Abstract
Bruton's tyrosine kinase (BTK) and the chemokine receptor CXCR4 are linked in various hematologic malignancies. The aim of the study was to understand the role of BTK in myeloma cell growth and metastasis using the stably BTK knockdown luciferase-expressing INA6 myeloma line. BTK knockdown had reduced adhesion to stroma and migration of myeloma cells toward stromal cell-derived factor-1. BTK knockdown had no effect on short-term in vitro growth of myeloma cells, although clonogenicity was inhibited and myeloma cell growth was promoted in coculture with osteoclasts. In severe combined immunodeficient-rab mice with contralaterally implanted pieces of bones, BTK knockdown in myeloma cells promoted their proliferation and growth in the primary bone but suppressed metastasis to the contralateral bone. BTK knockdown myeloma cells had altered the expression of genes associated with adhesion and proliferation and increased mammalian target of rapamycin signaling. In 176 paired clinical samples, BTK and CXCR4 expression was lower in myeloma cells purified from a focal lesion than from a random site. BTK expression in random-site samples was correlated with proportions of myeloma cells expressing cell surface CXCR4. Our findings highlight intratumoral heterogeneity of myeloma cells in the bone marrow microenvironment and suggest that BTK is involved in determining proliferative, quiescent or metastatic phenotypes of myeloma cells.
Highlights
Bruton’s tyrosine kinase (BTK) protein was markedly reduced in INA6 MM cells stably infected with lentiviral particles containing BTK short hairpin RNA (shRNA), as assessed by western blots (Supplementary Figure S1A); BTK gene expression was 96% lower in BTK-KD cells than in cells infected with control scrambled shRNA, and expression remained low after cells were engrafted in the severe combined immunodeficient (SCID)-rab mouse model for MM (Supplementary Figures S1B and C)
We demonstrated that, in INA6 MM cells growing in the supportive in vivo SCID-rab model for MM, BTK knockdown promoted MM cell growth but inhibited metastasis to new bone marrow (BM) niches
Our study revealed that the expression of BTK and CXCR4 is intratumorally heterogeneous in clinical MM samples from focal lesions and interstitial marrow of the myelomatous bone, suggesting that BTK is involved in determining proliferative, quiescent or metastatic phenotypes of MM cells (Figure 6c)
Summary
Cumulative evidence indicates that multiple myeloma (MM)emerges from its precursor disease, MGUS (monoclonal gammopathy of undetermined significance), and that alterations both in tumor cells and in their microenvironment likely mediate the conversion from MGUS and asymptomatic MM to overt, symptomatic MM.[1,2,3,4] In most cases, early-stage disease has MM cells within the interstitial bone marrow (BM) and active disease is characterized by the establishment of a MM niche in the form of focal growth that frequently converts to osteolytic lesions in later stages,[5,6] depending on molecular properties of the MM cells[7] and their unique interactions with and dependence on the BM microenvironment.[8,9] MM cells typically grow in BM, most patients with medullary MM have a small population of circulating MM plasma cells,[10,11] but the role of these cells in MM metastasis is only partially understood.[9]. A nonreceptor tyrosine kinase of the TEC family that is preferentially expressed in hematopoietic cells[14,15] including MM plasma cells,[16,17] BTK mediates chemotaxis of MM cells toward stromal cell-derived factor-1 (SDF-1),[16,17] which is secreted at high levels in the BM
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