A randomised phase II trial of hydroxychloroquine and imatinib versus imatinib alone for patients with chronic myeloid leukaemia in major cytogenetic response with residual disease.

In chronic-phase chronic myeloid leukaemia (CP-CML), residual BCR-ABL1+ leukaemia stem cells are responsible for disease persistence despite TKI. Based on in vitro data, CHOICES (CHlorOquine and Imatinib Combination to Eliminate Stem cells) was an international, randomised phase II trial designed to study the safety and efficacy of imatinib (IM) and hydroxychloroquine (HCQ) compared with IM alone in CP-CML patients in major cytogenetic remission with residual disease detectable by qPCR. Sixty-two patients were randomly assigned to either arm. Treatment 'successes' was the primary end point, defined as ≥0.5 log reduction in 12-month qPCR level from trial entry. Selected secondary study end points were 24-month treatment 'successes', molecular response and progression at 12 and 24 months, comparison of IM levels, and achievement of blood HCQ levels >2000 ng/ml. At 12 months, there was no difference in 'success' rate (p = 0.58); MMR was achieved in 80% (IM) vs 92% (IM/HCQ) (p = 0.21). At 24 months, the 'success' rate was 20.8% higher with IM/HCQ (p = 0.059). No patients progressed. Seventeen serious adverse events, including four serious adverse reactions, were reported; diarrhoea occurred more frequently with combination. IM/HCQ is tolerable in CP-CML, with modest improvement in qPCR levels at 12 and 24 months, suggesting autophagy inhibition maybe of clinical value in CP-CML.

Stockperson attitudes toward pig euthanasia.

Euthanasia is a necessary act for any facility keeping live animals. Nevertheless, the crucial role and responsibility of the stockperson in deciding and conducting on-farm euthanasia has been overlooked. Stockperson characteristics and knowledge that lead to appropriate decision-making and the skills to competently perform the procedure remain to be identified. An important component of the stockperson's characteristics that predict behavior is the stockperson's attitudes. This preliminary study investigated the factors that influence stockperson attitudes toward the practice of on-farm euthanasia in the pork industry. A total of 120 stockpeople from 10 Australian pig farms (ranging in size from 50 to 4,754 sows and from 2 to 32 employees) completed a questionnaire based on focus group input to assess their attitudes toward euthanasia and decision processes. Factors identified included stockperson attitudes and attributes (empathy affect, empathy attribution, feeling bad about euthanizing, and negative attitudes to pigs), beliefs about the working environment (perceived time constraints and relying on others), and factors related to decision-making (comfortable with euthanasia, trouble deciding and avoid if possible, confidence, insufficient knowledge, seeking knowledge, and using sources to get advice). Numerous significant correlations were found between these variables. Furthermore, regression analyses showed confidence as the only significant predictor of being comfortable with euthanasia (12.5% of the variance; < 0.001); insufficient knowledge and empathy attribution both as predictors of trouble deciding and avoid if possible (15.1% of the variance; = 0.001 and = 0.032, respectively); and empathy affect, insufficient knowledge, and perceived time constraints as predictors of feeling bad about euthanizing (23.2% of the variance; < 0.001, = 0.006, and = 0.022, respectively). Stockpeople reported seeking more knowledge if they had not euthanized an animal before working with pigs ( = 0.05), and women reported greater difficulty than men in conducting euthanasia ( < 0.01). The findings indicate that euthanasia, which comprises both a decision-making process and the act itself, can adversely affect stockpeople. This preliminary study offers insights for implementation of successful practical and humane pig euthanasia protocols on farm. This will benefit stockperson well-being and animal well-being alike.

Mtss1 is a critical epigenetically regulated tumor suppressor in CML.

Chronic myeloid leukemia (CML) is driven by malignant stem cells that can persist despite therapy. We have identified Metastasis suppressor 1 (Mtss1/MIM) to be downregulated in hematopoietic stem and progenitor cells from leukemic transgenic SCLtTA/Bcr-Abl mice and in patients with CML at diagnosis, and Mtss1 was restored when patients achieved complete remission. Forced expression of Mtss1 decreased clonogenic capacity and motility of murine myeloid progenitor cells and reduced tumor growth. Viral transduction of Mtss1 into lineage-depleted SCLtTA/Bcr-Abl bone marrow cells decreased leukemic cell burden in recipients, and leukemogenesis was reduced upon injection of Mtss1-overexpressing murine myeloid 32D cells. Tyrosine kinase inhibitor (TKI) therapy and reversion of Bcr-Abl expression increased Mtss1 expression but failed to restore it to control levels. CML patient samples revealed higher DNA methylation of specific Mtss1 promoter CpG sites that contain binding sites for Kaiso and Rest transcription factors. In summary, we identified a novel tumor suppressor in CML stem cells that is downregulated by both Bcr-Abl kinase-dependent and -independent mechanisms. Restored Mtss1 expression markedly inhibits primitive leukemic cell biology in vivo, providing a therapeutic rationale for the Bcr-Abl-Mtss1 axis to target TKI-resistant CML stem cells in patients.

