Search

Head and neck squamous cell carcinomas (HNSCCs) are heterogeneous tumors, well known for their frequent relapsing nature. To counter recurrence, biomarkers for early diagnosis, prognosis, or treatment response prediction are urgently needed. miRNAs can profoundly impact normal physiology and enhance oncogenesis. Among all of the miRNAs, the miR-30 family is frequently downregulated in HNSCC. Here, we determined how levels of the 3p passenger strands of miR-30a and miR-30e affect tumor behavior and clarified their functional role in LA-HNSCC. In a retrospective study, levels of miR-30a-3p and miR-30e-3p were determined in 110 patients and correlated to overall survival, locoregional relapse, and distant metastasis. miR-30a/e-3p were expressed in HNSCC cell lines and HNSCC patient-derived tumoroids (PDTs) to investigate their effect on tumor cells and their microenvironment. Both miRNAs were found to have a prognosis value since low miR-30a/e-3p expression correlates to adverse prognosis and reduces overall survival. Low expression of miR-30a/e-3p is associated with a shorter time until locoregional relapse and a shorter time until metastasis, respectively. miR-30a/e-3p expression downregulates both TGF-βR1 and BMPR2 and attenuates the survival and motility of HNSCC. Results were confirmed in PDTs. Finally, secretomes of miR-30a/e-3p-transfected HNSCC activate M1-type macrophages, which exert stronger phagocytic activities toward tumor cells. miR-30a/e-3p expression can discriminate subgroups of LA-HNSCC patients with different prognosis, making them good candidates as prognostic biomarkers. Furthermore, by targeting members of the TGF-β family and generating an immune-permissive microenvironment, they may emerge as an alternative to anti-TGF-β drugs to use in combination with immune checkpoint inhibitors.

PDF file - 27K, Relationship between Alpha5 mRNA level and p53 status in human biopsies and in human xenografts in nude mice

PDF file - 25K, Mean fluorescence intensities of Alpha5 and Beta1 integrin subunits at the cell membrane

PDF file - 85K

Integrins play a role in the resistance of advanced cancers to radiotherapy and chemotherapy. In this study, we show that high expression of the α5 integrin subunit compromises temozolomide-induced tumor suppressor p53 activity in human glioblastoma cells. We found that depletion of the α5 integrin subunit increased p53 activity and temozolomide sensitivity. However, when cells were treated with the p53 activator nutlin-3a, the protective effect of α5 integrin on p53 activation and cell survival was lost. In a functional p53 background, nutlin-3a downregulated the α5 integrin subunit, thereby increasing the cytotoxic effect of temozolomide. Clinically, α5β1 integrin expression was associated with a more aggressive phenotype in brain tumors, and high α5 integrin gene expression was associated with decreased survival of patients with high-grade glioma. Taken together, our findings indicate that negative cross-talk between α5β1 integrin and p53 supports glioma resistance to temozolomide, providing preclinical proof-of-concept that α5β1 integrin represents a therapeutic target for high-grade brain tumors. Direct activation of p53 may remain a therapeutic option in the subset of patients with high-grade gliomas that express both functional p53 and a high level of α5β1 integrin. Cancer Res; 72(14); 3463–70. ©2012 AACR.

PDF file - 48K, Nutlin-3a decreases the total expression of Beta1 integrin subunit in U87MG cells but not its expression at the cell membrane

PDF file - 131K, The effect of Alpha5 integrin on p53 activity and resistance to temozolomide is restricted to p53wt expressing cells

PDF file - 32K, Nutlin-3a does not modulate the Alpha5 integrin expression in U373 cells

International audience; Glioblastoma (GBM) is the most deadly type of malignant brain tumor, despite extensive molecular analyses of GBM cells. In recent years, the tumor microenvironment (TME) has been recognized as an important player and therapeutic target in GBM. However, there is a need for a full and integrated understanding of the different cellular and molecular components involved in the GBM TME and their interactions for the development of more efficient therapies. In this review, we provide a comprehensive report of the GBM TME, which assembles the contributions of physicians and translational researchers working on brain tumor pathology and therapy in France. We propose a holistic view of the subject by delineating the specific features of the GBM TME at the cellular, molecular, and therapeutic levels.

Cancer stem cells (CSCs) represent a difficult to treat cellular niche within tumours due to their unique characteristics, which give them a high propensity for resistance to classical anti-cancer treatments and the ability to re-populate the tumour mass. An attribute that may be implicated in the high rates of recurrence of certain tumours. However, other characteristics specific to these cells, such as their high dependence on mitochondria, may be exploited for the development of new therapeutic agents that are effective against the niche. As such, a previously described phosphorescent N-heterocyclic carbene iridium(III) compound which showed a high level of cytotoxicity against classical tumour cell lines with mitochondria-specific effects was studied for its potential against CSCs. The results showed a significantly higher level of activity against several CSC lines compared to non-CSCs. Mitochondrial localisation and superoxide production was confirmed. Although the cell death involved caspase activation, their role in cell death was not definitive, with a potential implication of other, non-apoptotic pathways shown. A cytostatic effect of the compound was also displayed at low mortality doses. This study thus provides important insights into the mechanisms and the potential for this class of molecule in the domain of anti-CSC therapeutics. Keywords: Anti-tumoral drug; Cancer stem cells; Cell death; Cytostatic effect; Glioblastoma; Mitochondria; N-heterocyclic carbene iridium.

Tumor metabolism is emerging as a potential target for cancer therapies. This new approach holds particular promise for the treatment of glioblastoma, a highly lethal brain tumor that is resistant to conventional treatments, for which improving therapeutic strategies is a major challenge. The presence of glioma stem cells is a critical factor in therapy resistance, thus making it essential to eliminate these cells for the long-term survival of cancer patients. Recent advancements in our understanding of cancer metabolism have shown that glioblastoma metabolism is highly heterogeneous, and that cancer stem cells exhibit specific metabolic traits that support their unique functionality. The objective of this review is to examine the metabolic changes in glioblastoma and investigate the role of specific metabolic processes in tumorigenesis, as well as associated therapeutic approaches, with a particular focus on glioma stem cell populations.

