CYLD介导的非经典RelB NF-κB转录轴在多发性骨髓瘤中的作用

Introduction: Multiple myeloma (MM), a plasma B cell neoplastic disease, has been studied widely for its dependency on NF-κB signaling, yet the crosstalk between canonical (RelA/p50) and non-canonical (RelB/p52) arms remains incompletely defined. Tumor suppressor protein CYLD, an established negative regulator of canonical NF-κB, emerged in our studies as an enforcer of RelB-driven transcription. We define a CYLD-mediated RelB enforced signaling that promotes the disease pathogenesis by supporting survival and migration in tumor cells. Methods: Primary CD138+ plasma cells were isolated from bone marrow aspirates of 74 patients with MM using MACS based separation. Cells were used for NF-κB DNA binding assay (n=15) and quantitative RT-PCR based gene expression studies (n=42). For in vitro studies, patient derived MM cell lines (MMCLs) JIM3 and KP-6 were used for biochemical studies. CYLD deficient isogenic MMCLs were generated using CRISPR-Cas9 gene editing. The RNA-seq metadata available with the Multiple Myeloma Research Foundation (MMRF) database IA13a were used for in silico transcriptomic analyses. Results: In our study, using primary myeloma cells, we established the presence of increased nuclear RelB (nRelB) activity in newly diagnosed patients with MM. This increased nRelB activity was shown to be positively correlated with high RelB expression. MMRF transcriptomic data allowed us to show association of high RelB expression with poor prognosis. With NEMO kinase activity assay and NF-κB DNA binding assay on MMCLs, we showed that CYLD deficient KP-6 has higher canonical NF-κB activity as compared to CYLD sufficient JIM3 cells. This absence of deubiquitinase CYLD was associated with high RelB mRNA and protein levels at basal state that were further augmented by stimulation with BAFF, a non-canonical NF-κB activating cytokine. The re-enforced RelB activity in the absence of CYLD also provided advantage to CYLD deficient cells in survival and migration activities. This phenotypic advantage was mapped onto RelB mediated transcription of pro-survival factors such as BCL2, BIRC2, BIRC3, TRAF1 & c-FLIP and pro-migratory chemokine receptors such as CXCR4 & CXCR7. Deletion of CYLD using CRISPR-Cas9 editing to generate JIM3 sg CYLD cells, provided the causal role of CYLD mediated RelB enforced transcriptional axis that supports survival and migration in these malignant plasma cells. Conclusions: The tumor microenvironment and cancer-associated mutations activate both canonical and non-canonical NF-κB pathways in MM. More so, these NF-κB pathways are intimately interlinked via a number of biochemical mechanisms. Our data signifies that RelB plays a role in the survival and migration of myeloma cells and mutational inactivation of CYLD exacerbates this disease-associated RelB phenotype, thus suggesting that NF-κB crosstalk mechanisms may provide prognostic evidence and therapeutic opportunities in the future.