Enhancing the Functional Activity of the OCT-1 Influx Pump May Overcome the Negative Impact of Low OCT-1 Activity in Imatinib Treated CML Patients

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 723-723 ◽  
Author(s):  
Deborah White ◽  
Phuong Dang ◽  
Kelvin Groot Obbink ◽  
Amity Frede ◽  
Chung Kok ◽  
...  
Keyword(s):  
Cell Lines ◽  
Functional Activity ◽  
Blood Cells ◽  
Negative Impact ◽  
Response To Therapy ◽  
Molecular Response ◽  
Mrna Levels ◽  
Dose Increase ◽  
Use Of Resources ◽  
The Impact

Abstract The human organic cation transporter-1 (hOCT-1) is the major active influx protein responsible for the transport of imatinib into blood cells 1,2. The functional activity of the OCT-1 protein is defined as the intracellular uptake and retention (IUR) of 14-C labelled imatinib into patient pre therapy mononuclear blood cells over a two hour period, which is inhibited by OCT-1 inhibitors such as prazosin or procainamide. The level of OCT-1 activity is a key determinant of the interpatient variation observed in intrinsic sensitivity to imatinib induced kinase inhibition (IC50imatinib3). We have previously demonstrated that a significantly greater proportion of de-novo CML patients with high functional activity of OCT-1, achieve a major molecular response (MMR: 3 log reduction in BCR-ABL mRNA from standardised baseline) when treated with imatinib, than patients with low OCT-1 Activity 4. We have also identified a link between dose and OCT-1 Activity, demonstrating that the negative impact of low OCT-1 Activity could be overcome to a variable extent by imatinib dose increase. However, not all patients can dose increase, largely because of tolerability issues. While the transport of second-generation ABL-kinase inhibitors (nilotinib and dasatinib5) is not OCT-1 mediated, the long term effect of these drugs is not yet known. Hence, we sought to identify strategies to increase OCT-1-mediated imatinib uptake. We queried the drug gene expression signatures in version 1 of the Connectivity Map (CMAP; Lamb J, Nat. Rev. Cancer7; 54–60, 2007: http://www.broad.mit.edu/cmap) with 3 transporters including OCT-1. This identified the Rho kinase inhibitor fasudil and COX-2 inhibitor / celecoxib analogue LM1685 as potential up-regulators of OCT-1 mRNA. The impact of these drugs on OCT-1 mRNA expression and IC50imatinib (fasudil alone to date) has been analysed in two bcr-abl positive cell lines (K562 and KU812). The effect of these two candidate OCT-1 enhancers on OCT-1-mediated imatinib uptake was also assessed in 10 newly diagnosed chronic phase CML patients, previously demonstrated to have low OCT-1 Activity (4 with no demonstrable OCT-1 Activity), using the IUR assay. Table 1: Assessing the effects of fasudil and LM1685 on the intracellular transport of imatinib. These data demonstrate a statistically significant increase in OCT-1 Activity with LM1685, and show a strong trend towards significance with fasudil. Importantly, we show that patients with no demonstrable OCT-1 Activity (0ng/200,000 cells) have detectable Activity in the presence of both fasudil (Range 1.5 to 2ng/200,000 cells) and LM1685 (Range 1.5 to 4.5 ng/200,000 cells). We have previously demonstrated that patients with no detectable OCT-1 Activity universally fail to achieve imatinib therapeutic response milestones (imatinib failure), whereas 54% of patients with low, but detectable OCT-1 Activity achieve these milestones4. The ability to enhance the functional activity of the OCT-1 protein may therefore be of significant clinical relevance in this group. In addition we demonstrate an increase in imatinib IUR which, along with the increase in OCT-1 Activity, is likely associated with increased OCT-1 mRNA levels. Significantly, in the two CML cell lines tested we show a marked reduction in the IC50imatinib indicating that the observed increase in IUR and OCT-1 Activity translates to an increase in the kinase inhibitory activity of imatinib. Preliminary analysis in one patient analysed to date also indicates a reduction in IC50 from 0.48 to 0.35μM in the presence of fasudil. In the clinical scenario the use of such OCT-1 enhancers may improve the response of some imatinib treated patients to both standard and increased dose imatinib. Importantly, these findings validate the use of resources such as C-MAP to identify candidate drugs that may mediate desired changes in the levels of key proteins resulting in improved response to therapy. Fasudil (10μM) %increase from control LM1685 (1μM) % increase from control IUR of imatinib OCT-1 Activity IC50imatinib IUR of imatinib OCT-1 Activity K562 76% (n=5) 163% (n=5) 51% reduction (n=3) 41% (n=2) 122% (n=2) KU812 10.7% (n=2) 75% (n=2) 15% reduction (n=2) NA NA CML patients n=10 8% 89% 9% 114% p value >0.05 0.08 >0.05 0.03

scholarly journals Impact of sialyltransferase ST6GAL1 overexpression on different colon cancer cell types