Overexpression of EGFR drives glioblastomas (GBM) cell invasion but these tumours remain resistant to EGFR-targeted therapies such as tyrosine kinase inhibitors (TKIs). Endocytosis, an important modulator of EGFR function, is often dysregulated in glioma cells and is associated with therapy resistance. However, the impact of TKIs on EGFR endocytosis has never been examined in GBM cells. In the present study, we showed that gefitinib and other tyrosine kinase inhibitors induced EGFR accumulation in early-endosomes as a result of an increased endocytosis. Moreover, TKIs trigger early-endosome re-localization of another membrane receptor, the fibronectin receptor alpha5beta1 integrin, a promising therapeutic target in GBM that regulates physiological EGFR endocytosis and recycling in cancer cells. Super-resolution dSTORM imaging showed a close-proximity between beta1 integrin and EGFR in intracellular membrane compartments of gefitinib-treated cells, suggesting their potential interaction. Interestingly, integrin depletion delayed gefitinib-mediated EGFR endocytosis. Co-endocytosis of EGFR and alpha5beta1 integrin may alter glioma cell response to gefitinib. Using an in vitro model of glioma cell dissemination from spheroid, we showed that alpha5 integrin-depleted cells were more sensitive to TKIs than alpha5-expressing cells. This work provides evidence for the first time that EGFR TKIs can trigger massive EGFR and alpha5beta1 integrin co-endocytosis, which may modulate glioma cell invasiveness under therapeutic treatment.

Simple Summary Glioblastomas are the deadliest brain tumours. The standard of care associates surgery, radio- and chemotherapy with Temozolomide as the reference drug. Despite this treatment, most of the tumours recur. The characterization of resistance mechanisms is of paramount importance to enable the proposal of more effective therapies. In this work we aimed to evaluate the molecular changes occurring during and after Temozolomide treatment in a glioma cell line. A high plasticity in the integrin repertoire exists in these cells. As an example, variations of the α5β1 integrin expression were observed with a reduction during the treatment and re-expression after removal of the drug. The association of integrin antagonists with p53 reactivators appears to be efficient in recurrent tumours. Specific integrins may thus be particularly targetable at different time points of glioblastoma treatment and combination therapies evaluated according to their time-dependent expression. Abstract Despite extensive treatment, glioblastoma inevitably recurs, leading to an overall survival of around 16 months. Understanding why and how tumours resist to radio/chemotherapies is crucial to overcome this unmet oncological challenge. Primary and acquired resistance to Temozolomide (TMZ), the standard-of-care chemotherapeutic drug, have been the subjects of several studies. This work aimed to evaluate molecular and phenotypic changes occurring during and after TMZ treatment in a glioblastoma cell model, the U87MG. These initially TMZ-sensitive cells acquire long-lasting resistance even after removal of the drug. Transcriptomic analysis revealed that profound changes occurred between parental and resistant cells, particularly at the level of the integrin repertoire. Focusing on α5β1 integrin, which we proposed earlier as a glioblastoma therapeutic target, we demonstrated that its expression was decreased in the presence of TMZ but restored after removal of the drug. In this glioblastoma model of recurrence, α5β1 integrin plays an important role in the proliferation and migration of tumoral cells. We also demonstrated that reactivating p53 by MDM2 inhibitors concomitantly with the inhibition of this integrin in recurrent cells may overcome the TMZ resistance. Our results may explain some integrin-based targeted therapy failure as integrin expressions are highly switchable during the time of treatment. We also propose an alternative way to alter the viability of recurrent glioblastoma cells expressing a high level of α5β1 integrin.

Over the last few decades, deciphering the alteration of molecular pathways in brain tumors has led to impressive changes in diagnostic refinement. Among the molecular abnormalities triggering and/or driving gliomas, alterations in the MAPK pathway reign supreme in the pediatric population, as it is encountered in almost all low-grade pediatric gliomas. Activating abnormalities in the MAPK pathway are also present in both pediatric and adult high-grade gliomas. Across those alterations, BRAF p.V600E mutations seem to define homogeneous groups of tumors in terms of prognosis. The recent development of small molecules inhibiting this pathway retains the attention of neurooncologists on BRAF-altered tumors, as conventional therapies showed no significant effect, nor prolonged efficiency on the high-grade or low-grade unresectable forms. Nevertheless, tumoral heterogeneity and especially molecular alteration(s) associated with MAPK-pathway abnormalities are not fully understood with respect to how they might lead to the specific dismal prognosis of those gliomas and/or affect their response to targeted therapies. This review is an attempt to provide comprehensive information regarding molecular alterations related to the aggressiveness modulation in BRAF-mutated gliomas and the current knowledge on how to use those targeted therapies in such situations.

Pediatric high-grade gliomas (pHGG) represent childhood and adolescent brain cancers that carry a rapid dismal prognosis. Since there is a need to overcome the resistance to current treatments and find a new way of cure, modeling the disease as close as possible in an in vitro setting to test new drugs and therapeutic procedures is highly demanding. Studying their fundamental pathobiological processes, including glutamatergic neuron hyperexcitability, will be a real advance in understanding interactions between the environmental brain and pHGG cells. Therefore, to recreate neurons/pHGG cell interactions, this work shows the development of a functional in vitro model co-culturing human-induced Pluripotent Stem (hiPS)-derived cortical glutamatergic neurons pHGG cells into compartmentalized microfluidic devices and a process to record their electrophysiological modifications. The first step was to differentiate and characterize human glutamatergic neurons. Secondly, the cells were cultured in microfluidic devices with pHGG derived cell lines. Brain microenvironment and neuronal activity were then included in this model to analyze the electrical impact of pHGG cells on these micro-environmental neurons. Electrophysiological recordings are coupled using multielectrode arrays (MEA) to these microfluidic devices to mimic physiological conditions and to record the electrical activity of the entire neural network. A significant increase in neuron excitability was underlined in the presence of tumor cells.

