sign in sign up
<<
Home , Asparaginase

ANTICANCER RESEARCH 30: 2119-2124 (2010)

Differential Ex Vivo Activity of in Newly Diagnosed Paediatric Acute Lymphoblastic and Myeloblastic Leukaemia

JOANNA SZCZEPANEK1, MONIKA POGORZALA1, BENIGNA KONATKOWSKA2, EDYTA JURASZEWSKA3, WANDA BADOWSKA4, IGOR OLEJNIK5, MARTA KUZMICZ6, ELZBIETA STANCZAK7, IWONA MALINOWSKA7, JOLANTA STEFANIAK8, GRAZYNA SOBOL9, TOMASZ SZCZEPANSKI10, KRZYSZTOF CZYZEWSKI1, MARIUSZ WYSOCKI1 and JAN STYCZYNSKI1

1Department of Pediatric Hematology and Oncology, Collegium Medicum, Nicolaus Copernicus University, Bydgoszcz, Poland; 2Department of Oncology, Hematology and Pediatric Transplantology, Medical University, Poznan, Poland; 3Department of Pediatric Hematology and Oncology, Jagiellonian University, Collegium Medicum, Cracow, Poland; 4Department of Pediatric Hematology and Oncology, Children Hospital, Olsztyn, Poland; 5Department of Pediatric Hematology and Oncology, Pediatric Center, Chorzow, Poland; 6Department of Pediatric Oncology, Medical University, Bialystok, Poland; 7Department of Pediatric Hematology and Oncology, Medical University, Warsaw, Poland; 8Department of Pediatric Hematology and Oncology, Medical University, Lublin, Poland; 9Department of Pediatrics, Division of Oncology, Hematology and , Medical University, Katowice, Poland; 10Department of Pediatric Hematology and Oncology, Medical University, Zabrze, Poland

Abstract. The , bortezomib, is known to thioguanine. Bortezomib was the only compound which was be effective in the therapy of various neoplasms. The objective more active in T-ALL than in common/pre-B-ALL paediatric of this study was the analysis of the ex vivo activity of samples. In conclusion, bortezomib had good ex vivo activity bortezomib in paediatric acute lymphoblastic leukaemia (ALL), in paediatric T-ALL samples. in comparison to paediatric acute myeloid leukaemia (AML). A total of 159 patients entered the study, including 106 ALL Bortezomib is a selective proteasome inhibitor and a potent (including 86 precursor-B-cell ALL, and 20 T-cell ALL) and 53 compound that shows strong activity in in vitro and in vivo AML children. The ex vivo sensitivity to bortezomib and 16 laboratory studies against many solid and haematological other drugs was studied by MTT assay. Paediatric AML tumour types (1). Through inhibition of the nuclear factor samples were more resistant than paediatric ALL samples to (NF)-κB pathway, it has a chemosensitising effect when most of the tested drugs, except for and thioguanine. administered together with other antitumoural drugs (2). With respect to immunophenotype, ex vivo drug resistance in Bortezomib is the first proteasome inhibitor approved for use in T-cell ALL (T-ALL) was higher for most of the drugs. No humans (3). Its efficacy and safety have been documented in differences in drug resistance between T-ALL and common/pre- multiple myeloma (4, 5). In vitro and in vivo studies have B-cell-ALL were found for , and 6- demonstrated the activity of bortezomib in prostate (6), breast, lung (7, 8), colon (9), bladder (10), ovary (6) and pancreatic cancer (11), squamous cell carcinoma (12) and acute leukaemia (13, 14). Bortezomib activity has been shown in human Correspondence to: Jan Styczynski, MD, Ph.D., Department of melanoma (15), human neuroendocrine tumour cell lines (16) Pediatric Hematology and Oncology, Collegium Medicum, Nicolaus and in glioblastoma (17). Preclinical studies have demonstrated Copernicus University, ul. Curie-Sklodowskiej 9, 85-094 the ability of bortezomib to chemosensitize and overcome Bydgoszcz, Poland. Tel: +48 525854860, Fax: +48 525854867, chemotherapy resistance (18, 19). In in vitro studies, NF-κB e-mail: [email protected] inhibition specifically induced apoptosis in acute myeloid Key Words: Drug resistance, individualised tumour response testing, leukaemia (AML) cells and potentiated the apoptotic response bortezomib, children, acute lymphoblastic leukaemia, acute myeloid to and cytarabine, while sparing normal CD34+ leukaemia. hematopoietic precursors (20). Bortezomib activity has been

