Glioma Cell Lines Research Articles

Phospholipid scramblase 1 acts through the IL-6/JAK/STAT3 pathway to promote the malignant progression of glioma

BackgroundPhospholipid scramblase 1 (PLSCR1) is a calcium-dependent endofacial plasma-membrane protein that plays an essential role in multiple human cancers. However, little is known about its role in glioma. This study aimed to investigate PLSCR1 function in glioma, and elucidate its underlying molecular mechanisms. MethodsPLSCR1 expression in human glioma cell lines (U87MG, U251, LN229, A172 and T98G) and human astrocytes was detected by western blot and qRT-PCR. PLSCR1 was silenced using si-PLSCR1-1 and si-PLSCR1-2 in LN229 and U251 cells. PLSCR1 was overexpressed using the pcDNA-PLSCR1 plasmid in T98G cells. Colony formation, 5-ethynyl-2′-deoxyuridine, flow cytometry and transwell assays were employed for measuring cell proliferation, apoptosis and mobility after PLSCR1 knockdown or overexpression. PLSCR1 function in glycolysis in glioma cells was determined through measuring the extracellular acidification rate, oxygen consumption rate, glucose consumption and lactate production. Besides, immunohistochemistry, western blot and qRT-PCR were utilized to assess mRNA and protein expression. Besides, the effect of PLSCR1 silencing on subcutaneous tumor was also monitored. ResultsPLSCR1 expression was upregulated in glioma. The downregulation of PLSCR1 repressed the proliferation, mobility, epithelial-to-mesenchymal transition (EMT) and glycolysis; however, it facilitated apoptosis in glioma cells. Whereas, PLSCR1 upregulation had the opposite effect. Moreover, PLSCR1 promoted the activation of the IL-6/JAK/STAT3 pathway in glioma cells. Besides, IL-6 treatment significantly reversed the function of PLSCR1 silencing on cell proliferation, mobility, EMT, apoptosis and glycolysis. In a nude mouse tumor model, silencing PLSCR1 suppressed tumor growth via inactivating IL-6/JAK/STAT3 signaling. ConclusionOur results indicated that PLSCR1 could facilitate proliferation, mobility, EMT and glycolysis, but repress apoptosis through activating IL-6/JAK/STAT3 signaling in glioma. Therefore, PLSCR1 may function as a potential therapeutic target for glioma.

UBA2 as a Prognostic Biomarker and Potential Therapeutic Target in Glioma.

Gliomas are characterized by aggressive behavior, leading to severe disability and high mortality. Ubiquitin-like modifier activating enzyme 2 (UBA2) is a subunit of the E1-activating enzyme involved in the SUMOylation (SUMO, small ubiquitin-related modifier) of numerous proteins. Although the abnormality of UBA2 is linked to the progression of various tumor types, the role of UBA2 in glioma is still unknown. A bioinformatic analysis using several public databases was conducted to examine the expression level, clinicopathological correlations, and prognostic significance of UBA2 in glioma. The correlation between UBA2 expression and drug sensitivity in cancers was also explored. Multiple cellular experiments were conducted to validate the role of UBA2 in glioma. Analysis of multiple databases and cellular experiments revealed that UBA2 was overexpressed in glioma tissues and cell lines, respectively. UBA2 expression in gliomas correlated with World Health Organization (WHO) grade, IDH gene status, 1p19q deletion, histological type, and immune cell infiltration in glioma. UBA2 expression in carcinomas also correlated with drug sensitivity. Kaplan-Meier analysis revealed that high expression of UBA2 predicted poorer survival in glioma patients. A nomogram model containing UBA2 expression was constructed for clinical practice. Knockdown of UBA2 was observed to suppress glioma cell progression and sensitize glioma cells to irradiation in vitro. Overall, this research showed that UBA2 might be involved not only in the development of glioma but also in the regulation of immunity, drug sensitivity, and radiosensitivity. Therefore, UBA2 may be a potential target for therapy and a candidate biomarker for glioma diagnosis and prognosis.

Tumor associated microglia/macrophages utilize GPNMB to promote tumor growth and alter immune cell infiltration in glioma

Tumor-associated microglia and blood-derived macrophages (TAMs) play a central role in modulating the immune suppressive microenvironment in glioma. Here, we show that GPNMB is predominantly expressed by TAMs in human glioblastoma multiforme and the murine RCAS-PDGFb high grade glioma model. Loss of GPNMB in the in vivo tumor microenvironment results in significantly smaller tumor volumes and generates a pro-inflammatory innate and adaptive immune cell microenvironment. The impact of host-derived GPNMB on tumor growth was confirmed in two distinct murine glioma cell lines in organotypic brain slices from GPNMB-KO and control mice. Using published data bases of human glioma, the elevated levels in TAMs could be confirmed and the GPNMB expression correlated with a poorer survival.

Sodium fluorescein uptake by the tumor microenvironment in human gliomas and brain metastases.

