Galectin-3 Is the Molecular Target for Overcoming Multidrug Resistance Due to the Cell Protection by Bone Marrow Leukemia Microenvironment in Chronic Myeloid Leukemia,

Abstract 3746

Recent advances in molecular targeted therapy using tyrosine kinase (TK) inhibitors (TKIs) for Bcr-Abl TK have greatly improved treatment outcomes for chronic myeloid leukemia (CML). However, complete elimination of CML clones has been rarely achieved by TKIs due to both intrinsic and extrinsic cell mechanisms. In this study, we investigated the molecular mechanisms for bone marrow microenvironment (BMME)-mediated leukemia progression and drug resistance in CML. CML cell lines MYL and K562 acquired drug resistance to imatinib mesylate (IM), dasatinib (Das), doxorubicin (DOX), cytarabine (CA), etoposide (VP16), or vincristine (VCR) through co-culture with HS-5, an immortalized human bone marrow stromal cell (BMSC)-derived cell line. We used microarray-based assays to investigate the changes in gene expression profiles in Philadelphia (Ph)-positive MYL as a result of co-culture with HS-5 and of adhesion to fibronectin (FN). In MYL with HS-5 or with FN, 902 and 910 genes, respectively, were upregulated more than 2.0-fold compared with control. Among the 284 genes commonly upregulated in MYL with HS-5 and with FN, we focused on galectin-3 (Gal-3) as one of the candidate mediators of BMME-mediated leukemia progression because of its pleiotropic cellular function. The levels of galectin-3 mRNA increased 3.84-fold as a result of co-culture with HS-5, and 2.83-fold as a result of adhesion to FN in MYL. The induction of Gal-3 by the co-culture with HS-5 or the adhesion to FN was also confirmed at the protein level, not only in MYL, but also in all Ph+ leukemic cell lines examined (K562, KBM5, KCL22, BV173), while Gal-3 protein expression was either absent or extremely low in normal liquid culture. To investigate the clinical significance of Gal-3 in CML, we examined its expression in BM-derived primary CML cells. Approval was obtained from the institutional review board at Kyoto Prefectural University of Medicine for a study using patient-derived samples, and the study was conducted in accordance with the ethical principles of the Declaration of Helsinki. Of the leukemic cells of 20 CML patients, those of all but one CML-blast crisis phase patient were positive for Gal-3. Ph⁺ cells from the CML-chronic phase were especially highly positive for Gal-3. In contrast, the frequency of Gal-3-positive cells from most acute leukemia patients was as low as that of BM hematopoietic cells from healthy volunteers. These results suggest that in the clinical setting Gal-3 expression in the BM milieu is more predominant in CML. To further characterize the role of Gal-3 in CML, Gal-3 overexpressing subcell lines of MYL and K562 were generated by transfection of pEF1Galec3.neo plasmid and designated as MYL/G3 and K562/G3, respectively. Gal-3 overexpression was found to confer moderately higher in vitro proliferation potency in medium containing 10% FCS as well as in low nutrient 1% FCS-containing medium compared with the cells transfected with the mock plasmid. We also examined the impact of Gal-3 overexpression on cell death induced by chemotherapeutic agents. Both MYL/G3 and K562/G3 were less sensitive than their parental cells to cell death induced by chemotherapeutic agents. This diminished sensitivity to cell death caused by chemotherapeutic agents was due to a reduction in apoptosis, as reflected by the reduced frequency of subG1 populations detected in DNA content analyses (FACS SubG1 analysis of results for 48-hour treatment: MYL 54.9%, MYL/G3 15.7% with IM 0.25uM; MYL 47.1%, MYL/G3 23.1% with Das 1.0nM; MYL 40.2%; MYL/G3 13.4% with DOX 200nM; MYL 45.7%; MYL/G3 19.4% with CA 150nM; MYL 50.9%; MYL/G3 19.1% with VP16 4uM; MYL 75.0%; MYL/G3 49.5% with VCR 8nM). We next examined the effect of an inhibitor for Gal-3, fractionated citrus pectin powder (FPP), on MYL and MYL/G3. As expected, MYL and MYL/G3 showed similar sensitivity to cell death induced by FPP, while the addition of FPP overcame resistance to IM-induced cell death in MYL/G3. Furthermore, the addition of FPP was found to have overcome HS-5-induced resistance against IM in MYL. Molecular sequelae of Gal-3 overexpression in leukemic cells were also investigated. Both co-culture with HS-5 and Gal-3 overexpression activated Akt and Erk, induced accumulation of Mcl-1 in MYL and K562. Collectively, our study disclosed that leukemia microenvironment-specific Gal-3 may be a candidate therapeutic target to help overcome BMME-mediated therapeutic resistance in CML.