthesis. KOPT-K1 TM cells were treated with GSI (1 µM compound E) for 48 h to permit accumulation of the -secretase substrate N *. Cells were then washed and refed medium containing GSI (mock washout), or medium lacking GSI (washout) with or without 20 µM cycloheximide (CHX). c-Myc RNA levels were determined after4hof additional culture by qPCR. Each sample was assayed in triplicate; error bars correspond to standard deviations. Similar results were obtained in four independent experiments. (B) Notch1 stimulates c-myc transcription. Nuclei were prepared from KOPT-K1 cells treated with vehicle (0.01% DMSO) for 48 h; treated with GSI (1 µM compound E) for 48 h; treated with GSI for 48 h, then washed thrice and cultured2hin fresh medium without GSI (washout); or treated with GSI for 48 h, then washed three times and cultured2hin fresh medium with GSI (mock washout). RNAs isolated from runoff reactions were hybridized to slot-blots containing probes specific for c-myc and GAPDH. Bound radioactivity was quantified using a PhosphorImager. (Top panel) Phosphorimages. (Bottom panel) Calculated c-myc/GAPDH transcript ratios. Results of a representative experiment are shown. (C) Notch1 binds to the c-myc promoter through a region containing a conserved CSL consensus sequence. Chromatin immunoprecipitates were performed on cross-linked fragmented DNAs prepared from T6E cells treated with DMSO or 1 µM compound E (GSI) for 24 h. Eluted DNAs were then analyzed by qPCR performed with primers flanking putative CSL-binding sites A and B. The amount of DNA amplified from immunoprecipitated DNAs was normalized to that amplified from input DNA. (D) CSL binds to the putative target sequence in the c-myc promoter. Oligonucleotides labeled with carboxyfluorescein (FAM) were mixed with buffer alone or buffer containing purified CSL and Notch1 polypeptides. Following electrophoresis in 10% native gels, fluorescently labeled probes were detected with an 860 Storm FluorImager (Amersham Pharmacia Biotech). GENES & DEVELOPMENT 2099 Downloaded from genesdev.cshlp.org on November 4, 2021 - Published by Cold Spring Harbor Laboratory Press Weng et al. potential CSL-binding sites that are conserved between c-myc mimicked the effects of ICN1. T6E cells were humans and mice: (1) site A (TTCCCAA), which lies transduced with either empty MigRI, or with c-myc, within the putative Notch-responsive element identified ICN1, other Notch1 targets (hes1 and hes5), the anti- by Satoh et al. (2004); and (2) site B (TTGGGAAA), apoptotic gene bcl-x ,or cyclinD3, a gene implicated in which is within intron 2 of c-myc (Fig. 2C). Chromatin the growth and proliferation of normal T-cell progenitors immunoprecipitates prepared from T6E cells revealed (Sicinska et al. 2003). Only ICN1 and c-myc fully rescued that ICN1 associated with DNA fragments containing T6E cells from GSI-induced growth arrest (Fig. 3A); a site A, and that GSI treatment depleted ICN1 from this small but significant survival advantage was imparted by site (Fig. 2C). In contrast, ICN1 did not associate with bcl-x (data not shown), whereas hes1, hes5, and cyclinD3 site B. As anticipated, GSI treatment also depleted ICN1 did not have effects significantly different than empty from CSL-binding sites in the hes1 promoter, a well- virus. Like ICN1, c-myc also maintained the cell cycle characterized Notch1 target (Jarriault et al. 1995). Elec- progression of T6E cells in the presence of GSI (Fig. 3B). trophoretic mobility shift assays (EMSA) showed that To further explore the significance of c-myc as a target, CSL and CSL/ICN1 complexes bind to an oligonucleo- T6E cells were simultaneously transduced with ICN1 tide that contains site A (Fig. 2D), albeit with an affinity and either mad1 or dominant-negative (DN) max (A- that is about fivefold lower than that of CSL for a con- max) (Krylov et al. 1997) to antagonize the action of c- sensus binding site (Supplementary Fig. S1). Taken to- myc (Fig. 3C). Upon GSI treatment, the substantial gether, these data suggest that activated Notch1 up-regu- growth advantage of ICN1 transduced cells (compared lates c-myc directly. with nontransduced cells) was abrogated by mad1 and DN max, indicating that c-myc function is necessary for ICN1 to rescue T6E cells (Fig. 3C). c-Myc is necessary and sufficient to rescue T6E cells from Notch1 withdrawal c-Myc rescues a subset of human T-ALL cell lines Treatment of T6E cells with GSI leads to a G /G cell 0 1 from Notch1 withdrawal cycle arrest that is prevented by transduction of ICN1 (Weng et al. 2003). Given the potent progrowth effects of To investigate whether c-myc is generally sufficient to c-myc (for recent review, see Oskarsson and Trumpp rescue T-ALL cell lines from Notch withdrawal, we 2005), we investigated whether enforced expression of transduced five human Notch-dependent T-ALL cell Figure 3. c-Myc is necessary and sufficient to rescue T6E cells from withdrawal of Notch1 signals. (A) c-Myc rescues growth. T6E cells, which express a membrane-tethered form of Notch1 that requires cleavage by -secretase for activation, were transduced with MigRI retroviruses expressing GFP alone (MigRI) or GFP and the indicated polypeptides, and then treated with GSI (1 µM compound E) for up to 6 d. The numbers of GFP cells at each time point are shown. (B) c-Myc restores cell cycle kinetics. The DNA content of T6E cells transduced with the indicated MigRI retroviruses was measured by flow cytometry at baseline and after6dof treatment with GSI. (C) Inhibitors of c-myc function prevent ICN1 from rescuing T6E cells treated with GSI. T6E cells were cotransduced with retroviruses expressing ICN1 and either A-max or mad1. Cells were then cultured in the presence of 1 µM compound E for 6 d. Cells transduced with ICN1 alone (top left quadrants) are enriched over the course of the experiment, while cells expressing either A-Max or Mad1 alone (bottom right quadrants) or one of these two c-myc inhibitors and ICN1 (top right quadrant) are selected against. 2100 GENES & DEVELOPMENT Downloaded from genesdev.cshlp.org on November 4, 2021 - Published by Cold Spring Harbor Laboratory Press Notch1 induces c-Myc in T-ALL lines with c-myc. Like ICN1 (Weng et al. 2004), c-myc Activated Notch1 can rescue T-ALL cells that depend provided a partial or complete rescue of three of these on transgenic c-myc cell lines (KOPT-K1, DND-41, TALL-1) from GSI treat- ment, as judged by proliferation (Fig. 4A) and cell cycle If Notch1 is a major upstream regulator of c-myc in T- parameters (Fig. 4B), whereas two cell lines (HPB-ALL ALL cells, activated Notch1 should replace functions and ALL-SIL) were not growth-rescued by c-myc. All of provided by trangenic c-myc. This idea was tested using the GSI-treated Notch1-dependent cell lines also showed the cell line 8946, which is derived from a murine T-ALL a significant decrease in cell size that was partially or induced with a doxycycline-repressible human c-myc completely abrogated by c-myc (Fig. 4C). Thus, although transgene (Felsher and Bishop 1999). 8946 cells undergo c-myc appears to be a consistent target of activated growth arrest and apoptosis when treated with doxycy- Notch1, the degree to which Notch-dependent T-ALL cline, but are unaffected by treatment with GSI (Fig. 5A). cell lines are rescued by c-myc alone is variable. Transduction of 8946 cells with ICN1 or E (a mem- Figure 4. Transduction of c-myc leads to variable rescue of human Notch-sensitive T-ALL cell lines from withdrawal of NOTCH signals. Rescue of growth (A), cell cycle progression (B), and cell size (C) were assessed in control T-ALL cell lines or the same lines transduced with c-myc and a GFP marker (c-myc) or GFP only (GFP). Cells were treated with DMSO vehicle (mock) or 1 µM compound + + E (GSI) for the indicated time periods. (A) The fraction of live GFP cells was monitored by flow cytometry. An increasing GFP fraction indicates a growth/survival advantage conferred by the retrovirus over untransduced GFP cells. (B) The DNA content of control cell lines was compared with that of sorted populations of cells transduced with c-myc by staining with Hoechst 33342 followed by flow cytometry. The DNA histograms shown were derived from >20,000-gated events. (C) Cell size in c-myc transduced cells and untrans- duced control cells treated with GSI (compound E, 1 µM) or DMSO vehicle (V) for the indicated time was determined by forward scatter of live cells. The relative cell sizes for each population (normalized to a value of 100 for vehicle-treated, untransduced control cells ± 1 SD) are depicted. The decreases in size upon addition of GSI in untransduced cells (*), and the increases in size upon transduction with c-myc in GSI-treated cultures (**, as compared with untransduced GSI-treated cells), were both significant (P < 0.01) in all cell lines tested, as measured by the Bonferroni post-test after two-way ANOVA (unweighted means) analysis. GENES & DEVELOPMENT 2101 Downloaded from genesdev.cshlp.org on November 4, 2021 - Published by Cold Spring Harbor Laboratory Press Weng et al. Figure 5. Activated Notch1 rescues the T-ALL cell line 8946 from withdrawal of transgenic c-myc and conveys sensitivity to Notch pathway inhibitors. (A) Basal 8946 cell growth is not affected by treatment with GSI (1 µM compound E). (B,C) Transduction of 8946 cells with retroviruses encoding two different forms of activated Notch1, ICN1, or E retroviruses, rescue the cells from the effects of doxycycline (20 ng/mL), whereas empty MigRI does not. 8946 cell rescue was judged variously by forward and side scatter (B, which shows the rescue by ICN1), or by the number of GFP positive cells on day 6 post-treatment (C, which shows the rescue by E). (D) When rescued with E from doxycycline treatment, 8946 cells become newly sensitized to GSI. 8946 cells transduced with E were treated for 6 d with doxycline (Dox, 20 ng/mL) ± 1 µM compound E (GSI). Effects of GSI treatment were assessed by flow cytometry. (E) 8946 cells rescued from doxycycline (Dox, 20 ng/mL) by E up-regulate endogenous c-myc and other Notch and c-myc target genes. 