Galectin-9 Overcomes Various Types of Treatment Resistance through ATF-3/Noxa Pathway-Mediated Apoptosis In Chronic Myelogenous Leukemia

Abstract 4930

Tyrosine kinase inhibitors (TKIs) against Bcr-Abl fusion oncoprotein, such as imatinib mesylate (IM), nilotinib, or dasatinib, are the first-line molecular targeted therapeutics for chronic myelogenous leukemia (CML). However, the resistance to Bcr-Abl TKIs is induced in leukemic cells not only by loss of sensitivity to TKIs through Bcr-Abl-related molecular mechanisms, such as the acquisition of Abl mutation or the overexpression of Bcr-Abl, but also by loss of addiction to Bcr-Abl TK activity by acquiring Bcr-Abl-unrelated additional oncogenic mutations. Therefore, a new treatment approach that induces an anti-leukemic effect via Bcr-Abl-unrelated molecular pathways is urgently needed for achievement of a complete cure and to overcome TKI resistance. Galectins are a family of animal lectins that show specific affinity for beta-galactosides. Among fourteen mammalian galectins, galectin-9 (Gal9) has been shown to possess the anti-cancer properties by regulating various cellular functions, such as cell adhesion, cell proliferation, or apoptosis. These prompted us to investigate whether Gal9 can have an anti-CML effect via signaling cascades distinct from the pathway utilized by Bcr-Abl TKIs or by other commonly utilized anti-cancer agents. Modified human Galectin-9 (hGal9) inhibits the proliferation of six CML-derived cell lines, BV173, KT-1, KCL22, K562, KBM5 and MYL, by inducing apoptosis at their IC₅₀s from 17.5 to 164.9 nM, with the activation of caspases-3, -4, - 8 and -9. The addition of 25 mM lactose prevented the growth inhibitory effect by hGal9 in K562, indicating the essential role of beta-galactoside binding activity in the anti-CML activity of hGal9. Because hGal9 treatment caused upregulation of Noxa, a pro-apoptotic BH3-only protein of Bcl-2 family proteins, and Mcl-1, a member of anti-apoptotic Bcl-2 proteins, in CML cell lines, we next investigated the involvement of Bcl-2-regulated apoptosis pathway in cell death by hGal9. K562 sublines overexpressing Bcl-2, Bcl-X_L, or Mcl-1, showed resistance to cell death induced by IM, but were as sensitive to hGal9-induced cell death as the parental cells, suggesting the involvement of a pathway which is independent of Bcl-2 family proteins. These results also indicate that the accumulation of Mcl-1 following hGal9 treatment does not hamper apoptotic induction by hGal9. Besides, the expression of dominant-negative FADD protein did not hamper the effect of hGal9, also indicating that the death receptor pathway was not responsible for apoptosis induced by hGal9. In contrast, our study revealed that hGal9 caused the upregulation of activating transcription factor 3 (ATF3), a member of the ATF/CREB family transcription factors, within 3 hour treatment, and the gene knockdown experiments using RNA interference (RNAi) technique revealed that ATF3 is the critical mediator for cell killing by hGal9. Moreover, RNAi experiments indicated that Noxa is one of the downstream effector molecules of ATF3, and that Noxa partly mediates cell death induction by hGal9. Bim, on the other hand, the BH3-only protein essential for apoptosis by Bcr-Abl TKIs, was not associated with hGal9-induced cell death. Considering that the activation of caspase-4 and caspase-8 is involved in ER stress-induced apoptosis, and that Noxa induction by ATF3 has been shown to be crucial in the cell death induced by inhibitors for ER-associated protein degradation, we suggest that hGal9-induced cell death may at least partly involve ER stress. ATF3-mediated cell death by hGal9 was not hampered by the absence of p53, the presence of mutant Abl^T315I, or by P-glycoprotein overexpression. In addition, hGal9 showed the additive growth inhibitory effect with IM on CML cell lines. Collectively, hGal9 is a candidate agent that may overcome various kinds of resistance to treatment for CML, and suggest that ATF3 may be a new target molecule for the development of new treatment modalities that can overcome resistance to currently available chemotherapeutics.