ABSTRACT
The dysregulated tyrosine kinase BCR-ABL causes chronic myelogenous leukemia in humans and forms a large multiprotein complex that includes the Src-homology 2 (SH2) domain-containing phosphatase 2 (SHP2). The expression of SHP2 is necessary for BCR-ABL-dependent oncogenic transformation, but the precise signaling mechanisms of SHP2 are not well understood. We have developed binding proteins, termed monobodies, for the N- and C-terminal SH2 domains of SHP2. Intracellular expression followed by interactome analysis showed that the monobodies are essentially monospecific to SHP2. Two crystal structures revealed that the monobodies occupy the phosphopeptide-binding sites of the SH2 domains and thus can serve as competitors of SH2-phosphotyrosine interactions. Surprisingly, the segments of both monobodies that bind to the peptide-binding grooves run in the opposite direction to that of canonical phosphotyrosine peptides, which may contribute to their exquisite specificity. When expressed in cells, monobodies targeting the N-SH2 domain disrupted the interaction of SHP2 with its upstream activator, the Grb2-associated binder 2 adaptor protein, suggesting decoupling of SHP2 from the BCR-ABL protein complex. Inhibition of either N-SH2 or C-SH2 was sufficient to inhibit two tyrosine phosphorylation events that are critical for SHP2 catalytic activity and to block ERK activation. In contrast, targeting the N-SH2 or C-SH2 revealed distinct roles of the two SH2 domains in downstream signaling, such as the phosphorylation of paxillin and signal transducer and activator of transcription 5. Our results delineate a hierarchy of function for the SH2 domains of SHP2 and validate monobodies as potent and specific antagonists of protein-protein interactions in cancer cells.
Subject(s)
Fusion Proteins, bcr-abl/antagonists & inhibitors , Protein Tyrosine Phosphatase, Non-Receptor Type 11/antagonists & inhibitors , Adaptor Proteins, Signal Transducing/metabolism , Amino Acid Sequence , Cell Transformation, Neoplastic , Crystallography, X-Ray , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Fusion Proteins, bcr-abl/chemistry , Fusion Proteins, bcr-abl/genetics , HEK293 Cells , Humans , K562 Cells , Models, Molecular , Peptide Library , Peptides/chemistry , Peptides/genetics , Peptides/pharmacology , Protein Binding , Protein Interaction Domains and Motifs , Protein Tyrosine Phosphatase, Non-Receptor Type 11/chemistry , Protein Tyrosine Phosphatase, Non-Receptor Type 11/genetics , Signal Transduction , src Homology DomainsABSTRACT
In this article, we are reviewing the molecular mechanisms that lead to kinase inhibitor resistance. As the oncogenic BCR-ABL kinase is the target of the first approved small-molecule kinase inhibitor imatinib, we will first focus on the structural and mechanistic basis for imatinib resistance. We will then show ways how next generations of BCR-ABL inhibitors and alternative targeting strategies have helped to offer effective treatment options for imatinib-resistant patients. Based on these insights, we discuss commonalities and further mechanisms that lead to resistance to other kinase inhibitors in solid tumors. This article is part of a Special Issue entitled: Inhibitors of Protein Kinases (2012).
Subject(s)
Drug Resistance, Neoplasm/drug effects , Fusion Proteins, bcr-abl/antagonists & inhibitors , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/drug therapy , Protein Kinase Inhibitors/therapeutic use , Benzamides/chemistry , Benzamides/pharmacology , Benzamides/therapeutic use , Drug Resistance, Neoplasm/genetics , Fusion Proteins, bcr-abl/chemistry , Fusion Proteins, bcr-abl/genetics , Humans , Imatinib Mesylate , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/genetics , Models, Molecular , Molecular Structure , Piperazines/chemistry , Piperazines/pharmacology , Piperazines/therapeutic use , Protein Binding , Protein Kinase Inhibitors/chemistry , Protein Kinase Inhibitors/pharmacology , Protein Structure, Tertiary , Pyrimidines/chemistry , Pyrimidines/pharmacology , Pyrimidines/therapeutic useABSTRACT
Multidrug resistance remains a significant obstacle to successful chemotherapy. The ability to determine the possible resistance mechanisms and surmount the resistance is likely to improve chemotherapy. Nilotinib is a very effective drug in the treatment of imatinib-sensitive or -resistant patients. Although very successful hematologic and cytogenetic responses have been obtained in nilotinib-treated patients, in recent years cases showing resistance to nilotinib have been observed. We aimed to examine the mechanisms underlying nilotinib resistance and to provide new targets for the treatment of chronic myeloid leukemia (CML). There was an up-regulation of antiapoptotic BCR/ABL, GCS and SK-1 genes and MRP1 transporter gene and down-regulation of apoptotic Bax and CerS1 genes in nilotinib-resistant cells. There was no mutation in the nilotinib-binding region of BCR/ABL in resistant cells. Inhibiton of GCS and SK-1 restored nilotinib sensitivity. Targeting the proteins that are involved in nilotinib resistance in addition to the inhibition of BCR/ABL could be a better method of treatment in CML.
Subject(s)
Antineoplastic Agents/pharmacology , Drug Resistance, Neoplasm , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/metabolism , Protein Kinase Inhibitors/pharmacology , Pyrimidines/pharmacology , Antineoplastic Agents/metabolism , Apoptosis/drug effects , Apoptosis/genetics , Base Sequence , Binding Sites , Caspase 3/metabolism , Cell Line, Tumor , Ceramides/metabolism , Fusion Proteins, bcr-abl/chemistry , Fusion Proteins, bcr-abl/genetics , Fusion Proteins, bcr-abl/metabolism , Gene Expression Regulation, Leukemic , Humans , K562 Cells , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/genetics , Mitochondria/drug effects , Mitochondria/metabolism , Molecular Sequence Data , Protein Binding , Protein Interaction Domains and Motifs , Protein Kinase Inhibitors/metabolism , Pyrimidines/metabolismABSTRACT
In this study, we aimed to increase the sensitivity of human K562 and Meg-01 chronic myeloid leukemia (CML) cells to nilotinib by targeting bioactive sphingolipids, in addition to investigating the roles of ceramide metabolizing genes in nilotinib induced apoptosis. Cytotoxic effects of nilotinib, C8:ceramide, glucosyle ceramide synthase (GCS) and sphingosine kinase-1 (SK-1) inhibitors were determined by XTT cell proliferation assay and synergism between the agents was determined by isobologram analysis. Also, quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) results demonstrated that expression levels of longevity assurance (LASS) genes in response to nilotinib were correlated with sensitivity to nilotinib. For the first time, The results of this study showed for the first time that nilotinib induces apoptosis through upregulating ceramide synthase genes and downregulating SK-1 in CML cells in addition to inhibition of BCR/ABL. On the other hand, manipulating bioactive sphingolipids toward generation/accumulation of ceramides increased the apoptotic effects of nilotinib in CML cells.
