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, 38 (3), 180-90

Impact of CXCR4 Inhibition on FLT3-ITD-positive Human AML Blasts

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Impact of CXCR4 Inhibition on FLT3-ITD-positive Human AML Blasts

Angela Jacobi et al. Exp Hematol.

Abstract

Objective: Internal tandem duplication (ITD) mutations of the FLT3 receptor are associated with a high incidence of relapse in acute myeloid leukemia (AML). Expression of the CXCR4 receptor in FLT3-ITD-positive AML is correlated with poor outcome, and inhibition of CXCR4 was shown to sensitize AML blasts toward chemotherapy. The aim of this study was to evaluate the impact of FLT3-ITD on cell proliferation and CXCR4-dependent migration in human hematopoietic progenitor cells and to investigate their response to CXCR4 inhibition.

Materials and methods: We used primary blasts from patients with FLT3-ITD or FLT3 wild-type AML. In addition, human CD34(+) hematopoietic progenitor cells were transduced to >70% with retroviral vectors containing human FLT3-ITD.

Results: We found that FLT3-ITD transgene overexpressing human hematopoietic progenitor cells show strongly reduced migration toward stromal-derived factor-1 in vitro and display significantly reduced bone marrow homing in nonobese diabetic severe combined immunodeficient mice. Cocultivation of FLT3-ITD-positive AML blasts or hematopoietic progenitor cells on bone marrow stromal cells resulted in a strong proliferation advantage and increased early cobblestone area-forming cells compared to FLT3-wild-type AML blasts. Addition of the CXCR4 inhibitor AMD3100 to the coculture significantly reduced both cobblestone area-forming cells and proliferation of FLT3-ITD-positive cells, but did not affect FLT3-wild-type cells-highlighting the critical interaction between CXCR4 and FLT3-ITD.

Conclusion: CXCR4 inhibition to decrease cell proliferation and to control the leukemic burden may provide a novel therapeutic strategy in patients with advanced FLT3-ITD-positive AML.

Conflict of interest statement

Conflict of Interest Disclosure

No financial interest/relationships with financial interest relating to the topic of this article have been declared.

