Johan van Nes

Sequencing of neuroblastoma identifies chromothripsis and defects in neuritogenesis genes

Neuroblastoma is a childhood tumour of the peripheral sympathetic nervous system. The pathogenesis has for a long time been quite enigmatic, as only very few gene defects were identified in this often lethal tumour. Frequently detected gene alterations are limited to MYCN amplification (20%) and ALK activations (7%). Here we present a whole-genome sequence analysis of 87 neuroblastoma of all stages. Few recurrent amino-acid-changing mutations were found. In contrast, analysis of structural defects identified a local shredding of chromosomes, known as chromothripsis, in 18% of high-stage neuroblastoma. These tumours are associated with a poor outcome. Structural alterations recurrently affected ODZ3, PTPRD and CSMD1, which are involved in neuronal growth cone stabilization. In addition, ATRX, TIAM1 and a series of regulators of the Rac/Rho pathway were mutated, further implicating defects in neuritogenesis in neuroblastoma. Most tumours with defects in these genes were aggressive high-stage neuroblastomas, but did not carry MYCN amplifications. The genomic landscape of neuroblastoma therefore reveals two novel molecular defects, chromothripsis and neuritogenesis gene alterations, which frequently occur in high-risk tumours.

LIN28B induces neuroblastoma and enhances MYCN levels via let-7 suppression

LIN28B regulates developmental processes by modulating microRNAs (miRNAs) of the let-7 family. A role for LIN28B in cancer has been proposed but has not been established in vivo. Here, we report that LIN28B showed genomic aberrations and extensive overexpression in high-risk neuroblastoma compared to several other tumor entities and normal tissues. High LIN28B expression was an independent risk factor for adverse outcome in neuroblastoma. LIN28B signaled through repression of the let-7 miRNAs and consequently resulted in elevated MYCN protein expression in neuroblastoma cells. LIN28B–let-7–MYCN signaling blocked differentiation of normal neuroblasts and neuroblastoma cells. These findings were fully recapitulated in a mouse model in which LIN28B expression in the sympathetic adrenergic lineage induced development of neuroblastomas marked by low let-7 miRNA levels and high MYCN protein expression. Interference with this pathway might offer therapeutic perspectives.

Functional MYCN signature predicts outcome of neuroblastoma irrespective of MYCN amplification

Neuroblastoma is a pediatric tumor of the sympathetic nervous system. MYCN (V-myc myelocytomatosis viral-related oncogene, neuroblastoma derived [avian]) is amplified in 20% of neuroblastomas, and these tumors carry a poor prognosis. However, tumors without MYCN amplification also may have a poor outcome. Here, we identified downstream targets of MYCN by shRNA-mediated silencing MYCN in neuroblastoma cells. From these targets, 157 genes showed an expression profile correlating with MYCN mRNA levels in NB88, a series of 88 neuroblastoma tumors, and therefore represent in vivo relevant MYCN pathway genes. This 157-gene signature identified very poor prognosis tumors in NB88 and independent neuroblastoma cohorts and was more powerful than MYCN amplification or MYCN expression alone. Remarkably, this signature also identified poor outcome of a group of tumors without MYCN amplification. Most of these tumors have low MYCN mRNA levels but high nuclear MYCN protein levels, suggesting stabilization of MYCN at the protein level. One tumor has an MYC amplification and high MYC expression. Chip-on-chip analyses showed that most genes in this signature are directly regulated by MYCN. MYCN induces genes functioning in cell cycle and DNA repair while repressing neuronal differentiation genes. The functional MYCN-157 signature recognizes classical neuroblastoma with MYCN amplification, as well as a newly identified group marked by MYCN protein stabilization.

Neuroblastoma is composed of two super-enhancer-associated differentiation states

Neuroblastoma and other pediatric tumors show a paucity of gene mutations, which has sparked an interest in their epigenetic regulation. Several tumor types include phenotypically divergent cells, resembling cells from different lineage development stages. It has been proposed that super-enhancer-associated transcription factor (TF) networks underlie lineage identity, but the role of these enhancers in intratumoral heterogeneity is unknown. Here we show that most neuroblastomas include two types of tumor cells with divergent gene expression profiles. Undifferentiated mesenchymal cells and committed adrenergic cells can interconvert and resemble cells from different lineage differentiation stages. ChIP–seq analysis of isogenic pairs of mesenchymal and adrenergic cells identified a distinct super-enhancer landscape and super-enhancer-associated TF network for each cell type. Expression of the mesenchymal TF PRRX1 could reprogram the super-enhancer and mRNA landscapes of adrenergic cells toward a mesenchymal state. Mesenchymal cells were more chemoresistant in vitro and were enriched in post-therapy and relapse tumors. Two super-enhancer-associated TF networks, which probably mediate lineage control in normal development, thus dominate epigenetic control of neuroblastoma and shape intratumoral heterogeneity.