8946 cells transduced with MigRI or MigRI-E were treated with doxycline (20 ng/mL) ± GSI (1 µM compound E) for 24 h Expression of deltex1 (a gene that is a up-regulated by Notch1), transgenic human c-myc, endogenous murine c-myc, and the c-myc target cad were monitored by RT–PCR. brane-tethered form of activated Notch1 resembling mal maturation of thymocytes (for review, see Radtke et TM N *) rescued these cells from c-myc withdrawal, al. 2004). The earliest steps of intrathymic T-cell devel- whereas empty virus did not (Fig. 5B,C). Significantly, opment are characterized by four hierarchical develop- the rescue by Notch1 was associated with the up-regu- mental stages, termed DN1–4. Notch1 signaling peaks in − − − − lation of endogenous murine c-myc (Fig. 5E). Further, DN3 thymocytes (defined as CD3 , CD4 , CD8 , c-kit , − + when rescued with E, 8946 cells became GSI-sensitive CD44 , CD25 ), and then declines as cells progress to the − − − − − − (Fig. 5D) and up-regulated the expression of both Notch1 DN4 stage (CD3 , CD4 , CD8 , c-kit , CD44 , CD25 ). (dtx1, c-myc) and c-myc (cad) target genes (Fig. 5E). Recently, Rothenberg’s group noted that DN3 thymo- Taken together, these data are consistent with a model cytes could be subdivided into DN3a and DN3b cells in which c-myc acts downstream of Notch to promote (Taghon et al. 2006). These cell types are positioned re- the growth of T-ALL cells. spectively just prior to or following -selection, which is characterized by the onset of signaling through the pre- TCR receptor and increased cell division. Of note, the Notch regulates c-myc in normal thymocytes expression of Notch1 and downstream Notch target In addition to its role in T-ALL, Notch1 is required for genes such as hes1 and deltex1 falls dramatically as cells induction of T cell differentiation and subsequent nor- progress from the DN3a to DN3b, and in keeping with 2102 GENES & DEVELOPMENT Downloaded from genesdev.cshlp.org on November 4, 2021 - Published by Cold Spring Harbor Laboratory Press Notch1 induces c-Myc in T-ALL regulates c-myc directly, rather than through a Notch1- dependent intermediate. c-Myc is a particularly attractive target to explain the progrowth effects of Notch1 in transformed T-cell pro- genitors. c-Myc drives cell cycle progression and regu- lates the expression of key enzymes that control cellular metabolism (for review, see Grandori et al. 2000; Levens 2003), and stimulates ribosome biogenesis and protein synthesis through interactions with RNA polymerase III (Felton-Edkins et al. 2003) and RNA polymerase I (Arabi et al. 2005; Grandori et al. 2005; Grewal et al. 2005). c-Myc also controls cell size (Johnston et al. 1999; Schuhmacher et al. 1999), presumably through its ability to stimulate protein synthesis (Oskarsson and Trumpp 2005). These functional attributes of c-myc are consis- tent with the growth arrest and decrease in cell size that are observed upon withdrawal of Notch signals from T- Figure 6. Notch signaling up-regulates c-myc in normal thy- ALL cells. mocytes at the DN3 stage. Sorted DN3 thymocytes from three to five mice were incubated for 16 h on OP9 or OP9-DLL1 feeder cells in the presence of the GSI compound E (1 µM) or DMSO Implications for prior studies linking Notch and c-myc vehicle. Murine T6E T-ALL cells cultivated in the presence or Retroviral mutagenesis studies conducted with c-myc absence of GSI for 24 h served as positive and negative controls. c-Myc transcript levels were determined by qPCR in these cells (MMTV /myc) transgenic mice showed that a high fre- and in freshly sorted DN3a and DN3b thymocytes. Expression quency of T-ALLs occurring with shortened latencies of c-myc was determined in three independent experiments. had proviral integrations into Notch1 (Girard et al. 1996; Mean expression levels ± 1 SD are shown. Hoemann et al. 2000). The simplest explanation for this observation (that c-myc and Notch1 act through inde- pendent, complementary pathways to promote pre-T- this, DN3a cells are dependent on Notch signals for cell transformation) is at odds with our functional data, growth and survival, whereas DN3b cells are not which places c-myc downstream of Notch1. (Taghon et al. 2006). How might these seemingly conflicting data be Based on these observations, we performed experi- brought into register? One explanation lies in the differ- ments to determine if Notch signaling affects c-myc ex- ent contexts in which the experiments were performed. pression in DN3 thymocytes. Sorted DN3 cells were co- In vivo models permit detection of oncogenic comple- cultured on OP9 cells or OP9 cells expressing the Notch mentation at different stages of tumor development and ligand dll1, which are capable of directing T-cell devel- progression, whereas our experiments are focused only opment from progenitors ex vivo (Schmitt et al. 2004). on growth maintenance in vitro. In mice, retroviral in- DN3 cells cultivated on OP9-DLL1 cells had higher ex- sertions into Notch1 in multipotent bone marrow pro- pression of c-myc than DN3 cells grown on control OP9 genitors are predicted to expand the pool of pre-T cells cells, and this difference was abrogated by a GSI (Fig. 6). and thereby increase the likelihood that some cell Further, freshly isolated DN3a cells had significantly within this pool will acquire other rate-limiting onco- higher expression of c-myc than did DN3b cells. Thus, genic aberrations. This ability to expand the pool of “at- Notch signaling correlates with c-myc expression at a risk” cells may underlie Notch1’s remarkable capacity critical stage of normal  T-cell development. to complement many (if not all) of the other genetic le- sions implicated in human (Weng et al. 2004) and murine T-ALL (Girard et al. 1996; Feldman et al. 2000; Dumor- tier et al. 2006; Lin et al. 2006; O’Neil et al. 2006). Much Discussion less commonly, retroviral insertions into c-myc occur in ic T-ALLs arising in lck-activated-Notch1 (lck-N ) trans- c-Myc is a Notch1 target in T-ALL cells genic mice (Beverly and Capobianco 2003). Although c- ic Although the Notch signaling pathway is best known for myc should already be up-regulated in lck-N mice, su- its ability to influence differentiation, inappropriate praphysiologic expression of c-myc due to proviral inser- gains in Notch function can have profound effects on tion may confer an additional selective advantage. This growth in specific contexts (Berry et al. 1997; Weng et al. idea is in line with studies suggesting that tumor latency 2003, 2004). Using genomic, biochemical, and functional is shortened in homozygous transgenic c-myc mice rela- approaches, we identified c-myc as an important media- tive to heterozygous animals (Sidman et al. 1993). Fi- tor of Notch’s progrowth effects in T-ALL cells. Impor- nally, the failure of c-myc to rescue all Notch-dependent tantly, the ability of Notch1 to stimulate c-myc tran- cell lines clearly points to the existence of other Notch1 scription in the presence of the protein synthesis inhibi- target genes that contribute to growth and survival, and tor cycloheximide is persuasive evidence that it which also likely provide a selective advantage to tu- GENES & DEVELOPMENT 2103 Downloaded from genesdev.cshlp.org on November 4, 2021 - Published by Cold Spring Harbor Laboratory Press Weng et al. mors in MMTV /myc transgenic mice bearing Notch1- binding site for Ikaros (Molnar and Georgopoulos 1994), proviral insertions. It will be important to define the mo- a transcriptional repressor that regulates pre-T-cell de- lecular basis for the variation in c-myc dependency among velopment (Georgopoulos et al. 1994; Winandy et al. cell lines, and to identify other genes downstream of 1999). Ikaros loss-of-function is associated with T-ALL Notch1 that contribute to T-ALL cell growth and survival. development (Winandy et al. 1995), and it has been hy- pothesized that Ikaros and CSL compete for access to the promoter sequences of genes that determine T-ALL de- Importance of Notch/myc interaction in normal velopment in mouse models (Beverly and Capobianco and neoplastic pre-T cells 2003); c-myc could be one such gene. Conversely, un- known cis-acting factors could enhance site-occupancy T-ALLs appear to resemble various stages of pre-T-cell by CSL and/or transcriptional activation by the CSL/ development (Ferrando et al. 2002; Asnafi et al. 2003), ICN1/MAML ternary complex. Recent work has identi- during which Notch signaling outcomes may differ dra- fied cis-acting elements that contribute to the activation matically. Notch1 is required for both T-lineage specifi- of a number of Notch-responsive elements (Cave et al. cation from multipotent progenitors (Pui et al. 1999; 2005; Ong et al. 2006), providing support for this model. Radtke et al. 1999; Sambandam et al. 2005) and subse- quent pre-T-cell development up to and including the -selection checkpoint (Wolfer et al. 2002; Tanigaki et Summary and implications al. 2004; Ciofani and Zuniga-Pflucker 2005), which oc- The existence of a direct link between Notch and c-myc curs during the maturation of “Notch-high” DN3a to in T-ALL cell lines and normal thymocytes has thera- “Notch-low” DN3b cells (Taghon et al. 2006). Of note, peutic as well as basic implications. Most mutated normal -selection is accompanied by an expansion of Notch1 receptors found in T-ALL cells depend on T-cell progenitors that requires both Notch1 and c-myc -secretase to transduce signals (Weng et al. 2004; (Douglas et al. 2001; Iritani et al. 2002). Our ex vivo Malecki et al. 2006), and the withdrawal of c-myc trans- studies show that Notch signals contribute to c-myc ex- gene expression “cures” ∼50% of mice with T-ALL pression in DN3a cells, and that there is a precipitous (Felsher and Bishop 1999). Recognition that the primary drop in c-myc expression in post--selection DN3b thy- effect of Notch1 signals in T-ALL cells appears to be on mocytes, which also down-regulate pre-T expression. proliferation and metabolism, rather than differentiation Of interest, Notch-associated T-ALLs are often associ- or survival, point to rational combinations of Notch ated with persistent expression of pre-T (see Fig. 1A; pathway inhibitors and other therapeutic agents. This Bellavia et al. 2000), which has been taken as evidence could take the form of agents that also impinge on pro- that these cells are arrested at a stage resembling normal tein synthesis through independent mechanisms, or -selection. Together, these findings suggest that the drugs that target parallel pathways, such as those that Notch/c-myc signaling axis found in Notch-dependent regulate cell survival directly. T-ALL cells reflects the persistence of a stage-specific relationship that is involved in the development of nor- mal T cells. Materials and methods Cell lines How are Notch1 targets restricted to specific contexts? All lymphoid lines were grown in RPMI 1640 (Invitrogen) supplemented with 10% fetal bovine serum (Hyclone), 2 mM The outcome of Notch signaling varies dramatically L-glutamine, 1 mM sodium pyruvate, and antibiotics. 293T from context to context. One probable basis for these cells were maintained in Dulbecco’s modified Eagle medium different outcomes is the existence of context-specific (DMEM) (Invitrogen) with the same supplements except so- target genes, such as pre-T (Deftos et al. 2000; Reizis dium pyruvate. Cells were grown at 37°C under 5% CO . The and Leder 2002). Since physiologic Notch signals do not T6E murine cell line has been described previously (Pear et al. generally lead to increased growth, it seems likely that 1996). 8946 is a murine T-ALL cell line derived from a tumor c-myc will also prove to be a context-specific target. created with a tetracycline-dependent human c-myc transgene How might this be achieved? Our data suggest that (Felsher and Bishop 1999). c-myc is targeted by Notch1 in T-ALL cells via a con- served site (TTGGGAA) located within the 5 c-myc pro- Plasmids and probes moter region immediately upstream of the TATA box cDNAs encoding murine and human c-myc, murine Mad1, HA- that was shown to contain a CSL-responsive element tagged A-Max (kind gift of Dr. Charles Vinson, NCI, Bethesda, previously (Satoh et al. 2004). This sequence differs from MD), and Flag-tagged cyclin D3 (kind gift of Dr. Alan Diehl, the canonical CSL recognition sequence YGTGRGAA University of Pennsylvania, Philadelphia, PA) were subcloned (Tun et al. 1994; Nellesen et al. 1999), and binds CSL into MigR1, a retroviral vector containing an internal ribosomal relatively weakly, which may have a role in restricting entry site (IRES) and GFP marker (Pui et al. 1999). The retroviral CSL occupancy to particular contexts. Other factors may constructs MigRI–ICN1 and MigRI–MAML1(13–74)-GFP have further modulate occupancy positively and negatively. been described (Weng et al. 2003). All constructs were se- In this regard, the putative CSL-binding site in the c-myc quenced and tested for expression and followed by detection on promoter is a high similarity match to the consensus Western blots stained with either anti-Mad1 (#4682, Cell Sig- 2104 GENES & DEVELOPMENT Downloaded from genesdev.cshlp.org on November 4, 2021 - Published by Cold Spring Harbor Laboratory Press Notch1 induces c-Myc in T-ALL naling), anti-HA (clone 6E2, Cell Signaling) to detect the A-Max conditions, then exposed