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short report Centrosome aberrations after nilotinib and imatinib treatment in vitro are associated with mitotic spindle defects and genetic instability Alice Fabarius,1 Michelle Giehl,1 Oliver Frank,1 Birgit Spiess,1 Chun Zheng,1 Martin C. Müller,1 Christel Weiss,2 Peter Duesberg,1, 3 Rüdiger Hehlmann,1 Andreas Hochhaus1 and Wolfgang Seifarth1 1 III. Medizinische Universitätsklinik, 2Abteilung für Medizinische Statistik, Medizinische Fakultät Mannheim der Universität Heidelberg, Mannheim, Germany, and 3Department of Molecular and Cell Biology, Donner Laboratory, University of California, Berkeley, CA, USA Received 3 April 2007; accepted for publication Summary Centrosomes play fundamental roles in mitotic spindle organisation, chromosome segregation and maintenance of genetic stability. Recently, we have demonstrated that the tyrosine kinase inhibitor imatinib induces centrosome and chromosome aberrations in vitro. Here, we comparatively investigated the effects of imatinib and the more potent successor drug nilotinib on centrosome, mitotic spindle and karyotype status in primary human fibroblasts. Therapeutic doses of imatinib and/or nilotinib administered separately, consecutively or in combination similarly induced centrosome, mitotic spindle, and karyotype aberrations. Our data suggest that distinct tyrosine kinases likewise targeted by both drugs are essential actuators in maintenance of centrosome and karyotype integrity. Keywords: chronic myeloid leukaemia, centrosomes, mitotic spindle, nilotinib, imatinib. 11 May 2007 Correspondence: Dr Alice Fabarius, PhD, III. Medizinische Universitätsklinik, Fakultät für Klinische Medizin Mannheim der RuprechtKarls-Universität Heidelberg, Wiesbadener Straße 7-11, 68305 Mannheim, Germany. E-mail: [email protected] Imatinib is an ATP-mimicking inhibitor targeting the tyrosine kinase activity of BCR-ABL-associated human leukaemias. Despite the success of imatinib in chronic myeloid leukaemia (CML) first line treatment, response rates of patients in accelerated or blastic phase are significantly decreased. Thus, there is a need for novel BCR-ABL tyrosine kinase inhibitors with greater potency and with the capability to overcome imatinib-resistance. Computer modelling has led to the development of nilotinib (AMN107; Novartis Pharma, Basel, Switzerland), an imatinib derivative with a more selective and at least 30-fold increased inhibitory potency when compared to imatinib (Kantarjian et al, 2006). Nilotinib has been described to act synergistically when combined simultaneously or sequentially with imatinib, making it highly promising for future therapy regimens to overcome BCR-ABL-related imatinib resistance in advanced phases of CML (Weisberg et al, 2005, 2006). However, emergence of Ph-negative clones with aberrant karyotypes distinctly different from the Ph-positive clone has been reported under continuous imatinib therapy, raising the controversial issue whether CML genetic instability might be caused or solely augmented by drug-related c-ABL inhibition (Bacher et al, 2005). Recently, we showed that imatinib treatment of normal human and mammalian cells in vitro caused unexpected centrosome and chromosome aberrations resembling lesions observed in CD34+ haematopoietic stem and progenitor cells concurring with the CML transformation process (Giehl et al, 2005). This supports the hypothesis that imatinib itself may play a role in the emergence of karyotype aberrations in Ph-negative cells (Fabarius et al, 2005). Albeit one can surmise a mechanism of action similar to that of imatinib, there is in fact a complete lack of comparable data on the effects of ª 2007 The Authors Journal Compilation ª 2007 Blackwell Publishing Ltd, British Journal of Haematology, 138, 369–373 doi:10.1111/j.1365-2141.2007.06678.x Short Report nilotinib on normal human primary cells as most in vitro studies have been performed on aberrant tumour cell lines (Weisberg et al, 2005). To address the question whether and to what extent nilotinib might affect genomic stability through induction of centrosomal and mitotic spindle aberrations, we comparatively investigated the effects of imatinib and nilotinib on centrosome, mitotic spindle and karyotype status in primary human fibroblasts after administration of therapeutic doses of both drugs in vitro. Materials and methods Primary cells Normal human dermal fibroblasts (NHDF; Promocell GmbH, Heidelberg, Germany) were grown as described previously (Fabarius et al, 2005). spindles were examined. Asymmetric and tri-, tetra- or multipolar patterns were considered abnormal. Cytogenetics Twenty metaphases were analysed by G-banding technique as described (Schoch et al, 2002). Statistical analysis Analyses of variance (anovas) and multiple regression analyses were