High-Level Expression of Mastermind-Like 2 Contributes to Aberrant Activation of the NOTCH Signaling Pathway in Human Lymphomas

Oncogene (2011) 30, 1831–1840 & 2011 Macmillan Publishers Limited All rights reserved 0950-9232/11 www.nature.com/onc ORIGINAL ARTICLE High-level expression of Mastermind-like 2 contributes to aberrant activation of the NOTCH signaling pathway in human lymphomas

KKo¨chert1,2, K Ullrich1,2, S Kreher1,2, JC Aster3, M Kitagawa4,KJo¨hrens5, I Anagnostopoulos5, F Jundt1,2, B Lamprecht1,2, U Zimber-Strobl6, H Stein3, M Janz1,2,BDo¨rken1,2 and S Mathas1,2

1Max-Delbru¨ck-Center for Molecular Medicine, Berlin, Germany; 2Hematology, Oncology and Tumorimmunology, Charite´ University Medical School, CVK, Berlin, Germany; 3Department of Pathology, Brigham and Women’s Hospital, Boston, MA, USA; 4Department of Molecular and Tumor Pathology, Chiba University Graduate School of Medicine, Chiba, Japan; 5Institute of Pathology, Charite´ University Medical School, CBF, Berlin, Germany and 6Department of Gene Vectors, Helmholtz Center Munich, German Research Center for Environmental Health, Munich, Germany

Inappropriate activation of the NOTCH signaling path- In Mammalia there are four NOTCH receptors that way, for example, by activating mutations, contributes to interact with a still growing list of ligands (Kopan the pathogenesis of various human malignancies. Here, and Ilagan, 2009). Upon ligand binding, cleavage of we demonstrate that aberrant expression of an essential the NOTCH receptor produces a membrane-anchored NOTCH coactivator of the Mastermind-like (MAML) form of NOTCH termed NOTCH transmembrane family provides an alternative mechanism to activate (NOTCHTM). NOTCHTM, in turn is cleaved by the NOTCH signaling in human lymphoma cells. We detected g-secretase membrane protease complex, releasing in- high-level MAML2 expression in several B cell-derived tracellular NOTCH (ICN), which then translocates to lymphoma types, including classical Hodgkin lymphoma the nucleus and activates transcription. (cHL) cells, relative to normal B cells. Inhibition of ICN does not directly bind to DNA, but interacts MAML-protein activity by a dominant negative form of with the enhancer-binding protein member CSL (CBF1, MAML or by small hairpin RNAs targeting MAML2 in Suppressor of Hairless, LAG-1) (Kopan and Ilagan, cHL cells resulted in downregulation of the NOTCH 2009). In the absence of ICN, CSL binds to DNA in target genes HES7 and HEY1, which we identified as a sequence-dependent manner and recruits various overexpressed in cHL cells, and in reduced proliferation. corepressor molecules. Interaction of ICN with CSL Furthermore, a NOTCH gene-expression signature in converts CSL from a transcriptional repressor to an cHL cells confirmed their cell-autonomous NOTCH activator (Lubman et al., 2007) by creating a dual ICN/ activity. Finally, in line with the essential role of MAML CSL binding interface for transcriptional coactivators proteins for assembly and activity of the NOTCH of the Mastermind-like (MAML) family (Fryer et al., transcriptional complex (NTC), we show that MAML- 2002; Jeffries et al., 2002; Del Bianco et al., 2008). derived small-peptide constructs block NOTCH activity Three MAML members exist (MAML1, MAML2 and and disrupt NTC formation in vitro. These data strongly MAML3) (Lin et al., 2002; Wu et al., 2000, 2002), all of suggest direct targeting of the NTC as treatment strategy which contain an N-terminal region of approximately 60 for NOTCH-dependent malignancies. amino acids (AA) that binds an extended groove formed Oncogene (2011) 30, 1831–1840; doi:10.1038/onc.2010.544; by CSL and the ankyrin–domain of ICN (Nam et al., published online 29 November 2010 2006; Wilson and Kovall, 2006). Binding of MAML proteins to the ICN–CSL complex is essential for Keywords: Mastermind-like; NOTCH; lymphoma; Hodgkin NOTCH-mediated transcriptional activation (Fryer et al., 2002; Nam et al., 2003, 2006; Maillard et al., 2004). The mechanisms by which MAML proteins regulate the fine-tuning of NOTCH signaling remain to Introduction be elucidated. However, several lines of evidence, for example, the (11;19)(q21;p13) translocation found in Numerous studies revealed a fundamental role of the mucoepidermoid carcinoma that creates a MAML2 NOTCH signaling pathway for key processes such as fusion protein and activates NOTCH target genes cellular differentiation, growth and apoptosis regulation (Tonon et al., 2003), point to the oncogenic potential of (Koch and Radtke, 2007; Kopan and Ilagan, 2009). MAML dysregulation. Deregulated NOTCH signaling results in pathological disorders, including developmental syndromes and various Correspondence: Dr S Mathas, Max-Delbru¨ck-Center for Molecular malignancies (Grabher et al., 2006; Koch and Radtke, Medicine and Charite´ University Medical School, Hematology/ 2007). Different mechanisms leading to aberrant NOTCH Oncology, Robert-Ro¨ssle-Str. 10, Berlin D-13125, Germany. E-mail: [email protected] activation have been unraveled so far, including activating Received 7 April 2010; revised 28 September 2010; accepted 20 October mutations of the NOTCH1 receptor itself (Weng et al., 2010; published online 29 November 2010 2004; Grabher et al., 2006) as well as alterations in proteins NOTCH activation by high MAML2 expression KKo¨chert et al 1832 that control ICN turnover (O’Neil et al., 2007). Such various cell lines. These data were confirmed at the defects are found in more than 50% of childhood and protein level (Figure 1b). In contrast, MAML2 mRNA adult T-cell acute lymphoblastic leukemia (T-ALLs). was abundant in all HRS cell lines, whereas except for We and others have previously shown that aberrant Reh and BL-60 cells, it was undetectable in the other NOTCH activity is involved in the pathogenesis of non-Hodgkin lymphoma cell lines (Figure 1b, upper human B cell-derived malignancies, including multiple panel). MAML2 protein expression correlated with myeloma, B-cell chronic lymphocytic leukemia, diffuse the mRNA expression data (Figure 1b, lower panel). large B-cell lymphoma and the malignant Hodgkin–/ As only few data regarding MAML expression in Reed–Sternberg (HRS) cells of classical Hodgkin lym- human lymphoid cells were available (Lin et al., 2002; phoma (cHL) (Kapp et al., 1999; Jundt et al., 2002, Wu et al., 2002), we analyzed MAML mRNA and 2004, 2008; Lee et al., 2009; Rosati et al., 2009; Stanelle protein expression in purified CD19 þ B cells and CD3 þ et al., 2010). Given the important function of MAML T cells (Figure 1c). Robust expression of MAML1 and coactivators for NOTCH activation (Wu et al., 2002) MAML3 mRNA was found in all lymphoid samples and for the development of distinct B cell subsets (Figure 1c, upper panel). In contrast, MAML2 was (Oyama et al., 2007; Wu et al., 2007), we analyzed the strongly expressed only in CD3 þ T cells, whereas it contribution of MAML to the deregulated NOTCH was hardly detectable in CD19 þ B cells, which was activity in human B cell-derived lymphomas. confirmed at protein level (Figure 1c, lower panel). We concluded that MAML2 is expressed at an aberrantly high level in several B cell-derived lymphoma cell lines, most commonly in those of cHL origin. Results

