15 research outputs found
Inhibition of MDR1 does not sensitize primitive chronic myeloid leukemia CD34<sup>+</sup> cells to imatinib
No full text<p><b>Objective:</b> To investigate the interaction of imatinib mesylate (IM) with the clinically relevant adenosine triphosphate-binding cassette efflux transporter MDR1 (ABCB1) in cells from patients with chronic myeloid leukemia (CML) and to explore whether inhibition of this transporter would improve IM's efficacy in the elimination of CML CD34<sup>+</sup> cells by increasing cell-associated drug accumulation.</p>
<p><b>Materials and Methods:</b> Cells from newly diagnosed chronic-phase CML patients were harvested by leukapheresis and enriched to >95% CD34<sup>+</sup>. Expression of the transporter gene MDR1 was performed by quantitative reverse transcription polymerase chain reaction. Interaction of IM with MDR1 was analyzed by substrate (rhodamine 123) displacement assay. Cell-associated levels of IM in CML CD34<sup>+</sup> cells were measured by high-pressure liquid chromatography. Intracellular phospho-CrkL levels, apoptosis in total CML CD34<sup>+</sup> cells and high-resolution tracking of cell division were assayed by flow cytometry.</p>
<p><b>Results:</b> Measurements of cell-associated IM uptake showed significantly lower drug levels in CD34<sup>+</sup> cells, particularly the CD38<sup>-</sup> subpopulation, as compared to IM-sensitive K562 cells. MDR1 was expressed at low level and dye efflux studies demonstrated very little MDR1 activity in CML CD34<sup>+</sup> cells. Furthermore, combination treatment of primitive CML cells with IM and the MDR1 inhibitor PSC833 did not result in elevated cell-associated IM levels. Although we observed slightly enhanced cytostasis with IM when combined with PSC833, this was independent of BCR-ABL inhibition because no associated decrease in phospho-CrkL was observed.</p>
<p><b>Conclusions:</b> Our findings demonstrate that inhibition of MDR1 neither enhances the effect of IM against BCR-ABL activity, nor significantly potentiates IM's efficiency in eliminating primitive CML cells.</p>
Abcg2 overexpression represents a novel mechanism for acquired resistance to the multi-kinase inhibitor Danusertib in BCR-ABL-positive cells in vitro.
No full textThe success of Imatinib (IM) therapy in chronic myeloid leukemia (CML) is compromised by the development of IM resistance and by a limited IM effect on hematopoietic stem cells. Danusertib (formerly PHA-739358) is a potent pan-aurora and ABL kinase inhibitor with activity against known BCR-ABL mutations, including T315I. Here, the individual contribution of both signaling pathways to the therapeutic effect of Danusertib as well as mechanisms underlying the development of resistance and, as a consequence, strategies to overcome resistance to Danusertib were investigated. Starting at low concentrations, a dose-dependent inhibition of BCR-ABL activity was observed, whereas inhibition of aurora kinase activity required higher concentrations, pointing to a therapeutic window between the two effects. Interestingly, the emergence of resistant clones during Danusertib exposure in vitro occurred considerably less frequently than with comparable concentrations of IM. In addition, Danusertib-resistant clones had no mutations in BCR-ABL or aurora kinase domains and remained IM-sensitive. Overexpression of Abcg2 efflux transporter was identified and functionally validated as the predominant mechanism of acquired Danusertib resistance <i>in vitro</i>. Finally, the combined treatment with IM and Danusertib significantly reduced the emergence of drug resistance <i>in vitro</i>, raising hope that this drug combination may also achieve more durable disease control <i>in vivo</i>
PKA and PDE4D3 anchoring to AKAP9 provides distinct regulation of cAMP signals at the centrosome
No full textPrevious work has shown that the protein kinase A (PKA)–regulated phosphodiesterase (PDE) 4D3 binds to A kinase–anchoring proteins (AKAPs). One such protein, AKAP9, localizes to the centrosome. In this paper, we investigate whether a PKA–PDE4D3–AKAP9 complex can generate spatial compartmentalization of cyclic adenosine monophosphate (cAMP) signaling at the centrosome. Real-time imaging of fluorescence resonance energy transfer reporters shows that centrosomal PDE4D3 modulated a dynamic microdomain within which cAMP concentration selectively changed over the cell cycle. AKAP9-anchored, centrosomal PKA showed a reduced activation threshold as a consequence of increased autophosphorylation of its regulatory subunit at S114. Finally, disruption of the centrosomal cAMP microdomain by local displacement of PDE4D3 impaired cell cycle progression as a result of accumulation of cells in prophase. Our findings describe a novel mechanism of PKA activity regulation that relies on binding to AKAPs and consequent modulation of the enzyme activation threshold rather than on overall changes in cAMP levels. Further, we provide for the first time direct evidence that control of cell cycle progression relies on unique regulation of centrosomal cAMP/PKA signals
Nilotinib concentration in cell lines and primary CD34+ chronic myeloid leukemia cells is not mediated by active uptake or efflux by major drug transporters
No full textImatinib 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<sup>+</sup> cells. One possible explanation is that CD34<sup>+</sup> ells 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<sup>+</sup>) 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<sup>+</sup> 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<sup>+</sup>) 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
Nilotinib exerts equipotent antiproliferative effects to imatinib and does not induce apoptosis in CD34<sup>+</sup> CML cells
