ro5126766 and trametinib

Disialoganglioside (GD2)-specific chimeric antigen receptor (CAR)-T cells (GD2-CAR-T cells) have been developed and tested in early clinical trials in patients with relapsed/refractory neuroblastoma. However, the effectiveness of immunotherapy using these cells is limited, and requires improvement. Combined therapy with CAR-T cells and molecular targeted drugs could be a promising strategy to enhance the antitumor efficacy of CAR T cell immunotherapy. Here, we generated GD2-CAR-T cells through piggyBac transposon (PB)-based gene transfer (PB-GD2-CAR-T cells), and analyzed the combined effect of these cells and a MEK inhibitor in vitro and in vivo on neuroblastoma. Trametinib, a MEK inhibitor, ameliorated the killing efficacy of PB-GD2-CAR-T cells in vitro, whereas a combined treatment of the two showed superior antitumor efficacy in a murine xenograft model compared to that of PB-GD2-CAR-T cell monotherapy, regardless of the mutation status of the MAPK pathway in tumor cells. The results presented here provide new insights into the feasibility of combined treatment with CAR-T cells and MEK inhibitors in patients with neuroblastoma.

Topics: Animals; Antineoplastic Agents; Cell Line, Tumor; Combined Modality Therapy; Coumarins; DNA Transposable Elements; Drug Resistance, Neoplasm; Female; Gangliosides; Genetic Therapy; Humans; Immunotherapy, Adoptive; Mice; Mice, SCID; Mitogen-Activated Protein Kinase Kinases; Mutation; Neoplasm Recurrence, Local; Neuroblastoma; Protein Kinase Inhibitors; Pyridones; Pyrimidinones; ras Proteins; Receptors, Chimeric Antigen; T-Lymphocytes; Xenograft Model Antitumor Assays

It was reported that almost 80% of relapsed neuroblastomas showed MAPK pathway mutations. In our previous study, both trametinib (MEK inhibitor) and CH5126766 (RAF/MEK inhibitor) showed in vitro antitumor effects on neuroblastoma cells with ERK phosphorylation (pERK). In this study, we analyzed the in vivo effects of MAPK pathway inhibition in neuroblastoma xenografts.. Xenograft mice with IMR5, CHP-212, or SK-N-AS received daily oral administration of either trametinib or CH5126766 for two weeks (short term) or eight weeks (long term). The tumors were measured twice weekly and harvested after treatment for histopathological analyses, including pERK and Ki67 immunohistochemistry.. In short-term treatment, both inhibitors showed significant growth inhibition in CHP-212 and SK-N-AS xenografts, which were pERK-positive before treatment. The number of pERK- and Ki67-positive cells decreased after treatment. Conversely, IMR5 xenografts, which were pERK-negative, were resistant to treatment. During long-term treatment, SK-N-AS xenografts started to regrow from about six weeks with partial differentiation. pERK-positive cells reincreased in these regrown tumors.. MAPK pathway inhibition was effective for treating pERK-positive neuroblastoma in vivo. Therefore, pERK immunohistochemistry might be a convenient biomarker for MAPK pathway inhibition in neuroblastoma treatment. However, neuroblastomas developed acquired drug resistance after long-term treatment. Further studies to overcome acquired resistance are needed.

Topics: Animals; Cell Line, Tumor; Coumarins; eIF-2 Kinase; Female; Humans; Immunohistochemistry; Ki-67 Antigen; Mice; Mitogen-Activated Protein Kinases; Neoplasm Recurrence, Local; Neuroblastoma; Protein Kinase Inhibitors; Pyridones; Pyrimidinones; Xenograft Model Antitumor Assays

A recent study reported that relapsed neuroblastomas had frequent RAS-ERK pathway mutations. We herein investigated the effects and pathways of MEK inhibitors, which inhibit the RAS-ERK pathway, as a new molecular-targeted therapy for refractory neuroblastomas.. Five neuroblastoma cell lines were treated with trametinib (MEK inhibitor) or CH5126766 (RAF/MEK inhibitor). Growth inhibition was analyzed using a cell viability assay. ERK phosphorylation and the MYCN expression were analyzed by immunoblotting or immunohistochemistry. RAS/RAF mutations were identified by direct sequencing or through the COSMIC database.. Both MEK inhibitors showed growth inhibition effects on cells with ERK phosphorylation, but almost no effect on cells without. In immunoblotting analyses, ERK phosphorylation and MYCN expression were suppressed in ERK active cells by these drugs. Furthermore, phosphorylated-ERK immunohistochemistry corresponded to the drug responses. Regarding the relationship between RAS/Raf mutations and ERK phosphorylation, ERK was phosphorylated in one cell line (NLF) without RAS/Raf mutations.. MEK inhibitors are a promising molecular-targeted therapeutic option for ERK active neuroblastomas. The efficacy of MEK inhibitors corresponds to ERK phosphorylation, while RAS/RAF mutations are not always detected in drug-sensitive cells. Phosphorylated-ERK immunohistochemistry is thus a useful method to analyze ERK activity and predict the therapeutic effects of MEK inhibitors.

Topics: Cell Line, Tumor; Coumarins; Humans; MAP Kinase Signaling System; Molecular Targeted Therapy; Neoplasm Recurrence, Local; Neuroblastoma; Phosphorylation; Protein Kinase Inhibitors; Pyridones; Pyrimidinones; Signal Transduction

MEK inhibitors are clinically active in BRAF(V600E) melanomas but only marginally so in KRAS mutant tumors. Here, we found that MEK inhibitors suppress ERK signaling more potently in BRAF(V600E), than in KRAS mutant tumors. To understand this, we performed an RNAi screen in a KRAS mutant model and found that CRAF knockdown enhanced MEK inhibition. MEK activated by CRAF was less susceptible to MEK inhibitors than when activated by BRAF(V600E). MEK inhibitors induced RAF-MEK complexes in KRAS mutant models, and disrupting such complexes enhanced inhibition of CRAF-dependent ERK signaling. Newer MEK inhibitors target MEK catalytic activity and also impair its reactivation by CRAF, either by disrupting RAF-MEK complexes or by interacting with Ser 222 to prevent MEK phosphorylation by RAF.

Topics: Animals; Benzamides; Cell Line; Coumarins; Diphenylamine; Drug Resistance, Neoplasm; Extracellular Signal-Regulated MAP Kinases; HEK293 Cells; Humans; Indoles; MAP Kinase Kinase 1; MAP Kinase Signaling System; Melanoma; Mice; Mice, Nude; Phosphorylation; Protein Kinase Inhibitors; Proto-Oncogene Proteins; Proto-Oncogene Proteins B-raf; Proto-Oncogene Proteins p21(ras); Pyridones; Pyrimidinones; raf Kinases; ras Proteins; RNA Interference; RNA, Small Interfering; Sulfonamides; Surface Plasmon Resonance; TNF Receptor-Associated Factor 3; Vemurafenib