Glycobiology ◽  
2019 ◽  
Vol 29 (10) ◽  
pp. 684-695 ◽  
Author(s):  
Giulia Venturi ◽  
Inês Gomes Ferreira ◽  
Michela Pucci ◽  
Manuela Ferracin ◽  
Nadia Malagolini ◽  
...  
Keyword(s):  
Cell Lines ◽  
Cell Types ◽  
Soft Agar ◽  
Response To Therapy ◽  
The Cancer Genome Atlas ◽  
Braf Mutations ◽  
Altered Expression ◽  
Retroviral Transduction ◽  
Experimental Systems ◽  
The Impact

AbstractCancer-associated glycan structures can be both tumor markers and engines of disease progression. The structure Siaα2,6Galβ1,4GlcNAc (Sia6LacNAc), synthesized by sialyltransferase ST6GAL1, is a cancer-associated glycan. Although ST6GAL1/Sia6LacNAc are often overexpressed in colorectal cancer (CRC), their biological and clinical significance remains unclear. To get insights into the clinical relevance of ST6GAL1 expression in CRC, we interrogated The Cancer Genome Atlas with mRNA expression data of hundreds of clinically characterized CRC and normal samples. We found an association of low ST6GAL1 expression with microsatellite instability (MSI), BRAF mutations and mucinous phenotype but not with stage, response to therapy and survival. To investigate the impact of ST6GAL1 expression in experimental systems, we analyzed the transcriptome and the phenotype of the CRC cell lines SW948 and SW48 after retroviral transduction with ST6GAL1 cDNA. The two cell lines display the two main pathways of CRC transformation: chromosomal instability and MSI, respectively. Constitutive ST6GAL1 expression induced much deeper transcriptomic changes in SW948 than in SW48 and affected different genes in the two cell lines. ST6GAL1 expression affected differentially the tyrosine phosphorylation induced by hepatocyte growth factor, the ability to grow in soft agar, to heal a scratch wound and to invade Matrigel in the two cell lines. These results indicate that the altered expression of a cancer-associated glycosyltransferase impacts the gene expression profile, as well as the phenotype, although in a cancer subtype-specific manner.

scholarly journals Investigation of the criteria for increasing the operational reliability of building frames

2021 ◽  
Vol 1 (47) ◽  
pp. 52-63
Author(s):  
G. Tonkacheev ◽  
I. Rudnieva
Keyword(s):  
Bearing Capacity ◽  
Negative Impact ◽  
Operational Reliability ◽  
Building Structures ◽  
Social Culture ◽  
Building Frames ◽  
Cultural Aspects ◽  
Use Of Resources ◽  
Range Of Functions ◽  
The Impact

Analysis and generalization of previous studies showed that there is no comprehensive solution to the problem of increasing the operational reliability of building frames, reconstruction and modernization of buildings and structures, taking into account a sustainable approach. After the completion of construction, the frame of the building is constantly and steadily changing and wearing out, as a result, requiring an increase in operational reliability, and these processes are associated with the correct decision-making on changing the bearing capacity of structures and on the technology and organization of the corresponding work. To solve this problem, it is necessary to create a general methodology for a system for modeling constructive and technological solutions. The system's methodology is intended to reduce costs while increasing the reliability of frame structures, taking into account social culture, optimal impact on society, efficient use of resources and environmental principles. Particularly acute is the problem of introducing innovative and effective organizational and constructive-technological solutions to ensure the required performance and functionality with a minimum negative impact on the environment, while taking into account the improvement of economic, social, cultural aspects at the local, regional and global levels. In addition, the necessary criteria are an increase of the bearing capacity, a decrease in deformability and the recovery of the operational suitability of building structures. The article investigates a comprehensive solution to the problem of increasing the operational reliability of building frames during the reconstruction and modernization of buildings and structures. Identified internal and external factors of changes, as well as the main criteria for increasing the operational reliability of building frames. Consideration of these factors as a range of functions makes it possible to predict the long-term needs and behavior of the building frame throughout the entire life cycle, taking into account the degradation of building components and the need to restore or strengthen the elements of the building frames. After collecting and analyzing information on the identified factors, a system of technological solutions is proposed that takes into account a sustainable approach to reconstruction and categories such as organizational and technological solutions, structures, materials, economics, management, ecology, social culture, in particular the impact on society and the environment.

scholarly journals Imatinib resistance: diagnostic and therapeutic choices

2015 ◽  
Vol 4 (2S) ◽  
pp. 21-25
Author(s):  
Marianna De Muro ◽  
Odoardo Maria Olimpieri ◽  
Rosa Greco ◽  
Lidia Altomare
Keyword(s):  
Chronic Myeloid Leukemia ◽  
Plasma Level ◽  
Adverse Events ◽  
Myeloid Leukemia ◽  
Response To Therapy ◽  
Molecular Response ◽  
Dose Increase ◽  
Imatinib Resistance ◽  
Optimal Response ◽  
Complete Molecular Response