The difficulty involved in the treatment of many tumours due to their recurrence and resistance to chemotherapy is tightly linked to the presence of cancer stem cells (CSCs). This CSC sub-population is distinct from the majority of cancer cells of the tumour bulk. Indeed, CSCs have increased mitochondrial mass that has been linked to increased sensitivity to mitochondrial targeting compounds. Thus, a platinum-based polyethylenimine (PEI) polymer–drug conjugate (PDC) was assessed as a potential anti-CSC therapeutic since it has previously displayed mitochondrial accumulation. Our results show that CSCs have increased specific sensitivity to the PEI carrier and to the PDC. The mechanism of cell death seems to be necrotic in nature, with an absence of apoptotic markers. Cell death is accompanied by the induction of a protective autophagy. The interference in the balance of this pathway, which is highly important for CSCs, may be responsible for a partial reversion of the stem-like phenotype observed with prolonged PEI and PDC treatment. Several markers also indicate the cell death mode to be capable of inducing an anti-cancer immune response. This study thus indicates the potential therapeutic perspectives of polycations against CSCs.

Integrin α5β1 was suggested to be involved in glioblastoma (GBM) aggressiveness and treatment resistance through preclinical studies and genomic analysis in patients. However, further protein expression data are still required to confirm this hypothesis. In the present study, we investigated by immunofluorescence the expression of integrin α5 and its prognostic impact in a glioblastoma series of patients scheduled to undergo the Stupp protocol as first-line treatment for GBM. The integrin α5 protein expression level was estimated in each tumor by the mean fluorescence intensity (MFI) and allowed us to identify two subpopulations showing either a high or low expression level. The distribution of patients in both subpopulations was not significantly different according to age, gender, recursive partitioning analysis (RPA) prognostic score, molecular markers or surgical and medical treatment. A high integrin α5 protein expression level was associated with a high risk of recurrence (HR = 1.696, 95% CI 1.031–2.792, p = 0.0377) and reduced overall survival (OS), even more significant in patients who completed the Stupp protocol (median OS: 15.6 vs. 22.8 months, HR = 2.324, 95% CI 1.168–4.621, p = 0.0162). In multivariate analysis, a high integrin α5 protein expression level was confirmed as an independent prognostic factor in the subpopulation of patients who completed the temozolomide-based first-line treatment for predicting OS over age, extent of surgery, RPA score and O-6-methylguanine-DNA methyltransferase (MGMT) promoter methylation (p = 0.029). In summary, for the first time, our study validates that a high integrin α5 protein expression level is associated with poor prognosis in GBM and confirms its potential as a therapeutic target implicated in the Stupp protocol resistance.

Nucleic acid aptamers are often referred to as chemical antibodies. Because they possess several advantages, like their smaller size, temperature stability, ease of chemical modification, lack of immunogenicity and toxicity, and lower cost of production, aptamers are promising tools for clinical applications. Aptamers against cell surface protein biomarkers are of particular interest for cancer diagnosis and targeted therapy. In this study, we identified and characterized RNA aptamers targeting cells expressing integrin α5β1. This αβ heterodimeric cell surface receptor is implicated in tumor angiogenesis and solid tumor aggressiveness. In glioblastoma, integrin α5β1 expression is associated with an aggressive phenotype and a decrease in patient survival. We used a complex and original hybrid SELEX (selective evolution of ligands by exponential enrichment) strategy combining protein-SELEX cycles on the recombinant α5β1 protein, surrounded by cell-SELEX cycles using two different cell lines. We identified aptamer H02, able to differentiate, in cyto- and histofluorescence assays, glioblastoma cell lines, and tissues from patient-derived tumor xenografts according to their α5 expression levels. Aptamer H02 is therefore an interesting tool for glioblastoma tumor characterization., Graphical Abstract

Osteosarcoma (OS) is the most common primary bone cancer, where the overall 5-year surviving rate is below 20% in resistant forms. Accelerating cures for those poor outcome patients remains a challenge. Nevertheless, several studies of agents targeting abnormal cancerous pathways have yielded disappointing results when translated into clinic because of the lack of accurate OS preclinical modeling. So, any effort to design preclinical drug testing may consider all inter-, intra-, and extra-tumoral heterogeneities throughout models mimicking extracellular and immune microenvironment. Therefore, the bioengineering of patient-derived models reproducing the OS heterogeneity, the interaction with tumor-associated macrophages (TAMs), and the modulation of oxygen concentrations additionally to recreation of bone scaffold is proposed here. Eight 2D preclinical models mimicking several OS clinical situations and their TAMs in hypoxic conditions are developed first and, subsequently, the paired 3D models faithfully preserving histological and biological characteristics are generated. It is possible to shape reproducibly M2-like macrophages cultured with all OS patient-derived cell lines in both dimensions. The final 3D models pooling all heterogeneity features are providing accurate proliferation and migration data to understand the mechanisms involved in OS and immune cells/biomatrix interactions and sustained such that engineered 3D preclinical systems will improve personalized medicine.

Simple Summary The EGFR-targeting antibody cetuximab (CTX) combined with radiotherapy has been proven effective for the treatment of locally advanced head and neck squamous cell carcinoma (LA-HNSCC). Due to resistance to CTX, some patients do not benefit from the treatment and recurrence is observed. As caveolin-1 (Cav1) has been reported to affect the EGFR pathway, we aimed to elucidate how it might affect the response to CTX-radiotherapy. We showed that Cav1 expression conferred surviving, growing and motile capacities that protect cells against the combination of CTX-radiotherapy. The protecting effects of Cav1 are mediated by the Cav1/EREG/YAP axis. We also showed in a retrospective study that a high expression of Cav1 was predictive of locoregional relapse of LA-HNSCC. Cav1 should be taken into consideration in the future as a prognosis marker to identify the subgroup of advanced HNSCC at higher risk of recurrence, but also to help clinicians to choose the more appropriate therapeutic strategies. Abstract The EGFR-targeting antibody cetuximab (CTX) combined with radiotherapy is the only targeted therapy that has been proven effective for the treatment of locally advanced head and neck squamous cell carcinoma (LA-HNSCC). Recurrence arises in 50% of patients with HNSCC in the years following treatment. In clinicopathological practice, it is difficult to assign patients to classes of risk because no reliable biomarkers are available to predict the outcome of HPV-unrelated HNSCC. In the present study, we investigated the role of Caveolin-1 (Cav1) in the sensitivity of HNSCC cell lines to CTX-radiotherapy that might predict HNSCC relapse. Ctrl- and Cav-1-overexpressing HNSCC cell lines were exposed to solvent, CTX, or irradiation, or exposed to CTX before irradiation. Growth, clonogenicity, cell cycle progression, apoptosis, metabolism and signaling pathways were analyzed. Cav1 expression was analyzed in 173 tumor samples and correlated to locoregional recurrence and overall survival. We showed that Cav1-overexpressing cells demonstrate better survival capacities and remain proliferative and motile when exposed to CTX-radiotherapy. Resistance is mediated by the Cav1/EREG/YAP axis. Patients whose tumors overexpressed Cav1 experienced regional recurrence a few years after adjuvant radiotherapy ± chemotherapy. Together, our observations suggest that a high expression of Cav1 might be predictive of locoregional relapse of LA-HNSCC.