to Kodak BioMax MS autoradiography construct, or anti-Flag (#2368, Cell Signaling) to detect the cy- film with intensifying screens. clin D3 construct (#2368, Cell Signaling). For the experiments in which ICN was cotransduced with either Mig A-Max or Mig Quantitative RT–PCR (qRT–PCR) Mad1, ICN1 was expressed from a MSCV-based retroviral vec- tor that coexpresses the truncated nerve growth factor receptor Oligo-d(T)-primed total RNAs (2 µg per cell line sample and 0.5 (tNGFR) as a surrogate marker (Tu et al. 2005). This approach µg for thymocytes) were reverse-transcribed with SuperScript II allows detection of GFP in the FL1 channel and tNGFR in the (Invitrogen). For cell line samples, an appropriate dilution of FL2 channel using a NGFR antibody (Pharmingen). The c-myc cDNA and gene-specific primers were combined with iQ SYBR and 18S rRNA probes used for Northern blot hybridization were Green Supermix (Bio-Rad) and amplified in an iCycler iQ real- generated by random hexamer priming of a ∼2.2-kb human c- time PCR machine (Bio-Rad). All qPCR reactions were per- myc cDNA (MGC-5183) and a 644-bp PCR product spanning formed in triplicate. Ct (threshold cycle number) and expression residues 548–1191 of GenBank accession M10098, respectively. values with standard deviations were calculated using the Gene Expression Macro for Excel (Bio-Rad). Primer sequences for real- time PCRs were as follows: c-myc forward, 5-CTTCTCTCC Retroviral transduction and GSI rescue GTCCTCGGATTCT-3; c-myc reverse, 5-GAAGGTGATCC Production of high-titer ecotropic (Pear et al. 1996) and pseudo- AGACTCTGACCTT-3; -actin forward, 5-CGCGAGAAGA typed (Weng et al. 2003) retroviruses and transduction of target TGACCCAGAT-3; -actin reverse, 5-GATAGCACAGCCT cells have been described. For the GSI rescue experiments, GGATAGCAAC-3. c-Myc and -actin primers were used at 5 5 5 × 10 to 7.5 × 10 cells were centrifuged with the appropriate 0.25 µM final and exhibited PCR efficiencies of 95.7% and amount of viral supernatant and 4 µg/mL hexadimethidrine bro- 92.9%, respectively. Real-time amplification was performed mide (Sigma) for 50 min at 2500 rpm (day −2). Transduction with initial denaturation at 95°C for 3 min, followed by 40 efficiency was measured 48 h post-infection (day 0) by GFP fluo- cycles of two-step amplification (95°C for 15 sec, 65°C for 1 rescence on a FACScan flow cytometer (Becton Dickinson). Af- min). For thymocyte samples, real-time RT–PCR was performed ter assessing GFP percentage, cells were immediately counted with TaqMan universal PCR Master Mix (Applied Biosystems) and prepared for treatment with GSI (1 µM compound E, which and analyzed on the ABI Prism 7900 (Applied Biosystems). was synthesized in the laboratory of M.S. Wolfe). Changes in the Notch1, c-myc, and deltex1 Taqman primers were obtained percentage of GFP cells and other cellular parameters were from Applied Biosystems. assessed with a FACScan flow cytometer and total cell number was obtained using a hemocytometer. The number of GFP cells DNA content/flow cytometric analysis was normalized to the number of GFP cells at day 0 to correct for differences in the initial retroviral transduction. Average cell Live or fixed cells were stained for DNA content with Hoechst counts and standard deviations were calculated for each experi- 33342 (4 µM, Sigma B2261) or propidium iodide (40 µg/mL), ment (carried out in triplicate), and each experiment was carried respectively. Flow cytometry was performed using an Influx out at least twice. Where appropriate, a Student’s t-test was Analyzer (Cytopeia) equipped with 488 nm and UV lasers to used to assess statistical significance. measure GFP and Hoechst 33342 in live cells, while FACSCali- bur cytometers (BD Biosciences) were used to measure prop- idium iodide in fixed cells or GFP in live cells. Data analysis Expression profiling/analysis was performed using FlowJo software (Treestar). Total RNA prepared using Trizol (Invitrogen) was used to gen- erate labeled cRNA as described (Shipp et al. 2002). cRNAs were Chromatin immunoprecipitation (ChIP) assay hybridized/scanned on AffyMetrix U74Av2 and/or Expression Set 430 A/B, chips, and raw fluorescence data were analyzed ChIP was performed using ChIP assay kits (Upstate Biotechnol- using dChip software. GSI-treated cells were cultured in the ogy). T6E cells were treated with DMSO or GSI (1 µM Com- presence of 1.0 µM compound E for 3, 6, 24, or 72 h. Mock- pound E) for 24 h prior to fixation. Cells were fixed with 1% treated cells were cultured in the presence of DMSO vehicle paraformaldehyde at room temperature for 10 min, washed, and (0.01% final) for 120 h. DN-MAML1 transduced cells were lysed with SDS lysis buffer (50 mM Tris-HCl, 1% SDS, 10 mM sorted at 72 h following exposure to retrovirus to purities of EDTA, protease inhibitor cocktail, Sigma). The lysates were + + 87% GFP (DN-MamA) and 98% GFP (DN-MamB); GFP-only sonicated to reduce DNA lengths to between 200 and 600 bp. controls were sorted to purities of 95% GFP (GFPposA) and The soluble fraction was diluted, precleared with salmon sperm 98% GFP (GFPposB); GFP-negative controls were sorted from DNA/protein A-agarose, then divided into two tubes and incu- cells exposed to, but not transduced by, DN-Mam retroviruses bated with 5 µL of antiserum specific for Notch1 TAD domain − − to purities of >98% GFP (GFPnegA) and >99% GFP (GFPnegB). (Aster et al. 2000) or normal rabbit IgG. The immune complexes 6 6 Sorted cells (2 × 10 to 4 × 10 ) were harvested for RNA imme- were then precipitated with protein A-agarose and eluted with diately following sorting. Cells transduced with ICN were cul- an elution buffer (0.1 M NaHCO containing 1% SDS). The tured in the presence of 0.5 µM compound E continuously for eluted material was reverse-cross-linked and treated with pro- several weeks prior to RNA harvest (>99% GFP ). teinase K (20 µg/mL). DNA was purified using a PCR purifica- tion kit (Qiagen) and eluted by water (5 × 10 cell equivalents/ 50 µL). Quantitative PCR (qPCR) was performed using the SYBR Northern blots Green system on the ABI 7900HT Sequence Detection System Total RNAs (10 µg per sample) were run on 1.2% agarose/2.2 M (Applied Biosystems) with the following primers: (1) c-myc pro- formaldehyde gels and transferred to Hybond-XL nylon mem- moter-forward, 5-TGAGGCTCCTCCTCCTCTTTC-3; (2) branes (Amersham Biosciences) with UV cross-linking (Stra- c-myc promoter reverse, 5-GCAGACCCCCGGAATATAAA- talinker, Stratagene). Blots were hybridized with radiolabeled 3; (3) c-myc intron2 forward, 5-CACGGGACCTGAAAG P-random-primed probes at 65°C and washed under stringent GTTCT-3; (4) c-myc intron2 reverse, 5-GGGTTAGGGCAC GENES & DEVELOPMENT 2105 Downloaded from genesdev.cshlp.org on November 4, 2021 - Published by Cold Spring Harbor Laboratory Press Weng et al. AGGTGAGA-3; (5) hes1 promoter forward, 5-CGTGTCT er’s recommendations (Miltenyi Biotec). Following staining CTTCCTCCCATTG-3; (6) hes1 promoter reverse, 5-CCAG with antibodies against lineage markers (TCR, TCR, CD3, GACCAAGGAGAGAGGT-3. Primers for the hes1 promoter CD4, NK1.1, CD19, Gr-1, CD11b), CD44, and CD25 antigen −/lo hi − sequence flank two CSL-binding sites that lie just 5 of the (Pharmingen), DN3 thymocytes (CD44 CD25 Lin ) were TATA box (Jarriault et al. 1995). Each sample was indepen- purified by cell sorting on a FACS Moflo (Cytomation). DN3a −/lo hi − lo −/lo hi dently prepared at least two times and run in duplicate. The (CD44 CD25 Lin CD27 ) and DN3b (CD44 CD25 − lo relative DNA amount was calculated using the standard curve Lin CD27 ) cells were purified by sorting after staining with method as described in the ABI 7900HT Sequence Detection anti-CD27 antibody (eBioscience) as described (Taghon et al. System manual. The input DNA was defined as an aliquot of 2006). Cells were either used for RNA extraction directly, or sheared chromatin prior to immunoprecipitation, and was used were cocultured with OP9 or OP9-DLL1 stromal cells in the to normalize the sample to the amount of chromatin added to presence or absence of 1 µM compound E for 16 h as described each ChIP. (Schmitt et al. 2004). RNA was isolated from thymocytes using the RNEasy kit according to the manufacturer’s recommenda- tions (Qiagen). EMSA Oligonucleotides fluorescently 5-labeled with carboxyfluores- Statistical analysis cein (FAM) and unlabeled complementary oligonucleotides ANOVA and t-test analyses were performed using the Prism were obtained from Integrated DNA Technologies. Sequences 4.03 software package (GraphPad Software). of the FAM-labeled oligonucleotides were as follows: c-myc pro- moter, 5-FAM-CCCCTCCCGGGTTCCCAAAGCAGAGGG CGT-3; mutated c-myc promoter, 5-FAM-CCCCTCCCGGG Acknowledgments TTCAAAAAGCAGAGGGCGT-3; consensus CSL-binding site, 5-FAM-TCCAAATTTTTTCCCACGGCGTGT-3. CSL We thank Alan Diehl and Charles Vinson for providing re- and the RAM-ANK domains of Notch1 were prepared as de- agents, and Gerd Blobel, Tom Kadesch, David Levens, Steve scribed (Nam et al. 2003). Nonradioisotopic EMSAs were per- McMahon, Gary Koretzky, Craig Thompson, and members of formed by incubating 2 pmol of probes for 30 min at 30°Cin the Pear and Aster laboratories for helpful suggestions. J.M. was binding buffer (10% glycerol, 20 mM HEPES at pH 7.9, 60 mM supported by NIH Training Grant (T32 CA 09140-31-35). C.D.B. KCl, 10mM DTT, 5 mM MgCl , 250 ng dGdC, 0.2 mg/mL bo- is supported by a Long-Term Fellowship from the Human Fron- vine serum albumin) in the presence or absence of 10 µg of CSL tier Science Program Organization, and M.L.A. is supported by and RAM-ANK. Gel electrophoresis was performed in 10% na- the Foundation pour la Recherche Medicale (FRM). A.P.W., tive gels at 4°C and 180 V. Following electrophoresis, gels were D.W.F., S.C.B., W.S.P., and J.C.A. are supported by grants from immediately analyzed by blue-excitation fluorescence scanning the NIH. with a Storm 860 FluorImager (Amersham Pharmacia Biotech). To compare the affinity of CSL binding to sequences of interest, oligonucleotides with 5 overhangs were incubated with P-- References dCTP (Perkin-Elmer) and the Klenow fragment of Escherichia coli DNA polymerase I (New England Biolabs). 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Runoff reactions were performed with opment. Science 284: 770–776. - P UTP (Perkin-Elmer), followed by DNase I (500 U/sample, Asnafi, V., Beldjord, K., Boulanger, E., Comba, B., Le Tutour, P., Invitrogen) and proteinase K digestion. RNA was isolated using Estienne, M.H., Davi, F., Landman-Parker, J., Quartier, P., RNeasy Mini columns (Qiagen). A Minifold II appartus (Schlei- Buzyn, A., et al. 2003. Analysis of TCR, pT , and RAG-1 in cher & Schuell BioScience) was used to transfer 5 µg of linear- T-acute lymphoblastic leukemias improves understanding ized, NaOH-denatured plasmid DNA per slot onto positively of early human T-lymphoid lineage commitment. Blood charged nylon membranes (Hybond-XL, Amersham Biosci- 101: 2693–2703. ences). Following UV cross-linking (70,000 µJ/cm ; UV Stra- Aster, J.C., Xu, L., Karnell, F.G., Patriub, V., Pui, J.C., and Pear, talinker 2400, Stratagene), 5 × 10 cpm of each runoff RNA W.S. 2000. 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Wolfer, A., Wilson, A., Nemir, M., MacDonald, H.R., and Radtke, F. 2002. Inactivation of Notch1 impairs VDJ rear- rangement and allows pre-TCR-independent survival of early  Lineage Thymocytes. Immunity 16: 869–879. Wu, L., Aster, J.C., Blacklow, S.C., Lake, R., Artavanis-Tsako- nas, S., and Griffin, J.D. 2000. MAML1, a human homologue of Drosophila mastermind, is a transcriptional co-activator for NOTCH receptors. Nat. Genet. 