STEM-19. NON-REDUNDANT, ISOFORM-SPECIFIC ROLES OF HDAC1 IN THE REGULATION OF THE GLIOMA STEM CELL PHENOTYPE

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. There is a temporal and cell-type specific requirement of HDAC1 and 2 during normal brain development, with HDAC2 being indispensable in neural stem cells. Here, we specifically investigated the functional importance of HDAC1 in glioma stem cells, an HDAC isoform whose expression increases with brain tumor grade and is correlated with decreased survival. Using cell-based and biochemical assays, transcriptomic analyses and patient-derived xenograft models, we report that knockdown of HDAC1 alone has profound effects on the glioma stem cell (GSC) phenotype and survival in a p53-dependent manner. HDAC1 is the essential class I deacetylase in glioma stem cells, and its loss is not compensated for by its paralogue HDAC2 or other HDACs. Loss of HDAC1 expression significantly suppresses viability of GSCs harboring functional p53, and that HDAC2 expression is completely dispensable in GSCs. In addition, HDAC1 silencing but not HDAC2, stabilizes and acetylates p53 in GSCs, resulting in upregulation of key p53 target genes and induction of programmed cell death. Furthermore, ablation of HDAC1 function alone results in histone hyperacetylation and a collapse of the stemness state in GSCs. We demonstrate significant 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 HDAC inhibitor drugs.

Chi3l1 is a modulator of glioma stem cell states and a therapeutic vulnerability for glioblastoma

Chi3l1 (Chitinase 3-like 1) is a secreted protein highly expressed in glioblastoma. Here, we show that exposure of glioma stem cells (GSCs) to Chi3l1 reduces the CD133+/SOX2+ cells and increases the CD44+/Chi3l1+ cells. Chi3l1 binds to CD44 and induces phosphorylation and nuclear translocation of beta-catenin, Akt and STAT3. Single cell RNA-seq and RNA velocity following incubation of GSCs with Chi3l1 show significant changes in GSC state dynamics driving GSCs towards a mesenchymal expression profile and reducing transition probabilities towards terminal cellular states. ATAC-seq reveals that Chi3l1 increases accessibility of promoters containing MAZ transcription factor footprint. Inhibition of MAZ directly regulates genes with highest expression in cellular clusters exhibiting significant cell state transitions. Finally, targeting Chi3l1 in vivo with a blocking antibody, resets the transcriptomic profile of glioblastoma and inhibits tumor growth. Our work implicates Chi3l1 as modulator of GSC cellular states and demonstrates pre-clinical efficacy of anti-Chi3l1 antibody treatment.

V-ATPase as a mediator of treatment resistance in Glioblastoma Multiforme: an in vitro study to investigate new therapeutic strategies

Recent evidences suggest the involvement of the Vacuolar H+ ATPase (V-ATPase) in the development and/or progression of Glioblastoma Multiforme (GBM). This proton pump could be a valid therapeutic target but more in-depth studies are necessary. The aim of this study is to better define the in vitro effects on Glioma Stem Cell (GSC) primary cultures viability of single and combined treatment with Bafilomycin-A1 (Baf-A1), a V-ATPase inhibitor, and Temozolomide (TMZ), the chemotherapeutic agent currently used to treat GBM patients. We found out that GSC were resistant to TMZ and more sensitive to treatments with Baf-A1 and that the two drugs exerted a synergistic effect when administered together.

P04.18 Heterogeneity and plasticity of integrin α5β1 expression in glioblastoma stem cells

BACKGROUND Glioblastoma (GBM) is the most frequent and deadliest type of central nervous system tumors. Despite the treatment by the Stupp protocol, almost all patients relapse and new therapeutic protocols have been unsuccessful for ameliorating patient survival. Molecular heterogeneity of GBM and existence of glioma stem cells (GSC) may be linked to therapy resistance and recurrence. We demonstrated earlier that α5β1 integrin is a GBM therapeutic target which participate to therapy resistance; a high expression in patient tumors is linked to a worse prognosis. Expression of α5β1 integrin is heterogeneous inter- and intra-tumorally. We particularly addressed the role of glioma stem cell plasticity in the modulation of the integrin expression. Stem cells reside in specific niches (perivascular or hypoxic niches) in the tumor and are at the origin of the more differentiated tumor cell bulk. Metabolism is known to change between the different GSC states and may be affected by or may affect the integrin expression. The aim of our work is therefore to consider the expression of the integrin α5β1 in relationship with GSC differentiation or in hypoxic environment and with cell metabolism. MATERIAL AND METHODS Ten different patient-derived glioma stem cell lines were investigated. Cell culture in stem cell medium (neurospheres) or differentiation medium (adherent cell monolayer) was made in normoxia (21% O2) or hypoxia (1%O2). Alternatively, chemically-induced hypoxia (cobalt chloride/desferoxiamine) was used. Integrin expression was kinetically checked at the mRNA (RT-qPCR) or protein (Western blot) levels. Cell metabolism was investigated with the Seahorse Xfp technology and by HRMAS-NMR. RESULTS No GSC lines (neurospheres) expressed the α5β1 integrin. Interestingly, only half of them did after differentiation suggesting a first level of heterogeneity. A second level of heterogeneity was observed in hypoxic conditions provoking induction of integrin α5β1 expression in only some non-differentiated GSC. Three categories of GSC were thus characterized: one able to express the integrin in hypoxia and after differentiation, one never expressing it and the third one only after differentiation. Cell metabolism differed between GSC before and after differentiation and in presence of integrin α5β1 antagonists. Specific glioma regulator network analysis revealed new targets to be inhibited concomitantly with the integrin. CONCLUSION Data suggest that α5β1 integrin expression may be induced by different signaling pathways. Molecular switches may occur either when stem cells differentiate to tumor cells but also directly in stem cells in hypoxic niches. Characterization of α5β1 integrin expression drivers may help to find new therapeutic targets but also to delineate subpopulation of patients who would benefit from an anti-integrin strategy.