Dual glutathione-S-transferase-θ1 and -µ1 gene deletions determine imatinib failure in chronic myeloid leukemia.

Approximately 40% of patients with chronic myeloid leukemia (CML) receiving imatinib fail treatment. There is an increased risk of CML in subjects with (i) deletions of genes encoding glutathione-S-transferase (GST)-θ1 (GSTT1) and -µ1, (GSTM1) and (ii) the GST-π1 (GSTP1) single-nucleotide polymorphism (SNP) Ile105Val (GSTP1*B; rs1695); however, their effects on imatinib treatment outcome are not known. Here, we assess the role of these GSTs in relation to imatinib treatment outcome in 193 CML patients. Deletion of GSTT1 alone, or in combination with deletion of the GSTM1 gene, significantly increased the likelihood of imatinib failure (P = 0.021 and P < 0.001, respectively). The GSTP1*B SNP was not associated with time to imatinib failure. Losses of the GSTT1 and GSTM1 genes are therefore important determinants of imatinib failure in CML. Screening for GSTT1 and GSTM1 gene deletions during diagnosis may identify patients who may be better treated using an alternative therapy.

Synergistic effects of proteasome inhibitor carfilzomib in combination with tyrosine kinase inhibitors in imatinib-sensitive and -resistant chronic myeloid leukemia models.

The tyrosine kinase inhibitor (TKI) imatinib has transformed the treatment and outlook of chronic myeloid leukemia (CML); however, the development of drug resistance and the persistence of TKI-resistant stem cells remain obstacles to eradicating the disease. Inhibition of proteasome activity with bortezomib has been shown to effectively induce apoptosis in TKI-resistant cells. In this study, we show that exposure to the next generation proteasome inhibitor carfilzomib is associated with a decrease in ERK signaling and increased expression of Abelson interactor proteins 1 and 2 (ABI-1/2). We also investigate the effect of carfilzomib in models of imatinib-sensitive and -resistant CML and demonstrate a potent reduction in proliferation and induction of apoptosis in a variety of models of imatinib-resistant CML, including primitive CML stem cells. Carfilzomib acts synergistically with the TKIs imatinib and nilotinib, even in imatinib-resistant cell lines. In addition, we found that the presence of immunoproteasome subunits is associated with an increased sensitivity to carfilzomib. The present findings provide a rational basis to examine the potential of carfilzomib in combination with TKIs as a potential therapy for CML, particularly in imatinib-resistant disease.

Targeting survival pathways in chronic myeloid leukaemia stem cells.

UNLABELLED: Chronic myeloid leukaemia (CML) is a clonal myeloproliferative disorder characterized by the presence of a fusion oncogene BCR-ABL, which encodes a protein with constitutive TK activity. The implementation of tyrosine kinase inhibitors (TKIs) marked a major advance in CML therapy; however, there are problems with current treatment. For example, relapse occurs when these drugs are discontinued in the majority of patients who have achieved a complete molecular response on TKI and these agents are less effective in patients with mutations in the BCR-ABL kinase domain. Importantly, TKI can effectively target proliferating mature cells, but do not eradicate quiescent leukaemic stem cells (LSCs), therefore allowing disease persistence despite treatment. It is essential that alternative strategies are used to target the LSC population. BCR-ABL activation is responsible for the modulation of different signalling pathways, which allows the LSC fraction to evade cell death. Several pathways have been shown to be modulated by BCR-ABL, including PI3K/AKT/mTOR, JAK-STAT and autophagy signalling pathways. Targeting components of these survival pathways, alone or in combination with TKI, therefore represents an attractive potential therapeutic approach for targeting the LSC. However, many pathways are also active in normal stem cells. Therefore, potential targets must be validated to effectively eradicate CML stem cells while sparing normal counterparts. This review summarizes the main pathways modulated in CML stem cells, the recent developments and the use of novel drugs to target components in these pathways which may be used to target the LSC population. LINKED ARTICLES: This article is part of a themed section on Emerging Therapeutic Aspects in Oncology. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2013.169.issue-8.

A pathway from leukemogenic oncogenes and stem cell chemokines to RNA processing via THOC5.

