Threshold levels of ABL tyrosine kinase inhibitors retained in chronic myeloid leukemia cells determine their commitment to apoptosis.

The imatinib paradigm in chronic myelogenous leukemia (CML) established continuous BCR-ABL inhibition as a design principle for ABL tyrosine kinase inhibitors (TKI). However, clinical responses seen in patients treated with the ABL TKI dasatinib despite its much shorter plasma half-life and the apparent rapid restoration of BCR-ABL signaling activity following once-daily dosing suggested acute, potent inhibition of kinase activity may be sufficient to irrevocably commit CML cells to apoptosis. To determine the specific requirements for ABL TKI-induced CML cell death for a panel of clinically important ABL TKIs (imatinib, nilotinib, dasatinib, ponatinib, and DCC-2036), we interrogated response of CML cell lines and primary CML cells following acute drug exposure using intracellular fluorescence-activated cell sorting and immunoblot analyses of BCR-ABL signaling, apoptosis measurements, liquid chromatography/tandem mass spectrometry of intracellular drug levels, and biochemical TKI dissociation studies.

The BCR-ABL35INS insertion/truncation mutant is kinase-inactive and does not contribute to tyrosine kinase inhibitor resistance in chronic myeloid leukemia.

Chronic myeloid leukemia is effectively treated with imatinib, but reactivation of BCR-ABL frequently occurs through acquisition of kinase domain mutations. The additional approved ABL tyrosine kinase inhibitors (TKIs) nilotinib and dasatinib, along with investigational TKIs such as ponatinib (AP24534) and DCC-2036, support the possibility that mutation-mediated resistance in chronic myeloid leukemia can be fully controlled; however, the molecular events underlying resistance in patients lacking BCR-ABL point mutations are largely unknown. We previously reported on an insertion/truncation mutant, BCR-ABL(35INS), in which structural integrity of the kinase domain is compromised and all ABL sequence beyond the kinase domain is eliminated.

Appl1 is dispensable for Akt signaling in vivo and mouse T-cell development.

Appl1 (Adaptor protein containing pleckstrin homology [PH], phosphotyrosine binding [PTB], and Leucine zipper motifs) is an adaptor that participates in cell signaling by interacting with various signaling molecules including Akt, PI3-kinase (PI3K), Rab5, adiponectin receptor, and TrkA. By using RNA knockdown technology, Appl1 has been implicated in zebrafish development and murine glucose metabolism. To investigate the unambiguous role of Appl1 in vivo, we generated a knockout mouse in which exon1 of the Appl1 gene was disrupted using gene trap methodology.

Appl1 is dispensable for mouse development, and loss of Appl1 has growth factor-selective effects on Akt signaling in murine embryonic fibroblasts.

The adaptor protein APPL1 (adaptor protein containing pleckstrin homology (PH), phosphotyrosine binding (PTB), and leucine zipper motifs) was first identified as a binding protein of AKT2 by yeast two-hybrid screening. APPL1 was subsequently found to bind to several membrane-bound receptors and was implicated in their signal transduction through AKT and/or MAPK pathways. To determine the unambiguous role of Appl1 in vivo, we generated Appl1 knock-out mice.

Akt and 14-3-3 control a PACS-2 homeostatic switch that integrates membrane traffic with TRAIL-induced apoptosis.

TRAIL selectively kills diseased cells in vivo, spurring interest in this death ligand as a potential therapeutic. However, many cancer cells are resistant to TRAIL, suggesting the mechanism mediating TRAIL-induced apoptosis is complex. Here we identify PACS-2 as an essential TRAIL effector, required for killing tumor cells in vitro and virally infected hepatocytes in vivo.

HIV-1 Nef binds PACS-2 to assemble a multikinase cascade that triggers major histocompatibility complex class I (MHC-I) down-regulation: analysis using short interfering RNA and knock-out mice.

Human immunodeficiency virus, type 1, negative factor (Nef) initiates down-regulation of cell-surface major histocompatibility complex-I (MHC-I) by assembling an Src family kinase (SFK)-ZAP70/Syk-phosphoinositide 3-kinase (PI3K) cascade through the sequential actions of two sites, Nef EEEE(65) and PXXP(75). The internalized MHC-I molecules are then sequestered in endosomal compartments by a process requiring Nef Met(20). How Nef assembles the multikinase cascade to trigger the MHC-I down-regulation pathway is unknown.

Human and mouse mesotheliomas exhibit elevated AKT/PKB activity, which can be targeted pharmacologically to inhibit tumor cell growth.

Malignant mesotheliomas (MMs) are very aggressive tumors that respond poorly to standard chemotherapeutic approaches. The phosphatidylinositol 3-kinase (PI3K)/AKT pathway has been implicated in tumor aggressiveness, in part by mediating cell survival and reducing sensitivity to chemotherapy. Using antibodies recognizing the phosphorylated/activated form of AKT kinases, we observed elevated phospho-AKT staining in 17 of 26 (65%) human MM specimens.

Role of extracellular signal-regulated kinase pathway in RRR-alpha-tocopheryl succinate-induced differentiation of human MDA-MB-435 breast cancer cells.

RRR-alpha-tocopheryl succinate (vitamin E succinate, VES) induces differentiation of human breast cancer cells. Previous studies ruled out transforming growth factor-beta and c-jun N-terminal kinase involvement in VES-induced differentiation but implicated extracellular signal-regulated kinases (ERKs). Here we show that dominant-negative mutants of either mitogen-activated protein kinase kinase (MEK) 1 or ERK1 blocked VES-induced differentiation of MDA-MB-435 cells, as measured by induction of cytokeratin 18 and p21 (Waf1/Cip1) proteins.