scholarly journals Modulation of FLT3-ITD Localization and Targeting of Distinct Downstream Signaling Pathways as Potential Strategies to Overcome FLT3-Inhibitor Resistance

Cells ◽  
2021 ◽  
Vol 10 (11) ◽  
pp. 2992
Author(s):  
Maximilian Fleischmann ◽  
Mike Fischer ◽  
Ulf Schnetzke ◽  
Colin Fortner ◽  
Joanna Kirkpatrick ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Signaling Pathways ◽  
Cell Lines ◽  
Kinase Inhibitors ◽  
Combined Treatment ◽  
Leukemia Cell ◽  
Surface Expression ◽  
Patient Specific ◽  
Downstream Signaling ◽  
Cellular Compartments

OBJECTIVES: Internal tandem duplications (ITDs) of the Fms-like tyrosine kinase 3 (FLT3) represent the most frequent molecular aberrations in acute myeloid leukemia (AML) and are associated with an inferior prognosis. The pattern of downstream activation by this constitutively activated receptor tyrosine kinase is influenced by the localization of FLT3-ITD depending on its glycosylation status. Different pharmacological approaches can affect FLT3-ITD-driven oncogenic pathways by the modulation of FLT3-ITD localization. AIMS: The objective of this study was to investigate the effects of N-glycosylation inhibitors (tunicamycin or 2-deoxy-D-glucose) or the histone deacetylase inhibitor valproic acid (VPA) on FLT3-ITD localization and downstream activity. We sought to determine the potential differences between the distinct FLT3-ITD variants, particularly concerning their susceptibility towards combined treatment by addressing either N-glycosylation and the heat shock protein 90 (HSP90) by 17-AAG, or by targeting the PI3K/AKT/mTOR pathway by rapamycin after treatment with VPA. METHODS: Murine Ba/F3 leukemia cell lines were stably transfected with distinct FLT3-ITD variants resulting in IL3-independent growth. These Ba/F3 FLT3-ITD cell lines or FLT3-ITD-expressing human MOLM13 cells were exposed to tunicamycin, 2-deoxy-D-glucose or VPA, and 17-AAG or rapamycin, and characterized in terms of downstream signaling by immunoblotting. FLT3 surface expression, apoptosis, and metabolic activity were analyzed by flow cytometry or an MTS assay. Proteome analysis by liquid chromatography–tandem mass spectrometry was performed to assess differential protein expression. RESULTS: The susceptibility of FLT3-ITD-expressing cells to 17-AAG after pre-treatment with tunicamycin or 2-deoxy-D-glucose was demonstrated. Importantly, in Ba/F3 cells that were stably expressing distinct FLT3-ITD variants that were located either in the juxtamembrane domain (JMD) or in the tyrosine kinase 1 domain (TKD1), response to the sequential treatments with tunicamycin and 17-AAG varied between individual FLT3-ITD motifs without dependence on the localization of the ITD. In all of the FLT3-ITD cell lines that were investigated, incubation with tunicamycin was accompanied by intracellular retention of FLT3-ITD due to the inhibition of glycosylation. In contrast, treatment of Ba/F3-FLT3-ITD cells with VPA was associated with a significant increase of FLT3-ITD surface expression depending on FLT3 protein synthesis. The allocation of FLT3 to different cellular compartments that was induced by tunicamycin, 2-deoxy-D-glucose, or VPA resulted in the activation of distinct downstream signaling pathways. Whole proteome analyses of Ba/F3 FLT3-ITD cells revealed up-regulation of the relevant chaperone proteins (e.g., calreticulin, calnexin, HSP90beta1) that are directly involved in the stabilization of FLT3-ITD or in its retention in the ER compartment. CONCLUSION: The allocation of FLT3-ITD to different cellular compartments and targeting distinct downstream signaling pathways by combined treatment with N-glycosylation and HSP90 inhibitors or VPA and rapamycin might represent new therapeutic strategies to overcome resistance towards tyrosine kinase inhibitors in FLT3-ITD-positive AML. The treatment approaches addressing N-glycosylation of FLT3-ITD appear to depend on patient-specific FLT3-ITD sequences, potentially affecting the efficacy of such pharmacological strategies.

