Abstract Medulloblastoma (MB) is the most common malignant brain tumor in children, and is classified into four genetic subgroups: WNT, SHH, Group 3 and Group 4. These subgroups are driven by major canonical driver genes (CDG). The OTX2 (Orthodenticle homeobox 2) oncogene is overexpressed or amplified in Group 3 and 4 MBs promoting self-renewal and tumor growth leading to enhanced tumorigenesis. Leptomeningeal disease/metastasis (LMD) is a hallmark of medulloblastomas. Despite several comprehensive studies on MB tumor development through CDG, understanding of the molecular mechanism(s) of LMD and possible contributions of the MB CDG on the development of LMD has been particularly limited. In the present study, we therefore performed gain-of-function experiments to investigate the possible contribution of the OTX2 gene in LMD metastasis. Using a human MB patient-derived Group 3 relevant cell line called D425 and transducing this cell line with an OTX2 gene-carrying lentivirus vector, we found that OTX2OE-D425 cell motility is ~2 fold higher (0.4891 um^2/min, 0.2244 um^2/min; p<0.0001), and displays enhanced polarization compared to control cells on a 4.6 kPa polyacrylamide hydrogel (PAG) coated with collagen type I. In addition, OTX2OE-D425 cells moved faster than control cells on mouse cerebellum slices (0.01981um^2/min vs 0.01489 um^2/min, p<0.00001). We next implanted the human-MB D425 cell line overexpressing the OTX2 gene into the cisterna magna of immune-deficient nude mice and observed that these mice had a lower median survival than that of the vehicle group (20 days vs. 33 days, log-rank test, p<0.00014). Moreover, we found that the mice implanted with OTX2OE-D425 cells also developed spinal cord and brain metastases, whereas almost no metastases were observed in the control cell-implanted mouse group. Taken together, our data indicate that the overexpression of OTX2 gene induces tumor growth and triggers spinal and brain metastases in Group 3 MB. In order to investigate the molecular mechanism by which the OTX2 gene regulates metastasis in MB, we performed bulk RNA sequencing studies and found that the overexpression of OTX2 gene activates the mTOR signaling pathway in D425 cells. Next, we treated MB cells with two mTOR inhibitors, AZ8055 and PQR620, and found a significant reduction in cell viability of D425 cells compared to control solvent (DMSO) treated cells. We are currently investigating the possible effect of PQR620 (that can pass through the Blood Brain Barrier) on our orthotopic MB mouse model. Citation Format: Elisabet Ampudia Mesias, Charles Scott Cameron, Sarah Anderson, Juan E. Abrahante Lloréns, Christopher L. Moertel, David J. Odde, Okay Saydam. The OTX2 gene induces tumor growth and triggers leptomeningeal metastasis by regulating the mTOR signaling pathway in medulloblastomas [abstract]. In: Proceedings of the AACR Special Conference on Brain Cancer; 2023 Oct 19-22; Minneapolis, Minnesota. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_1):Abstract nr B003.

INTRODUCTION: The role of resection for recurrent glioblastoma remains unclear. METHODS: Patients who underwent repeat resection for recurrent IDH-wild-type glioblastoma were prospectively matched with 1:1 control cohort (n = 30:30). The two groups signed the consent and were matched for age, gender, comorbidities, eloquent vs. non-eloquent location, preoperative tumor volume and extent of resection in first surgery, neuropathological features as in WHO 2021 classification, time to progression, tumor volume at the time of recurrence, Park Score, ventricular wall involvement, use of steroids or multiple antiseizure medications and adjuvant therapy. Primary aim was to evaluate the overall survival in two cohorts. The secondary aim was to determine predictive factors for long term survival after repeat resection. RESULTS: After matching, patients had a long term follow up until death. Median overall survival times for patients who underwent repeat and one resection was 23.8 months vs 9.1 months, respectively (Log-Rank p < 0.001). Two predictive factors for long term survival after repeat resection were preoperative Karnofsky Performance Score >70 and Glasgow Coma Scale >13 (Fisher p = 0.07 and p < 0.001, respectively) while age, time to surgery, preoperative volume, extend of resection in repeat resection, location of tumor, its extension to ventricular wall, ependymal enhancement, comorbidities, tumor histological and molecular features did not correlate with postoperative survival. CONCLUSIONS: This is the first prospective matched case-control study revealing survival benefit of repeat resection for recurrent gliomas with long term follow up in a select group.