We report a case of a 42-year-old woman with t(9;22) positive chronic myeloid leukemia (CML) who developed a sub-optimal response to therapy with imatinib mesylate due to M351T mutation and low plasma level of imatinib. Dose increase of imatinib resulted in toxicity. She obtained a complete molecular response to therapy with nilotinib, without adverse events.

scholarly journals The impact of high hydrostatic pressure (40 MPa and 60 MPa) on the apoptosis rates and functional activity of cryopreserved porcine mesenchymal stem cells

2018 ◽  
Vol 18 (1) ◽  
pp. 69-86
Author(s):  
Joanna Romanek ◽  
Jolanta Opiela ◽  
Zdzisław Smorąg
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Hydrostatic Pressure ◽  
Functional Activity ◽  
High Hydrostatic Pressure ◽  
Negative Impact ◽  
Caspase 8 ◽  
Significant Difference ◽  
The Impact

AbstractThe aim of the present study was to examine the influence of two varied high hydrostatic pressure (HHP) values on the apoptosis (assessing caspase-8, survivin, CAD, Bax, BclxL and BclxS) and functional activity (using cocultures with bovine embryos) of porcine mesenchymal stem cells (pBMSCs). pBMSCs were isolated from porcine bone marrow and cultured in vitro. Before cryopreservation and storage in liquid nitrogen, pBMSCs were subjected to HHP values of 40 MPa and 60 MPa for 1 h at 24°C. After thawing, the cells were analysed for caspase-8 activity and protein expression of survivin, CAD, Bax, BclxL and BclxS. To indirectly test the influence of HHP on the functional activity of pBMSCs, in vitro maturated bovine oocytes were fertilized in vitro, and the obtained embryos were cultured under 4 different conditions: 1. monoculture in SOF medium; 2. coculture with pBMSCs in SOF medium; 3. coculture with pBMSCs subjected to 40 MPa HHP in SOF medium and 4. coculture with pBMSCs subjected to 60 MPa HHP in SOF medium. The quality of the developed blastocysts was analysed by TUNEL assay. HHP did not induce apoptosis in pBMSCs, as no significant difference was noted in the expression of any of the analysed apoptosis- related proteins between pBMSCs subjected to HHP (40 MPa or 60 MPa) and control. The highest number of obtained blastocysts was observed when the embryos were cultured in SOF. A highly significant difference (P<0.005) was noted between embryos cultured in SOF and embryos cultured in the presence of pBMSCs subjected to 60 MPa HHP or untreated pBMSCs. A significant difference (P<0.05) was noted between embryos cultured in SOF and embryos cultured in the presence of pBMSCs subjected to 40 MPa HHP. In conclusion, HHP does not induce apoptosis in pBMSCs. The obtained results of the blastocysts cocultured in vitro with pBMSCs (HHP-treated and untreated cells) imply that coculture with pBMSCs has a negative impact on the developmental rates of blastocysts.

scholarly journals MSI2 and TGFβR1 Expression Levels in CML Patients and Hematopoietic Cell Lines

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4533-4533
Author(s):  
Jaspal S Kaeda ◽  
Simone Bonecker ◽  
Iris von Wunsch-Rolshoven ◽  
Frauke Ringel ◽  
Michaela Schwarz ◽  
...  
Keyword(s):  
Stem Cell ◽  
Cell Lines ◽  
Essential Thrombocythemia ◽  
Hematopoietic Cell ◽  
Molecular Response ◽  
Mrna Levels ◽  
Major Molecular Response ◽  
Significant Difference ◽  
Post Therapy ◽  
Hematopoietic Cell Lines