scholarly journals Registered report: Widespread potential for growth factor-driven resistance to anticancer kinase inhibitors

eLife ◽  
2014 ◽  
Vol 3 ◽  
Author(s):  
Edward Greenfield ◽  
Erin Griner ◽  
Keyword(s):  
Growth Factor ◽  
Tyrosine Kinase ◽  
Signaling Pathways ◽  
Receptor Tyrosine Kinase ◽  
Cancer Biology ◽  
Kinase Inhibitors ◽  
Open Science ◽  
Downstream Signaling ◽  
Rtk Inhibitors ◽  
Block Sensitivity

The Reproducibility Project: Cancer Biology seeks to address growing concerns about reproducibility in scientific research by conducting replications of 50 papers in the field of cancer biology published between 2010 and 2012. This Registered Report describes the proposed replication plan of key experiments from ‘Widespread potential for growth-factor-driven resistance to anticancer kinase inhibitors’ by Wilson and colleagues, published in Nature in 2012 (<xref ref-type="bibr" rid="bib20">Wilson et al., 2012</xref>). The experiments that will be replicated are those reported in Figure 2B and C. In these experiments, Wilson and colleagues show that sensitivity to receptor tyrosine kinase (RTK) inhibitors can be bypassed by various ligands through reactivation of downstream signaling pathways (Figure 2A; <xref ref-type="bibr" rid="bib20">Wilson et al., 2012</xref>), and that blocking the receptors for these bypassing ligands abrogates their ability to block sensitivity to the original RTK inhibitor (Figure 2C; <xref ref-type="bibr" rid="bib20">Wilson et al., 2012</xref>). The Reproducibility Project: Cancer Biology is a collaboration between the Center for Open Science and Science Exchange, and the results of the replications will be published by eLife.

scholarly journals Prolonged exposure to FLT3 inhibitors leads to resistance via activation of parallel signaling pathways

Blood ◽  
2006 ◽  
Vol 109 (4) ◽  
pp. 1643-1652 ◽  
Author(s):  
Obdulio Piloto ◽  
Melissa Wright ◽  
Patrick Brown ◽  
Kyu-Tae Kim ◽  
Mark Levis ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Signaling Pathways ◽  
Cell Lines ◽  
Kinase Inhibitors ◽  
Prolonged Exposure ◽  
Cellular Transformation ◽  
Mapk Signaling ◽  
Resistant Cell ◽  
Myelogenous Leukemia ◽  
Continuous Treatment

Abstract Continuous treatment of malignancies with tyrosine kinase inhibitors (TKIs) may select for resistant clones (ie, imatinib mesylate). To study resistance to TKIs targeting FLT3, a receptor tyrosine kinase that is frequently mutated in acute myelogenous leukemia (AML), we developed resistant human cell lines through prolonged coculture with FLT3 TKIs. FLT3 TKI-resistant cell lines and primary samples still exhibit inhibition of FLT3 phosphorylation on FLT3 TKI treatment. However, FLT3 TKI-resistant cell lines and primary samples often show continued activation of downstream PI3K/Akt and/or Ras/MEK/MAPK signaling pathways as well as continued expression of genes involved in FLT3-mediated cellular transformation. Inhibition of these signaling pathways restores partial sensitivity to FLT3 TKIs. Mutational screening of FLT3 TKI-resistant cell lines revealed activating N-Ras mutations in 2 cell lines that were not present in the parental FLT3 TKI-sensitive cell line. Taken together, these data indicate that FLT3 TKI-resistant cells most frequently become FLT3 independent because of activation of parallel signaling pathways that provide compensatory survival/proliferation signals when FLT3 is inhibited. Anti-FLT3 mAb treatment was still cytotoxic to FLT3 TKI-resistant clones. An approach combining FLT3 TKIs with anti-FLT3 antibodies and/or inhibitors of important pathways downstream of FLT3 may reduce the chances of developing resistance.

