piperidines and Leukemia--Myelogenous--Chronic--BCR-ABL-Positive

The tyrosine kinase inhibitors (TKIs) have revolutionized the management of chronic myeloid leukemia (CML) and BCR-ABL1 inhibitors form the mainstay of CML treatment. Although patients with CML generally do well under TKI therapy, there is a subgroup of patients who are resistant and/or intolerant to TKIs. In these group of patients, there is the need of additional treatment strategies. In this review, we provide an update on the current knowledge of these novel treatment approaches that can be used alone and/or in combination with TKIs.

Topics: Antineoplastic Agents; Clinical Trials as Topic; Drug Resistance, Neoplasm; Everolimus; Fusion Proteins, bcr-abl; Gene Expression; Histone Deacetylase Inhibitors; Homoharringtonine; Humans; Immunotherapy; Interferon-alpha; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Molecular Targeted Therapy; Niacinamide; Piperidines; Polyethylene Glycols; Protein Kinase Inhibitors; Pyrazoles; Pyridines; Quinolones; Recombinant Proteins

Topics: Animals; Benzamides; Chromosome Aberrations; Drug Resistance, Neoplasm; Fusion Proteins, bcr-abl; Hematopoietic Stem Cell Transplantation; Humans; Imatinib Mesylate; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Piperazines; Piperidines; Pyridines; Pyrimidines; Quinolones; Randomized Controlled Trials as Topic; Thionucleotides

Lonafarnib is an orally bioavailable nonpetidomimetic farnesyl transferase inhibitor with significant activity against BCR-ABL-positive cell lines and primary human chronic myeloid leukemia (CML) cells. Lonafarnib can inhibit the proliferation of imatinib-resistant cells and increases imatinib-induced apoptosis in vitro in cells from imatinib-resistant patients.. The authors conducted a phase 1 study of lonafarnib in combination with imatinib in patients with CML who failed imatinib therapy. The starting dose level for patients with chronic phase (CP) disease was imatinib, 400 mg/day, plus lonafarnib at a dose of 100 mg twice daily. The starting dose levels for accelerated phase (AP) and blast phase (BP) disease were 600 mg/day and 100 mg twice daily, respectively.. A total of 23 patients were treated (9 with CP, 11 with AP, and 3 with BP) for a median of 25 weeks (range, 4-102 weeks). Of those with CP disease, 2 patients had grade 3 (according to the National Cancer Institute Common Toxicity Criteria [version 2.0]) dose-limiting toxicities (DLTs) at the 400 + 125-mg dose, including diarrhea (2 patients), vomiting (1 patient), and fatigue (1 patient). In patients with AP/BP disease, DLTs were observed at the 600 + 125-mg dose and was comprised of diarrhea (1 patient) and hypokalemia (1 patient). Eight patients (35%) responded; 3 with CP disease achieved a complete hematologic response (CHR) (2 patients) and a complete cytogenetic response (1 patient). Three patients with AP disease responded (2 CHR, 1 partial cytogenetic response), and 2 patients with BP disease demonstrated hematologic improvement. Pharmacokinetics data suggest no apparent increase in exposure or changes in the pharmacokinetics of either lonafarnib or imatinib when they are coadministered.. The results of the current study indicate that the combination of lonafarnib and imatinib is well tolerated and the maximum tolerated dose of lonafarnib is 100 mg twice daily when combined with imatinib at a dose of either 400 mg or 600 mg daily.

Topics: Adult; Aged; Antineoplastic Combined Chemotherapy Protocols; Benzamides; Diarrhea; Dose-Response Relationship, Drug; Drug Administration Schedule; Fatigue; Female; Humans; Imatinib Mesylate; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Male; Middle Aged; Piperazines; Piperidines; Pyridines; Pyrimidines; Treatment Failure; Treatment Outcome; Vomiting