Abstract Musashi 2 (also known as MSI2), a mRNA binding protein is reported to control critical stem cell fate decisions by binding to the 3’untranslated region of target mRNAs, thereby inhibiting translation. MSI2 is preferentially expressed in hematopoietic tissue, in particular early myeloid progenitors. Moreover, investigators suggest upregulated MSI2 disrupts regulatory pathway/s leading to hematopoietic stem cell proliferation, impaired myeloid differentiation and worse clinical prognosis in CML and AML (Kharas et al. Nat Med. 2010; 16:903; Ito et al. Nature. 2010; 466:765). Indeed we have confirmed increased MSI2 levels in CML patients in blast crisis (BC) compared with those in chronic phase (CP), irrespective of lymphoid or myeloid transformation. Furthermore, we have shown MSI2 and BCR-ABL1 expressions correlate. Here we report data implying MSI2 functions viaTGFβ1 signalling pathway. We retrospectively studied 54 CML cDNA samples from 34 patients (M:15 ; F:19) in CP with median age 55.5 years (range 12-74). Apart from 3 patients treated with interferon+AraC, the remainder were prescribed Imatinib mesylate. Of the 54 samples 29 were collected at diagnosis (Dx) and 25 were obtained at 3 months post therapy (3M). For 20 of the patients, samples collected at Dx and 3M were available. Eight of the patients failed to achieve major molecular response (MMR) within 12 months post therapy. In addition to patient samples we included 19 normal cDNA controls from healthy blood donors (M:10 ; F:9), with 41 years median age (range 20-61). We also included 20 cDNA samples derived from hematopoietic cell lines (lymphoma: 7; AML: 3; CML:6 ; essential thrombocythemia: 1 ; hyperesoinophilic syndrome: 1 ; Acute lymphoblastic leukemia: 1). The cDNA was synthesized using reverse transcriptase and random hexamers. All the samples were subjected to quantitative real time PCR using TaqMan assay to quantify MSI2, TGFRβ1 and GUSβmRNA levels. Furthermore, we subjected protein isolated from the cells lines to Western blot analysis to assess TGFβR1 expression. MSI2 mRNA median levels in patient samples were significantly decreased compared with the NC group at Dx and at 3M, p=0.002 and p=0.013, respectively. But we found no significant difference in MSI2 mRNA levels at Dx nor at 3M, between those who achieved major molecular response and those who did not within 12 months of starting therapy, p=0.215 and p=1.871, respectively. Also we observed no significant difference between the patient samples and the NC group TGFβR1 transcript numbers at Dx nor at 3M, p=0.057 and p=0.097, respectively. Equally we observed no significant difference in TGFβR1 levels, either at Dx or 3M, between those who did and failed to achieve MMR. But we found strong to moderate correlation between MSI2 and TGFβR1 expression when we compared the Dx and 3M data as determined by Spearman correlation coefficient, r=0.6975 and 0.5715, respectively. More importantly, we observed 9 of the cell lines, with increased (>6.7%) or detectable MSI2 mRNA expression, of which 6 express BCR-ABL1 (BV173, Lama87, K562, KCL22, KU812 and SupB15), the TGFβR1 protein was considerably decreased. The BCR-ABL1 positive cell line Meg01 was the one exception with increased MSI2 mRNA levels (7.1%) and clearly detectable TGFβR1 protein. Conversely, of the 10 BCR-ABL1 negative cell lines with decreased or undetectable MSI2 transcripts the TGFβR1 protein was clearly detectable in 8. Of these 7 were lymphoid cell lines (L-248, KM-H2, L-1236, HDLM-2, BJAB, U-266, U-2932) and one was essential thrombocythemia, SET2. The 2 exceptions were AML cell lines OCI-AML-2 and OCI-AML-3 with considerably decreased MSI2mRNA levels and markedly decreased TGFβR1 protein expression. We suggest MSI2 expression is significantly decreased in CML patients in CP because the early progenitors expressing it are masked by large bulk of mature myeloid cells in these patients. Therefore to assess the prognostic value of MSI2 levels at diagnosis it should be quantified in CD34+ enriched samples at diagnosis. More importantly, finding cell lines expressing MSI2, with the exception of 3 cell lines (OCI-AML-2, OCI-AML-3 and Meg01) had decreased TGFβR1 protein is consistent with its role as a post transcription regulator. These findings combined with the previous data showing MSI2 mRNA levels are increased in CML BC support the notion that it functions via TGFβR1 signaling pathway to influence CML transformation. Disclosures le Coutre: Novartis: Honoraria.

Activation of Stem-Cell Specific Genes by HOXA9 and HOXA10 Homeodomain Proteins in CD34+ Human Cord Blood Cells.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3227-3227
Author(s):  
H. Jeffrey Lawrence ◽  
Christina M. Ferrell ◽  
Sheri T. Dorsam ◽  
Hideaki Ohta ◽  
R. Keith Humphries ◽  
...  
Keyword(s):  
Stem Cell ◽  
Cord Blood ◽  
Cell Lines ◽  
Blood Cells ◽  
Hematopoietic Cells ◽  
Mrna Levels ◽  
Hematopoietic Differentiation ◽  
Hematopoietic Stem ◽  
Over Expression ◽  
Cord Blood Cells

