Combined treatment of CpG-oligodeoxynucleotide with Nutlin-3 ...

Uncorrected Version. Published on November 12, 2007 as DOI:10.1189/jlb.0707459

Combined treatment of CpG-oligodeoxynucleotide with Nutlin-3 induces strong immune stimulation coupled to cytotoxicity in B-chronic lymphocytic leukemic (B-CLL) cells Paola Secchiero,*,1 Elisabetta Melloni,* Mario Tiribelli,† and Giorgio Zauli* *Department of Morphology and Embryology, University of Ferrara, Ferrara, Italy; and †Department of Medical and Morphological Researches, Division of Hematology and Bone Marrow Transplantation, University Hospital, Udine, Italy

Abstract: We have investigated the effect of combined treatment with CpG-oligodeoxynucleotide (CpG-ODN) plus Nutlin-3, a small molecule inhibitor of the murine double minute 2/p53 interaction, on the immune activation, cell cycle progression, and apoptosis of peripheral blood B chronic lymphocytic leukemia (B-CLL) cells. CpG-ODN induced a robust up-regulation of immune activation markers (CD54, CD69, CD80, CD86, MHC-II) in Zap70high and Zap70low B-CLL samples. Although cotreatment of B-CLL cells with CpG-ODN ⴙ Nutlin-3 did not interfere with such immune activation, CpG-ODN potentiated the Nutlin-3-mediated induction of the death receptors CD95 and TRAIL receptor 2. Importantly, treatment with CpG-ODN did not interfere with the ability of Nutlin-3 to inhibit cell cycle progression and to induce apoptosis. Thus, a therapeutic regimen including CpGODN plus Nutlin-3 might have the advantage to preserve the immune activation of B-CLL cells while restraining the prosurvival/proliferative potential of CpG-ODN treatment. J. Leukoc. Biol. 83: 000 – 000; 2008. Key Words: leukemia 䡠 p53 䡠 immune activation 䡠 apoptosis

B chronic lymphocytic leukemia (B-CLL) is characterized by the relentless accumulation of monoclonal mature CD5⫹/ CD19⫹ B cells in lymphoid organs, bone marrow, and peripheral blood [1]. Virtually all circulating B-CLL lymphocytes are arrested in the G0/early G1 phase of the cell cycle; however, a proliferating compartment is present in specialized structures called pseudo-follicles that are located in lymph nodes and bone marrow of B-CLL patients [1, 2]. Engagement of the TNFR superfamily member 5 (CD40), which is expressed throughout B cell development, has been shown to up-regulate the expression of costimulatory surface molecules on malignant B cells, which results in increased antigen-presenting capacity of indolent B cell malignancies [3]. However, a major adverse effect of CD40 ligation is its ability to up-regulate the expression of several antiapoptotic genes and to induce B-CLL proliferation [4 – 6]. In this respect, also, synthetic CpG-oligodeoxynucleotides (CpG-ODN), which mimic CpG motifs present in unmethylated bacterial DNA and are recognized by TLR9, 0741-5400/08/0083-0001 © Society for Leukocyte Biology

up-regulate the expression of costimulatory surface molecules of malignant B cells in vitro [7–10]. At variance to CD40 ligation, immune stimulation with CpG-ODN has been described to be accompanied by antiapoptotic and proapoptotic effects in indolent B cell malignancies [7–11], and a phase I trial of CpG-ODN is ongoing in B-CLL patients, irrespective of cytogenetic or mutational status [12]. Recent studies have demonstrated that the small molecules, Nutlins, which represent potent and selective inhibitors of p53/murine double minute 2 interaction [13], effectively induce apoptosis in BCLL when used alone or in combination with chemotherapeutic agents [14, 15] and also impair angiogenesis, at least in vitro [16]. On these bases, the aim of our study was to investigate the effect of combined treatment of CpG-ODN plus Nutlin-3 on relevant clinical parameters, such as immune activation, cell cycle progression, and apoptosis in B-CLL cells. For this purpose, peripheral blood samples were collected in heparincoated tubes from 10 patients (Table 1) fulfilling diagnostic criteria for B-CLL in agreement with institutional guidelines. The B-CLL patients were characterized for a number of clinical parameters, such as stage of the disease, according to the Rai classification, doubling time, levels of Zap-70, whose high expression represents an important negative prognostic marker [17, 18], and p53 status (Table 1). PBMC from B-CLL patients were processed as described previously [14], with a purity ⬎90% of resulting CD19⫹ B cells. All samples were p53 wild-type (Table 1), a requisite for optimal response to Nutlin-3 cytotoxicity [14, 15]. B-CLL samples were cultured at a density of 1 ⫻ 106 cells/ml in RPMI containing 10% FBS. Cells were left untreated or treated with 5 ␮g/ml CpG-ODN (C-class ODN 2395, Coley Pharmaceutical, Wellesley, MA, USA) [19] in the presence of 10 ␮M Nutlin-3 (Cayman Chemical, Ann Arbor, MI, USA) or of control vehicle (0.1% DMSO). The expression levels of Zap-70, as well as the induction of p53 by Nutlin-3, were determined by Western blot analysis, using antibodies

