Epigenetic down-regulation of BIM expression is associated with reduced optimal responses to imatinib treatment in chronic myeloid leukaemia Edurne San Jose ´-Eneriz a,1 , Xabier Agirre a,1 , Antonio Jime ´nez-Velasco b , Lucia Cordeu a , VanesaMartı´n c , Victor Arqueros c , Leire Ga ´ rate a , Vicente Fresquet a , Francisco Cervantes d , Jose ´ A. Martı´nez-Climent a , Anabel Heiniger b , Antonio Torres c , Felipe Pro ´ sper a, * , Jose Roman-Gomez c, * a Hematology Department and Area of Cell Therapy, Clinica Universitaria, Foundation for Applied Medical Research, University of Navarra, Avda. Pı ´o XII 36, Pamplona 31008, Spain b Hematology Department, Carlos Haya Hospital, Malaga, Spain c Hematology Department, Reina Sofia Hospital, Avda. Menendez Pidal s/n, 14004 Cordoba, Spain d Hematology Department, Hospital Clinic, IDIBAPS, University of Barcelona, Spain ARTICLE INFO Article history: Received 7 January 2009 Received in revised form 23 March 2009 Accepted 1 April 2009 Available online 4 May 2009 Keywords: BIM CML Imatinib Methylation ABSTRACT Background: Expression of the pro-apoptotic BCL-2-interacting mediator (BIM) has recently been implicated in imatinib-induced apoptosis of BCR-ABL1 + cells. However, the mecha- nisms involved in the regulation of BIM in CML and its role in the clinical setting have not been established. Design and methods: We analysed the mRNA expression of BIM in 100 newly diagnosed patients with CML in chronic phase by Q-RT-PCR and the protein levels by Western blot analysis. Methylation status was analysed by bisulphite genomic sequencing and MSP. CML cell lines were treated with imatinib and 5-aza-2’-deoxycytidine, and were transfected with two different siRNAs against BIM and cell proliferation and apoptosis were analysed. Results: We demonstrated that down-regulation of BIM expression was present in 36% of the patients and was significantly associated with a lack of optimal response to imatinib as indicated by the decrease in cytogenetic and molecular responses at 6, 12 and 18 months in comparison with patients with normal BIM expression (p < 0.05). Expression of BIM was mediated by promoter hypermethylation as demonstrated by restoration of BIM expression after treatment of CML cells with 5-aza-2 0 -deoxycytidine. Using CML cell lines with low and normal expression of BIM we further demonstrated that the expression of BIM is required for imatinib-induced CML apoptosis. Conclusion: Our data indicate that down-regulation of BIM is epigenetically controlled by methylation in a percentage of CML patients and has an unfavourable prognostic impact, and that the combination of imatinib with a de-methylating agent may result in improved responses in patients with decreased expression of BIM. Ó 2009 Elsevier Ltd. All rights reserved. 0959-8049/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.ejca.2009.04.005 * Corresponding authors: Tel.: +34 948 255400; fax: +34 948 296500 (F. Pro ´ sper), tel.: +34 957 010250; fax: +34 957 010429 (J. Roman-Gomez). E-mail addresses: fprosper@unav.es (F. Pro ´ sper), peperosa@teleline.es (J. Roman-Gomez). 1 E.S.J.E. and X.A. contributed equally to this work. EUROPEAN JOURNAL OF CANCER 45 (2009) 1877 – 1889 available at www.sciencedirect.com journal homepage: www.ejconline.com