Mechanisms of efficacy of endonuclease FEN1 inhibition in neuroblastoma

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 (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: 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: FEN1 inhibition was effective against NB cells and was significantly more effective in MYCN-amplified NB cells causing 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 and responses to FEN1 inhibition were associated with markers of replication stress. Conclusions: We have discovered that 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 neuroblastoma.