scholarly journals Arsenic Trioxide Synergizes with FLT3 Tyrosine Kinase Inhibitors to Kill FLT3-ITD+ Leukemic Cells through Depressed Production and Degradation of FLT3 Protein

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1568-1568
Author(s):  
Kozo Nagai ◽  
Lihong Hou ◽  
Li Li ◽  
Bao Nguyen ◽  
Courtney M Shirley ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Arsenic Trioxide ◽  
Tyrosine Kinase Inhibitors ◽  
Cell Lines ◽  
Research Funding ◽  
Kinase Inhibitors ◽  
Leukemia Cell ◽  
Leukemic Cells ◽  
Potential Candidate ◽  
Leukemia Cell Lines

Abstract A number of selective FLT3 tyrosine kinase inhibitors (TKIs) have been tested for treatment of FLT3-ITD+ AML. However, monotherapy with FLT3 TKIs alone has achieved only transient and limited clinical responses due to several resistance mechanisms. Arsenic trioxide (ATO) has demonstrated significant efficacy in treating acute promyelocytic leukemia (APL). ATO has also shown some activity in treating non-APL myeloid leukemias. Recent studies have demonstrated that ATO can affect the degradation of oncogenic mutant proteins including mutant p53 and NPM through the ubiquitin-proteasome pathway (UPP). Here we investigated the feasibility of combining FLT3 TKIs with ATO in the treatment of FLT3-ITD+leukemia. Anti-leukemic effects against FLT3-ITD+ patient AML samples and leukemia cell lines (Molm14, MV4;11) by FLT3 TKIs (Sorafenib, quizartinib), ATO and the combination were examined by MTT, apoptosis, cell viability and colony forming assays. Our data revealed that the combination showed synergistic growth inhibition of the FLT3-ITD+ cell lines Molm14 and MV4;11, with combinatorial index (CI) values at ED50 below 1.0 for both cell lines (CI values were 0.46 and 0.56 for ATO + sorafenib, 0.65 and 0.57 for ATO + quizartinib in Molm14 and MV4;11 cells, respectively). In contrast, there was no synergy observed for the combination in treating leukemia cell lines that do not express mutant FLT3. Synergistic effects for the combination in inducing apoptosis and inhibiting colony formation were also observed for the FLT3-ITD+ cell lines. Furthermore, when the combination was used to treat primary FLT3-ITD+ patient samples, there was also significant reduction of viability and clonogenicity. In contrast, normal BM MNCs showed very limited responses to the combination. Western blot (WB) analysis of Molm14 and FLT3-ITD+ patient samples revealed the combination of ATO and sorefenib potently reduced phosphorylation of FLT3 and its downstream targets (STAT5, MAPK, and AKT). In vivoexperiments using the combination to treat NSG mice engrafted with Molm14 cells demonstrated a significant reduction in the level of leukemic cells. We further investigated the mechanism by which ATO contributes to an anti-leukemic effect on FLT3-ITD+ cells. Morphologic and flow cytometric analysis showed that ATO promoted the differentiation of Molm14 cells. The expression of C/EBPα and PU.1, two key regulators for myeloid differentiation, was increased in ATO-treated Molm14 cells at both the mRNA and protein levels. These data suggest ATO is capable of inducing the differentiation of leukemic cells. We also found that, in FLT3-ITD+ cells, ATO decreased expression of FLT3 protein. This could result from reduced FLT3 production and/or increased protein degradation. Further quantitative PCR analysis revealed ATO decreased expression of FLT3 and its upstream regulators HoxA9 and meis1. Co-immunoprecipitation assay showed that ATO facilitated poly-Ubiquitination and degradation of FLT3 in a dose- and time-dependent fashion. These results indicate that ATO exerts its anti-leukemic effects in FLT3-ITD+AML cell lines and primary samples at least partly through reducing the level of FLT3 protein. These studies together demonstrate that ATO has a unique activity towards FLT3-ITD+ leukemia cells. Based on these findings, ATO is a potential candidate to work in combination with FLT3 TKIs to improve the outcome of FLT3-ITD+ AML patients. Disclosures Levis: Millennium: Consultancy, Research Funding; Daiichi-Sankyo: Consultancy, Honoraria; Astellas: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding.