Intravenous sodium fluorescein (SF) is increasingly used during surgery of gliomas and brain metastases to improve tumor resection. Currently, SF is believed to permeate the brain regions where the blood-brain barrier (BBB) is damaged and to accumulate in the extracellular space but not in tumor or healthy cells, making it possible to demarcate tumor margins to guide resection. By evaluating the immune contexture of a number of freshly resected gliomas and brain metastases from patients undergoing SF-guided surgery, the authors recurrently observed fluorescence-positive cells. Therefore, the aim of this study was to determine if SF accumulates inside the cells of the tumor microenvironment (TME), and if so, in which type of cells, and whether incorporation can also be observed in the leukocytes of peripheral blood. Freshly resected tumor specimens were dissociated to single cells and analyzed by multiparametric flow cytometry. Peripheral blood leukocytes, macrophages, and a glioma cell line were treated with SF in vitro, and their cell uptake was assessed by multiparametric and imaging flow cytometry and by confocal microscopy. The ex vivo and in vitro analyses revealed that SF accumulates intracellularly in leukocytes as well as in tumor cells, but with a high variability of incorporation in the different cell subsets analyzed. Myeloid cells showed the highest level of fluorescence. In vitro uptake experiments showed that SF accumulation increases over time. The imaging analyses confirmed the internalization of the compound inside the cells. SF is not just a marker of BBB damage, but its intracellular detection suggests that it selectively accumulates intracellularly. Future efforts should target the mechanisms of its differential uptake by the different TME cell types in depth.

235 IL-4/13 Blocking Peptide (KROS-401) Inhibits M2 Macrophage Polarization in vitro and Demonstrates Anti-tumor Activity in a Murine Model of Glioblastoma

INTRODUCTION: Glioblastoma (GBM) is the most aggressive primary brain malignancy with a median survival of 14 months. Tumor-associated macrophages (TAMs) comprise up to 40% of the GBM tumor mass and are significant enablers of an immunosuppressive tumor microenvironment (TME), limiting immunotherapeutic efficacy. TAMs are generally classified as M2 macrophages that demonstrate a pro-tumor phenotype and increased CD206 surface marker expression. Reducing the M2 macrophage population in tumors has shown benefit in preclinical studies across various cancers. Interleukins (IL)-4 and -13 both play a role in polarizing macrophages to an M2 state. We developed a peptide inhibitor (KROS-401) that targets the shared IL-4Ra subunit of the IL-4 and -13 receptor complex to inhibit macrophage polarization to an M2 phenotype and reprogram the immunosuppressive TME. METHODS: Human peripheral blood mononuclear cell (PBMC)-derived macrophages were isolated and treated with KROS-401 in the presence of IL-4 and IL-13 cytokines. M2 (CD206) surface markers were assessed by flow cytometry. The efficacy of KROS-401 peptide was examined using an intracranially implanted syngeneic murine model with luciferase-expressing GL261, a glioma cell line. Mice were inspected daily for survival and tumor growth was tracked by non-invasive in vivo bioluminescent imaging (BLI). RESULTS: Human PBMC-derived macrophages treated with KROS-401 displayed a dose-dependent decrease in M2 (CD206) cell surface markers. KROS-401 peptide inhibitor demonstrated anti-tumor activity in GL261 glioma-bearing mice with a significantly increased probability of survival compared to control (p = 0.0046) with a median survival of >40 days versus 27 days, respectively. CONCLUSIONS: KROS-401 inhibits M2 polarization of human macrophages in vitro and tumor growth in vivo. These findings highlight this IL-4/IL-13 peptide inhibitor as a potential immunomodulator of the TME for the treatment of GBM.

Mirna-383-5p Functions as an Anti-oncogene in Glioma through the Akt/mTOR Signaling Pathway by Targeting VEGFA.

Previously, we have screened 59 differentially expressed miRNAs and 419 mRNAs in the glioblastoma samples that have been compared to the peritumoral tissues using bioinformatics analyses, which included miRNA-383-5p and vascular endothelial growth factor A (VEGFA). miRNA-383-5p and VEGFA/Akt/mTOR pathway play important regulatory roles in the malignant biological behavior of glioma. Glioma cell lines, U87 and U251, were collected for in vitro experiments. miRNA-383-5p and VEGFA expression levels were detected with qRT-PCR and WB. The protein expressions of Akt, mTOR, and VEGFR in U87 and U251 were detected with WB. The effect of miRNA-383-5p on the VEGFA activity was verified by dual-luciferase reporter assay. CCK-8 was used to examine the U87 and U251 cells' inhibition. Flow cytometry and transwell assays were used to detect cell apoptosis and invasion, respectively. Our research data indicated overexpression of miRNA-383-5p to suppress malignant biological behavior, which was manifested as promoting the apoptosis of U87 and U251 cells and inhibiting invasion, proliferation, and metastasis. VEGFA is one of the downstream target genes of miRNA-383- 5p. miRNA-383-5p could inhibit the expression of VEGFA and Akt/mTOR signaling pathways. Overexpression of VEGFA can reverse the inhibitory effect of miRNA-383-5p and reactivate the Akt/mTOR signaling pathway. Our results indicate that miRNA-383-5p functions as an anti-oncogene by inhibiting the VEGFA/Akt/mTOR signaling pathway in glioma cells. These data provide potential therapeutic targets for glioblastoma.

