Abstract
CXCR4/SDF-1 axis regulates the trafficking of normal stem cells to and from the bone marrow (BM) microenvironment. SDF-1 is a chemokine widely expressed by many tissues especially BM stromal cells and osteoblasts. AMD3100 (AMD) is a novel bicyclam molecule that is a competitive inhibitor of SDF-1/CXCR4 binding and has been used to enhance stem cell mobilization when combined with G-CSF in mouse, dog and man. We are interested in evaluating whether leukemic cells “mobilize” similar to normal stem cells after treatment with AMD, and if so, whether this mobilization increases the efficacy of chemotherapy. Therefore, we utilized a mouse model of human acute promyelocytic leukemia (APL) in which the PML-RARα transgene was knocked into a single allele of the murine cathepsin G locus. To more efficiently track the leukemic cells, we transduced banked APL tumors with a dual function reporter gene that encodes a fusion protein comprised of click beetle red (CBR) luciferase, a bioluminescence imaging (BLI) optical reporter gene, and EGFP for ex vivo cell sorting (CBR/EGFP). We generated large numbers of CBR/EGFP+ APL cells by isolating EGFP+ cells using a MoFlo cell sorter, and passaging them in secondary syngeneic recipients. Importantly, the secondary recipients developed a rapidly fatal acute leukemia after intravenously (iv) or intraperitoneal injection, which displayed an APL phenotype (CD34/GR1 co-expression) and exhibited luciferase activity. Upon iv injection into syngeneic recipients, the CBR/EGFP+ APL cells rapidly migrated to the BM microenvironment, as evidenced by the significantly increased BLI signal in the femurs, spine, ribs, and skull of recipients at 4 days after injection. Over the next 2–3 days the CBR/EGFP+ cells migrated to the spleen followed rapidly by widespread dissemination and death due to leukostasis by 14–16 days. To our knowledge, this represents the only mouse leukemia model in which leukemia cells home preferentially to the BM microenvironment in a manner that is similar to what is seen in human AML. Therefore, we used this model to study the effect of AMD on the “mobilization” of APL cells into the peripheral blood (PB) and on their sensitivity to chemotherapeutic agents that are known to affect the proliferation of these cells. Surprisingly, injection of AMD (5 mg/kg) immediately at the time of APL infusion had no impact on the engraftment (short term or long term) of either normal BM stem cells or the leukemic cells. However, we observed rapid mobilization of the leukemic cells when AMD was administered 11 days after APL injection. In fact, 40% of mice that received a single dose of AMD on day +11 after APL injection died 2 to 4 hours after AMD injection as a result of the rapid and massive mobilization of blasts. Overall, we found that AMD treatment on day +11 induced a 3-fold increase in total WBC counts with a 10-fold increase in the leukemic blasts into PB. Interestingly, the administration of AMD concomitant with cytarabine (AraC) (200 mg/kg) on day +11 significantly prolonged the overall survival of mice, compared with mice treated only with AraC. In summary, we developed a mouse model to study the APL cell trafficking, and we have shown leukemia cell mobilize from the BM into PB after AMD administration. We propose that CXCR4/SDF-1 is a key regulator for leukemia migration and homing to the BM. In these preliminary results, we observed that AMD sensitizes APL cells to AraC.
Speciation of arsenic trioxide metabolites in peripheral blood and bone marrow from an acute promyelocytic leukemia patient