Poster Presentation Advances in Neuroblastoma Research Congress 2016

Growth advantage and oncogene addiction of neuroblastoma cells bearing an ALK mutation (#342)

Simon Durand 1 , Caroline Louis 1 , Cécile Pierre-Eugène 1 , Valérie Combaret 2 , Carine Giovannangelli 3 , Angela Bellini 4 , Virginie Raynal 1 5 , Sylvain Baulande 5 , Estelle Daudigeos 6 , Birgit Georger 6 , Gudrun Schleiermacher 7 , Olivier Delattre 1 , Isabelle Janoueix-Lerosey 1
  1. Institut Curie, PSL Research University, Inserm U830, Paris, France
  2. Centre Léon-Bérard, Laboratoire de Recherche Translationnelle, Lyon, France
  3. INSERM U1154, CNRS UMR7196, Structure and Instability of Genomes, Sorbonne Universités, Museum National d'Histoire Naturelle, Paris, France
  4. Laboratoire RTOP "Recherche Translationelle en Oncologie Pédiatrique"; INSERM U830, Institut Curie, Paris, France
  5. Institut Curie Genomics of Excellence (ICGex) Platform, Paris, France
  6. Vectorology and Anticancer Therapies, UMR 8203, CNRS, Univ. Paris-Sud, Gustave Roussy, Université Paris-Saclay, Villejuif, France
  7. Laboratoire RTOP "Recherche Translationelle en Oncologie Pédiatrique"; INSERM U830 and Department of Pediatric Oncology, Institut Curie, Paris, France

Activating ALK mutations have been observed at clonal or sub-clonal levels in patients affected by neuroblastoma, either at diagnosis or at relapse. To further decipher the specific role of ALK mutation in disease aggressiveness and progression, we sought to compare the properties of isogenic cell lines bearing the ALK mutation at different levels.

First, we characterized two neuroblastoma cell lines derived from a stage 4 patient at diagnosis, either from the primary abdominal tumor (PT) or from the bone marrow (BM). SNP array analysis showed that both cell lines exhibited similar genomic profiles with 1p deletion, 17q gain and MYCN amplification. Both samples presented with 2 copies of the ALK locus. Interestingly, by deep sequencing and digital PCR, we measured an ALK mutation rate (F1174L) of 2.4% in the PT-derived cell line, which likely corresponds to 5% of the cells bearing an heterozygous mutation. In contrast, a mutation rate of 50% was observed for cells from the BM-derived cell line indicating that all cells exhibited the mutation. The ALK mutation rate in the PT-derived cell line increased with successive passages. In vitro growth analysis showed that the BM-derived cell line had higher proliferation rate compared to the PT-derived cell line. Orthotopic injection of both cell lines in Nude mice resulted in tumor formation only for the BM-derived cell line. Second, we sought to knock-out the ALK gene in a neuroblastoma cell line bearing a R1275Q mutation. Among the clones that grew following CRISPR/Cas9 induced rearrangements in the first exon, we observed either in-frame rearrangements or knock-out of the wild-type ALK allele. No clone with an invalidation of the mutated allele could be obtained, suggesting an oncogene addiction of the cells to mutated ALK.

Altogether, our results suggest that the ALK mutation significantly contributes to disease aggressiveness and progression.