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Intra-tumour heterogeneity, lineage plasticity and drug resistance in neuroblastoma

In many tumour types, primary tumours respond to therapy by complete clinical remission, but ultimately relapse as therapy-resistant and lethal disease. My aim is to elucidate this enigmatic process and to devise new treatment options to prevent resistance and relapse. I focus on phenotypic tumour cell heterogeneity, the lineage plasticity of these cell types and their drug-resistance to understand the cellular and molecular mechanism(s) of relapse. My ultimate aim is to eradicate these therapy-resistant cells to prevent relapse and improve patient survival.

With my research group, I have developed a model system of lineage heterogeneity and cellular plasticity to study drug-resistance and relapse of neuroblastoma. We have identified that each neuroblastoma includes two phenotypically divergent tumour cell types with mesenchymal- (MES-type) and adrenergic (ADRN-type) lineage fates, respectively (van Groningen et al., Nature Genetics, 2017). MES and ADRN cell types have been extensively characterized for 1) mRNA expression, 2) the chromatin landscape of super-enhancers, 3) drug-resistance, 4) lineage identity and 5) transdifferentiation induced by lineage transcription factors (TFs).

Neuroblastoma includes MES and ADRN tumour cells

We study five isogenic pairs of MES and ADRN cell lines, with each pair derived from the tumour of a single patient. MES and ADRN cell lines shared identical genetic defects, but have highly divergent phenotypes and mRNA expression profiles. The MES-type cells expressed mesenchymal marker genes, were highly migratory and showed lamellipodia. In contrast, the ADRN-type cells were not migratory and expressed adrenergic marker genes, e.g. from the adrenalin-synthesis route (DBH, TH). Importantly, MES and ADRN cells can spontaneously transdifferentiate into one another, both in vitro and in vivo.

We characterized the chromatin landscape of MES and ADRN cells for H3K27ac and identified two sets of ~300 super-enhancers that were unique to either cell type. The MES-specific super-enhancers associated with MES-TFs, while ADRN-specific super-enhancers associated with ADRN TFs. These distinct sets of super-enhancer associated TFs constituted the core regulatory circuitries for each cell type and are proposed to determine lineage-identity. Indeed, overexpression of MES TFs in ADRN neuroblastoma cells reprogrammed ADRN cells towards the MES lineage (van Groningen et al., Nature Genetics, 2017; van Groningen et al., Nature Communications, 2019). This transition was accompanied by downregulation of ADRN super-enhancers and ADRN TFs while MES super-enhancers and MES TFs were induced. These data show that a core set of TFs can impose lineage identity in neuroblastoma.

Clinical relevance of MES and ADRN cells

We tested the sensitivity of MES and ADRN cells to chemotherapeutic drugs used in the clinical management of neuroblastoma and found that MES cells are chemo-resistant as compared to ADRN cells (van Groningen et al., Nature Genetics, 2017). In primary human neuroblastoma, we identified a minor population of MES cells. Analysis of pairs of pre- and post-chemotherapy biopsies revealed an enrichment of MES cells, suggesting selective pressure of therapy. Also, some relapse tumours predominantly consisted of MES cells. These data support a model in which a minor population of MES cells exists in primary neuroblastoma. This population can enrich after chemotherapy and may give rise to relapse.

We currently investigate the heterogeneity and plasticity of the divergent neuroblastoma cell types and study their contribution to relapses. We use combinations of single-cell RNA sequencing, bio-informatics, CRISPR-based cell lineage reporters and in vivo relapse models to identify the full spectrum and the dynamics of neuroblastoma cell types. New combinations of cell-type specific drugs are tested to prevent relapse and improve survival. 

Research team

Johan van Nes, PhD, group leader

Luciano Rago, PhD, post-doc

Nurdan Akogul, technician

Alvin Chan, technician

Tim van Groningen, PhD received his PhD from the University of Amsterdam (d.d. January 22^nd, 2020) for his work described in the thesis 'Intra-tumor heterogeneity and lineage plasticity in neuroblastoma' (promotor Prof. R. Versteeg, co-promotor and daily supervisor Johan van Nes, PhD).