Cooperation of cancer drivers with regulatory germline variants shapes clinical outcomes

Abstract

Pediatric malignancies including Ewing sarcoma (EwS) feature a paucity of somatic alterations except for pathognomonic driver-mutations that cannot explain overt variations in clinical outcome. Here, we demonstrate in EwS how cooperation of dominant oncogenes and regulatory germline variants determine tumor growth, patient survival and drug response. Binding of the oncogenic EWSR1-FLI1 fusion transcription factor to a polymorphic enhancer-like DNA element controls expression of the transcription factor MYBL2 mediating these phenotypes. Whole-genome and RNA sequencing reveals that variability at this locus is inherited via the germline and is associated with variable inter-tumoral MYBL2 expression. High MYBL2 levels sensitize EwS cells for inhibition of its upstream activating kinase CDK2 in vitro and in vivo, suggesting MYBL2 as a putative biomarker for anti-CDK2-therapy. Collectively, we establish cooperation of somatic mutations and regulatory germline variants as a major determinant of tumor progression and highlight the importance of integrating the regulatory genome in precision medicine.

Interaction between somatic mutations and germline variants contributes to clinical heterogeneity in cancer

“…One of the most prominent peaks mapped to a polymorphic GGAA-mSat located about 150kb telomeric of the MYBL2 gene. 4 Using reporter assays, we validated the EWSR1-FLI1-dependent enhancer activity of this GGAA-mSat in vitro and confirmed its regulatory effect on MYBL2 expression by Clustered Regularly Interspaced Short Palindromic Repeats interference (CRISPRi). 4 The regulatory potential of this GGAA-mSat on MYBL2 transcription was further supported by its expression quantitative trait loci (eQTL) properties.…”

“…4 Using reporter assays, we validated the EWSR1-FLI1-dependent enhancer activity of this GGAA-mSat in vitro and confirmed its regulatory effect on MYBL2 expression by Clustered Regularly Interspaced Short Palindromic Repeats interference (CRISPRi). 4 The regulatory potential of this GGAA-mSat on MYBL2 transcription was further supported by its expression quantitative trait loci (eQTL) properties. 4 In fact, we found in a whole-genome sequencing dataset comprising 35 primary EwS tumors with matched gene expression data, that EwS tumors exhibiting ≤13 GGAA-repeats at both alleles of the MYBL2-associated GGAA-mSat exhibited significantly lower MYBL2 mRNA levels than those with >13 GGAA-repeats at both alleles.…”

“…To identify clinically relevant EWSR1-FLI1 target genes that may potentially mediate inter-tumoral heterogeneity, we screened in a first step available 'omics'-datasets for genes that are on the one hand regulated by EWSR1-FLI1, and on the other hand associated with worse overall patient survival when being highly expressed. 4 This analysis revealed MYB proto-oncogene like 2 (MYBL2), encoding for a transcription factor involved in regulation of cell cycle, cell survival and cell differentiation, 8 as the top hit of our screen. 4 In a second step we analyzed published EWSR1-FLI1 chromatin immunoprecipitation and sequencing (ChIP-seq) data for peaks nearby MYBL2, which displayed histone marks indicative for active enhancers.…”

“…4 This analysis revealed MYB proto-oncogene like 2 (MYBL2), encoding for a transcription factor involved in regulation of cell cycle, cell survival and cell differentiation, 8 as the top hit of our screen. 4 In a second step we analyzed published EWSR1-FLI1 chromatin immunoprecipitation and sequencing (ChIP-seq) data for peaks nearby MYBL2, which displayed histone marks indicative for active enhancers. One of the most prominent peaks mapped to a polymorphic GGAA-mSat located about 150kb telomeric of the MYBL2 gene.…”

“…Hence, it is important to uncover mechanisms underlying inter-individual variability in oligo-mutated cancers, to improve diagnosis, risk-prediction and targeted therapies in the context of precision oncology. 3 In our recent study, we show in the Ewing sarcoma (EwS) model how the interplay between somatic mutations and regulatory germline variants impacts on tumor growth, patient survival, and drug response, thereby constituting a major cause of interindividual tumor heterogeneity 4 (Figure 1).…”