The discovery of novel anti-cancer drugs targeting anaplastic lymphoma kinase (ALK), an oncogenic tyrosine kinase, raises the need for in vitro assays suitable for screening compounds for ALK inhibition. To this aim we have developed and optimized an ALK-specific enzyme-linked immunosorbent assay that employs a novel ALK peptide substrate and purified ALK kinase domain

Donella-Deana, Arianna

Gambacorti-Passerini, Carlo

Gunby, Rosalind Helen

Porchia, Francesca

Scapozza, Leonardo

Tartari, Carmen Julia

GUNBY RH

TARTARI CJ

PORCHIA F

SCAPOZZA L

GAMBACORTI PASSERINI C.

DONELLA, ARIANNA

Archivio istituzionale della ricerca - Università di Padova

Correspondence: Emmanuel Raffoux, Département d’Hématologie,Hôpital Saint-Louis, 1, avenue Claude Vellefaux, 75010 Paris,France. Phone: international  +33.1.42499643. Fax: international+33.1.42499345. E-mail: emmanuel.raffoux@sls.aphp.fr References1. Duprez E, Lillehaug JR, Naoe T, Lanotte M. cAMP signalling isdecisive for recovery of nuclear bodies (PODs) during matura-tion of RA-resistant t(15;17) promyelocytic leukemia NB4 cellsexpressing PML-RARa. Oncogene 1996;12:2451-9.2. Gore SD, Weng LJ, Figg WD, Zhai S, Donehower RC, DoverG, et al. Impact of prolonged infusions of the putative differ-entiating agent sodium phenylbutyrate on myelodysplasticsyndromes and acute myeloid leukaemia. Clin Cancer Res2002;8:963-70.3. Phiel CJ, Zhang F, Huang EY, Guenther MG, Lazar MA, KleinPS.  Histone deacetylase is a direct target of valproic acid, apotent anticonvulsant, mood stabilizer, and teratogen. J BiolChem 2001;276:36734-41. 4. Göttlicher M, Minucci S, Zhu P, Krämer OH, Schimpf A,Giavara S, et al.  Valproic acid defines a novel class of HDACinhibitors inducing differentiation of transformed cells. EMBOJ 2001;20:6969-78.5. Kuendgen A, Strupp C, Aivado M, Bernhardt A, HildebrandtB, Haas R, et al.  Treatment of myelodysplastic syndromeswith valproic acid alone or in combination with all-transretinoic acid. Blood 2004;104:1266-9.6. Kieslich M, Schwabe D, Cinatl J, Driever PH. Increase of fetalhemoglobin synthesis indicating differentiation induction inchildren receiving valproic acid. Pediatr Hematol Oncol 2003;20:15-22.7. Cheson BD, Bennett JM, Kopecky KJ, Büchner T, Willman CL,Estey EH, et al.  Revised recommendations of the internation-al working group for diagnosis, standardization of responsecriteria, treatment outcomes, and reporting standards for ther-apeutic trials in acute myeloid leukemia. J Clin Oncol 2003;21:4642-9.8. Cheson B, Bennett J, Kantarjian H, Pinto A, Schiffer CA, NimerSD, et al. Report of an international working group to stan-dardize response criteria for myelodysplastic syndromes.Blood 2000;96:3671-4.9. Ichiyama T, Okada K, Lipton JM, Matsubara T, Hayashi T,Furukawa S. Sodium valproate inhibits production of TNF-aand IL-6 and activation of NF-kB. Brain Res 2000; 857:246-51.10. Tang R, Faussat AM, Majdak P, Perrot JY, Chaoui D, LegrandO, et al. Valproic acid inhibits proliferation and induces apop-tosis in acute myeloid leukemia cells expressing P-gp andMRP1. Leukemia 2004;18:1246-51.Malignant LymphomasAn enzyme-linked immunosorbent assay toscreen for inhibitors of the oncogenic anaplasticlymphoma kinaseThe discovery of novel anti-cancer drugs target-ing anaplastic lymphoma kinase (ALK), an onco-genic tyrosine kinase, raises the need for in vitroassays suitable for screening compounds for ALKinhibition. To this aim we have developed and opti-mized an ALK-specific enzyme-linked immunosor-bent assay that employs a novel ALK peptide sub-strate and purified ALK kinase domain.haematologica 2005; 90:988-990(http://www.haematologica.org/journal/2005/7/988.html)The anaplastic lymphoma kinase (ALK) is a receptortyrosine kinase normally expressed in the