THOC5 is a member of the THO complex that is involved in processing and transport of mRNA. We have shown previously that hematopoietic stem cells have an absolute requirement for THOC5 for survival and that THOC5 is phosphorylated on tyrosine 225 as a consequence of leukemogenic protein tyrosine kinase (PTK) action. We have investigated pathways for THOC5 phosphorylation to develop an understanding of THO complex modulation by tyrosine kinase (TK) oncogenes in leukemias. We demonstrate that THOC5 phosphorylation is mediated by Src PTK and CD45 protein tyrosine phosphatase action and that this event is sensitive to oxidative status. We show that THOC5 phosphorylation is elevated in stem cells from patients with chronic myeloid leukemia (CML) and that this phosphorylation is sensitive to the frontline drugs used in CML treatment. Further we show that THOC5 Y225 phosphorylation governs mRNA binding. In addition, CXCL12 is shown to induce THOC5 Y225 phosphorylation, and site-directed mutagenesis demonstrates that this modulates motile response. In conclusion, we delineate a signaling pathway stimulated by leukemogenic PTKs, chemokines and oxidative stress that can affect THO complex mediation of gene expression describing mechanisms for post-transcriptional regulation of protein levels.

Expression of p89(c-Mybex9b), an alternatively spliced form of c-Myb, is required for proliferation and survival of p210BCR/ABL-expressing cells.

The c-Myb gene encodes the p75(c-Myb) isoform and less-abundant proteins generated by alternatively spliced transcripts. Among these, the best known is p(c-Mybex9b), which contains 121 additional amino acids between exon 9 and 10, in a domain involved in protein-protein interactions and negative regulation. In hematopoietic cells, expression of p(c-Mybex9b) accounts for 10-15% of total c-Myb; these levels may be biologically relevant because modest changes in c-Myb expression affects proliferation and survival of leukemic cells and lineage choice and frequency of normal hematopoietic progenitors. In this study, we assessed biochemical activities of p(c-Mybex9b) and the consequences of perturbing its expression in K562 and primary chronic myeloid leukemia (CML) progenitor cells. Compared with p75(c-Myb), p(c-Mybex9b) is more stable and more effective in transactivating Myb-regulated promoters. Ectopic expression of p(c-Mybex9b) enhanced proliferation and colony formation and reduced imatinib (IM) sensitivity of K562 cells; conversely, specific downregulation of p(c-Mybex9b) reduced proliferation and colony formation, enhanced IM sensitivity of K562 cells and markedly suppressed colony formation of CML CD34(+) cells, without affecting the levels of p75(c-Myb). Together, these studies indicate that expression of the low-abundance p(c-Mybex9b) isoform has an important role for the overall biological effects of c-Myb in BCR/ABL-transformed cells.

Smouldering systemic mastocytosis presenting with cryptic life-threatening crises: case report and literature review.

Systemic mastocytosis (SM) is a rare, clonal disorder of the mast cell (MC) and its precursor cells. It is characterized by proliferation and accumulation of MCs within various organs, most commonly the skin. The clinical course is variable with indolent or smouldering and aggressive forms being described. We report the case of a middle-aged male patient with smouldering SM presenting with atypical recurrent life-threatening crises. The patient reported a 19-year history of chronic symptoms. The patient had four inpatient stays due to atypical life-threatening crises, during which he has shown end organ damage (cardiac and renal). With each crisis the patient reported acute symptoms. The management of each of these episodes was complex and made more challenging by the patient's longstanding history of hypertension and ischaemic heart disease. In short, SM can present with unexplained life-threatening crises which can be confused for an infectious disease being acute in nature.

Gfi-1 inhibits proliferation and colony formation of p210BCR/ABL-expressing cells via transcriptional repression of STAT 5 and Mcl-1.

Expression of the transcription repressor Gfi-1 is required for the maintenance of murine hematopoietic stem cells. In human cells, ectopic expression of Gfi-1 inhibits and RNA interference-mediated Gfi-1 downregulation enhances proliferation and colony formation of p210BCR/ABL expressing cells. To investigate the molecular mechanisms that may explain the effects of perturbing Gfi-1 expression in human cells, Gfi-1-regulated genes were identified by microarray analysis in K562 cells expressing the tamoxifen-regulated Gfi-1-ER protein. STAT 5B and Mcl-1, two genes important for the proliferation and survival of hematopoietic stem cells, were identified as direct and functionally relevant Gfi-1 targets in p210BCR/ABL-transformed cells because: (i) their expression and promoter activity was repressed by Gfi-1 and (ii) when constitutively expressed blocked the proliferation and colony formation inhibitory effects of Gfi-1. Consistent with these findings, genetic or pharmacological inhibition of STAT 5 and/or Mcl-1 markedly suppressed proliferation and colony formation of K562 and CD34+ chronic myelogenous leukemia (CML) cells. Together, these studies suggest that the Gfi-1STAT 5B/Mcl-1 regulatory pathway identified here can be modulated to suppress the proliferation and survival of p210BCR/ABL-transformed cells including CD34+ CML cells.