NOTCH1TM and cleaved ICN1 in human B cell-derived Aberrant MAML2 expression in distinct primary lymphoma cell lines lymphoma entities We first analyzed the expression levels of NOTCH1TM Next, we analyzed MAML2 expression in non-neoplas- and ICN1 in various cell lines by use of antibodies tic lymphoid tissue and 180 B cell-derived primary recognizing these different forms of NOTCH1 human lymphoma cases by immunohistochemistry (Figure 1a). As previously published (Jundt et al., (Figure 1d and Table 1). In contrast to normal tonsillar 2002), HRS cell lines (L428, L1236, KM-H2, L591, B cells, high MAML2 expression was detectable in HDLM-2, L540 and L540Cy) showed a prominent malignant cells of various primary lymphomas, in parti- expression of NOTCH1TM compared with most other cular, in all analyzed B lymphoblastic leukemias (8/8), non-Hodgkin lymphoma cell lines (Reh, Namalwa, 40% of mantle cell lymphoma cases (4/10), approxi- BL-60, BJAB and SU-DHL-4). However, although mately half of the marginal zone lymphoma cases and in ICN1 was detectable at variable levels in the cell lines the HRS cells of half of the cHL cases (30/61). Overall, (Figure 1a), ICN1 protein levels were not correlated these data revealed MAML2 deregulation as a common with NOTCH1TM protein levels (compare NOTCH1TM defect in B cell-derived lymphomas. Given the robust and ICN1 in, for example, L428, L1236 or BJAB cells). expression of MAML2 in the HRS cell lines (Figure 1), Furthermore, the ICN1 protein level did not predict the we investigated the function of MAML2 in these cell functional activity of the NOTCH transcriptional lines in more detail. complex (NTC), as the NTC was active in L428 cells with a low level of ICN1 but apparently inactive in MAML2 drives NOTCH-mediated transcriptional et al. BJAB cells (see also Figures 2–3; Weng , 2003; activation in lymphoid-derived cells and regulates the Jundt et al., 2002, 2008; Moellering et al., 2009). We NOTCH target genes HES7 and HEY1 in cHL cells hypothesized that altered expression or activity of NTC To study the function of MAML2 in lymphoid cell lines, components other than ICN1 might explain aberrant we first assessed the potential of MAML2 to potentiate activation of the NOTCH signaling pathway in certain NOTCH transcriptional activity in such cells, as cellular contexts. We therefore decided to analyze previous analyses were primarily performed with other expression and activity of NOTCH coactivators of the cell types (Wu et al., 2000, 2002; Lin et al., 2002). To MAML family in the various cell lines in detail. establish the experimental system, we cotransfected HEK293 cells with a NOTCH-dependent promoter High-level expression of MAML2 in human B cell-derived construct, and either ICN1 or MAML2 alone or lymphomas, in particular HRS cell lines in combination (Figure 2a). As previously published To this end, messenger RNA (mRNA) and protein (Wu et al., 2002), ICN1 and MAML2 synergistically expression of the three MAML family members activated the reporter construct. To extend this model MAML1–3 were analyzed in various HRS and non- system to one that more closely resembled HRS cell Hodgkin lymphoma cell lines (Figure 1b and Supple- lines, which display putative cell-autonomous constitu- mentary Figure 1). The expression levels of MAML1 did tive NOTCH1 signaling (Jundt et al., 2008; Stanelle not show significant differences between HRS and et al., 2010), we used the lymphoid cell line SUP-T1 non-Hodgkin cell lines, and MAML3 mRNA was, (Figure 2b), which has constitutive NOTCH1 activation apart from KM-H2, Reh and BJAB cell lines with more because of a (7;9)(q34;q34.3) translocation (Ellisen prominent expression, also similarly expressed in the et al., 1991). In combination with the inherent presence

Oncogene NOTCH activation by high MAML2 expression KKo¨chert et al 1833 Hodgkin cell lines

