Abstract
Background: Waldenstrom Macroglobulinemia (WM) is a low-grade lymphoma characterized by widespread involvement of the bone marrow (BM) and involvement of lymph nodes and hepatosplenomegaly (HSM) in about 20% of the patients. We have recently demonstrated that the presence of HSM is one of the most important adverse prognostic factors in WM. The mechanisms of trafficking of WM cells to and from the BM and lymphoid organs is not well defined. The PI3k/Akt pathway is constitutively activated in WM, and regulates migration/homing in cancer cells and B-cells. We hypothesized that the Akt inhibitor perifosine (NSC 639966; Keryx Biopharmaceuticals, NY) modulates homing of WM cells to the BM.
Methods: WM cell line (BCWM.1) was treated with perifosine 2 to 5uM for 2 hours. Surface adhesion receptors were studied using flow cytometry. The adhesion assay coated with fibronectin, a ligand of VLA-4 (EMD Biosciences, CA) was used to test in vitro adhesion. Migration was determined using the transwell migration assay (Costar, NY). We then studied homing of WM into the BM niches using in vivo flow cytometry and confocal microscopy in Balb/c mice. In brief, BCWM.1 were incubated with 5uM perifosine for 2 hrs (or control PBS). The cells were fluorescently labeled by incubation with the dialkylcarbocyanine membrane dye, “DiD” (Molecular Probes), 0.5uM dye for 30 minutes. Cells were then injected in the tail vein of the Balb/c mice, and in vivo confocal flow cytometry was performed on an artery from the ear lobe of the mice. Cell counts were obtained every 5 min. from the time of injection. In vivo confocal microscopy and two-photon microscopy was performed to study cells homing to BM vasculature of the skull (BM niches).
Results: WM cells expressed very high levels of VLA-4, with a median 95% expression. BCWM.1 demonstrated increased adhesion to fibronectin-coated wells as compared to BSA-coated wells. Perifosine inhibited adhesion in a dose dependent manner with 50% decrease in adhesion at 2uM. Perifosine 10uM did not change the level of surface expression of VLA-4 after 6 and 24 hrs, indicating an effect on intracellular signaling but not on surface adhesion molecules. Perifosine 5uM significantly inhibited migration of BCWM.1 in response to SDF-1, a ligand that induces migration of WM cells. Perifosine also demonstrated significant inhibition of in vivo homing of WM cells to the BM niches. The number of cells in the peripheral circulation decreased dramatically (75% decrease) after 1 hr in the control, indicating homing, whereas there was a 40% reduction in the cells at 1hr after perifosine treatment (p=0.001). We then looked at the images obtained from the BM niches. The number of cells that homed and adhered to these areas was lower in the perifosine-treated mouse compared to the control mouse, indicating that fewer cells homed to the BM of the treated mouse.
Conclusion: These results confirm that the PI3K/Akt pathway is important for migration, adhesion and homing of WM in vitro and in vivo.
The Akt pathway regulates survival and homing in Waldenstrom macroglobulinemia