developingnervous system.1 However, chromosomal translocationsinvolving the ALK gene (2p23) lead to the expression ofconstitutively activated ALK fusion proteins such asNPM/ALK, in tissues outside the nervous system.2,3 ALKfusion proteins stimulate mitogenic and anti-apoptotic sig-naling pathways, leading to malignant transformation incancers such as anaplastic large cell lymphoma (ALCL).4-6Therefore, ALK represents a valid target for pharmaceuti-cal intervention. In the present study, we describe thedevelopment of an enzyme-linked immunosorbent assay(ELISA) that can be used to rapidly screen compounds fortheir ability to inhibit ALK in vitro.His-tagged recombinant ALK (rALK) protein contain-ing the predicted ALK kinase domain spanning aminoacid residues 1116-1392 (NCBI Accession Code:Q9UM73), was expressed in Sf9 cells using a bac-ulovirus expression system. rALK protein was purifiedto apparent homogeneity as assessed by sodium dode-cylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) and silver staining and was shown to maintainautophosphorylation activity throughout the purifica-tion procedure, indicating the correct folding of thepurified protein (Figure 1A). Using purified rALK weidentified a specific peptide substrate of ALK (pep-tide#1: ARDIYRASFFRKGGCAMLPVK) resembling theALK activation loop (ARDIYRASYYRKGGCAMLPVK),which displayed a phosphorylation rate 3-fold higherthan that of the general substrate, polyGlu4Tyr (Figure1B). In the ALK-ELISA, peptide#1 was immobilized onan ELISA plate and phosphorylated by purified rALK inthe presence of ATP and co-factors. The immobilizedphosphorylated substrate was detected using anti-phos-photyrosine and horse-radish peroxidase (HRP)-conju-gated antibodies. Addition of HRP substrate stimulateda colorimetric reaction which was detected by spec-trophotometry. Assay conditions were optimized formeasuring ALK inhibition. A saturating amount of pep-tide#1 (2.5 mg/well) and an amount of rALK within thelinear range of the colorimetric response (15 – 150 ngprotein/well) were used. Plotting phosphorylation ofpeptide#1 by rALK against time indicated that there wasa linear increase in absorbance up to 16 minutes (Figure1C). No increase in absorbance was observed in theabsence of peptide or with a negative control peptide,indicating that background noise was negligible. A reac-tion time within the linear range (10 min) was chosenfor maximum sensitivity in measuring ALK inhibition. The reliability of the ALK-ELISA in measuring ALKinhibition was assessed using the general kinaseinhibitor, staurosporine. In a conventional in vitroradioactive kinase assay, performed in the presence of 30mM ATP, staurosporine inhibited phosphorylation of pep-tide#1 by rALK with an IC50 of 123 nM (Figure 2A). In theALK-ELISA performed under the same conditions, theIC50 value for staurosporine was identical to thatobtained in the radioactive assay (150 nM) (Figure 2B). Inthe presence of 300 mM ATP an IC50 value of approxi-mately 700 nM was obtained in the ALK-ELISA. Thisincrease in IC50 at higher concentrations of ATP reflectsthe competition between staurosporine and ATP forbinding the ALK kinase domain. As the majority ofkinase inhibitors developed so far are ATP competitors,7and considering the high concentrations of ATP observedin cells (>1 mM), it is desirable that in vitro screeningassays for ALK inhibitors are performed in the presenceof high concentrations of ATP. Unlike the radioactiveassay, which is restricted by specific activity considera-tions, the ALK-ELISA can be performed at ATP concen-trations approaching physiological concentrations, there-fore should generate results closer to those observed incells. Indeed, the IC50 value obtained in the presence of300 mM ATP (700 nM) in the ALK-ELISA