Letters to the editor 485 of GATA2 and biCEPBA mutations, the prognostic impact of is greatly appreciated. In addition, we are grateful for the data management support GATA2mut in dependence on biCEBPA mutations was analyzed. performed by Tamara Alpermann. Patients with biCEBPA mutations and additional GATA2mut (n ¼ 17) had a better 2-year OS compared with patients with A Fasan1, C Eder1, C Haferlach1, V Grossmann1, A Kohlmann1, biCEBPA mutations and GATA2wt (n ¼ 63) (100% vs 76.1% F Dicker1, W Kern1, T Haferlach1 and S Schnittger1 P ¼ 0.058) (Figure 2c). Interestingly, GATA2 had been shown to 1MLL Munich Leukemia Laboratory, Munich, Germany interact with CEPBA by forming protein complexes and this E-mail: [email protected] interaction is critical for the suppression of adipocyte differentia- tion.8 Thus, the coincidence of mutations in both genes could impair interaction and provoke a differentiation advantage REFERENCES of these cells resulting in the favorable prognosis of patients harboring both GATA2mut and biCEBPA mutations. 1 Vicente C, Conchillo A, Garcia-Sanchez MA, Odero MD. The role of the GATA2 transcription factor in normal and malignant hematopoiesis. Crit Rev Oncol Hematol In conclusion, we confirmed a strong association of GATA2mut 2012; 82:1–17. with biCEBPA mutations in a large set of intermediate-risk AML 2 Zhang SJ, Ma LY, Huang QH, Li G, Gu BW, Gao XD et al. Gain-of-function patients. Furthermore, we did not observe GATA2mut in patients mutation of GATA-2 in acute myeloid transformation of chronic myeloid leukemia. with monoCEPBA mutations underlining the different biology of Proc Natl Acad Sci USA 2008; 105: 2076–2081. AML with biallelic vs monoCEPBA mutated AML. For the first time, 3 Hahn CN, Chong CE, Carmichael CL, Wilkins EJ, Brautigan PJ, Li XC et al. this study also provides data on the frequency of GATA2mut Heritable GATA2 mutations associated with familial myelodysplastic syndrome and in CEPBAwt AML, in which a low frequency of only 3.3% was acute myeloid leukemia. Nat Genet 2011; 43: 1012–1017. detected. Analyses performed at relapse demonstrated that 4 Ostergaard P, Simpson MA, Connell FC, Steward CG, Brice G, Woollard WJ et al. GATA2mut are secondary events. Furthermore, GATA2mut are Mutations in GATA2 cause primary lymphedema associated with a predis- position to acute myeloid leukemia (Emberger syndrome). Nat Genet 2011; 43: associated with female sex and favorable impact on survival. 929–931. GATA2 thus seems to be a promising new marker to identify 5 Dufour A, Konstandin N, Ksienzyk B, Zellmeier E, Benthaus T, Yaghmaie M et al. patients with even more favorable prognosis in the subgroup of High frequency of GATA2 mutations in cytogenetically normal acute myeloid patients with the prognostically favorable biCEBPA mutated AML. leukemia with biallelic CEBPA mutations identified by exome sequencing. ASH Abstract 2011; 72. 6 Greif PA, Dufour A, Konstandin NP, Ksienzyk B, Zellmeier E, Tizazu B et al. CONFLICT OF INTEREST GATA2 zinc finger 1 mutations associated with biallelic CEBPA mutations define a CH, WK, TH and SuS are equity owners of and AF, CE, VG, AK and FD are employed by unique genetic entity of acute myeloid leukemia. Blood 2012; e-pub ahead of print the MLL Munich Leukemia Laboratory. 30 May 2012. 7 Grimwade D, Walker H, Oliver F, Wheatley K, Harrison C, Harrison G et al. The importance of diagnostic cytogenetics on outcome in AML: analysis ACKNOWLEDGEMENTS of 1,612 patients entered into the MRC AML 10 trial. The Medical Research We thank all clinicians for sending samples to our laboratory for diagnostic purposes, Council Adult and Children’s Leukaemia Working Parties. Blood 1998; 92: and for providing clinical information and follow-up data. In addition, we would like 2322–2333. to thank all co-workers at the MLL Munich Leukemia Laboratory for approaching 8 Tong Q, Tsai J, Tan G, Dalgin G, Hotamisligil GS. Interaction between GATA and the together many aspects in the field of leukemia diagnostics and research. Especially C/EBP family of transcription factors is critical in GATA-mediated suppression the technical assistance of Madlen Ulke, who performed Sanger Sequencing analyses, of adipocyte differentiation. Mol Cell Biol 2005; 25: 706–715.

