IMiDs mobilize acute myeloid leukemia blasts to peripheral blood through downregulation of CXCR4 but fail to potentiate AraC/Idarubicin activity in preclinical models of non del5q/5q- AML

Abstract

Treatment for acute myeloid leukemia (AML) remains suboptimal and many patients remain refractory or relapse upon standard chemotherapy based on nucleoside analogs plus anthracyclines. The crosstalk between AML cells and the BM stroma is a major mechanism underlying therapy resistance in AML. Lenalidomide and pomalidomide, a new generation immunomodulatory drugs (IMiDs), possess pleiotropic anti-leukemic properties including potent immune-modulating effects and are commonly used in hematological malignances associated with intrinsic dysfunctional BM such as myelodysplastic syndromes and multiple myeloma. Whether IMiDs may improve the efficacy of current standard treatment in AML remains understudied. Here, we have exploited in vitro and in vivo preclinical AML models to analyze whether IMiDs potentiate the efficacy of AraC/Idarubicin-based standard AML chemotherapy by interfering with the BM stroma-mediated chemoresistance. We report that IMiDs do not exert cytotoxic effects on either non-del5q/5q- AML cells nor BM-MSCs, but they enhance the immunomodulatory properties of BM-MSCs. When combined with AraC/Idarubicin, IMiDs fail to circumvent BM stroma-mediated resistance of non-del5q/5q- AML cells in vitro and in vivo but induce robust extramedullary mobilization of AML cells. When administered as a single agent, lenalidomide specifically mobilizes non-del5q/5q- AML cells, but not healthy CD34⁺ cells, to peripheral blood (PB) through specific downregulation of CXCR4 in AML blasts. Global gene expression profiling supports a migratory/mobilization gene signature in lenalidomide-treated non-del5q/5q- AML blasts but not in CD34⁺ cells. Collectively, IMiDs mobilize non-del5q/5q- AML blasts to PB through CXCR4 downregulation, but fail to potentiate AraC/Idarubicin activity in preclinical models of non-del5q/5q- AML.

Keywords: AML; AraC; BM-MSC; IMiDs; Idarubicin; lenalidomide; pomalidomide; xenografts.

Figure 1.

BM microenvironment protects HL60 and MOLM-13 cells from AraC+Idarubicin-based chemotherapy in vitro (A) and in vivo (B). (n = 3). *p < 0.05. **p < 0.01.

Figure 2.

Effects of IMiDs on primary AML blasts and BM-MSC. (A) Lenalidomide and pomalidomide do not have a cytotoxic effect on either AML cell lines (n = 3) (top panel), primary AML blasts (n = 9, bottom left panels) or BM-MSC (n = 3) (right bottom panels). (B) Left panel: IMiDs potentiate the immunosuppressive effect of BM-MSCs, measured as percentage of CFSE+ non-proliferating cells. CSFE-labeled PBMCs were stimulated with PHA and then co-cultured with BM-MSCs in the presence of lenalidomide or pomalidomide for 5 days (n = 3). Middle panels: representative flow-cytometry histograms of cycling (CSFE^low) PBMCs. Right panels: Concentration of the indicated cytokines in cell-culture supernatants determined by Luminex Multiplex assays. PBMCs were co-cultured with BM-MSCs in the presence/absence of IMiDs. Error bars indicate the SEM values of the n = 3 biological replicates. (C) IMiDs diminish IL6 production by BM-MSC (n = 3). *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Figure 3.

IMiDs combined with AraC-Idarubicin do not circumvent BM- microenvironment-mediated resistance of AML cells but induce robust extramedullary mobilization of AML cells. (A) Lenalidomide and pomalidomide do not overcome BM-MSC-mediated resistance of AML cell lines to intensive chemotherapy (n = 3). (B) IMiDs combined with AraC+Idarubicin highly mobilize AML blasts to PB and spleen in AML xenograft models (n = 3). *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Figure 4.

When administered as a single agent, lenalidomide highly mobilizes AML cells, but not healthy CD34⁺ cells, to PB. (A,B) Lenalidomide administered alone mobilizes AML cells to PB (A) and spleen (B). AML infiltration in PB and spleen was detected by FACS and immunohistochemistry, respectively. (C) Lenalidomide fails to mobilize healthy CD34⁺ cells (n = 3). *p < 0.05. **p < 0.01.

Figure 5.

IMiDs mobilizes AML cells to PB through CXCR4 downregulation. (A) Cartoon of the CXCR4-SDF1 crosstalk between leukemic blasts and BM stromal cells. (B) Effect of IMiDs on the expression levels of CXCR4 in AML cell lines, primary AML cells (top panels), PB-derived CD34⁺ cells and BM-derived CD34⁺ cells (bottom panels). (C) Effect of IMiDs on the expression (Left panel) and production levels (right panel) of SDF1 by BM-MSCs. The specific CXCR4 inhibitor AMD3100 was used as positive control.

Figure 6.

Global GEP reveals a migratory signature in lenalidomide-treated AML blasts. (A, B) Left panels: Heatmap representation of hierarchical clustering of genes differentially expressed between lenalidomide-treated and untreated AML blasts (n = 3 leukemias) (top panel) and BM-CD34⁺ cells (n = 2 healthy donors) (bottom panel). Right panel: Statistically significant biological functions identified using IPA on genes differentially expressed in lenalidomide-treated versus untreated AML blasts (top panel) and CD34+ cells (bottom panel). They are ranked by z-score. A z-score >2 indicates a predicted activation of that biological function. Biological functions associated with ‘cell migration/movement/motility’ are shown in black.

Figure 7.

Effects of Lenalidomide on primary blasts from patients with del5q/5q- AML. (A) Scheme depicting the experimental design. (B) Lenalidomide toxicity on primary del5q/5q- AML cells (n = 4). (C) When combined with standard chemotherapy lenalidomide largely overcomes BM-MSC-mediated resistance of del5q/5q- AMLs to AraC+Idarubicin (n = 4). (D) Effect of lenalidomide on the expression levels of CXCR4 in del5q/5q- AML cells (n = 4). AMD3100 was used as positive control. *p < 0.05; n.s, no statistical differences.