Prolonged Exposure to FLT3 Inhibitors Leads to Resistance Via Activation of Parallel Signaling Pathways.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1380-1380
Author(s):  
Obdulio Piloto ◽  
Melissa Wright ◽  
Patrick Brown ◽  
Kyu-Tae Kim ◽  
Mark Levis ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Signaling Pathways ◽  
Cell Lines ◽  
Kinase Inhibitors ◽  
Prolonged Exposure ◽  
Cellular Transformation ◽  
Mapk Signaling ◽  
Resistant Cell ◽  
Continuous Treatment

Abstract A number of tyrosine kinase inhibitors (TKI) have been developed to treat a variety of malignancies. However, continuous treatment with TKIs may select for resistant clones as has been seen with Gleevec treatment of CML. To study resistance to TKIs targeting FLT3, a receptor tyrosine kinase that is frequently mutated in AML, we developed resistant human cell lines through prolonged co-culture with FLT3 TKIs. Both FLT3 TKI sensitive and resistant cell lines exhibit inhibition of FLT3 phosphorylation upon FLT3 TKI treatment. However, FLT3 TKI resistant cell lines and primary samples often show continued activation of downstream PI3K/Akt and/or Ras/MEK/MAPK signaling pathways as well as continued expression of genes involved in FLT3-mediated cellular transformation. Inhibition of these pathways restores partial sensitivity to FLT3 TKIs. Mutational screening of FLT3 TKI resistant cell lines and primary samples failed to reveal any mutations in FLT3 or in 100 kinases/phosphatases tested but did reveal activating N-Ras mutations that were not present in the parental FLT3 TKI sensitive cell line. Taken together, these data indicate that FLT3 TKI resistant cells most frequently become FLT3 independent due to activation of parallel signaling pathways that provide compensatory survival / proliferation signals when FLT3 is inhibited. IMC-EB10, an unconjugated monoclonal antibody against FLT3, is still cytotoxic to FLT3 TKI resistant clones in vivo. An approach combining FLT3 TKIs with anti-FLT3 antibodies may prove superior and result in reduced chances of developing resistance.

Pharmacogenetic 3C interaction associated with the PDGFRA gene as a chromatin conformation marker for treatment with tyrosine kinase inhibitors.

2017 ◽  
Vol 35 (15_suppl) ◽  
pp. e23054-e23054
Author(s):  
Alexandre Akoulitchev ◽  
Aroul Ramadass ◽  
Jayne Green ◽  
Ulku Uzun ◽  
Jane Mellor ◽  
...  
Keyword(s):  
High Resolution ◽  
Tyrosine Kinase ◽  
Cell Line ◽  
Cell Lines ◽  
Kinase Inhibitors ◽  
Kinase Inhibitor ◽  
Leukemia Cell ◽  
Single Step ◽  
Chromosome Conformation ◽  
Insulator Function

