Canonical Wnt Pathway Is Activated in Myeloid Leukemia Stem Cells Via Hematopoietic Cell Kinase That Is Phosphorylated By an Autocrine TIM-3/Galectin-9 Signaling

(Introduction)

Activation of β-catenin is critical for self-renewal of cancer stem cells (CSCs). A number of recurrent mutations related to the aberrant accumulation and activation of β-catenin have been identified in CSCs in various types of cancer, but such somatic mutations are barely found in leukemic stem cells (LSCs) in acute myeloid leukemia (AML). We have recently identified the autocrine loop consisted of LSC-specific surface molecule T-cell immunoglobulin mucin-3 (TIM-3) and its ligand galectin-9 (Gal-9) in human myeloid malignancies. When Gal-9 binds to TIM-3, TIM-3 signaling induces accumulation of β-catenin in nucleus (Kikushige et al, Cell Stem Cell, 2015). In this study, we sought to identify the molecular mechanism of aberrant β-catenin activation in LSCs of AML.

(Results)

To test whether TIM-3 signaling can activate the canonical Wnt pathway, we evaluated the activation of LDL receptor-related protein 6 (LRP6), a key component of canonical Wnt pathway. Immunoprecipitation assay by using anti-LRP6 antibody revealed that Gal-9 stimulation promoted the formation of the "LRP6-signalosome", the protein complex consisted of Wnt components including LRP6, Axin and Dvl. Gal-9 stimulation also induced phosphorylation of LRP6at several sites including Thr¹⁴⁷⁹ and subsequent β-catenin accumulation in primary TIM-3⁺ AML cells. On the other hand, this effect on LRP6 induced by Gal-9 stimulation was completely canceled in the presence of an anti-TIM-3 antibody (F38-2E2), which can block the TIM-3/Gal-9 interaction. Since the Wnt ligand is not involved in this assay system, the TIM-3/Gal-9 interaction should directly activate the canonical Wnt pathway, not through the previously identified Wnt signaling machinery.

Gal-9 stimulation induced phosphorylation of cytoplasmic tail of TIM-3, which induces recruitment of several Src family kinases (SFKs). We found that Gal-9 ligation to TIM-3 activated several SFKs including hematopoietic cell kinase (HCK) by phosphorylation of the SH1 domain (Tyr⁴¹⁰ in HCK) in TIM-3⁺ AML cells. Furthermore, an inhibitor specific for HCK, but not inhibitors effective for other SFKs, inhibited LRP6 phosphorylation induced by TIM-3/Gal-9 signaling, indicating that HCK should be a critical mediator for TIM-3 signaling in human LSCs.

Next, we focused on p120-catenin because this molecule is known as not only a substrate for SFKs but also an indispensable molecule for the initial step of the LRP6-signalosome formation. We found that the phosphorylation of p120-catenin at Tyr²²⁸ was induced in response to Gal-9 stimulation. HCK inhibitors efficiently blocked the Tyr²²⁸ phosphorylation and subsequent activation of canonical β-catenin pathway. These results suggested that HCK activation induced by TIM-3/Gal-9 ligation could activate canonical β-catenin pathway through phosphorylation of p120-catenin.

(Conclusion)

In myeloid leukemia, TIM-3/Gal-9 signaling activates HCK, which induces p120-catenin phosphorylation, eventually acitavate the canonical β-catenin pathway, and these processes should be independent of Wnt ligands. Since TIM-3/Gal-9 autocrine loop signaling is constitutively active in AML, this novel machinery should contribute to the maintenance of AML LSCs through accumulation of β-catenin. Our study suggest that TIM-3 and Gal-9 as well as HCK should be important therapeutic targets in AML LCSs.