Modulation of the Immune Response by Deferasirox in Myelodysplastic Syndrome Patients

Abstract

Deferasirox (DFX) is an oral iron chelator used to reduce iron overload (IO) caused by frequent blood cell transfusions in anemic myelodysplastic syndrome (MDS) patients. To study the molecular mechanisms by which DFX improves outcome in MDS, we analyzed the global gene expression in untreated MDS patients and those who were given DFX treatment. The gene expression profiles of bone marrow CD34⁺ cells were assessed by whole-genome microarrays. Initially, differentially expressed genes (DEGs) were determined between patients with normal ferritin levels and those with IO to address the effect of excessive iron on cellular pathways. These DEGs were annotated to Gene Ontology terms associated with cell cycle, apoptosis, adaptive immune response and protein folding and were enriched in cancer-related pathways. The deregulation of multiple cancer pathways in iron-overloaded patients suggests that IO is a cofactor favoring the progression of MDS. The DEGs between patients with IO and those treated with DFX were involved predominantly in biological processes related to the immune response and inflammation. These data indicate DFX modulates the immune response mainly via neutrophil-related genes. Suppression of negative regulators of blood cell differentiation essential for cell maturation and upregulation of heme metabolism observed in DFX-treated patients may contribute to the hematopoietic improvement.

Keywords: deferasirox; iron chelation; molecular mechanisms; myelodysplastic syndrome.

Figure 1

Gene Ontology (GO) biological process (BP) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways (p < 0.05) identified by annotation and enrichment analysis of the differentially expressed genes between MDS patients with normal serum ferritin levels and those with iron overload. The bar chart represents the −log(p-value) of the GO term or signaling pathway.

Figure 2

GSEA enrichment plots for Hallmark gene sets: TNFA Signaling via NF-κB and Unfolded Protein Response. Genes involved in (a) HALLMARK TNFA Signaling via NF-κB and (b) HALLMARK Unfolded Protein Response show significant enrichment in iron-overloaded patients (overloaded) versus patients with normal ferritin levels (non-overloaded). Top, the running enrichment score for the gene set as the analysis walks along the ranked list; bottom, the plot of the ranked list of all genes. X-axis, the rank for all genes; Y-axis, value of the ranking metric. ES, enrichment score; NES, normalized enrichment score; FDR, false discovery rate.

Figure 3

Gene Ontology (GO) biological process (BP) terms (p < 0.01) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways (p < 0.05) identified by annotation and enrichment analysis of the differentially expressed genes between untreated MDS patients with iron overload and those treated with DFX. The bar chart represents the −log(p-value) of the GO term or signaling pathway.

Figure 4

GSEA enrichment plots for Hallmark gene sets: Heme Metabolism and TNFA Signaling via NF-κB. Genes involved in (a) HALLMARK Heme Metabolism and (b) HALLMARK TNFA Signaling via NF-κB show significant enrichment in iron-chelated patients (treated) versus untreated patients with iron overload (non-treated). Top, the running enrichment score for the gene set as the analysis walks along the ranked list; bottom, the plot of the ranked list of all genes. X-axis, the rank for all genes; Y-axis, value of the ranking metric. ES, enrichment score; NES, normalized enrichment score; FDR, false discovery rate.

Figure 5

Validation of the array data by RT-qPCR. The gene expression levels of (a) DEFA3 and (b) LEF1 genes were determined in MDS patients with normal serum ferritin levels (non-overloaded), MDS patients with iron overload (overloaded) and MDS patients treated with DFX (chelated) by RT-qPCR. The relative expression level was normalized to the level of B2M expression using the 2−ΔΔCT method. Significant changes in gene expression are indicated with an asterisk (* p ˂ 0.05, ** p ˂ 0.01).

Figure 6

Induced network module analysis of the DEGs generated using ConsensusPathDB by considering the genetic interactions, biochemical interactions and gene regulatory interactions with intermediate genes. Nodes with black labels are seed genes, while nodes with magenta labels are intermediate nodes. Blue boxes indicate genes, light blue boxes indicate proteins and orange boxes indicate RNAs. Blue interaction lines indicate genetic interactions, green interaction lines indicate biochemical interactions and light blue interaction lines indicate gene regulatory interactions.