No full textChronic myeloid leukemia (CML) stem and progenitor cells overexpress BcrAbl and are insensitive to imatinib mesylate (IM). We therefore investigated whether these cells were efficiently targeted by nilotinib. In K562, the inhibitory concentration (IC50) of nilotinib was 30 nM versus 600 nM for IM, consistent with its reported 20-fold-higher potency. However, in primary CD34<sup>+</sup> CML cells, nilotinib and IM were equipotent for inhibition of BcrAbl activity, producing equivalent but incomplete reduction in CrkL phosphorylation at 5 mu M. CML CD34<sup>+</sup> cells were still able to expand over 72 hours with 5 mu M of either drug, although there was a concentration-dependent restriction of amplification. As for IM, the most primitive cells (CFSEmax) persisted and accumulated over 72 hours with nilotinib and remained caspase-3 negative. Furthermore, nilotinib with IM led to further accumulation of this population, suggesting at least additive antiproliferative effects. These results confirmed that, like IM, the predominant effect of nilotinib is antiproliferative rather than proapoptotic
Uptake of synthetic low density lipoprotein by leukemic stem cells — a potential stem cell targeted drug delivery strategy
No full textChronic Myeloid Leukemia (CML) stem/progenitor cells, which over-express Bcr-Abl, respond to imatinib by a reversible block in proliferation without significant apoptosis. As a result, patients are unlikely to be cured owing to the persistence of leukemic quiescent stem cells (QSC) capable of initiating relapse. Previously, we have reported that intracellular levels of imatinib in primary primitive CML cells (CD34<sup>+</sup>38<sup>lo/−</sup>), are significantly lower than in CML progenitor cells (total CD34<sup>+</sup>) and leukemic cell lines. The aim of this study was to determine if potentially sub-therapeutic intracellular drug concentrations in persistent leukemic QSC may be overcome by targeted drug delivery using synthetic Low Density Lipoprotein (sLDL) particles. As a first step towards this goal, however, the extent of uptake of sLDL by leukemic cell lines and CML patient stem/progenitor cells was investigated. Results with non-drug loaded particles have shown an increased and preferential uptake of sLDL by Bcr-Abl positive cell lines in comparison to Bcr-Abl negative. Furthermore, CML CD34<sup>+</sup> and primitive CD34<sup>+</sup>38<sup>lo/−</sup> cells accumulated significantly higher levels of sLDL when compared with non-CML CD34<sup>+</sup> cells. Thus, drug-loading the sLDL nanoparticles could potentially enhance intracellular drug concentrations in primitive CML cells and thus aid their eradication
SEQUENTIAL POSITIVE REAL TIME PCR ASSAYS ARE HIGHLY PREDICTIVE OF INVASIVE FUNGAL INFECTIONS IN HIGH AND HIGH-INTERMEDIATE RISK HAEMATOLOGY PATIENTS
No full textAnalysis of imatinib in bone marrow and plasma samples of chronic myeloid leukaemia patients using solid phase extraction LC-ESI-MS
No full textThe LC-ESI-MS was developed and validated for the analysis of imatinib in plasma and bone marrow samples using deuterated imatinib (D(8)-IM) as an internal standard. The biological samples were extracted using Strata-X-C SPE cartridges and separated on C<sub>8</sub> column (50 x 3 mm, 3 µm), and methanol: 0.1% formic acid (70:30) was delivered at the rate of 0.7 ml/min as a mobile phase. Imatinib was quantified in samples by monitoring the ions m/z 494.3 for imatinib and 502.3 for D<sub>8</sub>-imatinib on mass spectrometer. The method was linear in the concentration range of 1-1500 ng/250 µl in spiked human plasma samples and limit of quantification was 5 ng/mL. Inter-day and intra-day variations in spiked human plasma spiked with 50, 250 and 500 ng /mL were less than 3.16%. The repeatability and reproducibility and other parameters of the methods were also validated. The method was employed for the analysis of the imatinib in human plasma and bone marrow samples. The drug levels in bone marrow and plasma samples were correlated to the degree of cytogenetic response. No significant difference of imatinib level between blood and bone marrow in IM-treated patients dosed to steady state was observed
Omacetaxine may have a role in chronic myeloid leukaemia eradication through downregulation of Mcl-1 and induction of apoptosis in stem/progenitor cells
No full textChronic 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<sup>+</sup>38<sup>lo</sup>) by downregulation of Mcl-1 protein. In contrast to our previous findings with TKIs, omacetaxine did not accumulate undivided cells <i>in vitro</i>. 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<sup>+</sup> 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
Chronic myeloid leukemia stem cells are not dependent on Bcr-Abl kinase activity for their survival
No full textRecent evidence suggests CML stem cells are insensitive to kinase inhibitors and responsible for minimal residual disease in treated patients. We investigated whether CML stem cells, in a transgenic mouse model of CML-like disease or derived from patients, are dependent on Bcr-Abl. In the transgenic model, following re-transplantation, donor-derived CML stem cells in which Bcr-Abl expression had been induced and subsequently shut off, were able to persist in vivo and re-initiate leukemia in secondary recipients upon Bcr-Abl re-expression. Bcr-Abl knockdown in human CD34+ CML cells cultured for 12 days in physiological growth factors achieved partial inhibition of Bcr-Abl and downstream targets p-CrkL and p-STAT5, inhibition of proliferation and colony forming cells, but no reduction of input cells. The addition of dasatinib further inhibited p-CrkL and p-STAT5, yet only reduced input cells by 50%. Complete growth factor withdrawal plus dasatinib further reduced input cells to 10%, however the surviving fraction was enriched for primitive leukemic cells capable of growth in long-term culture initiating cell assay and expansion upon removal of dasatinib and addition of growth factors. Together these data suggest that CML stem cell survival is Bcr-Abl kinase independent and suggest curative approaches in CML must focus on kinase-independent mechanisms of resistance