A NOTCH3 Transcriptional Module Induces Cell Motility in Neuroblastoma

Purpose: Neuroblastoma is a childhood tumor of the peripheral sympathetic nervous system with an often lethal outcome due to metastatic disease. Migration and epithelial–mesenchymal transitions have been implicated in metastasis but they are hardly investigated in neuroblastoma. Experimental Design: Cell migration of 16 neuroblastoma cell lines was quantified in Transwell migration assays. Gene expression profiling was used to derive a migration signature, which was applied to classify samples in a neuroblastoma tumor series. Differential expression of transcription factors was analyzed in the subsets. NOTCH3 was prioritized, and inducible transgene expression studies in cell lines were used to establish whether it functions as a master switch for motility. Results: We identified a 36-gene expression signature that predicts cell migration. This signature was used to analyse expression profiles of 88 neuroblastoma tumors and identified a group with distant metastases and a poor prognosis. This group also expressed a known mesenchymal gene signature established in glioblastoma. Neuroblastomas recognized by the motility and mesenchymal signatures strongly expressed genes of the NOTCH pathway. Inducible expression of a NOTCH intracellular (NOTCH3-IC) transgene conferred a highly motile phenotype to neuroblastoma cells. NOTCH3-IC strongly induced expression of motility- and mesenchymal marker genes. Many of these genes were significantly coexpressed with NOTCH3 in neuroblastoma, as well as colon, kidney, ovary, and breast tumor series. Conclusion: The NOTCH3 transcription factor is a master regulator of motility in neuroblastoma. A subset of neuroblastoma with high expression of NOTCH3 and its downstream-regulated genes has mesenchymal characteristics, increased incidence of metastases, and a poor prognosis. Clin Cancer Res; 19(13); 3485–94. ©2013 AACR.

Abstract IA10: Neuroblastoma is a biphasic tumor

Neuroblastoma is a pediatric tumor of the peripheral adrenergic lineage, which is neural crest derived. During embryogenesis cells delaminate from the neural crest, migrate ventrally, and differentiate to (nor)-adrenalin producing cells. Neuroblastoma typically expresses differentiation markers of the adrenergic lineage. High-stage neuroblastoma usually goes in complete remission upon therapy but often relapses as resistant disease. Several tumor types were recently found to include phenotypically divergent cell types, resembling lineage development stages. We have found that most neuroblastoma includes two types of tumor cells with shared genomic defects, but highly diverging gene expression profiles. Undifferentiated mesenchymal cells (MES-type) and more differentiated adrenergic cells (ADRN-type) can interconvert and may relate to normal lineage differentiation stages. ChIP-seq analysis of isogenic pairs of MES- and ADRN-type cells revealed distinct, highly consistent super-enhancer landscapes for each cell type. Lineage identity has been proposed to ensue super-enhancer (SE)-associated transcription factor (TF) networks. We identified two SE-associated TF networks that potentially master each cell type. Accordingly, the mesenchymal TF PRRX1 could reprogram the SE- and mRNA-profiles of ADRN-type cells towards an MES-state. Assessment of the clinical relevance of this biphasic system revealed that MES-type cells were more chemoresistant in vitro and were enriched in post-therapy and relapsed tumors. The super enhancer-associated TF networks, probably meant for lineage control in normal development, thus could impose two cellular states of neuroblastoma that shape intratumor heterogeneity. Citation Format: Rogier Versteeg, Tim van Groningen, Jan Koster, Linda J. Valentijn, Johan van Nes. Neuroblastoma is a biphasic tumor [abstract]. In: Proceedings of the AACR Special Conference: Pediatric Cancer Research: From Basic Science to the Clinic; 2017 Dec 3-6; Atlanta, Georgia. Philadelphia (PA): AACR; Cancer Res 2018;78(19 Suppl):Abstract nr IA10.

