Multikinase Inhibitor Sorafenib Induces Apoptosis, CD138-Downregulation, Actin Depolymerization and Inhibition of M210B4-Triggered Chemotaxis in Multiple Myeloma Cell Lines and Synergyzes with Proteasome Inhibitor Bortezomib
Authors: Udi Josefina , Wider Dagmar , Catusse Julie , Schnerch Dominik , Follo Marie , Ihorst Gabriele , Waesch Ralph , Engelhardt Monika 
Published: October 2019

Abstract
Abstract 2380

Introduction:
Sorafenib is an oral multikinase inhibitor that targets several cancer-specific pathways and directly affects tumor cell proliferation, cell survival and neovascularization. The Ras/Raf/MEK/ERK pathway is particularly known to be critical for proliferation of multiple myeloma (MM) cells. Moreover, its blockage may not only compromise MM cell survival and proliferation, but also influence cell adhesion and migration. We sought to elucidate the effects of sorafenib on proliferation, phenotype, specific signalling pathways, actin polymerization and chemotaxis, as well as cytotoxic interactions when combined with other anti-MM agents, such as bortezomib.

Methods:
L363, U266 and RPMI8226 were cultured with RPMI1640, 10% FCS and 0.2% penicillin/streptomycin. On day 0, cells were treated with increasing concentrations of sorafenib and/or bortezomib. Cell viability and cytotoxicity were assessed on days 3 and 6, in addition to day 1 or 2 in previous analyses. The cytotoxic effect for sorafenib and bortezomib combined was evaluated using Calcusyn Software, whereby a combination index =1, <1 or >1 indicated additive, synergistic and antagonistic effects, respectively. CD138 expression and morphologic changes were evaluated via flow cytometry, immunocytochemistry and confocal microscopy. The effect of sorafenib on ERK1/2 phosphorylation was investigated by western blot. Actin polymerization was studied by flow cytometry after labeling with FITC-phalloidin. Chemokine receptor expression was assessed by flow cytometry and chemotaxis of L363 cells with various chemoattractants was studied using 96-well chemotaxis chambers.

Results:
Our MM-in vitro model confirmed potent cytotoxicity for sorafenib single use and synergistic effects when combined with bortezomib. With 10 and 100μM sorafenib in L363, we observed increased median PI+ cells (62% and 94% on d3, respectively) compared to the control (median PI+ d0: 11%), with similar increases on d6 (median 81% and 92%, respectively). Combined sorafenib and bortezomib use showed additive effects and synergism at 10μM and 10nM bortezomib (combination index: 0.80). Similar to PI-results, viable cells and CD138 expression by flow cytometry substantially decreased with sorafenib in a dose- and time-dependent manner. Regarding the effects on the MAPK pathway, after incubating L363 cells with 1 and 10μM sorafenib for 6 and 24 hours, a dose-dependent downregulation of ERK1/2 phosphorylation was observed. After 3 days of incubation with increasing concentrations of sorafenib, MM cells were stained with DAPI, Phalloidin-Alexa594 and CD138-FITC and analyzed via confocal microscopy. L363 cells highly expressed CD138 in the absence of sorafenib. Of note, sorafenib not only affected cell proliferation, but also phenotype, morphology, actin metabolism and chemotaxis of MM cells. With sorafenib concentrations as low as 1μM, CD138 was downregulated and impressive morphologic changes with a reduction in F-actin content were observed. We could show CXCL12-stimulated actin polymerization and after treatment with sorafenib with concentrations of 10μM and 100μM its inhibition, as confirmed via flow cytometry after labeling with phalloidin-FITC. L363 cells showed high expression of the chemokine receptors CCR4 and CCR5 and underwent chemotaxis to their common ligand CCL5. Chemotaxis of L363 cells was even more evident with the use of supernatant from M210B4 bone marrow stromal cells. This M210B4-induced chemotaxis also occurred in the presence of the specific CXCR4-inhibitor AMD3100, supporting the involvement of chemokines other than CXCL12 in M210B4-induced MM cell migration. M210B4-triggered chemotaxis was substantially inhibited after 3 days of incubation with increasing concentrations of sorafenib in a dose-dependent manner.

Conclusions:
To the best of our knowledge this is the first analysis of the effects of sorafenib on phenotype, morphology, actin polymerization and migration of MM cells. Sorafenib induced down-regulation of phospho-ERK appeared responsible for the observed actin depolymerization and reduction in M210B4-triggered chemotaxis. Hence, further analysis of sorafenib and other novel anti-MM agents, both in MM cells and their microenvironment, should enable greater progress in this hematopoietic disease.

Disclosures:
No relevant conflicts of interest to declare.