26: 484–489. Ye, Y., Lukinova, N., and Fortini, M.E. 1999. Neurogenic phe- notypes and altered Notch processing in Drosophila Prese- nilin mutants. Nature 398: 525–529. GENES & DEVELOPMENT 2109 Erratum Genes & Development 20: 2096–2109 (2006) Andrew P. Weng, John M. Millholland, Yumi Yashiro-Ohtani, Marie Laure Arcangeli, Arthur Lau, Carol Wai, Cristina del Bianco, Carlos G. Rodriguez, Hong Sai, John Tobias, Yueming Li, Michael S. Wolfe, Cathy Shachaf, Dean Felsher, Stephen C. Blacklow, Warren S. Pear, and Jon C. Aster During a recent internal review of the data in the above-mentioned paper, the authors identified an inadvertent mistake in Figure 6 that they would like to correct. Figure 6 shows changes in c-myc expression in developing thymocytes. Within Figure 6, the authors included data showing that c-myc is reduced as cells progress from the DN3a to DN3b stage of thymocyte development. In assembling these two data points, they inadvertently substituted the Notch1 positive control data for the c-myc data. As shown in the revised figure below, the reduction in c-myc expression between DN3a and DN3b is closer to twofold rather than the fourfold difference shown in the published figure. This change is roughly equivalent to the reductions in c-myc expression that are produced in malignant T6E cells or normal DN3 thymocytes by withdrawal of Notch signals. The authors have also included the Notch1 expression data in the revised figure, as this was the positive control used by Rothenberg and coworkers (Fig. 6 in Taghon et al. 2006) to ascertain the purity of the DN3a and DN3b populations. Importantly, the revised figure fully supports the conclusions stated in the above-mentioned paper. However, the authors believe an Erratum is in order, both to correct their error for the record and to accurately depict the changes in c-myc expression that accompany transition from the DN3a to DN3b stage of thymocyte development. The authors apologize for their error. Figure 6. Notch signaling up-regulates c-myc in nor- mal thymocytes at the DN3 stage. Sorted DN3 thymo- cytes from three to five 4- to 6-wk-old B6 mice were incubated for 16 h on OP9 or OP9-DL1 feeder cells in the presence of the GSI compound E (1 µM) or DMSO vehicle. T6E T-ALL cells cultivated in the presence or absence of GSI for 24 h served as positive and negative controls. c-Myc transcript levels were determined by qPCR in these cells and in freshly sorted DN3a and DN3b thymocytes. Expression of notch1 served as the positive control for DN3a and DN3b thymocytes. Ex- pression of c-myc and notch1 was determined in three independent experiments. Mean expression levels ± 1 SD are shown. Reference Taghon, T., Yui, M.A., Pant, R., Diamond, R.A., and Rothen- berg, E.V. 2006. Developmental and molecular characteriza- tion of emerging - and -selected pre-T cells in the adult mouse thymus. Immunity 24: 53–64. GENES & DEVELOPMENT 21:625 © 2007 by Cold Spring Harbor Laboratory Press ISSN 0890-9369/07; www.genesdev.org 625 Downloaded from genesdev.cshlp.org on November 4, 2021 - Published by Cold Spring Harbor Laboratory Press c-Myc is an important direct target of Notch1 in T-cell acute lymphoblastic leukemia/lymphoma Andrew P. Weng, John M. Millholland, Yumi Yashiro-Ohtani, et al. Genes Dev. 2006, 20: Access the most recent version at doi:10.1101/gad.1450406 http://genesdev.cshlp.org/content/suppl/2006/07/14/gad.1450406.DC1 Supplemental Material Erratum Related Content Genes Dev. March , 2007 21: 625 This article cites 81 articles, 30 of which can be accessed free at: References http://genesdev.cshlp.org/content/20/15/2096.full.html#ref-list-1 Articles cited in: http://genesdev.cshlp.org/content/20/15/2096.full.html#related-urls License Receive free email alerts when new articles cite this article - sign up in the box at the top Email Alerting right corner of the article or click here. Service Copyright © 2006, Cold Spring Harbor Laboratory Press http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Genes & Development Unpaywall

c-Myc is an important direct target of Notch1 in T-cell acute lymphoblastic leukemia/lymphoma

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Downloaded from Downloaded from Downloaded from genesdev.cshlp.org genesdev.cshlp.org genesdev.cshlp.org on November 4, 2021 - Published by on November 4, 2021 - Published by on November 4, 2021 - Published by Cold Spring Harbor Laboratory Press Cold Spring Harbor Laboratory Press Cold Spring Harbor Laboratory Press c-Myc is an important direct target of Notch1 in T-cell acute lymphoblastic leukemia/lymphoma 1,2,8 3,8 3 3 Andrew P. Weng, John M. Millholland, Yumi Yashiro-Ohtani, Marie Laure Arcangeli, 2 2 1 3 3 4 Arthur Lau, Carol Wai, Cristina del Bianco, Carlos G. Rodriguez, Hong Sai, John Tobias, 5 6 7 7 1 Yueming Li, Michael S. Wolfe, Cathy Shachaf, Dean Felsher, Stephen C. Blacklow, 3,10 1,9 Warren S. Pear, and Jon C. Aster Department of Pathology, Brigham and Women’s Hospital and Harvard Medical School, Boston, Massachusetts 02115, USA; Department of Pathology and Lab Medicine, Terry Fox Laboratory, British Columbia Cancer Agency, Vancouver, British Columbia V5Z 1L3, Canada; Department of Pathology and Lab Medicine, Abramson Family Cancer Research Institute, Institute for Medicine and Engineering, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA; University of Pennsylvania Bioinformatics Core, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA; Molecular Pharmacololgy and Chemistry Program, Memorial Sloan-Kettering Cancer Center, New York, New York 10021, USA; Center for Neurologic Diseases, Harvard Medical School and Brigham and Women’s Hospital, Boston, Massachusetts 02115, USA; Division of Oncology, Departments of Medicine and Pathology, Stanford University, Stanford, California 94305, USA Human acute T-cell lymphoblastic leukemias and lymphomas (T-ALL) are commonly associated with gain-of-function mutations in Notch1 that contribute to T-ALL induction and maintenance. Starting from an expression-profiling screen, we identified c-myc as a direct target of Notch1 in Notch-dependent T-ALL cell lines, in which Notch accounts for the majority of c-myc expression. In functional assays, inhibitors of c-myc interfere with the progrowth effects of activated Notch1, and enforced expression of c-myc rescues multiple Notch1-dependent T-ALL cell lines from Notch withdrawal. The existence of a Notch1–c-myc signaling axis was bolstered further by experiments using c-myc-dependent murine T-ALL cells, which are rescued from withdrawal of c-myc by retroviral transduction of activated Notch1. This Notch1-mediated rescue is associated with the up-regulation of endogenous murine c-myc and its downstream transcriptional targets, and the acquisition of sensitivity to Notch pathway inhibitors. Additionally, we show that primary murine thymocytes at the DN3 stage of development depend on ligand-induced Notch signaling to maintain c-myc expression. Together, these data implicate c-myc as a developmentally regulated direct downstream target of Notch1 that contributes to the growth of T-ALL cells. [Keywords: Notch; Myc; leukemia; T cell; transformation] Supplemental material is available at http://www.genesdev.org. Received February 17, 2006; revised version accepted June 6, 2006. Notch receptors participate in a conserved signaling cells (HSCs) (Varnum-Finney et al. 2000; Calvi et al. pathway that regulates the development of diverse cell 2003; Duncan et al. 2005), T-cell specification from a and tissue types in metazoans. Outcomes resulting from multipotent precursor (Pui et al. 1999; Radtke et al. Notch signals are highly pleiotropic, depending on dose 1999; Sambandam et al. 2005; Tan et al. 2005), matura- and context (Artavanis-Tsakonas et al. 1999). Within the tion of double-negative (DN) thymocytes, especially at hematolymphoid compartment, Notch signaling affects the -selection checkpoint (Wolfer et al. 2002; Tanigaki lineage commitment at multiple developmental stages et al. 2004; Ciofani and Zuniga-Pflucker 2005; Taghon et (for review, see Radtke et al. 2004; Maillard et al. 2005). al. 2006), and the differentiation of CD4 T cells along Notch influences the self-renewal of hematopoietic stem either T 1orT 2 pathways (Amsen et al. 2004; Tani- H H gaki et al. 2004; Minter et al. 2005; Tu et al. 2005). Since the discovery of Notch1 through the analysis of a rare (7;9) chromosomal translocation in human T-cell acute These authors contributed equally to this work. lymphoblastic leukemia/lymphoma (T-ALL) (Ellisen et Corresponding authors. E-MAIL jaster@rics.bwh.harvard.edu; FAX (617) 264-5169. al. 1991), abundant evidence has also accumulated im- E-MAIL wpear@mail.med.upenn.edu; FAX (215) 746-6725. plicating Notch1 in the pathogenesis of this aggressive Article published online ahead of print. Article and publication date are online at http://www.genesdev.org/cgi/doi/10.1101/gad.1450406. cancer (Pear et al. 1996; Aster et al. 2000; Bellavia et al. 2096 GENES & DEVELOPMENT 20:2096–2109 © 2006 by Cold Spring Harbor Laboratory Press ISSN 0890-9369/06; www.genesdev.org Downloaded from genesdev.cshlp.org on November 4, 2021 - Published by Cold Spring Harbor Laboratory Press Notch1 induces c-Myc in T-ALL 2000). Recently, activating mutations in Notch1 were Results discovered in 55%–60% of human T-ALLs (Weng et al. Identification of c-myc as a putative Notch1 target 2004), and emerging data indicate that similar types of gene Notch1 mutations occur frequently in many different murine T-ALL models as secondary events (Dumortier et To identify potential Notch1 target genes, we performed al. 2006; Lin et al. 2006; O’Neil et al. 2006). expression profiling on a set of RNAs obtained from T6E Normal Notch signaling is initiated by the binding of T-ALL cells in which Notch was turned “off” (-secre- ligands of the Delta/Serrate/Lag-2 (DSL) family to the tase inhibitor [GSI]-treated or DN-MAML1 transduced) Notch ectodomain, which result in cleavage at a site just or left “on” (untreated and mock GSI-treated cells, external to the transmembrane domain by ADAM me- sorted GFP cells transduced with empty MigRI virus, talloproteases (Brou et al. 2000; Mumm et al. 2000). This and sorted GFP cells from MigRI-DN-MAML1 cul- TM event creates a short-lived Notch intermediate (N *) tures). RNAs were hybridized to Affymetrix U74Av2 that is recognized by nicastrin (Shah et al. 2005), a com- GeneChip arrays (∼12,000 genes). Raw data were ana- TM ponent of -secretase, which in turn, cleaves N * lyzed with dChip software (Li and Hung Wong 2001) by within its transmembrane domain (Schroeter et al. 1998; applying standard normalization and modeling routines, De Strooper et al. 1999; Kimberly et al. 2003). This final and by filtering gene lists based on “presence” call, ex- cleavage releases the intracellular domain of Notch pression level, and variation criteria. Hierarchical clus- (ICN) from the membrane, allowing it to translocate to tering performed using filtered gene sets resulted in un- the nucleus and form a transcriptional activation com- supervised discovery of the Notch “on” and “off” sample plex with a DNA-binding protein termed CSL (for CBF1, groups. Supervised analysis was then applied to a filtered Suppressor of Hairless, Lag-1) (Jarriault et al. 1995; Kopan list of ∼600 genes to identify genes for which the expres- et al. 1996; Struhl and Greenwald 1999; Ye et al. 1999) sion levels were most highly correlated with Notch ac- and transcriptional coactivators of the Mastermind-like tivation status. (MAML) family (Petcherski and Kimble 2000a,b; Wu et A total of 83 genes demonstrated multiple-compari- al. 2000). son-adjusted p-values of <0.05 (Fig. 1A; see Supplemen- Although signals mediated through Notch receptors tary Table S1 for the full list of genes, fold changes, and have