The target landscape of clinical kinase drugs Klaeger, Susan; Heinzlmeir, Stephanie; Wilhelm, Mathias ...
Science (American Association for the Advancement of Science),
12/2017, Letnik:
358, Številka:
6367
Journal Article
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Kinase inhibitors are important cancer therapeutics. Polypharmacology is commonly observed, requiring thorough target deconvolution to understand drug mechanism of action. Using chemical proteomics, ...we analyzed the target spectrum of 243 clinically evaluated kinase drugs. The data revealed previously unknown targets for established drugs, offered a perspective on the "druggable" kinome, highlighted (non)kinase off-targets, and suggested potential therapeutic applications. Integration of phosphoproteomic data refined drug-affected pathways, identified response markers, and strengthened rationale for combination treatments. We exemplify translational value by discovering SIK2 (salt-inducible kinase 2) inhibitors that modulate cytokine production in primary cells, by identifying drugs against the lung cancer survival marker MELK (maternal embryonic leucine zipper kinase), and by repurposing cabozantinib to treat FLT3-ITD-positive acute myeloid leukemia. This resource, available via the ProteomicsDB database, should facilitate basic, clinical, and drug discovery research and aid clinical decision-making.
The t(8;21) translocation is one of the most frequent cytogenetic abnormalities in acute myeloid leukaemia (AML) and results in the RUNX1/RUNX1T1 rearrangement. Despite the causative role of the ...RUNX1/RUNX1T1 fusion gene in leukaemia initiation, additional genetic lesions are required for disease development. Here we identify recurring ZBTB7A mutations in 23% (13/56) of AML t(8;21) patients, including missense and truncating mutations resulting in alteration or loss of the C-terminal zinc-finger domain of ZBTB7A. The transcription factor ZBTB7A is important for haematopoietic lineage fate decisions and for regulation of glycolysis. On a functional level, we show that ZBTB7A mutations disrupt the transcriptional repressor potential and the anti-proliferative effect of ZBTB7A. The specific association of ZBTB7A mutations with t(8;21) rearranged AML points towards leukaemogenic cooperativity between mutant ZBTB7A and the RUNX1/RUNX1T1 fusion.
Abstract
Chemistry teaching and learning bears some subject-specific challenges. For example, explanations and considerations of chemical phenomena drawing on the macroscopic, the sub-microscopic and ...the representational level. In this paper, we focus on the topic ‘acids and bases’ where the confusion of these levels leads to numerous misconceptions among learners. One possible source of these problems are textbooks, which can have an important impact on the quality of teaching and learning. To identify scientific and didactical appropriate textbooks for lower secondary classes, we draw on the work of Roseman, J. E., Stern, L. & Koppal, M. (2010), who developed an instrument to analyse textbooks using a conceptual coherence map. To develop our topic-specific instrument, big ideas of the topic were formulated, arranged in a conceptual coherence map, and set in relation with each other. Then we development a coding manual that describes precisely how to apply the different categories while analysing textbooks. The process described is part of a design-based research project with the aim to contribute to better chemistry teaching and learning. We give insight into the process of developing this instrument for analysing chemistry textbooks. Furthermore, it presents some examples for problematic representations from textbooks in the field of ‘acids and bases’.
•Separation of VLP from other extracellular vesicles by two step procedure.•Separation methods for bionanoparticles with similar surface properties.•Discrimination of particle populations by ...proteomics analysis.•In depth protocol including selection of the correct hardware.
Separation of enveloped virus-like particles from other extracellular vesicles is a challenging separation problem due to the similarity of these bionanoparticles. Without simple and scalable methods for purification and analytics, it is difficult to gain deeper insight into their biological function. A two-step chromatographic purification method was developed. In the first step, virus-like particles and extracellular vesicles were collected and separated from smaller impurities in a flow-through mode. Benzonase® treated HEK 293 cell culture supernatant was directly loaded onto a column packed with core-shell beads. The collected flow-through was further purified using heparin affinity chromatography. In heparin affinity chromatography 54% of the total particle load were found in the flow-through, and 15% of the particles were eluted during the salt linear gradient. The particle characterization, especially particle size distribution and mass spectrometry data, suggests that extracellular vesicles dominate the flow-through fraction and HIV-1 gag VLPs are enriched in the elution peak. This is in part in contradiction to other protocols where the extracellular vesicles are recovered by binding to heparin affinity chromatography. The developed method is easily scalable to pilot and process scale and allows a fast accomplishment of this separation within one day.
•Systematic comparison of performance of common purification methods for eVLPs.•Chromatin and eVLPs have similar size distribution in NTA measurements.•Heparin-affinity chromatography can separate ...eVLPs, host vesicles and chromatin.