Medulloblastoma (MB) encompasses diverse subgroups, and leptomeningeal disease/metastasis (LMD) plays a substantial role in associated fatalities. Despite extensive exploration of canonical genes in MB, the molecular mechanisms underlying LMD and the involvement of the orthodenticle homeobox 2 (OTX2) gene, a key driver in aggressive MB Group 3, remain insufficiently understood. Recognizing OTX2’s pivotal role, we investigated its potential as a catalyst for aggressive cellular behaviors, including migration, invasion, and metastasis. OTX2 overexpression heightened cell growth, motility, and polarization in Group 3 MB cells. Orthotopic implantation of OTX2-overexpressing cells in mice led to reduced median survival, accompanied by the development of spinal cord and brain metastases. Mechanistically, OTX2 acted as a transcriptional activator of the Mechanistic Target of Rapamycin (mTOR) gene’s promoter and the mTORC2 signaling pathway, correlating with upregulated downstream genes that orchestrate cell motility and migration. Knockdown of mTOR mRNA mitigated OTX2-mediated enhancements in cell motility and polarization. Analysis of human MB tumor samples (N = 952) revealed a positive correlation between OTX2 and mTOR mRNA expression, emphasizing the clinical significance of OTX2’s role in the mTORC2 pathway. Our results reveal that OTX2 governs the mTORC2 signaling pathway, instigating LMD in Group 3 MBs and offering insights into potential therapeutic avenues through mTORC2 inhibition.

Glioblastoma is one of the most devastating neoplasms of the central nervous system. This study focused on the development of serum extracellular vesicle (EV)-based glioblastoma tumor marker panels that can be used in a clinic to diagnose glioblastomas and to monitor tumor burden, progression, and regression in response to treatment. RNA sequencing studies were performed using RNA isolated from serum EVs from both patients (n = 85) and control donors (n = 31). RNA sequencing results for preoperative glioblastoma EVs compared to control EVs revealed 569 differentially expressed genes (DEGs, 2XFC, FDR < 0.05). By using these DEGs, we developed serum-EV-based biomarker panels for the following glioblastomas: wild-type IDH1 (96% sensitivity/80% specificity), MGMT promoter methylation (91% sensitivity/73% specificity), p53 gene mutation (100% sensitivity/89% specificity), and TERT promoter mutation (89% sensitivity/100% specificity). This is the first study showing that serum-EV-based biomarker panels can be used to diagnose glioblastomas with a high sensitivity and specificity.

Studies on tumor-associated antigens in brain tumors are sparse. There is scope for enhancing our understanding of molecular pathology, in order to improve on existing forms, and discover new forms, of treatment, which could be particularly relevant to immuno-oncological strategies. To elucidate immunological differences, and to provide another level of biological information, we performed antibody profiling, based on a high-density protein array (containing 8173 human transcripts), using IgG isolated from the sera of n = 12 preoperative and n = 16 postoperative glioblastomas, n = 26 preoperative and n = 29 postoperative meningiomas, and n = 27 healthy, cancer-free controls. Differentially reactive antigens were compared to gene expression data from an alternate public GBM data set from OncoDB, and were analyzed using the Reactome pathway browser. Protein array analysis identified approximately 350–800 differentially reactive antigens, and revealed different antigen profiles in the glioblastomas and meningiomas, with approximately 20–30%-similar and 10–15%-similar antigens in preoperative and postoperative sera, respectively. Seroreactivity did not correlate with OncoDB-derived gene expression. Antigens in the preoperative glioblastoma sera were enriched for signaling pathways, such as signaling by Rho-GTPases, COPI-mediated anterograde transport and vesicle-mediated transport, while the infectious disease, SRP-dependent membrane targeting cotranslational proteins were enriched in the meningiomas. The pre-vs. postoperative seroreactivity in the glioblastomas was enriched for antigens, e.g., platelet degranulation and metabolism of lipid pathways; in the meningiomas, the antigens were enriched in infectious diseases, metabolism of amino acids and derivatives, and cell cycle. Antibody profiling in both tumor entities elucidated several hundred antigens and characteristic signaling pathways that may provide new insights into molecular pathology and may be of interest for the development of new treatment strategies.