Abstract PR08: Neuroblastoma is biphasic and includes classical neuroepithelial cells and chemoresistant mesenchymal cells

Introduction: Most high stage neuroblastoma initially respond to chemotherapy, but ultimately relapse as therapy-resistant tumor. The mechanisms driving relapse and resistance remain elusive. We investigated whether neuroblastoma tumors include phenotypically and functionally divergent subsets of tumor cells that may underlie its clinical plasticity. Experimental Procedures: Fresh tumor cells were cultured in neural stem cell medium and analyzed by FACS, whole genome sequencing, mRNA profiling, motility assays and chemo-sensitivity assays. Lentivirally transduced inducible gene constructs were used to test state-transitions. Immunohistochemistry was used to define cellular subtypes in tumors. Results: We observed that new neuroblastoma cell lines always include two phenotypically divergent cell types. Both types share the same genetic defects, but have highly divergent phenotypes. One cell type has a neuro-epithelial (NE) phenotype and expresses all classical and diagnostically used neuroblastoma markers. The other type has a mesenchymal (MES) character, lacks all neuroblastoma markers and is highly motile. At low frequency, both cell types can spontaneously transdifferentiate in vitro. Immunohistochemistry (IHC) of primary neuroblastoma detected a small fraction of MES cells in most tumors. To analyze the clinical relevance of MES-type cells, we investigated their sensitivity to chemotherapeutics used in neuroblastoma treatment. In four isogenic pairs, MES cells were more resistant to the drugs than their NE-type counterparts. We investigated whether the chemo-resistance of MES cells may operate in vivo. We analyzed a series of primary neuroblastoma tumors surgically removed immediately after chemotherapy. The viable cells in the post-therapy samples were strongly enriched in MES-type cells as compared to the pre-treatment tumors of the same patients. We also compared primary, pre-treatment tumors with relapses emerging 4-5 years later in the same patients. Most strikingly, also the relapsed neuroblastoma tumors were highly enriched for MES-type cells. As these data suggest a role for MES-type cells in development of therapy-resistant relapses, we analyzed their key regulatory pathways. mRNA profiling of isogenic MES-NE cell line pairs identified consistent mRNA expression differences between both phenotypes. Major signaling routes and transcription factors were highly differentially expressed. MES-type cells had high expression and activation of NOTCH pathway genes and expression of the homeobox gene PRRX1. Induced expression of NOTCH or PRRX1 transgenes in multiple NE-type cell lines converted them into MES-type cell lines, including chemo-resistance. Analysis of the changes in gene expression and activity downstream of NOTCH or PRRX1 allowed reconstruction of the molecular wiring of MES-type cells. This identified several drugable key-players, like MEK and PDGFRβ. Targeting of them with small-molecule inhibitors specifically killed MES cells in vitro. Conclusions: Our data suggest that neuroblastoma is a bi-phasic tumor. MES and NE cells have very different characteristics, but can transdifferentiate into each other. It is tempting to speculate that the MES- and NE-phenotypes recapitulate two developmental stages of neuroblasts: MES cells may correspond to the migrating cell type that has delaminated from the neural crest, while NE cells could correspond to the more differentiated cell in the target organs expressing markers of the adrenalin synthesis route. MES cells strongly accumulate after chemo-therapy and in relapses. They may survive classical therapy and over time seed relapses, that ultimately become heterogeneous again. Targeted elimination of MES cells with small molecule inhibitors shows how cells with a potential key role in relapse development are amenable to therapy. This abstract is also presented as Poster B30. Citation Format: Rogier Versteeg, Tim van Groningen, Bart A. Westerman, Jan J. Molenaar, Ellen M. Westerhout, Mohamed Hamdi, Godelieve A. Tytgat, Jan Koster, Johan van Nes. Neuroblastoma is biphasic and includes classical neuroepithelial cells and chemoresistant mesenchymal cells. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Pediatric Cancer Research: From Mechanisms and Models to Treatment and Survivorship; 2015 Nov 9-12; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(5 Suppl):Abstract nr PR08.

Abstract 2453: Neuroblastoma is bi-phasic and includes classical neuro-epithelial cells and chemo-resistant mesenchymal cells

