Anti-Leukemic Activation of Immune Cells in a Leukemic-Induced Microenvironment By CXCR4 Inhibition

The bone marrow microenvironment (BMM) provides a protective niche that supports growth and survival of leukemic stem cells. Stromal-derived factor-1 (SDF-1)/CXC receptor 4 (CXCR4) plays pivotal roles in the cross-interactions between blasts and the BMM to prevent retention and mobilization of leukemic cells, as well as in normal hematopoiesis including the development of immune cells. Here, we show that the CXCR4 antagonist, Plerixafor, decreased the level of CXCR4 expression and inhibited SDF-1-induced migration of leukemic cells. Further, the inhibition of the interaction between leukemic cells and the BMM by the CXCR4 antagonist enhanced cytotoxic activity of immune cells as a result of increased susceptibility of leukemic cells to chemotherapeutic agents such as cytosine arabinoside (Ara-C) in a mouse model of acute myeloid leukemia (AML), suggesting biological effects of the BMM through immune cell activation. To examine the level of CXCR4 expressed by primary AML blasts and murine leukemic cells and the role of CXCR4 in migration at various concentration, 19 AML samples and murine C1498 and human Jurkat cells were subjected to FACS analysis and migration assay. CXCR4 expression in C1498 cells was about 31.29%, compared to 99.7% in the CXCR4⁺ Jurkat cell line and RNA expression was also confirmed. C57Bl/6J mice were used to construct a syngeneic AML animal model by infusion of 2x10⁶ C1498 cells, and peripheral blood from a facial vein and samples from multiple organs from the sacrificed mice were obtained until day 30 post-injection. Total RNA was extracted from BM cells, liver, and spleen of mice at day 15 and 1ug RNA was reverse transcribed into cDNA using a Transcriptor First Strand cDNA Synthesis Kit. All data were normalized to the amount of GAPDH expression, with samples run in triplicate. The quantitative real-time PCR assay for IFN-γ, perforin, and granzyme B, main cytokines produced by cytotoxic T and NK cells, was performed to examine the functional capacity of subsets. Data revealed that both normal human samples and AML mononuclear cells highly expressed CXCR4. CXCR4 expression in both groups was significantly decreased by CXCR4 inhibition, when treated with 5 µM Plerixafor. Our migration assay clearly showed the inhibitory effect of Plerixafor on SDF-1α-induced migration of primary AML and C1498 cells. Under SDF-1α conditions (100 ng/ml), C1498 cells were co-cultured with the CXCR4 antagonist at various concentrations. Migrations of both C1498 and primary AML cells were similarly inhibited. To test the direct role of the CXCR4 antagonist in apoptosis, C1498 cells were cultured with or without Ara-C. The Ara-C and Plerixafor dual-treated group (termed P+A group) displayed no significant difference in apoptosis when compared to the Ara-C only group, suggesting that apoptosis is exclusively controlled by Ara-C, but not Plerixafor, in vitro. However, leukemic blasts synergistically and significantly decreased in the P+A group, compared to those of the other groups, in a syngeneic leukemic mouse model experiments, suggesting an unexpected role for Plerixafor in leukemic blast suppression specifically in the AML niche. The frequency of CD4 and CD8 cells was maintained in the P+A group, compared to the Ara-C-only, Plerixafor treated only, and C1498 injected groups, implying that Plerixafor cannot independently alter the frequency of immune cells. Most of all, the expression levels of IFN-γ, perforin, and granzyme B in the spleens of the P+A group were significantly increased, compared to those of the control groups including the Plerixafor only group. This study demonstrates that the effects of CXCR4 inhibition on blast suppression and immune cell function in the tumor microenvironment and chemotherapy with Plerixafor represents an advanced therapeutic strategy of targeting the leukemic niche.