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

Glucocorticoid sensitivity of T-cell lymphoblastic leukemia/ lymphoma is associated with glucocorticoid receptor-mediated inhibition of Notch1 expression

Leukemia (2013) 27, 485–488; doi:10.1038/leu.2012.192 (T-ALL)-associated genetic lesion has been initially reported to result in deletion of Notch1 ectodomain and constitutive activa- Notch signaling can regulate a wide variety of cellular processes, tion of its intracellular region.5 Further studies have shown that including apoptosis1 differentiation,2 and drug resistance.3 At activating mutations in the NOTCH1 gene are present in over 50% the molecular level, Notch signaling has been demonstrated to of human T-ALL cases, making NOTCH1 the most prominent onco- function as a transcriptional activator.4 Receptor-ligand interaction gene specifically involved in the pathogenesis of this disease.5–7 renders Notch susceptible to cleavage by ADAM-type metallo- In T-ALL, activation of Notch signaling has been implicated in the proteases at site S2, which creates a short-lived intermediate, regulation of multiple cellular pathways, including those resulting which is in turn cleaved by g-secretase within the transmembrane in apoptosis,8 tissue infiltration9 and glucocorticoid resistance.10 domain at site S3. This frees the intracellular domain of Notch The link between Notch and glucocorticoid resistance is of parti- (Notch-IC), which translocates to the nucleus to form a trans- cular interest, as glucocorticoids were among the first drug classes criptional activation complex with the DNA-binding factor CSL. used in the treatment of patients with T-cell leukemia and are That the Notch signaling pathway has a crucial role in cancer is still the essential components of treatment (Inaba and Pui in11 and firmly established as a rare T-cell acute lymphoblastic leukemia references therein). They seem to exert their cytotoxic effects

Accepted article preview online 13 July 2012; advance online publication, 31 July 2012

& 2013 Macmillan Publishers Limited Leukemia (2013) 482 – 516 Letters to the editor 486 by binding to glucocorticoid receptors in the cytoplasm. These -resistant T cells. Thus, we compared Notch1 and Hes-1 levels in receptors can then form dimers, translocate to the nucleus and dexamethasone (DEX)-resistant cell lines (Jurkat and Molt3) interact with glucocorticoid-response elements to transactivate and the glucocorticoid-sensitive cell line (KE-37). We found that, gene expression, or they can remain as monomers and repress the in contrast with the presence of Notch1, Hes-1 expression was activity of transcription factors, such as the activating protein-1 or downmodulated in KE-37 cells (Figure 1). Interestingly, Jurkat and nuclear factor-kB.12–14 Published results suggest that inhibition of Molt3 cells are highly resistant to glucocorticoids, showing only Notch1 signaling enhances glucocorticoid receptor auto-upregulation a minimal loss of cell viability when treated with DEX concentra- and glucocorticoid sensitivity in otherwise glucocorticoid-resistant tions as high as 1 mM (Figure 1e). Conversely, treatment of KE-37 10 T-ALL cells. Short-hairpin RNA knockdown of HES-1 in T-ALL- cells with 100 nM DEX for 48 h effectively induces an apoptotic derived cells resulted in increased glucocorticoid receptor trans- response, as revealed by cell viability assay and poly (ADP-ribose) cript and protein levels and effective reversal of glucocorticoid polymerase (PARP) cleavage (Figures 1e and f). It has been resistance.15 In order to study the relationship between Notch1/ reported that inhibition of Notch1 target, Hes-1, can reverse Hes-1 expression and glucocorticoid sensitivity, we determined glucocorticoid resistance in leukemic lymphoblasts by inducing the relative expression of Notch1/Hes-1 by quantitative PCR upregulation of NR3C1 (nuclear receptor subfamily 3, group C, and western blot analysis in both glucocorticoid-sensitive and member 1, also known as GR or GCR);15 in agreement with this

Figure 1. Expression of Notch1/Hes-1 in T-ALL cells (a, b, c and d). Notch1 mutation in Jurkat, KE-37, Motl3 and DND-41 cell lines6 is summarized in supplementary Table 1. Notch1 and Hes-1 expression levels were analyzed by western blot and reverse transcription (RT)-PCR/ qRT-PCR in the indicated cell lines. Both actin and tubulin were used as the loading control. Analysis of apoptosis after DEX treatment. (e) Apoptosis was analyzed after incubation of cells for 48 h with the indicated amount of DEX by Annexin-V/PI staining and measured by flow cytometry, as shown for cell lines Jurkat, Molt3 and KE-37. Data are representative of three independent experiments. The result is expressed as % of viable cells and the number of viable cells in the corresponding untreated controls was set to 1. Note that DEX-induced apoptosis occurred only in the KE-37 cell lines. (f) Detection of apoptosis by detection of PARP cleavage. PARP cleavage is an established and reliable apoptosis indicator downstream of caspase activation. Cell extracts were prepared from the indicated cell lines untreated or treated with 1 mM DEX, as indicated in panel e, and analyzed by western blot with antibodies against PAPR protein. (g) Analysis of Notch1 protein expression by western blot, in Jurkat, Molt3 and KE-37 cells treated with the indicated amount of DEX for 48 h. (h) Analysis of NR3C1 protein expression by western blot, in Jurkat and KE-37 cells.

