TIM-3, a Leukemia Stem Cell Marker, Plays a Role In Leukemic Transformation Through Autocrine Stimulatory Signaling By Its Ligand, Galectin-9

Acute myeloid leukemia (AML) originates from self-renewing leukemic stem cells (LSCs), an ultimate therapeutic target for AML. We have reported that the T-cell immunoglobulin mucin-3 (TIM-3) is expressed on LSCs in most types of AML but not on normal hematopoietic stem cells (HSCs) (Kikushige et al, Cell Stem Cell, 2010). We extended the analysis of TIM-3 expression into various types of human hematological malignancies, and found that human TIM-3 is expressed in the vast majority of CD34⁺CD38^- LSCs of human myeloid malignancies including chronic myeloid leukemia, chronic myelomonocytic leukemia and myelodysplastic syndromes (MDS). Although CD34⁺CD38^- normal bone marrow stem cells do not express TIM-3, TIM-3 is expressed in the CD34⁺CD38^- population in MDS, and is further up-regulated with progression into leukemia. The average percentages of TIM-3⁺ cells in the CD34⁺CD38^- population was 7.8% in RCMD (n=10), 19.2% in RAEB-1 (n=10), 84.0% in RAEB-2 (n=10) and 92.2% in overt AML (n=10). The close association of TIM-3 expression with transformation into AML led us to hypothesize that TIM-3 itself has a function in AML stem cell development. TIM-3 is a type 1 cell-surface glycoprotein and has a structure that includes an N-terminal immunoglobulin variable domain followed by a mucin domain, a transmembrane domain and a cytoplasmic tail. Tyrosine residues are clustered in the cytoplasmic tail, suggesting that TIM-3 can induce signal transduction in TIM-3⁺ AML cells. To understand the function of TIM-3, we investigated the interaction between TIM-3 and its ligand galectin-9 in AML LSCs. We found that AML patients showed significantly higher serum galectin-9 concentration than healthy individuals (healthy controls: 18.3+4.3 pg/ml, AML patients: 139.1+33.4 pg/ml, P<0.05). Unexpectedly, we found that leukemic cells expressed a high level of galectin-9 protein, as compared to other hematopoietic cells including T cells, B cells and monocytes. Using KASUMI-3 (TIM-3⁺ AML cell line) and primary AML samples, we confirmed that AML cells could secrete galectin-9 after TLR stimulation in vitro. Furthermore, microarray analysis demonstrated that TIM-3 stimulation by the physiological concentration of galectin-9 induced significant gene expression changes toward pro-survival axis including up-regulation of MCL-1, the important survival factor for HSCs and LSCs. These results collectively suggest that AML cells can produce and secrete galectin-9, and galectin-9 can bind and stimulate TIM-3-expressing AML cells including LSCs in an autocrine manner to support their survival or leukemia progression.