pd-166326 and Leukemia--Myelogenous--Chronic--BCR-ABL-Positive

STI571 (imatinib; Gleevec) was developed to specifically target the tyrosine kinase activity of the Bcr-Abl protein in Philadelphia chromosome-positive chronic myeloid leukemia (CML). It also inhibits the activity of c-Kit and PDGFR. It is the first-line drug for newly diagnosed CML, with remarkable efficacy to patients in the chronic phase of this cancer. However, CML patients in the accelerated phase or blast crisis often relapse due to drug resistance. STI571 fails to eradicate leukemic stem cells, and BCR-ABL(+). cells remain detectable in the majority of patients. The necessity for alternative or additional treatment for STI571-resistant leukemia resulted in the development of a second generation of drugs for targeted therapies. In this review a literature overview of the alternative inhibitors which were designed to override STI571 resistance and decrease the aberrant kinase activity of Bcr-Abl protein with higher efficiency is presented.

Topics: Adenosine Triphosphate; Animals; Antineoplastic Agents; Benzamides; Dasatinib; Drug Resistance, Neoplasm; Fusion Proteins, bcr-abl; Humans; Hydroxamic Acids; Imatinib Mesylate; Indoles; Inhibitory Concentration 50; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Mice; Molecular Structure; Mutation; Panobinostat; Piperazines; Protein Kinase Inhibitors; Protein-Tyrosine Kinases; Pyridines; Pyridones; Pyrimidines; Thiazoles

Chronic myelogenous leukemia is characterized by the presence of the chimeric BCR-ABL gene, which is expressed as the constitutively active Bcr-Abl kinase. Although kinase activity is directly responsible for the clinical phenotype, current diagnostic and prognostic methods focus on a genetic classification system in which molecularly distinct subcategories are used to predict patient responses to small-molecule inhibitors of the Bcr-Abl kinase. Point mutations in the kinase domain are a central factor regulating inhibitor resistance; however, compensatory signaling caused by the activation of unrelated kinases can influence inhibitor efficacy. Kinase activity profiling can be used as a complementary approach to genetic screening and allows direct screening of small-molecule inhibitors. We developed a quantitative assay to monitor tyrosine kinase activities and inhibitor sensitivities in a model of chronic myelogenous leukemia using peptide reporters covalently immobilized on Luminex beads. Kinase activity is quantified by nonlinear regression from well-specific internal standard curves. Using optimized synthetic substrates and peptides derived from native substrates as probes, we measured kinase inhibition in cell lysates by the signal transduction inhibitors imatinib and dasatinib. Taking advantage of a convenient 96-well plate format, this assay also allows a straightforward and quantitative analysis of the differential effects of ATP and inhibitors on kinase activity. This method for analyzing a focused signaling network benefits from rigorous statistical analysis and short processing times, thereby offering a powerful tool for drug discovery and clinical testing.

Topics: Antineoplastic Agents; Calibration; Dose-Response Relationship, Drug; Drug Screening Assays, Antitumor; Fusion Proteins, bcr-abl; Humans; K562 Cells; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Microspheres; Models, Biological; Phosphorylation; Protein Kinase Inhibitors; Pyridines; Pyridones; Pyrimidines; Signal Transduction; Small Molecule Libraries; Time Factors

Imatinib is used to treat chronic myelogenous leukemia (CML), but resistance develops in all phases of this disease. The purpose of the present study was to identify the mode of resistance of newly derived imatinib-resistant (IM-R) and PD166326-resistant (PD-R) CML cells. IM-R and PD-R clones exhibited an increase in viability and a decrease in caspase activation in response to various doses of imatinib and PD166326, respectively, as compared with parental K562 cells. Resistance involved neither mutations in BCR-ABL nor increased BCR-ABL, MDR1 or Lyn expression, all known modes of resistance. To gain insight into the resistance mechanisms, we used pangenomic microarrays and identified 281 genes modulated in parental versus IM-R and PD-R cells. The gene signature was similar for IM-R and PD-R cells, accordingly with the cross-sensitivity observed for both inhibitors. These genes were functionally associated with pathways linked to development, cell adhesion, cell growth, and the JAK-STAT cascade. Especially relevant were the increased expression of the tyrosine kinases AXL and Fyn as well as CD44 and HMGA2. Small interfering RNA experiments and pharmacologic approaches identified FYN as a candidate for resistance to imatinib. Our findings provide a comprehensive picture of the transcriptional events associated with imatinib and PD166326 resistance and identify Fyn as a new potential target for therapeutic intervention in CML.

