Noxa upregulation is associated with apoptosis of chronic lymphocytic ...

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Sep 11, 2008 - The leukemic B-cells were purified (X95%) and immediately cultured as described previously.8. Chronic lymphocytic leukemia cells were ...
Letters to the Editor

594 22 Berman E, Nicolaides M, Maki RG, Fleisher M, Chanel S, Scheu K et al. Altered bone and mineral metabolism in patients receiving imatinib mesylate. N Engl J Med 2006; 354: 2006–2013. 23 Fitter S, Dewar AL, Kostakis P, To LB, Hughes TP, Roberts MM et al. Long term imatinib therapy promotes bone formation in CML patients. Blood 2007 [e-pub ahead of print].

24 Blake SJ, Bruce Lyons A, Fraser CK, Hayball JD, Hughes TP. Dasatinib suppresses in vitro natural killer cell cytotoxicity. Blood 2008; 111: 4415–4416. 25 Schade AE, Schieven GL, Townsend R, Jankowska AM, Susulic V, Zhang R et al. Dasatinib, a small-molecule protein tyrosine kinase inhibitor, inhibits T-cell activation and proliferation. Blood 2008; 111: 1366–1377.

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

Noxa upregulation is associated with apoptosis of chronic lymphocytic leukemia cells induced by hyperforin but not flavopiridol

Leukemia (2009) 23, 594–596; doi:10.1038/leu.2008.239; published online 11 September 2008

Chronic lymphocytic leukemia (CLL) is characterized by the accumulation of a CD5 þ B-lymphocyte clone resulting mainly from apoptosis deficiency. On account of the low response rate of the patients to chemotherapy, new therapeutic strategies based on apoptosis inducers including plant-derived compounds are under investigation (see Kolb et al.1 for review). Flavopiridol, a semisynthetic flavonoid, is a potent inducer of CLL cell apoptosis in vitro, and a promising phase II clinical trial was recently reported.2 The effect of flavopiridol is p53independent and involves caspase-3 activation through the mitochondrial pathway and the downregulation of antiapoptotic proteins, notably Mcl-1. This member of the Bcl-2 family seems to be crucial in CLL, because its high expression levels are related to apoptosis resistance in vitro and to chemoresistance in vivo.3 Hyperforin, a natural phloroglucinol, is another inducer of CLL cell apoptosis4 that exhibits antiangiogenic properties and impairs transporter activities involved in multidrug resistance (see Quiney et al.5 for review). Like flavopiridol, hyperforin acts through the mitochondrial caspase-dependent pathway of apoptosis, but decreases moderately Mcl-1 expression. However, the exact mechanisms whereby hyperforin and flavopiridol lead to the release of apoptogenic factors from mitochondria are not fully understood. Noxa is a proapoptotic BH3-only member of the Bcl-2 family, which binds specifically Mcl-1 (see Willis and Adam6 for review). When its BH3 domain inserts within the hydrophobic groove of Mcl-1, this displaces Mcl-1/Bak interaction, resulting in Bak freeing that leads to mitochondrial membrane permeabilization and finally to caspase activation. Recently, it was reported that Noxa expression levels are inversely related to CLL cell survival capacity.7 Here, we addressed the question of the possible role of Noxa in the apoptosis of CLL cells induced in vitro by hyperforin or flavopiridol. Peripheral blood samples of 12 untreated CLL patients (stage A, according to Binet’s classification) were obtained from the Hematology Department of Hoˆtel-Dieu hospital (Paris, France) after written informed consent, in accordance with the revised Helsinki protocol. Diagnosis was established according to the standard clinical criteria and to the International Workshop on CLL. The leukemic B-cells were purified (X95%) and immediately cultured as described previously.8 Chronic lymphocytic leukemia cells were induced to apoptosis by treatment for 18 h in culture with either 1 mM Leukemia

