The importance of Notch signaling in peripheral T-cell lymphomas
Maria Rørbæk Kamstrup1, Edyta Biskup1, Lise Mette Rahbek Gjerdrum2, Elizabeth Ralfkiaer2, Omid Niazi1
& Robert Gniadecki1
1Department of Dermatology, Bispebjerg Hospital, University of Copenhagen, Denmark and 2Department of Pathology, Rigshospitalet, University of Copenhagen, Denmark
Abstract T-cell lymphoma, not otherwise specified (PTL-NOS)
Peripheral T-cell lymphomas (PTLs) represent an area of high medical need. Previously, we demonstrated high expression of Notch, a known oncogene, in primary cutaneous anaplastic large cell lymphoma (ALCL). In this study, we performed immunohistochemical staining for Notch1 in lymph nodes from PTL not otherwise specified (PTL-NOS) and systemic ALCL (ALKti and ALKti ) and report a similar distribution among the three subgroups: Negative, moderate and strong expression was, respectively, 18%, 27% and 55% for PTL-NOS (33 cases), 20%, 0% and 80% for ALCL ALKti (10 cases) and 45%, 22% and 33% for ALCL ALKti (nine cases) (p ti 0.05). In the ALKti ALCL cell line, Karpas-299, pharmacological inhibition of Notch with g-secretase inhibitor (GSI) I was far more potent than with GSI IX, XX and XXI with regard to cell viability and apoptosis. In conclusion, PTL tumor cells have prominent Notch1 expression and treatment with Notch inhibitors has cytotoxic effects.
Keywords: Notch, systemic anaplastic large cell lymphoma,
peripheral T-cell lymphoma not otherwise specified, γ-secretase inhibitors
Introduction
The 2008 World Health Organization (WHO) classification of hematopoietic and lymphoid neoplasms divides anaplastic large cell lymphoma (ALCL) into primary cutaneous ALCL and nodal ALCL, of which the latter is separated into ALK (anaplastic lymphoma kinase) positive (ALKti) and ALK neg- ative (ALKti ) tumors. ALK refers to translocations involving the receptor tyrosine kinase (termed ALK) located on 2p23, of which the most frequent translocation is ALK fused with nucleophosmin (NPM) on 5q35 [1]. Whereas ALKti ALCL more frequently affects children and young adults, ALKti tumors are more commonly seen in the elderly and have a worse prognosis. Primary cutaneous ALCLs have an excel- lent prognosis (5-year overall survival ti 90%) and do not or rarely have the t(2;5)(p23;q35) translocation [3]. Peripheral
represents a heterogeneous category of nodal and extran- odal T cell lymphomas that cannot be grouped into defined entities, and it has been debated whether ALKti ALCL is distinguishable from these [2]. However, recent data justify separate categories due to clinical and immunophenotypic differences [3].
Notch proteins belong to a family of evolutionarily con- served transmembrane receptors (Notch1–4) that impinge on a wide array of cellular processes including differen- tiation, proliferation and apoptosis [4]. The receptors are triggered through direct cell contact with transmembrane ligands (Jagged-1, Jagged-2, Delta-like 1, -3 and -4) result- ing in two proteolytic cleavages by tumor necrosis factor α (TNF-α)-converting enzyme (TACE) and the γ-secretase/
presenilin complex. This results in release of the intracellular and functionally active part of the Notch receptor (N-IC), which translocates to the nucleus, where it engages other DNA-binding proteins and regulates gene expression. Dys- regulated Notch signaling is involved in the pathogenesis of both solid tumors and hematologic malignancies, such as acute lymphoblastic T-cell leukemia, acute myeloid leu- kemia and B-chronic lymphocytic leukemia [5–8]. We have previously demonstrated that Notch1 is expressed in primary cutaneous CD30 ti T-cell lymphoproliferative disorders (primary cutaneous ALCL and lymphomatoid papulosis), Sézary syndrome and mycosis fungoides, especially in the advanced stages, and that Notch inhibition by either chemical blockers or small-interfering RNA induces apoptosis [9–11].
The objective of the present study was to analyze the expression of Notch1 in ALKti and ALKti ALCL and PTL- NOS. We found that all three entities express Notch, but there was no significant difference in expression between the groups. In addition, we used the ALKti ALCL cell line Karpas- 299 to provide further evidence for a functional significance of Notch in these lymphomas and demonstrated that Notch inhibition by γ-secretase inhibitor I (GSI I) caused a partial G2/M phase cell cycle block and induced cell death.