Leukemia (2013) 482 – 516 & 2013 Macmillan Publishers Limited Letters to the editor 487

Figure 2. Binding of the NR3C1 proteins to the endogenous NOTCH1 promoter as assessed by chromatin immunoprecipitation. (a) Schematic representation of NOTCH1 promoter (human upper, mouse lower schema) NR3C1 binding sites is indicated. (b)DownregulationofNOTCH1 promoter activity by DEX. HEK293 cells were transfected with luciferase reporter plasmids carrying the NOTCH1 promoter and treated with DEX for the last 12 h of the experiment (48 h after transfection). Results are presented as mean±s.d. from triplicated samples. (c) KE-37 cells were left untreated or treated with 1 mM DEX for 24 h, then cells were processed for chromatin immunoprecipitation with antibodies against NR3C1, the immunoprecipitates were analyzed by PCR with oligonucleotide primers specific for the indicated region of the human NOTCH1 promoter (head- arrow); lanes 3, 4, 5 and 6 represent immunoprecipitate samples analyzed by PCR; lanes 2 and 3 represent input control; (lower panel) immunoprecipitated samples were analyzed by RT-PCR. The value of the immunoprecipitate samples in the corresponding controls was set to 1. (d) DEX-induced apoptosis of KE-37 cells is rescued by intracellular Notch1-IC expression. KE-37 cells were infected with either MSVC-empty retrovirus or a retrovirus that expresses human intracellular Notch1, then left untreated or treated with 100 nM and 1 mM DEX for 24 h, and the percentage of apoptotic cells was determined by PI/Annexin staining and FACS analysis. (e) Cell extracts were prepared from K3-37 cells infected with either MSVC-empty or MSVC-Notch-IC retrovirus; western blot analysis was performed with the indicated antibodies. (f, g) Jurkat, K3-37 and Molt3 cells were treated with 5 mM DAPT for 24 h. Cellular extracts were prepared and resolved by SDS-PAGE on 4–12% Tris-Glycine gel transferred to polyvinylidene fluoride membranes and sequentially analyzed with the indicated antibodies. In both panels f and g tubulin is shown as the loading control. Notch1 Val 1744 indicates the cleaved/activated Notch1. (h) Viability assay in GSI/glucocorticoid-treated cell lines. Jurkat, KE-37 and Molt3 cells were left untreated or treated with either 5 mMDAPTor1mM DEX, or treated with 5 mM DAPT plus 1 mM DEX.

model, we found a correspondence between Hes-1 expression package (Genomatix, Munchen, Germany; www.genomatix.de). A and GC resistance. We found that Hes-1 is expressed in both Jurkat scan of 2.5 kb of genomic sequence, located upstream of the and Molt3, and, as expected,15 we found that the level of NR3C1 predicted pre-NOTCH1 start site, identified several putative protein is low in both these cell lines (Figure 1h, only Jurkat cells glucocorticoid responsive elements (GRE) in both the human are shown). Conversely, we found that KE-37 cells did not express and the mouse NOTCH1 regulatory region (Figure 2a), suggesting Hes-1 but expressed a higher level of NR3C1 when compared with the involvement of a direct binding of GC to the NOTCH1 both Jurkat and Molt3 cells (Figure 1h). To elucidate the promoter. Thus, we examined the role of GC in the control of mechanism responsible for the GC-induced apoptosis in KE-37 NOTCH1 expression by generating a NOTCH1 promoter construct cells treated with glucocorticoids, we performed a western blot and testing it in a luciferase reporter assay. We found that the level analysis against Notch1 of the T leukemic cells treated with vehicle of NOTCH1 promoter activity was strongly reduced by GC or DEX. This analysis identified Notch1 as a gene that is regulated treatment in a dose-dependent manner with both human and by DEX (Figure 1g). Interestingly, dose–response analysis of KE-37 mouse promoter (Figure 2b, only the human promoter is shown). cells treated with DEX (100 nM and 1 mM) showed that DEX is To ascertain whether NR3C1 occupies the NOTCH1 promoter effective in promoting Notch1 down-modulation. Conversely, in vivo, we performed a chromatin immunoprecipitation assay analysis of Jurkat and Molt3 cells treated with DEX (100 nM and using formaldehyde cross-linked KE-37 cells. Chromatin extracts 1 mM) showed that Notch1 expression was unaffected by GC were subjected to immunoprecipitation with anti-NR3C1 anti- treatment. To understand at which level Notch1 expression was bodies. These samples were subjected to PCR amplification with regulated by GC treatment, we first characterized the NOTCH1 the use of specific oligos that target the GRE region of the promoter region by using the Genomatix MatInspector software NOTCH1 promoter. As shown in Figure 2c, anti-NR3C1 antibodies

