ALK kinase domain mutations in primary anaplastic large cell lymphoma: consequences on NPM-ALK activity and sensitivity to tyrosine kinase inhibitors

PLoS One. 2015 Apr 13;10(4):e0121378. doi: 10.1371/journal.pone.0121378. eCollection 2015.

Abstract

ALK inhibitor crizotinib has shown potent antitumor activity in children with refractory Anaplastic Large Cell Lymphoma (ALCL) and the opportunity to include ALK inhibitors in first-line therapies is oncoming. However, recent studies suggest that crizotinib-resistance mutations may emerge in ALCL patients. In the present study, we analyzed ALK kinase domain mutational status of 36 paediatric ALCL patients at diagnosis to identify point mutations and gene aberrations that could impact on NPM-ALK gene expression, activity and sensitivity to small-molecule inhibitors. Amplicon ultra-deep sequencing of ALK kinase domain detected 2 single point mutations, R335Q and R291Q, in 2 cases, 2 common deletions of exon 23 and 25 in all the patients, and 7 splicing-related INDELs in a variable number of them. The functional impact of missense mutations and INDELs was evaluated. Point mutations were shown to affect protein kinase activity, signalling output and drug sensitivity. INDELs, instead, generated kinase-dead variants with dominant negative effect on NPM-ALK kinase, in virtue of their capacity of forming non-functional heterocomplexes. Consistently, when co-expressed, INDELs increased crizotinib inhibitory activity on NPM-ALK signal processing, as demonstrated by the significant reduction of STAT3 phosphorylation. Functional changes in ALK kinase activity induced by both point mutations and structural rearrangements were resolved by molecular modelling and dynamic simulation analysis, providing novel insights into ALK kinase domain folding and regulation. Therefore, these data suggest that NPM-ALK pre-therapeutic mutations may be found at low frequency in ALCL patients. These mutations occur randomly within the ALK kinase domain and affect protein activity, while preserving responsiveness to crizotinib.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adolescent
  • Anaplastic Lymphoma Kinase
  • Animals
  • COS Cells
  • Child
  • Child, Preschool
  • Chlorocebus aethiops
  • Crizotinib
  • Drug Resistance, Neoplasm
  • Female
  • HEK293 Cells
  • Humans
  • INDEL Mutation
  • Infant
  • Lymphoma, Large-Cell, Anaplastic / drug therapy
  • Lymphoma, Large-Cell, Anaplastic / enzymology*
  • Lymphoma, Large-Cell, Anaplastic / genetics*
  • Male
  • Molecular Dynamics Simulation
  • Mutation*
  • Point Mutation
  • Protein Kinase Inhibitors / pharmacology*
  • Protein Structure, Tertiary
  • Protein-Tyrosine Kinases / antagonists & inhibitors
  • Protein-Tyrosine Kinases / chemistry
  • Protein-Tyrosine Kinases / genetics*
  • Protein-Tyrosine Kinases / metabolism
  • Pyrazoles / pharmacology*
  • Pyridines / pharmacology*
  • Receptor Protein-Tyrosine Kinases / chemistry
  • Receptor Protein-Tyrosine Kinases / genetics*
  • Receptor Protein-Tyrosine Kinases / metabolism

Substances

  • Protein Kinase Inhibitors
  • Pyrazoles
  • Pyridines
  • Crizotinib
  • p80(NPM-ALK) protein
  • ALK protein, human
  • Anaplastic Lymphoma Kinase
  • Protein-Tyrosine Kinases
  • Receptor Protein-Tyrosine Kinases

Grant support

This work was supported by "Associazione Italiana Leucemie Treviso", Italy (fellowship to FL), "Fondazione Cassa di Risparmio di Padova e Rovigo", Padua, Italy, and "Istituto di Ricerca Pediatrica Città della Speranza", Padua, Italy. The molecular modelling work has been carried out with financial support of the University of Padua, Italy, and the Italian Ministry for University and Research (MIUR), Rome, Italy. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.