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Aquired resistance mechanisms to EGFR-specific kinase inhibitors using chemical proteomics

ABSTRACT: Here we used kinase affinity enrichment (Kinobeads) and intensity-based mass spectrometry to elucidate aquired resistance mechanisms to EGFR inhibition in cancer cells. First, kinase expression differences between untreated control cells and long term treated resistant cells were investigated using Kinobeads and LC-MS/MS. Overexpressed Kinases were then further investigated by cellular assays to evaluate targets and development of combination treatment strategies.

INSTRUMENT(S): LTQ Orbitrap

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Epithelial Cell, Cell Culture

DISEASE(S): Non-small Cell Lung Carcinoma

SUBMITTER: Heiner Koch

LAB HEAD: Prof. Bernhard Kuster

PROVIDER: PXD001825 | Pride | 2015-05-20

REPOSITORIES: Pride

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			Action	DRS
	000249_A01_P001439_B00_A00_R1.RAW	Raw
	000249_B01_P001440_B00_A00_R1.RAW	Raw
	000249_C01_P001441_B00_A00_R1.RAW	Raw
	000249_D01_P001442_B00_A00_R1.RAW	Raw
	000249_E01_P001443_B00_A00_R1.RAW	Raw

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Publications

Chemical Proteomics Uncovers EPHA2 as a Mechanism of Acquired Resistance to Small Molecule EGFR Kinase Inhibition.

Koch Heiner H Busto M Estela Del Castillo ME Kramer Karl K Médard Guillaume G Kuster Bernhard B

Journal of proteome research 20150526 6

Tyrosine kinase inhibitors (TKIs) have become an important therapeutic option for treating several forms of cancer. Gefitinib, an inhibitor of the epidermal growth factor receptor (EGFR), is in clinical use for treating non-small cell lung cancer (NSCLC) harboring activating EGFR mutations. However, despite high initial response rates, many patients develop resistance to gefitinib. The molecular mechanisms of TKI resistance often remain unclear. Here, we describe a chemical proteomic approach co ...[more]

PMID: 25963923

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Differential kinobeads selectivity profiling

Project description:The goal of this project was to use differential kinobeads profiling for the discrimination of reversible and irreversible kinase targets of different covalent kinase inhibitors. For this, differences in target engagement measured in a lysate- and a cell-based kinobeads assay were compared in a kinome-wide scale.

2017-10-13 | PXD007920 | Pride

Defining the landscape of EPHA2 Inhibition by Clinical Kinase Inhibitors

Project description:The receptor tyrosine kinase EPHA2 plays important roles in oncogenesis, metastasis and treatment resistance but therapeutic targeting, drug discovery or investigation of EPHA2 biology is hampered by the lack of appropriate inhibitors and structural information on how these interact with the protein. Here, we used chemical proteomics to survey 235 clinical kinase inhibitors and identified 24 with sub-micromolar potency for EPHA2. We generated nine high resolution co-crystal structures to identify drug-protein interactions at the atomic level. The combination of compound selectivity, structure determinantion and kinome-wide sequence alignment allowed us to develop a classification system in which amino acids in the drug binding site are categorized into key, scaffold, potency and selectivity residues. This scheme should be generally applicable in kinase drug discovery and we anticipate that the provided information will greatly facilitate the development of selective EPHA2 inhibitors in particular and the repurposing of clinical kinase inhibitors in general.

2016-12-23 | PXD004112 | Pride

Chemical Proteomics Reveals Ferrochelatase as a Common Off-target of Kinase Inhibitors

Project description:Protein kinases are key components in signal transduction pathways and are established drug targets in oncology. Consequently, small molecule kinase inhibitors are on the rise but often show a rather broad target spectrum, potentially leading to toxic side effects. As a result, a broad assessment of the target space is desirable for proper interpretation of observed biological effects. The enzyme Ferrochelatase (FECH), which catalyzes the conversion of protoporphyrin IX into heme, was recently found to be an off-target of the BRAF inhibitor Vemurafenib potentially explaining the often severe phototoxicity associated with this drug in melanoma patients. However, the extent to which kinase inhibitors bind to FECH in general is currently unclear. Here, we used a chemical proteomics approach based on the kinobead technology to profile 226 clinical kinase inhibitors for their potential to bind FECH. Surprisingly, low or sub-micromolar FECH binding was detected for 29 (13%) of all compounds tested and isothermal dose response measurements confirmed drug binding to FECH in cells. We also show that Vemurafenib, Linsitinib, Neratinib and MK-2461 reduce heme levels in K562 cells, verifying that drug binding leads to loss of FECH activity. Further experiments identified the protoporphyrin pocket in FECH as one major binding site for small molecule inhibitors. Since genetic loss of FECH function leads to photosensitivity in humans, we suggest that FECH inhibition by kinase inhibitors is the molecular mechanism triggering photosensitivity in patients and should therefore be part of the pre-clinical tox package for kinase inhibitors.

