Abnormalities in Mitochondrial DNA Copy Number Have Pathogenetic and Prognostic Implications in Multiple Myeloma

Mitochondria are bioenergetic and biosynthetic organelles which control crucial biological pathways such as cell growth, proliferation and apoptosis among others. Over the last two decades, many studies have examined the role of mitochondrial DNA (mtDNA) alterations in carcinogenesis, via inactivating genetic mutations or dysregulation of the mtDNA copy number in the cell. However, the status of the mtDNA copy number and its implication in the pathogenesis of Multiple Myeloma (MM) remains unknown. Here, we have analyzed the mtDNA copy number in asymptomatic and symptomatic stages of MM and evaluated its clinical impact. We have estimated the mtDNA copy number in purified plasma cells from 139 patients across different stages of the disease (4 healthy donor, 19 MGUS, 14 Smoldering MM (SMM), 78 newly diagnosed MM (ndMM) and 24 relapse (rMM). The mtDNA copy number quantification was performed by RT-PCR using 2 Taqman probes which targeted the mitochondrial gene MT-RNR1 and the nuclear gene RPPH1. We have also used whole-genome or whole-exome sequencing data to examine changes in mtDNA copy number in newly-diagnosed or relapsed MM (n=64 samples) including paired sample from SMM patients progressing to ndMM (n=10). FastMitocalc and Samtools were used to extract reads aligning to mitochondrial genome from bam files and calculate de mtDNA copy number by comparing to the number of reads aligning to nuclear genome. Differential gene expression analysis was done by using canEvolve.org ocogenomics web portal (Multiple Myeloma, GSE6477 study). We observed a significant increase in the average mtDNA copy number in myeloma cells compared to healthy plasma cells (39 vs 228 copies; p-value = 0.02). Plasma cells from MGUS cells showed lower mtDNA content than more advanced stages of the disease (129 vs 242 mtDNA copies). Interestingly, amongst 10 paired samples from SMM progressing to MM an increase in the mtDNA copy number was observed in 8 of the 10 patients, being more evident when the time to progression was longer. Additionally, we also observed an increase in the mitochondrial content in 17 of the 21 patient samples examined pre and post relapse, including those patients with three consecutive relapsed samples available. We have evaluated the impact on survival of the mtDNA copy number in 69 newly diagnosed patients and we have found that patients with greater than 400 mtDNA copy number had significantly shorter progression free survival compared to the rest (p-value = 0.024). The differential expression analysis of plasma cells from MGUS, SMM, ndMM and rMM patients compared to normal plasma cells confirmed an overexpression of the mitochondrial biogenesis regulators (TFAM, TUFM, MYC and HNRNPK). We describe for the first time that mtDNA copy number increase across different stages of MM. Our data provides evidence that the malignant transformation of the myeloma cells from asymptomatic stages implies activation of mitochondrial biogenesis resulting in increased mtDNA levels. Functional experiments are ongoing to confirm the metabolic activation of myeloma cells over the time. These results highlight mitochondria as an important component of pathogenesis and progression of MM and as a potential target for therapy