Masae Kunitama

Active FKHRL1 overcomes imatinib resistance in chronic myelogenous leukemia‐derived cell lines via the production of tumor necrosis factor‐related apoptosis‐inducing ligand

FKHRL1 (also called FOXO3a) is a member of the Forkhead Box, class O (FOXO) subfamily of forkhead transcription factors and functions downstream of Bcr–Abl tyrosine kinase as a phosphorylated inactive form in chronic myelogenous leukemia (CML). The Bcr–Abl tyrosine kinase inhibitor imatinib induces cell cycle arrest and subsequent apoptosis via the conversion of FKHRL1 from the phosphorylated inactive form to the dephosphorylated active form in CML‐derived cell lines. In the present study, we examined whether active FKHRL1 can overcome resistance to imatinib. To this end, we generated a 4‐hydroxytamoxifen‐inducible active FKHRL1 (FKHRL1‐TM; a triple mutant of FKHRL1 in which all three Akt phosphorylation sites have been mutated)–estrogen receptor fusion protein expression system in CML‐derived imatinib‐resistant cell lines. 4‐Hydroxytamoxifen inhibited cell growth and cell cycle progression, and subsequently induced apoptosis, accompanied by upregulation of tumor necrosis factor‐related apoptosis‐inducing ligand (TRAIL). Thus, active FKHRL1 antagonized deregulated proliferation and induced apoptosis in these cell lines. In addition, imatinib‐resistant cells underwent apoptosis after transfection with full‐length TRAIL cDNA. Collectively, our results suggest that active FKHRL1 can overcome imatinib resistance in CML cells, in part via TRAIL production. (Cancer Sci 2007; 98: 1949–1958)

Inhibition of hypoxia-inducible factor-1 function enhances the sensitivity of multiple myeloma cells to melphalan

Abnormal activation of hypoxia-inducible factor-1 (HIF-1), one of the most important transcription factors for the adaptation of cells to hypoxia, is frequently observed in numerous types of solid tumors. Dysregulation of HIF-1 induces tumor angiogenesis and enhances the expression of anti-apoptotic proteins and glycolysis-associated enzymes in cancer cells, which in turn leads to the promotion of tumor growth. In the present study, we examined the pathophysiologic role of HIF-1 in multiple myeloma. Furthermore, we explored the possibility that HIF-1 may be a molecular target for myeloma therapy. We identified constitutive expression of the hypoxia-inducible factor-1 α (HIF-1α)-subunit in established myeloma cell lines and in primary myeloma cells. Treatment with insulin-like growth factor-1 (IGF-1) significantly increased HIF-1α expression through activation of the AKT and mitogen-activated protein kinase signaling pathways. Inhibition of HIF-1 function either by echinomycin, a specific HIF-1 inhibitor, or a siRNA against HIF-1α resulted in enhanced sensitivity to melphalan in myeloma cells. This inhibition of HIF-1 also reversed the protective effect of IGF-1 on melphalan-induced apoptosis. Inhibition of HIF-1 drastically reduced both basal and IGF-1–induced expression of survivin, one of the most important anti-apoptotic proteins in myeloma cells. We conclude that HIF-1 inhibition may be an attractive therapeutic strategy for multiple myeloma. [Mol Cancer Ther 2009;8(8):2329–38]

Erythropoietin Overcomes Imatinib-Induced Apoptosis and Induces Erythroid Differentiation in TF-1/bcr-abl Cells

Targeting BCR‐ABL tyrosine kinase by treatment with the selective inhibitor imatinib (formerly STI571, Gleevec) has proved to be highly efficient for inhibiting leukemic growth in vitro. In addition, in clinical trials, imatinib has produced high response rates in patients with chronic myeloid leukemia (CML) in chronic phase and blastic crisis. However, episodes of severe cytopenia were also frequently observed, leading to discontinuation of therapy in some cases. Therefore, it is important to examine whether administration of cytokines overcomes the adverse effects of imatinib in in vitro systems. In this study, we examine the effects of granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) and erythropoietin (EPO) on TF‐1/bcr‐abl (which was generated by transduction of a bcr‐abl fusion gene into the TF‐1 cell line) as a model system for CML with blastic crisis. Imatinib induced apoptosis in TF‐1/bcr‐abl cells but not in the parental TF‐1 cells. However, GM‐CSF, a survival factor of the parental TF‐1 cells, protected TF‐1/bcr‐abl cells from imatinib‐induced apoptosis in a dose‐dependent manner. Concomitantly, constitutive phosphorylation of Stat5 and FKHRL1 was significantly inhibited by imatinib, and the inhibition was canceled by the addition of GM‐CSF, accompanied by upregulation of Bcl‐xL and downregulation of p27/Kip1. In addition, although untreated TF‐1/bcr‐abl cells had lost responsiveness to both GM‐CSF and EPO and showed autonomous growth, GM‐CSF enhanced phosphorylation of Stat5 and FKHRL1 in these cells. Importantly, imatinib‐treated TF‐1/bcr‐abl cells differentiated into hemoglobin‐positive cells in the presence of EPO, as in the case for the parental TF‐1 cells. Taken together, imatinib‐treated CML cells may differentiate into mature cells in the presence of differentiation‐inducing cytokines such as EPO.