Simple Summary Pediatric high-grade gliomas are incurable brain tumors for which there is a critical need for new therapeutic strategies as well as treatment-predictive biomarkers. This study examined the expression of DNA repair and cell cycle genes in pediatric high-grade gliomas with distinct driving mutations. The aim is to propose a novel classification of these tumors based on sub-groups exposing therapeutic vulnerabilities. Several DNA repair factors were identified that might become new diagnostic markers. Abstract Background: Pediatric high-grade gliomas (pHGGs) are the leading cause of mortality in pediatric neuro-oncology, displaying frequent resistance to standard therapies. Profiling DNA repair and cell cycle gene expression has recently been proposed as a strategy to classify adult glioblastomas. To improve our understanding of the DNA damage response pathways that operate in pHGGs and the vulnerabilities that these pathways might expose, we sought to identify and characterize a specific DNA repair and cell-cycle gene expression signature of pHGGs. Methods: Transcriptomic analyses were performed to identify a DNA repair and cell-cycle gene expression signature able to discriminate pHGGs (n = 6) from low-grade gliomas (n = 10). This signature was compared to related signatures already established. We used the pHGG signature to explore already transcriptomic datasets of DIPGs and sus-tentorial pHGGs. Finally, we examined the expression of key proteins of the pHGG signature in 21 pHGG diagnostic samples and nine paired relapses. Functional inhibition of one DNA repair factor was carried out in four patients who derived H3.3 K27M mutant cell lines. Results: We identified a 28-gene expression signature of DNA repair and cell cycle that clustered pHGGs cohorts, in particular sus-tentorial locations, in two groups. Differential protein expression levels of PARP1 and XRCC1 were associated to TP53 mutations and TOP2A amplification and linked significantly to the more radioresistant pHGGs displaying the worst outcome. Using patient-derived cell lines, we showed that the PARP-1/XRCC1 expression balance might be correlated with resistance to PARP1 inhibition. Conclusion: We provide evidence that PARP1 overexpression, associated to XRCC1 expression, TP53 mutations, and TOP2A amplification, is a new theranostic and potential therapeutic target.

Simple Summary This review describes in a very detailed and exhaustive approach the literature of these last 20 years on glioblastoma targeted therapies in Phases II-IV of 257 clinical trials on adults with newly diagnosed or recurrent GBMs (excluding targeted immunotherapies and therapies targeting tumor cell metabolism, well documented in recent reviews). Divided in four Sections, are provided descriptions and lists (in 12 different tables) of, not only main but all drugs, targets, clinical trials and the results of targeted therapies when they are known. Abstract Glioblastoma (GBM), the most frequent and aggressive glial tumor, is currently treated as first line by the Stupp protocol, which combines, after surgery, radiotherapy and chemotherapy. For recurrent GBM, in absence of standard treatment or available clinical trials, various protocols including cytotoxic drugs and/or bevacizumab are currently applied. Despite these heavy treatments, the mean overall survival of patients is under 18 months. Many clinical studies are underway. Based on clinicaltrials.org and conducted up to 1 April 2020, this review lists, not only main, but all targeted therapies in phases II-IV of 257 clinical trials on adults with newly diagnosed or recurrent GBMs for the last twenty years. It does not involve targeted immunotherapies and therapies targeting tumor cell metabolism, that are well documented in other reviews. Without surprise, the most frequently reported drugs are those targeting (i) EGFR (40 clinical trials), and more generally tyrosine kinase receptors (85 clinical trials) and (ii) VEGF/VEGFR (75 clinical trials of which 53 involving bevacizumab). But many other targets and drugs are of interest. They are all listed and thoroughly described, on an one-on-one basis, in four sections related to targeting (i) GBM stem cells and stem cell pathways, (ii) the growth autonomy and migration, (iii) the cell cycle and the escape to cell death, (iv) and angiogenesis.

BRAF p.v600e mutation is encountered in brain tumors, mostly low grade pediatric diffuse gliomas (LGG) and epileptogenic glioneuronal tumors such as gangliogliomas (GG) or pleomorphic xanthoastrocytomas (PXA). Less frequently this mutation is present in high grade glial or glioneuronal tumors such as pleomorphic xanthoastrocytomas with anaplasia, anaplastic ganglioglioma, anaplastic diffuse astrocytomas or glioblastomas. Recently, few publications were highlighting differently the impact of BRAF mutation and CDKN2A deletion, as independent prognostic factors linked to a worst outcome in low grade forms. We studied retrospectively a monocentric cohort of 17 LGGs (14 GG and 3 pilocytic astrocytomas) and 7 BRAF pv600e HGG. The patients were aged below 20 years. We focused on extended tumors’ biology assessment (MethylEpic 850K, Next-Generation sequencing, RNA sequencing and metabolomics), as well as tumor immune microenvironment by immunohistochemistry. Among the LGGs, only one had a CDKN2A deletion and one a gain on chromosome 5. All except two LGGs had a complete surgical resection. Four of them were treated by chemotherapies but underwent relapses. All HGGs had a surgical resection followed by a first line chemotherapy (mainly Stupp protocol) and radiotherapy. Five patients relapsed rapidly, benefiting from targeted therapy with vemurafenib and/or biotherapy associating dabrafenib plus trametinib. Among those HGGs, we had both subgroups: “de novo” tumors and patients with a history of LGG tumors. Both were responding well to targeted treatments. The biology uncovers in all HGGs a loss of CDKN2A gene and/or protein. Additionally to this gene abnormality, specific transcriptomic expressions were associated to therapeutic response and immune microenvironment. Epigenetic modulation was linked to specific metabolic switches when BRAF p.v600e gliomas were getting higher grade features (e.g., glutaminolysis, serinolysis and phospholipidic metabolism). Those characteristics seem to be able to predict in LGG p.v600e potential evolution.

