P02 arginine deprivation induces acquisition of a senescent phenotype and favors genomic instability in multiple myeloma plasmacells

Multiple myeloma (MM) originates from a neoplastic clone of plasma cells which establish vicious interactions with the multicellular microenvironment, including myeloid derived suppressor cells that produce arginase leading to arginine (arg) deprivation. Our previous work showed that in vivo the treatment with arginase inhibitor could reduce MM growth while increasing serum arg concentrations independently from the infiltrates of myeloid cells. Thus, we aimed to investigate how arg deprivation can contribute to MM progression. We combined gene expression profiling with coupled metabolomic analysis performed by high performance liquid chromatography (HPLC), in three human myeloma cell lines (HMCLs, U266, NCI-H929 and OPM2). Cells were cultured for short (24h - 48h) and long term (10 days) in media with 50ug/ml of Arg (R low medium), which correspond to the concentration found in MM bone marrow, which is generally lower than in healthy and MGUS subjects, supplemented with 10% dialyzed fetal bovine serum. Progressive arg deprivation did not affect cell viability in vitro, but it was associated to proliferation slowing down and cell cycle arrest in G0-G1 phase, associated to reduced mitochondrial activity and a low-energy metabolic state, as detected by SeaHorse analysis. Differently from other cell lines, in H929 autophagy induction and the engagement of uncharged t-RNA tyrosin kinase GCN2 lacked, due to unchanged amount of available intracellular arg, as detected by HPLC. Conversely, H929 upregulated the expression of the arginine-succinate synthase (ASS1) enzyme, which catalyzes the conversion of nitrogen from ammonia and aspartate from glutamine to form argininosuccinate, a metabolite required to synthesis of urea, nitric oxide synthesis, polyamine, creatine synthesis, and the de novo synthesis of arginine. We used multiplex immune fluorescence immunohistochemical analysis to evaluate ASS1 in trephine BM biopsies from patients with MM (n=25), MGUS (n=10) and SMM (n=10). MM but not MGUS biopsies showed a multifocal pattern of ASS1 positivity, with dense clusters of tumor cells observed in MM but not MGUS biopsies. Combined flow cytometry and immunofluorescence microscopy analysis showed that γH2AX, a marker for DNA double-strand breaks, was increased and appeared as punctate spots in the cell nucleus and enriched in leaked DNAs, associated with genomic instability as showed by the increase of micronuclei percentage. Consistently, we also found the reduction of the Histone H3 lysine K4 (H3K4) and the increase of histone variant macro H2A1, recruited to DNA double-strand breaks to promote gene silencing and hampering DNA-repair mechanisms, as shown by a significant down-regulation of Fanconi Anemia (FA) pathway members BRCA2 and FANCI, master regulators of efficient replication DNA fork damage recovery. The acquisition of senescence-associated secretory phenotype was confirmed by the overexpression of of NLRP3, pro-Caspase 1, Caspase 1, interleukin-18 (IL-18) and cleaved IL-1□ as detected by WB analysis, associated to chromatin remodeling. Taken together, our findings suggest that arginine deprivation, while inducing immune dysfunction, conveys a complex adaptive response which causes a chromatin remodeling that leads to the acquisition of a senescence phenotype to select the most fit clone and favors genomic instability, providing new insights to improve immunotherapy and induce synthetic lethality in MM.