Mobilization of Normal Mouse Progenitors and Acute Promyelocytic Leukemia (APL) Cells with Inhibitors of CXCR4 and VLA-4 in Splenectomized and Unsplenectomized Mice.

Background: Hematopoietic stem cells (HSC) interact with stromal cells, osteoblasts and matrix proteins in the hematopoietic niche. This interaction plays an important role in HSC trafficking, proliferation and differentiation. Significant data support the roles of both the SDF-1/CXCR4 and the VCAM1/VLA-4 axes in HSC homing and mobilization. Little is known regarding the kinetics and effect of combining small molecule inhibitors of CXCR4 and VLA-4 on normal HSC and APL mobilization. Here we examine this question and assess the role that the spleen plays in HSC and APL mobilization and in the progression of APL cells in vivo. Methods: To evaluate mobilization of HSC progenitors, normal and splenectomized 8 week old 129/B6 F1 mice were treated with AMD3100 5 mg/kg sc and/or a small molecule inhibitor of VLA-4 (AMD15057 1mg/kg iv). Mobilization of APL cells was performed using the same doses and schedules of AMD3100 and/or AMD15057 by first injecting normal and splenectomized 8 week old 129/B6 mice iv with 106 banked APL cells from leukemic mice in which a single copy of PML-RARa was inserted into the murine cathepsin G locus (Westervelt, Blood. 2003;102:1857–65). These cells were transfected with murine oncoretroviruses carrying a CBR-luciferase-eGFP fusion gene to allow tracking by both bioluminescence (BLI) and FACS. Mobilization of APL cells was assessed 10 days after injection of normal mice and 12 days after injection of splenectomized mice when 1–2% of the peripheral blood of syngeneic recipients was leukemic as measured by FACS for the characteristic phenotypic markers of murine APL (CD34+/GR-1+). Engraftment and expansion of APL in normal and splenectomized mice was assessed by WBC, BLI and overall survival. Results: A single sc injection of AMD3100 or iv injection of AMD15057 resulted in maximum mobilization of HSC and APL in 3 hours in normal mice, and in 1–3 hours in splenectomized mice. Dramatic synergism of both normal HSC (130 fold over baseline as measured by CFU-GM/mL of peripheral blood) and APL (22 fold over baseline) mobilization was seen only when AMD3100 and AMD15057 were co-administered to normal and not splenectomized mice. Furthermore, the magnitude of AMD3100 or combination-induced mobilization of HSC and APL was dramatically greater from normal vs splenectomized mice (3.0 to 11 fold, CFU-GM) suggesting that the mouse spleen (vascular niche) represents the major site of both normal HSC and APL mobilization by AMD3100. In contrast, similar mobilization of both HSC and APL was seen in normal and splenectomized mice after treatment with AMD15057 suggesting that the marrow (osteoblastic niche) is the major site for mobilization after VLA4 blockade. After injection of 106 APL cells into syngeneic recipients, unsplenectomized controls compared to splenectomized mice exhibited: decreased overall leukemia burden (as measured by whole body BLI), increased expansion of APLLuc cells in the BM (femurs), and increased overall survival (median survival time increased from 14d to 22d; P=0.0003). Conclusions: Rapid mobilization of both normal progenitors and leukemia cells can be induced in vivo by AMD3100 and AMD15057. Co-administration of CXCR4 and VLA4 small molecule antagonists have a synergistic effect on the rapid mobilization of both HSC and APL from the vascular and osteoblastic niches, respectively. These results may have important clinical implications in patients undergoing chemotherapy for acute leukemia and stem cell transplantation.