Search

Supplementary Data from Mesenchymal-Type Neuroblastoma Cells Escape ALK Inhibitors

Cancer therapy frequently fails due to the emergence of resistance. Many tumors include phenotypically immature tumor cells, which have been implicated in therapy resistance. Neuroblastoma cells can adopt a lineage-committed adrenergic (ADRN) or an immature mesenchymal (MES) state. They differ in epigenetic landscape and transcription factors, and MES cells are more resistant to chemotherapy. Here we analyzed the response of MES cells to targeted drugs. Activating anaplastic lymphoma kinase (ALK) mutations are frequently found in neuroblastoma and ALK inhibitors (ALKi) are in clinical trials. ALKi treatment of ADRN neuroblastoma cells with a tumor-driving ALK mutation induced cell death. Conversely, MES cells did not express either mutant or wild-type ALK and were resistant to ALKi, and MES cells formed tumors that progressed under ALKi therapy. In assessing the role of MES cells in relapse development, TRAIL was identified to specifically induce apoptosis in MES cells and to suppress MES tumor growth. Addition of TRAIL to ALKi treatment of neuroblastoma xenografts delayed relapses in a subset of the animals, suggesting a role for MES cells in relapse formation. While ADRN cells resembled normal embryonal neuroblasts, MES cells resembled immature precursor cells, which also lacked ALK expression. Resistance to targeted drugs can therefore be an intrinsic property of immature cancer cells based on their resemblance to developmental precursors.Significance:In neuroblastoma, mesenchymal tumor cells lack expression of the tumor-driving ALK oncogene and are resistant to ALKi, but dual treatment with ALKi and mesenchymal cell–targeting TRAIL delays tumor relapse.

Neuroblastoma is a pediatric tumor of the adrenergic sympathetic lineage. Most high risk neuroblastoma go in complete clinical remission by chemotherapy, which is subsequently complemented by retinoic acid (RA) maintenance therapy. However, by unresolved mechanisms most tumors ultimately relapse as therapy-resistant disease. Neuroblastoma cell lines were recently found to include, besides lineage committed adrenergic (ADRN) tumor cells, also immature mesenchymal (MES) tumor cells. Here, we report that MES-type cells synthesize RA and require this metabolite for proliferation and motility. MES cells are even resistant to RA in vitro. MES cells appear to resemble Schwann Cell Precursors (SCP), which are motile precursors of the adrenergic lineage. MES and SCP cells express shared RA-synthesis and RA-target genes. Endogenous RA synthesis and RA resistance thus stem from normal programs of lineage precursors that are maintained in an immature tumor cell fraction. These cells are fully malignant in orthotopic patient-derived xenograft models and may mediate development of drug-resistant relapses.

Transition between differentiation states in development occurs swift but the mechanisms leading to epigenetic and transcriptional reprogramming are poorly understood. The pediatric cancer neuroblastoma includes adrenergic (ADRN) and mesenchymal (MES) tumor cell types, which differ in phenotype, super-enhancers (SEs) and core regulatory circuitries. These cell types can spontaneously interconvert, but the mechanism remains largely unknown. Here, we unravel how a NOTCH3 intracellular domain reprogrammed the ADRN transcriptional landscape towards a MES state. A transcriptional feed-forward circuitry of NOTCH-family transcription factors amplifies the NOTCH signaling levels, explaining the swift transition between two semi-stable cellular states. This transition induces genome-wide remodeling of the H3K27ac landscape and a switch from ADRN SEs to MES SEs. Once established, the NOTCH feed-forward loop maintains the induced MES state. In vivo reprogramming of ADRN cells shows that MES and ADRN cells are equally oncogenic. Our results elucidate a swift transdifferentiation between two semi-stable epigenetic cellular states., Neuroblastoma includes adrenergic and mesenchymal cell types that can interconvert. Here, the authors show that this transdifferentiation is driven by a NOTCH feedforward loop that allows a swift transition between two semi-stable cellular states.

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