+ #1 + #2 + #1 + #2 Cell line L428 L1236KM-H2L591 HDLM-2L540Reh NamalwaBL-60BJABSU-DHL-4293 mock293 ICN1SUP-T1 Cell line L428 NamalwaCD19 CD19- CD3 CD3 TM 120 kDa NOTCH1 MAML1 414 bp * 100 kDa MAML2 120 kDa 399 bp ICN1 ** 100 kDa MAML3 461 bp extracts RT-PCR whole cell 50 kDa β-actin GAPDH 257 bp 40 kDa

120 kDa MAML2 150 kDa ICN1 ** 100 kDa 120 kDa WB 120 kDa 50 kDa β-actin PARP1 40 kDa 100 kDa nuclear extracts

Hodgkin cell lines

Cell line L428 L1236KM-H2L591 HDLM-2L540 L540CyReh NamalwaBL-60 BJAB SU-DHL-4 MAML1 414 bp

MAML2 399 bp tonsil cHL

MAML3 461 bp RT-PCR

GAPDH 257 bp

MAML1 cHL 116 kDa B-ALL

MAML2 150 kDa

120 kDa WB

MAML3 cHL 116 kDa MCL 50 kDa β -actin 40 kDa

Figure 1 Analysis of NOTCH1TM, ICN1 and MAML expression in B cell-derived lymphoma cells. (a) Protein expression of NOTCH1TM and ICN1 in whole cell and nuclear extracts in various cell lines. Western blot (WB) analyses were performed with antibodies against total NOTCH1 (*; clone mN1A) or cleaved NOTCH1 (**; antibody specific for ICN1 created by cleavage between Gly1743 and Val1744). Extracts of HEK293 cells transfected with N-terminal FLAG-tagged ICN1 (293 ICN1) and of SUP-T1 cells served as controls. Note, that clone mN1A recognizes NOTCH1TM but not endogenous ICN1 in SUP-T1 cells, whereas the Val1744- specific antibody recognizes endogenous ICN1 in SUP-T1 cells but does not react with ectopically expressed Flag-tagged ICN1 in HEK293 cells, in which the ICN1 N-terminus is masked, nor with NOTCH1TM, confirming antibody specificity. b-actin and PARP1 were analyzed as controls. In case of the HEK293 control extracts only 2.5 mg protein were used. (b) Upper panel, analysis of MAML1-3 mRNA expression by RT–PCR. Lower panel, analysis of MAML1-3 protein expression by WB. GAPDH and b-actin were analyzed as controls. (c) Upper panel, analysis of MAML1-3 mRNA expression by RT–PCR in the L428 Hodgkin and Namalwa non-Hodgkin cell lines as well as in purified tonsillar CD19 þ B (CD19 þ #1 and #2) and CD3 þ T (CD3 þ #1 and #2) cells. Lower panel, analysis of MAML2 by WB in various cell types, as indicated. GAPDH and b-actin were analyzed as controls. (d) Representative immunohistochemistry of MAML2 in a germinal center of a normal human palatine tonsil (some dendritic cells and macrophages stain positive whereas all B cells are not stained), one case of B-ALL and MCL, respectively (note that staining is predominantly nuclear, but also cytoplasmic), and three cHL cases (some of the malignant HRS cells are marked by arrows; note, that the non-malignant small bystander cells do not express MAML2). of activated NOTCH1 in these cells (Figure 1a), ectopic To investigate the contribution of MAML2 to expression of MAML2 augmented NOTCH-dependent NOTCH activity in HRS cells, we first screened reporter activity 2.3-fold, similar to the super-induction microarray data from various cell lines for known by ectopically expressed ICN1. Ectopic expression of NOTCH target genes differentially expressed in HRS MAML2 together with ICN1 resulted in a synergistic cell lines. We identified the strongly NOTCH-dependent 5.2-fold activation of reporter activity (Figure 2b). We genes HES7 and HEY1 (Iso et al., 2003) as specifically concluded that overexpression of MAML2 is able to overexpressed in HRS compared with non-Hodgkin super-induce NOTCH activity even in cells where ICN1 lymphoma cell lines, a finding that was confirmed by is already present. reverse transcription PCR (Figure 2c). HES7 was

Oncogene NOTCH activation by high MAML2 expression KKo¨chert et al 1834 Table 1 Summary of MAML2 immunohistochemistry results of non-Hodgkin and classical Hodgkin lymphoma cases More than 90% of 50–90% of 0–49% of cells All cells Number of positive cells positive, cells positive, positive, negative, cases/number of case count case count case count case count cases examined

B lymphoblastic leukemia 7 1 0 0 8/8 B-cell chronic lymphocytic leukemia 2 0 3 2 5/7 Lymphoplasmacytic lymphoma 2 0 2 3 4/7 Plasma cell myeloma 0 0 2 6 2/8 Nodal marginal-zone lymphoma 3 0 1 6 4/10 Extranodal marginal-zone lymphoma 0 4 1 5 5/10 Follicular lymphoma (FL), grade 1 0 2 1 5 3/8 FL, grade 2 1 0 0 7 1/8 FL, grade 3a 0 0 0 8 0/8 FL, grade 3b 0 0 2 6 2/8 Mantle cell lymphoma 3 0 1 6 4/10 DLBCL 0 2 6 10 8/18 Burkitt lymphoma 0 0 1 8 1/9 Classical Hodgkin lymphoma 25 2 3 31 30/61

Abbreviation: DLBCL, diffuse large B cell lymphoma.

