PO-145 ERK5 pathway inhibitors inhibit the maintenance of chronic myeloid leukaemia stem cells

Abstract

IntroductionPancreatic ductal adenocarcinoma (PDAC) is associated with a 5 year survival rate of less than 4%, indicating the often poor success rate of chemotherapy and the need for novel treatment strategies based on targeted therapies. Neuropilins (NRP1, NRP2) are transmembrane glycoproteins that play important roles in angiogenesis and tumorigenesis. An aggressive phenotype and therapy resistance in PDAC is correlated with overexpression of the NRPs. The essential adaptor protein GAIP interacting protein C-terminus (GIPC1) is important for neuropilin signalling. Thus, in this project we focussed on the three different targets, by exploring their single and combined Inhibition.Material and methodsRNA interference (RNAi) was used as an efficient strategy for the specific target gene knockdown, mediated by small interfering RNAs (siRNAs). We analysed in detail the effects of RNAi-based transient GIPC1, NRP1 and NRP2 knockdown on the molecular and cellular level. This included anchorage-dependent and –independent cell proliferation (WST-1-assay), cell cycle distribution (flow cytometry), apoptosis (flow cytometry, caspase-3/7 activity-assay), colony formation (clonogenic assay) and changes in downstream signalling pathways.Results and discussionsIn various cell lines, siRNA mediated knockdown led to a specific downregulation of the selected target genes on mRNA and protein levels. No counter-upregulation of other members of the neuropilin axis was observed. Knockdown led to reduced cell viability, activation of apoptosis and decreased proliferation due to cell cycle inhibition. Spheroid assays showed altered shapes and densities after knockdown and in colony-forming assays decreased colony numbers were observed. In some cases, combined knockdown of two target genes in parallel led to enhanced tumour cell inhibition. Most importantly, treatment with nanoparticle-formulated siRNAs against these targets led to tumour growth inhibition in a xenograft mouse model, especially in the case of the NRP2.ConclusionOur data indicate that GIPC1, NRP1 and NRP2 are new promising targets in pancreatic cancer therapy, with a particular relevance of NRP2.