1 Correspondence: Department of Morphology and Embryology, University of Ferrara, Via Fossato di Mortara 66, 44100 Ferrara, Italy. E-mail: [email protected] Received July 12, 2007; revised October 24, 2007; accepted October 24, 2007. doi: 10.1189/jlb.0707459

Journal of Leukocyte Biology Volume 83, February 2008

Copyright 2007 by The Society for Leukocyte Biology.

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TABLE 1.

Clinical Features of the B-CLL Patients

Patient

Sex/Age

Stage

DTa

ZAP-70 statusb

p53 status

1 2 3 4 5 6 7 8 9 10

M/70 M/61 M/62 M/57 M/67 M/65 M/51 M/60 M/75 M/63

2 4 4 0 2 4 4 4 4 0

2 3 6 32 19 3 3 1 2 4

low high high low low low high low high high

wt wt wt wt wt wt wt wt wt wt

a Doubling time (DT; months); b ZAP-70 expression has been evaluated by Western blot on CD19⫹-purified B-CLL cells. wt, Wild-type.

from Upstate Biotechnology (Lake Placid, NY, USA) and Santa Cruz Biotechnology (Santa Cruz, CA, USA), respectively. Flow cytometry analysis of activation markers (CD54, CD69, CD80, CD86, MHC-II), of MHC-I, as well as of the death receptors CD95 and TRAIL receptor 2 (TRAIL-R2) was performed by using FITC- or PE-conjugated mAb from Becton Dickinson (San Jose, CA, USA), R&D Systems (Minneapolis, MN, USA), or Immunotech (Marseille, France). Assessment of cycle progression and apoptosis levels was analyzed by BrdU incorporation and annexin V/propidium iodide (PI), respectively, performed as described previously [14, 16]. CpG-ODN maximally up-regulated the expression of a variety of immune accessory molecules, such as CD54, CD69, CD80, CD86, and MHC-II, in all B-CLL samples after 48 h of treatment (Fig. 1A). Of note, CpG-ODN induced an efficient immune activation irrespectively of the Zap-70 status of the B-CLL samples, as exemplified in Figure 1A. Importantly, Nutlin-3 alone did not modulate the expression of activation

Fig. 1. Effects of the treatment with CpG-ODN ⫾ Nutlin-3 on immune accessory molecules and death receptors in B-CLL cells. B-CLL samples were left unstimulated (Unstim.) or treated with CpG-ODN in the absence (vehicle: 0.1% DMSO) or presence of Nutlin-3. After 48 h, the expression of the indicated surface antigens was analyzed by flow cytometry. (A) Flow cytometric profiles of expression/induction of immune accessory molecules in representative samples from patients characterized by low (#5) and high (#9) ZAP-70 levels. MFI, Mean fluorescence intensity. (B) After 48 h, the expression of surface CD95 and TRAIL-R2 was analyzed by flow cytometry, and results are expressed as means ⫾ SD of analyses performed in duplicate on all 10 patients of Table 1. *, P ⬍ 0.05, compared with cultures unstimulated ⫹ vehicle; **, P ⬍ 0.05, compared with cultures unstimulated ⫹ Nutlin.

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markers, and when added simultaneously to CpG-ODN, it did not interfere with the ability of CpG-ODN to up-regulate the surface activation markers (data not shown). On the other hand, Nutlin-3 alone significantly (P⬍0.05) up-regulated the expression of the death receptors CD95 and TRAIL-R2 (Fig. 1B, shaded bars), two known p53 targets [20], and the combined treatment of Nutlin-3 plus CpG-ODN significantly (P⬍0.05) enhanced CD95 expression further with respect to Nutlin-3 alone (Fig. 1B, open bars). A potential drawback of immune stimulation is the possibility to promote antiapoptotic pathways and thus, to increase the proliferative pool of B-CLL, especially in patients with worse prognostic factors [4 –9, 17, 18]. However, treatment with CpG-ODN has also been shown to elicit a heterogeneous response as a result of the simultaneous induction of B-CLL proliferation and apoptosis [10 –12]. Thus, to evaluate the net balance between proliferation and apoptosis, which represents a relevant biological parameter, we have analyzed the number of viable B-CLL cells after 48 –96 h of exposure to CpG-ODN (Fig. 2A). This time-frame was selected on the basis of preliminary experiments, in which a high variability, in terms of cell viability, became evident from sample to sample after Day 4 of culture, when most of the cultures rapidly died, irrespectively of the presence in culture of CpG-ODN (data not shown), hampering a reliable evaluation of the effects of Nutlin-3. Treatment with CpG-ODN induced a progressive decline in the total number of viable cells with respect to unstimulated cultures (Fig. 2A, open bars). As Nutlin-3 has been shown to inhibit the cell cycle progression and/or to induce apoptotic cell death depending on the cell model investigated [13–16], the cell cycle profile was investigated in B-CLL samples stimulated for 48 h with CpG-ODN in the absence or presence of