expressed exclusively in HRS cell lines, and HEY1 was NOTCH members, all three MAML members can strongly overexpressed. To evaluate the contribution of interact with all NOTCH members (Lin et al., 2002; MAML2 to the activation of these genes, we tested the Wu et al., 2002). Therefore, DnMAML1 will prevent effect of MAML2 knockdown. Initial attempts to do so binding of other MAML members including MAML2 by transfection of Dharmacon (Lafayette, CO, USA) into the NTC. To rule out possible general toxic effects ON-TARGETplus SMARTpool MAML2 small inter- of DnMAML1, we also investigated the B cell-derived fering RNA were confounded by weak MAML2 BJAB cell line. We observed a significant DnMAML1- knockdown (data not shown). To overcome this dependent growth inhibition of both HRS cell lines, limitation, three of the sequences from the MAML2 whereas BJAB cells were unaffected (Figure 3a). small interfering RNA SMARTpool were cloned into Similarly, specific knockdown of MAML2 resulted in the pSUPER expression vector (Brummelkamp et al., reduced [3H]-thymidine incorporation in both HRS 2002), which results in the efficient production of small cell lines but not BJAB cells (Figure 3b). We concluded hairpin RNA (shRNA) transcripts. Almost complete that cell-autonomous NOTCH signaling and aberrant knockdown of MAML2 was achieved using this MAML2 expression contribute to the proliferation of approach (Figure 2d). Significant downregulation of HRS cell lines. HES7 and HEY1 following MAML2 downregulation occurred in both HRS cell lines L428 and L591, suggesting that their expression was dependent on A NOTCH signature discriminates HRS from MAML2-mediated NOTCH activation (Figure 2e). non-Hodgkin cell lines To further substantiate a HRS-specific NOTCH activity, we hypothesized that there might be a NOTCH- MAML2-mediated NOTCH activity is essential for HRS dependent gene expression signature that separates HRS cell growth from non-Hodgkin cell lines. Based on an extensive Among other functions, NOTCH signaling regulates literature screen, the BioGRID (http://www.thebiogrid. proliferation in various cell types. To test the role of cell- org/) and KEGG PATHWAY (http://www.genome.jp/ autonomous NOTCH signaling for the growth of HRS kegg/pathway.html) databases, and inclusion of all cell lines, we transfected L428 and L591 cells with a well- probe sets present on the Affymetrix Human Genome characterized green fluorescent protein (GFP)-fused U133 Plus 2.0 array that Affymetrix NetAffx (Affyme- dominant-negative form of MAML1 described before trix, Santa Clara, CA, USA) classified under the gene (DnMAML1–pEGFP; Weng et al., 2003; Figure 3a). ontology term ‘NOTCH-signaling pathway’, we created DnMAML1–pEGFP inhibits NOTCH signaling by a gene set containing 102 genes associated with competing MAML coactivators off of the composite NOTCH signaling (see Supplementary Table 1). binding site formed upon ICN binding to CSL (Weng Intriguingly, using this set, principal component analysis et al., 2003; Nam et al., 2006). Among the different based on the microarray data resulted in a clear MAML proteins, the residues interacting with the separation of HRS from non-Hodgkin samples ankyrin domain of NOTCH and with CSL are highly (Figure 3c). Fisher’s Exact test (Po10À11) and GSEA conserved (Nam et al., 2006). Furthermore, dominant- (normalized enrichment score ¼ 1.34, FDR q-value ¼ negative mutants of MAML1, MAML2 or MAML3 0.016) confirmed significant deregulation of our can inhibit NOTCH activity (Maillard et al., 2004), and, NOTCH gene set. Furthermore, NOTCH1, NOTCH2, even though with different affinity for the different NOTCH3 and NOTCH4 expression levels were

Oncogene NOTCH activation by high MAML2 expression KKo¨chert et al 1835 7 HEK293 6 ** Hodgkin cell lines 5

4 Cell line ** L428 L1236KM-H2L591 HDLM-2L540 L540CyReh NamalwaBL-60BJABSU-DHL-4 3 HES7 451 bp

(fold induction) 2 ** HEY1 399 bp Luciferase activity

1 GAPDH 257 bp

MAML2 150 kDa transfection shControlshMAML2shControlshMAML2 ICN1 MAML2 150 kDa 100 kDa

β-actin β-actin 40 kDa 40 kDa ++++ Hes1-pGL2 L428 L591 MAML2 + + FLAG-ICN1 + + L428 shControl 1.4 6 SUP-T1 shMAML2 ** 1.2 5 1.0

GAPDH 0.8 4 ** 0.6 * 3 0.4 ** mRNA expression ** relative to 0.2 2 (fold induction)

Luciferase activity 0.0 1 L591 shControl 1.2 shMAML2 0 1.0

MAML2 0.8 ** 150 kDa GAPDH 0.6 ** 120 kDa ICN1 0.4 β mRNA expression -actin relative to 0.2 40 kDa Hes1-pGL2 ++ + + 0.0 MAML2 + + HES7 HEY1 FLAG-ICN1 + + Figure 2 MAML2-mediated super-induction of NOTCH-dependent transcription and transcriptional activation of HES7 and HEY1 in cHL cells. (a, b) HEK293 (a) and SUP-T1 cells (b) were transfected with the Hes1-pGL2 reporter construct and different combinations of expression plasmids encoding FLAG-tagged ICN1 or MAML2, as indicated. Luciferase activity was measured 48 h after transfection and is shown as fold induction compared with Hes1-pGL2 activity in control-transfected cells, which was set as 1. Data are represented as mean±s.d., P-values are stated for the comparisons of the control sample to each of the other samples. One out of six experiments is shown. Ectopic expression of the various cDNAs was verified by WB using MAML2 (HEK293 and SUP-T1), NOTCH1 (clone mN1A; HEK293) and FLAG antibody (detection of ICN1 in SUP-T1). b-actin was analyzed as control. (c) Analysis of HES7 and HEY1 mRNA expression by RT–PCR. GAPDH was analyzed as control. (d) MAML2 knockdown with shMAML2 constructs. The HRS cell lines L428 and L591 were transfected with either control shRNA (shControl) or shMAML2 constructs (shMAML2). After enrichment of transfected cells, MAML2 protein expression was analyzed by WB. b-actin was analyzed as a control. (e) HES7 and HEY1 mRNA expression depend on MAML2. L428 and L591 HRS cell lines were transfected as described in (d). Following purification of transfected cells, expression of HES7 and HEY1 mRNA was assessed by real-time PCR and calculated using the 2ÀDDCt method. Error bars denote 95% confidence intervals. One out of three experiments is shown. *Po0.05; **Po0.001. s.d., standard deviation.

