The Exosomal Transfer of Acid Sphingomyelinase Contributes to Drug Resistance in Multiple Myeloma
Authors: Oudaert Inge , Faict Sylvia , D'Auria Ludovic , Dehairs Jonas , Maes Ken , Vlummens Philip , Jacobs Lotte , De Veirman Kim , De Bruyne Elke , Fostier Karel , Vande Broek Isabelle , Schots Rik , Vanderkerken Karin , Swinnen Johannes V. , Menu Eline 
Published: November 2019

Introduction
Multiple myeloma (MM) is well-known for the development of drug resistance, leading to the need for multiple treatment lines at times of relapse or progression. Even then, most patients ultimately will succumb to this cancer. Therefore, there is a need for new therapeutic strategies to conquer this drug resistance. Lipidomics has recently gained more attention in the search for new cancer therapies. Lipids are mainly found in biological membranes and function as building blocks, but are also important metabolites that can influence energy, structure and signaling cascades. Lipid dysfunction has been correlated to other cancers, like prostate and breast cancer. In this study, we identified changes in lipid content in MM patients and further investigated this altered metabolism in vitro.
Methods
We performed a lipidomics assay to compare plasma from healthy volunteers to MM patients. For all in vitro experiments, we used four human MM cell lines (JJN3, OPM2, LP1, U266) and primary CD138+ patient samples, isolated by MACS. Differential mRNA expression (SMPD1 = acid sphingomyelinase, ASM) was measured by qRT-PCR and ASM protein levels by western blot. Exosomes were isolated by exoquick, while changes in secretion were measured by nanoparticle tracking analysis. Viability was measured by CellTiter Glo and apoptosis rates for melphalan, bortezomib and ASM inhibitor (amitriptyline) were measured by flow cytometry (annexinV-FITC/7-AAD staining). Drug efficacy of amitriptyline was also confirmed on primary CD138+ samples by CellTiter Glo.
Results
Lipidomics analysis revealed an increase in ceramides and a decrease in sphingomyelin. Therefore, we believe that the enzyme sphingomyelinase, which converts sphingomyelin into ceramide, is upregulated in MM, which we confirmed on primary CD138+ MM cells for ASM. We also observed an increase in SMPD1 expression by qRT-PCR and ASM levels by western blot after melphalan and bortezomib treatment. Furthermore, we also investigated effects of these drugs on exosome secretion, where we found an increase in the number of exosomes secreted, as well as higher ASM levels in these exosomes. U266-derived exosomes, containing high amounts of ASM, were able to transfer their resistance to ASM-low JJN3 cells as an increase in viability was measured. Inhibition of ASM by amitriptyline, combined with melphalan and bortezomib, increased apoptotic cell death by upregulating cleaved PARP and caspase 3 levels. Combination therapy of amitriptyline with melphalan and bortezomib was also successfully tested on primary CD138+ MM cells.
Conclusion
This study is the first to identify changes in sphingolipids in plasma of MM patients, where we matched the observed difference to an upregulation of ASM in MM cells. Standard-of-care drugs stimulated ASM expression and production, and cells were able to transfer their resistance to other cells by ASM-rich exosomes. Enzyme inhibition by amitriptyline, a cheap tricyclic antidepressant drug frequently used to combat neuropathic pain in MM, increased drug efficacy of standard-of-care drugs. This study therefore provides a rationale to add ASM-targeting drugs to current combination therapies in MM patients.

Disclosures
No relevant conflicts of interest to declare.

OffLabel Disclosure:
Amitriptyline is a tricyclic antidepressant, which is also often used to treat neuropathic pain in Multiple Myeloma patients. Here we describe additive effects of amitriptyline on bortezomib and melphalan treatment in MM.