Arginine Deprivation and Autophagy Sustain Bioenergetics in Multiple Myeloma

Background and Objective:

Multiple myeloma (MM) originates from a clone of transformed plasma cells (PCs) residing in the bone marrow (BM), which establish vicious interactions with the multicellular microenvironment. Autophagy is a highly conserved adaptive strategy that sustains metabolism under stress. In cancer, autophagy has been shown to mediate both onco-suppressive and pro-tumoral functions. Our group demonstrated that MM is addicted to autophagy for clonogenicity, survival, and drug resistance.

Little being known on metabolic changes during MM progression, we deployed a comprehensive metabolic analysis at different stages of disease.

Experimental design: First, we adopted ultra-high performance liquid and gas chromatography followed by mass spectrometry (UHPLC/GC-MS) on an independent series of 167 samples of bone marrow (BM) and peripheral plasma collected ad hoc from 125 individuals, comprising newly diagnosed MM (n=16), patients with relapsing or progressive disease (n=20), in clinical remission (n=13), with MGUS (n=30), smoldering MM (SMM) (n=17), and age-matched healthy volunteers (n=29). Upon exclusion of drug metabolites and xenobiotic compounds, 284 endogenous metabolites were processed by PCA (unsupervised) and OPLS-DA (supervised) multivariate analyses to identify myeloma-associated metabolites. Second, we explored the expression levels of key metabolic genes in a large series of highly purified BM PC samples from healthy donors (4N), 129 MM, 24 primary plasma cell leukemia (pPCL), 12 secondary PCL (sPCL) cases from a proprietary dataset (GSE66293).

Results

Metabolomics revealed the presence of robust metabolic differences in the progression from MGUS to MM, sustained by reduced amount of lysolipids and amino acids. These findings were validated by HPLC and ELISA in an independent cohort showing that arginine deprivation was associated to progression from MGUS through MM.

Upon MM progression, enolase-1 (ENO-1), phosphoglycerate kinase 1 (PGK-1), and dihydrolipoamide dehydrogenase (DLD) were increased, suggesting that branched chain amino acids, alpha-ketoglutarate, and glycine cleavage products are used in MM to sustain NADH availability and energy production. Moreover, clustering analysis of expression profiles of genes involved in tricarboxylic acid (TCA) cycle grouped the majority of pPCL and (t 14;16) cases, showing an up-regulated TCA cycle signature.

Using pharmacological inhibitors of glucose metabolism (2-deoxyglucose), lipolysis (atglistatin), and autophagy (bafilomycin-A1) we collected evidence that MM cells generally deploy TCA and oxidative phosphorylation, with glucose catabolism as a major source of ATP. Inhibition of glycolysis was partially compensated for by non-glycolytic oxidative phosphorylation; such compensation correlated with increased expression of transcripts encoding mediators of glutamine anaplerosis, lipid synthesis, lipid droplet biogenesis, lipolysis, and autophagy.

Next, we sought to determine whether the adaptation to amino acid starvation through autophagy altered cellular glucose dependence in three MM cell lines chosen on the basis of their cytogenetic alterations: U266 (t 11;14); H929 (t 4; 14) and MM.1S (t 14; 16).

Progressive arginine deprivation (range 1000-10 nM) did not affect proliferation in vitro, even in low-glucose media. However, long-term arginine deprivation, or short-term treatment with human recombinant arginase-1, altered the cellular dependence on mitochondrial ATP generation via oxidative phosphorylation and increased glutamine anaplerosis. Indeed, U266, MM.1S and H929 cell lines treated with oligomycin, an inhibitor of complex V in the electron transport chain, invariably showed reduced ATP and higher cell death.

Conclusion

Overall, our study reveals a substantial contribution of oxidative phosphorylation in MM bioenergetics, and point to decreased arginine availability in the microenvironment as the basis for the activation of metabolic compensatory pathways, offering new putative targets for synthetic lethality.