Drug Efflux Transporters and Imatinib Mesylate Insensitivity in Chronic Myeloid Leukemia

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 4424-4424
Author(s):  
Caroline Fátima Aquino Moreira-Nunes ◽  
Ana Cristina Simões Beltrão ◽  
Larissa Tatiana Valente Martins Francês ◽  
Rodrigo Guarischi Mattos Amaral Sousa ◽  
Israel Tojal Silva ◽  
...  
Keyword(s):  
Stem Cells ◽  
Chronic Myeloid Leukemia ◽  
Imatinib Mesylate ◽  
Differentially Expressed Genes ◽  
Myeloid Leukemia ◽  
Molecular Therapy ◽  
Differentially Expressed ◽  
Drug Efflux ◽  
Pool Sample ◽  
Cd34 Cells

Abstract Abstract 4424 Background. Chronic Myeloid Leukemia represents the first human cancer in which a molecular therapy produces an effective clinical response (Holtz Blood 99:3792-800, 2002). The imatinib mesylate (IM) (Novartis Pharmaceuticals™) is a drug, that was designated to inhibit selectively certain tyrosine kinase proteins involved in the process of oncogenesis (Guilhot, The Oncologist, 9:271-81, 2004). In 2005, Michor (Nature 435:1267-70), through mathematical model, concluded that imatinib efficiently reduces the differentiated leukemic cells population, but it has not the same effect on the cell population that drives this disease, the CD34+ leukemic stem cells, which can be kept alive during the treatment. The search for the main causes of imatinib resistance has been intensified in recent years, with a special focus on the possible role of drug transporters (Apperley, Lancet Oncol 8:1018–29, 2007; Baker and Reddy, Mount Sinai J Med, 77:573–86, 2010). These proteins are the main determinants of the intracellular drug concentration, and how they actively regulating the traffic of small molecules through the cell membrane (Melo, Blood 108:1116-7, 2006). Thus, a cell can be resistant to a drug, because the optimum amount of the drug does not stay inside it to a significant effect to be achieved (Kim, Toxicology 182:291-7, 2002). Purpose. Identify differentially expressed genes in CD34+ and CD66b+ cells as candidates for IM transport. Methods. Samples of bone marrow (BM) and peripheral blood (PB) were obtained from five patients with CML treated with imatinib in better then optimal response according to European LeukemiaNet criteria (Baccarani, Blood 108:1809-20, 2006). Cells Isolation and RNA extraction. CD34+ cells were isolated from BM of five patients with CML. Likewise, mature CD66b+ PB cells were isolated from the same patients. SOLiD sequencing and sequence analysis. cDNA was sequenced according to the manufacturer's protocols for the SOLiD Total RNA-Seq kit for whole transcriptome. Data Analysis. To characterize the class genes, we analyzed the Gene Ontology (GO) annotation (Ashburner, Nature Genetics 25:25-9, 2000), and the software Cufflinks (Trapnell, Nature Biotechnology 28:511–15, 2010) were used to identify the differential expression of genes in both samples, in patients (BM × PB) and in control (BM × PB). The difference in gene expression between compared samples, were calculated based on P < 0.05 significance, were called differentially expressed genes those who submit P ≤ 0.05. Results. In pool sample of patients, it was possible to identify the genes SLC22A1 (OCT1) – in both, BM and PB pool samples, without any significant change (p ≤ 0,05) - and SLCO1A2 (OATP1A2) – only in PB sample. Thus its presence could not be identified in any of the control samples, which may reinforce the fact that these channels are actually responsible for the influx of imatinib in cells from patients undergoing treatment (Crossman, Blood:1133-4, 2005; Hu, Clin Cancer Res 14:3141–8, 2008). The presence of ABC gene family (ABCB1; ABCG2; ABCC1), described in the literature as being responsible for imatinib efflux (Jordanides, Blood 108: 1370–3, 2006; Brendel, Leukemia 21:1267–75, 2007) were found only in BM cells of patients. The presence of other two genes responsible for the drug efflux was also found exclusively in BM pool sample of patients, SLC47A1 and SLC47A2. These genes known as Human multidrug and toxin extrusion (MATE1 and MATE2) have also being identified as one important efflux mechanism of various drugs (Yonezawa and Inui, British J Pharmacol, 164:1817–25, 2011; Minematsu and Giacomini, Mol Cancer Ther, 10:531–9, 2011). Conclusion. The presence of more drug influx channels - the SLC family (OCT1 and OATP1A2) in mature cell - and absence of drug efflux channels - family ABC (ABCB1, ABCG2, ABCC1) and MATE genes (SLC47A1 and SLC47A2) - and the reverse in stem cells (CD34+) of patients with CML analyzed in this study may be the answer of why the insensitivity of CD34+ cells to treatment with IM and consequent failure to eliminate minimal residual disease. These genes can be candidates to therapeutic targets in CML. Disclosures: Lemos: Novartis of Brazil: Research Funding.