analyzed at the mRNA level by quantitative real-time analyses demonstrated high-level NOTCH2 (in accor- PCR (Supplementary Figure 2A) and at the protein level dance with Kapp et al., 1999; Jundt et al., 2002) and by western blotting (Figure 1a and Supplementary JAG2 expression as a common feature of HRS cell lines Figures 2B–2D), and expression levels of the NOTCH in comparison with non-Hodgkin cell lines. These data ligands delta-like 1, delta-like 3, delta-like 4, jagged 1 suggested that at least NOTCH2 and JAG2 contribute and jagged 2 were analyzed by quantitative real-time to the cell-autonomous NOTCH activation in HRS PCR (Supplementary Figures 3A and 3B). These cell lines.

Oncogene NOTCH activation by high MAML2 expression KKo¨chert et al 1836 Short peptide constructs based on AAs 13–74 of MAML1 important in stable assembly of the NTC could be used inhibit NOTCH-mediated transcriptional activation as basis for the development of inhibitory peptides. The Our data suggested that disruption of the NTC by energetic characteristics of the NTC have been exten- inhibition of MAML coactivators might be a promising sively studied (Nam et al., 2006; Wilson and Kovall, strategy to block aberrant NOTCH activity. The 2006; Del Bianco et al., 2008). These studies revealed principle of such a strategy has been shown by use of that for NTC assembly the energetically most important DnMAML1 consisting of AAs 13–74 of MAML1 AA residues of MAML1 are R22, R25, R26, C30, R31 (Weng et al., 2003; Nam et al., 2006). We reasoned that and H34. On the basis of this knowledge, we constructed even shorter peptides containing AA residues most two peptide–GFP fusion constructs. Variant 1 encom- passes the MAML1 AA 13–50 and variant 2 AA 19–35 (Figure 4a). These constructs were tested in reporter 160000 assays in the T-ALL cell line SUP-T1 (Figure 4b). pEGFP Although inhibition of NOTCH-dependent reporter 140000 DnMAML1-pEGFP activity was not as pronounced as with the established DnMAML1 construct, DnMAML1 AA 13–50 dimin- 120000 ished reporter activity up to 77% and DnMAML1 AA ** 100000 NS 19–35 achieved a reduction of up to 45% (Figure 4b). To further substantiate these ﬁndings, we assessed 80000 the ability of the DnMAML1 constructs to disturb formation of the native NTC. We performed electro- 60000 phoretic mobility shift assays of nuclear extracts 40000 from HEK293 cells transfected with different combi- * nations of NTC components and the DnMAML1

H]-thymidine incorporation (cpm) 20000 3

[ constructs. Conﬁrming (Wu et al., 2000), ectopic 0 expression of MAML1, ICN1 and CSL sufﬁces for L428 L591 BJAB stable formation of the NTC (Figure 4c, lane 6). The DnMAML1 AA 13–74 construct forms a stable com- 80000 shControl plex and inhibits formation of the native NTC (Figure 4c, lane 7; Weng et al., 2003). Interestingly, 70000 shMAML2 NS the DnMAML1 AA 13–50 (Figure 4c, lane 8) and 60000 DnMAML1 AA 19–35 (Figure 4c, lane 9) constructs also prevented formation of a stable NTC, most likely 50000 by prevention of MAML binding to the ICN1/CSL * 40000 binding groove and destabilization of the complex. These results were in agreement with a recently 30000 published study (Moellering et al., 2009). ** 20000

H]-thymidine incorporation (cpm) 10000 3 [ 0 Figure 3 Proliferation of HRS cell lines depends on MAML2; L428 L591 BJAB HRS cell lines are characterized by a NOTCH signature. (a) L428 and L591 HRS, and BJAB non-Hodgkin cell lines were transfected with control plasmid (pEGFP) or with a dominant-negative MAML1 construct (DnMAML1–pEGFP). At 48 h after transfection, transfected GFP-positive cells were enriched and pulsed with 1 mCi [3H]-thymidine per well for a further 24 h, and [3H]-thymidine incorporation was determined. (b) L428 and L591 HRS and BJAB non-Hodgkin cell lines were transfected with control shRNA expression plasmid (shControl) or shMAML2 constructs (shMAML2). Further processing of the cells was as described in (a). (a, b), data are represented as mean±s.d. *Po0.01; PC3 **Po0.001; one out of three experiments is shown. (c) HRS cell lines show a distinct NOTCH-dependent gene expression signature. Expression data from 102 NOTCH-associated genes was extracted from microarray data of HRS (L428, L1236, KM-H2, HDLM-2, L540 and L540Cy) and non-Hodgkin (Reh, Namalwa and SU-DHL-4) cell lines for subsequent analyses. Principal PC1 component analysis showed a separation of HRS from non- Hodgkin control samples. PC1 accounts for 80.5%, PC2 for 7.9% and PC3 for 3.7% of explained variance. Fisher’s Exact test À11 PC2 (Po10 ) and GSEA (normalized enrichment score ¼ 1.34, FDR q-value ¼ 0.016) confirmed significant deregulation of our NOTCH HRS samples gene set. Note, that microarray analyses of cell lines were performed in duplicates, except for L540, L540Cy and SU-DHL-4. c.p.m., counts non-Hodgkin samples per minute; NS, not significant.