The MEK inhibitor PD184352 enhances BMS-214662-induced apoptosis in CD34+ CML stem/progenitor cells.

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.

Omacetaxine may have a role in chronic myeloid leukaemia eradication through downregulation of Mcl-1 and induction of apoptosis in stem/progenitor cells.

Chronic myeloid leukaemia (CML) is maintained by a rare population of tyrosine kinase inhibitor (TKI)-insensitive malignant stem cells. Our long-term aim is to find a BcrAbl-independent drug that can be combined with a TKI to improve overall disease response in chronic-phase CML. Omacetaxine mepesuccinate, a first in class cetaxine, has been evaluated by clinical trials in TKI-insensitive/resistant CML. Omacetaxine inhibits synthesis of anti-apoptotic proteins of the Bcl-2 family, including (myeloid cell leukaemia) Mcl-1, leading to cell death. Omacetaxine effectively induced apoptosis in primary CML stem cells (CD34(+)38(lo)) by downregulation of Mcl-1 protein. In contrast to our previous findings with TKIs, omacetaxine did not accumulate undivided cells in vitro. Furthermore, the functionality of surviving stem cells following omacetaxine exposure was significantly reduced in a dose-dependant manner, as determined by colony forming cell and the more stringent long-term culture initiating cell colony assays. This stem cell-directed activity was not limited to CML stem cells as both normal and non-CML CD34(+) cells were sensitive to inhibition. Thus, although omacetaxine is not leukaemia stem cell specific, its ability to induce apoptosis of leukaemic stem cells distinguishes it from TKIs and creates the potential for a curative strategy for persistent disease.

Nilotinib concentration in cell lines and primary CD34(+) chronic myeloid leukemia cells is not mediated by active uptake or efflux by major drug transporters.

Imatinib mesylate and nilotinib are highly effective at eradicating the majority of chronic myeloid leukemia (CML) cells; however, neither agent induces apoptosis of primitive CML CD34(+) cells. One possible explanation is that CD34(+) cells do not accumulate sufficient intracellular drug levels because of either inadequate active uptake or increased efflux. To determine the interaction of nilotinib with major clinically implicated drug transporters, we analyzed their interactions with MDR1 (ABCB1), MRP1 (ABCC1), ABCG2 (BCRP) and human organic cation transporter (hOCT)1 in CML cell lines and primitive (CD34(+)) primary CML cells. Nilotinib is neither dependent on active import by hOCT1, nor effluxed through the ATP-binding cassette transporters analyzed. Indeed, we found nilotinib to be an inhibitor of hOCT1, MDR1 and ABCG2. The efflux transporters MDR1, MRP1 and ABCG2 are expressed on CML CD34(+) cells at 13.5, 108 and 291% of control, respectively, although hOCT1 expression was absent; however, inhibition of efflux transporter activity did not potentiate the effect of nilotinib on apoptosis, Bcr-Abl inhibition or CML CD34(+) cell proliferation. Therefore, we have found no evidence for either active uptake of nilotinib through hOCT1 or efflux through MDR1, MRP1 or ABCG2, and it is therefore unlikely that these transporters will have any effect on the clinical response to this drug.

Chronic myeloproliferative diseases with and without the Ph chromosome: some unresolved issues.

Ph-positive chronic myeloid leukemia (CML) and Ph-negative chronic myeloproliferative diseases (MPDs), characterized in many cases by the presence of the JAK2(V617F) mutation, have many features in common and yet also show fundamental differences. In this review, we pose five discrete and related questions relevant to both categories of hematological malignancy, namely: What are the mechanisms that underlie disease progression from a relatively benign or chronic phase? By what therapeutic methods might one target residual leukemia stem cells in CML? Is JAK2(V617F) the original molecular event in MPD? What epigenetic events must have a role in dictating disease phenotype in MPDs? And finally, Will the benefits conferred by current or future JAK2(V617F) inhibitors equal or even surpass the clinical success that has resulted from the use of tyrosine kinase inhibitors in CML? These and others questions must be addressed and in some cases should be answered in the foreseeable future.

Successful use of National Cancer Registry data to monitor the effective use of imatinib for treating chronic myeloid leukaemia.