scholarly journals Differentially expressed genes responsible for insensitivity of CD34+ cells to kinase inhibitors in patients with chronic myeloid leukemia

2013 ◽  
Vol 7 (S2) ◽  
Author(s):  
Caroline FA Moreira-Nunes ◽  
Tereza CB Azevedo ◽  
Ana CS Beltrão ◽  
Larissa TVM Francês ◽  
Rodrigo GMA Sousa ◽  
...  
Keyword(s):  
Chronic Myeloid Leukemia ◽  
Differentially Expressed Genes ◽  
Myeloid Leukemia ◽  
Kinase Inhibitors ◽  
Differentially Expressed ◽  
Cd34 Cells

scholarly journals Comparison of the Transcriptomic Signatures in Pediatric and Adult CML

Cancers ◽  
2021 ◽  
Vol 13 (24) ◽  
pp. 6263
Author(s):  
Minyoung Youn ◽  
Stephanie M. Smith ◽  
Alex Gia Lee ◽  
Hee-Don Chae ◽  
Elizabeth Spiteri ◽  
...  
Keyword(s):  
Chronic Myeloid Leukemia ◽  
Rna Sequencing ◽  
Differentially Expressed Genes ◽  
High Throughput ◽  
Myeloid Leukemia ◽  
Differentially Expressed ◽  
Molecular Characteristics ◽  
Healthy Controls ◽  
Cd34 Cells ◽  
Transcriptomic Signature

Children with chronic myeloid leukemia (CML) tend to present with higher white blood counts and larger spleens than adults with CML, suggesting that the biology of pediatric and adult CML may differ. To investigate whether pediatric and adult CML have unique molecular characteristics, we studied the transcriptomic signature of pediatric and adult CML CD34+ cells and healthy pediatric and adult CD34+ control cells. Using high-throughput RNA sequencing, we found 567 genes (207 up- and 360 downregulated) differentially expressed in pediatric CML CD34+ cells compared to pediatric healthy CD34+ cells. Directly comparing pediatric and adult CML CD34+ cells, 398 genes (258 up- and 140 downregulated), including many in the Rho pathway, were differentially expressed in pediatric CML CD34+ cells. Using RT-qPCR to verify differentially expressed genes, VAV2 and ARHGAP27 were significantly upregulated in adult CML CD34+ cells compared to pediatric CML CD34+ cells. NCF1, CYBB, and S100A8 were upregulated in adult CML CD34+ cells but not in pediatric CML CD34+ cells, compared to healthy controls. In contrast, DLC1 was significantly upregulated in pediatric CML CD34+ cells but not in adult CML CD34+ cells, compared to healthy controls. These results demonstrate unique molecular characteristics of pediatric CML, such as dysregulation of the Rho pathway, which may contribute to clinical differences between pediatric and adult patients.

Functional ABCG2 is overexpressed on primary CML CD34+ cells and is inhibited by imatinib mesylate

Blood ◽  
2006 ◽  
Vol 108 (4) ◽  
pp. 1370-1373 ◽  
Author(s):  
Niove E. Jordanides ◽  
Heather G. Jorgensen ◽  
Tessa L. Holyoake ◽  
Joanne C. Mountford
Keyword(s):  
Chronic Myeloid Leukemia ◽  
Cell Line ◽  
Imatinib Mesylate ◽  
Cell Population ◽  
Myeloid Leukemia ◽  
Philadelphia Chromosome ◽  
Drug Transporter ◽  
Cd34 Cells ◽  
Considerable Debate ◽  
Intracellular Concentrations

Abstract Imatinib mesylate (IM) therapy for chronic myeloid leukemia (CML) has transformed the treatment of this disease. However, the vast majority of patients, despite major responses, still harbor Philadelphia chromosome–positive (Ph+) cells. We have described a population of primitive Ph+ cells that are insensitive to IM and may be a source of IM resistance. Cell line studies have suggested that the drug transporter ABCG2 may be a mediator of IM resistance, however there is considerable debate about whether IM is an ABCG2 substrate or inhibitor. We demonstrate here that primitive CML CD34+ cells aberrantly overexpress functional ABCG2 but that cotreatment with IM and an ABCG2 inhibitor does not potentiate the effect of IM. We definitively show that IM is an inhibitor of, but not a substrate for, ABCG2 and that, therefore, ABCG2 does not modulate intracellular concentrations of IM in this clinically relevant cell population.

