Molecular Mechanisms Involved in Homing and Migration of Plasma Cells in Response to CXCR4 Stimulation and Downstream Activation of the PI3K Pathway.
Authors: Ghobrial Irene M. , Ghobrial Joanna M. , Bramati Patricia , Kimlinger Terry , Timm Michael M. , Kumar Ashock , Hedin Karen E. , Witzig Thomas E. 
Published: October 2019

Abstract
Malignant plasma cells characteristically home to the bone marrow (BM). However, the mechanisms by which cells are recruited into and mobilized from the bone marrow into the peripheral blood (PB) are not well understood. In this study, we explore the molecular mechanisms involved in homing and migration of plasma cells in response to CXCR4 and investigated the role of the PI3K pathway in migration of MM cells in response to SDF-1. CXCR4 surface expression was determined by FACS analysis (PE CXCR4, Pharmingen) using samples from bone marrow and peripheral blood of patients diagnosed with multiple myeloma (MM), monoclonal gammopathy of undetermined significance (MGUS) and primary systemic amyloidosis (AL). All results were expressed as percent expression in gated CD38+ and CD45+ cells. Boyden chamber in vitro migration assays were performed using Kas6/1MM cells. In addition, live confocal microscopy was used to visualize changes in the subcellular location of YFP-fluorescent CXCR4 before and after SDF-1 stimulation. Cells were pretreated with 10mM LY294002 or 200nM rapamycin to assess the effect of inhibition of PI3K or mTOR on CXCR4 subcellular localization. Immunoblotting was performed to confirm inhibition of the PI3K pathway. Comparisons between groups was performed using Mann-Whitney testing. The median CXCR4 expression is described in table 1. There was a significant difference between expression of BM CXCR4 in MM and MGUS (p=0.02). SDF-1 induced dose dependent migration of Kas 6/1 cells indicating a functional CXCR4 receptor. MM cells transfected with YFP-CXCR4 demonstrated surface localization on the cells. 3-Dimensional and continuous live imaging after SDF-1 stimulation for 30 minutes demonstrated alterations in the CXCR4-YFP leading to its capping, internalization subcellularly, and production of pseudopodia in response to SDF-1. This process was abrogated with pretreatment with LY294002 and rapamycin. These data demonstrate for the first time that the surface expression of CXCR4 is markedly elevated in the peripheral blood as compared to the bone marrow. Once in the bone marrow, and with the presence of excess SDF-1, the receptor becomes internalized and downregulated. In contrast to MM, CXCR4 expression on plasma cells in the bone marrow of normal and MGUS patients was higher. In addition, we demonstrate that the process of CXCR4 subcellular localization is abrogated by the administration of PI3K inhibitors LY294002 and rapamycin. These data suggest that CXCR4 expression is required for MM cells to circulate, and that downregulation occurs in the BM leading to immobilization of the cells. Future clinical trials using CXCR4 inhibitors, or inhibitors of PI3K and mTOR to prevent the homing and migration of MM cells into the bone marrow may be explored. This was supported in part by CAP50 CA97274.
CXCR4 expression
N=45 Median CXCR4% expression of total plasma cells Range MM BM (n=23) 27 4.3–90.1 MM PB (n=5) 91 60–95.5 MGUS BM (n=6) 65 41–78.6 AL BM (n=7) 46 17.5–71.6 Normal BM (n=4) 59 6.3–69.5 Kas6/1 cell line 100 -