P01: ARGININE STARVATION IN MULTIPLE MYELOMA MICROENVIRONMENT CONVEYS BORTEZOMIB REFRACTORINESS VIA TLR4 PATHWAY.
Authors: A Romano, G Scandura, C Giallongo, E La Spina, A Barbato, D Tibullo, F Di Raimondo
Journal: HemaSphere (2022)

Background: In multiple myeloma (MM) malignant plasma cells (PC) can induce metabolic changes in the local microenvironment that are closely related to drug resistance and disease progression. Arginine (Arg) is a non-essential amino acid which plays an important role in the immune-escape mechanisms and in BTZ resistance. Since our group has previously demonstrated that BTZ- resistant PCs upregulate TLR4 pathway as a stress responsive mechanism, and TLR4-driven cytokine production in starvation is under control of general control nonderepressible 2 (GCN2), a serine/threonine-protein kinase that senses amino acid deficiency through binding to uncharged transfer RNA, we aimed to investigate TLR4 signalling in response toArg deprivation. Methods: Our in vitro studies are carried out on three human myeloma cell lines (HMCLs) (U266, NCI-H929, OPM2) cultured in medium with different Arg concentrations: R100 (114 μg/mL), R25 (28.5 μg/mL) and R10 (11.4 mg/mL), correspondent respectively to 100%, 25% and 10% of the arginine concentration in MM bone marrow, for 24h or 48h. Apoptosis and mitochondrial depolarization were measured by FACS. Protein expression was evaluated by western blot analysis. Gene expression was carried out by qRT-PCR. Results: In bortezomib-refractory patients Arg-1 was increased in peripheral blood. Sera of MM, but not MGUS, patients conferred bortezomib-resistance to U266 cell line in an Arg-1 dependent manner, an effect partially reverted by treatment with nor-NOHA (an aspecific Arg-1 inhibitor). Treatment with recombinant Arg-1 for 24 hours conferred protection to U266 after treatment with 20nM bortezomib for 48 hours, associated to increased expression of ASNS, ATF4 and p62. In HMCLs the progressive arginine deprivation induced delay in cell cycle until proliferation arrest (in total lack of arginine in culture media), with increase of G0-G1 length, associated decreased cell viability starting from R25. We next investigated if MM cells may adapt to Arg deprivation through unfolded protein response (UPR) system or GCN2 pathways. In the UPR system, higher levels of expression of metabolic-stress sensing protein PERK was observed only in NCI-H929 cells adapted to lower Arg deprivation (R25). Interestingly, we found an up-regulation of GCN2 protein expression in U266 cells already at R25 and in NCI-H929 only at R10 condition, associated to increased expression of TLR4 and its downstream effectors, involved in mitochondrial depolarization, suggesting a role of TLR4 pathway in preserving MM-PCs under arginine starvation. We finally tested the efficacy of bortezomib (BTZ) and carfilzomib (CFZ) in HMCLs cultured in R25 and R10 medium. Compared to MM cells treated with PIs in R100 medium, HMCLs exposed to BTZ and CFZ in R25 and R10 medium showed decreased PI-induced apoptosis and lower mitochondrial depolarization. Conclusions: Our study suggests that TLR4 pathway is part of adaptation to Arg deprivation in HMCLs and to convey bortezomib refractoriness.