Lonafarnib (SCH66336) is a nonpeptidomimetic farnesyl transferase inhibitor that has demonstrated significant preclinical activity against chronic myelogenous leukemia (CML) cells and in CML animal models.. In the current study, the efficacy of lonafarnib was investigated in patients with CML in the chronic or accelerated phase that was resistant or intolerant to imatinib. Thirteen patients with CML in the chronic (n = 6 patients) or accelerated (n = 7 patients) phase were treated with lonafarnib at a dose of 200 mg orally twice daily. Ten patients had failed therapy with imatinib and 3 patients were intolerant to imatinib. The median age of the patients was 62 years (range, 38-80 yrs) and the median time from the diagnosis of CML to therapy with lonafarnib was 5 years (range, 0.3-13 yrs). In addition to imatinib mesylate, all patients had received prior therapy with interferon-alpha and seven patients had received other treatments. The median duration of therapy with lonafarnib was 8 weeks (range, 2-41 wks).. Two patients responded. One patient in the accelerated phase of CML returned to the chronic phase, a response that lasted for 3 months. Another patient with chronic phase disease had lowering of the leukocyte count without the need for hydroxyurea and normalization of the differential count that lasted for 5 months. The most common adverse event was diarrhea, which was noted in 11 patients (84%) (Grade > or = 3 in 4 patients; 31%; toxicity was graded according to the National Cancer Institute Common Toxicity Criteria [version 2.0]). Therapy was discontinued in one patient because of diarrhea not responding to dose adjustments.. Single-agent lonafarnib appears to have clinical activity in a small proportion of patients with CML refractory to imatinib.

Topics: Adult; Aged; Aged, 80 and over; Antineoplastic Agents; Benzamides; Drug Resistance, Neoplasm; Farnesyltranstransferase; Female; Humans; Imatinib Mesylate; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Male; Middle Aged; Pilot Projects; Piperazines; Piperidines; Pyridines; Pyrimidines

The oncogenic role of ephrin type-B receptor 4 (EPHB4) has been reported in many types of tumors, including chronic myeloid leukemia (CML). Here, we found that CML patients have a higher EPHB4 expression level than healthy subjects. EPHB4 knockdown inhibited growth of K562 cells (a human immortalized myelogenous leukemia cell line). In addition, transient transfection of EPHB4 siRNA led to sensitization to imatinib. These growth defects could be fully rescued by EPHB4 transfection. To identify an EPHB4-specific inhibitor with the potential of rapid translation into the clinic, a pool of clinical compounds was screened and vandetanib was found to be most sensitive to K562 cells, which express a high level of EPHB4. Vandetanib mainly acts on the intracellular tyrosine kinase domain and interacts stably with a hydrophobic pocket. Furthermore, vandetanib downregulated EPHB4 protein via the ubiquitin-proteasome pathway and inhibited PI3K/AKT and MAPK/ERK signaling pathways in K562 cells. Vandetanib alone significantly inhibited tumor growth in a K562 xenograft model. Furthermore, the combination of vandetanib and imatinib exhibited enhanced and synergistic growth inhibition against imatinib-resistant K562 cells in vitro and in vivo. These findings suggest that vandetanib drives growth arrest and overcomes the resistance to imatinib in CML via targeting EPHB4.

Topics: Antineoplastic Agents; Apoptosis; Drug Resistance, Neoplasm; Ephrins; Humans; Imatinib Mesylate; K562 Cells; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Phosphatidylinositol 3-Kinases; Piperidines; Quinazolines

Multidrug resistance (MDR) is the main challenge in the treatment of chronic myeloid leukemia (CML), and P-glycoprotein (P-gp) overexpression is an important mechanism involved in this resistance process. However, some compounds can selectively affect MDR cells, inducing collateral sensitivity (CS), which may be dependent on P-gp. The aim of this study was to investigate the effect of piperine, a phytochemical from black pepper, on CS induction in CML MDR cells, and the mechanisms involved. The results indicate that piperine induced CS, being more cytotoxic to K562-derived MDR cells (Lucena-1 and FEPS) than to K562, the parental CML cell. CS was confirmed by analysis of cell metabolic activity and viability, cell morphology and apoptosis. P-gp was partially required for CS induction. To investigate a P-gp independent mechanism, we analyzed the possibility that poly (ADP-ribose) polymerase-1 (PARP-1) could be involved in piperine cytotoxic effects. It was previously shown that only MDR FEPS cells present a high level of 24 kDa fragment of PARP-1, which could protect these cells against cell death. In the present study, piperine was able to decrease the 24 kDa fragment of PARP-1 in MDR FEPS cells. We conclude that piperine targets selectively MDR cells, inducing CS, through a mechanism that might be dependent or not on P-gp.