0250-7005/2010 $2.00+.40 2119 ANTICANCER RESEARCH 30: 2119-2124 (2010) demonstrated against four acute lymphoblastic leukaemia 0.001-1 μg/ml), 4-HOO- (Asta Medica, Hamburg, (ALL) cell lines in an in vivo panel (21). However, although Germany; 0.096-100 μg/ml), 6-thioguanine (Sigma, nr A4882; 1.56- bortezomib treatment inhibited NF-κB activity, bortezomib had 50 μg/ml), (Bristol-Myers Squibb, Sermoneta, Italy; 0.048- little activity as a single agent in refractory ALL patients in a 50 μg/ml), cytarabine (Upjohn, Puurs, Belgium; 0.24-250 μg/ml), phosphate (Schering AG, Berlin, Germany; 0.019-20 phase I study (13). Since the NF-κB pathway is highly active in μg/ml), (Busilvex, Pierre-Fabre-Medicament, Castres, France; the subpopulation of T-cell ALL (T-ALL) carrying activating 1.17-1200 μg/ml), (Medac; 0.0005-1 μg/ml), mutations in the NOTCH1 gene (22), the present study (Lederle, Wolfratshausen, Germany, 0.032-100 μg/ml) and melfalan hypothesised that paediatric T-ALL samples may have good in (Glaxo Wellcome, Parma, Italy; 0.038-40 μg/ml). vitro sensitivity to bortezomib. The aim of this study was the analysis of in vitro resistance MTT assay. Cellular drug resistance was tested by MTT assay, as to bortezomib and 15 additional anticancer drugs in de novo described previously (14, 17, 24). The drug concentration that was inhibitory to 50% of the cells (IC ) was calculated from the dose- paediatric ALL, in comparison to AML patients, as well as to 50 response curve and was used as a measure for in vitro drug resistance T-ALL and chronic myeloid leukaemia (CML) cell lines. The in each sample. The relative resistance (RR) between groups analysed study was performed as an in vitro individualised tumour for each drug was calculated as the ratio of the mean values of IC50 of response testing (ITRT) (23, 24). the respective groups for this drug.

