Abstract
Hematopoietic stem cells (HSC) reside in the bone marrow (BM) and interact with stroma cells and extracellular matrix. CXCR4/SDF-1 axis regulates the trafficking of HSC to and from the BM. We utilized a PML-RARα knock-in mouse model of human acute promyelocytic leukemia (APL) to study APL interaction with the normal BM. We have previously shown there is a rapid mobilization of APL cells from the BM into peripheral blood (PB) after administration of AMD3100, a competitive inhibitor of CXCR4. We hypothesize that we can sensitize these tumor cells to chemotherapy by interrupting the interaction between APL and the BM stroma. We transduced banked APL cells with a dual function reporter gene that encodes a fusion protein comprised of Click Beetle Red luciferase, a bioluminescence imaging (BLI) optical reporter gene, and EGFP for ex vivo cell sorting (Luc/EGFP). Upon iv injection into genetically compatible recipients (F1 129/B6 mice), APL rapidly migrated to the BM with increased BLI signal in the femurs, spine, ribs, and skull, at 4 days after injection, followed by spleen infiltration and death due to leukostasis by day 15. 129/B6 F1 mice (n=28) were injected iv with 106 APL cells. By day 12 all mice had ±5% APL cells in PB. 8 mice received AraC (500mg/kg/sq) on days 12 and 13, and another 8 mice received AraC+AMD (5mg/kg/sq) 1 hour before and 3 hours after each AraC injection. 6 mice received only AMD and 6 control mice were observed. Total body BLI signal, WBC, and blasts per μl of blood on days 19 and 23 were higher in AraC versus AraC+AMD (p<0.004). Median survival for control, AMD, AraC and AraC+AMD groups were 18, 19, 23 and 30 days respectively (p<0.0006). Hemoglobin, platelet and granulocyte recovery post-chemotherapy was similar in both groups. We developed an in vitro mouse stroma system to study engraftment, ex vivo mobilization and sensitivity to chemotherapy. In vitro culture of APL cells showed no difference in APL survival between AraC versus AraC+AMD as measured by flow cytometry or BLI. Stroma offered a survival benefit versus no stroma (p<0.0001). We injected 4 genetically compatible mice with 106 APL cells iv and after 14 days mice were sacrificed. Blast percentage in blood, spleen and BM was 47, 58 and 40% respectively. We cultured cells from all three compartments ex vivo with AraC (25ng/ml). After 24 hours APL survival was 25, 80 and 60% respectively (p<0.006). We repeated the same experiment, but we did, in addition, a positive selection for CD34 to purify APL cells away from surrounding cells in the BM and spleen. Survival after ex vivo AraC incubation was 32, 30, 34% respectively (p=NS). In summary, CXCR4/SDF-1 is a key regulator for leukemia migration and homing to the BM. The interaction of APL cells with the BM and splenic microenvironments provides a survival benefit. Rapid mobilization of APL cells in vivo by AMD3100 interrupts APL-stromal interactions and sensitizes APL to chemotherapy. The impact of additional mobilizing agents on APL mobilization on sensitizing APL to chemo and radiotherapy will be presented. Finally, preliminary RNA profiling studies will be presented in an attempt to identify genes in APL cell that are differentially expressed when bound to and released from the BM.
The endosomal sorting adaptor HD-PTP is required for ephrin-B:EphB signalling in cell collapse and motor axon guidance