bms-214662 and Leukemia--Myelogenous--Chronic--BCR-ABL-Positive

Chronic myeloid leukemia (CML) is induced by the BCR-ABL oncogene, a product of Philadelphia (Ph) chromosome. The BCR-ABL kinase inhibitor imatinib is a standard treatment for Ph+ leukemia, and has been shown to induce a complete hematologic and cytogenetic response in most chronic phrase CML patients. However, imatinib does not cure CML, and one of the reasons is that imatinib does not kill leukemia stem cells (LSCs) in CML both in vitro and in vivo. Recently, several new targets or drugs have been reported to inhibit LSCs in cultured human CD34+ CML cells or in mouse model of BCR-ABL induced CML, including an Alox5 pathway inhibitor, Hsp90 inhibitors, omacetaxine, hedgehog inhibitor and BMS-214662. Specific targeting of LSCs but not normal stem cell is a correct strategy for developing new anti-cancer therapies in the future.

Topics: Animals; Antineoplastic Agents; Arachidonate 15-Lipoxygenase; Benzodiazepines; Harringtonines; Hedgehog Proteins; Homoharringtonine; HSP90 Heat-Shock Proteins; Humans; Imidazoles; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Lipoxygenase Inhibitors; Medical Oncology; Mice; Models, Biological; Neoplastic Stem Cells; Signal Transduction

Chronic myelogenous leukemia (CML) is a clonal myeloproliferative disorder that arises in the hematopoietic stem cell compartment. CML is one of the best-understood malignancies, as it results from a single genetic mutation, the fusion oncogene BCR-ABL, which has been widely studied. Specific tyrosine kinase inhibitors have been developed to target BCR-ABL in CML, but these agents fail to eliminate the CML stem cell population and thus are unlikely to cure CML. This article reviews recent developments in the biology and treatment of CML, specifically focusing on CML stem cells. Significant progress continues to be made in our understanding of CML stem cell biology, which has wider implications within the cancer stem cell field. We are also beginning to see the identification of novel therapies that specifically target the CML stem cell. These are exciting times in the quest to cure CML.

Topics: Antineoplastic Combined Chemotherapy Protocols; Benzodiazepines; Cell Lineage; Hematopoietic Stem Cells; Humans; Imidazoles; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Models, Biological; Neoplastic Stem Cells; Protein Kinase Inhibitors; Treatment Outcome

CML (chronic myeloid leukaemia) is a myeloproliferative disease that originates in an HSC (haemopoietic stem cell) as a result of the t(9;22) translocation, giving rise to the Ph (Philadelphia chromosome) and bcr-abl oncoprotein. The disease starts in CP (chronic phase), but as a result of genomic instability, it progresses over time to accelerated phase and then to BC (blast crisis), becoming increasingly resistant to therapy. bcr-abl is a constitutively active tyrosine kinase that has been targeted by TKIs (tyrosine kinase inhibitors), including IM (imatinib mesylate), nilotinib and dasatinib. We have developed various flow cytometry techniques to enable us to isolate candidate CML stem cells from CP patients at diagnosis that efflux Hoechst dye, express CD34, lack CD38 and are cytokine-non-responsive in culture over periods of up to 12 days in growth factors. These stem cells have been shown to regenerate bcr-abl-positive haemopoiesis in immunocompromised mice upon transplantation. We previously demonstrated that IM was antiproliferative for CML stem cells but did not induce apoptosis. Clinical experience now confirms that IM may not target CML stem cells in vivo with few patients achieving complete molecular remission and relapse occurring rapidly upon drug withdrawal. Our recent efforts have focused on understanding why CML stem cells are resistant to IM and on trying to find novel ways to induce apoptosis of this population. We have shown that CML stem cells express very high levels of functional wild-type bcr-abl; no kinase domain mutations have been detected in the stem cell population. Dasatinib, a more potent multitargeted TKI than IM, inhibits bcr-abl activity more efficiently than IM but still does not induce apoptosis of the stem cell population. Most recently, we have tested a number of novel drug combinations and found that FTIs (farnesyl transferase inhibitors) have activity against CML. BMS-214662 is the most effective of these and induces apoptosis of phenotypically and functionally defined CML stem cells in vitro, as a single agent and in combination with IM or dasatinib. The effect against CML stem cells is selective with little effect on normal stem cells. The drug is also effective against BC CML stem cells and equally effective against wild-type and mutant bcr-abl, including the most resistant mutant T315I. In association with apoptosis, there is activation of caspase 8 and caspase 3, inhibition of the MAPK pathway, IAP-1 (inhibitor

Topics: Antineoplastic Agents; Apoptosis; Benzamides; Benzodiazepines; Dasatinib; Drug Resistance, Neoplasm; Humans; Imatinib Mesylate; Imidazoles; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Neoplastic Stem Cells; Piperazines; Pyrimidines; Thiazoles