Topics: Alkaloids; Apoptosis; ATP Binding Cassette Transporter, Subfamily B; Benzodioxoles; Cell Survival; Cytochrome P-450 Enzyme Inhibitors; Drug Resistance, Multiple; Drug Resistance, Neoplasm; Humans; K562 Cells; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Piperidines; Polyunsaturated Alkamides

Our group designed and synthesized the N-phenyl-piperazine LQFM030 [1-(4-((1-(4-chlorophenyl)-1H-pyrazol-4-yl)methyl) piperazin-1-yl) ethanone], a small molecule derived from molecular simplification of the Nutlin-1, an inhibitor of the human homologue of murine double minute 2 (MDM2) protein that is expressed in several types of cancer. To better investigate the effects of LQFM030 regarding the p53 mutation status, this study investigated the antiproliferative activity of LQFM030 against the p53-null K562 leukemia cells as well as the cell death pathways involved. In addition, the effects of LQFM030 on the levels of the p53/MDM2 complex were also carried out using 3T3 cells as a p53 wild-type model. Our data suggest that LQFM030 triggered apoptosis in K562 cells via different mechanisms including cell cycle arrest, caspase activation, reduction of mitochondrial activity, decrease in MDM2 expression, and transcriptional modulation of MDMX, p73, MYC, and NF-ĸB. Additionally, it promoted effects in p53/MDM2 binding in p53 wild-type 3T3 cells. Therefore, LQFM030 has antiproliferative effects in cancer cells by a p53 mutation status-independent manner with different signaling pathways. These findings open new perspectives to the treatment of leukemic cells considering the resistance development associated with cancer treatment with conventional cytotoxic drugs.

Topics: Animals; Antineoplastic Agents; Apoptosis; Apoptosis Regulatory Proteins; BALB 3T3 Cells; Enzyme Inhibitors; Humans; K562 Cells; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Mice; Mutation; Piperidines; Proto-Oncogene Proteins c-mdm2; Pyrazoles; Signal Transduction; Tumor Suppressor Protein p53

Neurokinin-1 receptor (NK1R) antagonists are known for their anxiolytic, antiemetic, anticancer, and anti-inflammatory effects. Aprepitant is used in vomiting and nausea, which are the most common side-effects of patients undergoing chemotherapy for cancer. L-733,060 has been shown to have anxiolytic and antidepressant effects in animal studies and anticancer effect in in-vitro studies. Previous anticancer activity studies with NK1R antagonists have reported that NK-1 antagonists have an antitumoral activity on gastric carcinoma, larynx carcinoma, retinoblastoma, hepatocarcinoma, glioma, neuroblastoma, and osteoblastoma cells. In this study, we have aimed to show and compare the antileukemic effects of aprepitant and L-733,060 on acute and chronic myeloid leukemic cells by using in-vitro experiments, such as WST-1, cell imaging, annexin-V binding, soft agar colony formation, and Hoescht staining. As a result, we have determined that both aprepitant and L-733,060 had strong antiproliferative effects on K562 and HL-60 cells. Moreover, the two drugs caused significant apoptosis and decreased colony forming depending on concentration increase. These findings suggested that NK1R antagonists exhibited antileukemic activities and may be considered to have a novel therapeutic potential for acute and chronic myeloid leukemia.