UNLABELLED: Imatinib is a tyrosine kinase inhibitor, which selectively antagonises the BCR-ABL molecular pathway which causes chronic myeloid leukaemia (CML). Imatinib was first approved by the Scottish Medicines Consortium (SMC) in January 2002 with the recommendation that its use be audited. The cost of the drug has major financial implications for health resources. METHODS: All imatinib usage since its first prescription in Scotland in September 2000 to July 2003 was audited through pharmacy records and through the Scotland Leukaemia Registry (SLR), an existing national registry of patients with CML. RESULTS: One hundred and four patients in Chronic Phase (CP), 36 in Accelerated Phase (AP) and five in Blast Phase (BP) received imatinib. The median duration of therapy was not reached for CFP 17 months for AFP and two months for BP patients. Major (complete) cytogenetic response rates were 74% (63%) and 38% (24%) respectively for CP and APR Overall survival for all CP patients from the start of imatinib therapy was 94% at one year, 91% at two years and 83% at three years. An audit of the effectiveness of the SLR as an auditing agency, showed complete registration in 95% of cases. CONCLUSIONS: We believe such data collection should be an important ongoing resource for assessing outcomes in a rare form of leukaemia but one which already has major implications for health economics and will continue to do so given the future development of dual tyrosine kinase inhibitors for imatinib resistant cases.

On the use of lonafarnib in myelodysplastic syndrome and chronic myelomonocytic leukemia.

Lonafarnib is an orally bio-available farnesyltransferase inhibitor that prevents farnesylation of specific target proteins including Ras. In a multicenter study, 67 patients with advanced myelodysplastic syndrome (MDS) and chronic myelomonocytic leukemia (CMML) were treated with a continuous oral dose of 200-300 mg of lonafarnib and were evaluated for hematologic, pathologic and pharmacodynamic response. The median age of patients was 70 years (range 44-86). There were 32 patients with MDS (RAEB-20 and RAEB-t-12) and 35 with CMML. Overall 16 (24%) of the patients responded with two patients achieving a complete remission and one a partial response. Responses were seen in 6/32 and 10/35 patients with MDS and CMML, respectively. Of the 19 patients who were platelet transfusion-dependent prior to treatment, 5 (26%) became transfusion-free for a median duration of 185 days. A decrease in the farnesylation of the HDJ-2 protein measured in patient-derived cells was observed in the majority of patients during treatment with lonafarnib, but no clear correlation between changes in farnesylation and clinical effect could be made. Gastrointestinal toxicity was significant with 19% of patients discontinuing therapy due to diarrhea, nausea and/or anorexia. Lonafarnib has demonstrable activity in patients with advanced MDS and CMML.

Enhanced BCR-ABL kinase inhibition does not result in increased inhibition of downstream signaling pathways or increased growth suppression in CML progenitors.

The therapeutic success of imatinib in chronic myeloid leukemia (CML) is hampered by persistence of malignant stem cells. We investigated whether nilotinib, a more potent BCR-ABL kinase inhibitor could target CML primitive progenitors more effectively than imatinib. CML and normal progenitor cells were cultured with nilotinib or imatinib in growth factor supplemented medium. Nilotinib inhibited BCR-ABL kinase activity at lower concentrations than imatinib. Nilotinib inhibited mitogen-activated protein kinase (MAPK), AKT and STAT5 phosphorylation in CML CD34(+) cells in the absence of growth factors (GFs), but did not suppress AKT and STAT5 activity, and resulted in increased MAPK activity, in the presence of GFs. Nilotinib and imatinib resulted in similar suppression of CML primitive and committed progenitors in long-term culture-initiating cell and colony-forming cell assays. Inhibition of progenitor growth was related to marked reduction in proliferation, but only a modest increase in apoptosis. Nilotinib did not show increased efficacy in reducing nondividing CML progenitors compared with imatinib. These results indicate that more potent tyrosine kinase inhibitors by themselves will not be more effective in eliminating CML progenitors than imatinib and that additional mechanism required for maintenance of malignant stem cells need to be identified to improve targeting of leukemia stem cells.

Characterization of cancer stem cells in chronic myeloid leukaemia.

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 of apoptosis protein-1), NF-kappaB (nuclear factor kappaB) and iNOS (inducible nitric oxide synthase). Furthermore, BMS-214662 synergizes with MEK1/2 [MAPK (mitogen-activated protein kinase)/ERK (extracellular-signal-regulated kinase) kinase 1/2] inhibitors, suggesting a second mechanism other that RAS inhibition for induction of apoptosis. Our intentions are now to explore the activity of BMS-214662 in other cancer stem cell disorders and to move this preclinical work to a clinical trial combining dasatinib with BMS-214662 in CML.