Targeting the leukemia microenvironment by CXCR4 inhibition overcomes resistance to kinase inhibitors and chemotherapy in AML

Abstract

SDF-1alpha/CXCR4 signaling plays a key role in leukemia/bone marrow microenvironment interactions. We previously reported that bone marrow-derived stromal cells inhibit chemotherapy-induced apoptosis in acute myeloid leukemia (AML). Here we demonstrate that the CXCR4 inhibitor AMD3465 antagonized stromal-derived factor 1alpha (SDF-1alpha)-induced and stroma-induced chemotaxis and inhibited SDF-1alpha-induced activation of prosurvival signaling pathways in leukemic cells. Further, CXCR4 inhibition partially abrogated the protective effects of stromal cells on chemotherapy-induced apoptosis in AML cells. Fetal liver tyrosine kinase-3 (FLT3) gene mutations activate CXCR4 signaling, and coculture with stromal cells significantly diminished antileukemia effects of FLT3 inhibitors in cells with mutated FLT3. Notably, CXCR4 inhibition increased the sensitivity of FLT3-mutated leukemic cells to the apoptogenic effects of the FLT3 inhibitor sorafenib. In vivo studies demonstrated that AMD3465, alone or in combination with granulocyte colony-stimulating factor, induced mobilization of AML cells and progenitor cells into circulation and enhanced antileukemic effects of chemotherapy and sorafenib, resulting in markedly reduced leukemia burden and prolonged survival of the animals. These findings indicate that SDF-1alpha/CXCR4 interactions contribute to the resistance of leukemic cells to signal transduction inhibitor- and chemotherapy-induced apoptosis in systems mimicking the physiologic microenvironment. Disruption of these interactions with CXCR4 inhibitors represents a novel strategy of sensitizing leukemic cells by targeting their protective bone marrow microenvironment.

Figure 1

AMD3465 inhibits migration and intracellular signaling in AML cell lines. (A) Surface expression of CXCR4 was measured by flow cytometry, and the results are expressed as percent change in the mean fluorescent intensity (MFI) compared with control (untreated) cells. (B) U937 and MOLM13 cells (0.5 × 10⁶) were plated onto the upper chamber of transwell plates and exposed to 50 ng/mL SDF-1α in the lower chamber or to 0.1 × 10⁶ MS-5 cells preplated in the lower chamber with or without 1 μM AMD3465 for 24 hours. Migrating cells were counted after 24 hours of incubation. The results are expressed as a percentage of the migrating cells relative to the numbers of input cells. (C) Cells were pretreated with or without SDF-1α for 30 minutes, followed by exposure to 1 μM AMD3465 for 4 hours. Phosphorylation of Akt (pAkt) and Erk (pErk) was detected by Western blot analysis, and the intensity of the bands was quantified by densitometry and displayed as ratios of either phospho-proteins to total proteins. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control.

Figure 2

AMD3465 inhibits migration and enhances proaptotic effect of ara-C in primary AML.(A) AMD3465 suppresses SDF-1α–induced (n = 7 samples) and MS-5-induced (n = 14 samples) migration of primary AML cells. The error bars represent SEM. (B) Stromal cells protect primary AML cells (n = 21) from spontaneous and chemotherapy-induced apoptosis. Primary AML cells cultured alone (□) or cocultured with stroma (, ■) were treated with 3 μM ara-C for 24 hours. The percentage of the apoptotic cells (annexin V–positive cells) were analyzed by flow cytometry. (C) AMD3465 sensitized primary AML cells (n = 20) cocultured with stromal MS-5 cells to ara-C–induced apoptosis (24 hours). Apoptotic cells were detected by annexin V flow cytometry after gating on CD34⁺ leukemic cells. The specific apoptosis was calculated by the formula: % specific apoptosis = (test – control) × 100/(100 – control).