e23054 Background: IDH1 mutations detected in glioma cells impair the insulator function between FIPL1L1 and PDGFRA at 4q12 ( Flavahan et al. 2016). We have used a high-resolution chromosome-conformation capture 3C analysis platform, EpiSwitch, and quantitative PCR, to map, evaluate, and quantify the TKI-sensitive conformational juxtaposition between FIP1L1 and PDGFRA. Loss of the insulator function in glioma prompted us to investigate the same interaction in the context of insulator loss with interstitial deletions at 4q12 in eosinophilic leukemias and AML. Methods: We tested a total of 72 primers in temperature gradient PCRs, with concentration matched negative controls, using the AML cell lines EOL-1 and HL-60. Products were sequenced in forward and reverse order. Dual label 5’FAM-BHQ1-3’hydrolysis probe assays, entirely specific for the PCR products, targeted the junction region of the 3C fragments. A reference 3C interaction was used as an internal copy number control for 3C library production. Results: EpiSwitch predicted and identified six 3C FIP1L1-PDGFRA interactions in different sequence orientations, within the 3D organization of the PDGFRA locus. The interaction D7 identified by the EpiSwitch qPCR assay was detected reproducibly in EOL-1 cells and glioblastoma tissue using both single step PCR and qPCR. An imatinib-sensitive AML cell line EOL-1 was used as a positive control for qPCR assays. Both AML and glioma cell lines tested positive using the assay as did glioma patient biopsies. The glioblastoma cell line DBTRG-05MG also tested positive for the D7 interaction at a maximum of 8.92 copies per 20 ng of the template. Conclusions: We confirmed and characterized, at high resolution, the conformational deregulation of FIP1L1 and PDGFRA in glioma. Additionally, our group detected the interaction in TKI-sensitive leukemia cell lines. The analysis of 3C microstructural alterations is consistent with latest insights into epigenetic regulation of PDGFRA. It provides a promising approach to the stratification of patients for tyrosine kinase inhibitor treatment, which could not be provided diagnostically with conventional sequencing approaches.

scholarly journals Ampelopsin Inhibits Cell Proliferation and Induces Apoptosis in HL60 and K562 Leukemia Cells by Downregulating AKT and NF-κB Signaling Pathways

2021 ◽  
Vol 22 (8) ◽  
pp. 4265
Author(s):  
Jang Mi Han ◽  
Hong Lae Kim ◽  
Hye Jin Jung
Keyword(s):  
Signaling Pathways ◽  
Cell Lines ◽  
White Blood Cells ◽  
Leukemia Cell ◽  
Leukemia Cells ◽  
Ros Generation ◽  
Anticancer Activities ◽  
Nuclear Condensation ◽  
Leukemia Cell Lines ◽  
Antileukemic Effect

Leukemia is a type of blood cancer caused by the rapid proliferation of abnormal white blood cells. Currently, several treatment options, including chemotherapy, radiation therapy, and bone marrow transplantation, are used to treat leukemia, but the morbidity and mortality rates of patients with leukemia are still high. Therefore, there is still a need to develop more selective and less toxic drugs for the effective treatment of leukemia. Ampelopsin, also known as dihydromyricetin, is a plant-derived flavonoid that possesses multiple pharmacological functions, including antibacterial, anti-inflammatory, antioxidative, antiangiogenic, and anticancer activities. However, the anticancer effect and mechanism of action of ampelopsin in leukemia remain unclear. In this study, we evaluated the antileukemic effect of ampelopsin against acute promyelocytic HL60 and chronic myelogenous K562 leukemia cells. Ampelopsin significantly inhibited the proliferation of both leukemia cell lines at concentrations that did not affect normal cell viability. Ampelopsin induced cell cycle arrest at the sub-G1 phase in HL60 cells but the S phase in K562 cells. In addition, ampelopsin regulated the expression of cyclins, cyclin-dependent kinases (CDKs), and CDK inhibitors differently in each leukemia cell. Ampelopsin also induced apoptosis in both leukemia cell lines through nuclear condensation, loss of mitochondrial membrane potential, increase in reactive oxygen species (ROS) generation, activation of caspase-9, caspase-3, and poly ADP-ribose polymerase (PARP), and regulation of Bcl-2 family members. Furthermore, the antileukemic effect of ampelopsin was associated with the downregulation of AKT and NF-κB signaling pathways. Moreover, ampelopsin suppressed the expression levels of leukemia stemness markers, such as Oct4, Sox2, CD44, and CD133. Taken together, our findings suggest that ampelopsin may be an attractive chemotherapeutic agent against leukemia.