flavopiridol (a gift from Aventis Pharmaceuticals, Bridgewater, NJ, USA) or 10 mg/ml (18 mM) hyperforin (purified and kindly provided by Dr JD Fourneron, Faculte´ de Pharmacie, Marseille, France). These experimental conditions were previously demonstrated to allow optimal apoptosis induction (notably DNA fragmentation, dissipation of the mitochondrial transmembrane potential and caspase-3 activation).4,8 Noxa expression was analyzed by western blotting from equal amounts of cell lysates (30–50 mg of proteins in SDS-reducing buffer), as described previously.8 Two mAb against Noxa were used: IMG-249 from Imgenex (Clinisciences, Montrouge, France) and clone 6D619 from the US Biological (Euromedex, Mundolsheim, France). Caspase-dependent PARP-1 cleavage was evaluated simultaneously as a control of apoptosis. Expression of Mcl-1 and Bcl-2 was also analyzed, as well as two proteins, which are constitutively overexpressed in CLL cells and downregulated upon flavopiridol and hyperforin-triggered apoptosis: the inducible NO synthase that produces the antiapoptotic NO, and p27kip1 that is cleaved by caspases into a p23 fragment.4,8 Figure 1 shows that Noxa was expressed at low levels by CLL cells from eight patients (1–8). In all cases, hyperforin treatment resulted in a strong increase of Noxa expression and this effect was accompanied with stimulation of PARP cleavage (and p27 cleavage in some cases), as compared to untreated controls. In contrast, flavopiridol treatment, which also induced PARP and p27 cleavages, did not result in stimulation of Noxa expression; this latter was even decreased, likely due to the well-known inhibition of protein synthesis by the flavonoid, as revealed by a reduction in b-actin band intensity. Mcl-1 expression was strongly inhibited by flavopiridol, but occasionally by hyperforin. Regarding Bcl-2, the apparition of a cleaved fragment was observed especially after flavopiridol treatment. As expected, inducible NO synthase downregulation was induced by both compounds. The patient-dependent effects on Mcl-1, Bcl-2 and p27 were in accordance with previous data.4,8 Similar results were observed with four other patients (9–12). Indeed, in all 12 patients, the ratio of Noxa/actin band intensities increased after hyperforin treatment as compared to untreated controls (Figure 2a). This increase ranged from 2.9-fold (patient 4) to 40.3-fold (patient 2). The effect of hyperforin on Noxa/actin ratio was highly significant, whereas this ratio did not change upon flavopiridol exposure (Figure 2b). Our results show for the first time that the upregulation of the proapoptotic protein Noxa is associated with CLL cell apoptosis induced by the natural phloroglucinol hyperforin but not by flavopiridol, another plant-derived compound. Recently, Noxa

Letters to the Editor

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Noxa Mcl-1 Bcl-2

native cleaved

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p27 p23 Actin Patient No

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p27 p23 Actin Figure 1 Effects of flavopiridol and hyperforin on the expression of Noxa and other apoptosis-related proteins in CLL cells from eight patients (1–8). Freshly isolated leukemic cells from each patient were cultured for 18 h in the presence of 1 mM flavopiridol (F) or 10 mg/ml hyperforin (H) or in their absence (controls, C). Western blot experiments were then performed from cell lysates, as described in Materials and methods. Results were similar with the two different anti-Noxa antibodies used. Similar effects on Noxa expression were also found by immunofluorescence and flow cytometry analysis (data not shown). CLL, chronic lymphocytic leukemia.

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Controls Flavopiridol 1 µM Hyperforin 10 µg/ml

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Mean ±SD 0.18 ±0.07 (0.08-0.37) NS

Mean ±SD 1.63 ±0.82 (0.59-3.04) p=0.0002

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Mean ±SD 0.25 ±0.16 (0.06-0.58)

