The TAM receptor family in multiple myeloma

Abstract

Multiple myeloma (MM) is a haematological malignancy of antibody secreting monoclonal plasma cells (PCs) in the bone marrow (BM). MM PC growth and survival is supported by other cells within the BM microenvironment including osteoblasts, osteoclasts, bone marrow stromal cells and immune cells. Paracrine signalling between MM PCs and these other cell types is mediated by an array of cytokines, receptors and adhesion molecules. The TAM receptor family, Tyro3, Axl and Mer represent a distinct family of tyrosine kinase cell surface receptors, which have been implicated in the pathogenesis of cancers including MM. The studies presented in this thesis utilised single TAM receptor expressing 5TGM1 murine myeloma cell lines to further elucidate the roles of Axl and Mer in MM. The CRISPR Cas9 system and retroviral transduction were used to generate a 5TGM1 cell line expressing no TAM receptors (5TGM1 EV), a 5TGM1 cell line expressing only Axl (5TGM1 Axl) and a 5TGM1 cell line expressing only Mer (5TGM1 Mer). Dormant MM PCs that reside long term in the bone marrow of patients can be reactivated following therapy, giving rise to disease relapse. Given that Axl was highly expressed by dormant 5TGM1 MM cells in previous studies, the present study sought to determine whether high Axl expression alone was sufficient to initiate and maintain 5TGM1 MM cell dormancy. Features of dormancy in 5TGM1 Axl compared to 5TGM1 EV cells were assessed in vitro using cell cycle analysis and labelling with the heritable dye, DiD. 5TGM1 Axl and 5TGM1 EV cells were also inoculated into the C57BL/KaLwRij murine model of MM, and tumour burden was assessed. These studies provided no evidence that high Axl expression in 5TGM1 cells promote features of dormancy in vitro or in vivo when compared to the 5TGM1 EV cell line that does not express Axl. Studies in this thesis revealed that 5TGM1 Mer cells produce significantly greater myeloma tumour burden in vivo in comparison to 5TGM1 EV cells following intravenous inoculation into the C57BL/KaLwRij mouse model. However, following inoculation of cells directly into the bone marrow, Mer expression did not produce an increase in tumour burden. Additionally, when 5TGM1 Mer cells and 5TGM1 EV cells were inoculated into the immune compromised NSG mouse model, Mer expression had no effect on tumour burden. Immune checkpoint proteins PD-L1, Galectin 9 and PVR were upregulated in 5TGM1 Mer cells compared to 5TGM1 EV cells at the mRNA level. These findings indicate that the mechanism of action of Mer in potentiating MM tumour burden may be through increased 5TGM1 BM homing and regulating expression levels of immune checkpoint molecules in myeloma cells. Future studies should aim to fully characterise the possible role of Mer in MM immune suppression. Given that Mer and its ligand Gas6 are widely expressed by MM PCs of myeloma patients, Mer represents an attractive therapeutic target to limit MM disease progression.