Figures

Figure 1
Figure 1
Hematopoietic progenitor cells (HPC) overexpressing the FLT3-internal tandem duplication mutation (FLT3-ITD) transgene demonstrate constitutive phosphorylation of signal transducer and activator of transcription (STAT)5a, extracellular signal-regulated kinase (ERK)1/2, and STAT3. Using γ-retroviral vector transduction, we achieved unprecedented transgene overexpression of human FLT3-ITD in >70% of human cord blood hematopoietic progenitors (A). The functionality of the FLT3-ITD–transgene was demonstrated by constitutive phosphorylation of STAT5a (B), STAT3 (C), and ERK1/2 (D) in the FLT3-ITD overexpressing cells. Representative histograms are shown. GFP, green fluorescent protein.
Figure 2
Figure 2
Hematopoietic progenitor cells (HPC) overexpressing the FLT3-internal tandem duplication mutation (FLT3-ITD) transgene demonstrate significantly reduced CXCR4 expression and reduced in vitro and in vivo migration potential. Fewer FLT3-ITD transgene-positive HPCs (solid black line) show total CXCR4 expression compared to green fluorescent protein (GFP) control transduced cells (GFP Ctr, grey line; isotype control, dashed line) as analyzed by flow cytometry (A; 47% vs 77%). In addition, the level of CXCR4 expression as measured by the median fluorescence intensity was also significantly reduced for FLT3-ITD–positive HPC compared to control-transduced cells (87.7 vs 173.5). A representative histogram is displayed. Similarly, FLT3-ITD transgene-positive HPCs show significantly less CXCR4 expression (*<0.05%) as measured by quantitative reverse transcription polymerase chain reaction (RT-PCR) (B). Furthermore, compared to control-transduced cells, FLT3-ITD transgene-positive cells demonstrate reduced migration in vitro (C; ***p< 0.001) and reduced homing to the bone marrow of nonobese diabetic severe combined immunodeficient (NOD/SCID) mice (D; **p< 0.01). CFP = cyan fluorescent protein; GAPDH = glyceraldehyde 3-phosphate dehydrogenase; SDF-1 = stromal cell-derived factor 1.
Figure 3
Figure 3
Internal tandem duplication mutation of the receptor tyrosine kinase FLT3 (FLT3-ITD)–positive acute myeloid leukemia (AML) blasts show significantly reduced CXCR4 surface expression. Compared to FLT3 wild-type (FLT3-wt) AML blasts (A; 63.7% ± 20%), fewer FLT3-ITD–positive AML blasts show CXCR4 surface expression (B; 23% ± 8.6%), whereas total CXCR4 expression levels show no significant difference (dotted line: isotype; dashed line: surface expression, solid line: total expression). Cocultivation on human bone marrow stromal cells (hBMSC) resulted in a significantly increased number of FLT3-ITD–positive AML blasts with surface CXCR4 expression (C) compared to initiation of culture. Addition of AMD3100 reduces proportion of surface CXCR4 expression in FLT3-ITD–positive AML blasts, but not in FLT3-wt AML blasts (D), as determined by median fluorescence intensity (MDI). Representative histograms are shown. *p < 0.05.
Figure 4
Figure 4
Hematopoietic progenitor cells (HPC) overexpressing the FLT3-internal tandem duplication mutation (FLT3-ITD) transgene demonstrate upregulated expression of suppressor of cytokine signaling–3 (SOCS3). Using quantitative reverse transcription polymerase chain reaction, we found SOCS3, a known negative regulator of CXCR4 signaling, to be significantly (***p< 0.001) upregulated in FLT3-ITD transgene-positive HPC on days 3 and 7 after initiation of culture. GAPDH = glyceraldehyde 3-phosphate dehydrogenase.
Figure 5
Figure 5
The internal tandem duplication mutation of the receptor tyrosine kinase FLT3 (FLT3-ITD) mediates enhanced cell growth, which is selectively reduced by CXCR4 inhibition. Cyan fluorescent protein (CFP) transgene control-transduced hematopoietic progenitor cells (HPC) (A), FLT3-ITD transgene-positive HPC (B), FLT3-wild-type (wt) acute myeloid leukemia (AML) blasts (C), and FLT3-ITD–positive blasts (D) were cocultivated on stromal cells. Compared to control-transduced HPC and FLT3-wt blasts, FLT3-ITD–positive cells demonstrated significantly enhanced cell growth (open bars). The FLT3-ITD–mediated cell growth was significantly reduced (***p < 0.001) by the CXCR4 antagonist AMD3100 (B and D, filled bars). However, CXCR4 inhibition had no effect on cell growth of FLT3-wt cells (A and C, filled bars).
Figure 6
Figure 6
CXCR4 inhibition selectively reduces cell proliferation of cells expressing the internal tandem duplication mutation of the receptor tyrosine kinase FLT3 (FLT3-ITD) as measured by flow cytometric analyses of carboxyfluorescein succinimidyl ester (CFSE)–labeled cells on stromal cells. Both, adherent (A) and nonadherent (B) FLT3-ITD transgene-positive hematopoietic progenitor cells (HPC) show a greater proliferation rate compared to control (green fluorescent protein [GFP] Ctr, gray line) transduced adherent and nonadherent hematopoietic progenitor cells (HPC). CXCR4 inhibition with AMD3100 (dotted histograms) reduced proliferation of both, adherent (C) and nonadherent (D) FLT3-ITD transgene-positive HPC, in contrast to cultures without AMD3100 (continuous line in C and D). Similarly, while CXCR4 inhibition (dotted line) has no effect on cell proliferation of FLT3-wild-type (wt) acute myeloid leukemia (AML) blasts (E), AMD3100 reduces the proliferation of FLT3-ITD–positive AML blasts as seen in (F) (C–F: continuous line, without AMD3100; dotted line, with AMD3100). Representative histograms are shown. CFP = cyan fluorescent protein; CFSE = carboxyfluorescein succinimidyl ester.
Figure 7
Figure 7
CXCR4 inhibition reduces the formation of cobblestone area forming cells (CAFC) derived from cells positive for the internal tandem duplication mutation of the receptor tyrosine kinase FLT3 (FLT3-ITD) CAFC derived from green fluorescent protein (GFP)-control transduced hematopoietic progenitor cells (HPC) from three FLT3 wild-type (wt) donors appeared at day 12 and increased in number and size over the observation period (A, open bars). For FLT3-ITD transgene-positive HPC from three donors, CAFC were observed as early as 3 days after initiation of coculture (B, open bars). Inhibition of stromal-derived factor–1 (SDF-1)/CXCR4 signaling by addition of AMD3100 significantly (***p < 0.001) suppressed the early and overall formation of CAFC from FLT3-ITD–positive HPC (B, filled bars). Inhibition of CXCR4, however, had no effect on the number of CAFC from GFP-control transduced cells with FLT3-wt expression (A, filled bars). Similarly, for FLT3-ITD–positive AML blasts from five donors, CAFC were observed as early as 7 days after initiation of coculture (D, open bars). Inhibition of SDF-1/CXCR4 signaling by addition of AMD3100 significantly (***p < 0.001) suppressed the early formation of CAFC from FLT3-ITD–positive acute myeloid leukemia (AML) blasts (D, filled bars). In contrast, CXCR4 inhibition had no effect on the formation of CAFC of FLT3-wt AML blasts (C).

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