Pediatric midline high-grade gliomas (pmHGG) are aggressive and incurable tumors of the central nervous system. There is a pressing need for novel therapeutic approaches to treat them. Therefore, proactive translational studies wish to go further discovering new targetable proteins and pathways. Our objectives are then to focus on the modulation of microenvironmental extrinsic features like intra-tumor hypoxia. To do so, we looked first on expressions of hypoxia biomarkers in a pool of patient-derived preclinical models of pmHGG and tested oxygen modulations, as well as hypoxia drug targeting. We designed subsequently our work in those models H3.3 mutated to evaluate balance between HIF1 and HIF2 expressions (immunofluorescence, RTqPCR, RNAseq and metabolomics) and to evidence the impact of hypoxia targeting combined to irradiation on cell proliferation, migration and metabolism. Hypoxia is inducing mainly HIF1 expression and its upstream and downstream pathways and is stabilizing HIF2 expression. Both HIFs are part of crucial survival signaling and represent targets to combine with irradiation. The use of their specific inhibitors shows an antiproliferative effect when HIF1 is downregulated. HIF2 inhibitors are stopping HIF2 transcriptional effect letting us uncover new pathways that this hypoxic inducible factor is regulating in pmHGG (stemness, glycolytic and aminoacid metabolism and histone expression). Together with irradiation this anti-hypoxic strategy seems to be highly effective on cell arrest and migration. Those results are confirming central roles of HIFs in pmHGG and their potencies in pmHGG therapies. The therapeutic efficiency is independent from p53 abnormalities in our models.

Integrins are heterodimeric transmembrane proteins able to connect cells with the micro-environment. They represent a family of receptors involved in almost all the hallmarks of cancer. Integrins recognizing the Arg-Gly-Asp (RGD) peptide in their natural extracellular matrix ligands have been particularly investigated as tumoral therapeutic targets. In the last 30 years, intense research has been dedicated to designing specific RGD-like ligands able to discriminate selectively the different RGD-recognizing integrins. Chemists' efforts have led to the proposition of modified peptide or peptidomimetic libraries to be used for tumor targeting and/or tumor imaging. Here we review, from the biological point of view, the rationale underlying the need to clearly delineate each RGD-integrin subtype by selective tools. We describe the complex roles of RGD-integrins (mainly the most studied αvβ3 and α5β1 integrins) in tumors, the steps towards selective ligands and the current usefulness of such ligands. Although the impact of integrins in cancer is well acknowledged, the biological characteristics of each integrin subtype in a specific tumor are far from being completely resolved. Selective ligands might help us to reconsider integrins as therapeutic targets in specific clinical settings.

International audience; Osteosarcoma is the most frequent primary bone tumor diagnosed during adolescence and young adulthood. It is associated with the worst outcomes in the case of poor response to chemotherapy and in metastatic disease. While no molecular biomarkers are clearly and currently associated with those worse situations, the study of pathways involved in the high level of tumor necrosis and in the immune/metabolic intra-tumor environment seems to be a way to understand these resistant and progressive osteosarcomas. In this review, we provide an updated overview of the role of hypoxia in osteosarcoma oncogenesis, progression and during treatment. We describe the role of normoxic/hypoxic environment in normal tissues, bones and osteosarcomas to understand their role and to estimate their druggability. We focus particularly on the role of intra-tumor hypoxia in osteosarcoma cell resistance to treatments and its impact in its endogenous immune component. Together, these previously published observations conduct us to present potential perspectives on the use of therapies targeting hypoxia pathways. These therapies could afford new treatment approaches in this bone cancer. Nevertheless, to study the osteosarcoma cell druggability, we now need specific in vitro models closely mimicking the tumor, its intra-tumor hypoxia and the immune microenvironment to more accurately predict treatment efficacy and be complementary to mouse models.

Background: Osteosarcomas (OTS) represent the most common primary bone cancer diagnosed in adolescents and young adults. Despite remarkable advances, there are no objective molecular or imaging markers able to predict an OTS outcome at diagnosis. Focusing on biomarkers contributing broadly to treatment resistance, we examine the interplay between the tumor-associated macrophages and intra-tumor hypoxia. Methods: Radiological and immunohistochemical (IHC) data were correlated with the outcome in a retrospective and monocentric cohort of 30 pediatric OTS. We studied hypoxic (pS6, phospho-mTor, HIF-1α and carbonic anhydrase IX (CAIX)) and macrophagic (CD68 and CD163) biomarkers. Results: The imaging analyses were based on MRI manual volumetric measures on axial post-contrast T1 weighted images, where, for each tumor, we determined the necrotic volume and its ratio to the entire tumor volume. When they were above 50 cm3 and 20%, respectively, they correlated with a worse overall survival (p = 0.0072 and p = 0.0136, respectively) and event-free survival (p = 0.0059 and p = 0.0143, respectively). IHC assessments enable a significant statistical link between HIF-1α/CAIX hyper-expressions, CD68+ cells and a worse outcome, whereas activation of mTor pathway was linked to a better survival rate and CD163+ cells. Conclusions: This study evidenced the links between hypoxia and immunity in OTS, as their poor outcome may be related to a larger necrotic volume on diagnostic MRI and, in biopsies, to a specific IHC profile.

Basal cell carcinoma (BCC) is the most frequent human cancer and is becoming an important health problem in an aging population. Based on their clinical and histological characteristics, thick BCC are typically divided into low-risk nodular and high-risk infiltrative subtypes, although the underlying mechanisms are poorly understood. We have identified molecular mechanisms that explain the aggressiveness of high-risk infiltrative BCC, with a potential direct clinical impact. In this study, we first show that fibroblasts, transforming growth factor-β, and fibronectin are found preferentially in infiltrative human BCC. This allowed us to develop in vivo models for the study of infiltrative BCC, which in turn let us confirm the role of transforming growth factor-β in inducing peritumoral fibronectin deposition and tumor infiltration. We then show that fibronectin promotes adhesion and migration of BCC cell lines through integrin α5β1-mediated phosphorylation of focal adhesion kinase. Fittingly, both inhibition of integrin α5β1 and phospho-focal adhesion kinase prevent fibronectin-induced migration of BCC cells in vitro as well as BCC infiltration in vivo. Altogether, our results open important insights into the pathogenesis of aggressive infiltrative BCC and identify integrin α5β1 or focal adhesion kinase inhibition as promising strategies for the treatment of advanced BCC.

