Functional Effects of the Bcl-2/Bcl-xL Inhibitor ABT-737 on Primary Cells from Smoldering Multiple Myeloma.
Authors: Ricciardi Maria Rosaria , Calabrese Elisabetta , De Cave Fabiana , Santinelli Sara , Bergamo Paola , Decandia Samantha , Gregorj Chiara , Federico Vincenzo , Milella Michele , Foa Robert , Tafuri Agostino , Petrucci Maria Teresa 
Published: October 2019

Abstract
Smoldering multiple myeloma (MM) is an asymptomatic plasma-cell disorder with a high risk of progression to symptomatic MM. The time to progression and the underlying mechanisms are still unclear. The bcl-2 family proteins are key regulators of cell survival and are frequently found aberrantly expressed in MM, conferring resistance to chemotherapy. The activity of bcl-2 family inhibitors is currently under investigation in MM. In contrast, the activity in smoldering MM cells is still unexplored. We investigated the cell cycle and apoptotic effects of the bcl-2/bcl-xL inhibitor, ABT-737 (kindly provided by Abbott Laboratories) on primary CD138+ malignant plasma cells purified from smoldering MM samples. In addition, we extended this study to MM cell lines and to symptomatic MM. The MM cell lines (KMS18, ARH-77) exposed for 24, 48 and 72 hours to scalar concentrations of ABT-737 (from 0.1 to 1 μM) showed a dose- and time-dependent cell growth inhibition (IC50s 0.286 and 0.346 μM, respectively, at 72 hours) due to a significant increase of apoptotic cells. In addition, cell cycle analysis on KMS18 demonstrated at 72 hours a significant G1-phase depletion: from 54.6% ± 4.5 (DMSO) to 26.9% ± 1.4 (1 μM ABT-737) (p=0.021). The effects of ABT-737 were then examined on primary cells from 7 smoldering MM. Bone marrow cells, following CD138 enrichment (>80% of purity), were cultured in vitro with ABT-737 (at scalar concentrations from 0.1 to 1 μM) up to 72 hours. A statistically significant reduction in cell growth and increase in the percentage of apoptotic cells was observed in response to 0.1μM ABT-737. In particular, a statistically significant pro-apoptotic activity of ABT-737 was demonstrated by the increment of the subG0/1 peak (Acridine-Orange) at 24 hours from 24.0% ± 8.9 in DMSO to 51.7% ± 17.7 (p=0.02) and 77.6% ± 15.6 (p=0.001) in the presence of ABT-737 at 0.1 and 1 μM, respectively. The effects of ABT-737 on smoldering MM were then compared to those obtained following exposure of CD138+ cells from symptomatic MM (14 cases) to the bcl-2/bcl-xL inhibitor. The results obtained demonstrated that ABT-737 acts in smoldering MM as well as in symptomatic MM in a similar manner. In fact, a comparable increment of the subG0/1 peak was also recorded in symptomatic MM: from 31.2% ± 17.8 in DMSO to 56.4% ± 18.7 (p=0.017) and 69.9% ± 16.4 (p=0.0002) in the presence of ABT-737 at 0.1 and 1 μM, respectively, at 24 hours. These data were confirmed by measuring Annexin V+ cells. In conclusion, ABT-737 shows potent in vitro growth-inhibitory and pro-apoptotic activity at nanomolar concentrations in smoldering MM. This effects was similarly achieved in symptomatic MM. These data warrant a further pre-clinical/clinical development of the bcl-2 family inhibitor ABT-737 in patients with MM regardless of the disease status, aiming also at delaying disease progression.