Correspondence: Maria Rørbæk Kamstrup, Department of Dermatology D92, Bispebjerg Hospital, Bispebjerg Bakke 23, Copenhagen DK-2400, Denmark. Tel: ti 45-3531-6005. Fax: ti 45-3531-6010. E-mail: [email protected]
There is an accompanying commentary that discusses this paper. Please refer to the issue Table of Contents. Received 7 February 2013; revised 10 April 2013; accepted 16 May 2013
639
Materials and methods
Tissue samples and patient data
Fifty-two cases of PTL, including 33 patients with PTL-NOS and 19 patients with ALCL, were drawn from the archives at the Departments of Pathology at Rigshospitalet, Herlev and Aalborg hospitals. The patients were 32 males and 18 females. Data from two patients regarding sex and age could not be retrieved. The median age for the remaining patients was 57 years (range 5–85 years) at diagnosis. Histologic and immunohistochemical evaluation was performed by two pathologists (L.M.R.G. and E.R.) in accordance with the WHO classification [12]. Five cases had primary manifesta- tion of PTL in the skin. Of the 19 cases with systemic ALCL, 10 were positive and nine were devoid of the ALK protein. A multi-block, containing samples of tonsil, kidney and liver, was used as the control.
Cell culture and reagents
Karpas-299 derived from human ALCL with a t(2;5) trans- location [13] was cultured in RPMI 1640 medium with l-glutamine supplemented with 10% fetal calf serum at 37°C and with 5% CO2. The cell line was regularly tested to be negative for mycoplasma. GSI I (Z-Leu-Leu-Nle-CHO), GSI IX (DAPT, N-[N-(3,5-difluorophenacetyl-l-alanyl)]- S-phenylglycine t-butyl ester), GSI XX (dibenzazepine, (S,S)-2-[2-(3,5-difluorophenyl)acetylamino]-N-(5-methyl-6- oxo-6,7-dihydro-5H-dibenzo[b,d]azepin-7-yl)propionamide) and GSI XXI (Compound E, (S,S)-2-[2-(3,5-difluorophenyl)- acetylamino]-N-(1-methyl-2-oxo-5-phenyl-2,3-dihydro-1H- benzo[e][1,4]diazepin-3-yl)-propionamide) were purchased from Merck Calbiochem (Darmstadt, Germany). GSIs were dissolved in dimethyl sulfoxide (DMSO).
was obtained from the Developmental Studies Hybridoma Bank, developed under the auspices of the National Insti- tute of Child Health and Human Development (NICHD) and maintained by The University of Iowa, Department of Biology, Iowa City, IA. Anti-HES1 antibody was from Abcam (Cambridge, UK). Antibody against poly(ADP ribose) polymerase (PARP) was from Cell Signaling (Beverly, MA). Anti-actin antibody (Sigma Aldrich) functioned as a loading control. Secondary antibodies were labeled with 800IR dye (anti-rabbit) (Li-Cor, Lincoln, NE) or Alexa Fluor 680 (anti-mouse or anti-rat) (Molecular Probes, Invitrogen Corporation, Carlsbad, CA). Immunoreactivity was detected and quantified with the infrared Odyssey Imaging System (Li-Cor).