Abstract There is growing evidence for a role of HOX homeodomain (HD) proteins in normal hematopoiesis. Several HOX genes, including HOXA9 and HOXA10, are expressed in primitive hematopoietic cells, and that expression is down-regulated as cells mature, implying a role in early hematopoietic differentiation. In addition, retrovirally enforced over-expression of either of these two HD proteins results in dramatic expansion of hematopoietic stem cells (HSC). Our laboratory recently published a description of the HOXA9 transcriptome in human leukemic cell lines, using a transient over-expression strategy (Dorsam et al. Blood 2004). In that study we observed changes in the mRNA levels of a large number of genes within 24 hours of introduction of a HOXA9 expression vector in these cells. The modulated targets represented a wide variety of functional groups, including oncogenes, cell-cycle proteins, enzymes, membrane proteins and other transcription factors. Interestingly, a number of these putative targets are known to be part of the transcriptome of normal HSC. This previous study raises questions as to whether the HOXA9 targets identified in aneuploid immortalized myeloid cell lines would match the gene targets in normal hematopoietic cells. To answer this question and to compare target genes of two closely related HD proteins, human CD34+ umbilical cord blood cells were transduced with MSCV-based vectors expressing either HOXA9 or HOXA10, and RNA isolated from these cells was amplified and analyzed with cDNA microarrays. Statistical analysis using the Significance Analysis of Microarrays (SAM) algorithm revealed a common signature of several hundred modulated genes, demonstrating that the transcriptomes of HOXA9 and HOXA10 largely overlap in this cellular context. In a second step, CLUSTER analysis was performed, introducing threshold values to increase the likelihood of the identified genes being truly regulated and of biological significance. Using these more stringent criteria, 115 genes were modulated by one or both of the over-expressed genes. Several genes that were up-regulated by both HOX proteins were validated by quantitative real-time RT-PCR. HOXA9 and HOXA10 showed positive regulation of genes in the Wnt pathway, including Wnt 10b and two Wnt receptors Frizzled 1 and Frizzled 5, a important pathway for hematopoietic stem cell (HSC) self renewal. Other validated targets included Ets-related gene (ERG),, alcohol dehydrogenase 1 (ALDH1) and very long chain acyl-CoA synthetase (VLCS-H1), all of which are known to be expressed in normal CD34+ cells. One down-regulated gene in this survey is CYBB, a respiratory burst oxidase component pg91(phox), which is expressed in myeloid cells, and has previously been shown to be repressed by HOXA10. GenMAPP pathway analysis indicated that HOXA9 and HOXA10 repressed expression of several genes involved in heme biosynthesis and three globin genes, indicating a general suppression of the erythroid maturation program. HOXA9 and HOXA10 both appear to activate many HSC-specific genes while repressing genes involved in hematopoietic differentiation.

The Functional Activity of the OCT-1 Protein Is Predictive of Molecular Response and Survival in CP-CML Patients Treated with Imatinib: A 5 Year Update of the TIDEL Trial.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 507-507 ◽  
Author(s):  
Deborah L White ◽  
Verity A Saunders ◽  
Amity Frede ◽  
Phuong Dang ◽  
Stephanie Zrim ◽  
...  
Keyword(s):  
Board Of Directors ◽  
Functional Activity ◽  
Research Funding ◽  
Intracellular Concentration ◽  
Molecular Response ◽  
Advisory Committees ◽  
Significant Difference ◽  
The Impact ◽  
First Time

Abstract Abstract 507 The major active influx protein for imatinib into target BCR-ABL positive cells is the organic cation transporter OCT-1. We have previously demonstrated that the functional activity of the OCT-1 protein (OCT-1 activity) is predictive of molecular response in TIDEL (trial of imatinib 600 mg/day with selective dose intensification in untreated CP-CML) The OCT-1 activity (OA) is measured in mononuclear cells from untreated CML patients by calculating the intracellular concentration of 14-C imatinib less the intracellular concentration in the presence of OCT-1 inhibition. To address the question of whether OA is predicting only the rate of response, we now investigate the impact of OA on response and progression at 5 years. There is a significant difference in the achievement of MMR (p=0.007) and CMR by 60 months (p=0.032) (Table 1). Six patients developed kinase domain mutations over the course of this study. 5/6 had low OA. Significantly, for the first time addressing Event Free Survival (events defined as loss of CHR, MCR or CCR, progression to AP or BC or change of therapy due to unsatisfactory efficacy), we demonstrate that more patients with high OA are event free at 5 years when compared to patients with low OA (Table 1). To determine whether the detrimental effect of low OA on survival was more significant in those patients with OA in the lowest quartile (Q1) we compared the response of Q1 patients to all other patients (Table 2). These data demonstrate importantly, that patients in Q1 have significantly poorer outcomes, than the remainder of the patient cohort. In previous analyses we have shown that the effects of a low OA can be partially overcome by higher imatinib doses. Limiting the analyses to those patients receiving <600mg average daily dose over the first 12 months there was a significant difference in the achievement of MMR (low OA (n=11) 27%: high OA (n=12) 92% p=0.021) and EFS (36% vs 75% p=0.03). In patients receiving ≥600 mg there was no significant difference between the groups, reinforcing the importance of dose. In 45 patients we examined the expression of OCT-1 mRNA for prediction of MMR, CMR, EFS and mutation development. Dividing the patients into low and high OCT-1 expression about the median we found that the level of mRNA is not predictive of MMR (low–60% vs high 78 p=0.241) CMR (low–45% vs high 55 p=0.456) EFS (low–55% vs high 70 p=0.315) or mutation development (low–18% vs high 14% p=0.666). These data indicate that the level of OCT-1 mRNA is not sufficiently discriminating to predict response and progression. While our previous studies demonstrated that OA could predict the rate of decline in BCR-ABL over the first 12-24 months, this update demonstrates for the first time, that this assay can identify nearly all patients (>80%) who fail to achieve MMR in the long term. Most importantly OA is also strongly predictive of resistance and progression events. Functional assessment of OCT-1 Activity provides prognostic information that is more discriminating than assaying the level of OCT-1 mRNA. This long term study reinforces the notion that OA is an important predictive variable in CP-CML patients treated with IM. It provides further evidence that OA is a critical variable to consider in future trials of imatinib and a key factor to enable individualization of imatinib dose to optimize the long term outcome for CML patients. Disclosures: White: Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Research Funding. Manley:Novartis: Employment. Hughes:Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Flow Cytometry Detection of Intra-Cellular Tyrosine Kinase Inhibitors (TKI) Showed Variable Uptake in CML CD34+ Cells

