Loss of p16INK4a in neuroblastoma cells induces shift to an immature state with mesenchymal characteristics and increases sensitivity to EGFR inhibitors
Creators
- Nil A. Schubert1
- Sander R. van Hooff1
- Linda Schild1
- Kimberley Ober1
- Marjolein Hortensius1
- Kim van den Handel1
- Anke H.W. Essing1
- Bianca Koopmans1
- Manon Boeije2
- Natalie Proost2
- Marieke van de Ven2
- Selina Jansky3
- Sabine A. Stainczyk3
- Umut H. Toprak3
- Frank Westermann3
- Selma Eising1
- Jan J. Molenaar4
- Marlinde L. van den Boogaard1
- 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
Description
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.
Files
2021.10.27.465137v1.full.pdf
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