Patients and Methods Statistical analysis. IC50 values for cell lines were given as median±standard deviation, while for patient groups they were given Patients and leukaemic cells. The leukaemia cells of 159 children (86 as median and quartiles. The Mann-Whitney U-test was performed to male, 73 female, median age 7 years, range 0.1-18 years) with acute compare differences in drug resistance between groups. Correlations leukaemia were tested for their in vitro ITRT (drug-resistance profile). in resistance between groups were determined by Spearman’s rho The group included 106 de novo ALL and 53 de novo AML patients. coefficient. All tests were two-sided, with a p-value of 0.05 being According to the ALL immunophenotype, 86 children had pre- considered statisitically significant. B/common ALL phenotype and 20 had T-ALL phenotype. Samples of bone marrow (BM) were collected in a heparinised tube Results (or 15-20 U heparine was used for 1 ml BM). Before testing, the samples were diluted 1:1 or more with RPMI-1640 (Sigma, St Louis, AML de novo samples were more resistant than ALL de novo MO, USA). In the case of small clots, the BM sample was first filtered samples to most of the drugs tested, and only sensitivity to through a cell strainer (70 μm nylon filter; Falcon, Franklin Lakes, NJ, USA) using RPMI-1640 to rinse off the strainer. Leukaemic cells were cytarabine and thioguanine did not differ between both types separated on a Ficoll gradient at 540 ×g for 20 minutes at room of leukaemia (Table I). ALL cells were 1.8-fold more sensitive temperature. After centrifugation, cells were washed twice with RPMI- to bortezomib when compared to AML cells (p=0.005). 1640. The viability and recovery of the cells was tested by trypan blue With respect to ALL immunophenotype, it was found that ex exclusion assay. Cell morphology and percentage of blasts were vivo drug resistance was observed in T-ALL for prednisolone, analysed on cytospine slides stained using the May-Grunwald-Giemsa , L-asparaginase, , , 4-HOO- (MGG) method. Only samples with at least of 90% of leukaemia cyclophosphamide, cytarabine, fludarabine and etoposide. blasts, both in the beginning and at the end of the assay, were included in the study. The study was approved by the local Ethics Committee Drugs used in high-dose therapy before haematopoietic stem and written informed consent was obtained from all patients. cell transplantation (such as busulfan, treosulfan, thiotepa and melfalan) were less active in AML, however this only reached Cell lines. Four cell lines were used for the study: CCRF-CEM and statistical significance for melfalan (Table II). No differences in Jurkat (T-ALL), and K652 and KU-812 (CML). Cells were maintained drug resistance between T-ALL and common/pre-B-ALL were in RPMI 1640 medium (Sigma), supplemented with 2 mM found for daunorubicin, mitoxantrone and 6-thioguanine. The and 20% foetal bovine (FBS). The culture medium was only compound which was more active in T-ALL than in supplemented with 100 U/ml penicillin (Polfa, Tarchomin, Poland), common/pre-B-ALL was bortezomib. This drug was 1.25-fold 100 μg/ml streptomycin (Polfa), 200 μg/ml gentamicin (Krka, Slovenia) and 0.125 μg/ml amphotericin B. The culture was carried more active against T-ALL blasts (Figure 1). However, when out in conditions of 5% CO2, 37˚C, and 95% humidity. All the dosage of chemotherapy is taken into account, there is only experiments on the cell lines were performed at least 4 times. a 25% difference in dose of the drug. Thus, although the difference reached a statistical value, it probably has little Drugs. The following 17 drugs were used: bortezomib (Velcade, clinical significance. Janssen Pharmaceutica N.V., Beerse, Belgium; concentrations tested: The activity of bortezomib and the other 16 drugs tested was 19-2000 nM), prednisolone (Jelfa, Jelenia Gora, Poland; 0.0076-250 compared in samples of paediatric leukaemia and tested cell μg/ml), vincristine (Gedeon Richter, Budapest, Hungary; 0.019-20 lines (Table III). Sensitivity of both T-ALL cell lines was μg/ml), L-asparaginase (Medac, Hamburg, Germany; 0.0032-10 IU/ml), daunorubicin (Rhone-Poulenc Rorer, Paris, France; 0.0019-2 comparable with respect to most of the drugs. When compared μg/ml), doxorubicin (Pharmacia Italia S.p.A., Milan, Italy; 0.031-40 to the activity of the tested drugs in patient samples, T-ALL μg/ml), idarubicin (Pharmacia; 0.0019-2 μg/ml), mitoxantrone (Jelfa; cell lines were equally or more sensitive for all drugs. Finally,

2120 Szczepanek et al: Bortezomib in Paediatric ALL and AML

Table I. Drugs tested and comparison of their in vitro resistance in paediatric ALL and AML de novo.

Drug Median and quartiles of IC50 RR p-Value

ALL (n=106) AML (n=53)

