Summary In glioblastoma, invasion and proliferation are presumed to be mutually exclusive events; however, the molecular mechanisms that mediate this switch at the cellular level remain elusive. Previously, we have shown that phospho-OLIG2, a Central Nervous System-specific transcription factor, is essential for tumor growth and proliferation. Here, we show that modulation of OLIG2 phosphorylation can trigger a switch between proliferation and invasion. Glioma cells with unphosphorylated OLIG2S10, S13, S14 are highly migratory and invasive both in vitro and in vivo. Mechanistically, unphosphorylated OLIG2 induces TGFβ2 expression and promotes invasive mesenchymal properties in glioma cells. Inhibition of TGFβ2 pathway blocks this OLIG2-dependent invasion. Furthermore, ectopic expression of phosphomimetic Olig2 is sufficient to block TGFβ2 mediated invasion and reduce expression of invasion genes (ZEB1 and CD44). Our results not only provide a mechanistic insight into how cells switch from proliferation to invasion, but also offer therapeutic opportunities for inhibiting dissemination of gliomas.

Malignant gliomas are the most common, infiltrative, and lethal primary brain tumors affecting the adult population. The grim prognosis for this disease is due to a combination of the presence of highly invasive tumor cells that escape surgical resection and the presence of a population of therapy-resistant cancer stem cells found within these tumors. Several studies suggest that glioma cells have cleverly hijacked the normal developmental program of neural progenitor cells, including their transcriptional programs, to enhance gliomagenesis. In this review, we summarize the role of developmentally regulated signaling pathways that have been found to facilitate glioma growth and invasion. Furthermore, we discuss how the microenvironment and treatment-induced perturbations of these highly interconnected signaling networks can trigger a shift in cellular phenotype and tumor subtype.

Glioblastoma (GBM) is characterized by an aberrant yet druggable epigenetic landscape. One major family of epigenetic regulators, the histone deacetylases (HDACs), are considered promising therapeutic targets for GBM due to their repressive influences on transcription. Although HDACs share redundant functions and common substrates, the unique isoform-specific roles of different HDACs in GBM remain unclear. In neural stem cells, HDAC2 is the indispensable deacetylase to ensure normal brain development and survival in the absence of HDAC1. Surprisingly, we find that HDAC1 is the essential class I deacetylase in glioma stem cells, and its loss is not compensated for by HDAC2. Using cell-based and biochemical assays, transcriptomic analyses, and patient-derived xenograft models, we find that knockdown of HDAC1 alone has profound effects on the glioma stem cell phenotype in a p53-dependent manner. We demonstrate marked suppression in tumor growth upon targeting of HDAC1 and identify compensatory pathways that provide insights into combination therapies for GBM. Our study highlights the importance of HDAC1 in GBM and the need to develop isoform-specific drugs.

The perivascular niche (PVN) is a glioblastoma tumor microenvironment (TME) that serves as a safe haven for glioma stem cells (GSCs), and acts as a reservoir that inevitably leads to tumor recurrence. Understanding cellular interactions in the PVN that drive GSC treatment resistance and stemness is crucial to develop lasting therapies for glioblastoma. The limitations of in vivo models and in vitro assays have led to critical knowledge gaps regarding the influence of various cell types in the PVN on GSCs behavior. This study developed an organotypic triculture microfluidic model as a means to recapitulate the PVN and study its impact on GSCs. This triculture platform, comprised of endothelial cells (ECs), astrocytes, and GSCs, is used to investigate GSC invasion, proliferation and stemness. Both ECs and astrocytes significantly increased invasiveness of GSCs. This study futher identified 15 ligand‐receptor pairs using single‐cell RNAseq with putative chemotactic mechanisms of GSCs, where the receptor is up‐regulated in GSCs and the diffusible ligand is expressed in either astrocytes or ECs. Notably, the ligand–receptor pair SAA1‐FPR1 is demonstrated to be involved in chemotactic invasion of GSCs toward PVN. The novel triculture platform presented herein can be used for therapeutic development and discovery of molecular mechanisms driving GSC biology.

contributed RNA samples from pre-hypertrophic mouse models and assisted with computational analysis.

