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ALK signaling primes the DNA damage response sensitizing ALK-driven neuroblastoma to therapeutic ATR inhibition.

ABSTRACT: High-risk neuroblastoma (NB) is a significant clinical challenge. MYCN and Anaplastic Lymphoma Kinase (ALK), which are often involved in high-risk NB, lead to increased replication stress in cancer cells, suggesting therapeutic strategies. We previously identified an ATR (ataxia telangiectasia and Rad3-related)/ALK inhibitor (ATRi/ALKi) combination as such a strategy in two independent genetically modified mouse NB models. Here, we identify an underlying molecular mechanism, in which ALK signaling leads to phosphorylation of ATR and CHK1, supporting an effective DNA damage response. The importance of ALK inhibition is supported by mouse data, in which ATRi monotreatment resulted in a robust initial response, but subsequent relapse, in contrast to a 14-d ALKi/ATRi combination treatment that resulted in a robust and sustained response. Finally, we show that the remarkable response to the 14-d combined ATR/ALK inhibition protocol reflects a robust differentiation response, reprogramming tumor cells to a neuronal/Schwann cell lineage identity. Our results identify an ability of ATR inhibition to promote NB differentiation and underscore the importance of further exploring combined ALK/ATR inhibition in NB, particularly in high-risk patient groups with oncogene-induced replication stress.

SUBMITTER: Borenas M

PROVIDER: S-EPMC10769851 | biostudies-literature | 2024 Jan

REPOSITORIES: biostudies-literature

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ALK signaling primes the DNA damage response sensitizing ALK-driven neuroblastoma to therapeutic ATR inhibition.

Borenäs Marcus M Umapathy Ganesh G Lind Dan E DE Lai Wei-Yun WY Guan Jikui J Johansson Joel J Jennische Eva E Schmidt Alexander A Kurhe Yeshwant Y Gabre Jonatan L JL Aniszewska Agata A Strömberg Anneli A Bemark Mats M Hall Michael N MN Eynden Jimmy Van den JVD Hallberg Bengt B Palmer Ruth H RH

Proceedings of the National Academy of Sciences of the United States of America 20231228 1

High-risk neuroblastoma (NB) is a significant clinical challenge. MYCN and Anaplastic Lymphoma Kinase (ALK), which are often involved in high-risk NB, lead to increased replication stress in cancer cells, suggesting therapeutic strategies. We previously identified an ATR (ataxia telangiectasia and Rad3-related)/ALK inhibitor (ATRi/ALKi) combination as such a strategy in two independent genetically modified mouse NB models. Here, we identify an underlying molecular mechanism, in which ALK signali ...[more]

PMID: 38154064

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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

ALK signaling primes the DNA damage response sensitizing ALK-driven neuroblastoma to therapeutic ATR inhibition.

Publications

ALK signaling primes the DNA damage response sensitizing ALK-driven neuroblastoma to therapeutic ATR inhibition.

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