pd-184352 and Leukemia--Myelogenous--Chronic--BCR-ABL-Positive

Interactions between MEK1/2 inhibitors and the dual Abl/Src kinase inhibitor dasatinib (BMS-354825) were examined in chronic myeloid leukemia (CML) cell lines and primary specimens. Cotreatment of K562 or LAMA cells with subtoxic or marginally toxic concentrations of PD184352 (or U0126) and dasatinib synergistically potentiated mitochondrial damage, caspase activation, and apoptosis. Similar interactions were observed in CD34(+) cells from one CML patient-derived but not in a normal human CD34(+) bone marrow cell specimen. These interactions were associated with multiple perturbations in survival signaling pathways, including inactivation of Bcr/Abl, STAT5, and ERK1/2; down-regulation of Bcl-x(L) and Mcl-1; and dephosphorylation/activation of Bim. They were also associated with BAX/BAK conformational change, mitochondrial dysfunction, and caspase activation. Bim knockdown by shRNA suppressed BAX and BAK conformational change and protected cells from dasatinib/PD184352 lethality. Conversely, K562 cells ectopically expressing Mcl-1 or Bcl-x(L) were significantly less susceptible to dasatinib/PD184352 toxicity. Notably, the dasatinib/PD184352 regimen was active against leukemic cells exhibiting various forms of imatinib mesylate resistance, including Bcr/Abl overexpression, Lyn activation, and several Bcr/Abl kinase domain mutations (eg, E255K, M351T), but not T315I. Together, these findings suggest that strategies combining dasatanib with MEK1/2 inhibitors warrant further investigation in Bcr/Abl(+) malignancies, particularly in the setting of imatinib mesylate-resistant disease.

Topics: Benzamides; Butadienes; Dasatinib; Drug Resistance, Neoplasm; Drug Screening Assays, Antitumor; Drug Synergism; Fusion Proteins, bcr-abl; Gene Expression Regulation, Leukemic; Humans; Imatinib Mesylate; K562 Cells; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; MAP Kinase Kinase 1; MAP Kinase Kinase 2; Nitriles; Piperazines; Protein Kinase Inhibitors; Pyrimidines; src-Family Kinases; Thiazoles

The cytotoxic farnesyl transferase inhibitor BMS-214662 has been shown to potently induce mitochondrial apoptosis in primitive CD34+ chronic myeloid leukaemia (CML) stem/progenitor cells. Here, to enhance the BMS-214662 apoptotic effect, we further targeted the extracellular signal-regulated kinase (ERK) pathway, downstream of BCR-ABL, by treating CD34+ CML stem/progenitor cells with a highly selective adenosine triphosphate (ATP) non-competitive MEK inhibitor, PD184352. PD184352 increased the apoptotic effect of BMS-214662 in a CML blast crisis cell line, K562, and in primary chronic phase CD34+ CML cells. Compared with BMS-214662, after combination treatment we observed inhibition of ERK phosphorylation, increased Annexin-V levels, caspase-3, -8 and -9 activation and potentiated mitochondrial damage, associated with decreased levels of anti-apoptotic BCL-2 family protein MCL-1. Inhibition of K-RAS function by a dominant-negative mutant resulted in CML cell death and this process was further enhanced by the addition of BMS-214662 and PD184352. Together, these findings suggest that the addition of a MEK inhibitor improves the ability of BMS-214662 to selectively target CML stem/progenitor cells, notoriously insensitive to tyrosine kinase inhibitor treatment and presumed to be responsible for the persistence and relapse of the disease.

Topics: Antigens, CD34; Apoptosis; Benzamides; Benzodiazepines; Blast Crisis; Drug Screening Assays, Antitumor; Drug Synergism; Enzyme Inhibitors; Farnesyltranstransferase; Genes, Dominant; Genes, ras; Hematopoietic Stem Cells; Humans; Imidazoles; K562 Cells; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Leukemia, Myeloid, Chronic-Phase; MAP Kinase Kinase 1; MAP Kinase Kinase Kinases; Neoplasm Proteins; Neoplastic Stem Cells; Oncogene Protein p21(ras); Recombinant Fusion Proteins; Tumor Cells, Cultured

Although the BCR-ABL tyrosine kinase inhibitor Imatinib has undoubtedly revolutionized the therapy of chronic myeloid leukaemia (CML), acquired drug resistance remains a common problem in CML therapy. Resistance often arises from second-line mutations in BCR-ABL or overexpression of the BCR-ABL protein but in ~20% of CML cases resistance mechanisms do not involve altered BCR-ABL function. Imatinib-resistant CML cell lines have been widely used for comparative proteome/genome-wide expression screens in order to decipher resistance mechanisms but a clearcut molecular mechanism or molecular player in BCR-ABL-independent resistance to Imatinib has not yet evolved from those studies. Here, we report the identification of a novel mechanism for Imatinib resistance in CML cells with unaltered BCR-ABL function. Pharmacological analysis evidenced a constitutive, Imatinib-insensitive activation of the Erk-MAPK pathway in resistant cells. A systematic analysis of pathway constituents illustrated that Ras-GTP accumulation remained fully sensitive to Imatinib but c-Raf activity from serum-fed cultures was largely resistant to the drug's action. Sequencing excluded mutations in either B-Raf or c-Raf as the origin of resistance, indicating that a functional alteration in the regulation of c-Raf activity was responsible for this effect. Collectively, these findings highlight a novel mechanism of acquired Imatinib resistance based on the BCR-ABL and Ras-independent constitutive activation of the Erk-MAPK pathway through activated c-Raf, which could prove helpful for a better functional classification of the causes of Imatinib resistance in CML.