Oncogene NOTCH activation by high MAML2 expression KKo¨chert et al 1837 Discussion growth of lymphoma cells. Interestingly, no similar differences of MAML1 and MAML3 expression in the Given the importance of deregulated NOTCH activity various cell types were observed, supporting the inter- for a variety of malignancies and the central function of pretation that the various MAML proteins differ in MAML coactivators for formation and transcriptional their potential to fine-tune NOTCH signaling (Lin et al., activity of the NTC, surprisingly little is known about 2002). The particular importance of MAML2 for MAML expression and function in malignant cells. activation of the NTC is also supported by published We addressed these questions in human B cell-derived data, which demonstrate that MAML2 is more effective lymphomas and revealed an aberrant expression of regarding the amplification of NOTCH signaling than MAML2 in several entities, suggesting that MAML2 MAML1 or MAML3 (Lin et al., 2002; Wu et al., 2002). deregulation and subsequent NOTCH activation is a Furthermore, the involvement of MAML coactivators common pathogenetic principle in these lymphomas. in the regulation of signaling pathways other than Furthermore, our data demonstrate the importance NOTCH (McElhinny et al., 2008), including in the of MAML2 for NOTCH target gene regulation and several lymphoma types deregulated NF-kB pathway (Jin et al., 2010), has to be elucidated in future studies. With respect to cHL, we show that the NOTCH BD TAD1 TAD2 target genes HEY1 and HES7 are specifically upregu- MAML1 AA 1-1016 lated in HRS cell lines. Furthermore, these cell lines respond to disruption of the NTC by DnMAML1 or DnMAML1 AA 13-74 specific MAML2 knockdown, and a NOTCH-associated DnMAML1 AA 13-50 gene expression signature is sufficient to separate HRS from non-Hodgkin cell lines. These data indicate that DnMAML1 AA 19-35 NOTCH signaling is, in addition to its induction by ligands in situ in the lymph node (Jundt et al., 2002), 1.2 SUP-T1 activated in HRS cells in a cell-autonomous manner. 1.0 In support of such a cell-autonomous NOTCH activa- 0.8 tion, we detected high-level JAG2 and, in accordance 0.6 * with published data (Kapp et al., 1999; Jundt et al., 0.4 * 2002), NOTCH2 expression in HRS cell lines, providing

(fold induction) 0.2 Luciferase activity * further components of the NTC, which in turn will be 0.0 accessible for activation by MAML2, as suggested by 35 kDa anti-GFP our data. 25 kDa β-actin 50 kDa 40 kDa Hes1-pGL2 +++ + pEGFP + Figure 4 Small MAML1-derived peptide constructs inhibit DnMAML1-pEGFP AA 13-74 + NOTCH-dependent transcriptional activity. (a) Schematic of the DnMAML1-pEGFP AA 13-50 + design rationale of NOTCH inhibiting peptide constructs. Amino DnMAML1-pEGFP AA 19-35 + acids (AA) 1–123 are the binding domain (BD) of MAML1 necessary to bind to ICN1 and CSL. MAML1 also consists of two 135792 4 6 8 transactivation domains (TAD). (b) SUP-T1 cells were transfected with the NOTCH-dependent Hes1-pGL2 reporter construct and CSL/ICN1/ expression plasmids encoding pEGFP (control), DnMAML1– MAML1 * CSL/ICN1/ * pEGFP AA 13–74, DnMAML1–pEGFP AA 13–50 or * DnMAML1 DnMAML1–pEGFP AA 19–35, as indicated. Luciferase activity was measured 48 h after transfection and is shown compared with Hes1-pGL2 activity in pEGFP-transfected cells, which was set as 1. NS EMSA Data are represented as mean±s.d., P-values are stated for the comparisons of the control sample to each of the other samples. CSL One out of six experiments is shown. *Po0.001. Ectopic expression of the various constructs was veriﬁed by WB using GFP antibody. b-actin was analyzed as control. (c) EMSA analysis of the NTC MAML1 116 kDa and its disruption by different DnMAML1–pEGFP constructs, 120 kDa using the CSL binding site. HEK293 cells were transfected with ICN1 various combinations of MAML1, Flag-tagged ICN1, Myc-tagged CSL and the DnMAML1–pEGFP constructs, and nuclear extracts CSL 55 kDa

WB were prepared 48 h after transfection. Ectopic expression of the 35 kDa various cDNAs was veriﬁed by WB using MAML1 antibody, anti-GFP NOTCH1 antibody (clone mN1A), GFP antibody for detection 25 kDa of DnMAML1–pEGFP-fusion constructs and MYC antibody for PARP1 100 kDa detection of Myc-tagged CSL. PARP1 was analyzed as control. MAML1 + +++ + The CSL/ICN1/MAML1 complex in lane six is marked by one ICN1 + + ++++ asterisk, the CSL/ICN1/DnMAML1–pEGFP 13–74 complex in lane CSL + + + +++ seven by two asterisks. Note, that in the presence of DnMAML1– DnMAML1-pEGFP AA 13-74 + + pEGFP 13–50 (lane 8) or DnMAML1–pEGFP 19–35 (lane 9) stable DnMAML1-pEGFP AA 13-50 formation of a NTC complex binding to DNA is not possible. DnMAML1-pEGFP AA 19-35 + One out of three experiments is shown. NS, non-speciﬁc complex.

