P852: arginine deprivation induces acquisition of a senescent phenotype and favors genomic instability in multiple myeloma plasma cells

Topic: 13. Myeloma and other monoclonal gammopathies - Biology & Translational Research Background: 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, thus leading to arginine (arg) deprivation, and inducing a dynamic evolutionary process that depends on complex genomic and epigenetic landscapes, where micro-environment constraints play an active, supportive role. Our previous work showed that in vivo the treatment with arginase inhibitor CB1158 could reduce MM growth while increasing serum arg concentrations without affecting the infiltrates of myeloid cells. Aims: We aimed to investigate if tumor initiation can result in metabolite alterations in the tumor microenvironment which, in turn, contribute to tumor progression via an adaptive response to arg deprivation. Methods: We combined gene expression profiling with coupled metabolomic analysis performed by high performance liquid chromatography (HPLC, SeaHorses), 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. Results: 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 that persisted until 10 days, as detected by SeaHorse analysis. Since energy potential was preserved in the arg-deprived HMCLs without affecting cell survival or immunoglobulin secretion, but only delaying cell cycle progression, we aimed to investigate the acquisition of a quiescent profile following long-term arg-deprivation. Indeed, the acquisition of senescence-associated secretory phenotype (SASP) was confirmed by the overexpression of of NLRP3, pro-Caspase 1, Caspase 1, interleukin-18 (IL-18) and cleaved IL-1β as detected by WB analysis, associated to chromatin remodeling. 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. Conclusion: 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. Keywords: Senescence, Genomic instability, Arginine, Multiple myeloma