Evaluation of Ph+/CD34+ Residual Cells in Chronic Myeloid Leukemia Patients after Long Lasting Treatment with Imatinib Mesylate.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1999-1999
Author(s):  
Monica Bocchia ◽  
Elisabetta Abruzzese ◽  
Micaela Ippoliti ◽  
Simona Calabrese ◽  
Alessandro Gozzetti ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Chronic Myeloid Leukemia ◽  
Imatinib Mesylate ◽  
Median Time ◽  
False Positive ◽  
Myeloid Leukemia ◽  
Prolonged Treatment ◽  
Published Data ◽  
Imatinib Treatment ◽  
Cd34 Cells

Abstract Although the success of imatinib mesylate therapy represents an exciting advance in targeted cancer therapy, it has still to be determined whether responses to this p210 inhibitor in chronic myeloid leukemia (CML) patients will be durable. In fact most of clinical studies agree on the evidence of a persistent molecular disease in the majority of treated patients and altough the absolute level of bcr-abl transcript may vary over the treatment, yet a molecular complete response is of rare observation. In addition, discontinuation of imatinib exerts always in rapid loss of response. In accordance to this the persistence of malignant progenitors in patients in complete cytogenetic response (CCR) after short term imatinib treatment, has been recently demonstrated. In particular, Bathia et al. showed in 12/15 patients studied after a median time of 10 months of imatinib treatment a median of 11% of residual CML CD34+ progenitors in the bone marrow (by FISH Dual Fusion bcr/abl analysis)while only 3/15 patients had no detectable residual CD34+ cells. Less is known about residual Ph+/CD34+ cells surviving after a prolonged therapy with this targeting drug. Thus, we evaluated the amount of bone marrow residual CD34+ cells in 17 CML patients in stable CCR after a long lasting treatment with imatinib. At the time of evaluation, the patients were on conventional dose (400mg) Imatinib for a median time of 48 months (range 36–58 months) having achieved a CCR status (conventionally defined as the complete absence of t(9;22) on caryotypic analysis) within 3 to 6 months of treatment. However all of them still showed molecular disease as detected by nested RT-PCR. Bone marrow CD34+ cell-enriched populations were selected from mononuclear cells using immunomagnetic column separation and were evaluated after cytospin by FISH using a bcr-abl Dual Color Extra Signal Probe(LSI bcr-abl ES, Vysis), that is able to detect bcr-abl fusion in interphase nuclei with a false positive signal rate close to 0. A minimum of 100 CD34+ nuclei per each sample were evaluated. Interestingly, in 8/17 patients no Ph+/CD34+ cells were detected, while in the remaining 9/17 patients a median of 2% (range 0.5–11%) of bcr-abl positive progenitors were still observed. In this small selected serie of patients prolonged treatment with imatinib appears to be correlated with a lower, yet detectable, amount of residual bone marrow Ph+/CD34+ cells when compared to previously published data. This result could be partly explained with the different specificity and sensitivity of the probe used (bcr/abl ES<1% false positive; bcr-abl Dual Fusion 8–10% false positive) The clinical significance of these data as well as the role of this cell target to monitor minimal residual disease in CML needs to be evaluated on a larger serie of patients.

Gene Expression Signatures Associated with Treatment and Resistance to Imatinib Mesylate in Chronic Myeloid Leukemia Patients.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 433-433 ◽  
Author(s):  
Vivian Oehler ◽  
S. Branford ◽  
E. Pogosova-Agadjanyan ◽  
N. Shah ◽  
C. Sawyers ◽  
...  
Keyword(s):  
Gene Expression ◽  
Chronic Myeloid Leukemia ◽  
Differential Expression ◽  
Imatinib Mesylate ◽  
Myeloid Leukemia ◽  
Secondary Resistance ◽  
Total Rna ◽  
Expression Of Genes ◽  
Cd34 Cells ◽  
Study Gene Expression