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2747-2747
Author(s):  
Céline Bourgne ◽  
Mahchid Bamdad ◽  
Alexandre Janel ◽  
Frédéric Libert ◽  
Agnès Guerci ◽  
...  
Keyword(s):  
Stem Cells ◽  
Flow Cytometry ◽  
Cell Lines ◽  
Blood Cells ◽  
Second Generation ◽  
Molecular Response ◽  
Primary Cells ◽  
Uv Fluorescence ◽  
Cd34 Cells

Abstract Abstract 2747 Introduction Despite the major benefit of TKI in the treatment of Chronic Myeloid Leukemia (CML), patient response is heterogeneous and it is generally accepted that residual disease and relapse are due to persistent CML cells, considered as leukemic stem cells. Their resistance has been related to lower TKI uptake. The amount of drug penetrating the targeted cells is most likely a major parameter of targeted therapy efficacy since it is essential that the therapeutic molecule be as close as possible to the target molecule. We developed a flow cytometry technique to analyze primary cells. Method To evaluate intracellular imatinib (ICIM) uptake, we developed a patented method based on natural UV fluorescence related to chemical structure. Consequently, since the difference in UV fluorescence units between treated and control cells is proportional to the amount of intra-cellular drugs, we validated this method after incubating K562 and KCL22 cell lines with TKI. The flow cytometry technique was standardized by using Flow-Check Fluorosphere calibrated beads immediately before, and at the end of, each series of analyses with a Coulter Epics Elite™ flow cytometer (Beckman Coulter) equipped with an Innova I90C-4 UV laser (Coherent). Then we analyzed primary blood cells from CML patients in chronic phase before any treatment. After lysis of erythrocytes, nucleated cells were incubated at 1.106 cells/ml with different doses of imatinib (IMA) (n=22), Nilotinib (NIL) (n=20) and Dasatinib (DAS) (n=20) at different times. Whenever possible, CML stem cells were analyzed using CD34-FITC staining. Results In preliminary assays, we checked that there was a significant correlation between additional fluorescence measured by flow cytometry and the amount quantified by physico-chemical analysis after lysing a known number of cells (n=57, r2=0.73, p<0.001), which enabled us to convert UV fluorescence into pg of IMA per cell. Then we confirmed that IMI rapidly penetrated K562 and KCL22 cells (from 5 minutes of incubation) and reached a stable influx in viable cells from 1 hour (T1h). We chose this incubation time for further experiments. Similarly, we choose T2h for second generation TKI. We observed a dose-dependent accumulation in the two cells lines, but with differences at the lowest extra-cellular concentrations (1–5 μM) and not correlated with any membrane pump expression (OCT-1, ABCG2, ABCB1 and ABCC1). ICIM at T1h was correlated with cell sensitivity to IM at T24h expressed by the proportion of dead cells (r2=0.93 and 0.88 for K562 and KCL22 cells, respectively). We then applied our method to primary CML blood cells in comparison with normal blood cells. TKI penetrated all cell subsets, but amounts varied depending on cell sizes (FS/SS characteristics). The first data obtained with IM showed ICIM levels in CML cells that were relatively heterogeneous from one patient to another, ranging from 0.9 to 4 pg/cell for an extracellular concentration of 5 μM, i.e. a higher concentration (x 300) than in culture medium. The ability of the granulocyte cell lineage to store IMA was related to the Sokal prognostic index (p=0.05). We detected variable ICIM levels in CML CD34+ cells from 10/16 patients (0.04–0.7 pg/cell) and no signal for 6/16 patients. Surprisingly, the ability of CD34+ to store second generation TKIs is variable and not necessarily correlated to IMA uptake. Discussion We developed a simple, rapid flow cytometry method directly applicable to primary cells and requiring only few cells which makes it possible to identify target cell subsets, such as CML stem cells. The strong correlation between the ICIM amount and the sensitivity of CML cell lines to TKIs validated the method and suggested that ICIM could be a relevant biomarker for predicting the sensitivity of the CML clone. In our CML series, we observed striking inter-patient variability of the capacity of primary CML cells to store TKI. A correlation with the Sokal score suggests possible predictive value with regard to in vivo CML response to IMA, which could be taken into account when choosing TKI for first-line therapy. Furthermore, we observed marked heterogeneity between CML CD34+ cells for storing TKI that could partially explain the heterogeneity of in vivo response. The relationship between the ability of untreated CML CD34+ cells to store TKI and complete molecular response has to be established. Disclosures: No relevant conflicts of interest to declare.

