Transcriptional Changes Induced by Imatinib and Nilotinib in the Chronic Myelogenous Leukemia (CML) Cell Line K562.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4250-4250
Author(s):  
Daniela Bruennert ◽  
Ingmar Bruns ◽  
Norbert Gattermann ◽  
Ralf Kronenwett ◽  
Rainer Haas ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
Tyrosine Kinase ◽  
Tyrosine Kinase Inhibitors ◽  
Kinase Inhibitors ◽  
K562 Cells ◽  
Significant Differential Expression ◽  
Number Of Genes ◽  
In Vitro Treatment

Abstract Abstract 4250 Nilotinib is a selective bcr-abl tyrosine kinase inhibitor that is 30-fold more potent than Imatinib in vitro. To examine the molecular and functional effects of Nilotinib and Imatinib we performed gene expression and functional analyses in K562 cells following in vitro treatment with the two tyrosine kinase inhibitors. Affymetrix U133A 2.0 microarrays covering 21.722 probe sets were used to analyse the gene expression profile of 5×10 7 K562 cells after 24h in vitro treatment with Imatinib (0.5 μM) or Nilotinib (0.05 μM) (half maximal inhibitory concentration). Gene expression data of the treated cells were compared with data of untreated cells. In addition, proliferation (MTS Assay, Promega), apoptosis (Cell Death Detection ELISAPLUS, Roche) and cell cycle (FITC BrdU Flow Kit, BD Pharmingen) assays were performed. Protein levels of STAT5, pSTAT5(Thr694), PIM-1 and PIM-2 were detected with Western blots. Particular emphasis was put on 303 genes which we found to be differentially expressed in primary CD34+ cells from patients with CML in vivo during treatment with Imatinib (Bruennert et al, Leukemia, 2008). In K562 cells, we found that Imatinib led to a significant differential expression of 45 of those genes. In general, the effect of Nilotinib with regard to the number of genes affected and degree of suppression was more pronounced resulting in a significant differential expression of 120 genes of the aforementioned genes. Of note, genes affected by Nilotinib included all genes altered by Imatinib. Downregulation of genes involved in cell cycle was observed in 17 genes following Nilotinib exposure, but only in the PIM-1 gene following Imatinib exposure. This effect of Nilotinib is in line with the results of cell cycle experiments showing that Nilotinib exposed cells had the lowest proportion of actively cycling cells. The proportion of apoptotic K562 cells was 5.5-fold greater following treatment with Nilotinib in comparison to treatment with DMSO, whereas Imatinib treated K562 cells had a 1.3-fold higher apoptosis rate compared to DMSO treated 562 cells after 24 hours. The superiority of Nilotinib is also reflected by the results of protein analysis of STAT5, pSTAT5(Thr694), PIM-1 and PIM-2: Both tyrosine kinase inhibitors completely inhibit the phosphorylation of STAT5, but Nilotinib inhibits PIM-1 and PIM-2 stronger than Imatinib (by 85% vs. 70%) in K562 cells. In summary, on a molecular level Nilotinib is apparently more potent than Imatinib with regard to the number of genes affected and the degree of their suppression. Among the 45 genes that were significant differentially expressed with both drugs, genes of Imatinib treated K562 cells were downregulated 1.68fold (mean) whereas genes of Nilotinib treated K562 cells were downregulated 2.41fold (mean). The genes altered are mainly associated with cell cycle regulation. In addition to this Nilotinib has a stronger inhibitory effect on PIM 1 and 2. Disclosures: Gattermann: Novartis: Honoraria, Participation in Advisory Boards on deferasirox clinical trials.

Transcriptional Changes Induced by Imatinib and Nilotinib in the Chronic Myelogenous Leukemia (CML) Cell Line K562.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 4540-4540
Author(s):  
Frank Neumann ◽  
Daniela C. Bruennert ◽  
Anne-Marie Koch ◽  
Ingmar Bruns ◽  
Norbert Gattermann ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
Tyrosine Kinase ◽  
Cell Line ◽  
K562 Cells ◽  
Differentially Expressed ◽  
Cd34 Cells ◽  
Number Of Genes ◽  
In Vitro Treatment