Bortezomib 213.1 375.3 1.8 0.005 17.5-516 (n=56) 266->2000 (n=34) Prednisolone 17.7 130 7.3 <0.001 1.4-120 (n=106) 76->250 (n=40) Vincristine 0.1 0.5 4.5 0.003 0.07-0.63 (n=105) 0.11->20 (n=39) L-Asparaginase 0.4 1.6 4 0.003 0.2-1.6 (n=105) 0.6->10 (n=35) Daunorubicin 0.31 0.70 2.2 <0.001 0.25-0.40 (n=105) 0.35-1.22 (n=40) Doxorubicin 1.04 1.77 1.7 0.006 0.43-1.63 (n=71) 0.84-4.95 (n=28) Idarubicin 0.23 0.44 1.9 <0.001 0.10-0.35 (n=94) 0.31-1.09 (n=38) Mitoxantrone 0.13 0.46 3.5 <0.001 0.06-0.30 (n=66) 0.21->1 (n=28) 4-HOO-Cyclophosphamide 0.27 1.2 4.4 <0.001 0.16-0.84 (n=39) 0.26-5.13 (n=21) 6-Thioguanine 5.1 9.6 1.9 0.081 3.0-9.6 (n=76) 4.6->50 (n=36) Cytarabine 0.41 0.49 1.2 0.284 0.14-0.91 (n=92) 0.32-1.26 (n=38) Fludarabine phosphate 0.31 0.91 2.9 0.010 0.20-0.92 (n=69) 0.30->20 (n=29) Etoposide 2.04 11.2 5.5 0.042 0.88-5.98 (n=90) 0.9-45 (n=37) Busulfan 14.15 24.12 1.7 0.015 0.02->1200 (n=65) 1.17->1200 (n=32) Treosulfan 0.01 0.40 40 0.048 0.0005->1.0 (n=23) 0.093->1 (n=17) Thiotepa 1.04 3.72 3.6 0.041 0.47-2.02 (n=30) 1.78-12.61 (n=13) Melfalan 0.99 1.92 1.9 0.005 0.01->40 (n=69) 0.44-4.26 (n=35)

IC50: Value of in vitro resistance, given in IU/ml for L-asparaginase, ng/ml for bortezomib and μg/ml for the remaining drugs; RR: relative resistance=median IC50 (AML)/median IC50 (ALL); n: number of patients; p-value: Mann-Whitney U-test. both CML cell lines were more resistant to all tested drugs than Possible explanations for these findings include several were T-ALL cell lines, as well as the vast majority of paediatric factors. Firstly, bortezomib inhibits the NF-κB pathway. This ALL and AML samples. pathway is present in a high proportion of T-ALL samples, but not in precursor-B-lineage ALL samples. Constitutively Discussion active NOTCH1 activates the NF-κB pathway transcriptionally and via the IκB kinase complex in T-ALL, To date, data on the activity or use of bortezomib in acute showing that the NF-κB pathway is highly active in lymphoblastic leukaemia are very limited. This study showed established human T-ALL (22). Inhibition of the pathway can that the ex vivo activity of bortezomib is higher in paediatric efficiently restrict tumour growth both in vitro and in vivo. ALL samples than in paediatric AML samples. This observation Since most individuals with T-ALL carry activating mutations was true for most of the tested antileukaemia drugs. Moreover, in the NOTCH1 gene, inhibition of the NF-κB pathway may when ALL samples were divided into subgroups characterized be a potential target of future therapies of T-ALL (22). by immunophenotype, most of the drugs were more active Secondly, differences in the in vitro sensitivity of ALL cells to against common/pre-B-ALL samples than T-ALL samples. bortezomib are related to variability in the activity profiles of There was only one exception, bortezomib, which was more the individual proteasomal subunits among primary leukaemia active in paediatric T-ALL samples. cells (25, 26). Thirdly, bortezomib shows no cross-resistance

2121 ANTICANCER RESEARCH 30: 2119-2124 (2010)

Table II. Drugs tested and comparison of their in vitro resistance in paediatric T-ALL and common/pre-B-ALL de novo.

Drug Median and quartiles of IC50 RR p-Value

Common/pre-B-ALL (n=86) T-ALL (n=20)