used to compare control and drug treatment groups and different concentrations within the same group. P-values £0.001 were considered significant. All statistical computations were done with the sas software package, release 8.02 (SAS Institute Inc., Cary, NC, USA). Results In vitro drug exposure Cells were treated with: (i) 1–20 lmol/l nilotinib (Novartis, Basel, Switzerland, 3 weeks); (ii) 5–20 lmol/l imatinib (Novartis, 3 weeks); (iii) 5, 10 and 20 lmol/l imatinib (3 weeks) followed by 0.5, 1 and 2 lmol/l nilotinib respectively (3 weeks); (iv) 0.5, 1 and 2 lmol/l nilotinib followed by 5, 10 and 20 lmol/l imatinib (3 weeks each); and (v) imatinib and nilotinib simultaneously (3 weeks, identical concentrations). In vitro concentrations of 1–5 lmol/l represent pharmacological doses used therapeutically in vivo (Cwynarski et al, 2004). Centrosome and mitotic spindle staining Cells were grown on polylysine-coated slides, fixed and immunostained with an antibody to pericentrin. To confirm centrosome-specific staining, sample subsets were co-stained with gamma-tubulin directed antibody according to Giehl et al (2005). Mitotic spindles were co-stained using polyclonal antialpha-tubulin antibody (No. T6074; Sigma, Deisenhofen, Germany) under the same conditions. At least 20 mitotic All nilotinib- and imatinib-treated NHDF cultures were affected by both drugs and displayed similar centrosome and sporadic chromosome aberrations as well as spindle defects (Fig 1) when compared with controls (Fig 2) (P < 0.0001). Nilotinib induces genetic instability in an imatinibmimicking manner Cultures treated with nilotinib displayed centrosome alterations in 12–20% and spindle defects in 5.7–53%. Chromosome alterations could be detected in 30–45% (Fig 2A). However, cells treated with 20 lmol/l nilotinib showed lower aberration values. Cells under imatinib treatment displayed centrosome alterations in 9–19% and spindle defects in 27–32%. Chromosomal changes occurred in 20–45% (Fig 2B). Sequential drug treatment renders both drugs interchangeable After nilotinib treatment and with consecutive administration of imatinib, cells displayed centrosomal changes, chromosome (A) (B) Fig 1. Mitotic spindles in normal human dermal fibroblasts (NHDF) cells. Representative images showing normal (A) and aberrant (B) mitotic spindles were obtained by immunofluorescence staining of untreated and drug-treated NHDF cells respectively. Mitotic spindles were targeted with an antibody directed to alpha-tubulin, followed by a fluorescein isothiocyanate-conjugated secondary antibody. 370 ª 2007 The Authors Journal Compilation ª 2007 Blackwell Publishing Ltd, British Journal of Haematology, 138, 369–373 Short Report (A) (B) (C) (D) (E) Fig 2. Correlation between centrosome, mitotic spindle and chromosome aberrations in normal human dermal fibroblasts (NHDF) cultures treated with nilotinib (A), imatinib (B), sequential treatment with nilotinib followed by imatinib (C) and vice versa (D), and with a simultaneous combination of both drugs (E). Incubation times for NHDF cultures under mono- and combinatorial drug treatment were 3 weeks, for sequential drug regimen 6 weeks (3 weeks per drug). Various drug concentrations (x-axis) indicate a dose dependency of the observed aberrations (P < 0.0001 for all experiments). aberrations and spindle defects (Fig 2C). Inversely, imatinib treatment followed by nilotinib showed comparable results (Fig 2D). No differences in alteration patterns could be described after sequential treatment with both drugs compared to mono-treatment irrespective of the order of drug administration. Simultaneous drug treatment induces alterations and is toxic at high doses Simultaneous treatment with imatinib and nilotinib also led to centrosome and chromosome aberrations. Spindle defects could be observed in 9% of the 1 lmol/l/10 lmol/l imatinib/ nilotinib treated culture, but not in 0.5 lmol/l/5 lmol/l treated cells and in controls. Chromosome changes occurred in 15–35% (Fig 2E). Simultaneous high-dose treatment with 2 lmol/l/20 lmol/l nilotinib/imatinib proved deleterious for cells, suggesting synergistic effects in vitro. In all treated cultures, sporadic numerical chromosomal aberrations prevailed over sporadic structural alterations. Neither clonal changes nor a prevalence of specific chromosomes in aberrations were observed. Numerical and structural centrosome alterations occurred in an equal incidence. Our data demonstrated destabilising effects of both drugs in therapeutic doses (400 mg/800 mg equivalents) on centrosomes, chromosomes and mitotic spindle fidelity in vitro (P < 0.0001). The same outcome was achieved after culture treatment with imatinib, nilotinib or both (sequential or simultaneous drug treatment) (P ¼ 0.89, P ¼ 0.66 or P ¼ 0.63 respectively). Sequential drug treatment