Acadesine induces apoptosis in B cells from mantle cell ... - Nature

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Nov 18, 2004 - 6 Li MJ, McMahon R, Snyder DS, Yee JK, Rossi JJ. Specific killing of .... Castan˜o, Dr Gabriel Pons, Dr Jordi Llorens, Daniel Iglesias and.
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292 BAF15. Likewise, with 7.8 kcal/mol BAF22 has a very weak 50 -end of the antisense strand compared to 12.2 kcal/mol for BAF24. In addition, the 50 -end of the antisense strand is much weaker than the 50 -end of the sense strand (7.8 vs 12.1 kcal/ mol). According to the current models for siRNA activity, the thermodynamic properties of BAF22 can explain its superior activity compared to BAF24. In conclusion, the relatively high frequency of effective siRNA sequences observed in our experiments further supports the idea that translocations associated with human leukemias may serve as attractive targets for strategies applying RNAi.

Acknowledgements This work was financially supported by the Robert Bosch foundation Project No. 11.5.8002.0006.1.

Duality of Interest One of the authors (H-P Vornlocher) is employed by a company (Alnylam Europe AG) whose potential product (bcr-abl siRNA) was studied in this work.

L Wohlbold1 H van der Kuip1 A Moehring1 G Granot2 M Oren2 H-P Vornlocher3 WE Aulitzky4

1

Dr Margarete Fischer-Bosch Institute of Clinical Pharmacology, Stuttgart, Germany; 2 Department of Molecular Cell Biology, Weizmann Institute of Science, Rehovot, Israel; 3 Alnylam Europe AG, Kulmbach, Germany; and 4 2nd Department of Internal Medicine, Oncology and Hematology, Robert Bosch Hospital, Stuttgart, Germany

References 1 Novina CD, Sharp PA. The RNAi revolution. Nature 2004; 430: 161–164. 2 Kurzrock R, Kantarjian HM, Druker BJ, Talpaz M. Philadelphia chromosome-positive leukemias: from basic mechanisms to molecular therapeutics. Ann Intern Med 2003; 138: 819–830. 3 Wilda M, Fuchs U, Wossmann W, Borkhardt A. Killing of leukemic cells with a BCR/ABL fusion gene by RNA interference (RNAi). Oncogene 2002; 21: 5716–5724. 4 Scherr M, Battmer K, Winkler T, Heidenreich O, Ganser A, Eder M. Specific inhibition of bcr-abl gene expression by small interfering RNA. Blood 2003; 101: 1566–1569. 5 Wohlbold L, van der Kuip H, Miething C, Vornlocher HP, Knabbe C, Duyster J et al. Inhibition of bcr-abl gene expression by small interfering RNA sensitizes for imatinib mesylate (STI571). Blood 2003; 102: 2236–2239. 6 Li MJ, McMahon R, Snyder DS, Yee JK, Rossi JJ. Specific killing of Ph+ chronic myeloid leukemia cells by a lentiviral vector-delivered anti-bcr/abl small hairpin RNA. Oligonucleotides 2003; 13: 401–409. 7 Kronenwett R, Haas R, Sczakiel G. Kinetic selectivity of complementary nucleic acids: bcr-abl-directed antisense RNA and ribozymes. J Mol Biol 1996; 259: 632–644. 8 Khvorova A, Reynolds A, Jayasena SD. Functional siRNAs and miRNAs exhibit strand bias. Cell 2003; 115: 209–216. 9 Schwarz DS, Hutvagner G, Du T, Xu Z, Aronin N, Zamore PD. Asymmetry in the assembly of the RNAi enzyme complex. Cell 2003; 115: 199–208. 10 Freier SM, Kierzek R, Jaeger JA, Sugimoto N, Caruthers MH, Neilson T et al. Improved free-energy parameters for predictions of RNA duplex stability. Proc Natl Acad Sci USA 1986; 83: 9373–9377.