Bortezomib 232 185 0.8 0.042 21-516 (n=36) 17.5-208 (n=20) Prednisolone 12.5 94 7.5 <0.001 1.4-95 (n=86) 76-120 (n=20) Vincristine 0.10 0.25 2.5 0.011 0.07-0.33 (n=85) 0.14-0.63 (n=20) L-Asparaginase 0.35 1.24 3.4 0.002 0.20-0.95 (n=85) 0.40-1.60 (n=20) Daunorubicin 0.30 0.35 1.2 0.181 0.25-0.38 (n=85) 0.28-0.40 (n=20) Doxorubicin 0.88 1.35 1.5 0.016 0.43-1.61 (n=53) 0.84-1.63 (n=18) Idarubicin 0.23 0.44 1.9 <0.001 0.10-0.35 (n=76) 0.31-1.09 (n=18) Mitoxantrone 0.12 0.14 1.2 0.452 0.06-0.27 (n=50) 0.08-0.30 (n=16) 4-HOO-Cyclophosphamide 0.23 0.59 2.6 0.002 0.16-0.71 (n=28) 0.25-0.84 (n=11) 6-Thioguanine 4.6 7.2 1.6 0.275 3.0-7.7 (n=56) 4.1-9.6 (n=20) Cytarabine 0.31 0.72 2.3 0.004 0.14-0.65 (n=72) 0.22-0.91 (n=20) Fludarabine phosphate 0.30 0.74 2.5 0.008 0.20-0.83 (n=50) 0.28-0.92 (n=19) Etoposide 1.55 2.95 1.9 0.011 0.88-4.11 (n=70) 1.20-5.98 (n=20) Busulfan 11.30 23.28 2.1 0.065 0.02-1125 (n=47) 0.35->1200 (n=18) Treosulfan 0.005 0.02 4 0.123 0.0005->1.0 (n=16) 0.003->1 (n=7) Thiotepa 0.92 1.68 1.8 0.245 0.47-1.67 (n=19) 0.87-2.02 (n=11) Melfalan 0.87 2.05 2.4 0.002 0.01-17.2 (n=54) 0.04->40 (n=15)

IC50: Value of in vitro resistance, given in IU/ml for L-asparaginase, ng/ml for bortezomib and μg/ml for the remaining drugs; RR: relative resistance=median IC50 (T-ALL)/median IC50 (common/pre-B-ALL); n: number of patients; p-value: Mann-Whitney U-test. to daunorubicin, doxorubicin, and etoposide in ALL Activity of bortezomib in ALL is an important focus of cells (26); thus due to its different mechanism and research currently being investigated not only in vitro, but also independence from multidrug-resistance , it is more in vivo studies (13). Clinical studies confirmed that bortezomib active in subsets of ALL samples. Fourthly, it has been shown has antileukaemia activity and effective drug concentrations are recently that the valosin-containing (VCP/p97), part readily attainable in patients (29, 30). So far however, the of the ubiquitin proteasome degradation pathway (UPDP), clinical benefit has usually been minor, with only a modest and was identified as a differentially expressed protein in transient decrease of peripheral blood and BM blasts. prednisone good and poor responder ALL patients (27). This Significant interpatient heterogeneity in intrinsic drug may indicate that the UPDP is possibly altered and may be sensitivity was seen. Another tested option is to combine involved in multi-agent chemotherapy resistance in childhood bortezomib with standard chemotherapy or modern targeted ALL patients. Thus, possibly, VCP expression modulates drug therapy (31, 32). response in childhood ALL (28). Finally, proteasome In conclusion, bortezomib has good activity in paediatric inhibitors like bortezomib seem to be able to sensitise ALL samples, as obtained by ITRT results. These data glucocorticoid-resistant childhood ALL cells for prednisone provide a new insight into the spectrum of bortezomib treatment. Therefore, a drug targeting the proteasome may be activity and could assist in the design and rationale for future a novel therapeutic option in resistant ALL (28). clinical trials.

2122 Szczepanek et al: Bortezomib in Paediatric ALL and AML

Figure 1. Comparative values of median cytotoxicity of bortezomib with respect to diagnosis. AML: acute myeloid leukaemia; c/pre-B-ALL: common/pre-B-cell-acute lymphoblastic leukaemia; T-ALL: T-cell-acute lymphoblastic leukaemia.

Table III. In vitro drug sensitivity (IC50) in cell lines.

