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ATR inhibition enables complete tumour regression in ALK-driven NB mouse models.

ABSTRACT: High-risk neuroblastoma (NB) often involves MYCN amplification as well as mutations in ALK. Currently, high-risk NB presents significant clinical challenges, and additional therapeutic options are needed. Oncogenes like MYCN and ALK result in increased replication stress in cancer cells, offering therapeutically exploitable options. We have pursued phosphoproteomic analyses highlighting ATR activity in ALK-driven NB cells, identifying the BAY1895344 ATR inhibitor as a potent inhibitor of NB cell growth and proliferation. Using RNA-Seq, proteomics and phosphoproteomics we characterize NB cell and tumour responses to ATR inhibition, identifying key components of the DNA damage response as ATR targets in NB cells. ATR inhibition also produces robust responses in mouse models. Remarkably, a 2-week combined ATR/ALK inhibition protocol leads to complete tumor regression in two independent genetically modified mouse NB models. These results suggest that NB patients, particularly in high-risk groups with oncogene-induced replication stress, may benefit from ATR inhibition as therapeutic intervention.

SUBMITTER: Szydzik J

PROVIDER: S-EPMC8613282 | biostudies-literature | 2021 Nov

REPOSITORIES: biostudies-literature

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ATR inhibition enables complete tumour regression in ALK-driven NB mouse models.

Szydzik Joanna J Lind Dan E DE Arefin Badrul B Kurhe Yeshwant Y Umapathy Ganesh G Siaw Joachim Tetteh JT Claeys Arne A Gabre Jonatan L JL Van den Eynden Jimmy J Hallberg Bengt B Palmer Ruth H RH

Nature communications 20211124 1

High-risk neuroblastoma (NB) often involves MYCN amplification as well as mutations in ALK. Currently, high-risk NB presents significant clinical challenges, and additional therapeutic options are needed. Oncogenes like MYCN and ALK result in increased replication stress in cancer cells, offering therapeutically exploitable options. We have pursued phosphoproteomic analyses highlighting ATR activity in ALK-driven NB cells, identifying the BAY1895344 ATR inhibitor as a potent inhibitor of NB cell ...[more]

PMID: 34819497

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Project description:Phosphoproteomics DIA LC-MSMS analysis Phospho-enriched peptides were resuspended in aqueous formic acid and 0.2 ug of peptides subjected to LC-MS/MS analysis using an Orbitrap Exploris 480 Mass Spectrometer fitted with an Vanquish Neo (both Thermo Fisher Scientific) and a custom-made column heater set to 60C. Peptides were resolved using a RP-HPLC column (75um x 30cm) packed in-house with C18 resin (ReproSil-Pur C18-AQ, 1.9 um resin; Dr. Maisch GmbH) at a flow rate of 0.2 uLmin-1. Separation of peptides was achieved using the following gradient: 4% Buffer B to 10% Buffer B in 5 min, 10% Buffer B to 35% Buffer B in 45 min, 35% Buffer B to 50% Buffer in 10 min. Buffer A was 0.1% formic acid in water and buffer B was 80% acetonitrile, 0.1% formic acid in water. The mass spectrometer was operated in DIA acquisition mode with a total cycle time not exceeding approximately 3 s. For MS1, the following parameters were set: Resolution: 120,000 FWHM (at 200 m/z), Scan Range: 350-1400 m/z, Injection time: 25 ms, Normalized AGC Target: 300%. MS2 (SWATH) scans were acquired using the following parameters: Isolation Window: 8 m/z, HCD Collision Energy (normalized): 28%, Normalized AGC target: 1000%, Resolution: 15,000 FWHM (at 200 m/z), Precursor Mass Range: 400 - 1200 m/z, Max. Fill Time: 22 ms, DataType: Centroid. In total 100 DIA (MS2) mass windows per MS cycle followed by the one MS1 scan. Data analysis of phosphoproteomics DIA-MS data The acquired raw files were searched using SpectroNaut (v17.1, PTM workflow, default settings) against a human database (consisting of 20360 protein sequences downloaded from Uniprot on 20220222) and 392 commonly observed contaminants using the following search criteria: full tryptic specificity was required (cleavage after lysine or arginine residues, unless followed by proline); 3 missed cleavages were allowed; carbamidomethylation (C) was set as fixed modification; oxidation (M), N-acetlyation (N-term) and phosphorylation (STY) were applied as variable modifications. The normalized, phosphosite centric and statstically analysed quantiative results were exported as a csv-table from the SpectroNaut software using the candidate list export option without any filters applied.

Dataset Information

ATR inhibition enables complete tumour regression in ALK-driven NB mouse models.

Publications

ATR inhibition enables complete tumour regression in ALK-driven NB mouse models.

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