Abstract Imatinib mesylate (IM) has dramatically changed the treatment approach for patients with chronic myeloid leukemia (CML). However, ~20% of chronic phase (CP) patients are initially resistant to IM, and among patients who achieve a complete cytogenetic response (CCyR), a small minority relapse back into CP or progress to advanced disease. Abl tyrosine kinase domain mutations are the major cause of secondary resistance. Oligonucleotide microarray analysis was used to study gene expression patterns associated with primary IM resistance and relapse after initial successful response to IM. Samples included total RNA from diagnostic samples of 25 CML CP patients within 6 months of diagnosis and at the time of failure (7 patients); total RNA from 18 patients who relapsed after initial CCyR with documented Abl point mutations (12 CP and 6 BC); and 10 myeloid progenitor samples (sorted by CD34 and CD38 status) from an IM naïve and IM resistant (R) patient, both in blast crisis. Results: For primary IM resistance, analysis of paired samples before and after treatment revealed that primary failure was associated with the differential expression of genes associated with RNA post-transcriptional modification, protein synthesis, cellular growth and proliferation, and cell death. Two genes with the highest differential expression included the apotosis resistance genes API5 and TRAF5. TRAF5 was also increased in patients who relapsed after initial CCyR, while API5 showed significantly increased expression in sorted CD34+ cells from an IMR patient. In secondary resistance patients, a set of drug transporters including ABCA2, ABCA3, MDR1, and ABCC3 had increased expression and hOCT1 decreased expression relative to 42 IM naïve CP patients. In vitro experiments compared K562 resistant (R) and sensitive (S) cell lines over time exposed to IM. The K562 IMR cell line showed a 1.5 log higher expression of ABCG2 and a 1 log higher expression of TRAF5 compared to the K562 IMS cells. However, sequential clinical samples of 16 IM non-responders vs. 14 CCyR patients showed no change in ABCG2 expression, possibly because ABCG2 is expressed only in early progenitor cells, not differentiated cells. The importance of using CD34+ cells was demonstrated in 7 array studies comparing gene expression in CD34+ selected cells from an IMR patient compared to an IM naïve patient. IM resistance was associated with increased expression of genes associated with cell cycle, DNA recombination and repair, and proliferation. Genes associated with apoptosis resistance included increased expression of API5, survivin, and decreased expression of BAK1. Protein serine/threonine and tyrosine kinases associated with cell proliferation/survival and tumor progression with increased expression in the IMR patient included: AURKB, AKT3, BUB1B, CDC2, CHEK1, MAPK9, STK6, TTK, and WEE1. Conclusion: Gene expression studies suggest that primary resistance to IM therapy is associated with resistance to apoptosis; relapse on IM is associated with activation of drug transporter genes and genes associated with disease progression; in studying disease response and resistance, primitive hematopoetic cells are critical for analysis.

Individual Gene Expression Profiling of Bone Marrow CD34 Cells in Acquired Severe Aplastic Anemia (aSAA) in Children.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 978-978 ◽  
Author(s):  
Bernd Hubner ◽  
Sylvia Merk ◽  
Sonja Rauhut ◽  
Martin Dugas ◽  
Torsten Haferlach ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Bone Marrow ◽  
Differentially Expressed Genes ◽  
Expression Profiling ◽  
Cell Metabolism ◽  
Cell Communication ◽  
Differentially Expressed ◽  
Cd34 Cells ◽  
The Individual

