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.

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