Abstract Introduction: Nilotinib is a selective bcr-abl tyrosine kinase inhibitor that is 30-fold more potent than Imatinib in vitro. To examine the molecular and functional effects of Nilotinib and Imatinib we performed gene expression and functional analyses in K562 cells following in vitro treatment with the two tyrosine kinase inhibitors. Particular emphasis was put on 1539 genes which we found to be differentially expressed in primary CD34+ cells from patients with CML in first chronic phase in comparison to CD34+ cells from normal bone marrow (Diaz-Blanco et al., Leukemia 2006). Methods: Affymetrix U133A 2.0 microarrays covering 21.722 probe sets were used to analyse the gene expression profile of 5x107 K562 cells after 24h in vitro treatment with Imatinib (0.5 μM) or Nilotinib (0.05 μM) (half maximal inhibitory concentration, IC 50). FISH analysis confirmed the K562 cell line to be BCR-ABL positive. Gene expression data of the treated cells were compared with the data of untreated cells. In addition, proliferation (Cell Titer 96 AQueous One Solution Cell Proliferation Assay, Promega), apoptosis (Cell Death Detection ELISAPLUS, Roche) and cell cycle (FITC BrdU Flow Kit, BD Pharmingen) assays were performed. A colony assay was performed to see differences in cell growth. Results: Looking at those 1539 differentially expressed genes in K562 cells which distinguish patients with CML from healthy donors, we found that Imatinib led to a significant downregulation of 187 and upregulation of 45 genes. In general, Nilotinib had a more pronounced effect than Imatinib regarding the number of genes affected and the degree of suppression. It caused downregulation of 418 and upregulation of 41 genes. Of note, genes affected by Nilotinib included all genes altered by Imatinib such as those related to bcr-abl signalling (Lyn, BCL2, Myc, PIK3CB, G3BP2). Downregulation of genes involved in cell cycle (CDK2, ORC5L, MCM3, POLE2, CCNG1) was only observed following Nilotinib exposure. The stronger effect of Nilotinib is in line with the results of cell cycle experiments showing that Nilotinib exposed cells had the lowest proportion of actively cycling cells. The proportion of apoptotic K562 cells was 5.5 fold greater following treatment with Nilotinib in comparison to Imatinib after 24 hours. Treatment with either Imatinib or Nilotinib produced a similar apoptotic rate and similar decrease in cell numbers after 96 hours. In the colony forming assay, the controls (K562 cells incubated with DMSO only) displayed strong leukemic growth which was inhibited by both Nilotinib and Imatinib, allowing only small clusters to appear. Conclusion: Nilotinib is apparently more potent than Imatinib with regard to the number of genes downregulated and the degree of their suppression. Many of the suppressed genes are associated with bcr-abl signalling and cell cycle.

In vitro UGT1A1 inhibition by tyrosine kinase inhibitors and association with drug-induced hyperbilirubinemia

2018 ◽  
Vol 82 (5) ◽  
pp. 795-802 ◽  
Author(s):  
Hisham Qosa ◽  
Brittany R. Avaritt ◽  
Neil R. Hartman ◽  
Donna A. Volpe
Keyword(s):  
Tyrosine Kinase ◽  
Tyrosine Kinase Inhibitors ◽  
Kinase Inhibitors ◽  
Drug Induced

scholarly journals ABCB1 haplotypes do not influence transport or efficacy of tyrosine kinase inhibitors in vitro

2013 ◽  
pp. 63 ◽  
Author(s):  
Karin Skoglund ◽  
Samuel Boiso Moreno ◽  
Maria Baytar ◽  
Jan Ingvar Jönsson ◽  
Henrik Gréen
Keyword(s):  
Tyrosine Kinase ◽  
Tyrosine Kinase Inhibitors ◽  
Kinase Inhibitors

scholarly journals Combating Acquired Resistance to Tyrosine Kinase Inhibitors in Lung Cancer

2015 ◽  
pp. e165-e173 ◽  
Author(s):  
Christine M. Lovly
Keyword(s):  
Lung Cancer ◽  
Tyrosine Kinase ◽  
Tyrosine Kinase Inhibitors ◽  
Tyrosine Kinases ◽  
Kinase Inhibitors ◽  
Acquired Resistance ◽  
Initial Response ◽  
In Vitro Models ◽  
Tumor Biopsy

