P758: the mechanism of abnormal expression and mutation of rbpj gene promoting clone proliferation by regulating paf1 in paroxysmal nocturnal hemoglobinuria

Topic: 11. Bone marrow failure syndromes incl. PNH - Biology & Translational Research Background: Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired clonal disease of hematopoietic stem cells caused by somatic mutations. Our Previous studies used whole exome sequencing (WES) technology to perform deep sequencing on 13 patients with PNH(Fig.A), and also found that the relative expression of the gene is significantly correlated with the clinical condition of PNH. Therefore, we speculate that the high expression of RBPJ gene may be involved in the proliferation of PNH abnormal clones. Aims: To explore the mechanism of RBPJ gene mutation involved in PNH clone proliferation. Methods: We obtained blood samples from 6 PNH patients and 5 healthy individuals to use qRT-PCR to identify the presence of RBPJ. PNH cell line（KO）was constructed by knocking out PIGA by Crsiper/Cas9 technology on K562 cell line. Transfection of siRNA resulted in the creation of K562 and PNH cell lines with low expression of RBPJ. The EDU technique and flow cytometry were used to detect cell proliferation and cycle. The K562-KO cell line stably expressing flag-RBPJ was constructed, and the RBPJ interacting proteins in each group were identified by LC-MS after silver staining. CO-IP confirmed the relationship between RBPJ and its physical interaction. Western-blot analysis revealed the expression of RBPJ, PAF1, NOTCH1, Gapdh, and Tublin in the low-RBPJ-expressed K562 and PNH cell lines. PNH and K562 cell lines with low expression of PAF1 were constructed by siRNA transfection, and Western-Blot was used to detect RBPJ, PAF1, and Gapdh expression. Results: PNH patients had higher levels of RBPJ mRNA expression (FigB). According to this result, RBPJ expression was knocked down in K562 cell line and PNH cell line and the effect was verified (p< 0.05), indicating that the low-RBPJ-expressed K562 and PNH cell lines were successfully constructed. Flow cytometry showed that the proliferation of PNH cell line was decreased (Fig.C) and the apoptosis was increased (Fig.D) after RBPJ knockdown (p < 0.05). LC-MS was used to identify the RBPJ interacting proteins in each group. A total of 281 proteins interacting with RBPJ were identified (Fig.E), including the core components of the NOTCH pathway Notch1and other proteins that have been clearly reported to interact with RBPJ, as well as polymerase-related factor 1 (Paf1) and other polymerase-related factor 1 (Paf1) complex. Thus, we speculate that the polymerase-associated factor 1 (Paf1) complex may be involved in regulating the expression of RBPJ in PNH patients. We chose PAF1 from the interacting proteins for further investigation, and used immunoprecipitation experiments to further confirm the physical interaction between PAF1 and RBPJ in K562-KO cell line, whereas there is no physical interaction between PAF1 and RBPJ in K562 cell line (Fig.F). Knockdown RBPJ, PAF1 protein content in the K562 cell line increased, while it reduced in the K562-KO cell line. Knockdown RBPJ, NOTCH1 protein content also decreased in both cell lines (Fig.G). Knockdown of PAF1 expression in K562 and PNH cell lines and verification of the effect were confirmed (p<0.05), indicating that PAF1-low K562 and PNH cell lines were successfully created. In K562 and K562-KO cell lines, PAF1 knockdown decreased the amount of RBPJ protein (Fig.H). Summary/Conclusion: Patients with RBPJ mutations showed higher expression. After RBPJ was knocked out in PNH cell line, cell proliferation decreased and apoptosis rate increased; After PAF1 was knocked out, the amount of RBPJ protein and the expression of PAF1 protein decreased, and there was a physical interaction between PAF1 and RBPJ, while the expression of NOTCH1 protein decreased. RBPJ may regulate PAF1 through NOTCH signaling pathway, thus promoting PNH clone proliferation.Keywords: Proliferation, Paroxysmal nocturnal hemoglobinuria (PNH)