389 Combination Therapy With Paclitaxel, Carboplatin, and Low-Intensity Ultrasound for Recurrent Glioblastoma: Rationale for Ongoing Phase II Clinical Trial

INTRODUCTION: We recently participated in Phase 1/2 clinical trials where patients with recurrent glioblastoma received surgery for resection of the tumor followed by implantation of an ultrasound device (SonoCloud-9, Carthera, Lyon, France) into a window in the skull for subsequent repeated blood-brain barrier (BBB) opening and concomitant delivery of chemotherapy. Intraoperative experiments demonstrated a 4-6-fold increase in the brain concentration of paclitaxel (NCT04528680) and carboplatin (NCT03744026) after BBB disruption. METHODS: We analyzed the cytotoxicity of paclitaxel and carboplatin in vitro against five human cell lines and six patient-derived xenografts (PDX) as monotherapy and in combination at concentrations derived from our human intraoperative pharmacokinetic studies. Synergy was assessed using the Loewe model and survival benefit was evaluated in two xenografts where drugs were delivered with ultrasound. We also examined the effects on cell cycle progression, DNA damage, and apoptosis. RESULTS: Cell lines exhibited non-overlapping susceptibility to paclitaxel and carboplatin, with 27% being exclusively susceptible to one drug. The combination covered 81% of cell lines, demonstrating synergy in 55% of cell lines. Notably, paclitaxel, carboplatin, and ultrasound proved more effective than monotherapy in two xenograft models. Combination therapy led to distinct effects on cell cycle progression and enhanced DNA damage and apoptosis. CONCLUSIONS: Combination therapy may be more effective than monotherapy, as has been shown in other cancers, due to synergy and independent susceptibility to each drug in glioma cell lines. This strategy is currently under investigation in an ongoing phase II clinical trial to further evaluate the safety and efficacy of this approach in patients with recurrent glioblastoma (NCT04528680).

383 Epigenetic Potentiation of Anti-tumor Immune Responses Using Viral Mimicry for Gliomas

INTRODUCTION: Human endogenous retroviruses (HERVs) account for approximately 8% of our genome and are evolutionary relics of a germline retroviral infection. Although normally silenced by a master epigenetic regulator, np220, HERVs may be re-expressed in cellular states with marked epigenetic dysregulation and induce innate anti-viral inflammatory responses. This viral mimicry immune response sensitizes tumors to immunotherapy by eliciting double-stranded RNAs (dsRNA)-mediated interferon signaling cascades. Hence, viral mimicry could serve as a predictor of clinical responses to immunotherapy. METHODS: We conducted a preclinical study on the role of np220 in GBM and mediating viral mimicry responses. RESULTS: Using a tailored bulk transcriptional dataset from 71 newly diagnosed high-grade GBMs, we demonstrated significantly negative correlations between np220 expression and 48 HERV superfamilies. Additionally, using a transcriptional deconvolution pipeline of low-and high-grade gliomas, we demonstrated that np220 is negatively correlated with innate antiviral immune response signatures (TLR3) (RTLR3 = -0.300, pTLR3 = 0.00006). Our in vitro studies have shown that NP220 knockdown induces intracellular HERV-mediated dsRNA-signaling cascades via RIG-1 and TLR3. NP220 knockdown exponentially increases PD-L1 expression (p-value: <0.001), and, increases cytoplasmic accumulation of cellular endogenous dsRNA in established human and mouse glioma cell lines which may induce the interferon-mediated response. This response was recapitulated in recurrent GBM samples with radiographic responses to checkpoint inhibition (anti-PD1) with widely increased expression of dsRNA, PD-L1 and perivascular CD8 cell infiltration, suggesting dsRNA-signaling may mediate response to immunotherapy. Furthermore, we showed np220 is linked to reduced immune cell infiltration (CD8, Dendritic Cells) in the glioma microenvironment through deconvolution of RNA-seq data from TCGA (RCD8 = -.202, RDC = -0.198) (pCD8 = 8.79E-06, pDC = 1.34E-05). CONCLUSIONS: Our findings suggest that np220 could serve as a target to potentiate glioma ICI for immune response.

205 Microglia-Mediated Survival Outcomes in Preoperative Radiation for Glioblastoma: Insights From a Preclinical Model

INTRODUCTION: Glioblastoma (GBM) is the most common and devastating primary brain tumor in adults. The current standard of care includes maximal safe resection followed by concurrent radiation and temozolomide-based chemotherapy. However, preoperative radiotherapy is used in different cancer types and commonly used for brain metastasis to shrink tumors, but it has never been used in for primary GBM. METHODS: Thirty-nine immunocompromised adult athymic nude female rats were orthotopically engrafted with a GBM1A patient-derived glioma cell line and randomized to either receive radiotherapy prior to tumor resection (RTRE) or postoperative radiotherapy (RERT). The rats received 30 Gy hypofractionated stereotactic radiation in 5 fractions of 6 Gy. Kaplan-Meier curve was used to plot the survival. Hematoxylin-eosin staining, microglia/macrophages using IBA-1 and TMEM119 markers were used in resected and recurrent tumor tissues. Moreover, nuclei area was measured on all tumor samples. Student’s T-test and analysis of variance (ANOVA) were used for statistical analysis. RESULTS: Kaplan-Meier analysis showed a significant survival benefit in the RTRE group, with median survival of 18.28 weeks compared to 14.85 weeks for RERT (p < 0.001), which correlated with the time of recurrence. Simultaneously, an increase in the nuclei area was found in the resected tumor tissues from the RTRE group (p < 0.001) and RERT recurrent tumor tissues (p < 0.001). Interestingly, we found lower microglia/macrophage recruitment in RTRE in the recurrent tumors (p < 0.01) and throughout the tumor areas compared to the RERT group. CONCLUSIONS: Our study represents the first preclinical study demonstrating the feasibility and longer overall survival using RTRE instead of RERT in vivo. Moreover, RTRE shows an important effect in the macrophages/microglia recruitment and in the immune profile. This suggests strong superiority for new clinical radiation strategies.

