DNP73 PROMOTES DRUG RESISTANCE AND IMMUNE EVASION IN MULTIPLE MYELOMA VIA UPREGULATING MYC AND N‐MYC

Abstract

Introduction: Multiple myeloma (MM) remains an incurable malignancy of plasma cells. The complex bone marrow microenvironment promotes resistance to both current anti-myeloma agents and emerging immunotherapies. Crosstalk between the NF-κB and p53 can play a pivotal role in various hematologic malignancies. DNp73, an inhibitor of the p53 tumor suppressor family, drives drug resistance and cancer progression in several solid malignancies. However, the biological functions and molecular mechanisms of DNp73 in MM remain unclear. In this study, we investigated the role of DNp73 in the drug resistance of MM, and disclosed the corresponding mechanism of how DNp73 promotes the immune escape of MM cells. Methods: We constructed DNp73 overexpression and sh-RNA interference plasmids to obtain stable cell lines. The effects of DNp73 on proliferation and drug sensitivity were determined by flow cytometry and xenotransplantation model. RNA-seq and CHIP-seq were performed to detect the mechanisms of drug resistance in MM cells. The DNA damage repair and invasion ability of MM cells were detected by immunofluorescence and transwell assay. To validate the role of DNp73 in immune escape, we performed phagocytosis assays. Results: Our previous study reported that miR-15a was downregulated in MM cells and correlated with the inferior outcome of MM patients. Further analysis demonstrated that DNp73 was a direct target of p65, loss of miR-15a led to the activation of NF-κB-p65 pathway in MM cells. In addition, the level of miR-15a was negatively correlated with the expression of DNp73 in MM cells of patients (r = −0.672, p < 0.05). After DNp73 was down-regulated, cell proliferation and drug resistance were effectively inhibited. Further in vivo study indicated the tumor volume in DNp73 knockdown group was reduced, and increased sensitivity to the carfilzomib, epirubicin and pomalidomide was observed (p < 0.01). Of note, RNA-seq analysis indicated that DNp73 expression was positively correlated with MYCN, MYC and CDK7 transcriptional programs in MM. GSEA analysis showed that MYC targets and DNA damage repair pathways were significantly enriched in DNp73-OE cells. Compared to control group, DNp73-OE cells were resistant to irradiation-induced cell death (p < 0.01). Meanwhile, DNp73 knockdown significantly reduced the migration and invasion of cells (p < 0.01). CHIP-seq data showed that DNp73 could bind to the promoter region of MYCN. DNp73 overexpression protects MM cells from phagocytosis, treated with anti-human CD47 antibody increased phagocytosis of macrophages (p < 0.01). Conclusions: We demonstrated that miR-15a downregulation activated NF-κB, which promoted DNp73 expression at transcription level. DNp73 promotes drug resistance and immune escape in multiple myeloma by modulating Myc and N-Myc signal pathway. The research was funded by: the CAMS Innovation Fund for Medical Sciences (2022-I2M-1-022), the Natural Science Foundation of China (82170194), the Non-profit Central Research Institute Fund of the Chinese Academy of Medical Sciences (2018RC320012), the Natural Science Foundation of China (81920108006), the Non-profit Central Research Institute Fund of the Chinese Academy of Medical Sciences (2018PT31006) Keywords: multiple myeloma, tumor biology and heterogeneity No conflicts of interests pertinent to the abstract.