Abstract Acquired SAA in children is a rare, life-threatening disease characterized by pancytopenia and bone marrow hypocellularity. There is good clinical and laboratory evidence that a T-cell mediated immune attack against stem and progenitor cells plays an important role in the pathogenesis of SAA. However, due to the paucity of residual CD34 positive cells at diagnosis still only little is known about the stem cells and their response to the autoimmune attack in SAA in children. To further investigate the characteristics of CD34 cells in SAA we compared the individual transcriptomes of CD34 cells of 9 newly diagnosed, untreated pediatric SAA patients with 8 pediatric healthy controls. Hematopoietic stem cells were isolated with high efficiency from bone marrow by Ficoll density centrifugation and subsequent affinity purification using Dynabeads (Dynal, Invitrogen). Expression profiling experiments were performed using the two cycle amplification system and the HG-U133 plus 2.0 array (Affymetrix). Gene expression data were analyzed using R 2.3.0 and Bioconductor 1.8. packages (Affymetrix, multtest). Raw data were normalized using robust multiarray average (RMA) algorithm. Probe sets with “absent” calls in more than 50% of samples in the smaller group were identified and omitted from further analysis. To determine differentially expressed genes, t-test was applied. P value adjustments for multiple comparisons were done using the step-up false discovery rate (FDR) controlling method proposed by Benjamini and Hochberg. Overall 402 genes were differentially expressed in children with SAA compared to controls (p &lt; 0.05), 288 genes were downregulated and 114 were upregulated. Gene ontology analyses (FatiGO) indicated that biological processes in CD34 cells are significantly affected in pediatric SAA by mainly downregulation of genes for cell metabolism (78 down, 30 up), cell communication/adhesion (48 down, 25 up), growth and differentiation (15 down, 4 up) and stress response (16 down, 3 up). Unexpectedly only very few genes involved in cell death/apoptosis (5 down, 4 up) were differentially expressed. Genes encoding for DNA/RNA binding proteins (28 down, 14 up) and ion binding proteins (47 down, 18 up) were also mainly downregulated. Despite the extremely low numbers of residual CD34 cells present in the bone marrow of children with untreated SAA we were able to analyze the individual transcriptome pattern of single patients. These patterns showed homogeneously and significantly different gene expressions in the group of affected children when compared to controls. Genes involved in apoptosis seem to be less altered in there expression than expected from adult data. These observation might be consistent with the major clinical finding in these children of almost empty bone marrows where most of the apoptotic cell death has already taken place. In the tiny population of “survivors” most of the differentially expressed genes are involved in cell metabolism and cell communication or adhesion. These unexpected results provide new hints for further investigations regarding the involvement of CD34 cells in the pathogenesis of childhood aSAA.

p53 and c-Myc Are Critical Signaling Hubs That Maintain Chronic Myeloid Leukemia

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1465-1465
Author(s):  
Sheela A Abraham ◽  
Lisa Hopcroft ◽  
Emma Carrick ◽  
Andrew Williamson ◽  
Andrew Pierce ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Stem Cells ◽  
Flow Cytometry ◽  
Chronic Myeloid Leukemia ◽  
Cell Expansion ◽  
Myeloid Leukemia ◽  
Dose Effect ◽  
Dependent Manner ◽  
Cd34 Cells ◽  
Hub Proteins

Abstract Chronic myeloid leukemia (CML) is a clonal disorder of the hematopoietic system, leading to increased production of mature and progenitor myeloid cells. Although protein tyrosine kinase inhibitors (TKI) have been successful in managing the disease, there are exceptions where drug resistance and onset of blast crisis occur. Furthermore TKIs are ineffective against leukemic stem cells (LSC) that are responsible for disease initiation and maintenance. We have shown mRNA changes in primitive hematopoietic cells do not correlate directly to protein changes. Therefore to elucidate fundamental cellular differences between CML and normal cells we employed a proteomic approach (mass spectrometry with isobaric tagging for relative quantification). This approach permits unbiased analyses using direct comparative quantification of peptides and thus proteins from chronic phase CML and normal CD34+ human samples. Systematic data analysis identified that the majority of deregulated proteins are connected and regulated by two oncogenes with well defined roles in human disease, p53 and c-myc. The direction of regulation inferred suppression of p53 and up-regulation of c-myc. Altered expression of key proteins was validated using western blotting and immuno-fluorescence approaches. All (6/6) candidate/hub proteins identified using mass spectrometry were confirmed using these orthogonal approaches. Based on our systematic analysis, we targeted the candidate hubs using the drugs RITA (activates p53) and CPI-203 (inhibits c-myc expression; provided by Constellation Pharmaceuticals). In CML CD34+ cells, RITA reduced cell expansion in a concentration-dependent manner and induced significant levels of apoptosis as confirmed by positive staining of Annexin V and 4',6-diamidino-2-phenylindole (DAPI) using flow cytometry. CPI-203 also reduced cell expansion, but importantly induced differentiation in addition to apoptosis, as supported by flow cytometric monitoring of levels of carboxyfluorescein succinimidyl ester (CFSE) and CD34. Overlays of CFSE plots for untreated control vs. CPI-203 demonstrated that as cells divided in the presence of CPI-203, there was clear and rapid loss of CD34 expression which was not seen with RITA treatment. By measuring the dose-effect relationship of each drug alone and in combination, we demonstrated potent synergy with combination index (CI) values ranging from 0.034-0.286 based on loss of cell viability. Using flow cytometry we gated on CD34+38- CML cells to enable quantification of the differential effects of each drug alone and in combination against the most primitive and quiescent 1-5% of total CD34+ cells. Critically the apoptotic effect was inclusive of primitive CD34+38- cells and quiescent CFSEmax populations. In addition, experiments combining RITA and CPI-203 demonstrated undetectable colony forming cell units at the highest concentrations of drug used. Importantly, it appears that combining these two drugs has negligible effects on normal CD34+ cell counts, apoptosis and CFSE profiles. Currently NOD-SCID IL2R gamma null (NSG) repopulation assays are underway to determine if these drugs affect stem cells capable of engrafting immunocompromised mice. Our systems biology approach suggests that altered c-myc and p53 function underlie the most significant cellular differences within CML CD34+ cells, which has not been previously demonstrated. We confirm that in CML, p53 and c-myc hub proteins have the ability to modulate downstream defined target proteins thereby enhancing survival and proliferation and thus allowing maintenance of disease. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Establishment of a Reproducible Model of Chronic-Phase Chronic Myeloid Leukemia in NOD/SCID Mice Using Blood-Derived Mononuclear or CD34+ Cells