Tumor suppressor role of the complement inhibitor CSMD1 and its role in TNF-induced neuroinflammation in gliomas

BackgroundThe complement inhibitor CSMD1 acts as a tumor suppressor in various types of solid cancers. Despite its high level of expression in the brain, its function in gliomas, malignant brain tumors originating from glial cells, has not been investigated.MethodsThree cohorts of glioma patients comprising 1500 patients were analyzed in our study along with their clinical data. H4, U-118 and U-87 cell lines were used to investigate the tumor suppressor function of CSMD1 in gliomas. PDGFB-induced brain tumor model was utilized for the validation of in vitro data.ResultsThe downregulation of CSMD1 expression correlated with reduced overall and disease-free survival, elevated tumor grade, wild-type IDH genotype, and intact 1p/19q status. Moreover, enhanced activity was noted in the neuroinflammation pathway. Importantly, ectopic expression of CSMD1 in glioma cell lines led to decreased aggressiveness in vitro. Mechanically, CSMD1 obstructed the TNF-induced NF-kB and STAT3 signaling pathways, effectively suppressing the secretion of IL-6 and IL-8. There was also reduced survival in PDGFB-induced brain tumors in mice when Csmd1 was downregulated.ConclusionsOur study has identified CSMD1 as a tumor suppressor in gliomas and elucidated its role in TNF-induced neuroinflammation, contributing to a deeper understanding of glioma pathogenesis.

Enhancing the Antiproliferative Activity of Perillyl Alcohol against Glioblastoma Cell Lines through Synergistic Formulation with Natural Oils.

Due to its volatility, photostability, and gastrointestinal toxicity, Perillyl Alcohol (POH), a monoterpenoid component of various plant species, is a chemotherapeutic drug with insufficient efficacy. Many naturally occurring bioactive compounds have well-known antiproliferative properties, including sefsol, jojoba, tea tree, and moringa oils. This study sought to develop an oil-based Self Nanoemulsifying Drug Delivery System (SNEDDS) using tween 80 as the surfactant and Dimethyl Sulfoxide (DMSO) or Polyethylene Glycol (PEG) 400 as the cosurfactant; the oils were used in a range of 10-20% to boost POH's anticancer efficacy. The formulations' size, charge, and impact on the viability of glioma cell lines, ANGM-CSS and A172, were evaluated. The developed SNEDDS formulations ranged from 3 nm to 362 nm in size, with electronegative surface charges between 5.05 and 17.0 mV and polydispersity indices between 0.3 and 1.0. The findings indicated that the antiproliferative effect of POH-loaded Nanoemulsion (NE) could be used as a possible anticancer therapy for glioblastoma in vitro, particularly when paired with the tested natural oils. Before asserting that this delivery technique is appropriate for glioblastoma therapy, additional in vitro and in vivo investigations are required.

PSMC2 promotes glioma progression by regulating immune microenvironment and PI3K/AKT/mTOR pathway

BackgroundGlioma, the most frequent and malignant central nervous system (CNS) cancer, has a bad outcome. Proteasome 26S subunit ATPase 2 (PSMC2) is an essential part of the 26S proteasome and promotes the development of several tumors. However, the pathway and function of PSMC2 in glioma have not been unelucidated. MethodsThis study analyzed PSMC2 expression in glioma tissues and its predictive significance for patients. We examined the link between PSMC2 and DNA methylation, immune cell infiltration, tumor immune cycle, immune cell homeostasis, and immune checkpoints. Subsequently, immunohistochemistry and in vitro trials were employed to validate the expression, prognostic potential, and function of PSMC2 in glioma. The mechanisms of PSMC2 in glioma were further explored. ResultsOur study revealed that PSMC2 expression increased in glioma tissues contrasted with healthy tissues, and patients with high PSMC2 glioma exhibited poor overall survival (OS) compared to the low-PSMC2 group. Immune profile analysis revealed that PSMC2 was positively related to immunosuppressive cell infiltration and immune checkpoints and adversely related to the cancer immune cycle and immune cell homeostasis. In cell-based investigations, the inhibition of PSMC2 was found to effectively suppress the aggressiveness and proliferation of glioma cell lines while also enhancing cell cycle arrest and promoting cell death. Gene Set Enrichment Analysis (GSEA), Gene Set Variation Analysis (GSVA), and in vitro experiments showed that PSMC2 promoted glioma development through the PI3K/AKT/mTOR pathway. ConclusionsPSMC2 was upregulated in glioma and promoted cancer progression by modulating the tumor immune microenvironment, cancer cell biological behavior, immune cell homeostasis, and the PI3K/AKT/mTOR pathway, providing a new option to treat glioma.

