P870: DEFINING A NOVEL FUNCTION FOR THE POST-TRANSLATIONAL MODIFICATION UFMYLATION IN THE ADAPTIVE RESPONSE TO ARGININE DEPRIVATION IN MULTIPLE MYELOMA

Abstract

Background: Our previous work showed that arginine deprivation is associated to multiple myeloma (MM) bortezomib refractoriness, but the molecular basis of this adaptive response is still largely unknown. Aims: We designed an integrative transcriptional and metabolic study to explore the contemporary transcriptional and metabolic changes occurring upon arginine deprivation in four human myeloma cell lines (HMCLs, U266, NCI-H929, OPM2 and MM1.s). Methods: HMCLs were individually cultured with customized complete with different Arg concentrations, correspondent respectively to 100%, 25% and 10% of the arginine concentration in MM bone marrow. After 24-48 hours of culture, the cells were collected for global metabolomic analysis (Metabolon Inc) and transcriptome profiling by Illumina platform. Apoptosis and mitochondrial depolarization were measured by FACS. Protein expression was evaluated by western blot analysis. Results: In all tested HMCLs the progressive arginine deprivation (range 1124-0 µM) induced delay in cell cycle until proliferation arrest (in total lack of arginine in culture media), with increase of G0-G1 length, at specific timepoints for each cell line tested. However global protein synthesis was not affected and surprisingly, in all tested cell lines, the monoclonal component secretion in extracellular medium was preserved after 96 hours of arginine starvation. At 48 hours of arginine deprivation (range 1124-100 µM) GCN2 pathway was induced in HMCLs with consequent increase of basal levels of ATF4, p62, GABARAP and autophagy flux. After 48 hours of arginine starvation, coupled metabolomics and RNA-seq, disclosed an impairment in the pool of nucleotides and activation of salvage purine pathway, that could explain changes in cell cycle and proliferation. Arginine-deprived MM cells had higher levels of choline and phospho-ethanolamine with lower levels of glycerophosphorylcholine (GPC) compared to controls suggesting an effect of arginine-deprivation on membrane dynamics, associated to increased production of fatty acids, palmitate (16:0) and stearate (18:0), required for the generation of new membranes. Culture in arginine deficient media resulted in complete depletion of reduced glutathione (GSH) and oxidized glutathione (GSSG), with depletion of gamma-glutamylcysteine, indicating increased levels of oxidative stress. Gene set enrichment analysis (GSEA) showed deep transcriptome rearrangements, involving upregulation of genes required for the unfolded protein response, NF-kB response to TNF, p53 pathway and networks and proteosome degradation, with a minimal effect on metabolism features, except the upregulation of genes involved in lactate generation and degradation. In all cell lines, transcripts involved in the UFMylation pathway were affected, with reduction of UFSP2 and increase of UFMYlation targets RPL26, PKM2, ENO-1, PGK-1 and SQSTM1/p62. In a large series of primary samples (9N - 20MGUS - 33SMM - 170MM - 24PPCL - 12SPCL) and 18 MM cell lines, through bioinformatic analysis of previously published datasets (GENE1.0_BAv20_Neri-Gutierrez), we found that myeloma progression was associated to increased expression of UFMYlation targets ENO-1, PGK1 and DLD. Summary/Conclusion: Taken together, our data clearly indicate how arginine starvation can induce an heterogeneous adaptation processes occur in individual tumour clones. The ER stress adaptive response triggered by arginine deprivation promotes UFMylation activation, due to downregulation of UFSP2 and accumulation of RPN1/RPL26 targets.