Supplementary Table 1 from miRNA-7 Attenuation in Schwannoma Tumors Stimulates Growth by Upregulating Three Oncogenic Signaling Pathways

Supplementary Methods, Figure Legends 1-5 from miRNA-7 Attenuation in Schwannoma Tumors Stimulates Growth by Upregulating Three Oncogenic Signaling Pathways

Extracellular vesicles (EVs) may be used as a non-invasive screening platform to discover markers associated with early diagnosis, prognosis, and treatment response. Such an approach is invaluable for diseases such as glioblastoma, for which only a few non-invasive diagnostic or prognostic markers are available. We used mass spectrometry to analyze proteomics profiles of EVs derived from four glioblastoma cell lines and human primary astrocytes (HPAs) and found that SRPX is the only protein enriched in the majority of glioblastoma EVs that was absent in the HPA-derived EVs. Then, we evaluated the relationship between SRPX protein expression and tumor grade using immunohistochemical staining (IHC) and performed colony formation and viability assays to analyze the possible function of SRPX in glioblastoma. SRPX mRNA and protein expression were associated with tumor grade. Moreover, temozolomide (TMZ)-resistant tumor tissues showed highly positive SRPX staining, compared to all other tumor grades. Additionally, glioblastoma cells displayed enhanced SRPX gene expression when exposed to TMZ. Knockdown of SRPX gene expression via siRNA inhibited cell viability. Taken together, the results of this study suggest that SRPX can be used as a novel tumor marker for diagnostic and prognostic purposes and can also be a therapeutic target for glioblastomas.

Cancer metastasis is the major cause of death from cancer (Massague and Obenauf, 2016; Steeg, 2016). The extensive genetic heterogeneity and cellular plasticity of metastatic tumors set a prime barrier for the current cancer treatment protocols (Boumahdi and de Sauvage, 2020). In addition, acquired therapy resistance has become an insurmountable obstacle that abolishes the beneficial effects of numerous anti-cancer regimens (De Angelis et al., 2019; Boumahdi and de Sauvage, 2020). Here we report that deficiency of Ku leads to the exploitation of host cells in human cancer cell line models. We found that, upon conditional deletion of XRCC6 that codes for Ku70, HCT116 human colorectal cancer cells gain a parasitic lifestyle that is characterized by the continuous cycle of host cell exploitation. We also found that DAOY cells, a human medulloblastoma cell line, innately lack nuclear Ku70/Ku86 proteins and utilize the host-cell invasion/exit mechanism for maintenance of their survival, similarly to the Ku70 conditionally-null HCT116 cells. Our study demonstrates that a functional loss of Ku protein promotes an adaptive, opportunistic switch to a parasitic lifestyle in human cancer cells, providing evidence for a previously unknown mechanism of cell survival in response to severe genomic stress. We anticipate that our study will bring a new perspective for understanding the mechanisms of cancer cell evolution, leading to a shift in the current concepts of cancer therapy protocols directed to the prevention of cancer metastasis and therapy resistance.

