Previously it was reported that

Previously it was reported that the release of both Tim-3 and galectin-9 depends on PKCα and proteolysis (Chabot et al., 2002). Our results confirmed these findings. PMA treatments induced PKCα activation, which activated exocytosis of Tim-3 and galectin-9 as well as their mTOR-dependent production in THP-1 AML ldv (Fig. 6). Interestingly, natural and exogenous ligands of LPHN1, a G-protein coupled neuronal receptor expressed also in CD34-positive human stem cells (Supplementary Fig. 3) and AML cells but not healthy white blood cells, activated the PKCα pathway. They also induced both mTOR-dependent translation of Tim-3 and galectin-9 as well as their exocytosis. The effect was observed in THP-1 and primary human AML blasts. PKCα is known to provoke agglomeration of SNARE complex responsible for exocytosis (Stockli et al., 2011; Morgan et al., 2005). Since FLRT3, one of natural ligands of LPHN1, is present in bone marrow (Fig. 7) it might explain how LPHN1 causes PKCα activation. Interestingly, constitutively active PKCα in malignant primary AML cells correlates with a very poor prognosis and high mortality rate of patients (Kurinna et al., 2006). This suggests that AML cells constantly release high levels of Tim-3 and galectin-9. Bone marrow also expresses other PKCα-activating proteins. When we exposed THP-1 cells to mouse bone marrow extracts, galectin-9 release was significantly higher compared to resting THP-1 cells. FLRT3 neutralizing antibody significantly reduced but did not abolish FLRT3 induced PKCα-dependent galectin-9 release. This suggests that galectin-9 and Tim-3 are synthesized and exocytozed by AML cells in a PKCα and mTOR-dependent manner, using available plasma membrane-associated PKCα activating receptors (for example LPHN1) to induce the whole pathway. Galectin-9 prevents the delivery of granzyme B into AML cells (this is a perforin and mannose-6-phosphate receptor-dependent process (Supplementary Fig. 9)). Inside AML cells granzyme B performs cleavage of the protein Bid into tBid, thus inducing mitochondrial dysfunction and cytochrome C release followed by caspase 3 activation. Proteolytic activation of caspase 3, in addition to the classic pathway, might also be directly catalyzed by granzyme B (Lee et al., 2014). Our results with galectin-9 confirmed this concept (Fig. 11). Recently it was reported that galectin-9 induces interferon-gamma (IFN-γ) release from NK cells (Gleason et al., 2012). IFN-γ interacts with AML cells inducing the activity of indoleamine 2,3-dioxygenase (IDO1), an enzyme which converts L-tryptophan into formyl-L-kynurenine, which is then converted into L-kynurenine and released (Corm et al., 2009; Folgiero et al., 2015; Mabuchi et al., 2016). L-kynurenine affects the ability of NK cells to kill AML cells, an effect which was seen in our experiments and presented in Supplementary Fig. 9. Soluble Tim-3 was shown to significantly downregulate production of IL-2, a cytokine required for activation of NK cells and cytotoxic T lymphocytes. Taken together, our results show that human AML cells possess a secretory pathway which leads to the production and release of sTim-3 and galectin-9. Both proteins prevent the activation of NK cells and impair their AML cell-killing activity. This pathway, which involves the LPHN1-dependent activation of Tim-3 and galectin-9 production is summarized in Fig. 13. The described pathway presents both biomarkers for AML diagnostics and potential targets (both sTim-3 and galectin-9) for AML immune therapy and thus can be considered as a fundamental discovery.
Grant Support This work was supported by a Daphne Jackson Trust postdoctoral fellowship (to IMY), University of Kent Faculty of Sciences Research Fund (to VVS and YAU), Batzebär grant (to EFK and SB) and Oncosuisse grant KFS-3728-08-2015 (to LV and MB). Funders had no role in study design, data collection, data analysis, interpretation or writing of the report.