The Metabolic Phenotype of Myeloma Plasma Cells Differs Between Active and Residual Disease States
Authors: Koss Brian , Tevebaugh Whitney , Walker Brian A , Schinke Carolina , van Rhee Frits , Zangari Maurizio , Thanendrarajan Sharmilan , Davies Faith E , Epstein Joshua , Yaccoby Shmuel , Morgan Gareth J , Johnson Sarah K. 
Published: October 2019

Abstract
Background
Understanding the basis for drug resistance and relapse from treatment is a key aim and studying cells left after treatment exposure can inform the mechanisms underlying these features.Tumor cells are characterized by high ROS levels, increased glycolytic capacity, and low mitochondrial content. Cancer stem cells, however are reported to utilize oxidative phosphorylation, not glycolysis, to produce ATP. This metabolic phenotype is associated with features of stemness and constitutes a platform with which to understand the biology of residual and drug resistant cancer cells to elucidate novel targets.While this phenotype has not been extensively studied in myeloma,Bortezomib resistant cell lines have increased mitochondrial function as measured by membrane potential, oxygen consumption rate, ATP level, and mitochondrial Ca2+ concentrations.
We have shown that ROS levels in CD138-selected myeloma cells are variable at diagnosis and are lower in plasma cells (PCs) from focal lesions (FLs) than PCs from random bone marrow (BM) aspirates consistent with greater features of stemness in FLs. MM cases with low ROS levels have increased oxidative phosphorylation pathway activation as determined by pathway enrichment analysis.We have previously demonstratedin MM cell lines (MMCLs) that ROSLo cells have a significantly greater percentage of cells in G1 and in S phase and an increased clonogenic capacity than ROSHi cells. Based on these observations we hypothesized that quiescent ROSLo cells differ in their mitochondrial function from bulk tumor cells, possibly indicating their stemness. We tested this hypothesis in PCs from active and residual disease as well as in MMCLs.
Methods
PCs from active and residual (< 10% PCs in BM) MM cases treated on our Total Therapy protocols were stained for ROS level (H2DCFDA) or mitochondrial content (Mitotracker) and detected by FACS. Mitochondrial content was also assessed by electron microscopy. We examined oxidative capacity, mitochondrial content, and ATP content in ROS-sorted MMCLs, H929 and U266. We cultured MMCLs with varying mitochondrial content and oxidative capacity in high glucose growth media or in media with galactose substituted for glucose to force cells to use oxidative phosphorylation. Mitochondrial content, ATP levels, and chemosensitivity (Cell Titer Glo and Annexin V/PI) were compared.
Results
Residual MMPCs contain more mitochondria/cell than normal PCs (NPCs, p=0.0183). PCs from active disease have an intermediate number of mitochondria/cell. Residual MMPCs and NPCs had higher mitochondrial content than MMPCs from active disease as assessed by mitotracker staining. To correlate mitochondrial content with ROS levels, H2DCFDA staining of residual MMPCs, NPCs, and PCs from the same cases used for mitotracker staining was performed. Residual MMPCs have lower ROS levels than MMPCs from active disease (p<0.0001) but were similar to NPCs.
We linked these metabolic characteristics to subpopulations of MMCLs with differing ROS levels. From these analyses we found that ROSHi cells had increased ATP content and lower oxygen consumption rate than ROSLo cells. Interestingly ROSHi cells from lines had higher mitochondrial content by mitotracker staining (p<0.01), whereas electron microscopy showed fewer mitochondria/cell in ROSHi cells than bulk tumor cells from active MM cases, highlighting differences between highly proliferating MM cell lines grown in normoxic conditions and freshly obtained MMPCs.
In an effort to recapitulate the metabolic characteristics of residual PCs, MMCLs were cultured in galactose and exhibited reduced ROS levels, increased mitochondrial content, and increased basal ATP levels compared to cells cultured in glucose. MMCLs grown in galactose were also more sensitive to metformin (targets OxPhos) and navitoclax (targets Bcl-2, Bcl-XL, and Bcl-w) and more resistant to 2-DG (blocks glycolysis) and bortezomib than cells grown in glucose.
Conclusions
Residual PCs have higher mitochondrial content and lower ROS levels, markers of stemness, than normal PCs and PCs from active MM. Cell lines forced to use oxidative phosphorylation for energy production acquire ROSLo MitoHi phenotype and are associated with chemoresistance, and representative of residual PCs. These ROSLoMitoHi cells may be functionally similar to MMPCs held in a BM niche that are dormant and have been described as chemoresistant.

Disclosures
Davies: Janssen: Consultancy, Honoraria; Celgene: Consultancy, Honoraria; Takeda: Consultancy, Honoraria. Morgan:Univ of AR for Medical Sciences: Employment; Janssen: Research Funding; Bristol Meyers: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria.