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Supplementary Data from Mesenchymal-Type Neuroblastoma Cells Escape ALK Inhibitors

Supplementary Legends - PDF file 80K, This file contains the legends to supplementary figures, supplementary movies and supplementary tables

Supplementary Tables - XLS file 163K. This file contains 6 supplementary tables

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.

Supporting video 2 - MOV file 1047K, IMR32-NOTCH3IC cells cultured with doxycycline. Note the fast-moving presence of different cells.

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.

Supplementary Figures - PDF file 2758K, This file contains supplementary figure 1-7