Enhancing Transcriptional Reprogramming of Mesenchymal Glioblastoma with Grainyhead-like 2 and HDAC Inhibitors Leads to Apoptosis and Cell-Cycle Dysregulation.

Glioblastoma (GBM) tumor cells exhibit mesenchymal properties which are thought to play significant roles in therapeutic resistance and tumor recurrence. An important question is whether impairment of the mesenchymal state of GBM can sensitize these tumors to therapeutic intervention. HDAC inhibitors (HDACi) are being tested in GBM for their ability promote mesenchymal-to-epithelial transcriptional (MET) reprogramming, and for their cancer-specific ability to dysregulate the cell cycle and induce apoptosis. We set out to enhance the transcriptional reprogramming and apoptotic effects of HDACi in GBM by introducing an epithelial transcription factor, Grainyhead-like 2 (GRHL2), to specifically counter the mesenchymal state. GRHL2 significantly enhanced HDACi-mediated MET reprogramming. Surprisingly, we found that inducing GRHL2 in glioma stem cells (GSCs) altered cell-cycle drivers and promoted aneuploidy. Mass spectrometry analysis of GRHL2 interacting proteins revealed association with several key mitotic factors, suggesting their exogenous expression disrupted the established mitotic program in GBM. Associated with this cell-cycle dysregulation, the combination of GRHL2 and HDACi induced elevated levels of apoptosis. The key implication of our study is that although genetic strategies to repress the mesenchymal properties of glioblastoma may be effective, biological interactions of epithelial factors in mesenchymal cancer cells may dysregulate normal homeostatic cellular mechanisms.

Affinity Purification of NF1 Protein-Protein Interactors Identifies Keratins and Neurofibromin Itself as Binding Partners.

Neurofibromatosis Type 1 (NF1) is caused by pathogenic variants in the NF1 gene encoding neurofibromin. Definition of NF1 protein-protein interactions (PPIs) has been difficult and lacks replication, making it challenging to define binding partners that modulate its function. We created a novel tandem affinity purification (TAP) tag cloned in frame to the 3' end of the full-length murine Nf1 cDNA (mNf1). We show that this cDNA is functional and expresses neurofibromin, His-Tag, and can correct p-ERK/ERK ratios in NF1 null HEK293 cells. We used this affinity tag to purify binding partners with Strep-Tactin®XT beads and subsequently, identified them via mass spectrometry (MS). We found the tagged mNf1 can affinity purify human neurofibromin and vice versa, indicating that neurofibromin oligomerizes. We identify 21 additional proteins with high confidence of interaction with neurofibromin. After Metacore network analysis of these 21 proteins, eight appear within the same network, primarily keratins regulated by estrogen receptors. Previously, we have shown that neurofibromin levels negatively regulate keratin expression. Here, we show through pharmacological inhibition that this is independent of Ras signaling, as the inhibitors, selumetinib and rapamycin, do not alter keratin expression. Further characterization of neurofibromin oligomerization and binding partners could aid in discovering new neurofibromin functions outside of Ras regulation, leading to novel drug targets.

Multi-Omics Profiling for NF1 Target Discovery in Neurofibromin (NF1) Deficient Cells.

Loss of NF1 is an oncogenic driver. In efforts to define pathways responsible for the development of neurofibromas and other cancers, transcriptomic and proteomic changes are evaluated in a non-malignant NF1 null cell line. NF1 null HEK293 cells were created using CRISPR/Cas9 technology and they are compared to parental cells that express neurofibromin. A total of 1222 genes and 132 proteins are found to be differentially expressed. The analysis is integrated to identify eight transcripts/proteins that are differentially regulated in both analyses. Metacore Pathway analysis identifies Neurogenesis NGF/TrkA MAPK-mediated signaling alterations. Next, the data set is compared with other published studies that involve analysis of cells or tumors deficient for NF1 and it is found that 141 genes recur in the sample and others; only thirteen of these genes recur in two or more studies. Genes/proteins of interest are validated via q-RT-PCR or Western blot. It is shown that KRT8 and 14-3-3σ protein levels respond to exogenously introduced mNf1 cDNA. Hence, transcripts/proteins that respond to neurofibromin levels are identified and they can potentially be used as biomarkers.