diverse outcomes, only a fairly limited set of Notch p-values). Genes down-regulated in the Notch “off” target genes have been identified in various cellular/de- group included the majority of known/previously de- velopmental contexts. The hairy/enhancer of split (Hes) scribed Notch targets in immature T cells (hes1, hey1, genes are highly conserved target genes that are regu- pre-T, deltex1, and several interferon-induced genes), lated by Notch in multiple cell types (Preiss et al. 1988; and c-myc. We also performed an independent set of ex- Jarriault et al. 1995). On the other hand, investigators pression profiling experiments in which we sought to studying Notch1 function in immature T cells identified identify pathways that were perturbed by GSI treatment several likely T-cell-specific target genes, including of T6E. When analyzed with Ingenuity software, the CD25 and pre-T (Deftos et al. 2000; Reizis and Leder pathway most highly down-regulated by Notch inhibi- 2002). Other putative context-specific target genes that tion was that involving c-myc (data not shown). may promote cell growth include cyclinD1, which was The identification of c-myc as a potential Notch1 tar- identified as a transcriptional target in RKE cells get in studies using different inhibitors and several inde- (Ronchini and Capobianco 2001), and c-myc (Satoh et al. pendent analytical tools provided the impetus for more 2004), which was identified as a possible Notch target in focused studies. As an initial test, we assessed the effects hematopoietic stem cells. In this latter study, Notch re- of -secretase blockade on c-myc mRNA levels in five sponsiveness was linked to an ∼200-base-pair (bp) ele- human T-ALL cell lines that require Notch signals for ment lying immediately 5 of the c-myc transcriptional growth (Weng et al. 2004). Notch pathway blockade with start site, but neither direct association of Notch with a GSI led to the down-regulation of c-myc in all five of this site nor its functional importance was demon- the human T-ALL cell lines that require Notch signals, strated. Thus, the identities of the genes downstream of as well as murine T6E cells (Fig. 1B). Notch1 that maintain the growth of T-ALL cells have yet to be determined. c-Myc is a direct target of Notch1 To address this uncertainty, we used expression pro- filing to identify genes that are down-regulated by Notch Forms of Notch1 bearing activating mutations within pathway inhibitors in T6E, a murine T-ALL cell line the extracellular domain are susceptible to ligand-inde- whose growth depends on a membrane-tethered form of pendent cleavage by metalloproteases at site S2 TM Notch1 resembling N (Weng et al. 2003). Among the (Sanchez-Irizarry et al. 2004). The product of this cleav- TM potential target genes identified was c-myc, which has age, N *, is normally rapidly recruited to the -secre- been shown to induce T-ALL in animal models when tase complex and further processed into ICN1 (Shah et TM overexpressed (Girard et al. 1996; Felsher and Bishop al. 2005). However, we noted previously that N *in 1999; Langenau et al. 2003). This insight led to a series of T-ALL cells is stabilized and accumulates in the pres- TM functional studies, which showed that c-myc is an im- ence of GSI (Weng et al. 2003), creating a pool of N * portant target of Notch not only in T-ALL cells, but also that can be rapidly converted to ICN1 upon GSI wash- at a critical stage of normal pre-T-cell development. out. GENES & DEVELOPMENT 2097 Downloaded from genesdev.cshlp.org on November 4, 2021 - Published by Cold Spring Harbor Laboratory Press Weng et al. Figure 1. Withdrawal of Notch1 signals down-regulates c-myc expression in T-ALL cells. (A) Identification of Notch1-sensitive genes in T6E cells. Columns represent experimental samples, while rows represent genes. Each colored box indicates relative expression level (normalized for each gene), where red indicates high and blue indicates low. The seven columns on the left are samples from T6E cells with active Notch signaling, and the six columns on the right are samples from T6E cells in which Notch signaling was inhibited. Expression data from 82 genes significantly correlated with the Notch “on” (left) versus “off” (right) distinction (p < 0.05) are depicted; expression of the 25 genes in the upper cluster increased on inhibition of Notch signaling, expression of the 57 in the lower cluster decreased. Genes implicated in the literature as related to Notch signaling as well as the novel gene c-myc are highlighted. Sample label key: (GSI [1 µM compound E] + ICN1) sorted ICN1 transduced cells treated with GSI; (GSI mock) DMSO vehicle-treated cells; (GSI 0 h) untreated cells; (GFPposA/B) sorted GFP-only cells; (GFPnegA/B) sorted untransduced cells from the same cultures as transduced cells; (GSI) cells treated with GSI for the indicated number of hours; (DN-MamA/B) sorted dominant-negative MAML1 transduced cells. (B) Northern blot analysis demonstrating the down-regulation of c-myc following treatment with GSI (1 µM com- pound E). Notch-dependent T6E cells and human T-ALL cell lines were treated with carrier (DMSO) or GSI for the indicated time periods. Blots were hybridized with probes indicated in the right margin. 2098 GENES & DEVELOPMENT Downloaded from genesdev.cshlp.org on November 4, 2021 - Published by Cold Spring Harbor Laboratory Press Notch1 induces c-Myc in T-ALL We used this maneuver to determine if the transcrip- To determine if Notch stimulates the synthesis of c- tional up-regulation of c-myc by Notch1 requires protein myc transcripts, nuclear runoff experiments were per- synthesis. These experiments used the human T-ALL formed with KOPT-K1 nuclei obtained from cells treated cell line KOPT-K1, which bears a mutation in the extra- with DMSO or GSI (Fig. 2B). GSI treatment diminished cellular heterodimerization domain of Notch1 that re- c-myc transcription, whereas washout of GSI for as little sults in ligand-independent S2 cleavage (Weng et al. as 2 h produced a rebound in c-myc transcription com- 2004; Malecki et al. 2006). Washout of GSI in KOPT-K1 parable to that observed in the experiments conducted cells led to the rapid up-regulation of c-myc transcripts with cycloheximide. Importantly, the increase in c-myc even in the presence of cycloheximide, an inhibitor of pro- transcription was largely abrogated when cells were tein synthesis (Fig. 2A). The up-regulation of c-myc upon refed fresh medium containing GSI (mock washout). GSI washout was observed over a wide range of cyclohexi- Thus, Notch1 increases c-myc RNA in T-ALL cells by mide doses (5–40 µg/mL), all of which were sufficient to stimulating transcription. suppress the growth of KOPT-K1 cells (data not shown). Scanning of c-myc genomic sequences revealed two Figure 2. c-Myc is a direct target of Notch1. (A) c-Myc up-regulation by Notch does not require de novo protein synthesis. KOPT-K1 TM cells were treated with GSI (1 µM compound E) for 48 h to permit accumulation of the -secretase substrate N *. Cells were then washed and refed medium containing GSI (mock washout), or medium lacking GSI (washout) with or without 20 µM cycloheximide (CHX). c-Myc RNA levels were determined after4hof additional culture by qPCR. Each sample was assayed in triplicate; error bars correspond to standard deviations. Similar results were obtained in four independent experiments. (B) Notch1 stimulates c-myc transcription. Nuclei were prepared from KOPT-K1 cells treated with vehicle (0.01% DMSO) for 48 h; treated with GSI (1 µM compound E) for 48 h; treated with GSI for 48 h, then washed thrice and cultured2hin fresh medium without GSI (washout); or treated with GSI for 48 h, then washed three times and cultured2hin fresh medium with GSI (mock washout). RNAs isolated from runoff reactions were hybridized to slot-blots containing probes specific for c-myc and GAPDH. Bound radioactivity was quantified using a PhosphorImager. (Top panel) Phosphorimages. (Bottom panel) Calculated c-myc/GAPDH transcript ratios. Results of a representative experiment are shown. (C) Notch1 binds to the c-myc promoter through a region containing a conserved CSL consensus sequence. Chromatin immunoprecipitates were performed on cross-linked fragmented DNAs prepared from T6E cells treated with DMSO or 1 µM compound E (GSI) for 24 h. Eluted DNAs were then analyzed by qPCR performed with primers flanking putative CSL-binding sites A and B. The amount of DNA amplified from immunoprecipitated DNAs was normalized to that amplified from input DNA. (D) CSL binds to the putative target sequence in the c-myc promoter. Oligonucleotides labeled with carboxyfluorescein (FAM) were mixed with buffer alone or buffer containing purified CSL and Notch1 polypeptides. Following electrophoresis in 10% native gels, fluorescently labeled probes were detected with an 860 Storm FluorImager (Amersham Pharmacia Biotech). GENES & DEVELOPMENT 2099 Downloaded from genesdev.cshlp.org on November 4, 2021 - Published by Cold Spring Harbor Laboratory Press Weng et al. potential CSL-binding sites that are conserved between c-myc mimicked the effects of ICN1. T6E cells were humans and mice: (1) site A (TTCCCAA), which lies transduced with either empty MigRI, or with c-myc, within the putative Notch-responsive element identified ICN1, other Notch1 targets (hes1 and hes5), the anti- by Satoh et al. (2004); and (2) site B (TTGGGAAA), apoptotic gene bcl-x ,or cyclinD3, a gene implicated in which is within intron 2 of c-myc (Fig. 2C). Chromatin the growth and proliferation of normal T-cell progenitors immunoprecipitates prepared from T6E cells revealed (Sicinska et al. 2003). Only ICN1 and c-myc fully rescued that ICN1 associated with DNA fragments containing T6E cells from GSI-induced growth arrest (Fig. 3A); a site A, and that GSI treatment depleted ICN1 from this small but significant survival advantage was imparted by site (Fig. 2C). In contrast, ICN1 did not associate with bcl-x (data not shown), whereas hes1, hes5, and cyclinD3 site B. As anticipated, GSI treatment also depleted ICN1 did not have effects significantly different than empty from CSL-binding sites in the hes1 promoter, a well- virus. Like ICN1, c-myc also maintained the cell cycle characterized Notch1 target (Jarriault et al. 1995). Elec- progression of T6E cells in the presence of GSI (Fig. 3B). trophoretic mobility shift assays (EMSA) showed that To further explore the significance of c-myc as a target, CSL and CSL/ICN1 complexes bind to an oligonucleo- T6E cells were simultaneously transduced with ICN1 tide that contains site A (Fig. 2D), albeit with an affinity and either mad1 or dominant-negative (DN) max (A- that is about fivefold lower than that of CSL for a con- max) (Krylov et al. 1997) to antagonize the action of c- sensus binding site (Supplementary Fig. S1). Taken to- myc (Fig. 3C). Upon GSI treatment, the substantial gether, these data suggest that activated Notch1 up-regu- growth advantage of ICN1 transduced cells (compared lates c-myc directly. with nontransduced cells) was abrogated by mad1 and DN max, indicating that c-myc function is necessary for ICN1 to rescue T6E cells (Fig. 3C). c-Myc is necessary and sufficient to rescue T6E cells from Notch1 withdrawal c-Myc rescues a subset of human T-ALL cell lines Treatment of T6E cells with GSI leads to a G /G cell 0 1 from Notch1 withdrawal cycle arrest that is prevented by transduction of ICN1 (Weng et al. 2003). Given the potent progrowth effects of To investigate whether c-myc is generally sufficient to c-myc (for recent review, see Oskarsson and Trumpp rescue T-ALL cell lines from Notch withdrawal, we 2005), we investigated whether enforced expression of transduced five human Notch-dependent T-ALL cell Figure 3. c-Myc is necessary and sufficient to rescue T6E cells from withdrawal of Notch1 signals. (A) c-Myc rescues growth. T6E cells, which express a membrane-tethered form of Notch1 that requires cleavage by -secretase for activation, were transduced with MigRI retroviruses expressing GFP alone (MigRI) or GFP and the indicated polypeptides, and then treated with GSI (1 µM compound E) for up to 6 d. The numbers of GFP cells at each time point are shown. (B) c-Myc restores cell cycle kinetics. The DNA content of T6E cells transduced with the indicated MigRI retroviruses was measured by flow cytometry at baseline and after6dof treatment with GSI. (C) Inhibitors of c-myc function prevent ICN1 from rescuing T6E cells treated with GSI. T6E cells were cotransduced with retroviruses expressing ICN1 and either A-max or mad1. Cells were then cultured in the presence of 1 µM compound E for 6 d. Cells transduced with ICN1 alone (top left quadrants) are enriched over the course of the experiment, while cells expressing either A-Max or Mad1 alone (bottom right quadrants) or one of these two c-myc inhibitors and ICN1 (top right quadrant) are selected against. 