Chronic myeloid leukaemia (CML) arises in a haemopoietic stem cell and is driven by the Bcr-Abl oncoprotein. Abl kinase inhibitors (protein tyrosine kinase inhibitors) represent standard treatment for CML and induce remission in the majority of patients with early disease, however these drugs do not target leukaemic stem cells (LSCs) effectively, thus preventing cure. Previously, we identified the farnesyl transferase inhibitor BMS-214662 as a selective inducer of apoptosis in LSCs of CML patients relative to normal controls; however, the mechanism underlying LSC-specific apoptosis remains unclear. To identify pathways involved in the favourable effects of BMS-214662 in CML, we employed a proteomic approach (based on iTRAQ) to analyse changes in protein expression in response to drug treatment in the nuclear and cytoplasmic fractions of CD34(+) CML cells. The study identified 88 proteins as altered after drug treatment, which included proteins known to be involved in nucleic acid metabolism, oncogenesis, developmental processes and intracellular protein trafficking. We found that expression of Ebp1, a negative regulator of proliferation, was upregulated in the nucleus of BMS-214662-treated cells. Furthermore, proteins showing altered levels in the cytosol, such as histones, were predominantly derived from the nucleus and BMS-214662 affected expression levels of nuclear pore complex proteins. Validation of key facets of these observations suggests that drug-induced alterations in protein localisation, potentially via loss of nuclear membrane integrity, contributes to the LSC specificity of BMS-214662, possibly via Ran proteins as targets.

Topics: Adaptor Proteins, Signal Transducing; Antigens, CD34; Apoptosis; Benzodiazepines; Evaluation Studies as Topic; Farnesyltranstransferase; Fusion Proteins, bcr-abl; Gene Expression Regulation; Imidazoles; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Proteome; ran GTP-Binding Protein; RNA-Binding Proteins

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

Chronic myeloid leukemia (CML) is a hematopoietic stem cell disorder maintained by cancer stem cells. To target this population, we investigated the mechanism of action of BMS-214662, developed as a farnesyl transferase inhibitor (FTI) and unique in inducing apoptosis in these cells. By contrast, a related congener and equally effective FTI, BMS-225975 does not induce apoptosis, indicating a novel mechanism of action. BMS-214662 significantly and selectively induced apoptosis in primitive CD34(+)38(-) CML compared with normal cells. Apoptosis proceeded via the intrinsic pathway: Bax conformational changes, loss of mitochondrial membrane potential, generation of reactive oxygen species, release of cytochrome c, and caspase-9/3 activation were noted. Up-regulation of protein kinase Cbeta (PKCbeta), down-regulation of E2F1, and phosphorylation of cyclin A-associated cyclin-dependent kinase 2 preceded these changes. Cotreatment of CML CD34(+) and CD34(+)38(-) cells with PKC modulators, bryostatin-1, or hispidin markedly decreased these early events and the subsequent apoptosis. None of these events was elicited by BMS-214662 in normal CD34(+) cells or by BMS-225975 in CML CD34(+) cells. These data suggest that BMS-214662 selectively elicits a latent apoptotic pathway in CML stem cells that is initiated by up-regulation of PKCbeta and mediated by Bax activation, providing a molecular framework for development of novel therapeutics.

Topics: ADP-ribosyl Cyclase 1; Antigens, CD34; Apoptosis; bcl-2-Associated X Protein; Benzodiazepines; Bryostatins; Caspases; Cyclin A; Cyclin-Dependent Kinase 2; E2F1 Transcription Factor; Enzyme Activation; Enzyme Inhibitors; Farnesyltranstransferase; Humans; Imidazoles; In Vitro Techniques; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Membrane Glycoproteins; Microscopy, Electron, Transmission; Mitochondria; Neoplastic Stem Cells; Protein Kinase C; Protein Kinase C beta

Chronic myeloid leukemia (CML), a hematopoietic stem-cell disorder, cannot be eradicated by conventional chemotherapy or the tyrosine kinase inhibitor imatinib mesylate (IM). To target CML stem/progenitor cells, we investigated BMS-214662, a cytotoxic farnesyltransferase inhibitor, previously reported to kill nonproliferating tumor cells. IM or dasatinib alone reversibly arrested proliferation of CML stem/progenitor cells without inducing apoptosis. In contrast, BMS-214662, alone or in combination with IM or dasatinib, potently induced apoptosis of both proliferating and quiescent CML stem/progenitor cells with less than 1% recovery of Philadelphia-positive long-term culture-initiating cells. Normal stem/progenitor cells were relatively spared by BMS-214662, suggesting selectivity for leukemic stem/progenitor cells. The ability to induce selective apoptosis of leukemic stem/progenitor cells was unique to BMS-214662 and not seen with a structurally similar agent BMS-225975. BMS-214662 was cytotoxic against CML blast crisis stem/progenitor cells, particularly in combination with a tyrosine kinase inhibitor and equally effective in cell lines harboring wild-type vs mutant BCR-ABL, including the T315I mutation. This is the first report of an agent with activity in resistant and blast crisis CML that selectively kills CML stem/progenitor cells through apoptosis and offers potential for eradication of chronic phase CML.

Topics: Antigens, CD34; Antineoplastic Agents; Apoptosis; Benzamides; Benzodiazepines; Blast Crisis; Caspase 3; Cell Death; Cell Survival; Dasatinib; Drug Screening Assays, Antitumor; Drug Synergism; Farnesyltranstransferase; Fusion Proteins, bcr-abl; Humans; Imatinib Mesylate; Imidazoles; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Mutation; Neoplastic Stem Cells; Philadelphia Chromosome; Piperazines; Protein Kinase Inhibitors; Protein Structure, Tertiary; Pyrimidines; Thiazoles