Arginine Deprivation in Microenvironment Makes Multiple Myeloma Resistant to Proteasome Inhibitors Via Increased Lactate Release and Metabolic Rewiring

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. Our group previously demonstrated that Myeloid-derived suppressor cells produce arginase (ARG-1) that induce a reduction in the microenvironment of arginine (Arg), a non-essential amino acid, whose deficiency in the microenvironment plays an important role in the immune-escape mechanisms and in bortezomib (BTZ) resistance.

Aim

To explore the consequences of metabolic changes occurring after arginine deprivation on efficacy of proteasome inhibition (PI).

Methods

Our in vitro studies were carried out on two human myeloma cell lines (HMCLs) (U266, NCI-H929) cultured up to 10 days in medium with different arginine concentrations: R100 (114 μg/mL) and R25 (28.5 μg/mL) corresponding respectively to 100% and 25% of the arginine concentration in MM bone marrow, which is generally lower than in healthy and MGUS subjects. 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 (BTZ)-refractory patients, the arginine degrading enzyme Arginase 1 (ARG1) was increased in peripheral blood and bone marrow. Sera of MM, but not MGUS patients, conferred BTZ-resistance to U266 cell line in an ARG1 dependent manner, an effect partially reverted by treatment with nor-NOHA (an aspecific Arg-1 inhibitor). Treatment with recombinant ARG1 for 24 hours conferred protection to U266 after 48h treatment with 20nM bortezomib, associated to increased expression of ASNS, ATF4 and p62, but not autophagic flux. In HMCLs the progressive arginine deprivation induced delay in cell cycle until proliferation arrest, with increase of G0-G1 length. In cells cultured in R25, mitochondrial activity was accompanied by a low-energy metabolic state, associated to increased release of extracellular lactate. Compared to MM cells treated with PIs in R100 medium, HMCLs exposed to BTZ and CFZ in arginine deprivation (R25) showed a reduction of PI-induced apoptosis, associated to increased oxidative stress, HO-1 activation, induction of the inflammasome pathway and acquisition of a senescent phenotype.

To dissect better the effects of high concentrations of lactate in tumor microenvironment, we cultured HMCLs in medium with lactate for 3 days. After 72h, HMCLs chronically exposed to high lactate concentration were unable to increase oxygen consumption, as detected by Sea Horse analysis. Compared to controls, both U266 and NCI-H929 exhibited lower basal and maximal respiration, recapitulating metabolic rewiring observed in chronic arginine deprivation.

By modulating the lactate transporter MCT1, we found that the uptake of lactate by MM-PCs decreased the anti-myeloma efficacy of PIs, as further confirmed by higher colocalization of MCT1 within CD138+ PCs in relapsed/refractory MM compared to patients at diagnosis. To elucidate whether the protective effect of lactate was mediated by metabolic activity of lactate or also through the activation of GPR81 lactate receptor signaling, NCI-H929 cells were cultured in medium supplemented with 20% sera from different MM patients and treated with BTZ or carfilzomib (CFZ) alone or in combination with AZD3965, a selective inhibitor of MCT1, or 3-OBA, an antagonist of GPR81 receptor. MM-PCs treated with BTZ or CFZ in combination with AZD3965 showed higher apoptosis rate compared to PI-only treated cells. In contrast, 3- OBA decreased the anti-myeloma effect of BTZ. Since it is known that PIs exert their apoptotic effect by disrupting mitochondrial integrity and activity, we also measured mitochondrial polarization status. Compared to BTZ or CFZ alone, the combination with AZD3965 induced mitochondrial depolarization while the combination with 3-OBA significantly reduced the percentage of PI-induced mitochondrial depolarization.

Conclusions

Taken together, our data suggest that metabolic rewiring associated to arginine deprivation induces increased lactate availability in the microenvironment, which is in turn able to protect mitochondria against PI-induced cytotoxicity. This effect can be counteracted only blocking lactate import by MCT1 transporter using AZD3965, but not inhibiting the GPR81 signaling.