2100 GENES & DEVELOPMENT Downloaded from genesdev.cshlp.org on November 4, 2021 - Published by Cold Spring Harbor Laboratory Press Notch1 induces c-Myc in T-ALL lines with c-myc. Like ICN1 (Weng et al. 2004), c-myc Activated Notch1 can rescue T-ALL cells that depend provided a partial or complete rescue of three of these on transgenic c-myc cell lines (KOPT-K1, DND-41, TALL-1) from GSI treat- ment, as judged by proliferation (Fig. 4A) and cell cycle If Notch1 is a major upstream regulator of c-myc in T- parameters (Fig. 4B), whereas two cell lines (HPB-ALL ALL cells, activated Notch1 should replace functions and ALL-SIL) were not growth-rescued by c-myc. All of provided by trangenic c-myc. This idea was tested using the GSI-treated Notch1-dependent cell lines also showed the cell line 8946, which is derived from a murine T-ALL a significant decrease in cell size that was partially or induced with a doxycycline-repressible human c-myc completely abrogated by c-myc (Fig. 4C). Thus, although transgene (Felsher and Bishop 1999). 8946 cells undergo c-myc appears to be a consistent target of activated growth arrest and apoptosis when treated with doxycy- Notch1, the degree to which Notch-dependent T-ALL cline, but are unaffected by treatment with GSI (Fig. 5A). cell lines are rescued by c-myc alone is variable. Transduction of 8946 cells with ICN1 or E (a mem- Figure 4. Transduction of c-myc leads to variable rescue of human Notch-sensitive T-ALL cell lines from withdrawal of NOTCH signals. Rescue of growth (A), cell cycle progression (B), and cell size (C) were assessed in control T-ALL cell lines or the same lines transduced with c-myc and a GFP marker (c-myc) or GFP only (GFP). Cells were treated with DMSO vehicle (mock) or 1 µM compound + + E (GSI) for the indicated time periods. (A) The fraction of live GFP cells was monitored by flow cytometry. An increasing GFP fraction indicates a growth/survival advantage conferred by the retrovirus over untransduced GFP cells. (B) The DNA content of control cell lines was compared with that of sorted populations of cells transduced with c-myc by staining with Hoechst 33342 followed by flow cytometry. The DNA histograms shown were derived from >20,000-gated events. (C) Cell size in c-myc transduced cells and untrans- duced control cells treated with GSI (compound E, 1 µM) or DMSO vehicle (V) for the indicated time was determined by forward scatter of live cells. The relative cell sizes for each population (normalized to a value of 100 for vehicle-treated, untransduced control cells ± 1 SD) are depicted. The decreases in size upon addition of GSI in untransduced cells (*), and the increases in size upon transduction with c-myc in GSI-treated cultures (**, as compared with untransduced GSI-treated cells), were both significant (P < 0.01) in all cell lines tested, as measured by the Bonferroni post-test after two-way ANOVA (unweighted means) analysis. GENES & DEVELOPMENT 2101 Downloaded from genesdev.cshlp.org on November 4, 2021 - Published by Cold Spring Harbor Laboratory Press Weng et al. Figure 5. Activated Notch1 rescues the T-ALL cell line 8946 from withdrawal of transgenic c-myc and conveys sensitivity to Notch pathway inhibitors. (A) Basal 8946 cell growth is not affected by treatment with GSI (1 µM compound E). (B,C) Transduction of 8946 cells with retroviruses encoding two different forms of activated Notch1, ICN1, or E retroviruses, rescue the cells from the effects of doxycycline (20 ng/mL), whereas empty MigRI does not. 8946 cell rescue was judged variously by forward and side scatter (B, which shows the rescue by ICN1), or by the number of GFP positive cells on day 6 post-treatment (C, which shows the rescue by E). (D) When rescued with E from doxycycline treatment, 8946 cells become newly sensitized to GSI. 8946 cells transduced with E were treated for 6 d with doxycline (Dox, 20 ng/mL) ± 1 µM compound E (GSI). Effects of GSI treatment were assessed by flow cytometry. (E) 8946 cells rescued from doxycycline (Dox, 20 ng/mL) by E up-regulate endogenous c-myc and other Notch and c-myc target genes. 8946 cells transduced with MigRI or MigRI-E were treated with doxycline (20 ng/mL) ± GSI (1 µM compound E) for 24 h Expression of deltex1 (a gene that is a up-regulated by Notch1), transgenic human c-myc, endogenous murine c-myc, and the c-myc target cad were monitored by RT–PCR. brane-tethered form of activated Notch1 resembling mal maturation of thymocytes (for review, see Radtke et TM N *) rescued these cells from c-myc withdrawal, al. 2004). The earliest steps of intrathymic T-cell devel- whereas empty virus did not (Fig. 5B,C). Significantly, opment are characterized by four hierarchical develop- the rescue by Notch1 was associated with the up-regu- mental stages, termed DN1–4. Notch1 signaling peaks in − − − − lation of endogenous murine c-myc (Fig. 5E). Further, DN3 thymocytes (defined as CD3 , CD4 , CD8 , c-kit , − + when rescued with E, 8946 cells became GSI-sensitive CD44 , CD25 ), and then declines as cells progress to the − − − − − − (Fig. 5D) and up-regulated the expression of both Notch1 DN4 stage (CD3 , CD4 , CD8 , c-kit , CD44 , CD25 ). (dtx1, c-myc) and c-myc (cad) target genes (Fig. 5E). Recently, Rothenberg’s group noted that DN3 thymo- Taken together, these data are consistent with a model cytes could be subdivided into DN3a and DN3b cells in which c-myc acts downstream of Notch to promote (Taghon et al. 2006). These cell types are positioned re- the growth of T-ALL cells. spectively just prior to or following -selection, which is characterized by the onset of signaling through the pre- TCR receptor and increased cell division. Of note, the Notch regulates c-myc in normal thymocytes expression of Notch1 and downstream Notch target In addition to its role in T-ALL, Notch1 is required for genes such as hes1 and deltex1 falls dramatically as cells induction of T cell differentiation and subsequent nor- progress from the DN3a to DN3b, and in keeping with 2102 GENES & DEVELOPMENT Downloaded from genesdev.cshlp.org on November 4, 2021 - Published by Cold Spring Harbor Laboratory Press Notch1 induces c-Myc in T-ALL regulates c-myc directly, rather than through a Notch1- dependent intermediate. c-Myc is a particularly attractive target to explain the progrowth effects of Notch1 in transformed T-cell pro- genitors. c-Myc drives cell cycle progression and regu- lates the expression of key enzymes that control cellular metabolism (for review, see Grandori et al. 2000; Levens 2003), and stimulates ribosome biogenesis and protein synthesis through interactions with RNA polymerase III (Felton-Edkins et al. 2003) and RNA polymerase I (Arabi et al. 2005; Grandori et al. 2005; Grewal et al. 2005). c-Myc also controls cell size (Johnston et al. 1999; Schuhmacher et al. 1999), presumably through its ability to stimulate protein synthesis (Oskarsson and Trumpp 2005). These functional attributes of c-myc are consis- tent with the growth arrest and decrease in cell size that are observed upon withdrawal of Notch signals from T- Figure 6. Notch signaling up-regulates c-myc in normal thy- ALL cells. mocytes at the DN3 stage. Sorted DN3 thymocytes from three to five mice were incubated for 16 h on OP9 or OP9-DLL1 feeder cells in the presence of the GSI compound E (1 µM) or DMSO Implications for prior studies linking Notch and c-myc vehicle. Murine T6E T-ALL cells cultivated in the presence or Retroviral mutagenesis studies conducted with c-myc absence of GSI for 24 h served as positive and negative controls. c-Myc transcript levels were determined by qPCR in these cells (MMTV /myc) transgenic mice showed that a high fre- and in freshly sorted DN3a and DN3b thymocytes. Expression quency of T-ALLs occurring with shortened latencies of c-myc was determined in three independent experiments. had proviral integrations into Notch1 (Girard et al. 1996; Mean expression levels ± 1 SD are shown. Hoemann et al. 2000). The simplest explanation for this observation (that c-myc and Notch1 act through inde- pendent, complementary pathways to promote pre-T- this, DN3a cells are dependent on Notch signals for cell transformation) is at odds with our functional data, growth and survival, whereas DN3b cells are not which places c-myc downstream of Notch1. (Taghon et al. 2006). How might these seemingly conflicting data be Based on these observations, we performed experi- brought into register? One explanation lies in the differ- ments to determine if Notch signaling affects c-myc ex- ent contexts in which the experiments were performed. pression in DN3 thymocytes. Sorted DN3 cells were co- In vivo models permit detection of oncogenic comple- cultured on OP9 cells or OP9 cells expressing the Notch mentation at different stages of tumor development and ligand dll1, which are capable of directing T-cell devel- progression, whereas our experiments are focused only opment from progenitors ex vivo (Schmitt et al. 2004). on growth maintenance in vitro. In mice, retroviral in- DN3 cells cultivated on OP9-DLL1 cells had higher ex- sertions into Notch1 in multipotent bone marrow pro- pression of c-myc than DN3 cells grown on control OP9 genitors are predicted to expand the pool of pre-T cells cells, and this difference was abrogated by a GSI (Fig. 6). and thereby increase the likelihood that some cell Further, freshly isolated DN3a cells had significantly within this pool will acquire other rate-limiting onco- higher expression of c-myc than did DN3b cells. Thus, genic aberrations. This ability to expand the pool of “at- Notch signaling correlates with c-myc expression at a risk” cells may underlie Notch1’s remarkable capacity critical stage of normal  T-cell development. to complement many (if not all) of the other genetic le- sions implicated in human (Weng et al. 2004) and murine T-ALL (Girard et al. 1996; Feldman et al. 2000; Dumor- tier et al. 2006; Lin et al. 2006; O’Neil et al. 2006). Much Discussion less commonly, retroviral insertions into c-myc occur in ic T-ALLs arising in lck-activated-Notch1 (lck-N ) trans- c-Myc is a Notch1 target in T-ALL cells genic mice (Beverly and Capobianco 2003). Although c- ic Although the Notch signaling pathway is best known for myc should already be up-regulated in lck-N mice, su- its ability to influence differentiation, inappropriate praphysiologic expression of c-myc due to proviral inser- gains in Notch function can have profound effects on tion may confer an additional selective advantage. This growth in specific contexts (Berry et al. 1997; Weng et al. idea is