Abstract 3162: Antagonizing beta-2 adrenergic receptor’s function triggers death in glioma cells and down-regulates survivin

Abstract Introduction: Epidemiological studies showed that beta-adrenergic antagonists caused a low incidence of several cancer types. Glioblastoma highly expresses beta-2-adrenergic receptors. GBM accounts for 50% of central nervous system cancers. After standard-of-care therapeutic intervention, there is a survival time of 15 months. Our goal is to develop a translational therapy that will trigger the death of GBM cells while preserving healthy cells Methodology: Salmeterol xinafoate is a biased agonist that activates the G-protein pathway. Biased agonists activate signaling through the G-protein or the beta-arrestin pathway of G-protein coupled receptors. Dose-response experiments were performed using beta1 and beta2-adrenergic receptor antagonists and biased beta2-adrenergic receptor agonists to determine loss of glioma cell viability in GBM5, GBM12, and LN229 glioma cell lines. Western immunoblotting gauged survivin expression levels in all three lines. Epoxomicin was used to inhibit the proteasome complex. Annexin V and propidium iodide were employed to measure apoptotic or necrotic cell death. Results: Disrupting beta2-adrenergic receptor signaling by salmeterol xinafoate led to cell death of glioma cells but spared healthy neuron-glia cells using similar doses. Salmeterol xinafoate revealed greater efficacy for killing glioma cells than beta1 and beta2-adrenergic receptor antagonists. To determine the mechanism of glioma cell death, LN229 cells were treated with escalating doses of Salmeterol xinafoate, and at 24 hours, annexin V staining showed early apoptosis, and Annexin V/propidium iodide co-staining also showed late apoptosis. Propidium iodide revealed minor necrosis in LN229 glioma cells. Western immunoblotting showed salmeterol xinafoate downregulated survivin in a dose-dependent manner. However, the epoxomicin's inhibition of the proteasome complex prevented dose-escalated downregulation of survivin for all three glioma cell lines. Conclusion: The bitopic structure of salmeterol enables this drug to bind to the beta2-adrenergic receptors ligand binding site and an exosite site by its aryloxyalkyl tail that may contribute to the increased efficacy. Salmeterol xinafoate triggers glioma cell death by apoptosis at least in one glioma cell line. Salmeterol Xinafoate down-regulates survivin through protease degradation. Promising results show that the FDA-approved drug salmeterol xinafoate enables a favorable transition to the clinic. Repurposing this drug for GBMs may be combined with the current standard-of-care chemotherapies to enhance more significant loss of tumor volume and increase patient survival. Citation Format: Orli Algranatti, Ji Yae Lee, James M. Angelastro. Antagonizing beta-2 adrenergic receptor’s function triggers death in glioma cells and down-regulates survivin [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3162.

PH-sensitive polyacrylic acid /Fe3O4@SiO2 hydrogel nanocomposite modified with agarose for controlled release of quercetin

Cancer has consistently been a significant global concern as the leading second cause of death. Improving the performance of chemical methods using a drug delivery system is an alternative approach to cancer treatment. In this study, the delivery of an anti-cancer drug, quercetin (QC), was investigated by placing it in a pH-sensitive hydrogel nanocomposite made of polyacrylic acid (PAA), agarose, and Fe3O4@SiO2 particles. Then, the drug release control was improved by the double emulsion method. Spherical nanoparticles with a smooth surface morphology were observed in the images obtained through field-emission scanning electron microscopy (FESEM), their average size was determined to be 193 nm using dynamic light scattering (DLS) method, and they exhibited a zeta potential of 38 mV. Fourier transform infrared spectroscopy (FTIR) and X-ray diffraction (XRD) analyses provided evidence of the interactions and chemical bonds in the hydrogel structure. The magnetic properties of the nanocarrier were studied by vibrating-sample magnetometry (VSM). The amount of drug loaded in the PAA-Agarose/Fe3O4@SiO2 increased to 86 %, while the encapsulation efficiency increased to 48 %. Drug release kinetics at physiological (7.4) and acidic (5.4) pH were best fitted with the Korsmeyer-Peppas model, indicating a non-Fickian diffusion mechanism. The U-87 MG glioma cell line was evaluated for toxicity through cytotoxicity assay (MTT) and flow cytometry. A higher percentage of apoptosis was found in PAA-Agarose/Fe3O4@SiO2-QC compared to the free QC system. The findings corroborate that the approach developed herein is highly effective for pH-responsive drug delivery in cancer treatment applications.

Abstract 7110: Characterizing pY240-PTEN:Ki-67 interactions and development of a PPI disrupting peptide

Abstract Glioblastoma (GBM) is the most aggressive and common malignant primary brain tumor. Standard of care for GBM patients involves chemotherapy and ionizing radiation which induce DNA damage to kill tumor cells; however resistance to these treatment modalities commonly develop through various mechanisms. One novel mechanism of radioresistance is through enhanced homologous recombination (HR) DNA damage response (DDR) mediated by nuclear-localized tyrosine-phosphorylated PTEN (pY240-PTEN) recruited to chromatin through interaction with the PP1 Binding Domain (PP1BD) of Ki-67. PTEN is known to play an important tumor-suppressive role and is found to be mutated in approximately 40% of GBMs. A Y240F-PTEN knock-in mouse model has shown that loss of Y240 phosphorylation results in IR sensitivity. Ki-67 dependent, pY240-PTEN facilitated DDR may be regulated by currently uncharacterized interactions within the Ki-67 PP1BD and determination of these interactions may enable development of complex-disrupting peptides capable of radiosensitizing glioma cells. To this end, I have identified residues in the Ki-67 PP1BD that are essential for pY240-PTEN interaction via streptavidin pulldown of N-terminal biotinylated peptides synthesized with alanine mutations at all residues spanning the Ki-67 PP1BD. Orthogonally, glioma cell lines have been engineered that stably express Ki-67 minigene constructs harboring mutation of the identified PP1BD residues. The effects of these mutations on PTEN:Ki-67 interaction, as well as DDR, HR, chromatin accessibility, and colony formation efficiency are currently being interrogated. Competitor peptides have been designed based on candidate Ki-67 residues and evaluated for their ability to bind PTEN. Further optimization of these peptides has led to candidates that are being assessed for stability and potential to radiosensitize glioma cells. In addition, regulation of pY240-PTEN:Ki-67 interaction by posttranslational modification of the Ki-67 PP1BD by aurora B kinase and cyclin dependent kinase 1 has been investigated through pulldown of biotinylated peptides harboring phosphorylated candidate residues. Lastly, Ki-67 has been knocked out in U87 glioma cells, as well as TS528 glioma stem like cells, and the resulting effect on IR sensitivity and DDR is being investigated in the absence, or presence of PTEN. Overall, this project has begun to characterize the regulation and critical molecular interactions between pY240-PTEN and Ki-67, elucidating potential strategies that could disrupt these interactions to enhance the efficacy of radiotherapy. Citation Format: Brandon Jones, Sejal Patel, Frank Furnari. Characterizing pY240-PTEN:Ki-67 interactions and development of a PPI disrupting peptide [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7110.

