Efficacy of targeting replication stress in neuroblastoma.

Abstract

3116 Background: Children with high-risk and relapsed neuroblastoma (NB) need improved therapies, and recurrent cytogenetic abnormalities, such as MYCN oncogene amplification, represent candidate therapeutic targets. MYCN amplification and increased MYCN expression drive deregulated hyper-transcription that leads to development and growth of NB tumors, and MYCN amplifications, which can be found both within the linear genome on homogenously staining regions (HSR) and on circular extrachromosomal DNA (ecDNA), are associated with significantly worse survival rates for children with NB. MYCN overexpression has been linked to an increase in replication stress (RS), and RS and subsequent genome instability are important drivers of tumor initiation and progression. Flap Endonuclease 1 (FEN1), a non-essential DNA replication enzyme, was identified as a synthetic lethal target in BRCA1 / 2 -deficient cancers via induction of RS. Recent success of targeting RS-elevated cancers with replication enzyme inhibitors opens a new avenue to target MYCN -amplified NB. Methods: Associations of gene expression with patient survival and prognostic features were performed on available neuroblastoma tumor databases using the R2 Genomics Analysis and Visualization Platform. The efficacy of FEN1 inhibition was assessed using live cell imaging and cell viability assays, comparing results in MYCN -amplified to -nonamplified NB cells and in NB cells with inducible MYCN expression and repression. Mechanisms of cell death and impacts on replication stress in cells treated with FEN1 inhibitors were evaluated by Western blots. Results: We have found that FEN1 expression levels are associated with NB patient outcomes and with features of high-risk disease, including tumor stage and MYCN amplification. FEN1 inhibition was effective against NB cells and was significantly more effective in HSR- MYCN -amplified NB cells, but not ecDNA-MYCN amplified NB cells, with reduced cell growth and viability. Increased MYCN expression also led to increased sensitivity to FEN1 inhibition, and reduced MYCN expression reduced sensitivity. FEN1 inhibition led to the induction of apoptosis but not necroptosis in NB cells and responses to FEN1 inhibition were associated with markers of replication stress, including activation of the ATR-CHK1 and ATM-CHK2 pathways. Conclusions: We have discovered that HSR-mediated MYCN -amplified NB cells are hypersensitive to FEN1 inhibition, suggesting that FEN1 inhibition may be a promising therapeutic strategy for children with high-risk and relapsed NB.