Clinical Significance and Pathopysiological Function of the Tim-3/Galection-9 Pathway in Myelodysplastic Syndromes

Background: Galectin-9 (Gal-9), a member of the galectin family, plays a crucial role in inflammation and tumorigenesis involving angiogenesis, cell adhesion, immune escape, and cancer cell survival. In acute myeloid leukemia (AML), Gal-9 is highly expressed in some AML cell lines and binds with T-cell immunoglobulin- and mucin-domain-containing molecule-3 (Tim-3) on T cells and leukemic stem cells. The Tim-3/Gal-9 pathway may be associated with disease progression by inducing T-cell dysregulation and mediating autocrine proliferation of leukemic cells. However, the role of the pathway in myelodysplastic syndromes (MDS) remains unclear. In the current study, we investigated the prognostic impact of plasma Gal-9 levels and the pathophysiological roles of Gal-9 and Tim-3 in MDS.

Methods and results: 1) To investigate the prognostic impact of Gal-9, plasma Gal-9 levels were measured by ELISA in 92 MDS patients: 30 with refractory anemia (RA); 3 with RA with ringed sideroblasts (RARS); 18 with RA with excess blasts (RAEB); 14 with RAEB in transformation (RAEB-t); and 29 with acute leukemia transformed from MDS (AL-MDS). The plasma Gal-9 level increased with disease progression (mean ± SD: control 3.4 ± 0.9; RA, 9.3 ± 5.7; RARS, 10.2 ± 6.8; RAEB, 9.7 ± 6.5; RAEB-t, 17.2 ± 9.6; AML-MDS, 20.1 ± 14.1 ng/ml). Furthermore, patients with AL-MDS in International Prognostic Scoring System (IPSS) high-risk categories (Intermediate-2/High) had significantly higher Gal-9 levels compared with IPSS low-risk (Low/Intermediate-1) patients (Low/Intermediate-1 vs Intermediate-2/High, p = 0.002; Low/Intermediate-1 vs AML-MDS, p = 0.004). The overall survival of MDS patients with plasma Gal-9 higher than the normal level (10 ng/mL) was significantly shorter than that of other patients (log-rank p < 0.001) even in lower-risk (RA, RARS) cases (log-rank p = 0.0369). Moreover, multivariate analysis showed that a high level of Gal-9 was an independent predictor of shorter survival in MDS patients as well as IPSS. 2) Bone marrow mononuclear cells were isolated from some MDS patients, and Tim-3 expression on granulocytes, monocytes, lymphocytes, and blasts was analyzed using flow cytometry (FCM). Tim-3 expression on blasts tended to be higher in AL-MDS patients than in MDS patients. 3) To investigate whether MDS cells can produce Gal-9, we analyzed Gal-9 levels in supernatants of cell cultures of 3 human AL-MDS cell lines, F-36P, SKM-1, and MDS-L, and MDS blasts obtained from an AL-MDS patient by ELISA. Gal-9 was detected in all cell culture supernatants and its concentration was higher when cultured in medium containing 10% fetal bovine serum (FBS) than in 2.5% FBS medium in F-36P and SKM-1 cells. The concentration of Gal-9 produced from MDS blasts was increased time dependently in 2-6 day culture. Furthermore, recombinant human Gal-9 enhanced the cell proliferation of F-36P cells. 4) After analyzing Tim-3 expression in MDS cell lines using real-time quantitative PCR and FCM, all cell lines expressed relatively low levels of Tim-3 mRNA. Extracellular expression of Tim-3 was detected and induced by adding cell culture supernatants of the human stromal cell line HS-5 in F-36P cells. To elucidate the signaling pathway inducing Tim-3 expression, we investigated Tim-3 mRNA expression in F-36P cells treated with HS-5 supernatant with several signal transduction inhibitors (STAT3 inhibitor, U0126; MAPK/ERK; AG490; JAK2, LY294002; PI3K, PDTC; NF-κB inhibitor). Upregulation of Tim-3 gene expression in F-36P cells treated with HS-5 supernatant was inhibited by the MEK inhibitor U0126. These results suggest that the MAPK/ERK pathway is involved in Tim-3 expression induced by HS-5 supernatant.

Conclusions: In MDS, the elevation of plasma Gal-9 levels is associated with disease progression, including leukemic transformation, and with shorter survival. Furthermore, its receptor Tim-3 is upregulated on blasts in AL-MDS patients. Our data suggest that the Gal-9/Tim-3 pathway plays a key role in MDS disease progression. Although further study is required to clarify the detailed functions of the interaction between Gal-9 and Tim-3, the current study could lead to the development of a new immunotherapeutic strategy via the blockade of this pathway in MDS.