SIRT3 Modulates Mitochondrial Stress Response in Bone Marrow Mesenchymal Stromal Cells of Multiple Myeloma Patients
Authors: Berenstein Rimma , Blau Igor Wolfgang , Nogai Axel , Waechter Marlies , Schmidt-Hieber Martin , Pezzutto Antonio , Doerken Bernd , Blau Olga 
Published: October 2019

Abstract
Introduction: Multiple myeloma (MM) cells strongly rely on the interaction with bone marrow mesenchymal stromal cells (BMMSCs) for proliferation and survival. We previously reported that MM cells influence the mitochondrial function and senescence of surrounding BMMSCs amongst others via NAD-dependent deacetylase sirtuin-3 (SIRT3) (Blood, 2014; BMC Cancer, 2015). In the further course we examined the function of SIRT3 in BMMSCs and possible underlying mechanisms of SIRT3 activation.
Methods: In this study we transfected 2 healthy donor BMMSCs (HD-BMMSCs) for transient knock-down of SIRT3 using 4 different siRNAs (GeneSolution, Qiagen). We analysed mitochondrial membrane potential (ΔΨM) and reactive oxygen species (ROS) by FACS. Cell cycle analysis was performed using "cell cycle assay kit" (Abcam). Senescence was examined using FACS and senescence-associated β-galactosidase activity. Apoptosis was analysed using Annexin V-FITC Kit (Miltenyi) and protein expression was examined by western blot. In addition we analysed the influence of MCT transporter interaction on SIRT3 expression in 6 BMMSCs of myeloma patients (MM-BMMSCs) using α-cyano-4-hydroxycinnamic acid (α-CN).
Results: SIRT3 knock-down in HD-BMMSCs induced 1.4- to 1.9-fold increase in ROS levels (p<0.05). This was associated with dissipation of ΔΨM between 1.4- to 1.8-fold depending on the siRNA that was used for transient knock-down of SIRT3 (p<0.04). Furthermore inhibition of SIRT3 mimicked cell cycle arrest in S phase previously reported in BMMSCs of myeloma patients. Percentage of BMMSCs in S phase increased upon SIRT3 knock-down between 6.7%-9.6% (p<0.039). In addition, reduction of SIRT3 led to 1.5-fold increased senescence-associated β-galactosidase activity in transfected HD-BMMSCs indicating that this protein amongst others could be responsible for premature senescence of BMMSCs of myeloma patients (p<0.03). We further supposed that interaction of BMMSCs with MM cells via MCT transporters could influence SIRT3 levels in BMMSCs of myeloma patients. Inhibition of MCT interaction induced apoptosis in MM cells but not BMMSCs (p<0.04). Furthermore, suppression of MCT interaction between MM cells and MM-BMMSCs reduced activation of SIRT3 around 1.7-fold in MM-BMMSCs.
Conclusion: SIRT3 seems to be a major regulator of mitochondrial functions in BMMSCs. Thus, reduced expression of SIRT3 that was previously reported in MM-BMMSCs could be the reason for increased ROS levels, cell cycle arrest in S phase and premature senescence-like state of MM-BMMSCs. In addition, our data show that inhibition of MCT transporters in MM cells and MM-BMMSCs induces apoptosis in MM cells and inhibits increased expression of SIRT3 in MM-BMMSCs. Disabling of MCT transporter interaction could possibly inhibit sustained induction of mitochondrial stress response in MM-BMMSCs and circumvent induction of the premature senescence-like state.

Disclosures
Blau: MSD: Honoraria; JAZZ Pharma: Honoraria; Shire: Honoraria; Celgene: Honoraria, Research Funding; AMGEN: Honoraria; Janssen: Honoraria, Research Funding; Baxalta: Honoraria; BMS: Honoraria.