The prospective identification and therapeutic targeting of oncogenic tyrosine kinases with tyrosine kinase inhibitors (TKIs) has revolutionized the treatment for patients with non–small cell lung cancer (NSCLC). TKI therapy frequently induces dramatic clinical responses in molecularly defined cohorts of patients with lung cancer, paving the way for the implementation of precision medicine. Unfortunately, acquired resistance, defined as tumor progression after initial response, seems to be an inevitable consequence of this treatment approach. This brief review will provide an overview of the complex and heterogeneous problem of acquired resistance to TKI therapy in NSCLC, with a focus on EGFR-mutant and ALK-rearranged NSCLC. In vitro models of TKI resistance and analysis of tumor biopsy samples at the time of disease progression have generated breakthroughs in our understanding of the spectrum of mechanisms by which a tumor can thwart TKI therapy and have provided an important rationale for the development of novel approaches to delay or overcome resistance. Numerous ongoing clinical trials implement strategies, including novel, more potent TKIs and rational combinations of targeted therapies, some of which have already proven effective in surmounting therapeutic resistance.

scholarly journals MEK inhibition enhances the response to tyrosine kinase inhibitors in acute myeloid leukemia

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
María Luz Morales ◽  
Alicia Arenas ◽  
Alejandra Ortiz-Ruiz ◽  
Alejandra Leivas ◽  
Inmaculada Rapado ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Acute Myeloid Leukemia ◽  
Tyrosine Kinase ◽  
Tyrosine Kinase Inhibitors ◽  
Myeloid Leukemia ◽  
Kinase Inhibitors ◽  
Resistance Pathway ◽  
Acute Myeloid

AbstractFMS-like tyrosine kinase 3 (FLT3) is a key driver of acute myeloid leukemia (AML). Several tyrosine kinase inhibitors (TKIs) targeting FLT3 have been evaluated clinically, but their effects are limited when used in monotherapy due to the emergence of drug-resistance. Thus, a better understanding of drug-resistance pathways could be a good strategy to explore and evaluate new combinational therapies for AML. Here, we used phosphoproteomics to identify differentially-phosphorylated proteins in patients with AML and TKI resistance. We then studied resistance mechanisms in vitro and evaluated the efficacy and safety of rational combinational therapy in vitro, ex vivo and in vivo in mice. Proteomic and immunohistochemical studies showed the sustained activation of ERK1/2 in bone marrow samples of patients with AML after developing resistance to FLT3 inhibitors, which was identified as a common resistance pathway. We examined the concomitant inhibition of MEK-ERK1/2 and FLT3 as a strategy to overcome drug-resistance, finding that the MEK inhibitor trametinib remained potent in TKI-resistant cells and exerted strong synergy when combined with the TKI midostaurin in cells with mutated and wild-type FLT3. Importantly, this combination was not toxic to CD34+ cells from healthy donors, but produced survival improvements in vivo when compared with single therapy groups. Thus, our data point to trametinib plus midostaurin as a potentially beneficial therapy in patients with AML.

A Pathway-focused GSEA Platform In AML That Interrogates Interaction of Flt3ITD and tMLL with Their Epigenetic Targets (p16INK4a, DAPK1, and RUNX3), While Accurately Reporting Broad-based AML Prognosis and Targeting-agent Sensitivity.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1660-1660
Author(s):  
Tareq Al Baghdadi ◽  
Chirayu Goswami ◽  
Hamid Sayar ◽  
Katie J. Sargent ◽  
Larry Cripe ◽  
...  
Keyword(s):  
Tumor Suppressor ◽  
Tyrosine Kinase ◽  
Tyrosine Kinase Inhibitors ◽  
Hdac Inhibitor ◽  
Kinase Inhibitors ◽  
Normal Karyotype ◽  
Dual Inhibitor ◽  
Expression Levels ◽  
Kaplan Meier