Topics: Antineoplastic Agents; Apoptosis; Benzamides; Caspases; Cell Cycle; Cell Proliferation; Drug Resistance, Neoplasm; Flow Cytometry; Fusion Proteins, bcr-abl; Gene Expression Profiling; Humans; Imatinib Mesylate; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Microarray Analysis; Piperazines; Protein-Tyrosine Kinases; Proto-Oncogene Proteins c-fyn; Pyridines; Pyrimidines; RNA, Small Interfering; Tumor Cells, Cultured

Imatinib targets the Bcr-Abl oncogene that causes chronic myelogenous leukemia (CML) in humans. Recently, we demonstrated that besides triggering apoptosis in K562 cells, imatinib also mediated their erythroid differentiation. Although both events appear to proceed concomitantly, it is not known at present whether or not imatinib-induced apoptosis and differentiation are interdependent processes. Hence, we investigated the requirements for Bcr-Abl inhibitor-mediated apoptosis and erythroid differentiation in several established and engineered CML cell lines. Imatinib triggered apoptosis and erythroid differentiation of different CML cell lines, but only apoptosis exhibited sensitivity to ZVAD-fmk inhibition. Conversely, the p38 mitogen-activated protein (MAP) kinase inhibitor, SB202190, significantly slowed down erythroid differentiation without affecting caspase activation. Furthermore, imatinib and PD166326, another Bcr-Abl inhibitory molecule, triggered erythroid differentiation of K562 cell clones, nevertheless resistant to Bcr-Abl inhibitor-induced apoptosis. Finally, short hairpin RNA inhibitor (shRNAi) silencing of caspase 3 efficiently inhibited caspase activity but had no effect on erythroid differentiation, whereas silencing of Bcr-Abl mimicked imatinib or PD166326 treatment, leading to increased apoptosis and erythroid differentiation of K562 cells. Taken together, our findings not only demonstrate that Bcr-Abl inhibitor-mediated apoptosis and differentiation are fully distinguishable events, but also that caspases are dispensable for erythroid differentiation of established CML cell lines.

Topics: Apoptosis; Benzamides; Caspase Inhibitors; Caspases; Cell Differentiation; Cell Line, Tumor; Enzyme Activation; Enzyme Inhibitors; Erythroid Cells; Fusion Proteins, bcr-abl; Humans; Imatinib Mesylate; K562 Cells; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Piperazines; Protein-Tyrosine Kinases; Pyridines; Pyrimidines

In Philadelphia-positive (Ph(+)) leukemia, point mutations within the Bcr-Abl kinase domain emerged as a major mechanism of resistance to imatinib mesylate. We established a cell-based screening strategy for detection of clinically relevant point mutations using Bcr-Abl-transformed Ba/F3 cells. We identified 32 different single-point mutations within the kinase domain of Bcr-Abl. The pattern and frequency of mutations in this cell culture-based screen resembled the pattern and frequency observed in resistant patients. We then applied this screen to an alternative Abl kinase inhibitor. Using PD166326, the frequency of resistant colonies emerging at 5 to 10 times the median growth inhibition (IC50) of PD166326 was significantly lower than with imatinib. In addition, PD166326 produced a distinct pattern of Bcr-Abl mutations. The majority of mutations that came up with both imatinib and PD166326 could effectively be suppressed by increasing the dose of PD166326 to 50 to 500 nM. In contrast, only a few mutations could be suppressed by increasing the imatinib dose to 5 to 10 microM. However, 3 mutations affecting F317 displayed complete resistance to PD166326, but could be effectively inhibited by standard concentrations of imatinib. Thus, this robust and simple screening system provides a rational basis for combinatorial and sequential treatment strategies in targeted cancer therapy.

