RBPJ/PAF1 Signaling Axis Promotes Cloning Expansion in Paroxysmal Nocturnal Hemoglobinuria Through NOTCH Signaling

The pathogenesis of paroxysmal nocturnal hemoglobinuria (PNH) is closely related to additional somatic mutations besides PIG-A. Our previous research showed that some PNH patients had a high frequency mutation of RBPJ through whole genome exon sequencing, which played an important role in the pathogenesis of PNH, but its molecular mechanism is still unclear.

SiRNA transfection was employed to generate RBPJ-knockdown K562 and PNH cell lines. Cell proliferation and apoptosis were assessed via EDU and flow cytometry, respectively. LC-MS following silver staining identified RBPJ-interacting proteins, which were subsequently validated using CO-IP and gradient expression analysis. Western blot examined the relationship between RBPJ, PAF1, and the NOTCH signaling pathway in low-RBPJ-expressing PNH cell lines. The correlation between PAF1 expression, clonal PNH, and RBPJ expression was also analyzed.

Knocking Out RBPJ Leads to Reduced Cell Proliferation and Increased Apoptosis in PNH Cells. We Identified PAF1 as an Interacting Protein of RBPJ and Confirmed the Interaction Domain to Be the BTD Domain of RBPJ. Knocking Out RBPJ Resulted in a Decrease of PAF1 Protein in KO Cell Lines, While the NOTCH1 and HEY1 Protein Expression Related to the NOTCH Signaling Decreased. GSEA Demonstrated That the KO Cell Lines Exhibited a Decreased NOTCH Signaling Pathway Upon PAF1 Downregulation. Knocking Down PAF1 Results in Reduced Protein Levels of NOTCH1 and NOTCH2. Subsequently, After Knocking Out RBPJ With PAF1 Overexpression, NOTCH1 Expression Was Restored, Cell Apoptosis Rate Decreased, and Cell Proliferation Increased. Subsequently, we Observed Elevated PAF1 Expression in PNH Patients, Which Positively Correlated With the FLAER-CD14, FLAER-CD24 and RBPJ mRNA Expression in PNH Patients.

RBPJ/PAF1 may promote PNH clone proliferation by regulating the NOTCH signaling pathway.