Synergistic Induction of Apoptosis in Multiple Myeloma Cells by Simultaneous Inhibition of the Raf/MEK/ERK and BCL-2 Pathways
Authors: Ricciardi Maria Rosaria , Milella Michele , Libotte Francesco , Calabrese Elisabetta , Bergamo Paola , Santinelli Sara , Decandia Samantha , De Benedictis Cinzia , Levi Anna , Foà Robert , Tafuri Agostino , Petrucci Maria Teresa 
Published: April 2020

Abstract
The Raf/MEK/ERK pathway and the Bcl-2 family proteins are commonly overexpressed in hematologic malignancies, where they promote proliferation and survival of the neoplastic cells. We have previously demonstrated that selective MEK inhibitors (MEK-I) exert potent growth-inhibitory effects in preclinical models of both acute myeloid leukemia (AML) and multiple myeloma (MM) (Blood2006, 108:254). More recently, we have obtained evidence that ABT-737, a Bcl-2/Bcl-xL (BH3 mimetic) inhibitor (kindly provided by Abbott Laboratories), shows potent in vitro growth-inhibitory and pro-apoptotic activity on MM cell lines and primary CD138+ bone marrow cells from MM patients, regardless of the disease status (Libotte F, ASH 2008). Moreover, in AML models, we and others have reported a highly synergistic pro-apoptotic interaction between inhibitors of the Raf/MEK/ERK pathway and of the Bcl-2 family (Blood 2002, Cancer Cell 2006, Ricciardi ASH 2008). Here, we analyzed the impact of the simultaneous inhibition of these two pathways on cell proliferation and apoptosis in MM cell lines. To this purpose, we exposed different MM cell lines to increasing concentrations of MEK-I and ABT-737, alone and in combination. While single compounds dose-dependently inhibited cell growth, as assessed by trypan blue exclusion counting, we observed that their combination synergistically enhanced this effect with combination indexes (CI), as measured by isobologram analysis (Chou–Talalay method), of 0.28 and 0.12 for KMS18 and KMS27 cells, respectively. We next analyzed the effects of combined MEK and Bcl-2/Bcl-xL inhibition on apoptosis induction. Both MEK-I and ABT-737 induced apoptosis in MM cells at high concentrations, as determined by sub-G1 DNA peak and Annexin V staining. When used at concentrations that induced minimal apoptosis as single agents (7.55% and 6.8% net apoptosis induction in KMS18 cells after 72 hours of exposure to MEK-I and ABT-737, respectively), the combination of MEK-I and ABT-737 was able to induce substantial apoptosis (more than 50% net apoptosis induction after 72 hours in both KMS18 and KMS27 cell lines). Such pro-apoptotic interaction was highly synergistic in nature, with CI, as defined using isobologram analysis, of 0.2 and 0.17 for KMS18 and KMS27 cells, respectively. Mitochondrial membrane depolarization was similarly enhanced by the combination of MEK-I and ABT-737. Conversely, in the MEK-I resistant MM cell line ARH-77, ABT-737 was still able to induce apoptosis (up to 40% of the cells) but its effect was not significantly potentiated by MEK inhibition. Preliminary results on primary CD138+ MM cells exposed to both inhibitors confirmed the higher cell growth inhibition induced by combining MEK-I and Bcl-2/Bcl-xL inhibition. In conclusion, we demonstrated a striking synergistic pro-apoptotic activity with combined inhibition of Raf/MEK/ERK and Bcl-2 signaling in MM cell lines. Simultaneous disruption of these two pathways warrants further investigation as novel therapeutic strategy for this disease.