Topics: Antineoplastic Agents; Apoptosis; Aprepitant; Cell Proliferation; Drug Therapy, Combination; Humans; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Leukemia, Myeloid, Acute; Neurokinin-1 Receptor Antagonists; Piperidines; Tumor Cells, Cultured

Imatinib (IMA) is the standard treatment for CML; however, stopping IMA sometimes results in disease relapse, which suggests that leukemic stem cells (LSCs) remain in such patients, even after complete molecular remission has been achieved. Therefore, new strategies will be required to eradicate LSCs. The Janus kinase-signal transducer and activator of transcription (JAK-STAT) pathway is part of the BCR-ABL signaling network, and it is activated in CML, especially in LSCs. JAK2 is known to be associated with CML survival, but the role of JAK3 in CML remains unknown. The antitumor effects of IMA and a JAK3 inhibitor, tofacitinib were examined using the MTT assay in K562 and KCL22. To investigate the mechanisms of action of IMA and the JAK inhibitors in CML cells, we examined apoptosis, the cell cycle, and JAK-STAT signaling using flow cytometry, immunofluorescent microscopy, and Western blotting. The pharmacological inhibition of JAK3 by tofacitinib synergistically enhanced the antitumor effects of IMA in CML cells. Furthermore, the administration of IMA plus a JAK inhibitor reduced the expression of stem cells markers, such as ABCG2 and ALDH1A1. Co-blocking JAK3 with IMA and a JAK3 inhibitor might represent a new treatment strategy for eradicating LSCs and preventing CML relapses.

Topics: Antineoplastic Agents; Apoptosis; Cell Cycle; Cell Line, Tumor; Humans; Imatinib Mesylate; Janus Kinase 3; K562 Cells; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Neoplastic Stem Cells; Piperidines; Pyrimidines; Pyrroles; Signal Transduction

Topics: Adenine; Antineoplastic Agents; Biopsy; Disease Management; Exanthema; Humans; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Piperidines; Protein Kinase Inhibitors; Pyrazoles; Pyrimidines; Severity of Illness Index; Symptom Assessment

Topics: ADAMTS13 Protein; Adenine; Antibodies, Monoclonal; Antineoplastic Agents; Central Venous Catheters; fms-Like Tyrosine Kinase 3; Genetic Therapy; Graft vs Host Disease; Hematologic Diseases; Hemophilia B; Humans; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Leukemia, Myeloid, Acute; Multiple Myeloma; Mutation; Piperidines; Protein Kinase Inhibitors; Purpura, Thrombotic Thrombocytopenic; Pyrazoles; Pyrimidines; Recombinant Proteins; Societies, Medical; Thrombosis

Topics: Animals; Antineoplastic Agents; Benzamides; Breast Neoplasms; Cats; Disease Models, Animal; Dogs; Female; Humans; Imatinib Mesylate; Indoles; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Mast-Cell Sarcoma; Neoplasms; Piperazines; Piperidines; Protein Kinase Inhibitors; Pyridines; Pyrimidines; Pyrroles; Thiazoles; Transcriptome

Chronic myeloid leukemia (CML) is a hematopoietic neoplasm characterized by the Philadelphia chromosome and the related BCR-ABL1 oncoprotein. Acceleration of CML is usually accompanied by basophilia. Several proangiogenic molecules have been implicated in disease acceleration, including the hepatocyte growth factor (HGF). However, little is known so far about the cellular distribution and function of HGF in CML. We here report that HGF is expressed abundantly in purified CML basophils and in the basophil-committed CML line KU812, whereas all other cell types examined expressed only trace amounts of HGF or no HGF. Interleukin 3, a major regulator of human basophils, was found to promote HGF expression in CML basophils. By contrast, BCR-ABL1 failed to induce HGF synthesis in CML cells, and imatinib failed to inhibit expression of HGF in these cells. Recombinant HGF as well as basophil-derived HGF induced endothelial cell migration in a scratch wound assay, and these effects of HGF were reverted by an anti-HGF antibody as well as by pharmacologic c-Met inhibitors. In addition, anti-HGF and c-Met inhibitors were found to suppress the spontaneous growth of KU812 cells, suggesting autocrine growth regulation. Together, HGF is a BCR-ABL1-independent angiogenic and autocrine growth regulator in CML. Basophils are a unique source of HGF in these patients and may play a more active role in disease-associated angiogenesis and disease progression than has so far been assumed. Our data also suggest that HGF and c-Met are potential therapeutic targets in CML.