2017-03-22 | PXD003373 | Pride

Kinobeads affinity enrichment from lysate derived from stimulated cells

Project description:Investigate differential binding of kinases to Kinobeads depending on activation state

2018-05-02 | PXD008632 | Pride

Discovery of dedicated EPHA2 inhibitors - Medicinal chemistry meets chemical proteomics

Project description:The receptor tyrosine kinase EPHA2 has gained interest as therapeutical drug target in cancer and infectious diseases in the past years. However, EPHA2 research and EPHA2 based therapies have been hampered by the lack of selective small molecule EPHA2 inhibitors. We report on the synthesis and evaluation of dedicated EPHA2 inhibitors based on the clinical BCR-ABL/SRC inhibitor Dasatinib as lead structure. We designed hybrid structures of Dasatinib, CHEBI-513815, PD-173955 and a known EPHB4 inhibitor aiming at the exploitation of the ATP pocket entrance and the ribose pocket. Medicinal chemistry and inhibitor design was guided by a chemical proteomic approach allowing for early selectivity profiling of the newly synthesized inhibitor candidates. Additionally, protein crystallography delivered detailed insight into the molecular interactions that consitute structure-affinity-relationships. Finally, the anti-proliferative effect of the inhibitor candidates was confirmed in the glioblastoma cell line SF-268. In this work, we discovered a novel EPHA2 inhibitor candidate 4a comprising an improved selectivity profile while maintaining potency against EPHA2 and anti-cancer activity in SF-268 cells.

2017-05-30 | PXD006193 | Pride

Proteomics analysis with the Aurora-A PROTAC, JB170

Project description:The mitotic kinase Aurora-A is essential for cell cycle progression and considered a priority cancer target. Dozens of ongoing clinical trials are testing the anti-cancer potential of Aurora-A kinase inhibitors. While the catalytic activity of Aurora-A is essential for its function during mitosis, a growing body of evidence indicates an additional non-catalytic function, which is difficult to target by kinase inhibitors. We therefor developed a series of degrader molecules by connecting a kinase inhibitor of Aurora-A to the Cereblon-binding molecule thalidomide. One degrader, JB170, induced the rapid degradation of Aurora-A. We were able to show that JB170 mediated depletion is highly specific for Aurora-A, as degrader mediated complex formation is supported by cooperative binding between Cereblon and Aurora-A. Strikingly, JB-170 mediated depletion caused a strong S-phase arrest, which is not the cell cycle effect observed as a result of Aurora-A kinase inhibition, supporting an important non-catalytic role of Aurora-A during DNA replication. Finally, depletion of Aurora-A resulted in strong induction of apoptosis in various cancer cell lines. The tool compound JB170 presents a versatile starting point for developing new therapeutic drugs to counter Aurora-A function in cancer. In the presented datasets, we determined i) the binding selectivity profile of the Aurora-A PROTAC (JB170) using Kinobeads affinity matrix and ii) its intracellular degradation specificity. We therefore treated IMR5 cells with JB170, the inactive version JB211, or Alisertib and quantified the induced degradation using tandem mass tags.

2020-09-28 | PXD019585 | Pride

Chemoproteomic selectivity profiling of PIKK and PI3K kinase inhibitors

Project description:"Here, we report the development of a novel version of Kinobeads that extends kinome coverage to PIKK and PI3K kinases. This is achieved by inclusion of two affinity probes derived from the clinical PI3K/MTOR inhibitors Omipalisib and BGT226. The new affinity matrix was used to profile 13 clinical and pre-clinical PIKK/PI3K inhibitors. The large discrepancies between the PI3K affinity values obtained and reported results from recombinant assays led us to perform a phosphoproteomic experiment showing that the chemoproteomic assay is the better approximation of PI3K inhibitor action in-vivo."