Oncogene NOTCH activation by high MAML2 expression KKo¨chert et al 1838 Apart from indicating ongoing NOTCH signaling, to the manufacturer’s recommendation. Purity of CD19 þ B HEY1 and HES7 might contribute to lymphoma and CD3 þ T cells was greater than 95%, as determined by pathogenesis. Both genes encode for basic helix-loop- staining of purified cells with CD19- and CD3-specific helix factors able to repress transcription and to modify antibodies (#R0808 and #R0810, Dako, Hamburg, Germany) cellular differentiation (Iso et al., 2003). Furthermore, and subsequent fluorescent-activated cell sorter (FACS) HES7 represses transcription from E-box-containing analysis using a FACSCantoII flow cytometer and CantoDiva software (Becton Dickinson, Heidelberg, Germany). The use promoters by suppression of E2A/E47-activity (Bessho of human material was approved by the local ethics committee et al., 2001). Interestingly, inhibition of the basic helix- of the Charite´, and performed in accordance with the loop-helix transcription factor E2A has an important Declaration of Helsinki. Cells were electroporated in OPTI- role for NOTCH-mediated malignant transformation MEM I using a Gene-Pulser II (Bio-Rad, Munich, Germany) (Nie et al., 2003; Li et al., 2008) and E2A inhibition with 950 mF and 0.18 kV (L428, HEK293), 500 mF and 0.3 kV contributes to the pathogenesis of various human (L591), 500 mF and 0.28 kV (SUP-T1), 50 mF and 0.5 kV lymphomas (Murre, 2005; Mathas et al., 2006; Lietz (BJAB). Transfection efficiency was determined by pEGFP et al., 2007). (Clontech Laboratories, Mountain View, CA, USA) cotrans- Consistent with the essential function of MAML fection and FACS analysis or by FACS analysis of pEGFP- proteins for NOTCH transcriptional activity (Wu et al., fusion constructs, where indicated. Cells were transfected with 60 mg DnMAML1–pEGFP AA 13–74 fusion construct or 2002; Nam et al., 2003), a truncated form of MAML1 60 mg of pEGFP control plasmid, or with each 20 mg of three retaining the N-terminal ICN/CSL interaction domain different shMAML2-pSuper-based expression constructs or but lacking the C-terminal transactivation domain 60 mg of scrambled shRNA construct as control, both along blocks ICN function (Weng et al., 2003). Based on with 10 mg of pEGFP. At 48 h after transfection, GFP þ cells structural and mutational analyses of the NTC (Nam were enriched by FACS sorting and enriched cells were used for et al., 2006; Del Bianco et al., 2008), we followed up this proliferation assays as well as RNA and protein preparation. experimental approach and designed much shorter truncated MAML variants that still blocked NTC Reporter assays activity, most likely by preventing binding of full-length For measurement of luciferase activity, HEK293 and SUP-T1 MAML proteins into the groove formed by CSL and cells were transfected with 10 mg of the Hes1-pGL2 promotor NOTCH. In fact, (Moellering et al., 2009) showed construct and 100 ng pRL-TKLuc as an internal control. that the DnMAML1 AA 19–35 construct we used Where indicated, cells were cotransfected with 5 mg Flag- comprises the core a-helical AA motif that suffices tagged ICN1, 30 mg (HEK293) or 60 mg (SUP-T1) MAML2 for construction of a potent competitor. Using the expression constructs or both. Equal DNA loads were achieved by adding according amounts of empty plasmid. MAML1 AAs 21–36 as template sequence, they devel- For assaying the functional activity of MAML1-based oped stapled a-helical peptides, which effectively in- peptide-pEGFP constructs, SUP-T1 cells were cotransfected duced apoptosis of human NOTCH-dependent T-ALL with 80 mg of either pEGFP, or the various DnMAML1– cell lines in vitro and in vivo. Thus, the design of such pEGFP constructs, as indicated, along with 10 mg of the Hes1- small peptide inhibitors is a promising therapeutic pGL2 promotor construct and 100 ng pRL-TKLuc as an approach to functionally disrupt the NTC. Such an internal control. At 48 h after transfection, cells were lysed and approach would also be suitable for malignancies that the ratio of the two luciferases was determined (Dual luciferase depend on NOTCH activity but are resistant to kit; Promega, Mannheim, Germany). g-secretase inhibition, a major obstacle in the clinical use of g-secretase inhibitors. Plasmids and DNA constructs In summary, aberrant activation of MAML family MAML1, MAML3 and MYC-CSL constructs were described coactivators represents an as yet unknown alternative before (Wu et al., 2000, 2002). MAML2 was mobilized from mechanism of NOTCH activation. These data strongly the previously described MAML2-pEFBOS construct (Lin suggest that the development of new treatment ap- et al., 2002) by digestion with NotI and XhoI and cloned proaches interfering with NOTCH signaling should into pcDNA3.1( þ ) (Invitrogen, Karlsruhe, Germany). The explore, more than at present, direct targeting of the DnMAML1–pEGFP AAs 13–74 and the ICN1-pcDNA3.1- expression constructs as well as the Hes1-pGL2 reporter NTC itself. construct were described previously (Weng et al., 2003). The truncated versions DnMAML1 AA 13–50 and AA 19–35 were cloned into pEGFP through HindIII and KpnI restriction sites using high-performance liquid chromatography grade oligo- Materials and methods nucleotides (Biotez GmbH, Berlin, Germany). Sequences for the shMAML2 constructs were selected on the basis of small Cell lines, culture conditions and transfections interfering RNA sequences from the Dharmacon MAML2 HRS (L428, L1236, KM-H2, L591 (EBV þ ), HDLM-2, L540 ON-TARGETplus SMARTpool (#L-013568-00, Dharma- and L540Cy), pro-B lymphoblastic leukemia (Reh), Burkitt’s con). To generate shRNA expression constructs, target lymphoma (Namalwa, BL-60 and BJAB), diffuse large B-cell sequences 50-GGACAAAGTCAGATTATGT-30,50-CGAAA lymphoma (DLBCL; SU-DHL-4), T lymphoblastic lymphoma GTAATGGCTAACTA-30,50-AGACCAAATTTAACCCAT (SUP-T1) and HEK293 cells were cultured as described A-30 were cloned through BglII and HindIII restriction sites (Mathas et al., 2006). Primary CD19 þ B cells and CD3 þ T into pSUPER (Janz et al., 2006) using high-performance liquid cells were isolated from human tonsils with CD19 chromatography grade oligonucleotides (Biotez GmbH). The or CD3 MicroBeads (#130-050-301 or #130-050-101; both scrambled shRNA construct has been previously described Miltenyi Biotec, Bergisch Gladbach, Germany) according (Janz et al., 2006). All constructs were verified by sequencing.

