Interleukin-3 protects Bcr-Abl-transformed hematopoietic progenitor cells from apoptosis induced by Bcr-Abl tyrosine kinase inhibitors.

Bcr-Abl tyrosine kinase has been validated as a molecular target for the treatment of chronic myelogenous leukemia (CML). More recently, it has been reported that CML patients could develop resistance to the Bcr-Abl tyrosine kinase inhibitor, imatinib (STI571, Gleevec), pointing to the need for development of additional Bcr-Abl tyrosine kinase inhibitors or other therapeutic strategies. It was also found that a significant proportion of patients who received the Bcr-Abl inhibitor did not achieve complete cytogenetic response. Mechanisms for incomplete cytogenetic response to Bcr-Abl inhibition are not entirely clear. We report here three new pyrido[2,3-d]pyrimidine Bcr-Abl tyrosine kinase inhibitors, PD164199, PD173952, PD173958, that induced apoptosis of Bcr-Abl-dependent hematopoietic cells. An interleukin-3 (IL-3) autocrine loop was observed previously in primitive CD34(+)/Bcr-Abl(+) leukemic cells in CML patients. Using 32Dp210(Bcr-Abl)and Baf3p210(Bcr-Abl) cells as models, we tested whether IL-3 might protect Bcr-Abltransformed, IL-3-responsive cells from apoptosis caused by Bcr-Abl tyrosine kinase inhibition. Results of trypan blue exclusion, fluoroisothiocyanate-valyl-alanyl-aspartyl-[O-methyl] -fluoromethylketone (FITC-VAD-FMK), and Annexin-V/7-amino-actinomycin D (7-AAD) binding assays indicate that IL-3 could protect Bcr-Abl-transformed, IL-3 responsive hematopoietic progenitor cells from apoptosis induced by Bcr-Abl tyrosine kinase inhibitors. This finding raises the possibility that the IL-3 autocrine loop found in primitive CD34(+)/Bcr-Abl(+) cells in CML patients could contribute to the incomplete eradication of Bcr-Abl(+) cells by Bcr-Abl inhibition.

The pyrido[2,3-d]pyrimidine derivative PD180970 inhibits p210Bcr-Abl tyrosine kinase and induces apoptosis of K562 leukemic cells.

PD180970 is a novel pyrido[2,3-d]pyrimidine class of ATP-competitive inhibitor of protein tyrosine kinases. We found that PD180970 inhibited in vivo tyrosine phosphorylation of p210Bcr-Abl (IC50 = 170 nM) and the p210BcrAbl substrates Gab2 and CrkL (IC50 = 80 nM) in human K562 chronic myelogenous leukemic cells. In vitro, PD180970 potently inhibited autophosphorylation of p210Bcr-Abl (IC50 = 5 nM) and the kinase activity of purified recombinant Abl tyrosine kinase (IC50 = 2.2 nM). Incubation of K562 cells with PD180970 resulted in cell death. Results of nuclear staining, apoptotic-specific poly(ADP-ribose) polymerase cleavage, and annexin V binding assays indicated that PD180970 induced apoptosis of K562 cells. In contrast, PD180970 had no apparent effects on the growth and viability of p210Bcr-Abl-negative HL60 human leukemic cells. Thus, PD180970 is among the most potent inhibitors of the p210Bcr-Abl tyrosine kinase, which is present in almost all cases of human chronic myelogenous leukemia. These findings indicate that PD180970 is a promising candidate as a novel therapeutic agent for Bcr-Abl-positive leukemia.

Requirement of SHP2 binding to Grb2-associated binder-1 for mitogen-activated protein kinase activation in response to lysophosphatidic acid and epidermal growth factor.

