Molecular Mechanism of c-Myc Nuclear Output Mediated By m5C Binding Protein Alyref in c-Myc+ DLBCL

Objective: Diffuse large B-cell lymphoma (DLBCL) represents the most prevalent form of adult lymphoma. Elevated c-myc expression within DLBCL is strongly correlated with a poor prognosis and an aggressive disease course. In recent years, the significance of m5C epigenetic modification in the pathogenesis and progression of disease has increasingly been recognized. This study seeks to investigate the molecular mechanism of m5C modification in c-myc+ DLBCL, with the aim of identifying new therapeutic targets to enhance treatment response and improve patient prognosis.

Methods:We investigated the association between m5C regulators and c-myc overexpressed DLBCL by examining transcriptome data from the Gene Expression Omnibus (GEO) database. Data were sourced from two cohorts within the GEO database, comprising 420 and 223 patients respectively. Each cohort categorized patients based on the expression level of c-myc, assessed the differential levels of m5C regulators between these groups, and examined their correlation with overall survival. Additionally, a separate cohort was subjected to testing. The accuracy of gene expression levels was confirmed through quantitative Polymerase Chain Reaction (qPCR), Western Blot (WB) in cell lines, and Immunohistochemistry (IHC) in patients. Furthermore, we examined the regulatory impact of potential m5C targets on c-myc using cell transfection experiments.

Result:We divided the transcriptome data of two cohorts of DLBCL patients from the GEO database into high and low c-myc expression groups. Kaplan-Meier survival analysis confirmed that a high expression of c-myc is associated with poor prognosis. We then examined the enrichment and expression of m5C regulatory factors between these groups, identifying a positive correlation between NSUN2 and ALYREF expression and c-myc. Notably, NSUN2 expression was associated with poor prognosis. We elucidated the relationship between ALYREF, NSUN2, and c-myc using qPCR and WB experiments in cell lines, including normal lymphocyte line (GM12875) and DLBCL cell lines (WSU-DLCL-2, OCI-LY3, DB, and SUDHL-4). The expression levels of ALYREF, NSUN2, and c-myc were consistent across these cell lines, with particularly high expression in OCI-LY3, DB, and SUDHL-4 cell lines. Upon knocking down ALYREF in the DB and SUDHL-4 cell line, we observed a decrease in both NSUN2 and c-myc levels, suggesting a regulatory relationship between ALYREF and both NSUN2 and c-myc. Furthermore, we collected tumor tissues from 36 DLBCL patients for immunohistochemistry, dividing them into c-myc expression and non-expression groups. The results once again confirmed the correlation between ALYREF and NSUN2 and c-myc expression, with NSUN2 expression being associated with poor prognosis.

Conclusion:Our research highlights ALYREF/NSUN2 as a promising therapeutic target for DLBCL, demonstrating its correlation with poor prognosis in this disease. The development of drugs that specifically target ALYREF/NSUN2 could offer more effective treatment options for patients diagnosed with c-myc overexpressed DLBCL.

Key Words: ALYREF; NSUN2; c-myc; Diffuse large B-cell lymphoma; Targeted therapy.

No relevant conflicts of interest to declare.