DNp73 enhances tumor progression and immune evasion in multiple myeloma by targeting the MYC and MYCN pathways.

Introduction: Multiple myeloma (MM) is an incurable hematological malignancy with high chromosome instability and heavy dependence on the immunosuppressive bone marrow microenvironment. P53 mutations are adverse prognostic factors in MM; however, clinically, some patients without P53 mutations also exhibit aggressive disease progression. DNp73, an inhibitor of TP53 tumor suppressor family members, drives drug resistance and cancer progression in several solid malignancies. Nevertheless, the biological functions of DNp73 and the molecular mechanisms in myelomagenesis remain unclear. Methods: The effects of DNp73 on proliferation and drug sensitivity were assessed using flow cytometry and xenograft models. To investigate the mechanisms of drug resistance, RNA-seq and ChIP-seq analyses were performed in MM cell lines, with validation by Western blot and RT-qPCR. Immunofluorescence and transwell assays were used to assess DNA damage and cell invasion in MM cells. Additionally, in vitro phagocytosis assays were conducted to confirm the role of DNp73 in immune evasion. Results: Our study found that activation of NF-κB-p65 in multiple myeloma cells with different p53 mutation statuses upregulates DNp73 expression at the transcriptional level. Forced expression of DNp73 promoted aggressive proliferation and multidrug resistance in MM cells. Bulk RNA-seq analysis was conducted to assess the levels of MYCN, MYC, and CDK7. A ChIP-qPCR assay was used to reveal that DNp73 acts as a transcription factor regulating MYCN gene expression. Bulk RNA-seq analysis demonstrated increased levels of MYCN, MYC, and CDK7 with forced DNp73 expression in MM cells. A ChIP-qPCR assay revealed that DNp73 upregulates MYCN gene expression as a transcription factor. Additionally, DNp73 promoted immune evasion of MM cells by upregulating MYC target genes CD47 and PD-L1. Blockade of the CD47/SIRPα and PD-1/PD-L1 signaling pathways by the SIRPα-Fc fusion protein IMM01 and monoclonal antibody atezolizumab significantly restored the anti-MM activity of macrophages and T cells in the microenvironment, respectively. Discussion: In summary, our study demonstrated for the first time that the p53 family member DNp73 remarkably induces proliferation, drug resistance, and immune escape of myeloma cells by directly targeting MYCN and regulating the MYC pathway. The oncogenic function of DNp73 is independent of p53 status in MM cells. These data contribute to a better understanding of the function of TP53 and its family members in tumorigenesis. Moreover, our study clarified that DNp73 overexpression not only promotes aggressive growth of tumor cells but, more importantly, promotes immune escape of MM cells through upregulation of immune checkpoints. DNp73 could serve as a biomarker for immunotherapy targeting PD-L1 and CD47 blockade in MM patients.

LILRB4 represents a promising target for immunotherapy by dual targeting tumor cells and myeloid-derived suppressive cells in multiple myeloma.

Multiple myeloma (MM) remains an incurable hematologic malignancy. Despite tremendous advances in the treatment of this disease, about 10% of patients still have very poor outcomes with a median overall survival of less than 24 months. Our study aimed to underscore the critical mechanisms pertaining to rapid disease progression and provide novel therapeutic choices for these ultrahigh-risk patients. We utilized single-cell transcriptomic sequencing to dissect the characteristic bone marrow niche of patients who survived less than 2 years (EM24). Notably, enrichment of a LILRB4high pre-mature plasma-cell cluster was observed in EM24 patients compared to patients with durable remission. This cluster exhibited aggressive proliferation and a drug-resistance phenotype. High levels of LILRB4 promoted MM clonogenicity and progression. Clinically, high expression of LILRB4 was correlated with poor prognosis in both newly diagnosed MM patients and relapsed/ refractory MM patients. ATAC-sequencing analysis identified that pronounced chromosomal accessibility caused the elevation of LILRB4 on MM cells. CRISPR-Cas9 deletion of LILRB4 alleviated the growth of MM cells, inhibited the immunosuppressive function of myeloid-derived suppressive cells (MDSC), and further rescued T-cell dysfunction in the MM microenvironment. Greater infiltration of MDSC was observed in EM24 patients. We therefore generated an innovative T-cell receptor-based chimeric antigen receptor T cell, LILRB4-STAR-T. Cytotoxicity experiments demonstrated that LILRB4-STAR-T cells efficaciously eliminated tumor cells and impeded MDSC function. In conclusion, our study elucidates that LILRB4 is an ideal biomarker and promising immunotherapy target for high-risk MM. LILRB4-STAR-T-cell immunotherapy is promising against both tumor cells and the immunosuppressive tumor microenvironment in MM.