Project description: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.
Project description:Bulk RNA-Seq of PC9 xenograft tumors in different stages of response and resistance to Erlotinib treatment. A combination treatment is proposed in order to overcome Erlotinib drug resistance.
Project description:Approximately half of children diagnosed with neuroblastoma have aggressive high-risk disease, against which current multimodal therapy has little success. New targets for therapeutic intervention are urgently needed. In a systems approach, we combined time-resolved mRNA-Seq with miRNA profiling in a model of neuroblastoma differentiation treated with or without the clinically approved histone deacetylase inhibitor, panobinostat. Treatment most strongly regulated miR-630, which strongly downregulated AVEN, an inhibitor of apoptosis. Here we characterized AVEN for the first time in neuroblastoma pathogenesis, disclosed its epigenetic regulation and discovered correlations between AVEN expression and resistance to chemotherapy. High-level AVEN expression in 649 primary neuroblastomas negatively correlated with patient survival. Stably enforcing AVEN expression in BE(2)-C cells markedly promoted growth as subcutaneous xenografts in mice, while CRISP/Cas9-mediated AVEN knockout in BE(2)-C cells substantially reduced xenograft growth and enhances the efficacy of systemic doxorubicin treatment. Cell-based assays, flow cytometric cell cycle analyses and SILAC experiments demonstrate that AVEN is required for neuroblastoma proliferation.
Project description:Some neuroblastoma patients relapse after chemotherapy. Here, a Th-MYCNCPM32 mouse model usually have spontaneously tumours which are sensitive to chemotherapy. By treating mice with consecutive cycles of sublethal cyclophosphamide (CPM) using a personal dose escalation protocol (PDE), resistant tumours may develop. This experiment aims to compare the expression profiles (RNA-Seq) of sensitive and resistant MYCN driven tumours in order to understand the mechanisms behind resistance. Samples also presented cross-resistance to vincristine and doxorubicin.
Project description:Approximately half of children diagnosed with neuroblastoma have aggressive high-risk disease, against which current multimodal therapy has little success. New targets for therapeutic intervention are urgently needed. In a systems approach, we combined time-resolved mRNA-Seq with miRNA profiling in a model of neuroblastoma differentiation treated with or without the clinically approved histone deacetylase inhibitor, panobinostat. Treatment most strongly regulated miR-630, which strongly downregulated AVEN, an inhibitor of apoptosis. Here we characterized AVEN for the first time in neuroblastoma pathogenesis, disclosed its epigenetic regulation and discovered correlations between AVEN expression and resistance to chemotherapy. High-level AVEN expression in 649 primary neuroblastomas negatively correlated with patient survival. Stably enforcing AVEN expression in BE(2)-C cells markedly promoted growth as subcutaneous xenografts in mice, while CRISP/Cas9-mediated AVEN knockout in BE(2)-C cells substantially reduced xenograft growth and enhances the efficacy of systemic doxorubicin treatment. Cell-based assays, flow cytometric cell cycle analyses and SILAC experiments demonstrate that AVEN is required for neuroblastoma proliferation.
Project description:Most cancer deaths result from progression of therapy resistant disease, yet our understanding of this phenotype is limited. Cancer therapies generate stress signals that act upon mitochondria to initiate apoptosis. Mitochondria isolated from neuroblastoma cells were exposed to tBid or Bim, death effectors activated by therapeutic stress. Multidrug resistant tumor cells obtained from children at relapse had markedly attenuated Bak and Bax oligomerization and cytochrome c release (surrogates for apoptotic commitment) in comparison with patient-matched tumor cells obtained at diagnosis. Electron microscopy identified reduced endoplasmic reticulum-mitochondria contacts (ERMCs) in therapy resistant cells, and genetically or biochemically reducing ERMCs in therapy sensitive tumors phenocopied resistance. ERMCs serve as platforms to transfer Ca2+ and bioactive lipids to mitochondria. Reduced Ca2+ transfer was found in some but not all resistant cells, and inhibiting transfer did not attenuate apoptotic signaling. In contrast, reduced ceramide synthesis and transfer was common to resistant cells and its inhibition induced stress resistance. We identify ERMCs as physiologic regulators of apoptosis via ceramide transfer and uncover a previously unrecognized mechanism for cancer multidrug resistance.
Project description:We utilize the syngeneic 9464D-GD2 mouse model to investigate the role of neuroblastoma-derived small extracellular vesicles (sEVs) in developing resistance to the anti-GD2 monoclonal antibody dinutuximab. RNA-sequencing and flow cytometry analysis of whole tumors revealed that neuroblastoma-derived sEVs modulate immune cell tumor infiltration upon dinutuximab treatment to create an immunosuppressive tumor microenvironment that contains more tumor-associated macrophages (TAMs) and fewer tumor-infiltrating NK cells. Importantly, tipifarnib, a farnesyltransferase inhibitor that inhibits sEV secretion, drastically enhanced the efficacy of dinutuximab and reversed the immunosuppressive effects of neuroblastoma-derived sEVs.