Cell viability and apoptosis
GSI-induced cytotoxicity was measured by propidium iodide (PI) staining. Briefly, unfixed cells were incubated with increasing concentrations of GSI, stained with 5 μg/mL PI for 10 min, and the percentage of viable (i.e. PI-negative cells) was assessed by flow cytometry (Beckman Coulter, Fullerton, CA) and normalized versus the control (vehicle-treated cells). Dose–response curves were generated, and the half maximal effective concentration (EC50) was calculated using the trend line tool (Excel, Microsoft, Redmond, WA). In addition, we performed cell counting for selected GSI concentrations. Cell suspensions were diluted four times with sterile phos- phate buffered saline (PBS) and cell density was estimated by means of flow cytometry. The Caspase-Glo 3/7 (Promega Cooperation, Madison, WI) assay, which measures caspase 3/7 activities, was used according to the manufacturer’s rec- ommended conditions. Luminescence was measured with a Wallac 1420 Victor3™ II microplate reader (PerkinElmer, Wellesley, MA). Apoptosis was furthermore assessed by
Immunohistochemistry fluorescein isothiocyanate–annexin-V/PI staining accord-
Immunohistochemical staining was performed manually as described [10]. In brief, sections were subjected to microwave heat-induced epitope retrieval and incubated for 60 min at room temperature with a dilution 1:200 of Notch1 (affinity- purified monoclonal rabbit anti-human Notch1 c-20; Santa Cruz Biotechnology, Santa Cruz, CA) followed by staining using the DAKO EnVisionti Detection System-HRP for use with rabbit primary antibodies (DakoCytomation, Glostrup, Denmark). Upon diaminobenzidine (DAB) treatment, the slides were incubated in 0.5% copper sulfate and counter- stained in Mayer’s hematoxylin. L.M.R.G. and E.R. scored the Notch1 immunolabeling in tumor cells as negative (no visible staining or positive staining in ti 10%), moderately positive (positive staining in 10–50% of the tumor cells) and positive in a majority (ti 50%) of the tumor cells.
Western blotting
Whole-cell extracts were prepared as described previously [10]. Equal amounts of total cellular protein were separated by 12% Bis-Tris gel electrophoresis at 200 V, followed by elec- trophoretic transfer to a nitrocellulose membrane (Bio-Rad Laboratories, Hercules, CA). Antibodies specific for Notch1, Notch3, Notch 4 and p21 were from Santa Cruz Biotechno- logy. The Notch2 antibody developed by Artavanis-Tsakonas
ing to the manufacturer’s protocol (Beckman Coulter) and analyzed using Cell Lab Quanta SC MPL (Beckman Coulter) and Cell Lab Quanta SC MPL Analysis Software Version 1.0.
Cell cycle analysis and p21CIP1/WAF1 measurement
Cell cycle analysis and p21CIP1/WAF1 measurement were performed as previously described [11]. In brief, cytospin preparations of cells were fixed in ethanol, permeabilized with 0.25% Triton X-100 and, in the case of p21CIP1/WAF1 measurement, incubated with anti-p21 fluorescein con- jugate (Merck Calbiochem). Before being subjected to flow cytometry analysis, the cells were resuspended in 7-aminoactinomycin (7-AAD; Beckman Coulter). Each analysis was performed on at least 10 000 events.
Statistics
Notch1 immunostaining data were analyzed with the χ2 test. Continuous data were reported as mean plus or minus SD, and differences were evaluated by Student t-test. All experi- ments were repeated three times unless otherwise stated. p-Values less than 0.05 were considered to be statistically sig- nificant. Statistical analysis was performed using GraphPad Prism Version 4.03 (GraphPad Software Inc., San Diego, CA) or Excel.
Results
Notch1 is expressed in tumor cells in PTL-NOS and systemic ALCL
To address whether PTL-NOS and ALCL (ALKti and ALKti ) express Notch1 and to compare the degree of expression in these different entities, we performed immunohistochemi- cal staining of lymph node biopsies from patients with PTL-NOS and ALCL. All specimens were suitable for immu- nohistochemical analysis. Forty of 52 (77%) cases displayed positivity for anti-Notch1 in the neoplastic cells, as recognized by polymorphic nuclei, prominent nucleoli and/or irregular nuclear contours [Figure 1(a)]. In cases with PTL-NOS, 18 specimens displayed strong immunolabeling for anti-Notch1 and nine cases expressed the protein moderately strongly. The remaining six cases were devoid of Notch1. Eight patients with ALKti ALCL had positive tumor cells and two cases had negative neoplastic cells. In patients with ALKti ALCL, five cases displayed positive neoplastic cells of which two cases had moderately strong immunolabeling and three cases were strongly positive. The remaining four cases showed no immunoreaction for Notch1 in the neoplastic cells [Figure 1(b)]. A χ2 test showed no statistically significant difference between the different groups (p ti 0.15).
In tumor specimens, scattered reactive lymphocytes stained for Notch1. In the control materials, some lympho- cytes in the paracortex and intrafollicular regions of reactive tonsils were positive, as well as some scattered lymphocytes in the germinal centers. Immunolabeling of neoplastic and reactive lymphocytes were similar, and mainly cytoplasmic and on occasion nuclear. Microscopy of control liver and kidney tissues displayed no reactivity for Notch1.
