Piotr Waranecki

Culture methods of diffuse intrinsic pontine glioma cells determine response to targeted therapies

Abstract Diffuse intrinsic pontine glioma (DIPG) is an aggressive type of brainstem cancer occurring mainly in children, for which there currently is no effective therapy. Current efforts to develop novel therapeutics for this tumor make use of primary cultures of DIPG cells, maintained either as adherent monolayer in serum containing medium, or as neurospheres in serum‐free medium. In this manuscript, we demonstrate that the response of DIPG cells to targeted therapies in vitro is mainly determined by the culture conditions. We show that particular culture conditions induce the activation of different receptor tyrosine kinases and signal transduction pathways, as well as major changes in gene expression profiles of DIPG cells in culture. These differences correlate strongly with the observed discrepancies in response to targeted therapies of DIPG cells cultured as either adherent monolayers or neurospheres. With this research, we provide an argument for the concurrent use of both culture conditions to avoid false positive and false negative results due to the chosen method. HighlightsDIPG cells can be cultured as either adherent monolayers or neurospheres.Culture conditions induce major changes in pathway activation of DIPG cells.Culture conditions induce major changes in gene expression of DIPG cells.These induced changes influence the response of DIPG cells to targeted therapies.

An efficient method for the transduction of primary pediatric glioma neurospheres

Graphical abstract

ATRT-05. PRECLINICAL EFFICACY OF RADIATION-FREE TREATMENT OF ATYPICAL TERATOID/RHABDOID TUMORS BY COMBINED MEK AND MELK INHIBITION

AbstractAtypical teratoid/rhabdoid tumors (AT/RT) are highly malignant tumors that occur both in- and outside the central nervous system, predominantly in young children. Although therapeutic interventions, combining surgery with intensive chemotherapy and conformal radiotherapy, have shown curative potential, the long-term consequences of these treatment modalities in young children are often severe. Therefore, there is a dire need for novel, targeted therapeutic strategies that potentially reduce or eliminate chemo- and radiotherapy, especially for children under three years of age. Genetically, AT/RT is characterized by a biallelic inactivation of SMARCB1 or SMARCA4, causing aberrant chromatin remodeling and thereby expression of a variety of oncogenes. Recently, the importance of the mitogen-activated protein kinase (MAPK) pathway in the pathogenesis of AT/RT has been demonstrated, with preclinical efficacy of MEK inhibition in AT/RT cells. Preclinical patient-derived AT/RT models are largely lacking. We developed a patient-derived primary cell line and xenograft model, VUMC-AT/RT-01, from a surgical specimen of an AT/RT patient that had not received prior therapy. Immunohistochemistry was performed on xenograft tumors, revealing typical loss of SMARCB1 expression as well as heterogeneous expression of glial, epithelial, mesenchymal and neuronal differentiation markers. In silico analysis of gene expression revealed strong upregulation of MELK in AT/RT tumors compared to normal brain tissues. These high levels of MELK were subsequently confirmed in our xenograft model. Inhibition of MELK with the small molecule OTSSP167 effectively reduced proliferation and induced cell death in primary AT/RT neurospheres. Combined treatment of primary AT/RT cells with OTSSP167 and the MEK inhibitor Trametinib showed strongly synergistic cytotoxicity at low nanomolar concentrations. Treatment of mice carrying VUMC-AT/RT-01-Fluc xenografts with OTSSP167 and Trametinib confirmed this synergy in vivo. We conclude that combined MEK and MELK inhibition represents a promising targeted therapy strategy for AT/RT that might ultimately reduce or eliminate the need for radiation and intensive chemotherapy.