Over the last decades, several techniques have been developed to study cell adhesion, however they present significant shortcomings. Such techniques mostly focus on strong adhesion related to specific protein-protein associations, such as ligand/receptor binding in focal adhesions. Therefore, weak adhesion, related to less specific or non-specific cell/substrate interactions, are rarely addressed. Hence, we propose in this paper a complete investigation of cell adhesion, from highly specific to non-specific adhesiveness, using variable-angle Total Internal Reflection Fluorescence (vaTIRF) nanoscopy. This technique allows us to map in real time, cell topography with a nanometric axial resolution, along with cell cortex refractive index. These two key parameters allow us to distinguish high and low adhesive cell/substrate contacts. Furthermore, vaTIRF provides cell/substrate binding energy; thus, revealing a correlation between cell contractility and cell/substrate binding energy. Here, we highlight the quantitative measurements achieved by vaTIRF on U87MG glioma cells expressing different amounts of α5 integrins, and distinct motility on fibronectin. Regarding integrins expression level, data extracted from vaTIRF measurements, such as the number and size of high adhesive contacts per cell, corroborate the adhesiveness of U87MG cells as intended. Interestingly enough, we found that cells overexpressing α5 integrins, present a higher contractility and lower adhesion energy.

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.

BRAFv600e mutation is encountered mostly in low-grade pediatric gliomas (LGG) and epileptogenic glioneuronal tumors, such as gangliogliomas (GG). Less frequently this mutation is present in high-grade glial (HGG) or glioneuronal tumors. Recent publications were highlighting BRAF mutation and CDKN2A deletion, as independent prognostic factors linked to a worst outcome in LGGs. We studied retrospectively a monocentric cohort of 12 LGGs (9 GG and 3 pilocytic astrocytomas) and 9 HGG (5 “de novo” tumors and 4 with a long past of LGG evolution) with BRAFv600e positivity. The patients were aged from 1 to 47 years. LGGs were under 20 years and only 3 patients with HGGs had less than 18 years. We focused on extended tumors’ biology assessment by DNA single-cell analyses, RNAsequencing, NGS, metabolomics, radiology (MRI, PET-scanning and spectroscopy) and correlated them to tumor’s data. One LGG had a CDKN2A deletion. Six had a complete surgical resection, 2 had a minimal residue and 4 had chemotherapies after partial surgery and relapsed. All HGGs had a surgical resection followed by chemotherapy and radiotherapy and additional CDKN2A deletion. Two pediatric HGGs relapsed rapidly. Only one benefited positively from targeted therapy. Specific radiological and spectroscopic signs were linked to the BRAF mutation itself and those different groups (LGGs, HGGs and LGGs with long term evolution of HGG), where specific molecular pathways and metabolomic profiles are associated. Currently, we are going further in the correlations to be able to predict in LGG their potential long-term evolution, where MAPK pathway modulations might be involved.

Background: Pediatric high-grade gliomas (pHGGs) are facing a very dismal prognosis and representative pre-clinical models are needed for new treatment strategies. Here, we examined the relevance of collecting functional, genomic, and metabolomics data to validate patient-derived models in a hypoxic microenvironment. Methods: From our biobank of pediatric brain tumor-derived models, we selected 11 pHGGs driven by the histone H3.3K28M mutation. We compared the features of four patient tumors to their paired cell lines and mouse xenografts using NGS (next generation sequencing), aCGH (array comparative genomic hybridization), RNA sequencing, WES (whole exome sequencing), immunocytochemistry, and HRMAS (high resolution magic angle spinning) spectroscopy. We developed a multicellular in vitro model of cell migration to mimic the brain hypoxic microenvironment. The live cell technology Incucyte© was used to assess drug responsiveness in variable oxygen conditions. Results: The concurrent 2D and 3D cultures generated from the same tumor sample exhibited divergent but complementary features, recreating the patient intra-tumor complexity. Genomic and metabolomic data described the metabolic changes during pHGG progression and supported hypoxia as an important key to preserve the tumor metabolism in vitro and cell dissemination present in patients. The neurosphere features preserved tumor development and sensitivity to treatment. Conclusion: We proposed a novel multistep work for the development and validation of patient-derived models, considering the immature and differentiated content and the tumor microenvironment of pHGGs.

BACKGROUND Integrin α5β1 was suggested to be involved in glioblastoma (GBM) aggressiveness through preclinical studies and genomic analysis of several cohorts of patients. However, protein expression data are still missing to confirm this hypothesis. Our aim was to investigate the prognostic value of integrin α5 protein expression level in GBM. MATERIAL AND METHODS We retrospectively determined the protein expression level of integrin α5 using immunochemistry in tumors from patients treated in 6 French centers. Paraffin sections of GBM were labeled by immunofluorescence and analyzed by confocal microscopy. The corresponding clinical and survival data have been identified and analyzed. The primary end-point was overall survival (OS). RESULTS Out of 297 patients newly diagnosed with GBM between 2006 and 2013, 152 met the inclusion criteria (scheduled for initial treatment with the Stupp protocol, age > 18 years) and 95 tumor samples were suitable for immunohistochemical analysis. The median age is 58 years, (64 men, 34 women). Most of patients received macroscopic (43%) or partial (36%) surgery. In univariate analysis using the Log Rank test, high integrin α5 expression level was associated with poor prognosis (PFS: hazard ratio (HR) = 1,696, p=0,0355; OS: HR=1,598, p = 0,0508). Corresponding median OS were 15,6 versus 19,2 months. Similarly, OS was significantly reduced with age (> 60 years), lower resection degree, higher RPA (recursive partitioning analysis) score and non-methylated MGMT (O-6-methylguanine-DNA methyltransferase) promoter. In the subgroup of patients who received the full initial protocol (temozolomide treatment together with radiotherapy and later as adjuvant treatment; n=58) mean OS was strongly reduced when integrin α5 expression level was high (15,6 versus 22,8 months, p=0,0162) suggesting an impact of integrin signaling on temozolomide response in GBM. CONCLUSION Our study validates for the first time that the high protein level expression of α5 integrin is associated with poor prognosis in GBM. It also confirms its potential as a therapeutic target and its likely role in resistance to temozolomide as previously shown in preclinical study.