Drug CCRF-CEM Jurkat K-562 KU-812

Bortezomib 210±109 241±78 1260±355 1655±482 Prednisolone 11.66±5.78 18.86±1.31 152.7±9.61 193±17.64 Vincristine 0.27±0.10 0.31±0.11 1.07±0.42 1.68±0.51 L-Asparaginase 0.65±0.36 0.24±0.04 >10 >10 Daunorubicin 0.08±0.01 0.13±0.02 0.52±0.15 0.80±0.03 Doxorubicin 0.28±0.08 0.47±0.19 4.31±1.10 5.17±2.70 Idarubicin 0.15±0.01 0.19±0.09 1.24±0.31 1.24±0.43 Mitoxantrone 0.13±0.07 0.14±0.07 0.81±0.34 0.73±0.25 4-HOO-Cyclophosphamide 0.17±0.07 0.18±0.0.8 4.53±1.89 8.46±3.63 6-Thioguanine 2.6±0.6 3.2±0.7 24.7±6.6 35.6±11.2 Cytarabine 0.13±0.04 0.10±0.08 6.52±2.83 2.72±0.68 Fludarabine phosphate 0.56±0.30 0.32±0.42 19.5±4.1 15.3±3.2 Etoposide 3.73±1.22 1.64±0.73 17.49±4.62 18.64±3.15 Busulfan 9.3±2.2 17.1±4.8 54.6±12.5 70.4±6.7 Treosulfan 0.041±0.034 0.036±0.026 0.773±0.445 0.763±0.347 Thiotepa 0.48±0.11 1.09±0.49 9.51±1.49 18.50±2.12 Melfalan 0.76±0.23 0.49±0.16 18.11±1.91 24.05±6.37

In vitro sensitivity IC50 is given in IU/ml for L-asparaginase, ng/ml for bortezomib and μg/ml for the remaining drugs; values are expressed as median±standard deviation (of at least 4 independent experiments).

Acknowledgements summary for bortezomib for injection in the treatment of multiple myeloma. Clin Cancer Res 10: 3954-3964, 2004. This study was supported by grants: MNiSW N407 078 32/2964 and 4 Richardson PG, Barlogie B, Berenson J, Singhal S, Jagannath S, UMK 10/2009. The Authors thank Beata Kolodziej, Beata Rafinska- Irwin D, Rajkumar SV, Srkalovic G, Alsina M, Alexanian R, Siegel Kurylo and Malgorzata Kubicka for their technical assistance. D, Orlowski RZ, Kuter D, Limentani SA, Lee S, Hideshima T, Esseltine DL, Kauffman M, Adams J, Schenkein DP and Anderson KC: A phase 2 study of bortezomib in relapsed, refractory myeloma. References N Engl J Med 348: 2609-2617, 2003. 5 Richardson PG, Sonneveld P, Schuster MW, Irwin D, Stadtmauer 1 Wojcik C and DeMartino GN: Intracellular localization of EA, Facon T, Harousseau JL, Ben-Yehuda D, Lonial S, Goldschmidt proteasomes. Int J Biochem Cell Biol 35: 579-589, 2003. H, Reece D, San-Miguel JF, Blade J, Boccadoro M, Cavenagh J, 2 Ludwig H, Khayat D, Giaccone G and Facon T: Proteasome Dalton WS, Boral AL, Esseltine DL, Porter JB, Schenkein D and inhibition and its clinical prospects in the treatment of hematologic Anderson KC: Bortezomib or high-dose dexamethasone for relapsed and solid malignancies. Cancer 104: 1794-1807, 2005. multiple myeloma. N Engl J Med 352: 2487-2498, 2005. 3 Bross PF, Kane R, Farrell AT, Abraham S, Benson K, Brower ME, 6 Frankel A, Man S, Elliott P, Adams J and Kerbel RS: Lack of Bradley S, Gobburu JV, Goheer A, Lee SL, Leighton J, Liang CY, multicellular drug resistance observed in human ovarian and Lostritto RT, McGuinn WD, Morse DE, Rahman A, Rosario LA, prostate carcinoma treated with the proteasome inhibitor PS-341. Verbois SL, Williams G, Wang YC and Pazdur R: Approval Clin Cancer Res 6: 3719-3728, 2000.

