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Early and late effects of pharmacological ALK inhibition on the neuroblastoma transcriptome.

ABSTRACT: Neuroblastoma is an aggressive childhood malignancy of the sympathetic nervous system. Despite multi-modal therapy, survival of high-risk patients remains disappointingly low, underscoring the need for novel treatment strategies. The discovery of ALK activating mutations opened the way to precision treatment in a subset of these patients. Previously, we investigated the transcriptional effects of pharmacological ALK inhibition on neuroblastoma cell lines, six hours after TAE684 administration, resulting in the 77-gene ALK signature, which was shown to gradually decrease from 120 minutes after TAE684 treatment, to gain deeper insight into the molecular effects of oncogenic ALK signaling.Here, we further dissected the transcriptional dynamic profiles of neuroblastoma cells upon TAE684 treatment in a detailed timeframe of ten minutes up to six hours after inhibition, in order to identify additional early targets for combination treatment.We observed an unexpected initial upregulation of positively regulated MYCN target genes following subsequent downregulation of overall MYCN activity. In addition, we identified adrenomedullin (ADM), previously shown to be implicated in sunitinib resistance, as the earliest response gene upon ALK inhibition.We describe the early and late effects of ALK inhibitor TAE684 treatment on the neuroblastoma transcriptome. The observed unexpected upregulation of ADM warrants further investigation in relation to putative ALK resistance in neuroblastoma patients currently undergoing ALK inhibitor treatment.

SUBMITTER: Claeys S

PROVIDER: S-EPMC5739776 | biostudies-literature | 2017 Dec

REPOSITORIES: biostudies-literature

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Early and late effects of pharmacological ALK inhibition on the neuroblastoma transcriptome.

Claeys Shana S Denecker Geertrui G Cannoodt Robrecht R Kumps Candy C Durinck Kaat K Speleman Frank F De Preter Katleen K

Oncotarget 20171106 63

<h4>Background</h4>Neuroblastoma is an aggressive childhood malignancy of the sympathetic nervous system. Despite multi-modal therapy, survival of high-risk patients remains disappointingly low, underscoring the need for novel treatment strategies. The discovery of <i>ALK</i> activating mutations opened the way to precision treatment in a subset of these patients. Previously, we investigated the transcriptional effects of pharmacological ALK inhibition on neuroblastoma cell lines, six hours afte ...[more]

PMID: 29290991

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Project description:Survival rates among patients with high-risk neuroblastoma remain low and novel therapies for recurrent neuroblastomas are required. ALK is commonly mutated in primary and relapsed neuroblastoma tumors and ALK tyrosine kinase inhibitors (TKI) are promising treatments for ALK-driven neuroblastoma; however, innate or adaptive resistance to single-agent ALK-TKIs remain a clinical challenge. Recently, SHP2 inhibitors have been shown to overcome ALK-TKI resistance in lung tumors harboring ALK rearrangements. Here, we have assessed the efficacy of the SHP2 inhibitor TNO155 alone and in combination with the ALK-TKIs crizotinib, ceritinib, or lorlatinib for the treatment of ALK-driven neuroblastoma using in vitro and in vivo models. In comparison to wild-type, ALK-mutant neuroblastoma cell lines were more sensitive to SHP2 inhibition with TNO155. Moreover, treatment with TNO155 and ALK-TKIs synergistically reduced cell growth and promoted inactivation of ALK and MAPK signaling in ALK-mutant neuroblastoma cells. ALK-mutant cells engrafted into larval zebrafish and treated with single agents or dual SHP2/ALK inhibitors showed reduced growth and invasion. In murine ALK-mutant xenografts, tumor growth was likewise reduced or delayed, and survival was prolonged upon combinatorial treatment of TNO155 and lorlatinib. Finally, we show that lorlatinib-resistant ALK-F1174L neuroblastoma cells harbor additional RAS-MAPK pathway alterations and can be resensitized to lorlatinib when combined with TNO155 in vitro and in vivo. Our results report the first evaluation of TNO155 in neuroblastoma and suggest that combinatorial inhibition of ALK and SHP2 could be a novel approach to treating ALK-driven neuroblastoma, potentially including the increasingly common tumors that have developed resistance to ALK-TKIs.SignificanceThese findings highlight the translatability between zebrafish and murine models, provide evidence of aberrant RAS-MAPK signaling as an adaptive mechanism of resistance to lorlatinib, and demonstrate the clinical potential for SHP2/ALK inhibitor combinations for the treatment of ALK-mutant neuroblastoma, including those with acquired tolerance or potentially resistance to ALK-TKIs.

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

Early and late effects of pharmacological ALK inhibition on the neuroblastoma transcriptome.

Publications

Early and late effects of pharmacological ALK inhibition on the neuroblastoma transcriptome.

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