Acadesine induces apoptosis in B cells from mantle cell lymphoma and splenic marginal zone lymphoma

Leukemia (2005) 19, 292–294. doi:10.1038/sj.leu.2403593 Published online 18 November 2004 TO THE EDITOR

Mature B-cell neoplasms include B-cell chronic lymphocytic leukemia (B-CLL), follicular lymphoma (FL), mantle cell lymphoma (MCL) and splenic marginal zone B-cell lymphoma (SMZL), among others.1 These neoplasms have an increasing incidence in Western countries, where they account for about 75% of all lymphoid neoplasms. Therapeutic options for mature B-cell neoplasms include alkylating agents, purine nucleoside analogues, combination chemotherapy, interferon, and monoclonal antibodies. Although most patients respond to chemotherapy, these neoplasms are characterized by a continuous pattern of relapse and there is no standard treatment.2–4 Moreover, current chemotherapeutic agents are not selective for tumor cells and consequently they have several negative effects on the quality of life of those treated. For instance, purine analogues induce apoptosis of T cells, which leads to immunosuppression. Thus, new therapeutic drugs with a differential effect on B and T lymphocytes are of great interest. Correspondence: Dr J Gil, Departament de Cie`ncies Fisiolo`giques II, Universitat de Barcelona, C/Feixa Llarga s/n, E-08907 L’Hospitalet de Llobregat, Spain; Fax: þ 34 93 4024268; E-mail: [email protected] Received 22 July 2004; accepted 7 October 2004; Published online 18 November 2004 Leukemia

Blood samples from patients with peripheral blood manifestation of the disease contain both tumor and normal lymphocytes. Thus, analysis of drug-induced apoptosis in the B-cell and T-cell population by flow cytometry is a useful method to study the cell-type selectivity of anticancer drugs or combinations.5 We have previously reported that acadesine (5-aminoimidazole-4carboxamide riboside, AICAR) induces apoptosis in B cells from B-CLL patients.5 This apoptotic effect is caspase-dependent and p53-independent. Normal B lymphocytes are as sensitive as B-CLL cells to acadesine-induced apoptosis, while T lymphocytes are resistant. Here, we examine whether acadesine induces apoptosis in cells from other mature B-cell neoplasms. After informed consent, peripheral blood samples of SMZL, MCL, and FL patients from the Hematology Unit at the Hospital Universitari de Bellvitge (Barcelona, Spain) were collected. Diagnosis was based on clinical, morphologic, immunophenotypic and molecular criteria following the WHO classification.1 A total of 34 patients were included (21 SMZL, eight MCL and five FL). Eight of the 21 SMZL samples and six of the eight MCL samples were positive for CD5, determined by flow cytometry. All MCL samples were positive for cyclin D1 expression, as measured by immunohistochemistry. All FL samples were positive for CD10 and Bcl-2, negative for CD5, and all of them were low grade FL (with no transformation). All samples were positive for CD19, CD20 and presented clonality of surface immunoglobulins.

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293 Table 1

Characteristics of the B-cell neoplasm samples LK/ml (  106)

LY/ml (  106)

Ba (%)

Ta (%)

TH

SMZL 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21

12.3 33.5 9.0 12.7 10.0 10.7 11.8 78.8 5.2 9.2 4.3 34.1 51.0 22.8 11.3 9.3 13.2 18.9 9.0 8.5 9.0

5.9 25.1 8.5 6.9 2.4 4.7 8.7 71.7 3.4 7.6 1.9 31.0 41.3 13.8 4.6 5.2 6.2 17.5 4.4 3.9 2.5

31 80 50 7 45 40 30 78 39 55 24 45 74 20 15 50 ND 80 50 ND 10

40 12 20 5 30 30 43 15 30 15 25 23 12 30 40 32 ND 8 37 ND 27

F F F F S F S F F F F F F S F F S S, C S C S

MCL 22 23 24 25 26 27 28 29

11.7 12.0 41.6 94.7 18.6 39.6 21.4 13.0

7.1 9.6 33.7 86.2 10.2 38.8 18.0 11.4

50 52 80 80 80 80 65 75

20 6 12 6 7 4 23 13

C C C F F C S C

FL 30 31 32 33 34

11.5 35.5 55.0 33.0 10.6

7.8 31.6 53.9 30.7 3.2

59 75 74 62 50

20 6 6 10 11

F F F F C

N

N ¼ patient number; LK ¼ leukocytes; LY ¼ lymphocytes; B ¼ B cells; T ¼ T cells; TH ¼ therapy; ND ¼ nondetermined; S ¼ splenectomy; C ¼ chemotherapy with CHOP (cyclophosphamide + prednisone + doxorubicin + vincristine); F ¼ no treatment. a Percentage calculated from the total mononuclear cells after isolation.