2123 ANTICANCER RESEARCH 30: 2119-2124 (2010)

7 Teicher BA, Ara G, Herbst R, Palombella VJ and Adams J: The 22 Vilimas T, Mascarenhas J, Palomero T, Mandal M, Buonamici S, proteasome inhibitor PS-341 in cancer therapy. Clin Cancer Res 5: Meng F, Thompson B, Spaulding C, Macaroun S, Alegre ML, Kee 2638-2645, 1999. BL, Ferrando A, Miele L and Aifantis I: Targeting the NF-kappaB 8 Ling YH, Liebes L, Jiang JD, Holland JF, Elliott PJ, Adams J, signaling pathway in Notch1-induced T-cell . Nat Med 13: Muggia FM and Perez-Soler R: Mechanisms of proteasome 70-77, 2007. inhibitor PS-341-induced G(2)-M-phase arrest and apoptosis in 23 Bosanquet AG, Richards SM, Wade R, Else M, Matutes E, Dyer human non-small cell lung cancer cell lines. Clin Cancer Res 9: MJ, Rassam SM, Durant J, Scadding SM, Raper SL, Dearden CE 1145-1154, 2003. and Catovsky D: Drug cross-resistance and therapy-induced 9 Cusack JC Jr, Liu R, Houston M, Abendroth K, Elliott PJ, Adams resistance in chronic lymphocytic leukaemia by an enhanced J and Baldwin AS Jr: Enhanced chemosensitivity to CPT-11 with method of individualised tumour response testing. Br J Haematol proteasome inhibitor PS-341: implications for systemic nuclear 146: 384-395, 2009. factor-kappaB inhibition. Cancer Res 61: 3535-3540, 2001. 24 Styczynski J, Gil L, Derwich K, Wachowiak J, Balwierz W, 10 Caravita T, de Fabritiis P, Palumbo A, Amadori S and Boccadoro Badowska W, Krawczuk-Rybak M, Matysiak M, Wieczorek M, M: Bortezomib: efficacy comparisons in solid tumors and Balcerska A, Sonta-Jakimczyk D, Stefaniak J, Kowalczyk J, hematologic malignancies. Nat Clin Pract Oncol 3: 374-387, Urasinski T, Sobol G, Komarnicki M and Wysocki M: Comparison 2006. of activity in childhood and adult acute leukemia: 11 Nawrocki ST, Bruns CJ, Harbison MT, Bold RJ, Gotsch BS, individual tumor response study. Anticancer Res 29: 1643-1650, Abbruzzese JL, Elliott P, Adams J and McConkey DJ: Effects of 2009. the proteasome inhibitor PS-341 on apoptosis and angiogenesis in 25 Kraus M, Ruckrich T, Reich M, Gogel J, Beck A, Kammer W, orthotopic human pancreatic tumor xenografts. Mol Cancer Ther Berkers CR, Burg D, Overkleeft H, Ovaa H and Driessen C: 1: 1243-1253, 2002. Activity patterns of proteasome subunits reflect bortezomib 12 Ramadan KM, McKenna KE and Morris TC: Clinical response of sensitivity of hematologic malignancies and are variable in primary cutaneous squamous-cell carcinoma to bortezomib given for human leukemia cells. Leukemia 21: 84-92, 2007. myeloma. Lancet Oncol 7: 958-959, 2006. 26 Lu S, Chen Z, Yang J, Chen L, Gong S, Zhou H, Guo L and Wang 13 Horton TM, Pati D, Plon SE, Thompson PA, Bomgaars LR, J: Overexpression of the PSMB5 gene contributes to bortezomib Adamson PC, Ingle AM, Wright J, Brockman AH, Paton M and resistance in T-lymphoblastic /leukemia cells derived Blaney SM: A phase 1 study of the proteasome inhibitor bortezomib from Jurkat line. Exp Hematol 36: 1278-1284, 2008. in paediatric patients with refractory leukemia: a Children's 27 Lauten M, Schrauder A, Kardinal C, Harbott J, Welte K, Oncology Group study. Clin Cancer Res 13: 1516-1522, 2007. Schlegelberger B, Schrappe M and von Neuhoff N: Unsupervised 14 Gil L, Styczynski J, Dytfeld D, Debski R, Kazmierczak M, proteome analysis of human leukaemia cells