in line with studies suggesting that tumor latency 2003, 2004). Using genomic, biochemical, and functional is shortened in homozygous transgenic c-myc mice rela- approaches, we identified c-myc as an important media- tive to heterozygous animals (Sidman et al. 1993). Fi- tor of Notch’s progrowth effects in T-ALL cells. Impor- nally, the failure of c-myc to rescue all Notch-dependent tantly, the ability of Notch1 to stimulate c-myc tran- cell lines clearly points to the existence of other Notch1 scription in the presence of the protein synthesis inhibi- target genes that contribute to growth and survival, and tor cycloheximide is persuasive evidence that it which also likely provide a selective advantage to tu- GENES & DEVELOPMENT 2103 Downloaded from genesdev.cshlp.org on November 4, 2021 - Published by Cold Spring Harbor Laboratory Press Weng et al. mors in MMTV /myc transgenic mice bearing Notch1- binding site for Ikaros (Molnar and Georgopoulos 1994), proviral insertions. It will be important to define the mo- a transcriptional repressor that regulates pre-T-cell de- lecular basis for the variation in c-myc dependency among velopment (Georgopoulos et al. 1994; Winandy et al. cell lines, and to identify other genes downstream of 1999). Ikaros loss-of-function is associated with T-ALL Notch1 that contribute to T-ALL cell growth and survival. development (Winandy et al. 1995), and it has been hy- pothesized that Ikaros and CSL compete for access to the promoter sequences of genes that determine T-ALL de- Importance of Notch/myc interaction in normal velopment in mouse models (Beverly and Capobianco and neoplastic pre-T cells 2003); c-myc could be one such gene. Conversely, un- known cis-acting factors could enhance site-occupancy T-ALLs appear to resemble various stages of pre-T-cell by CSL and/or transcriptional activation by the CSL/ development (Ferrando et al. 2002; Asnafi et al. 2003), ICN1/MAML ternary complex. Recent work has identi- during which Notch signaling outcomes may differ dra- fied cis-acting elements that contribute to the activation matically. Notch1 is required for both T-lineage specifi- of a number of Notch-responsive elements (Cave et al. cation from multipotent progenitors (Pui et al. 1999; 2005; Ong et al. 2006), providing support for this model. Radtke et al. 1999; Sambandam et al. 2005) and subse- quent pre-T-cell development up to and including the -selection checkpoint (Wolfer et al. 2002; Tanigaki et Summary and implications al. 2004; Ciofani and Zuniga-Pflucker 2005), which oc- The existence of a direct link between Notch and c-myc curs during the maturation of “Notch-high” DN3a to in T-ALL cell lines and normal thymocytes has thera- “Notch-low” DN3b cells (Taghon et al. 2006). Of note, peutic as well as basic implications. Most mutated normal -selection is accompanied by an expansion of Notch1 receptors found in T-ALL cells depend on T-cell progenitors that requires both Notch1 and c-myc -secretase to transduce signals (Weng et al. 2004; (Douglas et al. 2001; Iritani et al. 2002). Our ex vivo Malecki et al. 2006), and the withdrawal of c-myc trans- studies show that Notch signals contribute to c-myc ex- gene expression “cures” ∼50% of mice with T-ALL pression in DN3a cells, and that there is a precipitous (Felsher and Bishop 1999). Recognition that the primary drop in c-myc expression in post--selection DN3b thy- effect of Notch1 signals in T-ALL cells appears to be on mocytes, which also down-regulate pre-T expression. proliferation and metabolism, rather than differentiation Of interest, Notch-associated T-ALLs are often associ- or survival, point to rational combinations of Notch ated with persistent expression of pre-T (see Fig. 1A; pathway inhibitors and other therapeutic agents. This Bellavia et al. 2000), which has been taken as evidence could take the form of agents that also impinge on pro- that these cells are arrested at a stage resembling normal tein synthesis through independent mechanisms, or -selection. Together, these findings suggest that the drugs that target parallel pathways, such as those that Notch/c-myc signaling axis found in Notch-dependent regulate cell survival directly. T-ALL cells reflects the persistence of a stage-specific relationship that is involved in the development of nor- mal T cells. Materials and methods Cell lines How are Notch1 targets restricted to specific contexts? All lymphoid lines were grown in RPMI 1640 (Invitrogen) supplemented with 10% fetal bovine serum (Hyclone), 2 mM The outcome of Notch signaling varies dramatically L-glutamine, 1 mM sodium pyruvate, and antibiotics. 293T from context to context. One probable basis for these cells were maintained in Dulbecco’s modified Eagle medium different outcomes is the existence of context-specific (DMEM) (Invitrogen) with the same supplements except so- target genes, such as pre-T (Deftos et al. 2000; Reizis dium pyruvate. Cells were grown at 37°C under 5% CO . The and Leder 2002). Since physiologic Notch signals do not T6E murine cell line has been described previously (Pear et al. generally lead to increased growth, it seems likely that 1996). 8946 is a murine T-ALL cell line derived from a tumor c-myc will also prove to be a context-specific target. created with a tetracycline-dependent human c-myc transgene How might this be achieved? Our data suggest that (Felsher and Bishop 1999). c-myc is targeted by Notch1 in T-ALL cells via a con- served site (TTGGGAA) located within the 5 c-myc pro- Plasmids and probes moter region immediately upstream of the TATA box cDNAs encoding murine and human c-myc, murine Mad1, HA- that was shown to contain a CSL-responsive element tagged A-Max (kind gift of Dr. Charles Vinson, NCI, Bethesda, previously (Satoh et al. 2004). This sequence differs from MD), and Flag-tagged cyclin D3 (kind gift of Dr. Alan Diehl, the canonical CSL recognition sequence YGTGRGAA University of Pennsylvania, Philadelphia, PA) were subcloned (Tun et al. 1994; Nellesen et al. 1999), and binds CSL into MigR1, a retroviral vector containing an internal ribosomal relatively weakly, which may have a role in restricting entry site (IRES) and GFP marker (Pui et al. 1999). The retroviral CSL occupancy to particular contexts. Other factors may constructs MigRI–ICN1 and MigRI–MAML1(13–74)-GFP have further modulate occupancy positively and negatively. been described (Weng et al. 2003). All constructs were se- In this regard, the putative CSL-binding site in the c-myc quenced and tested for expression and followed by detection on promoter is a high similarity match to the consensus Western blots stained with either anti-Mad1 (#4682, Cell Sig- 2104 GENES & DEVELOPMENT Downloaded from genesdev.cshlp.org on November 4, 2021 - Published by Cold Spring Harbor Laboratory Press Notch1 induces c-Myc in T-ALL naling), anti-HA (clone 6E2, Cell Signaling) to detect the A-Max conditions, then exposed to Kodak BioMax MS autoradiography construct, or anti-Flag (#2368, Cell Signaling) to detect the cy- film with intensifying screens. clin D3 construct (#2368, Cell Signaling). For the experiments in which ICN was cotransduced with either Mig A-Max or Mig Quantitative RT–PCR (qRT–PCR) Mad1, ICN1 was expressed from a MSCV-based retroviral vec- tor that coexpresses the truncated nerve growth factor receptor Oligo-d(T)-primed total RNAs (2 µg per cell line sample and 0.5 (tNGFR) as a surrogate marker (Tu et al. 2005). This approach µg for thymocytes) were reverse-transcribed with SuperScript II allows detection of GFP in the FL1 channel and tNGFR in the (Invitrogen). For cell line samples, an appropriate dilution of FL2 channel using a NGFR antibody (Pharmingen). The c-myc cDNA and gene-specific primers were combined with iQ SYBR and 18S rRNA probes used for Northern blot hybridization were Green Supermix (Bio-Rad) and amplified in an iCycler iQ real- generated by random hexamer priming of a ∼2.2-kb human c- time PCR machine (Bio-Rad). All qPCR reactions were per- myc cDNA (MGC-5183) and a 644-bp PCR product spanning formed in triplicate. Ct (threshold cycle number) and expression residues 548–1191 of GenBank accession M10098, respectively. values with standard deviations were calculated using the Gene Expression Macro for Excel (Bio-Rad). Primer sequences for real- time PCRs were as follows: c-myc forward, 5-CTTCTCTCC Retroviral transduction and GSI rescue GTCCTCGGATTCT-3; c-myc reverse, 5-GAAGGTGATCC Production of high-titer ecotropic (Pear et al. 1996) and pseudo- AGACTCTGACCTT-3; -actin forward, 5-CGCGAGAAGA typed (Weng et al. 2003) retroviruses and transduction of target TGACCCAGAT-3; -actin reverse, 5-GATAGCACAGCCT cells have been described. For the GSI rescue experiments, GGATAGCAAC-3. c-Myc and -actin primers were used at 5 5 5 × 10 to 7.5 × 10 cells were centrifuged with the appropriate 0.25 µM final and exhibited PCR efficiencies of 95.7% and amount of viral supernatant and 4 µg/mL hexadimethidrine bro- 92.9%, respectively. Real-time amplification was performed mide (Sigma) for 50 min at 2500 rpm (day −2). Transduction with initial denaturation at 95°C for 3 min, followed by 40 efficiency was measured 48 h post-infection (day 0) by GFP fluo- cycles of two-step amplification (95°C for 15 sec, 65°C for 1 rescence on a FACScan flow cytometer (Becton Dickinson). Af- min). For thymocyte samples, real-time RT–PCR was performed ter assessing GFP percentage, cells were immediately counted with TaqMan universal PCR Master Mix (Applied Biosystems) and prepared for treatment with GSI (1 µM compound E, which and analyzed on the ABI Prism 7900 (Applied Biosystems). was synthesized in the laboratory of M.S. Wolfe). Changes in the Notch1, c-myc, and deltex1 Taqman primers were obtained percentage of GFP cells and other cellular parameters were from Applied Biosystems. assessed with a FACScan flow cytometer and total cell number was obtained using a hemocytometer. The number of GFP cells DNA content/flow cytometric analysis was normalized to the number of GFP cells at day 0 to correct for differences in the initial retroviral transduction. Average cell Live or fixed cells were stained for DNA content with Hoechst counts and standard deviations were calculated for each experi- 33342 (4 µM, Sigma B2261) or propidium iodide (40 µg/mL), ment (carried out in triplicate), and each experiment was carried respectively. Flow cytometry was performed using an Influx out at least twice. Where appropriate, a Student’s t-test was Analyzer (Cytopeia) equipped with 488 nm and UV lasers to used to assess statistical significance. measure GFP and Hoechst 33342 in live cells, while FACSCali- bur cytometers (BD Biosciences) were used to measure prop- idium iodide in fixed cells or GFP in live cells. Data analysis Expression profiling/analysis was performed using FlowJo software (Treestar). Total RNA prepared using Trizol (Invitrogen) was used to gen- erate labeled cRNA as described (Shipp et al. 2002). cRNAs were Chromatin immunoprecipitation (ChIP) assay hybridized/scanned on AffyMetrix U74Av2 and/or Expression Set 430 A/B, chips, and raw fluorescence data were analyzed ChIP was performed using ChIP assay kits (Upstate Biotechnol- using dChip software. GSI-treated cells were cultured in the ogy). T6E cells were treated with DMSO or GSI (1 µM Com- presence of 1.0 µM compound E for 3, 6, 24, or 72 h. Mock- pound E) for 24 h prior to fixation. Cells were fixed with 1% treated cells were cultured in the presence of DMSO vehicle paraformaldehyde at room temperature for 10 min, washed, and (0.01% final) for 120 h. DN-MAML1 transduced cells were lysed with SDS lysis buffer (50 mM Tris-HCl, 1% SDS, 10 mM sorted at 72 h following exposure to retrovirus to purities of EDTA, protease inhibitor cocktail, Sigma). The