Briefly, mononuclear cells were isolated from peripheral blood by centrifugation on a Ficoll/Hypaque gradient and cultured in RPMI 1640 medium, 10% heat-inactivated fetal bovine serum, 2 mmol/l glutamine, 100 U penicillin and 100 ng/ ml streptomycin, as previously described.5 The percentage of B and T cells in the isolated mononuclear cells was determined by flow cytometry by coincubation with APC-conjugated anti-CD3 and PE-conjugated anti-CD19 antibodies (Becton Dickinson, Mountain View, CA, USA) immediately after isolation (Table 1). Cells from each sample were incubated for 24 h with increasing doses of acadesine (Toronto Research Chemicals Inc., North York, Canada) ranging from 0.2 to 2 mM, 3 mM fludarabine or 10 mM chlorambucil, and apoptosis in B and T cells was quantified by surface annexin V staining as previously described.5 A live-gate for the CD19 þ /CD3 cells (B-cell population) and for the CD19/CD3 þ cells (T-cell population)

Figure 2 Effect of acadesine on B cells from SMZL, MCL and FL. Peripheral lymphocytes from patients were incubated for 24 h with increasing doses of acadesine ranging from 0.2 to 2 mM. The EC50 (half-maximal effective concentration) for each sample is shown in the figure. We consider that a sample is resistant when there is no apoptotic effect at 2 mM anadesine.

Figure 1 Acadesine induces apoptosis in B cells without affecting T cells. Mononuclear cells from a MCL sample (patient 24) were treated with 0.5 mM of acadesine for 24 h. Phosphatidylserine exposure was analysed in B cells (CD19 þ and CD3) and T cells (CD19 and CD3 þ ) by flow cytometry. The percentages of non-apoptotic (black dots) and apoptotic (grey dots) cells are determined for each population. The figure shows the distribution of these populations in 3D. Leukemia

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294

Figure 3 Effect of acadesine, fludarabine and chlorambucil on SMZL cells. Mononuclear cells from 4 SMZL samples were cultured with 0.5 mM (0.5), 1 mM (1) or 2 mM (2) acadesine, 3 mM fludarabine (F) or 10 mM chlorambucil (Clb) for 24 h. Viability was measured by analysis of phosphatidylserine exposure in the CD19 þ population, as described in the text. Cell viability is expressed as the percentage of non-apoptotic cells with respect to control cells at 24 h of culture.

was drawn and the percentages of nonapoptotic and apoptotic cells were determined for each population (Figure 1). Viability was expressed as the percentage of nonapoptotic cells. Samples were acquired and data were analyzed using Cell Quest software (Becton Dickinson). We observed a dose-dependent decrease in B-cell viability in the 21 SMZL samples treated with acadesine, and the mean EC50 (half-maximal effective concentration) for SMZL B cells was 1.270.7 mM (Figure 2). Moreover, acadesine induced apoptosis in B cells from seven of the eight MCL patients and one sample from FL. B cells from one MCL sample (patient 29) and four of the five FL samples were resistant to acadesine at doses up to 2 mM. At 2 mM, acadesine did not induce apoptosis in T cells from any of the 34 samples from mature B-cell neoplasms (results from one sample are shown in Figure 1), which is consistent with our previous report on T cells from B-CLL patients or healthy donors.5 The alkylating agent chlorambucil, and the nucleoside analogue fludarabine have been used in the treatment of SMZL patients.6 Thus, we analyzed the apoptotic effect of 3 mM fludarabine and 10 mM chlorambucil, which correspond to the therapeutically achievable plasmatic levels of these drugs. Cells from the four patients studied responded to acadesine in a dose-dependent manner. However, only one of the four samples (patient 17) responded to fludarabine and chlorambucil ex vivo (Figure 3). The mechanism of acadesine-induced apoptosis is unknown. Incorporation of acadesine into the cell and its subsequent phosphorylation to AICA ribotide (ZMP) are necessary to induce apoptosis in B-CLL cells.5 The only molecular target of ZMP described to date is adenosine monophosphate-activated protein kinase (AMPK), and acadesine induces the activation of AMPK in B-CLL cells. In contrast, in Jurkat cells, inhibition of ZMP synthesis does not inhibit acadesine-induced apoptosis.7 The involvement of AMPK in acadesine-induced apoptosis in B cells requires further investigation. Preliminary data suggest that acadesine does not induce p53 accumulation in SMZL or MCL cells (data not shown), as described for B-CLL cells.5 Most FL samples were resistant to acadesine-induced apoptosis. All FL analyzed were low-grade FL, thus we cannot rule out that other types could be more sensitive to acadesine. Acadesine enters FL cells as it induces phosphorylation of AMPK (results not shown). Bcl-2 is overexpressed in both FL and B-CLL; however, B-CLL cells are sensitive to acadesine-induced apoptosis. It would be very interesting to analyze this resistance as it could provide clues to understand the mechanism of acadesine-induced apoptosis. Previous clinical studies in patients show that acadesine is well tolerated by healthy individuals when administered Leukemia

