IDENTIFICATION OF NOVEL COMBINATORIAL TREATMENTS TO DRIVE GLIOBLASTOMA STEM CELLS OUT OF STEMNESS IN 2D AND 3D ORGANOID MODELS

Glioblastoma multiforme (GBM) is a highly aggressive brain tumour with poor survival due to recurrence. This is driven by therapy-resistant glioblastoma stem-like cells (GSCs), hijacking developmental programs to maintain stemness. Our previous 2D work showed that the basic helix–loop–helix (bHLH) transcription factor ASCL1 promotes GSC differentiation into neuronal-like cells, an effect enhanced by phospho-defective ASCL1 variant (SA ASCL1) but fails to engage full differentiation programme in this context. We therefore designed physiologically more relevant 3D brain organoid models of glioblastoma expressing either wild-type (WT) or SA ASCL1. We found a marked reduction in tumour size, associated with increased cell death rather than with reduced proliferation of ASCL1-expressing GSCs. This is in contrast to previous work in 2D GSCs cultures where ASCL1 expression induced a reduction in proliferation. These findings indicate that the 3D microenvironment and cell–cell interactions might better reflect tumour behaviour and mimic recurrence in patients more truthfully than 2D cultures. For potential treatment application, we screened combinations of developmental factors and kinase inhibitors promoting expression and de-phosphorylation of ASCL1. Three treatments, all containing a MEK inhibitor in combination with other inhibitors, significantly reduced GSC proliferation without inducing cytotoxicity. When tested in 3D organoids, these treatments induced tumour reduction via cell death but did not decrease proliferation, similar to the observations obtained upon ASCL1 overexpression. Together, findings highlight the importance of the 3D tumour microenvironment in shaping GSC responses, providing a model to test new treatments. Here, targeting ASCL1 expression might reflect a promising treatment approach.