Oncogene NOTCH activation by high MAML2 expression KKo¨chert et al 1839 RNA preparation, semi-quantitative and real-time PCR analyses Ficoll was used instead of glycerol. Electrophoretic mobility RNA preparation and complementary DNA synthesis were shift assays were performed with nuclear extracts. For performed as described (Mathas et al., 2006). Primer sequences preparation of nuclear extracts, HEK293 were transfected are listed in Supplementary Table 2. Real-time PCR analyses with different combinations of 10 mg Myc-tagged CSL, 10 mg were performed using Power SYBR Green Mastermix and the Flag-tagged ICN1, 20 mg MAML1, 40 mg DnMAML1– ABI StepOnePlus real-time PCR system (Applied Biosystems, pEGFP AA 13–74, 40 mg DnMAML1–pEGFP AA 13–50, Darmstadt, Germany). Relative quantities were calculated 70 mg of DnMAML1–pEGFP AA 19–35 and total DNA using the 2ÀDDCt method. All PCR products were veriﬁed by amount was adjusted with empty plasmid. Cells were harvested sequencing. after 48 h.

Preparation of whole-cell and nuclear extracts and western Statistical analyses of experimental and microarray data blotting All statistical analyses were done in R v2.9.1 (http://www. Whole-cell and nuclear-extract preparation, and western r-project.org/). Independent Student’s t-test was used to analyze blotting were performed as described (Mathas et al., 2006). data from proliferation- or real-time PCR-experiments. For For western blotting analyses, 30 mg of whole cell or nuclear analyses of luciferase assays, one-way analysis of variance was extracts were used. Primary antibodies were: anti-MAML1 done before applying Tukey’s Honestly Significant Differences and anti-MAML2 (#4608 and #4618, Cell Signaling, Boston, test with 95% family-wise confidence level. For microarray MA, USA), anti-MAML3 (NB100-2129, Novus Biologicals, analysis of the various cell lines, RNA processing and Cambridge, UK), anti-cleaved NOTCH1 (#2421; Cell Signaling), hybridization to Human Genome U133 Plus 2.0 arrays anti-NOTCH1 (clone mN1A; #N6786), anti-b-actin (#A5441) (Affymetrix) were performed according to the manufacturer’s and anti-FLAG (#F3165; all from Sigma-Aldrich, Taufkirchen, recommendation. Raw microarray data was processed using Germany), anti-NOTCH2 (C651.6DbHN; developed by S Bioconductor v2.4. RMA background correction and quantile Artanavis-Tsanakonas, Developmental Studies Hybridoma normalization were applied. The LIMMA (Linear Models for Bank, University of Iowa, Iowa City, IA, USA), anti-NOTCH3 Microarray Data) framework was used for determination of and anti-NOTCH4 (#34465 and #2423, Cell Signaling), anti- significantly differentially-expressed genes and Fisher’s Exact PARP1 (#sc-8007; Santa Cruz Biotechnology, Santa Cruz, CA, test for testing significance of deregulation of the NOTCH gene set. USA), anti-GFP (#BA-0702; Vector Laboratories, Burlingame, Unrotated principal component analysis was applied for analysis of CA, USA), anti-myc (#46-0603; Invitrogen). Membranes were intensity values of NOTCH-associated genes. Gene set enrichment incubated with horseradish peroxidase-conjugated secondary analysis (GSEA) was done as described in (Subramanian et al., antibodies. 2005). Microarray data are available through Gene Expression Omnibus accession number GSE20011. Proliferation assays Proliferation of cells was determined by measurement of DNA synthesis following [3H]-thymidine incorporation assays using standard protocols. Proliferation assays were measured 72 h Conflict of interest and 96 h after transfection of cells. The authors declare no conflict of interest. Immunohistochemistry All cases were drawn from the files of the Consultation and Reference Center for Haematopathology at the Institute of Acknowledgements Pathology, Campus Benjamin Franklin, Medical University Berlin, Germany. Diagnoses were established according to the We thank Caroline Ga¨rtner (Berlin), Melanie Manzke (Berlin) world health organization criteria. The primary antibody used and Simone Kressmann (Berlin) for outstanding technical was anti-MAML2 (#4618, Cell Signaling). Bound antibodies assistance, and Peter Rahn (Berlin) for cell sorting. We thank were made visible using the streptavidin-biotin-alkaline Raphael Kopan (Washington) for the Hes1-pGL2 reporter phosphatase method and FastRed as chromogen (all from construct, Lizi Wu (Gainesville, Florida) for the MAML3 expres- DakoCytomation, Glostrup, Denmark). sion construct, Karin Zimmermann (Berlin) and Ulf Leser (Berlin) for helpful suggestions in regard to gene set analysis, Electrophoretic mobility shift assay and Ariane Buchal and Benedikt Sedlmaier (both Berlin, CSL electrophoretic mobility shift assay was performed as Germany) for providing human tonsil material. This work was described previously (Fryer et al., 2002) using the high-affinity supported in part by grants from the Deutsche Forschungsge- CSL-binding site from the HES1 promoter (50-CTAGGTTAC meinschaft, the Berliner Krebsgesellschaft and the Wilhelm TGTGGGAAAGAAAGTCC-30). As for the shift buffer, 8% Sander-Stiftung.

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Supplementary Information accompanies the paper on the Oncogene website (http://www.nature.com/onc)

Oncogene