Published online before print November 12, 2007
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* 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
1 Correspondence: Department of Morphology and Embryology, University of Ferrara, Via Fossato di Mortara 66, 44100 Ferrara, Italy. E-mail: secchier{at}mail.umbi.umd.edu
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 + 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 CpG-ODN 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.
Key Words: leukemia • p53 • immune activation • 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 5 6 ]. In this respect, also, synthetic CpG-oligodeoxynucleotides (CpG-ODN), which mimic CpG motifs present in unmethylated bacterial DNA and are recognized by TLR9, up-regulate the expression of costimulatory surface molecules of malignant B cells in vitro [7 8 9 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 8 9 10 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 B-CLL 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 heparin-coated 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 x 106 cells/ml in RPMI containing 10% FBS. Cells were left untreated or treated with 5 µg/ml CpG-ODN (C-class ODN 2395, Coley Pharmaceutical, Wellesley, MA, USA) [19 ] in the presence of 10 µM 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 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 ].
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Table 1. Clinical Features of the B-CLL Patients
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Figure 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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Figure 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).
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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 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.
ACKNOWLEDGEMENTS
This work was supported by grants from the Italian Association for Cancer Research (AIRC) and Carife Foundation.
Received July 12, 2007; revised October 24, 2007; accepted October 24, 2007.
REFERENCES
This article has been cited by other articles:
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  F. Corallini and C. Celeghini The potential role of Nutlins in the treatment of B-chronic lymphocytic leukemia (B-CLL) J. Leukoc. Biol., September 1, 2008; 84(3): 651 - 651. [Full Text] [PDF]
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