N = 12 Patient No 1 2 3 4 5 6 7 8 9 10 11 12

0.0 1 2 3 4 5 6 7 8 9 10 11 12 Patient No

C

Figure 2 Effects of flavopiridol and hyperforin on the Noxa/actin ratio in CLL cells from the 12 patients of the study. The intensities of Noxa and b-actin bands visualized in the western blots with patients 1–8 shown in Figure 1, and those obtained with four additional patients (9–12) were quantified with the NIH Image 1.62b software, and the Noxa/actin ratios were calculated. (a) Analysis for each of the 12 patients of the Noxa/actin ratio in CLL cells after treatment for 18 h with 1 mM flavopiridol (F) or 10 mg/ml hyperforin (H) and in untreated controls (C). (b) Statistical analysis using the paired t-test, providing a P-value of 0.17 for flavopiridol (NS, not significant) and a highly significant P-value for hyperforin. CLL, chronic lymphocytic leukemia.

upregulation has been demonstrated to mediate apoptosis triggered by the proteasome inhibitor bortezomib in melanoma and two B-cell malignancies, multiple myeloma and mantle cell

lymphoma9,10 and to be involved in bortezomib-elicited apoptosis of CLL cells.7 Similarly, histone deacetylase inhibitors were found to induce CLL cell apoptosis through Noxa Leukemia

Letters to the Editor

596

upregulation.11 Noxa induction was suggested to be transcriptionally regulated in melanoma cells.9 Although the mechanisms controlling Noxa gene expression are not fully defined (see Smit et al.7 for discussion), they appear to be p53-independent in malignant B-cells.9,10 The protein Noxa induced by either bortezomib or histone deacetylase inhibitors was actually found to bind Mcl-1 in CLL cells.7,11 Thus, as Noxa is known to displace Mcl-1/Bak interaction, its upregulation could be a major mechanism by which hyperforin triggers the mitochondrial pathway of caspase activation in CLL cells. Indeed, only marginal effects of the phloroglucinol on Mcl-1 or Bcl-2 were detected here; in addition, Mcl-1 cleavage seems rather a secondary mechanism inasmuch as it was previously found to be caspase-dependent.4 Our observation that flavopiridol did not stimulate Noxa expression indicates that the flavonoid does not act through the same mechanism as hyperforin or other apoptosis inducers in CLL cells. The strong inhibition of Mcl-1 expression by flavopiridol, as a result of either protein degradation or transcriptional inhibition, may be sufficient to allow a sustained Bak activity, together with convergent downregulation of other antiapoptotic proteins.4,8 Moreover, the effects of certain apoptosis inducers could involve proapoptotic Bcl-2 familly members distinct from Noxa and Bak, such as Bak or the BH3-only Puma and Bim.11 Finally, our results favor the concept that hyperforin and agents having the capacity to induce or mimic Noxa represent a novel attractive therapeutic strategy for CLL, as recently suggested.12

Acknowledgements This work was supported by INSERM, Universite´ Pierre et Marie Curie-Paris 6 and by a grant from Canceropole Ile-de-France. We are indebted to Dr JD Fourneron for his gift of hyperforin and to Professor JP Marie and colleagues for providing the blood samples of patients.

M Zaher, I Akrout, M Mirshahi, J-P Kolb and C Billard Centre de Recherches des Cordeliers, Equipe 18, UMRS 872 INSERM; Universite´ Pierre et Marie Curie-Paris 6; Universite´ Paris-Descartes, Paris, France E-mail: [email protected]

