Implication of BMP7 in Human Mantle Cell Lymphoma (MCL) Secondary Drug Resistance.

Abstract Despite a high response rate to 1st line chemotherapy, the probability of cure is very low in MCL. Secondary drug resistance invariably develops and the identification of mechanisms involved in this secondary drug resistance is a major challenge. We choose to track at a tumor level which genes are associated to the appearance of secondary drug resistance. The strategy was based on the use of paired samples from the same patient in order to control inter-patients variability. Five MCL patients had tumor samples collected from the same tissue before therapy and after failure. Two patients had refractory disease (non responders) and three achieved an objective response to therapy (responders). For each patient, pairs of samples from the same patient were co-hybridized on Agilent dual color DNA chips. Four DNA-chips were processed per patient (dye swap + replicate). The statistical analysis concentrated on variations in gene expression between the two samples from each patient. Each gene was analysed independently from the others. A multiple regression model (GLM procedure, SAS 8.2, Cary, NC, USA) was used to analyse the relation between the fold change in expression between the two samples of the same patient and several parameters including the response to treatment. Genes were selected on the basis of the absolute value of the coefficient associated to response to treatment. This absolute value was then expressed as a fold-change ratio (FCR), or ratio between the mean fold changes in responders and in non responders. Nineteen genes with a FCR greater than 2 and a P value < 10–6 were selected. FCR values larger than 5 were observed for CD69 (FCR=7.7) and BMP7 (FCR=7.0). Seventeen of the selected genes (89%) had a decreased expression at relapse in responders and an increased expression in non responders. The variation in expression according to response to therapy was in the reverse direction for the two remaining genes (BMP7 and TRAF5). The expression of TRAF5 decreased in non responders and remained unchanged in responders. In responders, the expression of BMP7, which was very low before treatment, was multiplied by about 8 after relapse, and did not vary in non responders. Because of this increase in expression, observed only in responders, BMP7 was considered as a major gene probably involved in secondary drug resistance. The possibility to interfere with its activity using miRNA was tested on JEKO cell line. JEKO cell line (EBV - MCL cell line) spontaneously express BMP7 and do not show growth inhibition when exposed in vitro to high concentration of BMP7. BMP7 RNA interference markedly increased necrosis (from 8.3% to 31.5%; P<0.05) of JEKO exposed to Bortezomide pointing BMP7 as a key gene involved in drug resistance. In conclusion, the patient oriented strategy is extremely powerful and permits relevant gene selection with a limited number of samples. Biologic validation with RNA interference confirms the relevance of this approach. Further investigations concerning the role of BMP7 and its possible use as a target for therapy are under investigation.