Nutlin-3. As shown in Figure 2B, treatment of B-CLL cells with CpG-ODN induced a significant (P⬍0.05) increase of BrdU uptake (from 2.9⫾2.3% in unstimulated to 16.2⫾8.3% in CpG-ODN-stimulated B-CLL samples; means⫾SD of five separate experiments). Of note, the increase in BrdU uptake induced by CpG-ODN stimulation was counteracted significantly (P⬍0.05) by Nutlin-3 (4.5⫾2.3% in B-CLL samples treated with CpG-ODN⫹Nutlin-3; means⫾SD of five separate experiments; Fig. 2B). In parallel, the degree of apoptosis was evaluated by annexin-V/PI double-staining (Fig. 2C). Nutlin-3 promoted apoptosis significantly (P⬍0.05) in unstimulated (from 5⫾2.3% in vehicle-treated to 22⫾5.5% in Nutlin-3treated B-CLL samples; means⫾SD of 10 separate experiments) and CpG-ODN-stimulated B-CLL cultures (from 7⫾3.1% in vehicle-treated to 25⫾7.3% in Nutlin-3-treated B-CLL samples; means⫾SD of 10 separate experiments). Current treatment regimens for indolent B cell malignancies use alkylating agents, purine analogs, mAb, or combinations thereof [21]. Such combination therapies result in greater response rates than those seen with single, agent-based therapy alone, but none of these therapies is curative. Among innovative, therapeutic approaches, the use of CpG-ODN for immunotherapy has recently entered the phase I trial in B-CLL patients [12]. In this respect, the central finding of this study was that Nutlin-3, a potent, nongenotoxic activator of the p53 pathway [13], induced a comparable cytoxicity in quiescent B-CLL cells as well as in B-CLL cells immune-stimulated with CpG-ODN, without interfering with the ability of CpG-ODN to induce immune activation. Moreover, we found that the treatment with CpG-ODN plus Nutlin-3 did not interfere and rather potentiated the expression of the death receptors CD95 and TRAIL-R2. Therefore, our current findings expand previous

Fig. 2. Effects of the treatment of CpG-ODN ⫹ Nutlin-3 on B-CLL cell viability, BrdU incorporation, and apoptosis. B-CLL samples were left unstimulated or treated with CpG-ODN in the absence (vehicle: 0.1% DMSO) or presence of Nutlin-3. (A) Percentage of cell viability was evaluated by Trypan blue after 48 h of exposure to Nutlin-3 in cultures cotreated with CpG-ODN or prestimulated (for 48 h) with CpG-ODN. Results are expressed as means ⫾ SD of analyses performed in duplicate on all 10 patients of Table 1. *, P ⬍ 0.05, compared with cultures unstimulated ⫹ vehicle (set to 100). (B) Representative flow cytometric cell cycle analysis performed by BrdU incorporation in unstimulated and CpG-ODN-stimulated B-CLL cells (from Patient #8). (C) Representative flow cytometric apoptosis measurement by annexin-V/PI in unstimulated and CpG-ODN-prestimulated B-CLL cells (from Patient #7).

Secchiero et al. Effect of CpG-ODN plus Nutlin-3 on B-CLL

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data showing that CD40-mediated immune activation sensitizes B-CLL cells to CD95 ligand (CD95L) and TRAIL-mediated killing [22, 23], suggesting that the combination of immune activation plus Nutlin-3 might further increase the susceptibility of B-CLL to CD95L and TRAIL. Thus, our present data suggest that a strategy of association between CpG-ODN and Nutlin-3 might be effective in achieving an efficient immune activation and to restrain the proliferative potential of CpG-ODN treatment in B-CLL samples, irrespective of its Zap-70 status.

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11.

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ACKNOWLEDGMENT This work was supported by grants from the Italian Association for Cancer Research (AIRC).

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