The overall aim of this study was to find ways to sensitize treatment resistant glioma-initiating cell (GIC) clones to conventional therapy. Heterogeneity is substantial in glioblastoma multiforme (GBM) and through establishment of clonal GIC cultures from patient biopsies we have demonstrated a wide variety in the responses to drugs and radiation. A multitherapy resistance phenotype was linked to proneural-mesenchymal transition (PMT) in the transcriptome. The variety in therapy response was observed as a continuum of phenotypes. The distribution of phenotypes resembled a normal distribution and multitherapy resistance was associated with low DNA methylation grade in promoter regions of mesenchymal master regulators (FOSL2, RUNX1). Our data thus implied that the transition is bi-directional and epigenetically regulated (Segerman et al, Cell Reports - accepted in principle).

To investigate if spontaneous changes in drug and radiation response occur, we have derived subclones from a resistant clone. Both subclones with higher and lower therapy resistance than the parental clone were generated. Also molecularly the subclones largely reconstituted the original clonal variation.

PMT shows similarities to epithelial-mesenchymal transition (EMT), which is induced by extrinsic factors. We therefore specifically analyzed the gene expression data for signaling receptors differentially expressed in resistant vs. sensitive clones as well as cognate ligands. To estimate the importance of a particular signaling pathway, expression of co-receptors and ligands was taken into account. We found several cases of coherent upregulation of receptor and ligand indicative of autocrine loops. Regarding pathways that appeared to be overactive in resistant vs. sensitive clones, there was a prominent overlap with EMT. It was also apparent that several pathways were activated concomitantly.

We are currently focusing on identifying combinations of drugs (and antibodies) that sensitize resistant clones to conventional treatment through modulation of cell signaling patterns. In the initial screen temozolomide (TMZ) response is used as an indicator of achieved sensitization. The strategy is to iteratively combine primarily antagonists of signaling receptors connected to resistance. We are also exploring the effect of stimulating pathways with apparently higher activity in sensitive clones (e.g. addition of ligands).The concept of sensitizing glioma and other types of cancer cells by targeting the mesenchymal character through usage of e.g. signaling receptor inhibitors is not new and has shown promise. Indeed, our preliminary data look encouraging.

In conclusion, our data show that multitherapy resistance is connected to a plastic cell-state. Also, receptors and ligands that are differentially expressed in resistant and sensitive clones engage pathways regulating EMT.

Citation Format: Mia Niklasson, Malin Jarvius, Caroline Haglund, Efthymia Chantzi, Tobias Bergström, Frida Nyberg, Annika Hermansson, Mårten Fryknäs, Mats Gustafsson, Bo Segerman, Rolf Larsson, Bengt Westermark, Anna Segerman. Targeting of a mesenchymal profile in order to sensitize multitherapy resistant glioblastoma clones [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4175. doi:10.1158/1538-7445.AM2017-4175