The Gene Expressions of hOCT1 and ABCB1 Change During the Course of CML in Relation to Imatinib Therapy

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2495-2495
Author(s):  
Katerina Machova Polakova ◽  
Jitka Koblihova ◽  
Zuzana Ondrackova ◽  
Filip Razga ◽  
Vaclava Polivkova ◽  
...  
Keyword(s):  
Cell Lines ◽  
Blood Count ◽  
Transcript Level ◽  
Molecular Response ◽  
Mrna Levels ◽  
Elevated Expression ◽  
Travel Grant ◽  
Immature Cells ◽  
Lower Expression

Abstract Abstract 2495 Background. The intracellular concentration of imatinib (IM) in patients with chronic myeloid leukemia (CML) is supposed to be influenced by the expression of its main cellular transporters, hOCT1 (influx) and ABCB1 (efflux). The assessment of the genes expression may be potentially important in clinical practice to effectively manage the therapy. Our recent results showed the necessity to cautiously interpret results from gene expression measurements of both transporters as their detected mRNA levels are affected by the proportion of different cell types in the sample analyzed (Racil et al. 2010 Am J Hem 85:525, Racil et al. 2011 Leuk & Lymph 52:331). Aims. In this report, we aimed to comprehensively assess differences in the expression of hOCT1 and ABCB1 in total leukocytes of peripheral blood (PB) in relation to the blood cell lineage in CML patients with different responses to IM. Methods. Kruskall Wallis's and Dunn's multiple comparison tests were applied to calculate differences in transcript levels of hOCT1 and ABCB1 in patients at diagnosis (Dg=43), in major molecular response (MMR=27), complete molecular response (BCR-ABL log 4.5 reduction; CMR4.5 =15), therapy failure (TF=13), accelerated phase (AP=12) and in 75 healthy controls. CMR4.5 is defined here as either a detectable disease ≤0.0032% BCR-ABLIS or as undetectable by nested PCR. TF is defined as non CCgR achievement. Additionally, we used the Spearman's correlation test to investigate relationship between expressions and percentage of immature cells and neutrophils in patients with non-physiological blood count (Dg and AP). In patients with normal blood count (CMR4.5, MMR, TF), we calculated correlation with BCR-ABL transcript level. Finally, we performed in vitro experiments with BCR-ABL negative (SKM-1, MOLM-13) and positive cell lines (K562, MOLM-7) treated with IM to study its effect on ABCB1 expression. Results. We found a significantly lower expression of hOCT1 and ABCB1 at Dg (P<0.001) and in AP (P<0.001 and P<0.01, respectively) in comparison to controls. The lower expression probably depends indirectly on the immature cells count (hOCT1 r = −0.4850, P=0.0002; ABCB1 r = −0.6451, P< 0.0001). Interestingly, while a significant positive correlation of neutrophil count was found with hOCT1 mRNA levels (r=0.6090, P< 0.0001), no correlation was observed with ABCB1. In patients with physiological blood count, we observed significantly elevated expression of ABCB1 in MMR and CMR4.5 in comparison to controls (P< 0.01). As MMR and CMR4.5 samples are characterized by the BCR-ABL transcript level <0.1%, this result led us to perform in vitro experiments to test the effect of IM on ABCB1 expression in BCR-ABL negative cell lines showing an elevated expression after incubation with IM. We assume this effect to be a natural defense of BCR-ABL negative cells to get rid of IM. By contrast, we found a drop in ABCB1 expression after BCR-ABL positive cell lines incubation with IM, suggesting inhibitory effects of IM. A significantly reduced expression of hOCT1 was found in TF in comparison to MMR and CMR4.5 (P< 0.01). These samples did not differ in blood count, but we observed a significant negative correlation of hOCT1 with BCR-ABL transcript levels (r= − 0.5117; P< 0.0001). Conclusion. Cell composition in PB changes during the CML treatment with IM which influences the measurement of hOCT1 and ABCB1 mRNA levels in total leukocytes. However, hOCT1 expression was found to be significantly lower in patients who failed to achieve CCgR but had normal blood count. Interestingly, our data suggest that ABCB1 is differently expressed in BCR-ABL positive and negative cells due to the inhibitory and enhancing effects of IM, respectively. Supported by IGA NT11555 and MZOUHKT2005 Disclosures: Machova Polakova: Novartis: travel grant. Ondrackova:BMS: travel grant.