2019-03-29 | PXD011719 | Pride

Identification of common features proffered by KIT and PDGFRA mutations in gastro-intestinal stromal tumors

Project description:Mutations in KIT proto-oncogene receptor tyrosine kinase (KIT) or platelet derived growth factor receptor alpha (PDGFRA) are responsible for more than 85% of the gastrointestinal stromal tumors (GIST). The introduction of imatinib in the therapy scheme of GIST revolutionized the patient outcome. Unfortunately, the therapy allows a disease stabilization rather than curation. Resistance to the inhibitor arises in most cases within the two first years of therapy. The identification of new targets to treat GIST is now essential. We propose a thorough investigation of the activating mechanisms derived from the main PDGFRA and KIT mutants encountered in the GIST landscape. We identified striking differences among the different KIT mutants while PDGFRA mutants delivered a very uniform picture. KIT Exon 11 deletion mutant exhibited the highest intrinsic kinase activity and all KIT mutants were, in addition to their constitutive activation, responsive to stem cell factor (SCF) stimulation. This highlights the importance of evaluating the SCF expression profile in GIST patients. In contrast, PDGFRA mutants were not responsive to their ligand, PDGFAA, and displayed a very high intrinsic kinase activity. At the transcriptomic level, the mitogen-activated protein kinase (MAPK) pathway was established as the most prominent activated pathway, commonly up-regulated by all PDGFRA and KIT mutants. Inhibition of this pathway using the MEK inhibitor PD0325901 reduced the proliferation of GIST primary cells in the nanomolar range. This demonstrates the high value of MEK inhibitors for combination therapy in GIST treatment. This experiment contains expression data from HEK-293 cells expressing wild-type and mutant KIT. The mutants included in the study correspond to the main mutations found in GIST, mainly KIT Ex9 exhibits a duplication of AY502-503 and KIT Ex11 a deletion of residues 553 to 557.

2017-07-05 | E-MTAB-4548 | biostudies-arrayexpress

Active kinome in human skeletal muscle insulin resistance

Project description:Obesity-related insulin resistance (OIR) is one of the main contributors to type 2 diabetes and other metabolic diseases. Protein kinases are implicated in key signaling steps including insulin signaling and glucose metabolism. Molecular mechanisms underlying OIR involving global active kinases remain less understood. Herein, we report findings from an in vivo active kinase profiling study in skeletal muscle from lean control (Lean) and OIR human participants, which identified the 1st active kinome comprised of 54 active protein kinases in human skeletal muscle. The activities of 23 kinases were different in OIR compared to Lean muscle (11 hyper- and 12 hypo-active), while their protein abundance was the same between the two groups. The activities of multiple kinases involved in AMPK and p38 signaling were lower in OIR compared to Lean. On the contrary, multiple kinases in JNK signaling pathway exhibited higher activity in OIR vs. Lean muscle. The kinase-substrate-prediction based on experimental data further confirmed a potential down-regulation of insulin signaling (e.g., inhibited phosphorylation of insulin receptor substrate-1 and AKT1/2). These findings provide a global view of the active kinome in OIR and Lean muscle, pinpoint novel specific impairment in kinase activities in multiple signaling pathways important for skeletal muscle insulin resistance, and provide potential drug targets (i.e., abnormal active kinase) to prevent and/or reverse skeletal muscle insulin resistance in humans.

2020-01-23 | PXD011909 | Pride

Single methyl group positioning opens new chemical space for EPH receptor kinase inhibition

Project description:EPHrin receptors (EPH) are transmembrane receptor tyrosine kinases. Their extracellular domains bind specifically to ephrin A/B ligands, and this binding modulates the intracellular kinase activity. EPHs are key players in bidirectional intercellular signalling, controlling cell morphology, adhesion and migration. They are increasingly recognized as cancer drug targets. We analysed the binding of the patented Novartis inhibitor NVP-BHG712 (NVP) to EPHA2 and EPHB4. Unexpectedly, all tested commercially available NVP samples were regioisomers NVPiso with a single methyl group binding to an adjacent nitrogen atom compared to the patented compound. The two compounds of identical mass reveal different binding modes. Further, both, in vitro and in vivo experiments show that the isomers differ in their kinase affinity and selectivity.

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