Topics: Basophils; Cell Line, Tumor; Cell Movement; Cell Proliferation; Crizotinib; Fusion Proteins, bcr-abl; Gene Expression Regulation, Leukemic; Hepatocyte Growth Factor; Human Umbilical Vein Endothelial Cells; Humans; Interleukin-3; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Neoplastic Stem Cells; Piperidines; Proto-Oncogene Proteins c-met; Pyrazoles; Pyridines

The Bcr-Abl kinase inhibitor, imatinib mesylate, is the front line treatment for chronic myeloid leukaemia (CML), but the emergence of imatinib resistance has led to the search for alternative drug treatments and the examination of combination therapies to overcome imatinib resistance. The pro-apoptotic PBOX compounds are a recently developed novel series of microtubule targeting agents (MTAs) that depolymerise tubulin. Recent data demonstrating enhanced MTA-induced tumour cell apoptosis upon combination with the cyclin dependent kinase (CDK)-1 inhibitor flavopiridol prompted us to examine whether this compound could similarly enhance the effect of the PBOX compounds. We thus characterised the apoptotic and cell cycle events associated with combination therapy of the PBOX compounds and flavopiridol and results showed a sequence dependent, synergistic enhancement of apoptosis in CML cells including those expressing the imatinib-resistant T315I mutant. Flavopiridol reduced the number of polyploid cells formed in response to PBOX treatment but only to a small extent, suggesting that inhibition of endoreplication was unlikely to play a major role in the mechanism by which flavopiridol synergistically enhanced PBOX-induced apoptosis. The addition of flavopiridol following PBOX-6 treatment did however result in an accelerated exit from the G2/M transition accompanied by an enhanced downregulation and deactivation of the CDK1/cyclin B1 complex and an enhanced degradation of the inhibitor of apoptosis protein (IAP) survivin. In conclusion, results from this study highlight the potential of these novel series of PBOX compounds, alone or in sequential combination with flavopiridol, as an effective therapy against CML.

Topics: Antineoplastic Agents; Apoptosis; Benzamides; Down-Regulation; Drug Resistance, Neoplasm; Drug Synergism; Flavonoids; Humans; Imatinib Mesylate; Inhibitor of Apoptosis Proteins; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Microtubule-Associated Proteins; Oxazepines; Piperazines; Piperidines; Protein Kinase Inhibitors; Pyrimidines; Pyrroles; Survivin

Mutation in the target oncoprotein is a common mechanism of resistance to tyrosine kinase inhibitors, as exemplified by the many BCR/ABL mutations that thwart imatinib activity in patients with chronic myelogenous leukemia. It remains unclear whether normal cellular protein targets of chemotherapeutics will evolve drug resistance via mutation to a similar extent. We conducted an in vitro screen for resistance to lonafarnib, a farnesyl protein transferase inhibitor that blocks prenylation of a number of proteins important in cell proliferation, and identified 9 mutations clustering around the lonafarnib binding site. In patients treated with a combination of imatinib and lonafarnib, we identified farnesyl protein transferase mutations in residues identified in our screen. Substitutions at Y361 were found in patients prior to treatment initiation, suggesting that these mutants might confer a proliferative advantage to leukemia cells, which we were able to confirm in cell culture. In vitro mutagenesis of normal cellular enzymes can be exploited to identify mutations that confer chemotherapy resistance to novel agents.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Benzamides; Cell Proliferation; Drug Resistance, Neoplasm; Enzyme Inhibitors; Farnesyltranstransferase; Humans; Imatinib Mesylate; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Mice; Mutagenesis; Mutagenesis, Site-Directed; Mutation; Pilot Projects; Piperazines; Piperidines; Protein Conformation; Protein Prenylation; Pyridines; Pyrimidines; Tumor Cells, Cultured