Abstract 3907: Novel public and tumor-wide neoantigens arising from clonal aberrant splicing events drive tumor-specific T-cell responses across diverse cancer types

Abstract BACKGROUND: Immunotherapy in gliomas is limited by intratumor heterogeneity (ITH) and low mutational burden. We developed a novel comprehensive in silico pipeline for detecting tumor-specific splicing events (neojunctions), and across multiple cancer types, we successfully identified a new class of tumor-wide, public, alternatively spliced neoantigens (ASNs) that elicit CD8+ T-cell-mediated immune responses. METHODS: Our pipeline identified recurring public neojunctions expressed in TCGA RNA-seq (positive sample rate (PSR) > 10%) and not in GTEx normal tissue RNA-seq data (PSR < 1%) across ten cancer types. To characterize tumor-wide neojunctions, we utilized available multi-site RNA sequencing across diverse cancer types. With gliomas, our in-house dataset comprised of 56 patients with approximately 10 maximally-distanced intratumoral biopsy sites per patient (n=535). Tumor-wide public neojunction expression was subsequently validated in RNA-sequencing and mass spectrometry (MS) data from patient-derived cell lines (n=68) and samples (n=447). Two independent algorithms then predicted peptide processing likelihood and HLA-binding affinity of ASN candidates. TCRs identified by in vitro sensitization (IVS) of donor-derived CD8+ T-cells followed by 10x VDJ scRNA-seq, were transduced into CD8+ T-cells and co-cultured with tumor cells to evaluate TCR functionality and cytotoxicity. RESULTS: Pan-cancer analysis identified large subsets of neojunctions that were interpatiently and intratumorally conserved. Our glioma-specific analysis identified 789 public neojunctions, and 8 ASNs were validated in transcriptomic and proteomic glioma data and predicted to be presented by HLA-A*02:01 with high confidence. IVS and subsequent 10x VDJ scRNA-seq on expanded PBMC-derived CD8+ populations cultured against ASN-pulsed dendritic cells identified TCR clonotypes reactive against neojunctions in RPL22 (n=7) and GNAS (n=1), the latter being highly intratumorally-conserved (detected in > 90% of spatially-mapped biopsies in 17/56 patients (26.78%)). TCR-transduced T-cells demonstrated recognition and immunogenic activation against endogenously processed and presented neoantigens in both pan-cancer cell lines and transfected COS7 cells and cytotoxicity against glioma cell lines. Pan-cancer analysis revealed the detection of both neojunctions in various tumor types beyond gliomas, with the GNAS neojunction notably found tumor-wide in mesothelioma, prostate cancer, and liver hepatocellular carcinoma spatial data. CONCLUSION: Our unique integrative pipeline identified a novel class public tumor-wide splice-derived neoantigen candidates and ASN-specific TCRs, offering a promising off-the-shelf immunotherapy approach for diverse cancer types and tackling the critical challenge of ITH in immunotherapy resistance. Citation Format: Darwin Kwok, Nicholas O. Stevers, Takahide Nejo, Lee H. Chen, Inaki Etxeberria, Jangham Jung, Kaori Okada, Maggie Colton Cove, Senthilnath Lakshmanachetty, Marco Gallus, Abhilash Barpanda, Chibo Hong, Gary KL Chan, Samuel H. Wu, Emilio Ramos, Akane Yamamichi, Jerry YZ Liu, Payal Watchmaker, Hirokazu Ogino, Atsuro Saijo, Aidan Du, Nadia Grishanina, James Woo, Aaron Diaz, Susan M. Chang, Joanna J. Phillips, Arun P. Wiita, Christopher A. Klebanoff, Joseph F. Costello, Hideho Okada. Novel public and tumor-wide neoantigens arising from clonal aberrant splicing events drive tumor-specific T-cell responses across diverse cancer types [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3907.

