Defects in 8-oxo-guanine repair pathway cause high frequency of C > A substitutions in neuroblastoma

Marlinde L Van Den Boogaard,M Emmy M Dolman,Lieke L Hoyng,Rogier Versteeg,Thomas Helleday,Jan J Molenaar,Anke H W Essing,Rurika Oka,Jarno Drost,Danny A Zwijnenburg,Anne Hakkert,Bianca Koopmans,Linda Schild,Marli E Ebus,Piet Molenaar,Ruben Van Boxtel,Jan Koster,Michael R Van Gerven

doi:10.1073/pnas.2007898118

Abstract

Neuroblastomas are childhood tumors with frequent fatal relapses after induction treatment, which is related to tumor evolution with additional genomic events. Our whole-genome sequencing data analysis revealed a high frequency of somatic cytosine > adenine (C > A) substitutions in primary neuroblastoma tumors, which was associated with poor survival. We showed that increased levels of C > A substitutions correlate with copy number loss (CNL) of OGG1 or MUTYH Both genes encode DNA glycosylases that recognize 8-oxo-guanine (8-oxoG) lesions as a first step of 8-oxoG repair. Tumor organoid models with CNL of OGG1 or MUTYH show increased 8-oxoG levels compared to wild-type cells. We used CRISPR-Cas9 genome editing to create knockout clones of MUTYH and OGG1 in neuroblastoma cells. Whole-genome sequencing of single-cell OGG1 and MUTYH knockout clones identified an increased accumulation of C > A substitutions. Mutational signature analysis of these OGG1 and MUTYH knockout clones revealed enrichment for C > A signatures 18 and 36, respectively. Clustering analysis showed that the knockout clones group together with tumors containing OGG1 or MUTYH CNL. In conclusion, we demonstrate that defects in 8-oxoG repair cause accumulation of C > A substitutions in neuroblastoma, which contributes to mutagenesis and tumor evolution.

Highlights

Neuroblastomas are childhood tumors with frequent fatal relapses after induction treatment, which is related to tumor evolution with additional genomic events
Signature 18 or signature 36 has the highest relative contribution in 90% of the tumors with OGG1 or MUTYH copy number loss (CNL), while this is only 31% in tumors without defective 8-oxoG repair. These results indicate that the OGG1 and MUTYH knockout clones have a high contribution of C > A mutational signatures 18 and 36, respectively, and cluster together with neuroblastoma tumors with OGG1 or MUTYH CNL
We show that defects in the 8-oxoG repair pathway result in high levels of C > A substitutions in neuroblastoma tumors, which is correlated with a poor prognosis

Summary

Introduction

Neuroblastomas are childhood tumors with frequent fatal relapses after induction treatment, which is related to tumor evolution with additional genomic events. We showed that increased levels of C > A substitutions correlate with copy number loss (CNL) of OGG1 or MUTYH Both genes encode DNA glycosylases that recognize 8-oxo-guanine (8-oxoG) lesions as a first step of 8-oxoG repair. We show that neuroblastoma tumors with high C > A substitution frequencies were enriched for copy number loss (CNL) of OGG1 and MUTYH. To mimic this phenotype, we used CRISPR-CAS9 to engineer defects in the 8-oxoG repair genes OGG1 and MUTYH in neuroblastoma cells, resulting in an increased accumulation of C > A substitutions in single-cell knockout clones and a high contribution of C > A mutational signatures 18 and 36.

Methods

Results

Conclusion