& 2013 Macmillan Publishers Limited Leukemia (2013) 482 – 516 Letters to the editor 488 recovered a 300 bp fragment of the NOTCH1 promoter containing glucocorticoid-sensitive T-ALL. To conclude, our data indicate that the GRE site. These results demonstrate that NR3C1 directly any factors that modulate NR3C1 signaling will also affect Notch1 associate with the NOTCH1 promoter region containing the expression and response. Thus, this mechanism could be functional GRE-binding site. The GC-induced downmodulation of exploited to protect cells from an excessive Notch1 response, Notch1 in KE-37 cells suggests that the GC-induced apoptosis of particularly relevant in those diseases where Notch1 expression is KE-37 cells might be mediated by the inhibition of a Notch1- likely to have a key role in pathogenesis. mediated anti-apoptotic response. However, the possibility exists that the apoptosis observed in GC-treated KE-37 cells may be CONFLICT OF INTEREST mediated only in part by downmodulation of Notch1 expression The authors declare no conflict of interest. and alternatively, may be mediated by inhibition of other GC- sensitive pathways. To explore this possibility and analyze whether in KE-37 cells GC-mediated apoptosis requires Notch1 ACKNOWLEDGEMENTS downmodulation, we tested whether intracellular Notch1 expres- ´ ´ ´ sion can rescue the GC-induced apoptosis in KE-37 cells. Thus, We thank Dr Cathrin Brisken, Ecole Polytechnique Federale De Lausanne, for kindly providing the Notch1-IC retroviral construct. This work was supported by NotchIT ITN KE-37 cells were infected with retroviruses containing pMSCV- Project; FP7-MC-ITN 215761. Notch1-IC or control vector pMSCV. Infected cells were then G418 selected and treated with either vehicle or DEX. At 24 h after S Cialfi1,2,3, R Palermo1,3, S Manca1, S Checquolo1, D Bellavia1, treatment, apoptosis/viability was evaluated with the use of 1 1 2 1 Annexin-V. The presence of the active form of Notch1 in KE-37 M Pelullo , R Quaranta , C Dominici , A Gulino , I Screpanti1 and C Talora1 cells increased resistance to GC-induced apoptosis (Figure 2d), and 1 this was associated with the increased expression of Hes-1 and Department of Molecular Medicine, Sapienza University of Rome, Rome, Italy and NR3C1 downmodulation as compared with KE-37 cells infected 2 with the control virus (Figure 2e). Taken together, these data Department of Pediatrics and Infantile Neuropsychiatry, demonstrate that Notch1-IC expression driven by a heterologous Sapienza University of Rome, Rome, Italy E-mail: [email protected] or [email protected] promoter confers DEX resistance to KE-37 cells by promoting 3 glucocorticoid receptor downmodulation, thus inhibiting the DEX- These authors contributed equally to this work. mediated apoptotic response. We observed that, although KE-37 cells expressing endogenous Notch1 were sensitive to GC-induced apoptosis (Figure 1), conversely, Notch1-IC-enforced expression in REFERENCES KE-37 cells had an effect on GC susceptibility. An ensuing question 1 Dang TP. Notch, apoptosis and cancer. Adv Exp Med Biol 2012; 727: 199–209. was why does not endogenous NOTCH1 confer GC resistance on 2 Watt FM, Estrach S, Ambler CA. Epidermal Notch signalling: differentiation, cancer KE-37 cells? To address this question we analyzed the level of and adhesion. Curr Opin Cell Biol 2008; 20: 171–179. activated Notch1 in Jurkat, Molt3 and KE-37 cells. We found that 3 Wang Z, Li Y, Ahmad A, Azmi AS, Banerjee S, Kong D et al. Targeting Notch signaling pathway to overcome drug resistance for cancer therapy. Biochim Bio- both Jurkat and Molt3 cells have functionally active Notch1, as phys Acta 2010; 1806: 258–267. revealed by anti-val1744 Notch1 antibody reactivity, whereas KE-37 4 Kopan R, Ilagan MX. The canonical Notch signaling pathway: unfolding the acti- cells do not express cleaved Notch1 (Figure 