Analysis of the Metabolic State of Residual Multiple Myeloma Plasma Cells Reveals a Proliferative Subset with Glycolytic Gene Expression

Background

Tumor cells are characterized by high reactive oxygen species (ROS) levels, low mitochondrial content and increased glycolytic capacity. Whereas cancer stem cells are reported to utilize oxidative phosphorylation (OxPhos) to produce ATP, a metabolic phenotype associated with stemness that constitutes a platform to understand the biology of residual and drug resistant cancer cells.

We have shown that residual multiple myeloma plasma cells (MMPCs) contain more mitochondria than normal PCs and have lower ROS levels than MMPCs from active disease. To recapitulate the metabolic characteristics of residual MMPCs, myeloma cell lines (MMCLs) were cultured in medium with galactose substituted for glucose to force cells to use oxidative phosphorylation. These cells exhibited reduced ROS levels, increased mitochondrial content, and were more sensitive to drugs that target OxPhos and more resistant to those that target glycolysis than cells grown in glucose. To detect the transition from dormancy to proliferative disease at the earliest stage, we defined the metabolic state of residual MMPCs.

Methods

We used FACS to analyze ROS (H₂DCFDA), superoxide (DHE) and mitochondrial content (Mitotracker) in residual MMPCs (0.28-8.9%) in whole bone marrow from 98 patients receiving therapy. Serum LDH was used as a marker of glycolytic activity and proliferation. Ki67, a proliferation marker, was detected by FACS. We compared glucose uptake (NBDG) by FACS in MMCLs cultured in either glucose or galactose media. Expression of genes associated with glycolysis (HK2, MYC) or OxPhos (PPARGC1a) were detected by qRT-PCR in MMCLs and by gene expression profiling (GEP) in clinical samples.

Results

A subset of residual MMPCs have higher ROS (p=0.0165) and superoxide (p<0.0001) than normal PCs; in most cases this was mutually exclusive. Superoxide levels were lower in residual MMPCs from cases in remission (n=16) than those not in remission (n=82, p=0.0261). Of the cases with archived GEP data; HK2 (p=0.0004) and MYC (p=0.001) expression were weakly correlated with ROS level and PPARGC1a was negatively correlated with mitochondrial content (p=0.003, suggesting low mitochondrial activity). Similarly, MMCLs cultured in galactose had lower expression of MYC and HK2, but unlike residual MMPCs,higher expression of PPARGC1a . MMCLS cultured in galactose had lower glucose uptake than cells cultured in glucose.

In an effort, to understand the subset of residual cases with higher ROS levels we developed a metabolic score (MS) by defining quartiles for mitochondrial content, superoxide, ROS and LDH level from measurements of residual MMPCs. High superoxide and mitochondrial content (lower score) were considered to indicate dormancy and high ROS and LDH (higher score) indicate glycolytic and proliferative activity. Of the cases analyzed, the MS of 29% indicated dormancy (≤ -2), 48% had an intermediate MS, and 23% had an MS ≥ 2 indicating glycolytic activity and possible transition to the proliferative state. Residual MMPCs from 10 cases were examined for Ki67 production; 0.1- 12.2% of cells were positive for Ki67, with 3 samples > 4%. The MS for these samples ranged from -3 to +4 including one outlier (low MS, high Ki67), however there is clear potential to phenotype residual cases. Repeated measurements after 3 months in 3 cases showed that the MS for Pt 1 improved from 2 to -3, Pt 2 remained at 2, and Pt 3 had a slight change from -1 to 0, suggesting that in the absence of external stimuli the metabolic state of MMPCs is dynamic and most likely responds during dormancy to cues from the microenvironment.

Conclusions

We examined metabolic and proliferative parameters in residual MMPCs. Our data suggest that in a dormant state, most residual MMPCs have lower ROS and higher superoxide and mitochondrial content. However, some residual cases had higher ROS levels, possibly indicating loss of dormancy and transition to active disease. We developed a metabolic score to define the metabolic and proliferative activity for each case to allow precision targeting of residual MMPCs and will test this in an additional cohort to determine its sensitivity and specificity. Additionally, these parameters will be used to predict risk of progression prior to clinical relapse, and to test drugs targeting dormant residual cells prior to progression.