Figure 3

AMD3465 induces mobilization of A20 cells in vivo and enhances antitumor effects of ara-C. (A) A20-luc/YFP cells were injected IV into BALB/c mice, and bone marrow engraftment was confirmed by bioluminescence imaging on day 5 (top row). Mice were injected with ara-C, AMD3465, or ara-C plus AMD3465 on day 7 and 8 at the indicated doses described in “A20-luc/YFP leukemia murine model.” On days 5, 7, 10, and 14, mice were imaged after D-luciferin injection. Serial images of 3 representative mice are shown on day 7, 10, and 14. (B) AMD3465 was administered on day 7 after tumor cell injection, and percentages of circulating A20-luc/YFP cells in peripheral blood before and after 1 hour of AMD3465 were examined by flow cytometry (left panel). Percentage of circulating A20-luc/YFP-positive cells in control mice, in mice mobilized with AMD3465, or in mice treated with ara-C ± AMD3465 was detected by flow cytometry on day 8 after tumor cell injection (right panel). (C) Bioluminescence imaging results on day 14 were averaged from the peak light-emitting exposure from each group and displayed as photons per second. Error bars represent the SEM of each group.

Figure 4

AMD3465 inhibits SDF-1α– or stroma-induced migration and suppresses prosurvival signaling pathways in FLT3-mutated cells. (A) Migration of either Ba/F3-ITD or Ba/F3-FLT3 cells in response to 4 hours SDF-1α was examined as described in “Chemotaxis studies” in the presence or absence of 1 μM AMD3465 and/or 10 nM sorafenib. (B) The effects of AMD3465 on the SDF-1α–induced or MS-5–induced up-regulation of pAKT, pERK, and pCXCR4 were analyzed by Western blot analysis in Ba/F3-ITD cells. (C) The combined effects of sorafenib and AMD3465 on AKT, ERK, and CXCR4 phosphorylation were examined in Ba/F3-ITD cells in the presence of MS-5 cells. (D) Primary AML cells with FLT3-ITD mutation grown alone or cocultured with MS-5 cells were exposed to the indicated concentration of AMD3465 alone, 1 μM sorafenib alone, or AMD3465 combined with sorafenib. Phosphorylation of AKT, ERK, and CXCR4 was analyzed by immunoblotting after 24 hours of treatment, and (E) the inhibitory effects of AMD3465 with or without sorafenib on cell migration was measured after 4 hours. The intensity of the phosphorylation bands was quantified by densitometry and displayed as ratios of phosphoproteins either to total proteins or to the loading control GAPDH.

Figure 5

AMD3465 sensitizes FLT3-mutated cells to FLT3 inhibitor–induced apoptosis. (A) The average percentage of annexin V⁺ Ba/F3-ITD and Ba/F3-FLT3 cells after exposure to AMD3465 alone, sorafenib alone, or sorafenib in combination with AMD3465 in the absence or presence of MS-5 cells for 24 hours. (B) Ba/F3-ITD and Ba/F3-FLT3 cells were treated with AMD3465, AG1296 (FLT3 inhibitor), or AG1296 in combination with AMD3465 in the absence or presence of MS-5 cells, and apoptotic cells were detected by annexin V flow cytometry. (C) MOLM13 carrying FLT3-ITD cells were treated with AMD3465, sorafenib, or their combination in the absence or presence of MS-5 cells for 24 hours, under normoxic (21% O₂) or hypoxic (2% O₂) conditions. Induction of apoptosis was measured by annexin V flow cytometry. (D) Blasts from primary AML samples with FLT3 mutations (n = 7) were treated with sorafenib alone or in combination with AMD3465 in coculture with MS-5 cells for 96 hours, and apoptosis induction was measured by annexin V flow cytometry after gating on CD34⁺ cells. The specific apoptosis was calculated using the formula described above. Error bars represent the SEM of each group.

Figure 6

In vivo effects of AMD3465/G-CSF and sorafenib in a mouse xenograft model of FLT3-ITD mutant leukemia. (A) Serial bioluminescence images of mice in the groups receiving sorafenib, AMD3465/G-CSF, sorafenib combined with AMD3465/G-CSF, or in the group without any treatment (control) were taken on days 6, 9, and 12 after tumor cell injection. Deceased mice are identified by “X” sign. (B) Overall survival in each group was estimated by Kaplan-Meier method. Statistical significance was calculated using the log-rank test. (C,D) Histologic sections of liver, spleen, and bone marrow of mice stained with H&E (C) or anti-GFP antibody (D) in untreated mice (day 13), AMD3465/G-CSF (day 14), sorafenib (day 14), or AMD3465/G-CSF + sorafenib (day 14) treated mice.