is closer to the| 988 | haematologica/the hematology journal | 2005; 90(7)Letters to the Editorhaematologica/the hematology journal | 2005; 90(7) | 989 |Letters to the EditorIC50 value (1 mM) observed for full length NPM/ALKexpressed in BaF3 cells assessed by anti-phosphotyrosineimmunoblotting and densitometry (Figure 2C).Ten compounds derived from the tyrphostin, AG957,developed as a Bcr/Abl inhibitor,8 were screened usingthe ALK-ELISA. These compounds were mostly inactiveABCIc50 = 1.08±0.04 mMStaurosporine (mM)Log10 [Staurosporine] (mM)% Activity0 0.125 0.25 0.5 1.0 5.0100% 126% 113% 82% 52% 3%93 kDa –Anti-p TyrAnti-ALK1-2.5 -2.0 -1.5 -1.0 -0.5 0.0 0.5 1.0RRRRRRRR RR125100755025030 mM ATP: IC50 = 150±41 nM300 mM ATP: IC50 = 678±21 nMStaurosporine (mM)Peptide phosphorylation(% of control)IC50=123 nM0 0.2 0.4 0.6 0.8 1.010080604020RRRR RFigure 2. The effect of staurosporine on ALK activity.Staurosporine concentration-response curves showing inhibitionof peptide#1 phosphorylation by purified rALK determined in theradioactive assay (A) and the ALK-ELISA (B). A. The kinase reac-tion was performed as described in Figure 1B in the presence ofstaurosporine, for 10 mins. B. The ALK-ELISA was performed asdescribed in Figure 1C in the presence of 30 or 300 µM ATP andstaurosporine. Results are normalized to vehicle control andexpressed as the mean ± s.dev. (n=3). IC50 values were deter-mined using GraphPad prism software fitting data using nonlinearregression. C. Inhibition of NPM/ALK autophosphorylation by stau-rosporine. BaF3 pro-B murine cells transfected with NPM/ALKwere treated for 2h with staurosporine. NPM/ALK tyrosine phos-phorylation was determined by anti-phosphotyrosine (anti-pTyr)immunoblotting and protein loading was controlled by anti-ALK1immunoblotting. Band density was determined by densitometryand expressed as a percent of the control. ABCi) Silver stainii) ImmunoblotAnti-ALK1iii) AutoradiographrALK97 kDa –45 kDa –1 2ARDIYRASFFRKGGCAMLPGVKPoly (Glu4 Tyr)No peptideALK peptide 2Control peptideGGGG0 10 20 30 40Time (min)0 20 40 60Time (min)3.0¥1052.0¥1051.0¥10501.51.00.50.0ALKactivity (dpm)R R R RRRRRRA450 nmXXXXXXXFigure 1. A. Production and purification of recombinant (6x)His-taggedALK (rALK). rALK containing amino acids 1073–1459 (NCBI AccessionCode: Q9UM73) was expressed in Sf9 cells using the MaxBac®2.0 bac-ulovirus expression system (Invitrogen). rALK was purified on a Q-sepharose Fast Flow column followed by a HiTrap™-nickel column(Amersham-Pharmacia Biotech). Q-sepharose (lane 1) and HiTrap frac-tions (lane 2) were assessed for: (i) purity by SDS-PAGE and silverstaining; (ii) the presence of ALK by anti-ALK1 immunoblotting; (iii)autophosphorylation activity by a radioactive kinase assay. B.Identification of a peptide substrate of rALK. Phosphorylation of theARDIYRASFFRKGGCAMLPVK peptide#1 (200 µM) and the randompolymer polyGlu4Tyr (0.1 mg/mL) by rALK was measured by an in vitroradioactive kinase assay. Reactions were performed in the presence of50 mM Tris/HCl pH7.5, 5 mM MnCl2, 10 µM Na-vanadate, 30 mM[g32P]ATP (specific activity 1000 cpm/pmol) and 10 units of rALK [1unit = amount of enzyme transferring 1 pmol phosphate/min topolyGlu4Tyr] for 10 min at 30°C. Reactions were spotted onto P81phosphocellulose paper, washed and labeled peptide measured byscintillation. Results are expressed as mean dpm ± s.dev. (n=3). C.Time course of the phosphorylation of peptide#1 in the ALK-ELISAcompared with negative control peptide (GILGFVFTL) or no peptide.Peptide (2.5 mg/well) was immobilized on NuncImmuno plates andphosphorylated by rALK (50 ng/well) in the presence of 300 mM ATP,50 mM Tris pH7.5, 5 mM MnCl2, and 5 mM MnCl2 at 30°C for 10 min.Phosphorylated peptide was detected using the monoclonal 4G10anti-phosphotyrosine antibody (UpstateBiotech Ltd.), an anti-mouseIgG-HRP linked antibody (Amersham Pharmacia Biotech) and HRPsubstrate (tetramethylbenzidine substrate solution, Endogen).Absorbance was read at 450 nm using an Ultrospec‚ 300 microtiterplate reader (Amersham-Pharmacia Biotech). Results are expressedas the mean ± s.dev. (n=3).