Abstract Abstract 1660 GSEA (gene-set enrichment array) analysis for expression levels in acute myeloid leukemia (AML) blasts of hoxA9/meis1, has afforded additional prognostic capacity to cytogenetically-defined subtypes. Signaling pathway (KEGG, GO, Ingenuity) analysis of Affymetrix gene expression data has also been used to predict prognosis of AML patients, identifying an adverse high MAPK gene signature. Genome-wide epigenetic (DNA methylation) profiling has added to AML prognostication. We analyzed patient prognosis and in vitro sensitivity for blasts by pathway-targeting agents, in relation to expression clusters defined by the results of GSEA profiling. Blast cell sensitivity was assayed using the agents: (Tyrosine kinase inhibitors for: Flt3: Sorafenib (Onyx/Bayer) and Syk/Flt3: R406 (AstraZeneca); Bortezomib (Millenium), a proteasome- and NFkB- inhibitor, inducer of endoplasmic reticulum (ER) stress; or the pan-histone deacetylase (HDAC) inhibitor, SBHA, Vorinostat analogue). A group of 70 AML cases was studied. GSEA's (Taqman/RQ-PCR, ABI) were performed on two different 31-gene platforms interrogating the interaction of tyrosine kinase survival pathways (eg. Flt3) with downstream epigenetic targets: tumor suppressor genes DAPK1, p16INK4a/CDKN2A, and RUNX3. Expression levels of these are responsive to input from c-jun/AP-1, non-canonical NFkB isoforms/HDAC's, polycomb genes, and ets/ERG, which were monitored. Analysis of overall patient survival (OS) by Kaplan-Meier plots revealed conformity with established outcomes for conventional cytogenetic categories. Also, poor-risk categories defined by normal karyotype Flt3ITD mutation, as well as high hoxA9 and meis1 expressions were recognized. Classification categories involving NKFlt3ITD/high hoxA9/meis1 or complex cytogenetics/high MAPK were most strictly separated by Kaplan-Meier curves, and by representation on the GSEA heatmaps, vs. CBF/PML-RAR translocations. Specific association, between repression of the tumor suppressor DAPK1 (normalized to c-jun, a transcriptional activator) with high expressions of hoxA9 and DAPK1-repressive, non-canonical NFkB/relB, was apparent in most NKFlt3ITD and tMLL cases. Also, a known functional interaction implicating high Id1 expression with its repression of p16INK4a(CDKN2A) was linked by their expression levels on GSEA. The DAPK1/CDKN2A clusters demonstrated significant overlap, often with additional RUNX3 repression. The DAPK1-repressed(R) clusters demonstrated independent prognostic importance. There was median OS for DAPK1-R cluster #1(lower MAPK signature) of 17 months vs. for DAPK1-R cluster #2 of 6 months. This latter cluster was described by higher MAPK signature evidenced by heightened transcript levels of BRCA1 [Bullinger et al. Blood, 2007], FoxM1, bcl-2, IL-1b. The dominant cytogenetic/molecularly-defined phenotypes represented in these DAPK1-R clusters were NKFlt3ITD+ve and tMLL. A very-high MAPK/complex cluster, evidenced median OS 4.5 months vs. good prognosis (low MAPK/DAPK1, and low hoxA9/meis1) group: median OS> 3 years. Interestingly, in vitro activity for the dual inhibitor R406 on blasts was greatest in the category with strongest repression of both p16INK4a/CDKN2A and DAPK1, and with lowest expression of syk transcripts (10-fold compared with high patient values), with a median IC50 of between 10 nM (tMLL)-100nM (Flt3ITD). By contrast, Sorafenib demonstrated its greatest activity in a high MAPK phenotype, including but not restricted to Flt3ITD+ve status. Bortezomib demonstrated median IC50 40 nM. However, high expression levels of FoxM1, putative Bortezomib target, were negatively-associated with its activity. SBHA, the HDAC inhibitor, demonstrated lower activity on a molar basis, but had important synergy with tyrosine kinase inhibitors, a property that was optimal in the context of Id1 hyperexpression. Bortezomib was strongly synergistic with Flt3/syk inhibitors, particularly on FltITD+ve blasts. Indeed, both Bortezomib and the HDAC inhibitor demonstrated by correlation analysis (coefficients=63.3 and 13.4, respectively) optimal activities with Id1 hyperexpression. In conclusion, a pathway-focused genetic and epigenetic prognostic classification that also reports targeting agent sensitivity was established and further validated in a phase I (Sorafenib/Vorinostat) trial in AML (H Sayar, these abstracts). Disclosures: Sayar: Onyx/Bayer: Research Funding.

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