BioID-Screening Identifies PEAK1 and SHP2 as Components of the ALK Proximitome in Neuroblastoma Cells

J Mol Biol. 2021 Sep 17;433(19):167158. doi: 10.1016/j.jmb.2021.167158. Epub 2021 Jul 15.

Abstract

Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase (RTK) that is mutated in approximately 10% of pediatric neuroblastoma (NB). To shed light on ALK-driven signaling processes, we employed BioID-based in vivo proximity labeling to identify molecules that interact intracellularly with ALK. NB-derived SK-N-AS and SK-N-BE(2) cells expressing inducible ALK-BirA* fusion proteins were generated and stimulated with ALKAL ligands in the presence and absence of the ALK tyrosine kinase inhibitor (TKI) lorlatinib. LC/MS-MS analysis identified multiple proteins, including PEAK1 and SHP2, which were validated as ALK interactors in NB cells. Further analysis of the ALK-SHP2 interaction confirmed that the ALK-SHP2 interaction as well as SHP2-Y542 phosphorylation was dependent on ALK activation. Use of the SHP2 inhibitors, SHP099 and RMC-4550, resulted in inhibition of cell growth in ALK-driven NB cells. In addition, we noted a strong synergistic effect of combined ALK and SHP2 inhibition that was specific to ALK-driven NB cells, suggesting a potential therapeutic option for ALK-driven NB.

Keywords: BirA*; RMC-4550; SHP099; lorlatinib; signaling.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Aminopyridines / pharmacology
  • Anaplastic Lymphoma Kinase / metabolism*
  • Animals
  • Cell Line, Tumor
  • Chromatography, Liquid
  • Drug Synergism
  • Gene Expression Regulation, Neoplastic / drug effects
  • HEK293 Cells
  • Humans
  • Lactams / pharmacology
  • Neuroblastoma / metabolism*
  • PC12 Cells
  • Phosphorylation
  • Piperidines / pharmacology
  • Protein Tyrosine Phosphatase, Non-Receptor Type 11 / metabolism*
  • Protein-Tyrosine Kinases / metabolism*
  • Proteomics / methods*
  • Pyrazoles / pharmacology
  • Pyrimidines / pharmacology
  • Rats
  • Tandem Mass Spectrometry

Substances

  • Aminopyridines
  • Lactams
  • Piperidines
  • Pyrazoles
  • Pyrimidines
  • SHP099
  • ALK protein, human
  • Anaplastic Lymphoma Kinase
  • PEAK1 protein, human
  • Protein-Tyrosine Kinases
  • PTPN11 protein, human
  • Protein Tyrosine Phosphatase, Non-Receptor Type 11
  • lorlatinib