GCN2 Activation Promotes PD-L1 Expression and Survival of RPMI-8226 Multiple Myeloma Cells: An In Vitro Study Under Arginine Deprivation

Background: Multiple myeloma (MM) features metabolic stress along with immune evasion in the bone marrow microenvironment. This study aimed to clarify the role of general control nonderepressible 2 (GCN2) kinase signaling in regulating MM cell survival and programmed death-ligand 1 (PD-L1) expression under amino acid deficiency.Methods: RPMI-8226 multiple myeloma cells were cultured under arginine-deprived conditions to mimic metabolic stress. Activation of the GCN2 signaling pathway and its downstream effectors (activating transcription factor 4 [ATF4], C/EBP homologous protein [CHOP]) was assessed by Western blotting. Cell viability and apoptosis were evaluated using Cell Counting Kit-8 (CCK-8) assays and Annexin V/PI flow cytometry. PD-L1 expression was measured using Western blotting, quantitative real-time PCR (qRT-PCR), and flow cytometry. Functional immune effects were further examined in a co-culture system with peripheral blood mononuclear cells (PBMCs). The role of GCN2 was validated using the selective inhibitor GCN2iB and the stress mimetic halofuginone.Results: Arginine deprivation markedly activated the GCN2–ATF4–CHOP signaling axis in MM cells, promoting cell viability and suppressing apoptosis. In contrast, pharmacological inhibition of GCN2 with GCN2iB significantly reduced cell viability and increased apoptosis. GCN2 activation also upregulated PD-L1 expression at both transcriptional and surface levels, whereas GCN2 inhibition attenuated this effect. In a co-culture system, GCN2-mediated PD-L1 upregulation impaired T-cell activation, as evidenced by decreased CD69/CD25 expression and reduced IFN-γ secretion; these effects were partially reversed by GCN2 inhibition or PD-L1 blockade. Additionally, GCN2 inhibition exhibited limited cytotoxicity in PBMCs, indicating relative selectivity toward tumor cells. Halofuginone recapitulated the effects of amino acid deprivation, further confirming the role of GCN2 in metabolic and immunoregulatory adaptation.Conclusion: GCN2 functions as a critical metabolic stress sensor in RPMI-8226 multiple myeloma cells, promoting tumor cell survival and immune evasion through PD-L1 upregulation under amino acid deprivation. Targeting the GCN2 pathway may represent a dual-acting therapeutic strategy to impair both metabolic adaptation and PD-L1 activation in MM.