Cell Death Mechanisms Induced by INNO-406, a Novel Tyrosine Kinase Inhibitor for Imatinib-Resistant Ph+ Leukemia.

The blockade of Bcr-Abl signaling suppresses cellular growth and induces cell death in Bcr-Abl-positive (Bcr-Abl+) cells. We herein assessed the cell death mechanisms induced by INNO-406 (formerly NS-187; Kimura et al, Blood 2005), in four CML-derived Bcr-Abl+ cell lines (K562, KT-1, BV173 and MYL), and Ba/F3 harboring wild type bcr-abl (Ba/F3/wt bcr-abl). When cells are treated by INNO-406, the accumulation of subG1 fraction was seen in all five cell lines. This cell death was accompanied by loss of mitochondrial membrane potential and was inhibited by over-expression of Bcl-2, indicating that INNO-406-induced cell death is mainly mediated by mitochondria-dependent apoptosis. Caspase-3 activation in INNO-406-treated cell was also common among all cell lines. However, the inhibition of caspase activity by ZVAD-fmk (ZVAD), a pan-caspase inhibitor, was variable in the cell lines tested. In K562, KT-1 and BV173 cells treated with INNO-406, ZVAD almost completely prevented apoptosis (i.e. showing atypical feature for apoptosis, no DNA fragmentation and no accumulation of subG1 fraction), with cell death resulting from morphologically non-apoptotic cell death. The percentages of non-apoptotic cells under ZVAD co-treated with INNO-406 varied among the three cell lines, suggesting that the dependence on non-apoptotic cell death is variable. While, in MYL and Ba/F3/wt bcr-abl cells, despite the sufficient inhibition of caspases’ activity, the inhibition of the cell death by ZVAD was only partial and these cell lines still underwent apoptosis (i.e. showing DNA fragmentation and the accumulation of subG1 population), suggesting the presence of caspase-independent apoptotic machineries. In addition, assay data for apoptosome activities (complex of Apaf-1, cytochrome c and caspase-9 that initiates and drives cysteine protease activities of caspase in mitochondrial-mediated pathway) suggested that cell types could be largely subdivided into two groups, namely those cells with high apoptosome activity (K562, KT-1 and BV173) that undergo non-apoptotic, and, those cells with low apoptosome activity (MYL and Ba/F3/wt bcr-abl.) that undergo caspase-independent apoptosis when caspase activity was blocked by ZVAD. These data indicate that there is a common initial pathway for cell death due to INNO-406, while the pathway for cell death commitment (i.e. dependence on apoptosome/caspases-mediated apoptosis pathway that has been commonly believed to be central for apoptosis execution) vary among cellular context in Bcr-Abl+ leukemic cells. Moreover, in a mouse model of primary human CML in blast crisis, INNO-406 caused cell death with fragmented nuclei typical to apoptosis and “necklace-like” nuclei not typical of apoptosis, further implicating the significance of involvement of caspase-independent, non-apoptotic cell death in vivo. Further studies of the role of caspase-independent cell death in patient-derived Bcr-Abl+ cells and the molecular mechanisms that lead to mitochondrial-depolarization and caspase-independent apoptotic and/or non-apoptotic cell death may help the development of novel therapeutic strategies against Bcr-Abl+ leukemias.