Topics: Animals; Benzamides; Cell Culture Techniques; Cell Line; Cell Line, Transformed; Cells, Cultured; Drug Resistance, Neoplasm; Fusion Proteins, bcr-abl; Genes, abl; Humans; Imatinib Mesylate; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Mice; Piperazines; Point Mutation; Protein Kinase Inhibitors; Protein Structure, Tertiary; Protein-Tyrosine Kinases; Pyridines; Pyrimidines; Transfection

Imatinib mesylate is highly effective in newly diagnosed chronic myeloid leukemia (CML), but BCR/ABL (breakpoint cluster region/abelson murine leukemia)-positive progenitors persist in most patients with CML treated with imatinib mesylate, indicating the need for novel therapeutic approaches. In this study, we have used the murine CML-like myeloproliferative disorder as a platform to characterize the pharmacokinetic, signal transduction, and antileukemic properties of PD166326, one of the most potent members of the pyridopyrimidine class of protein tyrosine kinase inhibitors. In mice with the CML-like disease, PD166326 rapidly inhibited Bcr/Abl kinase activity after a single oral dose and demonstrated marked antileukemic activity in vivo. Seventy percent of PD166326-treated mice achieved a white blood cell (WBC) count less than 20.0 x 10(9)/L (20,000/microL) at necropsy, compared with only 8% of imatinib mesylate-treated animals. Further, two thirds of PD166326-treated animals had complete resolution of splenomegaly, compared with none of the imatinib mesylate-treated animals. Consistent with its more potent antileukemic effect in vivo, PD166326 was also superior to imatinib mesylate in inhibiting the constitutive tyrosine phosphorylation of numerous leukemia-cell proteins, including the src family member Lyn. PD166326 also prolonged the survival of mice with imatinib mesylate-resistant CML induced by the Bcr/Abl mutants P210/H396P and P210/M351T. Altogether, these findings demonstrate the potential of more potent Bcr/Abl inhibitors to provide more effective antileukemic activity. Clinical development of PD166326 or a related analog may lead to more effective drugs for the treatment of de novo and imatinib mesylate-resistant CML.

Topics: Animals; Antineoplastic Agents; Benzamides; Cell Line; Cell Proliferation; Disease Models, Animal; Drug Resistance, Neoplasm; Fusion Proteins, bcr-abl; Imatinib Mesylate; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Mice; Molecular Structure; Mutation; Phosphorylation; Phosphotyrosine; Piperazines; Protein Kinase Inhibitors; Pyridines; Pyrimidines; Signal Transduction; Stem Cell Factor; Survival Rate; Time Factors

Topics: Benzamides; Fusion Proteins, bcr-abl; Humans; Imatinib Mesylate; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Piperazines; Pyridines; Pyrimidines

Imatinib mesylate (STI571, Glivec), a 2-phenylaminopyrimidine small-molecule ATP competitor-type kinase inhibitor, proved to be active in Philadelphia-positive leukemias. Resistance toward imatinib develops frequently in advanced-stage Philadelphia-positive leukemia, and is even observed in chronic-phase chronic myelogenous leukemia. Point mutations within the BCR-ABL kinase domain emerged as a major mechanism of resistance toward imatinib. Mutations occur at positions that determine specific contacts of imatinib to the ATP-binding site. We aimed to examine whether pyrido-pyrimidine-type kinase inhibitors were capable of inhibiting both wild-type and mutant forms of BCR-ABL. We screened 13 different pyrido-pyrimidine with cells expressing wild-type and mutant BCR-ABL. All of the substances specifically suppressed the Bcr-Abl dependent phenotype and inhibited Bcr-Abl kinase activity with higher potency than imatinib. Two of the most active compounds were PD166326 and SKI DV-M016. Interestingly, these compounds suppressed the activation loop mutant Bcr-Abl H396P as effectively as wild-type Bcr-Abl. In addition, nucleotide-binding loop mutations (Y253H, E255K, and E255V) were selectively and potently inhibited. In contrast, T315I, a mutant located at a position that makes a direct contact with imatinib, was not affected. This observation is consistent with the hypothesis that unlike imatinib, pyrido-pyrimidine inhibitors bind Bcr-Abl regardless of the conformation of the activation loop. We conclude that pyrido-pyrimidine-type kinase inhibitors are active against different frequently observed kinase domain mutations of BCR-ABL that cause resistance toward imatinib. Resistance as a consequence of selection of mutant BCR-ABL by imatinib may be overcome using second-generation kinase inhibitors because of their higher potency and their ability to bind Bcr-Abl irrespective of the conformation of the activation loop.

Topics: Animals; Apoptosis; Benzamides; Cell Division; Cell Line, Transformed; Enzyme Inhibitors; Fusion Proteins, bcr-abl; Humans; Imatinib Mesylate; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Mice; Piperazines; Point Mutation; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Protein Kinase Inhibitors; Pyridines; Pyrimidines