2f). Consistently with vation mechanism. Cell 2009; 137: 216–233. their expression of activated Notch1, Jurkat and Molt3 cells express 5 Ellisen LW, Bird J, West DC, Soreng AL, Reynolds TC, Smith SD et al. TAN-1, the theNotch1targetgeneHes-1(Figure1andFigure2f).Thisindicates human homolog of the Drosophila notch gene, is broken by chromosomal that endogenous Notch1 signaling is not active in the KE-37 cells, translocations in T lymphoblastic neoplasms. Cell 1991; 66: 649–661. which fits with the observation that KE-37 cells had a higher level 6 Weng AP, Ferrando AA, Lee W, JPt Morris, Silverman LB. Sanchez-Irizarry C et al. of NR3C1 protein as compared with Jurkat and Molt3 cells. Activating mutations of NOTCH1 in human T cell acute lymphoblastic leukemia. To inhibit NOTCH-mediated signal transduction, we treated Science 2004; 306: 269–271. 7 Thompson BJ, Buonamici S, Sulis ML, Palomero T, Vilimas T, Basso G et al. T-ALL cell lines with the GSI-IX (DAPT) at 5 mM, or with dimethyl The SCFFBW7 ubiquitin ligase complex as a tumor suppressor in T cell leukemia. sulfoxide (control vehicle). We did not observe any effects on J Exp Med 2007; 204: 1825–1835. proliferation or viability when the treatment was maintained for 8 Koch U, Radtke F. Notch in T-ALL: new players in a complex disease. up to 7 days in any of the cell lines (Figure 2h, only 48 h treatment Trends Immunol 2011; 32: 434–442. is shown). Importantly, treatment with DAPT as early as 12 h led to 9 Buonamici S, Trimarchi T, Ruocco MG, Reavie L, Cathelin S, Mar BG et al. CCR7 reduced levels of activated Notch1 and Hes-1 in each of the T-ALL signalling as an essential regulator of CNS infiltration in T-cell leukaemia. Nature cell lines in which the V1744 cleaved protein could be detected, 2009; 459: 1000–1004. indicating that the compound effectively inhibited NOTCH1 10 Real PJ, Ferrando AA. NOTCH inhibition and glucocorticoid therapy in T-cell acute cleavage and activation (Figure 2f, time-point at 24 h is shown). lymphoblastic leukemia. Leukemia 2009; 23: 1374–1377. 11 Inaba H, Pui CH. Glucocorticoid use in acute lymphoblastic leukaemia. Lancet Western blot analysis also demonstrated that the cell lines with Oncol 2010; 11: 1096–1106. activated Notch1 expressed low levels of NR3C1 (Figure 2g), 12 Tissing WJ, Meijerink JP, den Boer ML, Pieters R. Molecular determinants of reflecting the fact that aberrant activation of the NOTCH pathway glucocorticoid sensitivity and resistance in acute lymphoblastic leukemia. could be important in glucocorticoid receptor downmodulation. Leukemia 2003; 17: 17–25. However, after DAPT treatment, both Jurkat and Molt3 cell lines 13 Tsai SY, Carlstedt-Duke J, Weigel NL, Dahlman K, Gustafsson JA, Tsai MJ also expressed low levels of NR3C1, implying that glucocorticoid et al. Molecular interactions of steroid hormone receptor with its receptor expression is repressed in these cell lines through a enhancer element: evidence for receptor dimer formation. Cell 1988; 55: mechanism other than NOTCH1/Hes-1 activation. Consistently, GSI 361–369. treatment did not sensitize Jurkat and Molt3 cells to DEX 14 Ray A, Prefontaine KE. Physical association and functional antagonism between the p65 subunit of transcription factor NF-kappa B and the glucocorticoid treatment (Figure 2h), even though Hes-1 expression was reduced receptor. Proc Natl Acad Sci USA 1994; 91: 752–756. (Figure 2f). Thus, while confirming that glucocorticoid resistance of 15 Real PJ, Tosello V, Palomero T, Castillo M, Hernando E, de Stanchina E et al. T-ALL cells requires an intact Notch signaling, we showed that Gamma-secretase inhibitors reverse glucocorticoid resistance in T cell acute glucocorticoid treatment reduces Notch1 expression specifically in lymphoblastic leukemia. Nat Med 2009; 15: 50–58.

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

Leukemia (2013) 482 – 516 & 2013