Topics: Benzamides; Cell Growth Processes; Cell Line, Tumor; Clone Cells; Drug Resistance, Neoplasm; Enzyme Activation; Extracellular Signal-Regulated MAP Kinases; Fusion Proteins, bcr-abl; Humans; Imatinib Mesylate; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; MAP Kinase Kinase Kinases; Piperazines; Protein Kinase Inhibitors; Pyrimidines; raf Kinases; ras Proteins

We show that imatinib, nilotinib, and dasatinib possess weak off-target activity against RAF and, therefore, drive paradoxical activation of BRAF and CRAF in a RAS-dependent manner. Critically, because RAS is activated by BCR-ABL, in drug-resistant chronic myeloid leukemia (CML) cells, RAS activity persists in the presence of these drugs, driving paradoxical activation of BRAF, CRAF, MEK, and ERK, and leading to an unexpected dependency on the pathway. Consequently, nilotinib synergizes with MEK inhibitors to kill drug-resistant CML cells and block tumor growth in mice. Thus, we show that imatinib, nilotinib, and dasatinib drive paradoxical RAF/MEK/ERK pathway activation and have uncovered a synthetic lethal interaction that can be used to kill drug-resistant CML cells in vitro and in vivo.

Topics: Amino Acid Substitution; Animals; Antineoplastic Agents; Apoptosis; Benzamides; Cell Line, Tumor; Dasatinib; Drug Resistance, Neoplasm; Drug Synergism; Enzyme Activation; Extracellular Signal-Regulated MAP Kinases; Fusion Proteins, bcr-abl; Genes, ras; Humans; Imatinib Mesylate; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; MAP Kinase Kinase Kinases; MAP Kinase Signaling System; Mice; Mice, Nude; Piperazines; Proto-Oncogene Proteins B-raf; Proto-Oncogene Proteins c-raf; Pyrimidines; raf Kinases; Thiazoles; Tumor Cells, Cultured; Xenograft Model Antitumor Assays

Deregulated activation of protein tyrosine kinases, such as the epidermal growth factor receptor (EGFR) and Abl, is associated with human cancers including non-small cell lung cancer (NSCLC) and chronic myeloid leukemia (CML). Although inhibitors of such activated kinases have proved to be of therapeutic benefit in individuals with NSCLC or CML, some patients manifest intrinsic or acquired resistance to these drugs. We now show that, whereas blockade of either the extracellular signal-regulated kinase (ERK) pathway or the phosphatidylinositol 3-kinase (PI3K)-Akt pathway alone induced only a low level of cell death, it markedly sensitized NSCLC or CML cells to the induction of apoptosis by histone deacetylase (HDAC) inhibitors. Such enhanced cell death induced by the respective drug combinations was apparent even in NSCLC or CML cells exhibiting resistance to EGFR or Abl tyrosine kinase inhibitors, respectively. Co-administration of a cytostatic signaling pathway inhibitor may contribute to the development of safer anticancer strategies by lowering the required dose of cytotoxic HDAC inhibitors for a variety of cancers.

Topics: Analgesics; Benzamides; Carcinoma, Non-Small-Cell Lung; Drug Resistance, Neoplasm; ErbB Receptors; Extracellular Signal-Regulated MAP Kinases; Gefitinib; Histone Deacetylase Inhibitors; Humans; Imatinib Mesylate; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Lung Neoplasms; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Piperazines; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Pyrimidines; Quinazolines

Interactions between the histone deacetylase inhibitor SAHA and the pharmacologic MEK1/2 inhibitor PD184352 were examined in Bcr/Abl+ human leukemia cells. Coadministration of minimally toxic concentrations of SAHA (or sodium butyrate) and PD184352 (or U0126) resulted in a synergistic increase in mitochondrial damage, caspase activation, and apoptosis in K562 and LAMA 84 cells. Similar interactions were observed in CD34+ cells from two patients with CML and in imatinib mesylate-resistant K562 cells but not in normal human CD34+ bone marrow cells. These events were associated with a marked increase in ROS generation, inactivation of ERK and Akt, downregulation of p21CIP1, Bcr/Abl, and cyclin D1, and activation of JNK. Of these events, ROS generation, ERK inactivation, and cytochrome c/AIF release were largely caspase-independent, whereas the other phenomena displayed varying degrees of caspase-dependence. Using pharmacologic and genetic approaches, generation of ROS, p21CIP1 downregulation, and inactivation of Akt and MEK were found to play significant functional roles in SAHA/PD184352-mediated lethality, whereas JNK activation and Raf-1 downregulation were determined to represent secondary events. These findings indicate that interruption of the MEK/ERK pathway substantially lowers the threshold for HDAC inhibitor-mediated oxidative injury, mitochondrial dysfunction, and apoptosis, suggesting that this approach warrants further examination in Bcr/Abl+-related malignancies.

Topics: Antigens, CD34; Apoptosis; Benzamides; Bone Marrow Cells; Butadienes; Butyrates; Caspases; Cell Line, Tumor; Dose-Response Relationship, Drug; Drug Synergism; Enzyme Inhibitors; Fusion Proteins, bcr-abl; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; K562 Cells; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Mitochondria; Mitogen-Activated Protein Kinase Kinases; Nitriles; Vorinostat