A Fast and Efficient Approach to Obtaining High-Purity Glioma Stem Cell Culture

Glioma is the most common and malignant primary brain tumor. Patients with malignant glioma usually have a poor prognosis due to drug resistance and disease relapse. Cancer stem cells contribute to glioma initiation, progression, resistance, and relapse. Hence, quick identification and efficient understanding of glioma stem cells (GSCs) are of profound importance for therapeutic strategies and outcomes. Ideally, therapeutic approaches will only kill cancer stem cells without harming normal neural stem cells (NSCs) that can inhibit GSCs and are often beneficial. It is key to identify the differences between cancer stem cells and normal NSCs. However, reports detailing an efficient and uniform protocol are scarce, as are comparisons between normal neural and cancer stem cells. Here, we compared different protocols and developed a fast and efficient approach to obtaining high-purity glioma stem cell by tracking observation and optimizing culture conditions. We examined the proliferative and differentiative properties confirming the identities of the GSCs with relevant markers such as Ki67, SRY-box containing gene 2, an intermediate filament protein member nestin, glial fibrillary acidic protein, and s100 calcium-binding protein (s100-beta). Finally, we identified distinct expression differences between GSCs and normal NSCs including cyclin-dependent kinase 4 and tumor protein p53. This study comprehensively describes the features of GSCs, their properties, and regulatory genes with expression differences between them and normal stem cells. Effective approaches to quickly obtaining high-quality GSCs from patients should have the potential to not only help understand the diseases and the resistances but also enable target drug screening and personalized medicine for brain tumor treatment.

OPTC-2. Identification of enriched genes and pathways associated to hypoxia induced migration in patient-derived glioma stem cell lines by RNAseq

Glioblastomas (GBM), the most prevalent and lethal primary brain tumors, are characterized by high intertumoral heterogeneity, diffuse infiltration, and resistance to conventional therapies. Notably, the ability of tumor cells to invade surrounding tissues is one of their most damaging characteristics, it not only causes resistance to therapies such as surgery and radiotherapy but is ultimately the primary cause of death. Therapies that cause hypoxia (e.g. anti-angiogenic therapies) have been shown to increase invasiveness, leading to resistance to the therapy itself and further complications for the patients. Using patient-derived glioma stem cell lines (GSCL) we have discovered cell lines that display heterogeneous migratory behavior in response to hypoxia. As expected we observed that four GSCLs studied had increased migration in hypoxia. Strikingly, two other cell lines studied showed decreased migration in hypoxia. This unforeseen result reflects the heterogeneous nature of GBM and the difference between these GSCLs could be key to understanding this variable. To delve into the molecular context that could explain these differences we performed an exploratory RNAseq analysis on four of the GSCLs, two that showed hypoxia-induced migration and two with decreased migration in hypoxia, and evaluated genes differentially expressed in hypoxia versus normoxia. We also carried out gene ontology and pathway enrichment analysis to discover molecular and pathway patterns consistent with the migratory behaviors observed in each group of GSCLs. The results show how that a similar migratory response to hypoxia coincides with particular sets of enriched genes and pathways. Specifically, we found NOTCH and WNT signaling pathways upregulated in GSCLs which showed increased migration in hypoxia while the IFN-gamma pathway upregulated in GSCLs with decreased migration in hypoxia. Knowing the individual molecular mechanisms responsible for the migratory behavior could allow for tailor-made therapies that reduce the dissemination of these tumors.

Effects of the Anti-Tumorigenic Agent AT101 on Human Glioblastoma Cells in the Microenvironmental Glioma Stem Cell Niche

Glioblastoma (GBM) is a barely treatable disease due to its profound chemoresistance. A distinct inter- and intratumoral heterogeneity reflected by specialized microenvironmental niches and different tumor cell subpopulations allows GBMs to evade therapy regimens. Thus, there is an urgent need to develop alternative treatment strategies. A promising candidate for the treatment of GBMs is AT101, the R(-) enantiomer of gossypol. The present study evaluates the effects of AT101, alone or in combination with temozolomide (TMZ), in a microenvironmental glioma stem cell niche model of two GBM cell lines (U251MG and U87MG). AT101 was found to induce strong cytotoxic effects on U251MG and U87MG stem-like cells in comparison to the respective native cells. Moreover, a higher sensitivity against treatment with AT101 was observed upon incubation of native cells with a stem-like cell-conditioned medium. This higher sensitivity was reflected by a specific inhibitory influence on the p-p42/44 signaling pathway. Further, the expression of CXCR7 and the interleukin-6 receptor was significantly regulated upon these stimulatory conditions. Since tumor stem-like cells are known to mediate the development of tumor recurrences and were observed to strongly respond to the AT101 treatment, this might represent a promising approach to prevent the development of GBM recurrences.

Low-Intensity Pulsed Ultrasound-Generated Singlet Oxygen Induces Telomere Damage Leading to Glioma Stem Cell Awakening From Quiescence

The authors have requested that this preprint be withdrawn due to erroneous posting.