Blood ◽  
1998 ◽  
Vol 91 (2) ◽  
pp. 630-640 ◽  
Author(s):  
Ian D. Lewis ◽  
Louise A. McDiarmid ◽  
Leanne M. Samels ◽  
L. Bik To ◽  
Timothy P. Hughes
Keyword(s):  
Stem Cells ◽  
Chronic Myeloid Leukemia ◽  
B Cell ◽  
Blood Cells ◽  
Myeloid Leukemia ◽  
Scid Mice ◽  
Colony Stimulating Factor ◽  
Cell Engraftment ◽  
Cd34 Cells ◽  
Stimulating Factor

Abstract An animal model of chronic myeloid leukemia (CML) will help characterize leukemic and normal stem cells and also help evaluate experimental therapies in this disease. We have established a model of CML in the NOD/SCID mouse. Infusion of ≥4 × 107chronic-phase CML peripheral blood cells results in engraftment levels of ≥1% in the bone marrow (BM) of 84% of mice. Engraftment of the spleen was seen in 60% of mice with BM engraftment. Intraperitoneal injection of recombinant stem cell factor produced a higher level of leukemic engraftment without increasing Philadelphia-negative engraftment. Granulocyte colony-stimulating factor and granulocyte-macrophage colony-stimulating factor did not increase the level of leukemic or residual normal engraftment. Assessment of differential engraftment of normal and leukemic cells by fluorescence in situ hybridization analysis with bcr and abl probes showed that a median of 35% (range, 5% to 91%) of engrafted cells present in the murine BM were leukemic. BM engraftment was multilineage with myeloid, B-cell, and T-cell engraftment, whereas T cells were the predominant cell type in the spleen. BM morphology showed evidence of eosinophilia and increased megakaryocytes. We also assessed the ability of selected CD34+ CML blood cells to engraft NOD/SCID mice and showed engraftment with cell doses of 7 to 10 × 106 cells. CD34− cells failed to engraft at cell doses of 1.2 to 5 × 107. CD34+ cells produced myeloid and B-cell engraftment with high levels of CD34+ cells detected. Thus, normal and leukemic stem cells are present in CD34+ blood cells from CML patients at diagnosis and lead to development of the typical features of CML in murine BM. This model is suitable to evaluate therapy in CML.

Nilotinib exerts equipotent antiproliferative effects to imatinib and does not induce apoptosis in CD34+ CML cells

Blood ◽  
2007 ◽  
Vol 109 (9) ◽  
pp. 4016-4019 ◽  
Author(s):  
Heather G. Jørgensen ◽  
Elaine K. Allan ◽  
Niove E. Jordanides ◽  
Joanne C. Mountford ◽  
Tessa L. Holyoake
Keyword(s):  
Chronic Myeloid Leukemia ◽  
Progenitor Cells ◽  
Imatinib Mesylate ◽  
Myeloid Leukemia ◽  
Caspase 3 ◽  
Inhibitory Concentration ◽  
Antiproliferative Effects ◽  
Cd34 Cells ◽  
Stem And Progenitor Cells ◽  
Predominant Effect

Abstract Chronic 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+ CML cells, nilotinib and IM were equipotent for inhibition of BcrAbl activity, producing equivalent but incomplete reduction in CrkL phosphorylation at 5 μM. CML CD34+ cells were still able to expand over 72 hours with 5 μM 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.