The Clinical Significance of Early Imatinib Induced ABCB1 Overexpression in Chronic Phase CML Patients: A TIDEL II Sub-Study

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 348-348 ◽  
Author(s):  
Laura N Eadie ◽  
Timothy P. Hughes ◽  
Deborah L White
Keyword(s):  
Research Funding ◽  
Therapeutic Option ◽  
Blast Crisis ◽  
Drug Transporters ◽  
Chronic Phase ◽  
Response To Therapy ◽  
Molecular Response ◽  
The Impact ◽  
Post Therapy

Abstract Recent data from the TIDEL II trial indicate first-line imatinib (IM) treatment, with selective switching to nilotinib (NIL) for failure to meet specific molecular targets or for intolerance, results in excellent molecular response and overall survival in chronic phase Chronic Myeloid Leukaemia (CP-CML) patients. Drug transporters, particularly OCT-1 and ABCB1, have previously been demonstrated to impact IM response. Furthermore, ABCB1 overexpression has been implicated in IM and NIL resistance in vitro. In this TIDEL II sub-study the level of ABCB1 mRNA expression was retrospectively assessed by PCR at day 0 and day 22 post the start of IM therapy and where relevant, at cessation. A change in the level of expression of ABCB1 compared with baseline after 22 days exposure to IM was observed in all patients (Range: 0.6-12.3 fold; median: 2 fold), suggesting IM exposure resulted in altered ABCB1 expression that was variable between patients; this change was not related to the level of BCR-ABL1 expression (p=0.29 at day 0 and p=0.84 at 1 mo). Assessing the impact of >2 fold rise in ABCB1 expression after 22 days IM exposure on subsequent molecular response, and comparing this with patients where a <2 fold rise was observed revealed significant differences in outcome between the 2 groups (Table 1). Table 1.% of patients achieving:Over the first 22 days of treatmentEarly Molecular ResponseMMR by 12 moEvent Free SurvivalMR4.5<2 fold rise in ABCB1 (n=22)86%64%77%64%>2 fold rise in ABCB1 (n=22)60%14%37%5%p -value0.0060.0010.018<0.001 Importantly, change in ABCB1 mRNA over the first 22 days of treatment was predictive of MMR by 24 mo (Figure 1). These data support previous in vitro findings of ABCB1-mediated IM export and provide the first in vivo evidence for up regulation of ABCB1 in response to IM therapy. Because ABCB1-mediated export of NIL has also been demonstrated, we assessed the correlation between response to NIL therapy and changes in ABCB1 expression following 22 days IM exposure. Of the patients who demonstrated a >2 fold rise in ABCB1 compared with baseline, 16 of the remaining 20 patients (2/22 died at 3.2 mo (Cardiac event) and 3.7 mo (Blast Crisis)) switched to sequential NIL therapy because of sub-optimal response to IM (no prior MMR). Importantly, 0/16 patients receiving NIL subsequently achieved confirmed MMR, suggesting that NIL may provide a poor therapeutic option for patients with up-regulation of ABCB1. These data highlight the potential importance for monitoring increases in ABCB1 expression early in therapy in order to predict those patients likely to achieve poor molecular responses. We next determined the impact of ABCB1 expression on Event Free Survival (EFS) and demonstrated that the fold change in ABCB1 levels at day 22 was predictive for achieving EFS: 77% of patients with <2 rise in ABCB1 vs 37% of patients with >2 rise in ABCB1; p =0.018. Furthermore a sustained >2 fold rise in ABCB1 mRNA compared with levels at diagnosis was observed in 3/3 patients prior to progression to blast crisis and ABCB1 mRNA increased by >2 fold post therapy in 9/12 patients prior to development of KD mutations. Previous studies investigating the correlation between ABCB1 expression and response to therapy have focussed on absolute levels of ABCB1, usually at 12 mo post-therapy initiation when leukaemic cell burden would be low, and have observed no significant difference in ABCB1 expression in optimal vs sub-optimal responders. Conversely, our rapid PCR based assay performed pre and 22 days post the start of IM therapy in CP-CML patients provides a potent early predictor of subsequent IM response. The findings detailed here also suggest that NIL is likely a poor subsequent therapeutic option for patients with up-regulated ABCB1 expression in response to IM. Importantly, these data highlight the fact that better understanding of the effects of TKIs on factors with the potential to influence treatment outcome (such as drug transporter expression) can be used to personalise therapy. Overarchingly, these data exemplify the importance of drug transporters in the setting of TKI therapy and patient response, and, following validation in further studies, provide a new, effective and easily translatable prognostic biomarker based on ABCB1. Disclosures Hughes: Bristol-Myers Squibb: Honoraria, Research Funding; ARIAD: Honoraria, Research Funding; Novartis: Honoraria, Research Funding. White:Novartis: Honoraria, Research Funding; BMS: Honoraria, Research Funding.

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