Notch targeting by GSI induces cell death
Since there was no significant difference in Notch1 expres- sion between the different lymphoma entities, we focused
on a single ALKti ALCL cell line, Karpas-299, to study the functional significance of Notch. Karpas-299 cells were cultured with different GSIs, which interfere with the γ-secretase complex and thereby block the proteolytic cleavage of all Notch receptors [14]. Karpas-299 cells were incubated for 48 h with increasing concentrations of differ- ent GSIs (GSI I, IX, XX and XXI) or vehicle (DMSO), and cell viability was measured by propidium iodide exclusion. Sim- ilar to our previous results in common types of cutaneous T-cell lymphomas (mycosis fungoides, Sézary syndrome and primary cutaneous CD30 ti lymphoproliferative disorders), we observed that GSI I had a marked and dose-dependent effect on cell viability (EC50 ti 0.36 μM) [Figure 2(a)]. We confirmed that treatment with GSI I regulated the Notch pathway by detecting a dose-dependent down-regulation of Notch1–4 intracellular levels and the downstream target gene hairy enhancer of split (Hes1) [Figure 2(b)], which indi- cates the functional inactivation of Notch [14]. The cell line was largely unaffected by GSI IX, XX and XXI treatment at concentrations of 20 μM (EC50 ti 20 μM) after 48 h (data not shown). These results were confirmed by flow cytometric cell counting after 48 h [Figure 2(c)]. As Notch blockage can induce apoptosis, we next investigated the apoptotic effect of GSI I, IX, XX and XXI after 24 h of incubation by caspase 3/7 activity. GSI I was the most potent agent, with signifi- cant values observed at concentrations of 0.5 μM or greater at 24 h [Figure 2(d)]. GSI IX, XX and XXI produced a very mild increase in caspase activity [Figure 2(e)]. The caspase activity did not increase further after 48 h (data not shown). These results were confirmed by annexin-V/PI staining after incubation for 24 h that primarily demonstrated an increase in late apoptotic/necrotic cells after treatment with 0.4 μM GSI I [Figure 2(f)] and analysis of the 113 kDa PARP, which is the main substrate for activated caspase 3, which showed a dose-dependent increase in the 89 kDa apoptosis-specific cleavage product of PARP after treatment
(a)
(b)
>50% Notch1 10–50% Notch1
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PTL NOS ALCL ALK+ ALCL ALK–
Figure 1. Expression of Notch1 in PTL-NOS, ALKti and ALKti ALCL. (a) Hematoxylin and eosin (HE) staining (left) and immunostaining for Notch1 (right) in lymph node biopsies from a patient with ALKti ALCL (upper images) and a patient with PTL-NOS (lower images) (UplanSApo 40ti /0.90; lower images using Olympus BX51 microscope with camera Olympus UC30) showing, respectively, strong and moderate Notch1 expression in tumor cells. The arrowheads show Notch1 positive tumor cells and the arrow displays Notch1 negative reactive T-lymphocytes. (b) Quantification of Notch1 expression in lymph node biopsies from patients with PTL-NOS, ALCL ALKti and ALCL ALKti . The histograms show the percentage of patients classified into one of the three categories (negative, moderately positive and positive), as described in the “Materials and methods.” Comparison of the expression profiles shows no statistically significant difference in Notch expression (p ti 0.15).