Introduction Most high stage neuroblastoma initially respond to chemotherapy, but ultimately relapse as therapy-resistant tumor. The mechanisms driving relapse and resistance remain elusive. We investigated whether neuroblastoma tumors include divergent cell types that may underlie this plasticity. Experimental procedures Fresh tumor cells cultured in neural stem cell medium were analyzed by FACS, whole genome sequencing, Chip-seq, mRNA profiling, and motility and chemo-sensitivity assays. Inducible transgenes were used to test state-transitions. Tumors were analyzed by immunohistochemistry. Results New neuroblastoma cell lines always included two cell types, which share the same genetic defects but have highly divergent phenotypes. One type has a neuro-epithelial (NE) phenotype and expresses all classical neuroblastoma markers. The other type has a mesenchymal (MES) character, is motile and lacks all neuroblastoma markers. Immunohistochemistry (IHC) detected a small fraction of MES cells in most primary neuroblastoma. In four isogenic cell line pairs, we found that MES cells were more chemo-resistant than their NE-type counterparts. Indeed, comparison of primary neuroblastoma lesions before and after chemotherapy showed an accumulation of viable MES-type cells in post treatment samples. Moreover, comparison of primary, pre-treatment tumors with relapses emerging 4-5 years later in the same patients showed a strong enrichment for MES cells in the latter. As these data suggest a role for MES-type cells in relapse development, we analyzed their key regulatory pathways. The isogenic MES-NE cell line pairs showed consistent mRNA expression differences between both phenotypes, activating major signaling routes and transcription factors. Chip-seq identified divergent histone modifications. MES cells had high NOTCH pathway activity and PRRX1 expression. Induced expression of NOTCH or PRRX1 converted multiple NE-type cell lines into MES-type cells, including chemo-resistance. Further analysis of these routes reconstructed molecular wiring of MES-type cells. This identified key-players like MEK and PDGFRβ, which were successfully targeted by small molecules to specifically kill MES cells in vitro. Conclusions Our data suggest that neuroblastoma is a bi-phasic tumor. MES and NE cells differ in many characteristics, but can transdifferentiate into each other. MES and NE cells may correspond to developmental stages, i.e. mesenchymal migratory cells delaminated from the neural crest and more differentiated cells of the adrenergic lineage. MES cells accumulate after chemo-therapy and in relapses. They may survive classical therapy and over time seed relapses, that ultimately become heterogeneous again. Elimination of MES cells with small molecule inhibitors shows how cells with a potential key role in relapse development are amenable to therapy. Citation Format: Rogier Versteeg, Tim van Groningen, Linda J. Valentijn, Bart A. Westerman, Jan J. Molenaar, Ellen M. Westerhout, Mohamed Hamdi, Godelieve A. Tytgat, Jan Koster, Johan van Nes. Neuroblastoma is bi-phasic and includes classical neuro-epithelial cells and chemo-resistant mesenchymal cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2453.

Abstract LB-209: Neuroblastoma is biphasic with classical neuro-epithelial cells and chemoresistant mesenchymal cells controlled by PRRX1-NOTCH signaling

Most high stage neuroblastoma initially respond to chemotherapy, but ultimately relapse as therapy-resistant tumor. The mechanisms driving relapse and resistance remain elusive. We observed that new neuroblastoma cell lines cultured in defined medium always include two phenotypically divergent cell types. Whole genome sequencing showed that both types were genetically identical. One cell type has a neuro-epithelial (NE) phenotype and expresses all classical and diagnostically used neuroblastoma markers. The other type has a mesenchymal (MES) character, lacks all neuroblastoma markers and is highly motile. MES cells are more chemo-resistant in vitro as compared to NE cells. Immunohistochemistry (IHC) of primary neuroblastoma detected a small fraction of MES cells in most tumors. However, MES cells were strongly enriched in surgically removed post-chemotherapy samples. Moreover, neuroblastoma patients that had been tumor-free for several years but relapsed, also showed a strong accumulation of MES type cells in their relapses as compared to the primary tumors. As these data suggest a major role for this new neuroblastoma cell type in development of therapy-resistant relapses, we analyzed their key regulatory pathways. In multiple cell line models, the homeobox gene PRRX1 was identified as a master regulator that converted the NE phenotype in a MES phenotype. PRRX1 concomitantly induced a chemo-resistant phenotype in vitro. PRRX1 activated a cascade of MEK, NOTCH and PDGFRβ signaling. Also NOTCH was able to induce the mesenchymal phenotype, as well as chemo-resistance. Analysis of the PRRX1-induced downstream signaling pathway identified several drugable key-players, like MEK and PDGFRβ. Targeting them with small-molecule inhibitors specifically killed MES cells in vitro. Our data suggest that neuroblastoma is a bi-phasic tumor. MES and NE cells have very different characteristics, but can transdifferentiate into each other. MES cells strongly accumulate after chemo-therapy and in relapses. They may survive classical therapy and seed relapses, that ultimately become heterogeneous again. Targeted elimination of MES cells with small molecule inhibitors shows how cells with a potential key role in relapse development are amenable to therapy. Note: This abstract was not presented at the meeting. Citation Format: Tim van Groningen, Natalia E. Nowakowska, Nurdan Akogul, Marloes Broekmans, Johannes Bras, Jan Booij, Marli E. Ebus, Jan J. Molenaar, Ellen M. Westerhout, Mohamed Hamdi, Peter van Sluis, Jan Koster, Bart A. Westerman, Godelieve A. Tytgat, Rogier Versteeg, Johan van Nes. Neuroblastoma is biphasic with classical neuro-epithelial cells and chemoresistant mesenchymal cells controlled by PRRX1-NOTCH signaling. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-209. doi:10.1158/1538-7445.AM2015-LB-209