Project Summary.
Despite the numerous pharmacological and immunological approaches for multimodal glioma treatments
proposed in recent years, glioma phenotypic and genotypic spatial profiles remain heterogeneous and, therefore,
represent the biggest disadvantage for patient outcomes due to the development of treatment resistance.
Cell fusions through permanent cell-fusion and temporal tunneling nanotube (TNT) formations are novel,
recently discovered sources of intercellular gene transfers and glioma heterogeneity. In our grant, we will provide
a detailed analysis of cellular mechanisms essential for intercellular gene transfer via cell fusion and TNT
formations for different glioma subtypes in patient-derived tissue, mimicked glioma microenvironment in vitro,
and in mouse glioma models in vivo. Cell fusion and TNT formations in the context of tumor heterogeneity will
be detailed by following techniques: i) the high-resolution spatial cell transcriptional and histological profiling in
tissue slices (Visium platform); ii) the RNA-Seq and WES profiling (Illumina platform) on a single-cell level after
cell-type-specific enrichment by flow cytometry technique from dissociated tissue; iii) the proteomics (HCP, mass
spectrometry) analysis of molecular complexes involved in gene transfer at different stages of cell interaction.
Cell-specific Cas9/gRNA- directed gene knockdown in combination with the target rescue experiments and
quantitative Cre/fluorescence-based reporters of cell fusion and TNT formations will be utilized in vitro and in
vivo to complement RNA-Seq, WES, and proteomics data, allowing identification of new targets and providing
seeds for the development of novel pharmacological inhibitors of cell fusion and TNT formations.
Our preliminary data identified horizontal gene transfer via cell fusion and TNT formations between
glioma cells themselves and glioma/normal host cells in the hypoxic, inflammatory, mechanically stressed,
cytotoxic, and amino acid deprived microenvironmental glioma loci. We analyzed the transcriptomic and
proteomic signature of five PDGx cell lines of different molecular subtypes and confirmed that a central node of
the cellular stress response, the mRNA-binding protein HuR, is an essential regulator of cell fusion and TNT
formations in numerous stress conditions. The key biomarkers of TNT formations (TNFAIP2, GJA1) and
numerous endogenous fusogenes are direct HuR mRNA targets and overexpressed in gliomas in a HuR-
dependent manner. Therefore, we propose that pharmacological inhibitors of HuR function may serve as
suppressors of intercellular gene transfers evoked by cell fusion and TNT formations. In our grant, the impact of
the recently developed and patented by our group inhibitors of HuR dimerization (SRI42127 is a lead compound)
will be assessed in the regulation of glioma heterogeneity in vitro and in vivo. The high throughput genome-wide
in vivo screening of PDGx cell lines transduced with a Human GeCKOv2 CRISPR genome-wide knockout library
will be employed to reveal and compensate potential mechanisms of cell fusion tolerance to the HuR inhibitors.
