BCL-2/BCL-XL Inhibition in Smoldering Multiple Myeloma CD138+ Cells: Functional Effects and Protein Expression Levels
Authors: Libotte Francesco , Ricciardi Maria Rosaria , Calabrese Elisabetta , De Benedictis Cinzia , Santinelli Sara , Bergamo Paola , Decandia Samantha , Levi Anna , Mohamad Ali Fathi , Federico Vincenzo , Milella Michele , Foà Robert , Tafuri Agostino , Petrucci Maria Teresa 
Published: April 2020

Abstract
Smoldering multiple myeloma (SMM) is an asymptomatic plasma-cell disorder with a high risk of progression to a symptomatic multiple myeloma (MM). The time and mechanisms underlying the progression are still unclear. The Bcl-2 family proteins are frequently aberrantly expressed in MM and support proliferation, survival and drug-resistance. The activity of inhibitors of the anti-apoptotic Bcl-2 family members is currently under investigation in MM, while their therapeutic potential has never been explored in SMM. In this study, we investigated the constitutive protein expression profile of anti-apoptotic Bcl-2 family members and the functional effects of their pharmacological modulation by the Bcl-2/Bcl-xL inhibitor ABT-737 (kindly provided by Abbott Laboratories) on primary cells from SMM and symptomatic MM at different stages of the disease. Mononuclear cells isolated from BM aspirates of MM patients were fractionated into purified CD138+ (&gt;80%) and CD138- populations. Protein extracts were prepared and subjected to Western blot analysis for anti-apoptotic Bcl-2 family members. Within the CD138+ malignant plasma cells, SMM samples were characterized by elevated Mcl-1 levels and barely detectable expression of Bcl-xL, while symptomatic MM samples exhibited higher levels of both Mcl-1 and Bcl-xL. No significant differences in Bcl-2 level expression were instead observed between SMM and symptomatic MM samples. We, then, investigated the functional effects of ABT-737 on MM cell line and on CD138+ malignant plasma cells from SMM and symptomatic MM samples. MM cell lines exposed to increasing concentrations of ABT-737 (10–1000 nM) for up 72 hours showed a dose- and time-dependent cell growth inhibition (IC50 200–400 nM at 72 hours) due to the loss of mitochondrial membrane potential, caspase activation and significant increase in the proportion of apoptotic cells. In addition, cell cycle analysis demonstrated a significant G1-phase depletion in KMS18 cells exposed to 1000 nM ABT-737 for 72 hours (26.9%±1.4 vs 54.6%±4.5, p=0.021). Western Blot analysis demonstrated that ABT-737 dramatically reduces Bcl-2 protein levels, with appearance of cleaved forms, while Mcl-1 was down-regulated only at higher doses. No effects were seen on Bcl-xL expression. The ABT-737 activity was then examined on CD138+ purified primary cells from 9 SMM samples cultured with ABT-737 (10–1000 nM) for 72 hours. A remarkable reduction in cell growth and increase in the percentage of apoptotic cells was observed in response to 100 nM ABT-737. In particular, a statistically significant pro-apoptotic activity of 100 nM ABT-737 was demonstrated by the increment of the subG1 peak after 24 hours (25.0%±9.8 vs 60.1%±17.0, p=0.00011, and vs 78.1%±14.0, p=0.0003, in the presence of 100 and 1000 nM ABT 737, respectively). Similar results were obtained in the CD138+ fraction from symptomatic MM (7 newly diagnosed and in 9 relapsed/refractory) with a significant increment of the subG1 peak after 24 hours of ABT-737 exposure (27.9%±15.5 vs 53.4%±16.8, p&lt;0.0001, and vs 70.0%±15.4, p&lt;0.0001, in the presence of 100 and 1000 nM ABT 737, respectively). Preliminary results indicate that, as in cell lines, Bcl-2 cleavage also occurs in CD138+ primary cells in response to ABT–737. In conclusion, we present the first evidence that Bcl-2/Bcl-xL inhibition by ABT-737 enhances apoptosis in CD138+ primary cells from SMM. We therefore suggest that this strategy may be developed for therapeutic purposes in different disease stages of MM patients.