Downstream processing (DSP) of large bionanoparticles is still a challenge. The present study aims to systematically compare some of the most commonly used DSP strategies for capture and purification of enveloped viruses and virus-like particles (eVLPs) by using the same staring material and analytical tools. As a model, Human Immunodeficiency Virus-1 (HIV-1) gag VLPs produced in CHO cells were used. Four different DSP strategies were tested. An anion-exchange monolith and a membrane adsorber, for direct capture and purification of eVLPs, and a polymer-grafted anion-exchange resin and a heparin-affinity resin for eVLP purification after a first flow-through step to remove small impurities. All tested strategies were suitable for capture and purification of eVLPs. The performance of the different strategies was evaluated regarding its binding capacity, ability to separate different particle populations and product purity. The highest binding capacity regarding total particles was obtained using the anion exchange membrane adsorber (5.3 × 1012 part/mL membrane), however this method did not allow the separation of different particle populations. Despite having a lower binding capacity (1.5 × 1011 part/mL column) and requiring a pre-processing step with flow-through chromatography, Heparin-affinity chromatography showed the best performance regarding separation of different particle populations, allowing not only the separation of HIV-1 gag VLPs from host cell derived bionanoparticles but also from chromatin. This work additionally shows the importance of thorough sample characterization combining several biochemical and biophysical methods in eVLP DSP.
The baculovirus expression vector system is a very powerful tool to produce virus‐like particles and gene‐therapy vectors, but the removal of coexpressed baculovirus has been a major barrier for ...wider industrial use. We used chimeric human immunodeficiency virus‐1 (HIV‐1) gag influenza‐hemagglutin virus‐like particles produced in Tnms42 insect cells using the baculovirus insect cell expression vector system as model virus‐like particles. A fast and simple purification method for these virus‐like particles with direct capture and purification within one chromatography step was developed. The insect cell culture supernatant was treated with endonuclease and filtered, before it was directly loaded onto a polymer‐grafted anion exchanger and eluted by a linear salt gradient. A 4.3 log clearance of baculovirus from virus‐like particles was achieved. The absence of the baculovirus capsid protein (vp39) in the product fraction was additionally shown by high performance liquid chromatography‐mass spectrometry. When considering a vaccination dose of 109 particles, 4200 doses can be purified per L pretreated supernatant, meeting the requirements for vaccines with <10 ng double‐stranded DNA per dose and 3.4 µg protein per dose in a single step. The process is simple with a very low number of handling steps and has the characteristics to become a platform for purification of these types of virus‐like particles.
Highlights • DNA vaccine targeting affected humoral and cellular immunity. • Elevated Th1 immunity did not correlate with superior protection from sensitization. • Plasmid vaccination boosted Treg ...numbers within re-stimulated splenocyte cultures.
In acute myeloid leukemia (AML), the Fms-like tyrosine kinase 3 (FLT3) is one of the most frequently mutated genes. Recently, a new and recurrent juxtamembrane deletion mutation (p.Q569Vfs*2) ...resulting in a truncated receptor was identified. The mutated receptor is expressed on the cell surface and still binds its ligand but loses the ability to activate ERK signaling. FLT3 p.Q569fs-expressing Ba/F3 cells show no proliferation after ligand stimulation. Furthermore, coexpressed with the FLT3 wild-type (WT) receptor, the truncated receptor suppresses stimulation and activation of the WT receptor. Thus, FLT3 p.Q569Vfs*2, to our knowledge, is the first FLT3 mutation with a dominant negative effect on the WT receptor.
The fms-related tyrosine kinase 3 (FLT3) receptor has been extensively studied over the past two decades with regards to oncogenic alterations that do not only serve as prognostic markers but also as ...targets for therapy in acute myeloid leukemia (AML). Internal tandem duplications (ITDs) became of special interest in this setting as they are associated with unfavorable prognosis. Due to sequence-dependent protein conformation changes, FLT3-ITD tends to auto-phosphorylate and displays a constitutive intracellular localization. As tyrosine kinase inhibitors (TKIs) efficiently block FLT3 auto-phosphorylation, we set out to study the effect of TKI on the subcellular localization of the FLT3 receptor and its mutants.
We visualized FLT3 WT and its mutants (N676K, D835Y, W51 (ITD), NPOS (ITD), K644R, W51-K644R) in transiently transfected U2OS cells, using anti-FLT3 immunofluorescence in combination with confocal microscopy. Treatment with 50nM of the TKI AC220 for 6 hours restored the membrane localization of both ITD (Figure 1 A) and D835Y. The TKI-induced surface expression of FLT3-ITD was confirmed by flow cytometry (mean fluorescence intensity, MFI) of BaF3 cells stably expressing FLT3 WT or mutants and in the homozygous (ITD/ITD) AML cell line (MV4-11). Similar results were obtained in cells from an AML patient derived xenograft (pdx) with FLT3-ITD and loss of heterozygosity (LOH) (Figure 1 B). Western blot analysis showed increased glycosylation of FLT3-ITD after TKI-treatment (Figure 1 C) suggesting that auto-phosphorylation prevents physiological processing of the receptor, which is required for maturation and surface expression. To monitor the subcellular localization of FLT3 and its mutants in vivo we have generated a FLT3-GFP fusion. Proliferation assays in BaF3 cells confirmed that the C-terminal GFP-tag does not alter the function of FLT3. Upon FLT3-ligand (FL) stimulation of FLT3-GFP we observed FLT3-internalization by life cell imaging in U2OS cells. Conversely, TKI treatment increased cell surface expression of FLT3-GFP.