Transcriptome of Expressed Genes in CD34+ and CD66b+ Chronic Myeloid Leukemia Cells and Its Potential Role in the Transport of Kinase Inhibitors

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 4426-4426
Author(s):  
Caroline Fátima Aquino Moreira-Nnes ◽  
Ana Cristina Simões Beltrão ◽  
Tereza Cristina Brito Azevedo ◽  
Larissa Tatiana Valente Martins Francês ◽  
Rodrigo Guarischi Mattos Amaral Sousa ◽  
...  
Keyword(s):  
Stem Cells ◽  
Chronic Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Kinase Inhibitors ◽  
Leukemic Cells ◽  
Cancer Resistance ◽  
Optimal Response ◽  
Oncology Research ◽  
Cd34 Cells ◽  
Long Term Treatment

Abstract Abstract 4426 Background. Chronic Myeloid Leukemia (CML) is a clonal myeloproliferative disorder characterized by Phildaelphia chromosome and by formation of BCR-ABL fusion. Some studies have shown that residual cells are part of the leukemic cells undifferentiated compartment. In 2005, Michor et al. (Nature 435: 1267–1270), through mathematical model, concluded that imatinib efficiently reduces the differentiated leukemic cells population, but it has not the same effect on the cell population that drives this disease, the CD34+ leukemic stem cells, which can be kept alive during the treatment. However, Mahon et al. (Lancet Oncol 11: 1029–35, 2010) described a cohort in which patients remained disease free for 18 months after discontinued treatment. This finding is an indication that the leukemia stem cells (LSC) are not totally insensitive to kinase inhibitors (KIs). Purpose. Identify the expressed genes in CD34+ and CD66b+ cells as candidates for KIs transport. Methods. Samples of bone marrow (BM) and peripheral blood (PB) were obtained from five patients with CML treated with imatinib in optimal response in according to European LeukemiaNet criteria. Cells Isolation and RNA extraction. CD34+ cells were isolated from BM of five patients with CML. Mature CD66b+ PB cells were isolated from the same patients. SOLiD sequencing. cDNA was sequenced according to the manufacturer's protocols for the SOLiD. Transcriptome. Two libraries were constructed for this purpose and approximately 120 millions of beads were deposited on a half slide for each library and sequenced using the Opti Fragment Library Sequencing kit-Master Mix 50 on a SOLiD machine (Ver 3+). To characterize the genes showing differential regulation, we analyzed the Gene Ontology (GO) annotation associated with transporters genes exhibiting a greater than 2-fold difference in expression by RNA-seq. Analysis using the functional annotation clustering feature of DAVID. Results. We have sequenced 14.133 genes in CD34+ pool cells and 3.379 genes in CD66b+ pool cells, with 2.883 genes expressed in both. Of these, 1.201 genes from membrane transport were functionally annotated, and we have found 560 genes expressed exclusively in CD34+, and 99 genes in CD66b+, and 542 genes in both, as shown below. Regarding imatinib transportation, two major classes of transporters are widely recognized for its importance in drug influx and efflux inside the cell, the ATP-biding cassette transporters (ABC family) and Solute Carrier family (SLC family). Studies have demonstrated that the organic cation transporter 1 (OCT-1, also known as SLC22A1) is the major active influx transporter for imatinib in CML cells, in our study we find no evidence of OCT-1 in any of our samples, which may indicate that this is not the channel for the influx of drugs into cells. Within ABC family is already recognized the important function of at least three genes in multidrug-resistance cancer through the mechanism of drug efflux through the membrane, they are: ABCB1 (MDR1); ABCG2 (Breast Cancer Resistance Protein – BCRP) e ABCC1 (MRP1). These genes were found exclusively in the CD34+ lineage of CML patients, reinforcing the theory that HSC are resistant to treatment with imatinib, through the expression of efflux channels. Conclusion. The efflux channel genes exclusively expressed in CD34+ cells represents a major barrier to maintaining optimal response to KIs in long-term treatment of CML patients. Those genes should be investigated to achieve the development of drugs with potential to block the efflux channels and improve outcome for cancer patients. Disclosures: Lemos: Novartis Oncology: Research Funding.

Sign in / Sign up

Export Citation Format

Share Document