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(f) Control GIS I 0.4 µM GSI IX 20 µM GSI XX 20 µM GSI XXI 20 µM
Figure 2. Inhibition of Notch by γ-secretase inhibitors affects cell viability and apoptosis. (a) Effect of 24 h GSI I (0.2–1 μM) treatment on cell viability in the ALKti ALCL cell line Karpas-299. Cell viability was expressed as the percentage of propidium iodide (PI)-negative cells measured by flow cytometry. *p-Value ti 0.05 compared with vehicle treatment. Means with SD. (b) Western blot analysis of whole-cell lysates from Karpas- 299 treated with GSI I (0.05–5 μM) or vehicle (DMSO) and analyzed with Western blotting. Blots were probed with antibodies against Notch1–4 (intracellular domains are visualized as bands at ~110 kDa [Notch1 and ti2], ~90 kDa [Notch3] and ~52 kDa [Notch4]) and Hes, the transcription factor induced by Notch signaling. (c) Karpas-299 cultured with GSI I (0.4 μM), GSI IX, XX or XXI (20 μM) for 48 h followed by flow cytometric cell counting and compared to vehicle. Bars represent data from four independent experiments with each experiment performed in triplicate. Means with SD. (d, e) Induction of proapoptotic caspases 3 and 7 after treatment of Karpas-299 by increasing concentrations of different GSIs: I, IX, XX and XXI for 24 h. Data are shown as a fold-increase over residual caspase 3/7 activity in control, vehicle-treated cells. p ti 0.05 for all concentrations of GSI I compared to vehicle treatment. p ti 0.05 for concentrations of GSI IX of 40 and 80 μM, for GSI XX of 5–40 μM and for GSI XXI for all tested concentrations. Means with SD. (f) Karpas-299 was treated with 0.4 μM GSI I or 20 μM GSI IX, XX or XXI for 48 h and stained with annexin-V and propidium iodide for flow cytometry, as described in “Materials and methods.” Cells were gated according to annexin-V (green FL1 channel, x-axis) and PI-specific (red, FL3 channel, y-axis) fluorescence. Values in the quadrants represent the percentages of cells; the experiment was repeated twice with similar results. (g) PARP cleavage in Karpas-299 cells treated with 0.05-5 μM GSI I for 24 h. Whole-cell extracts were prepared for Western blot as in Figure 1 and blots were probed with antibody against PARP detecting its intact form (116 kDa) and the caspase-cleaved 89 kDa fragment.
for 18 h with GSI I at concentrations of 0.5 μM or greater [Figure 2(g)].
Treatment with GSI I induces G2/M cell cycle arrest
The effect of GSI on the cell cycle was assessed in the GSI- treated cells by flow cytometry. Figure 3(a) shows a repre- sentative result of Karpas-299 incubated for 24 h with GSI I at 0.2, 0.4 or 0.6 μM, resulting in a marked dose-dependent accumulation of cells in the G2/M cell cycle phase (from 15% [0 μM] to 42% [0.6 μM]). As the percentage of cells in G2/M increased, the percentage of cells in G0/G1 phase decreased (from 61% [0 μM] to 36% [0.6 μM]). A similar trend was seen at 4 h of incubation at a higher concentration (5 μM). GSI IX, XX or XXI at 20 μM did not alter the cell cycle distribution (data not shown). Further, we demonstrated by flow cytom- etry and Western blotting that the GSI I-induced cell cycle restraint was associated with an up-regulation of p21CIP1/WAF1 [Figures 3(b) and 3(c)]. p21CIP1/WAF1 is an inhibitor of most of
the cyclin-dependent kinases, and is known to induce cell cycle arrest at G1 as well as G2 [15]. Since Karpas-299 cells have a non-functional point mutation of the p53 gene, this up-regulation indicates a p53-independent mechanism [16]. Expression of another cyclin-dependent inhibitor, p27KIP1, did not change in response to GSI I treatment (data not shown).
Discussion
Notch signaling has emerged as a target for the treatment of both solid and hematopoietic cancers [5–8,17–19], and its inhibition can be exploited through promising pharmaco- logical drugs identified as GSIs [14]. Previous investigations have suggested that Notch1 plays a critical role in the patho- genesis of cutaneous T-cell lymphoma, including primary cutaneous ALCL [9–11,20]. In the present study we focused on systemic ALCL (ALKti and ALKti) and PTL-NOS, and
Figure 3. Effect of GSI I treatment on the cell cycle and p21CIP1/WAF1 expression. (a) Karpas-299 cells were treated with GSI I 0.2–06 μM or vehicle for 24 h and stained with 7-aminoactinomycin (7-AAD) (FL3 channel) for cell cycle analysis. Representative histograms of a set of three independent experiments show the DNA content. (b) Cells were stained with 7-AAD and p21CIP1/WAF1 as described in “Materials and methods.” Quantification of flow cytometric data showing an increase in p21CIP1/WAF1 expression after treatment with GSI I 0.4, 0.6 or 0.8 μM for 24 h. Bars represent data from two independent experiments. Means with SD. (c) Expression of p21CIP1/WAF1 determined by Western blotting performed as described in.
showed that the vast majority of these lymphomas displayed positivity for Notch1. The level of Notch expression has previously been shown to correlate inversely with clinical outcome in other types of cancer [21,22]. In systemic ALCL, positive ALK expression correlates with a favorable progno- sis, whereas negative ALK expression correlates with poorer overall survival, and PTL-NOS again has an inferior outcome compared with ALKti ALCL [3]. We found that the level of Notch1 expression was independent of the lymphoma sub- type, and this therefore does not add further to the under- standing of their distinct clinical behaviors.