OLIG2 is a central nervous system-specific transcription factor that is expressed in almost all diffuse gliomas. It is also one of the key core transcription factors that can reprogram differentiated glioma cells to highly tumorigenic glioma stem-like cells (GSCs). We have previously shown that expression of OLIG2 is critical for glioma growth both in a genetically relevant mouse model as well as in patient-derived xenograft models. Our work suggests that a small molecule inhibitor of OLIG2 could serve as a highly targeted therapy for high-grade glioma; however, transcription factors are generally very difficult to target because their interactions with DNA and co-regulatory proteins involve large and complex surface area contacts. Our laboratory has shown that OLIG2 functions are regulated through interactions with distinct co-regulator proteins in normal neural stem cells. However, there are currently no reports on interactors that promote the proto-oncogenic functions of OLIG2 in malignant glioma. In this study, we employed two independent proteomics screens identify tumor-specific, druggable OLIG2 co-regulators as possible surrogate targets to suppress OLIG2 function in glioma. These screens led to the identification of a novel OLIG2 partner protein: Histone Deacetylase 1 (HDAC1). We confirmed that this interaction occurs in both murine and human glioma models. Although HDACs are ubiquitously expressed and are known to be functionally redundant, we show that ablation of HDAC1 alone significantly decreases the stemness and proliferation capacity of patient-derived GSCs in a p53-dependent manner, while having a minimal impact on normal human neural stem cells and astrocytes. Furthermore, we demonstrate that knockdown of HDAC1, in combination with ionizing radiation treatment, significantly alters the growth pattern of intracranial tumors in vivo. We demonstrate that HDAC1 function is critical for GSC growth and provide a strong rationale for targeting the OLIG2-HDAC1 interaction in malignant glioma.

Glioblastoma (GBM), the most common and aggressive primary brain tumor affecting adults, is characterized by an aberrant yet druggable epigenetic landscape. Class I histone deacetylases (HDACs) mediate chromatin compaction and are frequently overexpressed in GBM. Hence, over the last decade, there has been considerable interest in HDAC inhibitors (HDACi) for the treatment of malignant brain tumors. However, to date almost all HDACi tested clinically have failed to provide significant therapeutic benefit to primary and recurrent GBM patients. These HDACi are broad-spectrum with poor or unknown pharmacokinetic profiles and have narrow therapeutic window. Isoform specificity for HDACi is important given that not all HDAC enzymes are equally expressed in GBM. Recently, we uncovered the functional importance of HDAC1 in therapy-resistant glioma stem cells, where we found that its expression increases with brain tumor grade and is correlated with decreased survival. While no class I HDAC isoform-specific inhibitors are currently available, the second-generation HDACi, quisinostat harbors high specificity for HDAC1. In this study, we assessed the pharmacokinetic, pharmacodynamic and radiosensitizing properties of quisinostat in preclinical models of human GBM. In vitro analyses conducted in patient-derived glioma stem cell (GSC) lines revealed that quisinostat exhibited potent growth inhibition in multiple GSC lines (IC50 ~ 50-86 nM), and induced global histone hyperacetylation, elevated DNA damage, cell death, cell cycle arrest and decreased expression of multiple key stem cell proteins. To determine the efficacy and drug distribution profile of quisinostat in vivo, athymic mice with orthotopic or flank tumors were treated with an optimized treatment schedule. At various timepoints during treatment, plasma and brain/tumor tissue were collected to measure total and unbound drug levels by liquid chromatography tandem mass spectrometry (LC-MS/MS) and assess pharmacodynamic changes within tumor tissues. We found that quisinostat reduces tumor burden in flank and orthotopic models of GBM and extends survival when administered in combination with radiation therapy in vivo. Together, these results provide a rationale for developing quisinostat as a potential combination therapy with radiation in the treatment of GBM. Citation Format: Costanza Lo Cascio, Tigran Margaryan, Ernesto Luna Melendez, James McNamara, William Knight, Sarah Himes, Connor White, Alexis Giff, Jesus Peralta, Nader Sanai, Artak Tovmasyan, Shwetal Mehta. Pharmacokinetics- and pharmacodynamics-based evaluation of quisinostat as a radiosensitizer in preclinical models of human glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 349.

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