Some tumors are dependent on the continued activity of a single oncogene for maintenance of their malignant phenotype. The best-studied example is the Bcr-Abl fusion protein in chronic myelogenous leukemia (CML). Although the clinical success of the Abl kinase inhibitor imatinib against chronic-phase CML emphasizes the importance of developing therapeutic strategies aimed at this target, resistance to imatinib poses a major problem for the ultimate success of CML therapy by this agent. We hypothesized a sequential blockade strategy that is designed to decrease the expression of the Bcr-Abl protein, with the goal of complementing the action of imatinib on kinase activity. In this study, flavopiridol, an inhibitor of transcription, homoharringtonine (HHT), a protein synthesis inhibitor, and imatinib were used singly and in combination against the Bcr-Abl-positive human CML cell line K562. Flavopiridol alone inhibited phosphorylation of the RNA polymerase II COOH-terminal domain, specifically reduced RNA polymerase II-directed mRNA synthesis, and decreased the Bcr-Abl transcript levels. HHT inhibited protein synthesis and reduced the Bcr-Abl protein level. Imatinib directly inhibited the kinase activity of Bcr-Abl. The combinations of flavopiridol and HHT and flavopiridol and imatinib synergistically decreased clonogenicity as evaluated by the median-effect method. Greater synergy was observed when HHT and imatinib were given sequentially compared with simultaneous administration. Imatinib-resistant Ba/F3 cells that were transfected to express the E255K and T315I mutations of Bcr-Abl were not cross-resistant to flavopiridol and HHT. These results provided a rationale for the combination of inhibitors of transcription and/or translation with specific kinase inhibitors.

Topics: Antineoplastic Combined Chemotherapy Protocols; Benzamides; Drug Synergism; Flavonoids; Fusion Proteins, bcr-abl; Harringtonines; Homoharringtonine; Humans; Imatinib Mesylate; K562 Cells; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Piperazines; Piperidines; Pyrimidines; RNA, Neoplasm

Recent studies indicate that a rare population of primitive quiescent BCR-ABL(+) cells are innately insensitive to imatinib mesylate (IM) and persist after IM therapy of patients with chronic myeloid leukemia (CML). New approaches to the eradication of these cells are therefore likely to be crucial to the development of curative therapies for CML. We have now found that Ara-C, LY294002 (a PI-3 (phosphatidylinositol-3' kinase) kinase inhibitor), 17AAG (a heat-shock protein (HSP)-90 antagonist) and lonafarnib (a farnesyltransfease inhibitor) all enhance the toxicity of IM on K562 cells and on the total CD34(+) leukemic cell population from chronic phase CML patients. However, for quiescent CD34(+) leukemic cells, this was achieved only by concomitant exposure of the cells to lonafarnib. Ara-C or LY294002 alone blocked the proliferation of these cells but did not kill them, and Ara-C, LY294002 or 17AAG in combination with IM enhanced the cytostatic effect of IM but did not prevent the subsequent regrowth of the surviving leukemic cells. These studies demonstrate the importance of in vitro testing of novel agents on the subset of primary leukemic cells most likely to determine long-term treatment outcomes in vivo.

Topics: Antigens, CD34; Antineoplastic Combined Chemotherapy Protocols; Benzamides; Benzoquinones; Cell Line, Tumor; Cell Proliferation; Chromones; Cytarabine; Dose-Response Relationship, Drug; Drug Resistance, Neoplasm; Drug Screening Assays, Antitumor; Drug Synergism; Female; Humans; Imatinib Mesylate; Lactams, Macrocyclic; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Male; Morpholines; Piperazines; Piperidines; Pyridines; Pyrimidines; Rifabutin