Gliomas are the most common type of pediatric brain tumors. One third of them are high grade gliomas (pHGG) with a significant dismal prognosis and a very poor therapeutic arsenal. So, new therapeutic ideas are needed to counterpart the high resistance to current treatments. Therefore, our team focused recently on the glutaminolysis switch, which was previously described in our work targeting mTor and HIF1 in pHGG cell lines. Therefore, to go further and understand its role in pHGG, we started with metabolomics analyses in our monocentric cohort of 34 pediatric high grade gliomas (14 DIPG, 10 thalamic and 10 sus-tentoriel high grade gliomas). First, we were testing 14 appropriate fresh-frozen samples at diagnosis for metabolomics and used the 34 FFPE specimens for validations. A second step of in vitro experimentation was performed to target specifically the different metabotropic glutamate receptors (mGluR) involved in pHGG glutamine metabolism. We used 6 HGG cell lines mutated or not for H3.3 K27M variant. In terms of results, metabolomics, done by HRMAS NMR, confirmed in tumors and in cell lines a high level of glutaminolysis, which is currently confirmed in all tumors by immunohistochemistry. This specific metabolic switch was also significantly observed by RNAsequencing analyses in our primary derived cell lines cultured in hypoxic conditions. Multiple genes, like mGluR8, mGluR2/3 and several subunits of the glutamate ionotropic receptors, were highly expressed. The in vitro blockade with mGluR2/3 NAM showed, finally, in our cell lines a significant decrease of proliferation, when using them as a single drug therapy, but also in association with temozolomide. This first in vitro work will be confirmed in our PDX mouse models of pHGG. Nevertheless, taking together, these promising results seem to be a scientifically-based rationale for testing mGluR antagonists as adjuvant therapy in pHGGs.

International audience; Integrins contribute to cancer progression and aggressiveness by activating intracellular signal transduction pathways and transducing mechanical tension forces. Remarkably, these adhesion receptors share common signaling networks with receptor tyrosine kinases (RTKs) and support their oncogenic activity, thereby promoting cancer cell proliferation, survival and invasion. During the last decade, preclinical studies have revealed that integrins play an important role in resistance to therapies targeting RTKs and their downstream pathways. A remarkable feature of integrins is their wide-ranging interconnection with RTKs, which helps cancer cells to adapt and better survive therapeutic treatments. In this context, we should consider not only the integrins expressed in cancer cells but also those expressed in stromal cells, since these can mechanically increase the rigidity of the tumor microenvironment and confer resistance to treatment. This review presents some of these mechanisms and outlines new treatment options for improving the efficacy of therapies targeting RTK signaling.

Pediatric high-grade gliomas (pHGGs), including diffuse intrinsic pontine gliomas (DIPGs), despite their low incidence, are the leading cause of mortality in pediatric neuro-oncology. Frequently, pHGGs, harboring mainly histone mutations, are or become resistant to standard therapies like irradiation or chemotherapies. Recent insights showed in adult HGG the predominant role of DNA damage repair (DDR) as a way of prognostic classification. Given the recent evidence that transcription conflicts like in histone-mutated gliomas can induce replication stress and be linked to DDR abnormalities, this study is aiming to establish a DDR signature able to classify specifically pHGGs and to cluster among them poor responders to radiation. Transcriptomic analyses were performed to discriminate seven pHGGs comparatively to a cohort of 10 pilocytic astrocytomas with specific DDR deregulations. The specific transcriptomic signature obtained from this differential gene expression analysis was compared to the aHGG signature already established. To strengthen, refine and finalize the DDR signature able to classify and cluster the pHGGs, we explored both signatures and their common genes in already published transcriptomic analyses of DIPGs and sus-tentorial pHGGs. To check DDR protein expressions correlated to loss of trimethylation as well as histone and TP53 mutations, an immunohistochemical assessment of several DDR markers was performed on a collection of 21 pHGG diagnostic samples and 9 paired relapses. To validate the DDR functional inhibition, we used 3 patient-derived cell lines bearing H3.3K27M mutations. A finalized signature of 28 genes involved in DNA repair and cell cycle machineries was used to cluster in two groups the pHGG cohorts. The differential protein expression of PARP1, XRCC1, p53 and stem cell markers was linked significantly to the more resistant pHGGs and the rapid progressions after radiotherapy. Those DDR makers might be used as theranostic and therapeutic targets, which were screened in PDCLs with promising results.

// Guillaume Renner 1 , Fanny Noulet 1 , Marie-Cecile Mercier 1 , Laurence Choulier 1 , Nelly Etienne-Selloum 1 , Jean-Pierre Gies 1 , Maxime Lehmann 1 , Isabelle Lelong-Rebel 1 , Sophie Martin 1 , Monique Dontenwill 1 1 UMR7213 CNRS, Team Tumoral Signaling and Therapeutic Targets, Universite de Strasbourg, Faculte de Pharmacie, Illkirch, France Correspondence to: Monique Dontenwill, email: monique.dontenwill@unistra.fr Keywords: α5β1 integrin, beta-catenin, migration, EMT, glioma Received: April 13, 2016 Accepted: August 10, 2016 Published: August 23, 2016 ABSTRACT The Wnt/beta catenin pathway has been highlighted as an important player of brain tumors aggressiveness and resistance to therapies. Increasing knowledges of the regulation of beta-catenin transactivation point out its hub position in different pathophysiological outcomes in glioma such as survival and migration. Crosstalks between integrins and beta-catenin pathways have been suggested in several tumor tissues. As we demonstrated earlier that α5β1 integrin may be considered as a therapeutic target in high grade glioma through its contribution to glioma cell migration and resistance to chemotherapy, we addressed here the potential relationship between α5β1 integrin and beta-catenin activation in glioma cells. We demonstrated that overexpression and activation by fibronectin of α5β1 integrin allowed the transactivation of beta-catenin gene targets included in an EMT-like program that induced an increase in cell migration. Hampering of beta catenin activation and cell migration could be similarly achieved by a specific integrin antagonist. In addition we showed that α5β1 integrin/AKT axis is mainly involved in these processes. However, blockade of beta-catenin by XAV939 (tankyrase inhibitor leading to beta-catenin degradation) did not synergize with p53 activation aiming to cell apoptosis as was the case with integrin antagonists. We therefore propose a dual implication of α5β1 integrin/AKT axis in glioma cell resistance to therapies and migration each supported by different signaling pathways. Our data thus suggest that α5β1 integrin may be added to the growing list of beta-catenin modulators and provide new evidences to assign this integrin as a valuable target to fight high grade glioma.

