Myeloma mesenchymal stem cells' bioenergetics afford a novel selective therapeutic target

Abstract

Bone-marrow mesenchymal stem cells (BM-MSCs) rely on glycolysis, yet their trafficked mitochondria benefit recipient cells' bioenergetics in regenerative and cancerous settings, most relevant to BM-resident multiple myeloma (MM) cells. Fission/fusion dynamics regulate mitochondria function. Proteomics demonstrates excessive mitochondrial processes in BM-MSCs from MM patients compared to normal donors (ND). Thus, we aimed to characterize BM-MSCs (ND, MM) mitochondrial fitness, bioenergetics and dynamics with a focus on therapeutics. MM-MSCs displayed compromised mitochondria evidenced by decreased mitochondrial membrane potential (ΔΨm) and elevated proton leak. This was accompanied by stimulation of stress-coping mechanisms: spare respiratory capacity (SRC), mitochondrial fusion and UPR^mt. Interfering with BM-MSCs mitochondrial dynamics equilibrium demonstrated their significance to bioenergetics and fitness according to the source. While ND-MSCs depended on fission, reducing MM-MSCs fusion attenuated glycolysis, OXPHOS and mtROS. Interestingly, optimization of mtROS levels is central to ΔΨm preservation in MM-MSCs only. MM-MSCs also demonstrated STAT3 activation, which regulates their OXPHOS and SRC. Targeting MM-MSC' SRC with Venetoclax diminished their pro-MM support and sensitized co-cultured MM cells to Bortezomib. Overall, MM-MSCs distinct mitochondrial bioenergetics are integral to their robustness. Repurposing Venetoclax as anti-SRC treatment in combination with conventional anti-MM drugs presents a potential selective way to target MM-MSCs conferred drug resistance.

Fig. 1. Mass spectrometry analysis of MM-MSCs demonstrated increase in mitochondrial-related metabolic pathways and proteins.

A Principal component analysis (PCA) graph presents clear segregation of MM-MSCs and ND-MSCs mass spectrometry data according to source. B The BM-MSCs (ND and MM-MSCs) mass spectrometry data (differentially expressed proteins, p < 0.05 (FDR), N = 3 each) was assessed for cellular component and pathways using WebGestalt. Pie graphs present the percentage of metabolism-related GO pathways (red) out of all cell pathways (Cyan) in BM-MSCs. C STRING functional enrichment analysis of BM-MSCs upregulated proteins (FC ≥ 1.25, p < 0.05) included in the “Mitochondrion” cellular component STRING category (p = 2.16E−17, red nodes). D Median values of 63 protein levels (mass spectrometry) included in the BM-MSCs “Mitochondrion” cellular component category (WebGestalt, GO:0005739, p = 0) presented individually by shape and color.

Fig. 2. MM-MSCs display increased bioenergetics, mitochondrial stress adaptation and elevated fusion.

ND (cyan) and MM (red) MSCs (n≧5) were subjected to (A) Assessment of cellular glycoATP, mitoATP and TotalATP production rates (Seahorse XF Real-Time ATP Rate Assay Kit). B Assessment of oxygen consumption rates (OCR) and determination of Basal respiration, Maximal respiration and SRC (Seahorse XF Cell Mito Stress Test Kit). C Representative graph of Seahorse XF Cell Mito Stress Test Kit. D Assessment of Proton leak (Seahorse XF Cell Mito Stress Test Kit). E Analysis of mitochondrial membrane potential (TMRE). F Quantification of UPR^mt factors’ immunoblotting (ClpX and ClpP). G Representative Immunoblots of UPR^mt factors (ClpX and ClpP) and Fusion/Fission regulators (OPA1, MFF and pDRP1^S616). H Representative confocal images of BM-MSCs mitochondria (ND and MM, Mitotracker Red CMXros). I Analysis of mitochondria Branches and Total Branch Length (ND and MM confocal images, Imagej/FUJI Mitochondrial Analyzer tool). J Quantification of mitochondrial fusion regulator OPA1 levels (immunoblotting). K Quantification of mitochondrial fission regulators MFF and pDRP1^S616 levels (immunoblotting). Immunoblots are cropped for visual ease. All immunoblotting results were normalized to tubulin and are expressed as percent (Mean ± SE, n ≥ 3) of protein levels in MM-MSCs compared to ND-MSCs. Asterisks depicted statistical significance *p < 0.05; **p < 0.01.

Fig. 3. Intervention with fission/fusion balance differentially affects BM-MSCs bioenergetics, mtROS and ΔΨm.

ND (cyan) and MM (red) MSCs (n≧3) were treated with Mdivi-1 (mitochondrial fission inhibitor, 50 µM; 24 h), MYLS22 (mitochondrial fusion inhibitor, 50 µM; 24 h), M1 (mitochondrial fusion promoter, 5 µM; 24 h), Ascorbic Acid (AA) (ROS scavenger, 500 µM; 24 h) and analyzed for (A) MM-MSCs glycoATP, mitoATP and TotalATP production rates following Mdivi-1 treatment (Seahorse XF Real-Time ATP Rate Assay Kit) (B) MM-MSCs SRC following Mdivi-1 treatment (Seahorse XF Cell Mito Stress Test Kit). C ND-MSCs glycoATP, mitoATP and TotalATP production rates following MYLS22 treatment (Seahorse XF Real-Time ATP Rate Assay). D ND-MSCs SRC following MYLS22 treatment (Seahorse XF Cell Mito Stress Test Kit). E ND and MM-MSCs mtROS levels following MYLS22 treatment (mitoSOX red). F MM-MSCs mitochondrial membrane potential following MYLS22 treatment (TMRE). G MM-MSCs mitochondrial membrane potential following AA treatment (TMRE). H ND-MSCs mitochondrial membrane potential following M1 treatment (TMRE). I ND-MSCs mtROS levels following M1 treatment (mitoSOX red). J ND-MSCs proton leak following Mdivi-1/MYLS22 treatment (Seahorse XF Cell Mito Stress Test Kit). Results are expressed as percentage (Mean ± SE, n ≥ 3) and normalized to untreated cells represented by control bar (100%) or dashed line. Asterisks depicted statistical significance *p < 0.05; **p < 0.01.

Fig. 4. Venetoclax mitigates MM-MSCs support of adjacent MM cells’ survival and sensitizes them to anti-MM drug Bortezomib.

ND and MM MSCs (n ≥ 3) were pre-treated with Venetoclax (Ven) (SRC inhibitor, 0.5 µM; 24 h) then co-cultured with RPMI-8226 or MM.1S MM cell lines (MM cells) in the presence of Ven for 48 h or added with Bortezomib (BTZ)(anti-MM drug, 5 nM; last) for the last 24 h of 72 h of co-culture. Untreated MM cells alone or in co-culture served as controls. A Ven’s effect on co-cultured (with ND/MM-MSCs, 72 h) MM cells’ Viability (Presto-Blue). B, C Ven ± BTZ effect on co-cultured (with MM-MSCs, 72 h) MM cells’ Viability (Presto-Blue) and total cell count (Trypan-Blue). Cell counts and viability were normalized to untreated MM cells cultured alone. Results are expressed as percentage (Mean ± SE, n ≥ 3). Asterisks depicted statistical significance *p < 0.05; **p < 0.005.