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Entrectinib Resistance in a Neuroblastoma Xenograft Model

ABSTRACT: TrkB with its ligand, brain-derived neurotrophic factor (BDNF), are overexpressed in the majority of high-risk neuroblastomas (NBs). Entrectinib is a novel pan-TRK, ALK, and ROS1 inhibitor that has shown excellent preclinical efficacy in NB xenograft models, and recently it has entered phase 1 trials in pediatric relapsed/refractory solid tumors. We examined entrectinib-resistant NB cell lines to identify mechanisms of resistance. Entrectinib-resistant cell lines were established from five NB xenografts initially sensitive to entrectinib therapy. Clonal cell lines were established in increasing concentrations of entrectinib and had >10X increase in IC50. Cell lines underwent genomic and proteomic analysis using whole-exome sequencing, RNA-Seq, and proteomic expression profiling with confirmatory RT-PCR and Western blot analysis. There was no evidence of NTRK2 (TrkB) gene mutation in any resistant cell lines. Inhibition of TrkB was maintained in all cell lines at increasing concentrations of entrectinib (target independent). PTEN pathway downregulation and ERK/MAPK pathway upregulation were demonstrated in all resistant cell lines. One of these clones also had increased IGF1R signaling, and two additional clones had increased P75 expression, which likely increased TrkB sensitivity to ligand. In conclusion, NB lines overexpressing TrkB developed resistance to entrectinib by multiple mechanisms, including activation of ERK/MAPK and downregulation of PTEN signaling. Individual cell lines also had IGF1R activation and increased P75 expression, allowing preservation of downstream TrkB signaling in the presence of entrectinib. An understanding of changes in patterns of expression can be used to inform multimodal therapy planning in using entrectinib in phase II/III trial planning.

ORGANISM(S): Homo sapiens

PROVIDER: GSE141636 | GEO | 2019/12/10

REPOSITORIES: GEO

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Project description:Decremental loss of PTEN results in cancer susceptibility and tumor progression. In turn this raises the possibility that PTEN elevation might be an attractive option for cancer prevention and therapy. We have generated several transgenic mouse lines with variably elevated PTEN expression levels, taking advantage of BAC (Bacterial Artificial Chromosome)-mediated transgenesis. Super-PTEN mutants are viable and show reduced body size due to decreased cell number, with no effect on cell size. Unexpectedly, PTEN elevation at the organism level results in healthy metabolism characterized by increased energy expenditure and reduced body fat accumulation. Cells derived from these mice show reduced glucose and glutamine uptake, increased mitochondrial oxidative phosphorylation, and are resistant to oncogenic transformation. Mechanistically we find that PTEN elevation orchestrates this metabolic switch by regulating PI3K-dependent and independent pathways, and negatively impacts two of the most pronounced metabolic features of tumor cells: glutaminolysis and the Warburg effect. In order to elucidate the pathophysiological impact of PTEN elevation, we generated transgenic mice carrying additional copies of this critical tumor suppressor gene (referred to as Super-PTEN mice). In order to maintain the regulation properties of the endogenous Pten gene, we made use of large genomic fragments containing the entire Pten locus carried by BACs (Bacterial Artificial Chromosomes). We next generated mouse embryonic fibroblasts (MEFs) and confirmed successful overexpression of PTEN by the BAC transgenic system. Primary cells derived from Super-PTEN mice represent a powerful tool to elucidate the molecular mechanisms underlying dose-dependent PTEN actions. We therefore performed microarray analysis in primary cells (MEFs) derived from day 13.5 embryos obtained by crossing Super-PTEN mice with C57BL6 mice. Three independent embryos from each genotype were analyzed (background: >98%C57BL6 / CBA). Gene expression profile analysis in these cells will reveal target genes and pathways differentially regulated upon PTEN elevation.

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Entrectinib Resistance in a Neuroblastoma Xenograft Model

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