| 990 | haematologica/the hematology journal | 2005; 90(7)Letters to the Editoron ALK with the most potent derivatives, adaphostin andNSC 689857, having IC50 values of 23 and 10 mM, respec-tively (data not shown). UCN-01, a 7-hydroxy stau-rosporine derivative, reported to have induced diseasestability in a patient with ALK-positive ALCL in a phaseI clinical trial,9 was also tested. UCN-01 inhibited ALK inthe presence of 30 mM ATP (IC50=5 mM), however noinhibition was observed at 300 mM ATP (data not shown).The low potency of UCN-01 and its lack of specificity forNPM/ALK transformed cells in proliferation assays (datanot shown) suggest that UCN-01 does not target ALK.Therefore, potent and specific ALK inhibitors still need tobe developed. The ALK-ELISA is a robust and accuratemethod suitable for middle and high-throughput screeningthat can be applied to the discovery of ALK inhibitors. Rosalind Helen Gunby,* Carmen Julia Tartari,* Francesca Porchia,*Arianna Donella-Deana,° Leonardo Scapozza,#Carlo Gambacorti-Passerini*@*Department of Experimental Oncology, National Cancer Institute,Milan, Italy; °Department of Biological Chemistry and CRIBI,National Research Centre, Institute of Neuroscience, University ofPadua, Italy; #Section des Sciences Pharmaceutiques, University ofGeneva, Switzerland; @McGill University, Montreal, Canada;ˆDepartment of Internal Medicine, University of Milan Bicocca,Milan, ItalyAcknowledgments: the authors gratefully acknowledge O. Marin(University of Padua, Italy) for peptide synthesis, L. Mologni(National Cancer Institute, Milan) for assistance with purification,Dr. P.G. Pellici (European Institute of Oncology, Milan) forproviding pcDNA3-NPM/ALK plasmid, Dr. K. Pulford(John Radcliffe Hospital, Oxford, UK) for providing themonoclonal anti-ALK1 antibody, Dr. E. Sausville (DevelopmentalTherapeutics Program, National Cancer Institute, Bethesda,MD, USA) for providing compounds for screening andG. Tasinato (University of Padova, Italy) for skilful technicalassistance.Funding: this work was supported by the Jose Carreras Foundation,Italian Association for Cancer Research (AIRC),Min. San. Ricerca Finalizzata (2003), CNR, MIUR-COFINand PRIN programs (2003, 2004), EU (Prokinase network,#503467) CIHR, NCI-C.Key words: ALCL, ALK, kinase assay, inhibitor screening.Correspondence: Rosalind Helen Gunby, Department ofExperimental Oncology, National Cancer Institute, via Venezian 1,20133 Milan, Italy. Phone: international +39.02.23902689.Fax: international +39.02.23903237.E-mail: rosalind.gunby@istitutotumori.mi.itReferences1. Morris SW, Naeve C, Mathew P, James PL, Kirstein MN, CuiX, et al. ALK, the chromosome 2 gene locus altered by thet(2;5) in non-Hodgkin's lymphoma, encodes a novel neuralreceptor tyrosine kinase that is highly related to leukocytetyrosine kinase (LTK). Oncogene 1997;14:2175-88.2. Morris SW, Kirstein M0l. Fusion of a kinase gene, ALK, to anucleolar protein gene, NPM, in non-Hodgkin's lymphoma.Science 1994;263:1281-4.3. Pulford K, Morris SW, Turturro F. Anaplastic lymphoma kinaseproteins in growth control and cancer. J Cell Physiol 2004;199:330-58.4. Nieborowska-Skorska M, Slupianek A, Xue L, Zhang Q,Raghunath PN, Hoser G, et al. Role of signal transducer andactivator of transcription 5 in nucleophosmin/ anaplastic lym-phoma kinase-mediated malignant transformation of lym-phoid cells. Cancer Res 2001;61:6517-23.5. Bai RY, Dieter P, Peschel C, Morris