To our knowledge, no publications exist regarding Notch expression in PTL-NOS. A study by Jundt et al. implicated oncogenic Notch signaling in systemic ALCL [23]. Using an antibody specific for the intracellular domain of Notch1, the authors found a high expression of Notch1 in tumor cells from all 12 patients with systemic ALCL included, of which nine were ALKti. In addition, the ligand Jagged-1 was expressed in malignant and bystander cells co-localizing with Notch1- positive tumor cells. In vitro experiments confirmed that Jagged-1 activated Notch1 and resulted in tumor cell prolif- eration. In our study we used an antibody specific for full- length and truncated Notch1, and found that 80% of ALKti ALCL and 56% of ALKti ALCL showed positivity for Notch1. Differences in results between the studies may arise from different staining characteristics of the antibodies used, and, in addition, Jundt et al. [23] did not address the definitions of weak and strong Notch1 protein expression, which makes direct comparisons difficult. Notch1 activating mutations such as the t(7;9)(q34;q34.3) chromosomal translocation, mutations in the extracellular heterodimerization domain and/or the C-terminal PEST-domain have been described in hematopoietic cancers of T-cell acute lymphoblastic leu- kemia (T-ALL) [5,8,24] and chronic lymphocytic leukemia
[25,26]. Mutations have also been described in other genes involved in the regulation of the Notch pathway, such as the FBXW7 gene in T-ALL [27]. As oncogenic Notch signaling has been implicated in many cancers, with the detection of acti- vating Notch mutations of the Notch receptor itself or altera- tions in proteins involved in N-IC turnover in only a minority of these [5,8,25–29], other mechanisms likely contribute to constitutive activation. Ligand-mediated activation of the pathway is believed to be an important mechanism in some cancers where Notch activating mutations have not been discovered, and based on previously published data, it is possible that this also applies to ALCL [9,23].
Several approaches for Notch inhibition have been devel- oped or are theoretically possible, but GSIs are currently the only form of Notch inhibitors tested in clinical trials of, for example, T-cell acute leukemias, breast cancer and glioblas- toma [14,30,31]. However, in general, the clinical effects of GSI treatment so far have not been very convincing, and have resulted in significant toxicity [30,31]. We performed func- tional studies on the ALKti ALCL cell line, Karpas-299, with different GSIs: I, IX, XX and XXI. Notch inhibition resulted in decreased cell viability and induction of apoptosis with a superior efficacy of GSI I compared to the three other tested compounds. As reported previously in other cancers, GSI I also promoted a dose-dependent G2/M cell cycle arrest [32,33]. The superiority of GSI I is in line with our previous findings in cutaneous T-cell lymphoma as well as those reported by others in, for example, breast cancer and Kaposi’s sarcoma [10,11,32,34]. It is conceivable that GSIs, apart from the specific inhibition of Notch, also have off-target effects [14]. GSI I has structural similarity to a well-known protea- some inhibitor, MG-132, and we and others have shown that proteasome inhibition is an important mechanism of GSI I in some tumor cells such as cutaneous T-cell lymphoma,
breast cancer and precursor-B acute lymphoblastic leuke- [15] Ando T, Kawabe T, Ohara H, et al. Involvement of the interaction
mia [35–37].
In conclusion, this study demonstrates the expression of Notch1 in tumor cells of ALCL (ALKti and ALKti ) and PTL- NOS. Since the expression of Notch1 was similar, differences in Notch signaling cannot explain diversities in the clinical behavior of these lymphomas. Furthermore, the induction of cell death by Notch inhibitors supports the proposal that inactivation of Notch is a future therapeutic target for patients suffering from peripheral T-cell lymphomas.
Acknowledgement
We thank Ms. Vibeke Pless for excellent technical assistance.
Potential conflict of interest: Disclosure forms provided
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