Published October 28, 2021 | Version v1
Preprint Open

Loss of p16INK4a in neuroblastoma cells induces shift to an immature state with mesenchymal characteristics and increases sensitivity to EGFR inhibitors

  • 1. Princess Máxima Center for Pediatric Oncology, Utrecht, The Netherlands
  • 2. Preclinical Intervention Unit of the Mouse Clinic for Cancer and Ageing (MCCA), Netherlands Cancer Institute, Amsterdam, The Netherlands
  • 3. Hopp Children's Cancer Center Heidelberg (KiTZ) and Division of Neuroblastoma Genomics, German Cancer Research Center (DKFZ), Heidelberg, Germany
  • 4. Jan J. Molenaar


Homozygous inactivation of the CDKN2A locus is one of the most common genomic aberrations in human cancer. The locus codes for two unrelated and distinctly regulated proteins: p14ARF and p16INK4a, which inhibit MDM2 and CDK4/6, respectively. Loss of CDKN2A is also a recurrent event in relapsed neuroblastoma, a childhood tumour that arises from neural crest cells. To examine the consequences of the loss of the two distinct gene transcripts in neuroblastoma, we used the CRISPR-Cas9 system to knockout p14, p16 and p14+p16 in SY5Y cells. RNA sequencing of the transcriptome revealed a striking shift towards an immature Schwann cell precursor-like phenotype with mesenchymal characteristics, specifically in the p16 and p14+p16 knockouts. High-throughput drug screening of p16 and p14+p16 knockout clones identified a large increase in sensitivity to EGFR inhibitors. On protein level, we were able to confirm that EGFR pathway activation is higher in p14+p16 knockout cells and that treatment with the EGFR inhibitor afatinib resulted in higher levels of apoptosis. Afatinib also reduced tumour growth in vivo in xenografts transplanted with p14+p16 knockout SY5Y cells. Overall, our study suggests that CDKN2A deletion in neuroblastoma relates to a phenotypic shift towards a more progenitor like state and increases sensitivity to EGFR inhibitors.



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iPC – individualizedPaediatricCure: Cloud-based virtual-patient models for precision paediatric oncology 826121
European Commission