SW, Duyster J. Nucleo-phosmin-anaplastic lymphoma kinase of large-cell anaplasticlymphoma is a constitutively active tyrosine kinase that uti-lizes phospholipase C-gamma to mediate its mitogenicity.Mol Cell Biol 1998;18:6951-61.6. Bai RY, Ouyang T, Miething C, Morris SW, Peschel C, DuysterJ. Nucleophosmin-anaplastic lymphoma kinase associatedwith anaplastic large-cell lymphoma activates the phos-phatidylinositol 3-kinase/Akt antiapoptotic signaling path-way. Blood 2000;96:4319-27.7. Fabbro D, Ruetz S, Buchdunger E, Cowan-Jacob SW, FendrichG, Liebetanz J, et al. Protein kinases as targets for anticanceragents: from inhibitors to useful drugs. Pharmacol Ther 2002;93:79-98.8. Svingen PA, Tefferi A, Kottke TJ, Kaur G, Narayanan VL,Sausville EA, et al. Effects of the bcr/abl kinase inhibitorsAG957 and NSC 680410 on chronic myelogenous leukemiacells in vitro. Clin Cancer Res 2000;6:237-49.9. Sausville EA, Arbuck SG, Messmann R, Headlee D, Bauer KS,Lush RM, et al. Phase I trial of 72-hour continuous infusion ofUCN-01 in patients with refractory neoplasms. J Clin Oncol2001;19:2319-33.Malignant LymphomasHighly active antiretroviral therapy and outcomeof AIDS-related Burkitt’s lymphoma or leukemia.Results of the PETHEMA-LAL3/97 studyShort, intensive cycles of chemotherapy haveresulted in improved survival in Burkitt’s lym-phoma/leukemia (BL) in adults.1 The prognosis ofpatients with immunodeficiency virus (HIV)-associ-ated BL is considered to be poor, but these patientshave seldom been treated with BL-specific proto-cols.2 However, a study (PETHEMA-LAL3/97) inwhich patients with BL were treated regardless oftheir HIV status failed to find differences betweenHIV-infected and immunocompetent individuals.3Furthermore, patients who received highly activeantiretroviral therapy (HAART) seemed to have aslightly better disease-free survival than those whodid not (p=0.051). We extended the follow-up analy-sis to elucidate the role of HAART in the survival ofHIV-infected patients included in the PETHEMA-LAL3/97 protocol.haematologica 2005; 90:990-992(http://www.haematologica.org/journal/2005/7/990.html)We present the longer-term results of the multicenterPETHEMA LAL3/97 study on Burkitt’s lymphoma/leu-kemia carried out by the Spanish PETHEMA group. Thediagnostic criteria, characteristics of treatment, responsecriteria and follow-up procedures were reported in theoriginal analysis.3 Briefly, patients over 15 years old  withnewly diagnosed advanced BL (leukemic disease, lym-phoma in stages III-IV or in stage II with a bulky mass)were treated, irrespectively of HIV status, with eightcycles of chemotherapy including alternating combina-tions of cytarabine, methotrexate, cyclophosphamide,ifosfamide, doxorubicin, teniposide, vincristine and dex-amethasone. Triple drug HAART,including at least oneprotease inhibitor and two nucleoside reverse transcrip-tase inhibitors,4 was recommended from diagnosis forHIV-positive patients if they were not receiving it alreadyand was continued thereafter. The present analysisincludes an extended follow-up of the 14 original HIV-positive patients reported3 and 5 additional patientsincluded in the protocol but previously excluded fromanalysis because of insufficient follow-up at that time.Overall survival (OS) and disease-free survival (DFS)were censored in July 2004 or date of last contact.Virological response to HAART was defined as having

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An enzyme-linked immunosorbent assay to screen for inhibitors of the oncogenic anaplastic lymphoma kinase

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An enzyme-linked immunosorbent assay to screen for inhibitors of the oncogenic anaplastic lymphoma kinase

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