Critical Roles of LILRB4 in Promoting PIM Kinase Mediated Cell Proliferation and Tumorigenesis in Multiple Myeloma

Introduction

Multiple myeloma (MM) is the second most common hematologic malignancy. Although multiple targets on MM cells are discovered and applied in clinical treatment, relapse is almost inevitable in MM patients. Our previous study found a high-risk cell cluster from MM patients with overall survival less than 2 years by single-cell RNA sequencing (scRNA seq). In this specific cluster, leukocyte immunoglobulin-like receptor B4 (LILRB4) was highly expressed, indicating the critical role of LILRB4 in myelomagenesis and drug resistance. However, the mechanisms of LILRB4 in MM development has not been fully understood. Here, we investigated the role of LILRB4 in tumorigenesis and MM cell proliferation.

Methods and Results

Our clinical data showed that MM patients with LILRB4 overexpression had poor prognosis and decreased overall survival. Compared with newly-diagnosed MM patients, expression of LILRB4 was higher in the MM patients relapsed after treatment, indicating the significance of LILRB4 in MM progression and drug resistance. In vitro experiment showed that MM cells with LILRB4 overexpression enhanced the cell colony-forming ability and promoted cell proliferation. Conversely, knocking-out LILRB4 induced cell apoptosis. We used MM cells with LILRB4 overexpression to establish myeloma xenograft model and results showed that compared with LILRB4^neg MM cells, mice injected with LILRB4^high MM cells displayed higher tumor formation rate and lower survival rate. Then, we analyzed RNA-seq data to investigate the mechanisms of LILRB4 in MM proliferation. Transcriptome analysis indicated that overexpression of LILRB4 in MM cell lines activated NF-kB, glycolysis, hypoxia and MTORC1 signaling pathways. Among these, NF-kB pathway has been proved in previous studies. More important, RNA-seq data showed that PIM kinase, correlating with cancer cell proliferation and apoptosis, was significantly upregulated in MM cells with LILRB4 overexpression, which has been confirmed in our in vitro experiments. We also found that LILRB4 upregulated the expression of PIM through activating Src-homology 2-containing protein tyrosine phosphatase (SHP) and signal transducer and activator of transcription (STAT) protein, indicating the underlying mechanism of MM cell proliferation. Interestingly, transcription factors promoting B cell program were upregulated, and the immunophenotypic marker related to B cell differentiation was downregulated after LILRB4 overexpression. These findings indicated that LILRB4 may induce myeloma cell dedifferentiation and promote tumorigenesis.

Conclusion

In conclusion, LILRB4 is highly associated with poor prognosis of MM patients and has the ability of tumorigenesis. LILRB4 promotes MM cell proliferation by activating SHP and STAT to upregulate the expression of PIM kinase, which plays critical roles in MM pathogenesis.