Our findings have translational implications when considering FLT3 as a target for immunotherapy in AML, since FLT3 surface expression might facilitate antigen recognition. Especially FLT3-ITD positive patients with LOH may benefit from a combined TKI-immunotherapy approach. Therefore, we currently study the effect of AC220 on FLT3 antibody-dependent cellular cytotoxicity (ADCC), mediated by natural killer (NK) cells. Taken together, we discovered that TKI alters the subcellular localization and protein processing of specific FLT3 mutants. However, the complex interplay between the individual posttranslational modifications and their role in FLT3 traffic remains elusive.
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Krupka:AMGEN Research (Munich): Research Funding. Subklewe:AMGEN Research (Munich): Research Funding.
Introduction
In Acute Myeloid Leukemia (AML) internal tandem duplications (ITD) in the fms-related tyrosine kinase 3 (FLT3) are a frequent event associated with an unfavorable prognosis. At ...diagnosis, the FLT3-ITD status is routinely assessed by fragment analysis of PCR-amplified cDNA. However, this assay only provides information on the length but not on the position and sequence of the insertion. Therefore, it is attractive to overcome this limitation by the use of high-throughput amplicon sequencing (HTAS) as an alternative strategy for FLT3-ITD detection. To proof the feasibility and accuracy of this approach we performed HTAS on 260 AML patients, of which 250 were FLT3-ITD positive according to routine diagnostics.
Patients and Methods
All samples were obtained from patients treated on the German Acute Myeloid Leukemia Cooperative Group (AMLCG) trials 1999, 2004 and 2008. All patients received intensive induction therapy with curative intent. At diagnosis, patients had a median age of 60 years (range, 18-80 years). Additional molecular marker were screened routinely: NPM1 mutation (62%), KMT2A-PTD (8%), CEBPA mutations (8%). According to the ELN-classification patients clustered into the following groups: ELN intermediate I (70%), intermediate II (21%) or adverse (9%). A normal karyotype was observed in 71%, while 29% had an aberrant karyotpye. All patient samples were analyzed routinely by FLT3 fragment analysis. For HTAS of FLT3 we used custom FLT3 cDNA primers including barcode and adapter-sequences enabling a one-step PCR-protocol. Sequencing was performed (2x250bp paired end) on a MiSeq (Illumina). Per run, up to 96 samples were sequenced, yielding a median of 79,110 reads per amplicon (range: 31,996 - 259,783). As controls, cDNA from FLT3-ITD positive (Molm-13) and negative (HL60) cell lines were used. Sequencing data were aligned to the FLT3 cDNA reference (NM_004119.2) and FLT3-ITDs were called using Pindel software (version 0.2.5a7).
Results
Based on the HTAS results obtained from the FLT3 wild-type control HL60, we set the cut-off for ITD detection at a variant allele frequency (VAF) of 0.5%. Using this threshold, all patients which were assessed to be FLT3-ITD negative in diagnostic routine, were also negative in HTAS. FLT3-ITDs were detected by HTAS in 242 out of 250 (97%) patients who were FLT3-ITD positive patients according to routine fragment analysis. For six out of the 8 remaining patients, no valid ITD was detected by HTAS possibly due to one of the following reasons: a low mutational burden resulting in a VAF below the cut-off level (n=4) or a deletion near the ITD (n=2) potentially interfering with ITD-detection. In total, 308 ITDs were detected in 242 patients by HTAS while in these patients 282 ITDs were detected by routine diagnostics. Of note, HTAS missed 13 subclonal ITDs reported in routine, while 39 additional subclonal ITDs were detected by HTAS only. Overall, HTAS detected a higher number of ITDs per patient (mean: 1.27; range: 1-4) compared to fragment analysis (mean: 1.17; range: 1-3). Patients with more than one ITD according to HTAS showed a trend towards shorter overall and relapse free survival (p=0.105, p=0.104 respectively; Figure 1A). The ITD position (i.e. affected FLT3 domain) based on HTAS did not impact on clinical outcome. There was a significant correlation between the FLT3-ITD levels detected by fragment analysis and HTAS (Pearson, R=0.801, p=0.01; Figure 1B). High FLT3-ITD levels measured by fragment analysis had a significant impact on RFS, whereas this effect was not observed for FLT3-ITD levels measured by HTAS. The quantification of FLT3-ITD by HTAS might be hampered by underestimation of the VAFs of long ITDs that are less likely to be mapped correctly compared to shorter ITDs.
Conclusion
In summary, our study demonstrates the feasibility of HTAS for FLT3-ITD detection in AML. In particular, the identification of subclonal ITDs with high sensitivity provides additional information with potential prognostic value. Thus, HTAS may serve as a robust tool that could be implemented in future diagnostic routines. However, bioinformatic algorithms for ITD detection may need further improvement, e.g. to optimize ITD quantification and to facilitate the detection of ITDs in combination with deletions.
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Hiddemann:Roche: Other: Grants; Genentech: Other: Grants; Roche: Membership on an entity's Board of Directors or advisory committees.
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