lysates were + + 87% GFP (DN-MamA) and 98% GFP (DN-MamB); GFP-only sonicated to reduce DNA lengths to between 200 and 600 bp. controls were sorted to purities of 95% GFP (GFPposA) and The soluble fraction was diluted, precleared with salmon sperm 98% GFP (GFPposB); GFP-negative controls were sorted from DNA/protein A-agarose, then divided into two tubes and incu- cells exposed to, but not transduced by, DN-Mam retroviruses bated with 5 µL of antiserum specific for Notch1 TAD domain − − to purities of >98% GFP (GFPnegA) and >99% GFP (GFPnegB). (Aster et al. 2000) or normal rabbit IgG. The immune complexes 6 6 Sorted cells (2 × 10 to 4 × 10 ) were harvested for RNA imme- were then precipitated with protein A-agarose and eluted with diately following sorting. Cells transduced with ICN were cul- an elution buffer (0.1 M NaHCO containing 1% SDS). The tured in the presence of 0.5 µM compound E continuously for eluted material was reverse-cross-linked and treated with pro- several weeks prior to RNA harvest (>99% GFP ). teinase K (20 µg/mL). DNA was purified using a PCR purifica- tion kit (Qiagen) and eluted by water (5 × 10 cell equivalents/ 50 µL). Quantitative PCR (qPCR) was performed using the SYBR Northern blots Green system on the ABI 7900HT Sequence Detection System Total RNAs (10 µg per sample) were run on 1.2% agarose/2.2 M (Applied Biosystems) with the following primers: (1) c-myc pro- formaldehyde gels and transferred to Hybond-XL nylon mem- moter-forward, 5-TGAGGCTCCTCCTCCTCTTTC-3; (2) branes (Amersham Biosciences) with UV cross-linking (Stra- c-myc promoter reverse, 5-GCAGACCCCCGGAATATAAA- talinker, Stratagene). Blots were hybridized with radiolabeled 3; (3) c-myc intron2 forward, 5-CACGGGACCTGAAAG P-random-primed probes at 65°C and washed under stringent GTTCT-3; (4) c-myc intron2 reverse, 5-GGGTTAGGGCAC GENES & DEVELOPMENT 2105 Downloaded from genesdev.cshlp.org on November 4, 2021 - Published by Cold Spring Harbor Laboratory Press Weng et al. AGGTGAGA-3; (5) hes1 promoter forward, 5-CGTGTCT er’s recommendations (Miltenyi Biotec). Following staining CTTCCTCCCATTG-3; (6) hes1 promoter reverse, 5-CCAG with antibodies against lineage markers (TCR, TCR, CD3, GACCAAGGAGAGAGGT-3. Primers for the hes1 promoter CD4, NK1.1, CD19, Gr-1, CD11b), CD44, and CD25 antigen −/lo hi − sequence flank two CSL-binding sites that lie just 5 of the (Pharmingen), DN3 thymocytes (CD44 CD25 Lin ) were TATA box (Jarriault et al. 1995). Each sample was indepen- purified by cell sorting on a FACS Moflo (Cytomation). DN3a −/lo hi − lo −/lo hi dently prepared at least two times and run in duplicate. The (CD44 CD25 Lin CD27 ) and DN3b (CD44 CD25 − lo relative DNA amount was calculated using the standard curve Lin CD27 ) cells were purified by sorting after staining with method as described in the ABI 7900HT Sequence Detection anti-CD27 antibody (eBioscience) as described (Taghon et al. System manual. The input DNA was defined as an aliquot of 2006). Cells were either used for RNA extraction directly, or sheared chromatin prior to immunoprecipitation, and was used were cocultured with OP9 or OP9-DLL1 stromal cells in the to normalize the sample to the amount of chromatin added to presence or absence of 1 µM compound E for 16 h as described each ChIP. (Schmitt et al. 2004). RNA was isolated from thymocytes using the RNEasy kit according to the manufacturer’s recommenda- tions (Qiagen). EMSA Oligonucleotides fluorescently 5-labeled with carboxyfluores- Statistical analysis cein (FAM) and unlabeled complementary oligonucleotides ANOVA and t-test analyses were performed using the Prism were obtained from Integrated DNA Technologies. Sequences 4.03 software package (GraphPad Software). of the FAM-labeled oligonucleotides were as follows: c-myc pro- moter, 5-FAM-CCCCTCCCGGGTTCCCAAAGCAGAGGG CGT-3; mutated c-myc promoter, 5-FAM-CCCCTCCCGGG Acknowledgments TTCAAAAAGCAGAGGGCGT-3; consensus CSL-binding site, 5-FAM-TCCAAATTTTTTCCCACGGCGTGT-3. CSL We thank Alan Diehl and Charles Vinson for providing re- and the RAM-ANK domains of Notch1 were prepared as de- agents, and Gerd Blobel, Tom Kadesch, David Levens, Steve scribed (Nam et al. 2003). Nonradioisotopic EMSAs were per- McMahon, Gary Koretzky, Craig Thompson, and members of formed by incubating 2 pmol of probes for 30 min at 30°Cin the Pear and Aster laboratories for helpful suggestions. J.M. was binding buffer (10% glycerol, 20 mM HEPES at pH 7.9, 60 mM supported by NIH Training Grant (T32 CA 09140-31-35). C.D.B. KCl, 10mM DTT, 5 mM MgCl , 250 ng dGdC, 0.2 mg/mL bo- is supported by a Long-Term Fellowship from the Human Fron- vine serum albumin) in the presence or absence of 10 µg of CSL tier Science Program Organization, and M.L.A. is supported by and RAM-ANK. Gel electrophoresis was performed in 10% na- the Foundation pour la Recherche Medicale (FRM). A.P.W., tive gels at 4°C and 180 V. Following electrophoresis, gels were D.W.F., S.C.B., W.S.P., and J.C.A. are supported by grants from immediately analyzed by blue-excitation fluorescence scanning the NIH. with a Storm 860 FluorImager (Amersham Pharmacia Biotech). To compare the affinity of CSL binding to sequences of interest, oligonucleotides with 5 overhangs were incubated with P-- References dCTP (Perkin-Elmer) and the Klenow fragment of Escherichia coli DNA polymerase I (New England Biolabs). P-labeled Amsen, D., Blander, J.M., Lee, G.R., Tanigaki, K., Honjo, T., and probes were incubated with 0–4000 ng of recombinant CSL as Flavell, R.A. 2004. Instruction of distinct CD4 T helper cell described above. Following electrophoresis, gels were exposed fates by different notch ligands on antigen-presenting cells. to PhosphorImager screens and analyzed with a Storm 860 Phos- Cell 117: 515–526. phorImager (Amersham Pharmacia Biotech). Arabi, A., Wu, S., Ridderstrale, K., Bierhoff, H., Shiue, C., Fatyol, K., Fahlen, S., Hydbring, P., Soderberg, O., Grummt, I., et al. 2005. c-Myc associates with ribosomal DNA and activates Nuclear runoff assays RNA polymerase I transcription. Nat. Cell Biol. 7: 303–310. Nuclei were prepared by hypotonic detergent lysis and centrifu- Artavanis-Tsakonas, S., Rand, M.D., and Lake, R.J. 1999. Notch gation (300 × g) from 50 × 10 cells/sample as described (Green- signaling: Cell fate control and signal integration in devel- berg and Bender 1997). Runoff reactions were performed with opment. Science 284: 770–776. - P UTP (Perkin-Elmer), followed by DNase I (500 U/sample, Asnafi, V., Beldjord, K., Boulanger, E., Comba, B., Le Tutour, P., Invitrogen) and proteinase K digestion. RNA was isolated using Estienne, M.H., Davi, F., Landman-Parker, J., Quartier, P., RNeasy Mini columns (Qiagen). A Minifold II appartus (Schlei- Buzyn, A., et al. 2003. Analysis of TCR, pT , and RAG-1 in cher & Schuell BioScience) was used to transfer 5 µg of linear- T-acute lymphoblastic leukemias improves understanding ized, NaOH-denatured plasmid DNA per slot onto positively of early human T-lymphoid lineage commitment. Blood charged nylon membranes (Hybond-XL, Amersham Biosci- 101: 2693–2703. ences). Following UV cross-linking (70,000 µJ/cm ; UV Stra- Aster, J.C., Xu, L., Karnell, F.G., Patriub, V., Pui, J.C., and Pear, talinker 2400, Stratagene), 5 × 10 cpm of each runoff RNA W.S. 2000. 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Wolfer, A., Wilson, A., Nemir, M., MacDonald, H.R., and Radtke, F. 2002. Inactivation of Notch1 impairs VDJ rear- rangement and allows pre-TCR-independent survival of early  Lineage Thymocytes. Immunity 16: 869–879. Wu, L., Aster, J.C., Blacklow, S.C., Lake, R., Artavanis-Tsako- nas, S., and Griffin, J.D. 2000. MAML1, a human homologue of Drosophila mastermind, is a transcriptional co-activator for NOTCH receptors. Nat. Genet. 26: 484–489. Ye, Y., Lukinova, N., and Fortini, M.E. 1999. Neurogenic phe- notypes and altered Notch processing in Drosophila Prese- nilin mutants. Nature 398: 525–529. GENES & DEVELOPMENT 2109 Erratum Genes & Development 20: 2096–2109 (2006) Andrew P. Weng, John M. Millholland, Yumi Yashiro-Ohtani, Marie Laure Arcangeli, Arthur Lau, Carol Wai, Cristina del Bianco, Carlos G. Rodriguez, Hong Sai, John Tobias, Yueming Li, Michael S. Wolfe, Cathy Shachaf, Dean Felsher, Stephen C. Blacklow, Warren S. Pear, and Jon C. Aster During a recent internal review of the data in the above-mentioned paper, the authors identified an inadvertent mistake in Figure 6 that they would like to correct. Figure 6 shows changes in c-myc expression in developing thymocytes. Within Figure 6, the authors included data showing that c-myc is reduced as cells progress from the DN3a to DN3b stage of thymocyte development. In assembling these two data points, they inadvertently substituted the Notch1 positive control data for the c-myc data. As shown in the revised figure below, the reduction in c-myc expression between DN3a and DN3b is closer to twofold rather than the fourfold difference shown in the published figure. This change is roughly equivalent to the reductions in c-myc expression that are produced in malignant T6E cells or normal DN3 thymocytes by withdrawal of Notch signals. The authors have also included the Notch1 expression data in the revised figure, as this was the positive control used by Rothenberg and coworkers (Fig. 6 in Taghon et al. 2006) to ascertain the purity of the DN3a and DN3b populations. Importantly, the revised figure fully supports the conclusions stated in the above-mentioned paper. However, the authors believe an Erratum is in order, both to correct their error for the record and to accurately depict the changes in c-myc expression that accompany transition from the DN3a to DN3b stage of thymocyte development. The authors apologize for their error. Figure 6. Notch signaling up-regulates c-myc in nor- mal thymocytes at the DN3 stage. Sorted DN3 thymo- cytes from three to five 4- to 6-wk-old B6 mice were incubated for 16 h on OP9 or OP9-DL1 feeder cells in the presence of the GSI compound E (1 µM) or DMSO vehicle. T6E T-ALL cells cultivated in the presence or absence of GSI for 24 h served as positive and negative controls. c-Myc transcript levels were determined by qPCR in these cells and in freshly sorted DN3a and DN3b thymocytes. Expression of notch1 served as the positive control for DN3a and DN3b thymocytes. Ex- pression of c-myc and notch1 was determined in three independent experiments. Mean expression levels ± 1 SD are shown. Reference Taghon, T., Yui, M.A., Pant, R., Diamond, R.A., and Rothen- berg, E.V. 2006. Developmental and molecular characteriza- tion of emerging - and -selected pre-T cells in the adult mouse thymus. Immunity 24: 53–64. GENES & DEVELOPMENT 21:625 © 2007 by Cold Spring Harbor Laboratory Press ISSN 0890-9369/07; www.genesdev.org 625 Downloaded from genesdev.cshlp.org on November 4, 2021 - Published by Cold Spring Harbor Laboratory Press c-Myc is an important direct target of Notch1 in T-cell acute lymphoblastic leukemia/lymphoma Andrew P. Weng, John M. Millholland, Yumi Yashiro-Ohtani, et al. Genes Dev. 2006, 20: Access the most recent version at doi:10.1101/gad.1450406 http://genesdev.cshlp.org/content/suppl/2006/07/14/gad.1450406.DC1 Supplemental Material Erratum Related Content Genes Dev. March , 2007 21: 625 This article cites 81 articles, 30 of which can be accessed free at: References http://genesdev.cshlp.org/content/20/15/2096.full.html#ref-list-1 Articles cited in: http://genesdev.cshlp.org/content/20/15/2096.full.html#related-urls License Receive free email alerts when new articles cite this article - sign up in the box at the top Email Alerting right corner of the article or click here. Service Copyright © 2006, Cold Spring Harbor Laboratory Press

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