The Bcl-2 protein family in the progression of multiple myeloma: constitutive expression, proapoptotic effects of the Bcl-2/Bcl-xl inhibitor ABT-737 and clinical correlations.

Introduction. Monoclonal plasma cells disorders (PCD) are clinically heterogeneous, including monoclonal gammopathy of undetermined significant (MGUS), smoldering multiple myeloma (SMM), symptomatic MM and plasma cell leukemia (PCL). Several molecular characteristics have been described in PCD, including the aberrant expression of the Bcl-2 protein family, which prompt innovative therapeutic approaches based on the Bcl-2 targeted inhibition. However, neither the expression pattern of the Bcl-2 protein family, nor differences in the sensitivity to specific inhibitors have been associated with the different clinical statuses. Aims. We therefore examined the constitutive expression of the Bcl-2 protein family (Bcl-2, Bcl-xL and Mcl-1) and the functional and molecular effects of the Bcl-2/Bcl-xL inhibitor ABT-737 (kindly provided by Abbott Laboratories) on purified primary CD138+ cells from 11 SMM, 20 symptomatic MM (8 at diagnosis and 12 at relapse) and 4 PCL samples. Methods. Expression of the Bcl-2 protein family was evaluated by western blot analysis. The cytotoxicity of ABT-737 on MM cells was established using the MTT assay. The drug concentration inducing 50% cell killing (IC-50) was calculated from the dose-response curve. Cell cycle inhibition and induction of apoptosis were analyzed by flow cytometry using the Acridine-Orange (AO) technique and by Annexin V binding assay. Results. Our data indicate that the constitutive protein expression of Mcl-1 is significantly higher (P=0.059) in MM samples at diagnosis compared to SMM (mean ratio Mcl-1/GAPDH: 1.70±0.51 vs 0.95±0.27) and slightly decreases in the more aggressive PCD disorders (1.1±0.21 and 1.21±0.83 in relapsed MM and PCL, respectively). Bcl-2 and Bcl-xL did not differ among samples from these different groups of patients. A marked ABT-737-induced pro-apoptotic activity was observed on purified CD138+ cells from primary samples. However, this effect was significantly (P=0.02) higher on CD138+ cells from SMM and on newly diagnosed MM compared to relapsed MM and PCL samples, which proved progressively less sensitive to ABT-737. In particular, the pro-apoptotic activity of ABT-737 gradually decreased from SMM, to newly diagnosed MM, relapsed MM and PCL, with a net apoptosis (sub-G0/1 peak) at 24 hours in the presence of 1000 nM ABT-737 of 55.9±15.2%, 49.3±21.3%, 37.6±10.6% and 33.3±17.9%, respectively. Conclusions. In summary, these data show that Mcl-1 is overexpressed in PCD, increasing from SMM to MM. Nevertheless, the activity of the Bcl-2 inhibitor ABT-737 is preserved, showing that in MM cells the interaction between Mcl-1 and other pro-apoptotic members, such as Bim, plays a major role in the response to ABT-737. Moreover, the highest levels of sensitivity of SMM to ABT-737, associated with the lowest levels of Mcl-1 expression, prompt new investigational approaches for the management of patients with SMM.