identifies the Valosin- Kolodziej B, Rafinska B, Kubicka M, Nowicki A, Komarnicki M containing protein as a putative marker for glucocorticoid and Wysocki M: Activity of bortezomib in adult de novo and resistance. Leukemia 20: 820-826, 2006. relapsed acute myeloid leukemia. Anticancer Res 27: 4021-4025, 28 Junk S, Lauten M, Cario G, Wittner N, Schrappe M, 2007. Schlegelberger B and von Neuhoff N: Proteasome inhibitor 15 Freudlsperger C, Thies A, Pfuller U and Schumacher U: The bortezomib induces apoptosis in prednisone-resistant childhood proteasome inhibitor bortezomib augments anti-proliferative effects acute lymphoblastic leukemia cells. Blood 114: 991 (abstract), of mistletoe lectin-I and the PPAR-gamma agonist rosiglitazone in 2009. human melanoma cells. Anticancer Res 27: 207-213, 2007. 29 Cortes J, Thomas D, Koller C, Giles F, Estey E, Faderl S, Garcia- 16 Larsson DE, Lovborg H, Rickardson L, Larsson R, Oberg K and Manero G, McConkey D, Ruiz SL, Guerciolini R, Wright J and Granberg D: Identification and evaluation of potential anticancer Kantarjian H: Phase I study of bortezomib in refractory or relapsed drugs on human neuroendocrine tumor cell lines. Anticancer Res acute . Clin Cancer Res 10: 3371-3376, 2004. 26: 4125-4129, 2006. 30 Orlowski RZ, Voorhees PM, Garcia RA, Hall MD, Kudrik FJ, 17 Styczynski J, Olszewska-Slonina D, Kolodziej B, Napieraj M and Allred T, Johri AR, Jones PE, Ivanova A, Van Deventer HW, Wysocki M: Activity of bortezomib in glioblastoma. Anticancer Gabriel DA, Shea TC, Mitchell BS, Adams J, Esseltine DL, Trehu Res 26: 4499-4503, 2006. EG, Green M, Lehman MJ, Natoli S, Collins JM, Lindley CM and 18 Voorhees PM, Dees EC, O'Neil B and Orlowski RZ: The Dees EC: Phase 1 trial of the proteasome inhibitor bortezomib and proteasome as a target for cancer therapy. Clin Cancer Res 9: 6316- pegylated liposomal doxorubicin in patients with advanced 6325, 2003. hematologic malignancies. Blood 105: 3058-3065, 2005. 19 Yang HH, Ma MH, Vescio RA and Berenson JR: Overcoming 31 Miller CP, Ban K, Dujka ME, McConkey DJ, Munsell M, drug resistance in multiple myeloma: the emergence of Palladino M and Chandra J: NPI-0052, a novel proteasome therapeutic approaches to induce apoptosis. J Clin Oncol 21: inhibitor, induces caspase-8 and ROS-dependent apoptosis alone 4239-4247, 2003. and in combination with HDAC inhibitors in leukemia cells. Blood 20 Frelin C, Imbert V, Griessinger E, Peyron AC, Rochet N, Philip P, 110: 267-277, 2007. Dageville C, Sirvent A, Hummelsberger M, Berard E, Dreano M, 32 Kurosu T, Ohki M, Wu N, Kagechika H and Miura O: Sorafenib Sirvent N and Peyron JF: Targeting NF-kappaB activation via induces apoptosis specifically in cells expressing BCR/ABL by pharmacologic inhibition of IKK2-induced apoptosis of human inhibiting its kinase activity to activate the intrinsic mitochondrial acute myeloid leukemia cells. Blood 105: 804-811, 2005. pathway. Cancer Res 69: 3927-3936, 2009. 21 Houghton PJ, Morton CL, Kolb EA, Lock R, Carol H, Reynolds CP, Keshelava N, Maris JM, Keir ST, Wu J and Smith MA: Initial testing (stage 1) of the proteasome inhibitor bortezomib by the Received February 13, 2010 pediatric preclinical testing program. Pediatr Blood Cancer 50: 37- Revised May 2, 2010 45, 2008. Accepted May 10, 2010

2124