Exosomes are small extracellular vesicles (sEVs), playing a crucial role in the intercellular communication in physiological as well as pathological processes. Here, we aimed to study whether the melanoma-derived sEV-mediated communication could adapt to microenvironmental stresses. We compared B16F1 cell-derived sEVs released under normal and stress conditions, including cytostatic, heat and oxidative stress. The miRNome and proteome showed substantial differences across the sEV groups and bioinformatics analysis of the obtained data by the Ingenuity Pathway Analysis also revealed significant functional differences. The in silico predicted functional alterations of sEVs were validated by in vitro assays. For instance, melanoma-derived sEVs elicited by oxidative stress increased Ki-67 expression of mesenchymal stem cells (MSCs); cytostatic stress-resulted sEVs facilitated melanoma cell migration; all sEV groups supported microtissue generation of MSC-B16F1 co-cultures in a 3D tumour matrix model. Based on this study, we concluded that (i) molecular patterns of tumour-derived sEVs, dictated by the microenvironmental conditions, resulted in specific response patterns in the recipient cells; (ii) in silico analyses could be useful tools to predict different stress responses; (iii) alteration of the sEV-mediated communication of tumour cells might be a therapy-induced host response, with a potential influence on treatment efficacy.

Meningiomas are primary central nervous system (CNS) tumors that originate from the arachnoid cells of the meninges. Recurrence occurs in higher grade meningiomas and a small subset of Grade I meningiomas with benign histology. Currently, there are no established circulating tumor markers which can be used for diagnostic and prognostic purposes in a non-invasive way for meningiomas. Here, we aimed to identify potential biomarkers of meningioma in patient sera. For this purpose, we collected preoperative (n = 30) serum samples from the meningioma patients classified as Grade I (n = 23), Grade II (n = 4), or Grade III (n = 3). We used a high-throughput, multiplex immunoassay cancer panel comprising of 92 cancer-related protein biomarkers to explore the serum protein profiles of meningioma patients. We detected 14 differentially expressed proteins in the sera of the Grade I meningioma patients in comparison to the age- and gender-matched control subjects (n = 12). Compared to the control group, Grade I meningioma patients showed increased serum levels of amphiregulin (AREG), CCL24, CD69, prolactin, EGF, HB-EGF, caspase-3, and decreased levels of VEGFD, TGF-α, E-Selectin, BAFF, IL-12, CCL9, and GH. For validation studies, we utilized an independent set of meningioma tumor tissue samples (Grade I, n = 20; Grade II, n = 10; Grade III, n = 6), and found that the expressions of amphiregulin and Caspase3 are significantly increased in all grades of meningiomas either at the transcriptional or protein level, respectively. In contrast, the gene expression of VEGF-D was significantly lower in Grade I meningioma tissue samples. Taken together, our study identifies a meningioma-specific protein signature in blood circulation of meningioma patients and highlights the importance of equilibrium between tumor-promoting factors and anti-tumor immunity.

Gene therapy involves the introduction of genes (termed transgenes) into cells to compensate for a deficiency or to make a beneficial protein. Gene therapy can used as a form of cancer treatment. A particularly attractive paradigm in this regard involves the selective introduction of transgenes into cancer cells that converts inactive prodrugs into active chemotherapeutic agents, thereby triggering the death of cancer cells. Since prodrugs are inactive, they tend not to cause significant side-effects and are well-tolerated by patients relative to conventional chemotherapy. Several viral and nonviral vectors have been used as delivery tools for suicide gene therapy. Extracellular vesicles (EVs) are now recognized as a promising class of nonviral delivery vectors. Here, we describe a method in which a suicide fusion gene construct is loaded into EVs derived from a non-tumorigenic cell line. Delivery of these modified EVs to glioblastoma cell lines and spheroids decreases glioblastoma cell viability, induces apoptotic cell death, and inhibits tumor growth in vivo.