Grb2-associated binder-1 (Gab1) is a multisite docking protein containing a pleckstrin homology (PH) domain, multiple potential tyrosine phosphorylation sites, and several proline-rich sequences. Gab1 becomes tyrosine-phosphorylated in cells stimulated with growth factors, cytokines, and ligands for G protein-coupled receptors. A major Gab1-binding protein detected in cells treated with extracellular stimuli is the tyrosine phosphatase, SHP2. Although the role of SHP2-Gab1 interaction in cell signaling has not yet been characterized, SHP2 is known to mediate mitogen-activated protein (MAP) kinase activation induced by the epidermal growth factor (EGF). However, the mechanism by which the SHP2 phosphatase exerts a positive signaling role remains obscure. In this study, we prepared Gab1 mutants lacking the SHP2 binding site (Gab1Y627F), the phosphatidylinositol 3-kinase (PI3K) binding sites (Gab1DeltaPI3K), and the PH domain (Gab1DeltaPH). Expression of Gab1Y627F blocked the extracellular signal-regulated kinase-2 (ERK2) activation by lysophosphatidic acid (LPA) and EGF. Conversely, expression of the wild-type Gab1 in HEK293 cells augmented the LPA receptor Edg2-mediated ERK2 activation. Whereas the PH domain was required for Gab1 mediation of ERK2 activation by LPA, it was not essential for EGF-induced ERK2 activation. Expression of Gab1DeltaPI3K had no apparent effect on ERK2 activation by LPA and EGF in the cells that we have examined. These results establish a role for Gab1 in the LPA-induced MAP kinase pathway and clearly demonstrate that Gab1-SHP2 interaction is essential for ERK2 activation by LPA and EGF. These findings also suggest that the positive role of SHP2 in the MAP kinase pathway depends on its interaction with Gab1.

Reversible regulation of SHP-1 tyrosine phosphatase activity by oxidation.

Increasing evidence indicates that redox regulation is an important signaling mechanism. Protein tyrosine phosphatases (PTPases) are sensitive to oxidative inactivation and are potential targets of redox regulation. In this study, we analyzed the reversibility of oxidative inactivation of the PTPase SHP-1, which negatively regulates protein tyrosine kinase signaling. H2O2 inactivated SHP-1 in vitro. Incubation of the H2O2-inactivated SHP-1 with dithiothreitol recovered 44-99% of the PTPase activity, depending on the H2O2 concentrations used to inactivate SHP-1. Glutathione and N-acetylcysteine also reactivated H2O2-treated SHP-1. Stimulation of SHP-1-transfected HeLa cells with H2O2 rapidly decreased SHP-1 activity, which was completely reversed within 15 min. Thus, oxidative inactivation of SHP-1 is a reversible process.

Role of tyrosine kinase activity of epidermal growth factor receptor in the lysophosphatidic acid-stimulated mitogen-activated protein kinase pathway.

Recent evidence indicates that the epidermal growth factor (EGF) receptor mediates a branch of lysophosphatidic acid (LPA)-induced signal transduction pathways that activate mitogen-activated protein (MAP) kinase. However, it is unclear whether the intrinsic tyrosine kinase activity of EGF receptor is involved. We previously showed that reactive oxygen species (ROS) were involved in the LPA-stimulated MAP kinase pathway. Here, we identify tyrosine phosphorylation of EGF receptor as an LPA signaling step that requires ROS. To evaluate the role of the tyrosine kinase activity of EGF receptor in the LPA-stimulated MAP kinase pathway, we examined the effects of an EGF receptor-specific tyrosine kinase inhibitor, PD158780. PD158780 potently inhibited the LPA-stimulated MAP kinase kinase 1/2 (MKK1/2) activation and EGF receptor tyrosine phosphorylation in HeLa cells, while it had no detectable effect on c-Src kinase activity. PD158780 also inhibited LPA-induced MKK1/2 activation and DNA synthesis in NIH 3T3 cells. Furthermore, we compared LPA-stimulated MKK1/2 and MAP kinase activation, transcriptional activity of the c-fos promoter, and DNA synthesis in B82L cells, which lack endogenous EGF receptor, and B82L cells expressing kinase-defective or wild-type human EGF receptor. Results obtained from analysis of these cell lines suggest that the EGF receptor tyrosine kinase contributes to the LPA-stimulated MAP kinase activation, c-fos transcription, and mitogenesis.