Abstract 701: Preclinical data of a novel DNA-PK inhibitor in combination with radiation therapy shows promise in the treatment of established GBM and lung carcinoma cell lines

Abstract Radiation therapy (RT) is a cornerstone of oncologic therapy for the majority of solid malignancies, and unrepaired DNA double-strand breaks are responsible for radiation-induced cytotoxicity. RT-induced DNA lesions can be repaired by multiple mechanisms including homologous recombination (HR) and non-homologous end joining (NHEJ), and disruption of either of these pathways can enhance cytotoxicity. A key component of the NHEJ pathway is the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs), and loss of kinase activity can significantly increase radiosensitivity. Herein we describe the results of our medicinal chemistry campaign to develop a potent and selective DNA-PKcs inhibitor, WNC0901. WNC0901 inhibits DNA-PKcs kinase activity in a cell free system with an IC50 of 0.071 nM and demonstrated at least 30-fold higher sensitivity than other family members (ATM, ATR, mTOR, PI3K). WNC0901 has limited aldehyde oxidase (AO) liability with a T1/2 >2000 min in liver cytosol and is stable for over 120 minutes in the presence and absence of an AO inhibitor. A preliminary pharmacokinetic analysis in wista han rat (IV=2mg/kg, PO=20mg/kg) demonstrated 116% apparent absolute oral bioavailability, 2.6 hour terminal half-life, 33.0 mL/min/kg clearance, and a 2.6% unbound brain-to-plasma partition coefficient (Kpuu). WNC0901 also had favorable pharmacokinetic properties in beagle dog (IV=2mg/kg, PO=20mg/kg) with moderate clearance (8.5 mL/min/kg), high apparent absolute oral bioavailability (131%) with good exposure (AUC=36092 h*ng/mL), moderate half-life (4.28 h), and low protein binding (74.3% fraction unbound). The volume of distribution was moderate in both species (Vss = 1.32 L/kg in wistar han rat and 1.87 L/kg in beagle dog). In cell culture, WNC0901 inhibited autophosphorylation of DNA-PKcs in HT29 cells irradiated with 10 Gy with an IC50 of 32.7 nM and robustly inhibited autophosphorylation in both U251 glioma and A549 lung cancer cell lines at 300 nM in combination with 5Gy. In a clonogenic assay, 5Gy irradiation (10% survival) combined with 100nM WNC0901 demonstrated modestly enhanced cell killing (1.5% survival), and maximal effects were seen at 300nM (0.04% survival, p<0.01). Similar radiosensitizing effects with 300 nM WNC0901 were seen in the A549 cell line (0.2% survival with combination compared to 19% with 5Gy alone, p<0.01). In summary, WNC0901 inhibits DNA-PK kinase activity and provides potent radiosensitization in a GBM and lung cancer cell line. Future studies will assess the combination of WNC0901 and RT in GBM patient-derived xenograft models in vivo. Citation Format: Ann C. Mladek, Danielle L. Burgenske, William F. Elmquist, Wei Zhong, Jann N. Sarkaria. Preclinical data of a novel DNA-PK inhibitor in combination with radiation therapy shows promise in the treatment of established GBM and lung carcinoma cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 701.

Abstract 2037: Enhanced chemosensitivity to DNA-damaging agents in human high-grade glioma through 2-DG treatment

Abstract Background: Human high-grade gliomas are aggressive brain cancers known for their resistance to treatment. Targeted therapies with single agents have shown limited success, and the presence of tumor heterogeneity complicates treatment. Combining multiple chemotherapeutic agents has emerged as a potential strategy to overcome these challenges. 2-deoxy-D-glucose (2-DG) is a glucose analog known for its ability to disrupt glycolysis, protein folding, and the pentose phosphate pathway. This disruption renders cancer cells more susceptible to further damage from reactive oxygen species and DNA damage. Previous studies have demonstrated synergistic effects when combining 2-DG with chemotherapeutic drugs in various cancers. Our study examines combining 2-DG with DNA-damaging agents for high-grade gliomas to uncover potential synergies, theorizing that this combination enhances cell death and metabolic stress, offering insights for better treatments against these tumors. Methods: The cell proliferation, clonogenic growth, and cell migration assays assessed viability, clonogenic formation, and migration. Additionally, the cell cycle and reactive oxygen species analysis were employed to evaluate cell division, oxidative stress, and antioxidant capacity. The Illumina NovaSeq 6000 platform measured transcript levels, and Liquid Chromatography-High-Resolution Mass Spectrometry analyzed metabolite changes in high-grade glioma cell lines following 2-DG treatment. The Agilent Seahorse Mito Stress Test evaluated alterations in cellular bioenergetics. SYNERGYFINDER 3 quantified synergistic effects of 2-DG with DNA-damaging agents, while the Annexin V-FITC Conjugates and a ROS multiplex assay assessed cell death and metabolic stress. Results: Our findings demonstrate that when used as a single agent, 2-DG induces cell senescence and limits cell migration but is insufficient to trigger cell death. Moreover, 2-DG treatment shifts glioma cells towards oxidative phosphorylation for energy production, elevates ROS levels, and hampers DNA synthesis and repair mechanisms. Our synergy study reveals promising synergistic effects when combining 2-DG with DNA-damaging agents, including 5-FU, Methotrexate, Gemcitabine, and Lomustine. This combined therapy leads to significant ROS generation and increased cell death compared to single-agent treatments. Furthermore, prolonged exposure to 2-DG leads to increased levels of both glucogenic and ketogenic amino acids within glioma cells, potentially serving as an alternative energy source. This finding hints at a prospective therapeutic approach for targeting drug-resistant cells. Conclusions: Our results suggest that the anticancer therapeutic effect of 2-DG is enhanced when used in combination with DNA-damaging agents, indicating this strategy as a potential approach for treating human high-grade glioma. Citation Format: Tracy Miller, Esther Jane, Matthew Halbert, Taylor Gatesman, Stacey Wendell, Dinesh Mohanakrishnan, Sameer Agnihotri, Daniel Premkumar, Ian Pollack. Enhanced chemosensitivity to DNA-damaging agents in human high-grade glioma through 2-DG treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2037.

