Overexpression of the toll-like receptor (TLR) signaling adaptor MYD88, but lack of genetic mutation, in myelodysplastic syndromes

Abstract

MYD88 is a key mediator of Toll-like receptor innate immunity signaling. Oncogenically active MYD88 mutations have recently been reported in lymphoid malignancies, but has not been described in MDS. To characterize MYD88 in MDS, we sequenced the coding region of the MYD88 gene in 40 MDS patients. No MYD88 mutation was detected. We next characterized MYD88 expression in bone marrow CD34+ cells (N = 64). Increased MYD88 RNA was detected in 40% of patients. Patients with higher MYD88 expression in CD34+ cells had a tendency for shorter survival compared to the ones with lower MYD88, which was significant when controlled for IPSS and age. We then evaluated effect of MYD88 blockade in the CD34+ cells of patients with lower-risk MDS. Colony formation assays indicated that MYD88 blockade using a MYD88 inhibitor resulted in increased erythroid colony formation. MYD88 blockade also negatively regulated the secretion of interleukin-8. Treatment of MDS CD34+ cells with an IL-8 antibody also increased formation of erythroid colonies. These results indicate that MYD88 plays a role in the pathobiology of MDS and may have prognostic and therapeutic value in the management of patients with this disease.

Figure 1. Barcode PCR-deep sequencing analysis MYD88 gene in bone marrow samples of 40 patients with MDS.

(A) Genomic Sequence of MYD88 is analyzed in this study. Coding regions are illustrated by capitalized characters, and locations of 10 pairs of primers used in the 1^st round of PCR are highlighted in different colors. Sequence encoding the TIR domain is underlined. First codon (ATG) and stop-codon are squared by red lines. (B) Graphic illustration of the barcode PCR-deep sequencing approach used in this study.

Figure 2. Analysis of MYD88 RNA expression in bone marrow CD34+ cells of patients with MDS.

(A) Q-PCR results of MYD88 RNA in MDS and control CD34+ cells. (B) Impact of MYD88 RNA level in overall MDS patient survival.

Figure 3. Effect of MYD88 blockade in cultured lower-risk MDS bone marrow CD34+ cells.

(A) Analysis of the activity of the MYD88 inhibitor in KG-1 cells. Luciferase assays indicates that MYD88 inhibitor reduces the NF-kB activity that is stimulated by the TLR2 agonists PAM3CSK4 and MALP2. (B) Representative microphotographs of colonies derived from MDS CD34+ cells in methocult plates after two weeks of culture, treated with control peptide or MYD88 inhibitor peptide. Arrows point to CFU-E. (C) Colony counting after two weeks of methocult culture and treatment of MYD88 inhibitor peptide in comparison to control peptide. (D) Q-PCR analysis indicated elevated RNA levels of erythroid differentiation genes EPOR, CD71 and Glycophorin A and B in MYD88 inhibitor peptide treated cells in comparison to control peptide treatment. Cells were collected from methocult plates after two weeks of culture. (E) Q-PCR analysis of GATA1, GATA2 or GATA2/GATA1 ratio in MYD88 inhibitor peptide treated cells.

Figure 4. Analysis of the implication of IL-8 in MDS.

(A) IL-8 protein concentration is elevated in bone marrow plasma samples of patients with MDS in comparison to healthy controls. (B). MYD88 inhibitor peptide treatment in cultured bone marrow CD34+ cells of patients with MDS leads to reduced IL-8 RNA levels. (C) Colony counting after two weeks of methocult culture in cells treated with IL-8 antibody or isotype control (ISO) indicates that treatment with IL-8 antibody leads to increased number of erythroid colonies in cultured CD34+ cells of patients with low-risk MDS. (D) Q-PCR analysis indicated elevated RNA levels of erythroid differentiation genes EPOR, and CD71 in IL-8 antibody treated cells in comparison to isotype control. Cells were collected from methocult plates after two weeks of culture.