intravenously, and plasma concentrations in the range of those producing apoptosis in B cells (0.3 mM)8 are achieved. In conclusion, on the basis of our results, we propose that acadesine may provide an effective treatment for mature B-cell neoplasms such as SMZL and MCL.

Acknowledgements This study was supported by a grant from the Ministerio de Ciencia y Tecnologı´a and FEDER (SAF 2001-3026) to J Gil and by Advanced In Vitro Cell Technologies, SL (Barcelona). We thank Dr Jose´ Manuel Lo´pez, Dr Montserrat Barraga´n, Dr Esther Castan˜o, Dr Gabriel Pons, Dr Jordi Llorens, Daniel Iglesias and Llorenc¸ Coll for helpful discussion and suggestions, and R Rycroft for language assistance. AF Santidria´n is recipient of a research fellowship from the Ministerio de Educacio´n y Ciencia.

C Campa`s1 AF Santidria´n1 A Domingo2 J Gil1

1 Departament de Cie`ncies Fisiolo`giques II, IDIBELL-Universitat de Barcelona, Hospitalet de Llobregat, Spain; and 2 Servei d’Hematologia, IDIBELL-Hospital Universitari de Bellvitge, Hospitalet de Llobregat, Spain

References 1 Jaffe ES, Harris NL, Stein H, Vardiman JW (eds). World Health Organization Classification of tumors. Pathology and Genetics Tumors of Hemopoietic and Lymphoid tissues. IARC Press, 2001. 2 Franco V, Florena AM, Iannitto E. Splenic marginal zone lymphoma. Blood 2003; 101: 2464–2672. 3 Bertoni F, Zucca E, Cotter FE. Molecular basis of mantle cell lymphoma. Br J Haematol. 2004; 124: 130–140. 4 Reiser M, Diehl V. Current treatment of follicular non-Hodgkin’s lymphoma. Eur J Cancer 2002; 38: 1167–1172. 5 Campa`s C, Lo´pez JM, Santidria´n AF, Barraga´n M, Bellosillo B, Colomer D et al. Acadesine activates AMPK and induces apoptosis in B-cell chronic lymphocytic leukemia cells but not in T lymphocytes. Blood 2003; 101: 3674–3680. 6 Lefrere F, Hermine O, Belanger C, Francois S, Tilly H, Lebas de La Cour JC et al. Fludarabine: an effective treatment in patients with splenic lymphoma with villous lymphocytes. Leukemia 2000; 14: 573–575. 7 Lo´pez JM, Santidria´n AF, Campa`s C, Gil J. 5-Aminoimidazole-4carboxamide riboside induces apoptosis in Jurkat cells, but the AMP-activated protein kinase is not involved. Biochem J 2003; 370: 1027–1032. 8 Dixon R, Gourzis J, McDermott D, Fujitaki J, Dewland P, Gruber H. AICA-riboside: safety, tolerance, and pharmacokinetics of a novel adenosine-regulating agent. J Clin Pharmacol 1991; 31: 342–347.