scholarly journals (Phenylamino)pyrimidine-1,2,3-triazole derivatives as analogs of imatinib: searching for novel compounds against chronic myeloid leukemia

2021 ◽  
Vol 17 ◽  
pp. 2260-2269
Author(s):  
Luiz Claudio Ferreira Pimentel ◽  
Lucas Villas Boas Hoelz ◽  
Henayle Fernandes Canzian ◽  
Frederico Silva Castelo Branco ◽  
Andressa Paula de Oliveira ◽  
...  
Keyword(s):  
Chronic Myeloid Leukemia ◽  
Tyrosine Kinase ◽  
Myeloid Leukemia ◽  
Kinase Inhibitors ◽  
Competitive Inhibition ◽  
Leukemia Cell ◽  
Docking Studies ◽  
Leukemia Cell Line ◽  
Inhibition Mechanism ◽  
Triazole Derivatives

The enzyme tyrosine kinase BCR-Abl-1 is the main molecular target in the treatment of chronic myeloid leukemia and can be competitively inhibited by tyrosine kinase inhibitors such as imatinib. New potential competitive inhibitors were synthesized using the (phenylamino)pyrimidine-pyridine (PAPP) group as a pharmacophoric fragment, and these compounds were biologically evaluated. The synthesis of twelve new compounds was performed in three steps and assisted by microwave irradiation in a 1,3-dipolar cycloaddition to obtain 1,2,3-triazole derivatives substituted on carbon C-4 of the triazole nucleus. All compounds were evaluated for their inhibitory activities against a chronic myeloid leukemia cell line (K562) that expresses the enzyme tyrosine kinase BCR-Abl-1 and against healthy cells (WSS-1) to observe their selectivity. Three compounds showed promising results, with IC50 values between 1.0 and 7.3 μM, and were subjected to molecular docking studies. The results suggest that such compounds can interact at the same binding site as imatinib, probably sharing a competitive inhibition mechanism. One compound showed the greatest interaction affinity for BCR-Abl-1 in the docking studies.

scholarly journals An overview on the role of FLT3-tyrosine kinase receptor in acute myeloid leukemia: biology and treatment

2012 ◽  
Vol 6 (1) ◽  
pp. 8 ◽  
Author(s):  
Tiziana Grafone ◽  
Michela Palmisano ◽  
Chiara Nicci ◽  
Sergio Storti
Keyword(s):  
Acute Myeloid Leukemia ◽  
Tyrosine Kinase ◽  
Signaling Pathways ◽  
Myeloid Leukemia ◽  
Molecular Therapy ◽  
Tyrosine Kinase Receptor ◽  
Hematopoietic Stem ◽  
Downstream Signaling ◽  
Proliferation And Differentiation ◽  
Acute Myeloid

Hematopoiesis, the process by which the hematopoietic stem cells and progenitors differentiate into blood cells of various lineages, involves complex interactions of transcription factors that modulate the expression of downstream genes and mediate proliferation and differentiation signals. Despite the many controls that regulate hematopoiesis, mutations in the regulatory genes capable of promoting leukemogenesis may occur. The <em>FLT3</em> gene encodes a tyrosine kinase receptor that plays a key role in controlling survival, proliferation and differentiation of hematopoietic cells. Mutations in this gene are critical in causing a deregulation of the delicate balance between cell proliferation and differentiation. In this review, we provide an update on the structure, synthesis and activation of the FLT3 receptor and the subsequent activation of multiple downstream signaling pathways. We also review activating FLT3 mutations that are frequently identified in acute myeloid leukemia, cause activation of more complex downstream signaling pathways and promote leukemogenesis. Finally, FLT3 has emerged as an important target for molecular therapy. We, therefore, report on some recent therapies directed against it.

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