revealed that both drugs were interchangeable in terms of their resulting effect. Discussion Emergence of Ph-negative clones with aberrant karyotypes under continuous imatinib therapy gives rise to the speculation that tyrosine kinase inhibitors themselves may trigger induction of genetic instability. As this adverse effect may be directly proportional to the drug’s therapeutic potency, this could have serious impact for patients possibly switching drugs upon emergence of imatinib resistance and who are advised to receive such medication lifelong. Applying therapeutic doses of both drugs in vitro, we found similar dose-dependent centrosome and karyotype alterations in NHDF cells. These alterations correlated with spindle defects (Figs 1 and 2) functionally linking tyrosine kinase inhibitors with loss of centrosomal integrity and karyotype stability. Our findings can help to explain why 2–17% of imatinibreceiving patients display karyotype changes in Ph-negative cells under long-term treatment (Bacher et al, 2005). In vitro emergence of tyrosine kinase inhibitor-related alterations in normal human primary cells points to drug-associated mechanisms of de novo induction of aberrations rather than to selection of pre-existing clonal aberrations of the Ph-negative haematopoiesis being uncovered by a simultaneous gradual ª 2007 The Authors Journal Compilation ª 2007 Blackwell Publishing Ltd, British Journal of Haematology, 138, 369–373 371 Short Report elimination of the Ph-positive clone under therapy (Cortes & O’Dwyer, 2004). The possibility that both inhibitors may simply augment genetic instability in cells already carrying hidden genetic defects, thus favouring the acquisition of further chromosome defects, including the BCR-ABL gene rearrangement, seems, albeit not impossible, fairly unlikely, as it would imply that normal human cells harbour mutations at considerable frequency with capability of centrosome and gross genomic destabilisation. To assess potential in vivo side effects of tyrosine kinase inhibitors on haematopoietic cells, nine patients (imatinib, n ¼ 5; nilotinib, n ¼ 4) were analysed and spindle defects, however, at much lower frequencies (range, 5–10%) were observed (data not shown). However, due to the lack of proper controls (patients lacking prodromal chemotherapy) these data have to be considered preliminary. Nevertheless, the discrepancy between the in vitro and in vivo situation, i.e. higher in vitro aberration rates in NHDF cells than in haematopoietic cells in vivo, could be explained by better drug availability in vitro or differing drug sensitivities. The complete lack of effective mechanism to eliminate or suppress aberrant clonal phenotypes in vitro is also conceivable. Imatinib has been reported to affect the function of normal non-malignant cells and concurs with altered gene expression and myelosuppression in treated patients (Mattiuzzi et al, 2003; Balabanov et al, 2005a,b). Thus, a resulting altered immunosurveillance could explain higher incidences of secondary malignancies and infections under imatinib (Mattiuzzi et al, 2003; Bacher et al, 2005). The molecular mechanisms for the observed detrimental effects of imatinib and nilotinib on centrosome and karyotype stability seem to involve one or more ABL-related tyrosine kinases operating on regulation of centrosome replication, DNA repair and cell cycle. Potential mechanisms include the RAD51 protein that plays a fundamental role in DNA double strand break repair and is essentially regulated by c-ABL (Bertrand et al, 2003). Moreover, inhibition of c-ABL involved in p53-dependent G1 arrest response could cause defects in DNA repair and G1 arrest, thus predisposing cells to aberrant centrosome and genome duplication (Kharbanda et al, 1998). Finally, drug-related inhibition of c-ABL may directly affect ubiquitination of centrosomal components leading to extra centrosomal duplication and mitotic spindle catastrophy (Parvin & Sankaran, 2006). In conclusion, our data demonstrate that therapeutic doses of nilotinib and imatinib in vitro similarly trigger the emergence of centrosomal, mitotic spindle and chromosomal abnormalities. As both drugs are ATP-competitive inhibitors targeting closely related tyrosine kinases, the same modes of action on molecular levels interfering with proper centrosome reduplication can be suggested. Acknowledgements The study was supported by the Albert und Anneliese KonanzStiftung, Heidelberg, Germany and the Competence Network 372 ‘Acute and chronic leukemias’, sponsored by the German Bundesministerium für Bildung und Forschung (Projektträger Gesundheitsforschung; DLR e.V.- 01 GI9980/6). Nilotinib and imatinib were kindly provided by Dr Paul Manley and Dr Elisabeth Buchdunger, Novartis Pharma, Basel, Switzerland. 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