Ape1 is the major apurinic/apyrimidinic (AP) endonuclease activity in mammalian cells, and a key factor in base-excision repair of DNA. High expression or aberrant subcellular distribution of Ape1 has been detected in many cancer types, correlated with drug response, tumor prognosis, or patient survival. Here we present evidence that Ape1 facilitates BRCA1-mediated homologous recombination repair (HR), while counteracting error-prone non-homologous end joining of DNA double-strand breaks. Furthermore, Ape1, coordinated with checkpoint kinase Chk2, regulates drug response of glioblastoma cells. Suppression of Ape1/Chk2 signaling in glioblastoma cells facilitates alternative means of damage site recruitment of HR proteins as part of a genomic defense system. Through targeting "HR-addicted" temozolomide-resistant glioblastoma cells via a chemical inhibitor of Rad51, we demonstrated that targeting HR is a promising strategy for glioblastoma therapy. Our study uncovers a critical role for Ape1 in DNA repair pathway choice, and provides a mechanistic understanding of DNA repair-supported chemoresistance in glioblastoma cells.

BACKGROUND:Overexpression of minichromosome maintenance (MCM) proteins 2, 3, and 7 is associated with migration and invasion in medulloblastoma (MB). However, expression profiling of all prereplication complex (pre-RC) has not been addressed in MBs. PROCEDURE:We performed mRNA expression profiling of a large set of pre-RC elements in cell lines and tumor tissues of MB. RNAi technology was employed for functional studies in MB cell lines. RESULTS:Our data showed that most of the pre-RC components are significantly overexpressed in MB. Among all pre-RC mRNAs, MCM10 showed the highest level of expression (∼500- to 1,000-fold) in MB cell lines and tissues compared to the levels detected in cerebellum. In addition, RNAi silencing of MCM10 caused reduced cell proliferation and cell viability in MB cells. CONCLUSIONS:Taken together, our study reveals that the pre-RC is dysregulated in MB. In addition, MCM10, a member of this complex, is significantly overexpressed in MB and is required for tumor cell proliferation.

Extracellular vesicles (EVs) are considered as important mediators of intercellular communication, which carry a diverse repertoire of genetic information between cells. This feature of EVs can be used and improved to advance their therapeutic potential. We have previously shown that genetically engineered EVs carrying the suicide gene mRNA and protein—cytosine deaminase (CD) fused to uracil phosphoribosyltransferase (UPRT)—inhibited schwannoma tumor growth in vivo . To further examine whether this approach can be applied to other cancer types, we established a subcutaneous xenograft glioblastoma tumor model in mice, as glioblastoma represents the most common primary brain tumor, which is highly aggressive compared with the original schwannoma tumor model. U87-MG glioblastoma cells were implanted into the flanks of nude SCID mice, and the animals were intratumorally injected with the EVs isolated from the cells expressing EGFP or CD-UPRT. After the intraperitoneal administration of the prodrug 5-fluorocytosine, the tumor growth was assessed by regular caliper measurements. Our data revealed that the treatment with the CD-UPRT-enriched EVs significantly reduced the tumor growth in mice. Taken together, our findings suggest that EVs uploaded with therapeutic CD-UPRT mRNA/protein may be a useful tool for glioblastoma treatment.

Extracellular vesicles (EVs) are different types of membrane-derived vesicles that originate from the endosomal pathway or the plasma membrane. These vesicles are used as "carriers" in intercellular communication, and are responsible for the transfer of biological cargo (lipids, proteins, RNA species, and DNA) between different cells. Despite the shortcomings in our knowledge of EV biology, attempts to employ EVs as natural delivery tools for therapeutic purposes have been partly successful in different settings. In this review, we highlight this unique potential of EVs, and discuss previous examples and future scenarios.