Pediatric high-grade gliomas (pHGGs) have a very dismal prognosis and need new innovative strategy for treatment. Despite the past discovery of histone H3 driver mutations, we are not able for instance to stop this induced epigenetic remodulation. Therefore, proactive translational studies wish to go further discovering new targetable proteins in pHGG. In our past clinical work, we were able to link significantly HIF-2alpha to a worse pHGG outcome and to their treatment resistance. We designed this new work to determine in several patient-derived cell lines (6 PDCLs) with or without H3.3 mutation the variation of HIF-2alpha, its role, its induction in normoxic and hypoxic microenvironment and its transcriptional targets using RNAseq, metabolomics and ChipSeq analyses. Complementary functional analyses were performed using siRNA strategy during cultures and migration assays. Finally, preclinical drug testing involving commercialized and non-commercialized HIF-2alpha specific inhibitors in the same PDCLs were evaluating their antiproliferative and pro-apoptotic effect. Our results confirmed the central role of HIF-2alpha in cell resistance to treatment, in pHGG stemness features and its direct link with metabolism adaptation and histone interaction. After the confirmation of its frequent presence in multiple PDCLs initiated from thalamic pHGGs and DIPG, we were using inhibitors in a single and combinatorial strategy targeting HIF-2alpha plus another hypoxia biomarker (mTor). This preclinical targeting was highly effective to favor cell arrest, apoptosis and to stop cell migration. In conclusion, HIF-2alpha seem to be a major biomarker in pHGGs that might be targeted giving a useful new opportunity for pHGG treatments.

Osteosarcoma is the most common primary malignant bone tumor in children and young adults. Its immune environment was described as predominantly involving M2 macrophages or osteoclasts. Previous studies were observing ganglioside GD2 expression on the osteosarcoma cell surface. Even more and more publications are deciphering the role of hypoxic biomarkers and its metabolic cell impact. Nevertheless, no links were, for instance, described clearly between mTor-HIF1 pathway and the macrophages in osteosarcomas through GD2 targeting. The aim of the study was to understand the role of all those molecular partners in osteosarcoma models. Material and methods: A cohort of 35 patients was explored to determine the expression by immunohistochemistry, as well as semi-quantitative PCR of several hypoxic and macrophages' biomarkers. Additionally, in 2D and 3D in vitro osteosarcoma models, analyses using metabolomics, western blotting, immunofluorescent imaging and RNA sequencing were performed to understand functional interactions between patient-derived osteosarcoma cells and the M2 macrophages. Endly, drug screening studies were testing 3D models (integrating matrix and hypoxic microenvironment) to assess efficacy of combinations using dinutuximab, mTor and HIF inhibitors. Results: Osteosarcoma samples are highly expressing CD163 and CD68 macrophage markers, as well as activated mTORC1 and GD2. Hypoxic biomarkers, when overexpressed, were statistically linked to a worst patient outcome. For macrophages and GD2 markers, a low expression was mostly linked to metastatic disease or poor response to chemotherapy. Specific metabolomic changes through glutaminolysis were frequently present and associated to a transcriptomic signature combining metabolism changes and specific immune macrophage environment. The targeting by dinutuximab associated to hypoxia inhibition was having a clear effect on osteosarcoma cell proliferation decrease, when associated to macrophages in 3D models mimicking osteosarcoma tumors. Conclusion: Macrophage immune environment is a key component of pediatric osteosarcomas and can be targeted in association with hypoxia inhibitors in 3D models recreating the osteosarcoma immune environment and bone matrix. Citation Format: Marina Pierrevelcin, Isabelle Lelong-Rebel, Quentin Fuchs, Monique Dontenwill, Natacha Entz-Werle. Integrative analyses in pediatric osteosarcomas to understand the role of GD2 associated to activated mTor for macrophage targeting [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4998.

Pediatric high-grade gliomas (pHGGs) represent a very dismal disease that is needing new innovative compound for treatment. Despite the past discovery of histone H3 driver mutations, we are not able for instance to stop this induced process on epigenetic remodulation. Therefore, there is since a decade proactive works aimed to discover new targetable proteins in these tumors. In our recent previous works in pHGGs, we highlighted HIF2alpha as a biomarker of worse prognosis and outcome and a key in treatment resistance. Therefore, this new project was designed to determine in several patient-derived cell lines (5 PDCLs) the presence of HIF2alpha, its role and its induction in normoxic and hypoxic microenvironment concomitantly by immunofluorescence and Western blot assessments and RNA sequencing analyses. Complementary ChipSeq was also performed using HIF2 antibodies to determine its role as a transmission factor. Specific promoters were involved. After the confirmation of its frequent presence in multiple PDCLs initiated from thalamic pHGGs and DIPG, we used allosteric inhibitors to target HIF2alpha. Surprisingly, this protein was expressed constantly in hypoxic and normoxic conditions and specifically in PDCLs bearing stem cell features. Specific expression associations were established between stemness markers and HIF2alpha. To go further, we tested specific HIF2A inhibitors, which were having an impact on cell proliferation and on the decrease of the target itself. In conclusion, HIF2 seem to be a major biomarker in pHGGs that might be targeted, and it is a useful new opportunity for pHGGs treatments. Citation Format: Quentin Fuchs, Anne Florence Blandin, Isabelle Lelong Rebel, Marina Pierrevelcin, Monique Dontenwill, Natacha Entz-Werlé. HIF2 in pediatric high-grade glioma and its targeting [abstract]. In: Proceedings of the AACR Special Conference on the Advances in Pediatric Cancer Research; 2019 Sep 17-20; Montreal, QC, Canada. Philadelphia (PA): AACR; Cancer Res 2020;80(14 Suppl):Abstract nr A33.