Abstract 2130: Glioblastoma-derived extracellular vesicle cargo as a reporter of drug delivery and effect

Abstract The selectivity of the blood-brain-barrier (BBB) can prevent therapeutics from reaching the brain, and this may contribute to the high failure rate of new drugs for tumors like glioblastoma (GBM) and brain metastasis. The capacity to detect BBB penetrance in real-time in a noninvasive manner would arguably aid the rate of therapeutic development. Extracellular vesicles, which are membrane bound particles secreted by all cell types in the body, have potential as a neuro pharmacodynamic reporter given that their protein and RNA cargo reflect the originating cell population. We hypothesized that the exposure of GBM cells to therapy results in altered EV cargo, and that the detection of this shift can be leveraged as a liquid biopsy from patients during early clinical trials to determine BBB penetrance and tumor engagement. To identify cargo shifts indicative of treatment, we exposed four short-term patient derived glioma cell lines to arsenic trioxide (an idiopathic toxin), MLN4924 (a neddylation inhibitor), and the alkylating agent temozolomide, then harvested EVs for unbiased mass spectrometry and RNA sequencing. Interestingly, we identified conserved upregulated and downregulated protein and RNA species across cell lines and across drug treatments, despite the diverse mechanisms of action. Candidate proteins were further validated by mesoscale immunoassays and exoview evaluation of tetraspanin-captured EVs. RNA species were validated by qRT-PCR. Overall, we demonstrate that GBM-derived EV cargo changes in response to therapy, supporting the development of EVs as a reporter of drug delivery in GBM. Ongoing and future work is focused on identifying enrichment handles in order to selectively evaluate GBM-derived EV cargo from patient biofluids. Thus far, B7H3, PTPRZ1, EGFRvII and IL13Ra2 have been screened as putative handles. Success in this pursuit could substantially enhance findings and accelerate drug testing in early-stage clinical trials in brain tumor patients by reporting drug accessibility to the tumor and engagement with drug targets. Citation Format: Valerie DeLuca, Nanyun Tang, Nathaniel Hansen, Anjali Raju, Charles Shaffer, Yue Hao, Terry C. Burns, Jann N. Sarkaria, Ian Parney, Patrick Pirrotte, Kendall Van Keuren-Jensen, Michael E. Berens. Glioblastoma-derived extracellular vesicle cargo as a reporter of drug delivery and effect [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2130.

Abstract 5110: Exploring the effects of Indirubins in pediatric diffuse high grade glioma models

Abstract Diffuse midline glioma (DMG) is an invasive pediatric brain tumor which grows in the pons of the brainstem. The current course of treatment is focal radiation however, DMG continues to have a very poor prognosis with a median survival of less than 10 months. A major barrier for chemotherapeutic treatment of DMG is the blood brain barrier (BBB) which tightly controls access of molecules to the brain. This limits the efficacy of drugs to treat brain cancer and other diseases of the central nervous system. Previous studies from the Lawler lab and others have shown that the indirubin derivative, 6-bromoindirubin-3′-acetoxime(BIO-acetoxime/BIA), has anti-invasive properties and can enhance survival in glioblastoma xenograft models. Here, we investigated the effects of BIA on pediatric glioma models using a range of relevant assays. In an in vitro collagen migration assay, BIA was observed to significantly slow cell migration at a concentration of 1µM in DIPG 36 cells, over 72 hours. Furthermore, we have also shown that BIA modulates the tumor vasculature, which could limit tumor growth, and improves drug delivery to tumors by targeting tight junctions in tumor associated endothelium. The effect on BBB drug penetration was assessed in a multicellular 3D in vitro BBB model and effects of BIA on endothelial barrier functions was determined using trans-endothelial resistance assays (TEER). BIA treatment increased dextran uptake into 3D BBB models, and also lowered endothelial cell permeability in vitro via a reduction in the expression of tight junction proteins as shown by staining and a decrease in TEER values. In order to assess potential synergistic interactions, a drug screen was performed on a panel of distinct pediatric glioma cell lines (DIPG 36 and KNS42). This identified several FDA approved drugs that combine well with BIA. From this screen, BIA was shown to synergize with DNA damaging chemotherapy to enhance toxicity and in combination with cisplatin promote DNA damage in pediatric glioma cell lines. Other drugs identified in the screen are currently under investigation and details will be reported. Thus, our hypothesis is that BIA may provide an effective approach for further development as a DMG therapeutic through targeting invasion and enhancing drug potency and delivery via modulation of endothelial cell tight junctions. Interrogating the mechanisms involved and developing drug formulations informed by the drug screen for in vivo studies to determine the translational potential of this approach is our focus for future studies. Citation Format: Jasmine S. Clark, Jorge Jimenez-Macias, Philippa Vaughn-Beaucaire, Shengliang Zhang, Wafik El-Deiry, Rishi Lulla, Sean Lawler. Exploring the effects of Indirubins in pediatric diffuse high grade glioma models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5110.