BACKGROUND Head and neck cancers comprise the sixth most common cancer type worldwide. One of the most remarkable malignancies of the head and neck is the cancer of the nasopharynx, with a strong metastatic tendency already in the early stage. Besides the conventional pathways of metastasis formation, the information content of exosomes produced by the cancer cells may play a key role in metastatic transformation. The aim of this study was to investigate how stressors alter the characteristic of tumor derived exosomes. METHODS In our experimental model, we compared the quantity and content of exosomes produced by a nasopharyngeal carcinoma cell line (5-8F) under conventional (chemotherapy) and alternative (Ag-TiO2 -catalyzed reactive oxygen species generation) cytostatic treatment. After isolation, exosomes were identified by atomic force microscopy and quantified with Nanosight NS500 device. MicroRNA content of them was analyzed using SOLiD 5500xl technology. The sequences were annotated in CLC Genomics Workbench version 5.5.1. RESULTS Beyond the classic chemotherapeutic agent (doxorubicin), Ag-TiO2 in a photo-catalytic process also showed cytostatic activity. Tumor cell damage induced by the cytostatic treatments significantly altered the number of released exosomes and led to the predominance of tumor suppressors in the exosomal miRNA profile. CONCLUSIONS Our results suggest that the intercellular communication between tumor cells and surrounding stroma cells can be altered by microenvironment which increased quantity of exosomes and diversity of miRNAs in this study. Imbalance of oncogenic and tumor suppressor miRNAs caused by cytostatic treatments may influence the antiproliferative and metastasis inhibitory effect of cytostatic agents.

The predominant human apurinic/apyrimidinic (AP) endonuclease 1, Ape1 (also known as Ref-1) plays a central role in the pathways of base excision repair (BER) of DNA. BER is the major system for correcting the oxidative DNA damage that arises endogenously as a result of metabolism. Spontaneous hydrolytic decay of DNA or DNA glycosylase-mediated hydrolysis of N-glycosyl bonds generates AP sites at an estimated frequency of >10,000 per day in each human cell. If unrepaired, non-instructional AP sites potently block DNA replication and thereby cause toxicity. Thus, the proper repair of AP sites is crucial for maintaining genome stability. Genetic studies in mice support the essential role of Ape1 in cellular viability. In this study, we have explored the molecular details of Ape1-mediated genome maintenance mechanisms, and applied glioblastoma multiforme (GBM) tumor model for investigation of the functional significance of the Ape1-mediated cell responses to chemotherapy. Here we present evidence that Ape1 links to a Chk2-associated tumor suppressor pathway, and is necessary for proper execution of DNA damage-induced checkpoints. Furthermore, our study revealed an unexpected role for Ape1 in BRCA1-mediated homologous recombination (HR) repair of DNA breaks, which is crucial for directing DNA repair pathway choices. The analyses of the TCGA data set for GBM showed that Ape1 and Chk2 are coordinately regulated in glioblastoma multiforme, and increased expression of APEX1 gene coding for Ape1 positively correlates with better survival of glioblastoma patients. GBM is the most common and deadly type of primary brain tumor. The current therapy remains relatively ineffective due to diffuse infiltration and intrinsic resistance of GBM cells. Thus, there is an urgent need for rational treatment protocols for the GBM cure. Our study provides evidence for critical importance of the survival network of Ape1 and Chk2 in GBM response to chemotherapy. Regulation of the equilibrium between Ape1 and Chk2 enables tumor cells switch between DNA repair pathways to favor cell survival. Identification of tumor-specific pre-dominant DNA repair pathways and interruption of such survival networks can be a useful strategy for designing novel therapies for GBM. Citation Format: Thomas Stroebel, Sibylle Madlener, Serkan Tuna, Sarah Vose, Daniella Morse, Bakhos A. Tannous, Tonny Lagerweij, Thomas Wurdinger, Christine Marosi, Irene Slavc, Klemens Vierlinger, Okay Saydam, Brendan D. Price, Bruce Demple, Nurten Saydam. Ape1/Ref-1 directs DNA repair pathway choice, linked to Chk2 signaling, critical for glioblastoma response to chemotherapy. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Drug Sensitivity and Resistance: Improving Cancer Therapy; Jun 18-21, 2014; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(4 Suppl): Abstract nr B26.