References 1 Kolb JP, Kern C, Quiney C, Roman V, Billard C. Re-establishment of a normal apoptotic process as a therapeutic approach in B-CLL. Curr Drug Targets Cardiovasc Hematol Disord 2003; 3: 261–286. 2 Byrd JC, Lin TS, Dalton JT, Wu D, Phelps MA, Fischer B et al. Flavopiridol administered using a pharmacologically derived schedule is associated with marked clinical efficacy in refractory, genetically high-risk chronic lymphocytic leukemia. Blood 2007; 109: 399–404. 3 Kitada S, Andersen J, Akar S, Zapata JM, Takayama S, Krajewski S et al. Expression of apoptosis-regulating proteins in chronic lymphocytic leukemia: correlations with in vitro and in vivo chemoresponses. Blood 1998; 91: 3379–3389. 4 Quiney C, Billard C, Faussat AM, Salanoubat C, Ensaf A, Naı¨t-Si Y et al. Pro-apoptotic properties of hyperforin in leukemic cells from patients with B-cell chronic lymphocytic leukemia. Leukemia 2006; 20: 491–497. 5 Quiney C, Billard C, Salanoubat C, Fourneron JD, Kolb JP. Hyperforin, a new lead compound against the progression of cancer and leukemia? Leukemia 2006; 20: 1519–1525. 6 Willis SN, Adams JM. Life in the balance: how BH3-only proteins induce apoptosis. Curr Opin Cell Biol 2005; 17: 617–625. 7 Smit L, Hallaert DYH, Spijker R, de Goeij B, Jaspers A, Kater AP et al. Differential Noxa/Mcl-1 balance in peripheral versus lymph node chronic lymphocytic leukemia cells correlate with survival capacity. Blood 2007; 109: 1660–1668. 8 Billard C, Kern C, Tang R, Ajchenbaum-Cymbalista F, Kolb JP. Flavopiridol downregulates the expression of both the inducible NO synthase and p27kip1 in malignant cells from B-cell chronic lymphocytic leukemia. Leukemia 2003; 17: 2345–2443. 9 Qin JZ, Ziffra J, Stennett L, Bodner B, Bonish BK, Chaturvedi V et al. Proteasome inhibitors trigger NOXA-mediated apoptosis in melanoma and myeloma cells. Cancer Res 2005; 65: 6282–6293. 10 Pe´rez-Galan P, Roue´ G, Villamor N, Montserrat E, Campo E, Colomer D. The proteasome inhibitor bortezomid induces apoptosis in mantle-cell lymphome through generation of ROS and Noxa induction activation independent of p53 status. Blood 2006; 107: 257–264. 11 Inoue S, Riley J, Gant TW, Dyer MJ, Cohen GM. Apoptosis induced by histone deacetylase inhibitors in leukemic cells is mediated by Bim and Noxa. Leukemia 2007; 21: 1773–1782. 12 Pe´rez-Galan P, Roue´ G, Lopez-Guerra M, Nguyen M, Villamor N, Montserrat E et al. BCL-2 phosphorylation modulates sensitivity to the BH3 mimetic GX15-070 (Obatoclax) and reduces its synergistic interaction with bortezomib in chronic lymphocytic leukemia cells. Leukemia, e-pub ahead of print 3 July 2008; doi: 10.1038/ leu.2008.175.

Polymorphisms in DNA repair and one-carbon metabolism genes and overall survival in diffuse large B-cell lymphoma and follicular lymphoma

Leukemia (2009) 23, 596–602; doi:10.1038/leu.2008.240; published online 2 October 2008

Diffuse large B-cell lymphoma (DLBCL) and follicular lymphoma (FL) are the two most commonly diagnosed subtypes of non-Hodgkin’s lymphoma (NHL) in the United States.1 Both diseases show variable progression, and identifying prognostic markers applicable in a clinical setting could greatly improve patient management. Established adverse prognostic factors in DLBCL as delineated in the International Prognostic Factor Index (IPI) include older age, higher stage, poor performance score, extranodal involvement and above normal lactate dehydrogenase. For FL, adverse prognostic factors are combined Leukemia

in the Follicular Lymphoma International Prognostic Index and include older age, higher stage, lower hemoglobin level, more nodal areas and above normal lactate dehydrogenase. As both IPI and Follicular Lymphoma International Prognostic Index predict risk incompletely, it is thought that molecular characteristics of the tumor and its microenvironment may improve predicting DLBCL and FL survival. Notably, recent gene expression studies suggest a molecular profile of DLBCL with poorer survival.2 New prognostic markers for FL have also been suggested,3 but none have yet been translated to the clinical setting. Evaluating host germline genetic variability offers another promising approach for refining prognosis of DLBCL and FL.