Interactions between the cyclin-dependent kinase (CDK) inhibitor flavopiridol and the proteasome inhibitor bortezomib were examined in Bcr/Abl(+) human leukemia cells. Coexposure of K562 or LAMA84 cells to subtoxic concentration of flavopiridol (150-200 nM) and bortezomib (5-8 nM) resulted in a synergistic increase in mitochondrial dysfunction and apoptosis. These events were associated with a marked diminution in nuclear factor kappaB (NF-kappaB)/DNA binding activity; enhanced phosphorylation of SEK1/MKK4 (stress-activated protein kinase/extracellular signal-related kinase 1/mitogen-activated protein kinase kinase 4), c-Jun N-terminal kinase (JNK), and p38 mitogen-activated protein kinase (MAPK); down-regulation of Bcr/Abl; and a marked reduction in signal transducer and activator of transcription 3 (STAT3) and STAT5 activity. In imatinib mesylate-resistant K562 cells displaying increased Bcr/Abl expression, bortezomib/flavopiridol treatment markedly increased apoptosis in association with down-regulation of Bcr/Abl and BclxL, and diminished phosphorylation of Lyn, Hck, CrkL, and Akt. Parallel studies were performed in imatinib mesylate-resistant LAMA84 cells exhibiting reduced expression of Bcr/Abl but a marked increase in expression/activation of Lyn and Hck. Flavopiridol/bortezomib effectively induced apoptosis in these cells in association with Lyn and Hck inactivation. The capacity of flavopiridol to promote bortezomib-mediated Bcr/Abl down-regulation and apoptosis was mimicked by the positive transcription elongation factor-b (P-TEFb) inhibitor DRB (5,6-dichloro 1-beta-d-ribofuranosylbenzinida-sole). Finally, the bortezomib/flavopiridol regimen also potently induced apoptosis in Bcr/Abl(-) human leukemia cells. Collectively, these findings suggest that a strategy combining flavopiridol and bortezomib warrants further examination in chronic myelogenous leukemia and related hematologic malignancies.

Topics: Antineoplastic Agents; Apoptosis; Benzamides; Boronic Acids; Bortezomib; DNA-Binding Proteins; Drug Resistance, Neoplasm; Drug Synergism; Flavonoids; Fusion Proteins, bcr-abl; Humans; I-kappa B Proteins; Imatinib Mesylate; In Vitro Techniques; JNK Mitogen-Activated Protein Kinases; K562 Cells; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Milk Proteins; Mitochondria; Mitogen-Activated Protein Kinases; NF-kappa B; Nuclear Proteins; Phosphoprotein Phosphatases; Phosphorylation; Piperazines; Piperidines; Pyrazines; Pyrimidines; RNA Polymerase II; src-Family Kinases; STAT3 Transcription Factor; STAT5 Transcription Factor; Trans-Activators

Topics: Alkyl and Aryl Transferases; Apoptosis; Benzamides; Drug Synergism; Enzyme Inhibitors; Farnesyltranstransferase; Fusion Proteins, bcr-abl; Humans; Imatinib Mesylate; K562 Cells; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Neoplasm Proteins; Piperazines; Piperidines; Pyridines; Pyrimidines; Research Design; Tumor Cells, Cultured

The development of chronic myeloid leukemia (CML) is dependent on the deregulated tyrosine kinase of the oncoprotein BCR-ABL. STI571 (imatinib mesylate), an abl tyrosine kinase inhibitor, has proven remarkably effective for the treatment of CML. However, resistance to STI571 because of enhanced expression or mutation of the BCR-ABL gene has been detected in patients. In the current study we show that the farnesyl transferase inhibitor (FTI) SCH66336 (lonafarnib) inhibits the proliferation of STI571-resistant BCR-ABL-positive cell lines and hematopoietic colony formation from peripheral blood samples of STI571-resistant patients with CML. Moreover, SCH66336 enhances STI571-induced apoptosis in STI571-sensitive cells and, in patients with STI571 resistance from gene amplification, cooperates with STI571 to induce apoptosis. Our data provide a rationale for combination clinical trials of STI571 and SCH66336 in CML patients and suggest that combination therapy may be effective in patients with STI571 resistance.

Topics: Alkyl and Aryl Transferases; Antineoplastic Combined Chemotherapy Protocols; Benzamides; Cell Survival; Drug Resistance, Neoplasm; Enzyme Inhibitors; Farnesyltranstransferase; Hematopoietic Stem Cells; Humans; Imatinib Mesylate; K562 Cells; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Piperazines; Piperidines; Pyridines; Pyrimidines

