CELF2-deficient mutations promote B-cell lymphoma development by inducing a germinal center B-cell fate Free

RNA-binding proteins (RBPs) are key regulators in the processes of RNA transcription and translation. As an important member of RBPs, ELAV-like family member 2 (CELF2) has been confirmed to regulate RNA splicing and embryonic hematopoietic development, and its expression is frequently dysregulated in B-cell lymphoma. However, the functional role of CELF2 in the initiation and progression of B-cell lymphoma has not been fully elucidated. This study found that loss-of-function mutations in CELF2 can lead to the expansion of germinal center B (GCB) cells and promote the progression of B-cell lymphoma.

Analysis of ICGC datasets revealed CELF2 genomic alterations in 36.08% of DLBCL cases. DLBCL harboring CELF2 mutations exhibited significantly reduced mRNA expression compared to wild-type counterparts, suggesting mutation-induced transcriptional silencing. To explore the impact of CELF2 deficiency on the differentiation of mature B cells, we immunized WT (Celf2^fl/fl) and B cell-specific Celf2 knockout mice (CD19-Cre; Celf2^fl/fl) with sheep red blood cells (SRBCs) and analyzed GC B-cell responses. After SRBC immunization, the spleen weight of CD19-Cre; Celf2^fl/fl mice was much greater than that of littermate controls. The percentage of GC B-cells in spleen from CD19-Cre; Celf2^fl/fl mice was significantly increased, while the percentage of plasma blast was decreased in spleen and peripheral blood when compared with that of littermate controls. To assess the proliferative capacity of CELF2-deficient germinal center B (GCB) cells in vivo, we performed EdU incorporation assays. Quantitative analysis revealed an increased proportion of EdU+ GCB cells in CELF2 knockout mice compared to littermate controls. Single-cell analysis also indicated an increased proportion of germinal center B (GCB) cells in the spleen of CD19-Cre; Celf2^fl/fl mice. Together, these data suggested that CELF2 loss might promote GCB cell proliferation through accelerating cell cycle progression.

To investigate CELF2's lymphoma-modulatory function in the context of MYC overexpression, we engineered B cell-specific Celf2 knockout mice within the Eμ-Myc transgenic background (Celf2^△;Eμ-Myc). Celf2^△;Eμ-Myc mice exhibited markedly accelerated lymphomagenesis versus Eμ-Myc controls. Notably, Celf2^△;Eμ-Myc mice manifested expansion of premalignant B220^low cells in peripheral blood (PB) compared to Eμ-Myc controls. To determine whether Celf2 deficiency potentiates MYC-driven lymphomagenesis in a cell-autonomous manner, we reconstituted lethally irradiated WT recipients with Celf2^△;Eμ-Myc or Eμ-Myc bone marrow cells. Kaplan-Meier analysis demonstrated a 2.6-fold increased mortality risk in Celf2^△;Eμ-Myc chimeras. Flow cytometric analysis of splenic lymphocytes demonstrated an expansion of premalignant B220^low B cells in Celf2^△;Eμ-Myc chimeras compared to Eμ-Myc chimeras.

Having established CELF2's tumor-suppressive function in B lymphoma mouse model, we extended these findings to human B-cell lymphoma. Over-expression of CELF2 (CELF2 OE) in Ly-7 and Raji cells suppressed proliferation and increased apoptosis. Clinically, DLBCL patient datasets revealed an inverse CELF2-MYC expression correlation, with CELF2^low/MYC^high tumors showing reduced overall survival. Together, these data demonstrated the tumor-suppressive roles of CELF2 in B-cell lymphomas.

Finally, to investigate the mechanistic role of CELF2 in B-cell lymphomagenesis, we performed RNA-seq analysis on sorted B lymphoma cells derived from Celf2^△;Eμ-Myc mice compared to control Eμ-Myc mice. This reciprocal expression pattern mechanistically substantiated CELF2's tumor-suppressive function via tonic repression of MYC/E2F networks in B-cell lymphomagenesis.