Effects and Molecular Mechanism of Inhibiting p53 Signaling Pathway By NSUN6 on the Resistance to BCL-2 Inhibitor for Diffuse Large B-Cell Lymphoma
Authors: Shi Yuanfei , Zhao Yanchun , Xu Yi , Sun Jianai , Zheng Xiaolong , Zhou De , Jin Jie , Tong Hongyan , Xie Wanzhuo 
Published: December 2024

Background: Most DLBCL patients with recurrent and refractory DLBCL have high BCL-2 expression, but some patients are resistant to BCL-2 inhibitors for unknown reasons, the mechanism of which is unknown yet. We constructed a DLBCL cell line resistant to venetoclax in the early stage to explore its molecular mechanism of resistance. We found that m5C methyltransferase NSUN6 was highly expressed in DLBCL resistant cell lines, and also highly expressed in lymph nodes of relapsed and refractory DLBCL patients. Those with high expression had poor prognosis. Knocking down NSUN6 can significantly reduce the tumor burden and prolong the survival cycle of experimental mice. Therefore, we speculate that NSUN6 may be an important target for venetoclax resistance in relapsed and refractory DLBCL patients. Molecular complex detection (MCODE) and RNA-seq analysis revealed that NSUN6 mainly exerts its effect by inhibiting the p53 signaling pathway, and the specific mechanism remains to be explored. This project aims to elucidate the roles of NSUN6 and p53 signaling pathways in venetoclax resistance at the molecular, cellular, and animal levels based on previous research. Exploring how m5C methylation modification mediates venetoclax resistance and regulates the p53 pathway, providing a theoretical basis for addressing ABT-199 resistance in relapsed and refractory DLBCL patients.

Methods: This study successfully constructed the venetoclax resistant strain OCI-Ly1 in DLBCL cell lines, established a cell line-derived xenograft (CDX) model in DLBCL mice, and identified the venetoclax resistant gene NSUN6 through RNA seq sequencing and MCODE method. NSUN6 knockdown and overexpression cell lines were successfully constructed using CRISPR-Cas9 technology and lentivirus methods. Using methods such as fluorescence quantitative PCR, Western blot, immunohistochemistry, co immunoprecipitation (Co-IP), flow cytometry, and cell proliferation experiments to explore the molecular mechanisms of venetoclax resistance at the cellular, animal, and primary patient levels, in order to systematically and comprehensively reveal the important role of NSUN6 in venetoclax resistance.

Results: We successfully constructed venetoclax-R in DLBCL and elucidated that NSUN6 is the main resistance gene of venetoclax, revealing that NSUN6 is an important adverse factor affecting patient prognosis. Elucidating that knocking down NSUN6 in OCI-Ly1-R can reverse the development of venetoclax resistance. We proving that the p53 pathway is an important pathway for NSUN6 to function. Search for target genes regulated by NSUN6 and investigate the effect of knocking out NSUN6 on the m5C modification level of target genes.

Conclusion: Our research takes DLBCL cell lines, primary cell samples, and BALB/C nude mice as research objects. Based on existing research, the biological effects of NSUN6 and the feasibility of reversing venetoclax resistance are verified in vitro and in vivo, and the specific mechanism of NSUN6 mediated venetoclax resistance is elucidated.