The goal of this study was to characterize interactions between the Bcr/Abl kinase inhibitor STI571 and the cyclin-dependent kinase inhibitor flavopiridol in Bcr/Abl(+) human leukemia cells.. K562 leukemia cells were exposed to STI571 +/- flavopiridol for 24 or 48 h, after which mitochondrial damage, caspase activation, expression/activation of signaling and cell cycle regulatory proteins, and apoptosis were assessed.. In K562 cells, coadministration of marginally toxic concentrations of STI571 (200 nM) and flavopiridol (150 nM) for 48 h resulted in a marked increase in mitochondrial damage (e.g., cytochrome c release), activation of caspase-3, caspase-8, and Bid, and apoptosis. Similar interactions were observed in Bcr/Abl(+) LAMA-84 cells but not in leukemic cells that fail to express Bcr/Abl (e.g., HL-60, U937, Jurkat). STI571/flavopiridol-mediated apoptosis was associated with the caspase-independent down-regulation of Bcl-x(L) and Mcl-1, activation of extracellular signal-regulated kinase and c-Jun NH(2)-terminal kinase, and the caspase-dependent release of Smac/DIABLO and loss of deltapsi(m). Coadministration of flavopiridol and STI571 did not result in changes in levels of expression of Bcl-2, phopho-Stat5, phospho-p34(cdc2), or Bcr/Abl. Finally, STI571/flavopiridol effectively induced apoptosis in STI571-resistant K562 cells displaying amplification of the Bcr/Abl protein.. Together, these findings indicate that the cyclin-dependent kinase inhibitor flavopiridol induces multiple perturbations in signaling pathways in STI571-treated Bcr/Abl(+) human leukemia cells that culminate in mitochondrial injury, caspase activation, and apoptosis. They also suggest that simultaneous disruption of survival signaling and cell cycle regulatory pathways may represent an effective strategy in Bcr/Abl(+) malignancies.

Topics: Apoptosis; Benzamides; Biomarkers, Tumor; Drug Resistance, Neoplasm; Drug Screening Assays, Antitumor; Drug Synergism; Flavonoids; Fusion Proteins, bcr-abl; HL-60 Cells; Humans; Imatinib Mesylate; Jurkat Cells; K562 Cells; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Mitochondria; Phosphorylation; Piperazines; Piperidines; Protein Processing, Post-Translational; Pyrimidines; Signal Transduction; U937 Cells

BCR/ABL, the oncoprotein responsible for chronic myeloid leukemia (CML), transforms hematopoietic cells through both Ras-dependent and -independent mechanisms. Farnesyl protein transferase inhibitors (FTIs) were designed to block mutant Ras signaling, but they also inhibit the growth of transformed cells with wild-type Ras, implying that other farnesylated targets contribute to FTI action. In the current study, the clinical candidate FTI SCH66336 was characterized for its ability to inhibit BCR/ABL transformation. When tested against BCR/ABL-BaF3 cells, a murine cell line that is leukemogenic in mice, SCH66336 potently inhibited soft agar colony formation, slowed proliferation, and sensitized cells to apoptotic stimuli. Quantification of activated guanosine triphosphate (GTP)-bound Ras protein and electrophoretic mobility shift assays for AP-1 DNA binding showed that Ras effector pathways are inhibited by SCH66336. However, SCH66336 was more inhibitory than dominant-negative Ras in assays of soft agar colony formation and cell proliferation, suggesting activity against targets other than Ras. Cell cycle analysis of BCR/ABL-BaF3 cells treated with SCH66336 revealed G2/M blockade, consistent with recent reports that centromeric proteins that regulate the G2/M checkpoint are critical farnesylated targets of FTI action. Mice injected intravenously with BCR/ABL-BaF3 cells developed acute leukemia and died within 4 weeks with massive splenomegaly, elevated white blood cell counts, and anemia. In contrast, nearly all mice treated with SCH66336 survived and have remained disease-free for more than a year. Furthermore, SCH66336 selectively inhibited the hematopoietic colony formation of primary human CML cells. As an oral, nontoxic compound with a mechanism of action distinct from that of ABL tyrosine kinase inhibition, FTI SCH66336 shows promise for the treatment of BCR/ABL-induced leukemia.

Topics: Alkyl and Aryl Transferases; Animals; Antineoplastic Agents; Apoptosis; Bone Marrow Cells; Cell Cycle; Cell Division; Enzyme Inhibitors; Farnesyltranstransferase; Genes, abl; Hematopoietic Stem Cells; Humans; Leukemia, Experimental; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Mice; Piperidines; Pyridines; Spleen; Survival Rate; Transformation, Genetic; Tumor Cells, Cultured