Human Glioblastoma Models Research Articles

Abstract 1151: CD6 isoforms improve CAR T cell fitness

Abstract The ability of chimeric antigen receptor (CAR) T cells to initiate and sustain an effective immune response entails a delicate balance between T-cell activation and deactivation and is key for complete tumor elimination. CD6 is a co-receptor that fine-tunes T cell receptor signal strength through a unique dual function intracellular domain. CD6 is alternatively spliced, creating isoforms that lack distinct regions which interact with activating or inhibitory adapter proteins, thereby delivering inhibitory or activating signals, respectively. We hypothesized that CAR T cell fitness, namely their ability to sustain their effector function, can be achieved by using alternatively spliced CD6 isoforms which lack exons responsible for inhibitory signaling. First, we discovered that overexpression of the canonical full-length CD6 molecule, isoform CD6A, resulted in marginal improvement of CAR T cell function, while its deletion using CRISPR/Cas9 depressed the antitumor CAR T cell functionality. This supported a net stimulatory role for CD6 in CAR T cell function. To study their effect on cellular fitness, we then synthesized and overexpressed known alternatively spliced cytoplasmic isoforms CD6B, CD6C, CD6D, CD6E, and CD6F on CAR T cells and, noted a significant tendency towards preserving the CAR T cell naïve and central memory compartments, upon tumor encounter. Overexpression of isoform CD6F, which lacks exon 9, and to a lesser extent isoforms CD6C and CD6E enhanced the short- and long-term cytotoxicity of HER2 and CD19 targeting CAR T cells when compared to CAR T cells alone or CAR T cells overexpressing, CD6A. Despite exhibiting a lower proliferation index, and lower CAR CD3ζ phosphorylation at rest, CD6F CAR T cells secreted significantly higher concentrations of the Th1 cytokine, IFN-γ, and mediated more sustained in vitro cytotoxicity long-term, with more complete elimination of large tumor cell loads. Lastly, in an orthotopic model of human glioblastoma, the adoptive transfer of CD6F HER2 CAR T cells induced a more significant but also more sustained anti-glioma effect that translated into a significant survival advantage for experimental animals. Our data support that the overexpression of the alternatively spliced CD6 isoforms improves CAR T cell fitness resulting in superior antitumor activity both in vitro and in vivo. Citation Format: Jessica S. Morris, Ahmed Z. Gad, Lea M. Godret-Miertschin, Ryan Fleischmann, Vita Salsman, Sujith K. Joseph, Nabil Ahmed. CD6 isoforms improve CAR T cell fitness [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1151.

893 Development of a Rabbit Human Glioblastoma Model for Testing of Endovascular Selective Intra-Arterial Infusion (ESIA) of Novel Stem Cell-Based Therapeutics

INTRODUCTION: Poor outcomes for glioblastoma (GBM) are partly due to the inability to deliver therapeutic agents to the tumor because of the blood brain barrier (BBB) as well as ineffective agents. We have evaluated bone marrow-derived mesenchymal stem cells (MSCs) as transport vehicles of biological therapeutics, particularly Delta-24-RGD (D24), an engineered tumor-selective oncolytic adenovirus that replicates and lyses only in human tumor cells. In mice, we have shown that MSCs loaded with D24 (MSCs-D24) home to GBMs after intra-arterial (IA) carotid infusion, resulting in widespread distribution in the tumor and oncolysis. Intravenous infusion is ineffective, rendering IA delivery the only effective systemic option. To understand the applicability of IA infusions of MSCs-D24 requires a large animal, human brain tumor model recapitulating the challenges of clinical use. METHODS: Rabbits underwent stereotactic xenoimplantation of human GBM cell lines. Tumor creation was confirmed on magnetic resonance imaging (MRI), histologic and immunohistochemistry analysis. Selective internal carotid artery MSCs-D24 infusion ipsilateral to the tumor was performed assess efficacy and safety. RESULTS: We report on successful xenoimplantation of human glioblastoma cell lines (U87, GSC17, and GSC8-11) in 28 immunosuppressed rabbits using Mycophenolate Mofetil, Dexamethasone, and Tacrolimus. The implanted rabbits were followed for 35 days prior to tumor assessment with MRI, angiography, and histological analysis. On MRI, the tumors were hyperintense on T2-weighted image and enhanced (evidence of BBB breakdown). On histological analysis, tumors showed phenotypic traits of human GBM and display varying levels of vascularity, an important feature for testing IA therapy. Selective internal carotid artery MSCs-D24 infusion was safe in the model, and MSCs-D24 homed to the implanted tumor at 24 hours. CONCLUSIONS: The intracranial immunosuppressed rabbit human GBM model allows testing of ESIA infusion of novel therapeutics (eg. MSCs-D24) in a clinically relevant fashion.

Key role of PIN1 in telomere maintenance and oncogenic behavior in a human glioblastoma model.

PIN1 is the only known enzyme capable of recognizing and isomerizing the phosphorylated Serine/Threonine‑Proline motif. Through this mechanism, PIN1 controls diverse cellular functions, including telomere maintenance. Both PIN1 overexpression and its involvement in oncogenic pathways are involved in several cancer types, including glioblastoma (GBM), a lethal disease with poor therapeutic resources. However, knowledge of the role of PIN1 in GBM is limited. Thus, the present work aimed to study the role of PIN1 as a telomere/telomerase regulator and its contribution to tumor biology. PIN1 knockout (KO) LN‑229 cell variant using CRISPR/Cas9 was developed and compared with PIN1 LN‑229 expressing cells. To study the effect of PIN1 absence, status of NF‑κB pathway was evaluated by luciferase reporter gene assay and quantitative PCR. Results revealed that PIN1 deletion in GBM cells diminished the active levels of NF‑κB and decrease the transcription of il‑8 and htert genes. Then, telomere/telomerase related processes were studied by RQ‑TRAP assay and telomere length determination by qPCR, obtaining a reduction both in telomerase activity as in telomere length in PIN1 KO cells. In addition, measurement of SA β‑galactosidase and caspase‑3 activities revealed that loss of PIN1 triggers senescence and apoptosis. Finally, migration, cell cycle progression and tumorigenicity were studied by flow cytometry/western blot, Transwell assay and invivo experiments, respectively. PIN1 deletion decreased migration as well as cell cycle progression by increasing doubling time and also resulted in the loss of LN‑229 cell ability to form tumors in mice. These results highlight the role of PIN1 in telomere homeostasis and GBM progression, which supports PIN1 as a potential molecular target for the development of novel therapeutic agents for GBM treatment.

A high-density 3-dimensional culture model of human glioblastoma for rapid screening of therapeutic resistance

Glioblastoma is among the most lethal cancers, with no known cure. A multitude of therapeutics are being developed or in clinical trials, but currently there are no ways to predict which patient may benefit the most from which drug. Assays that allow prediction of the tumor’s response to anti-cancer drugs may improve clinical decision-making. Here, we present a high-density 3D primary cell culture model for short-term testing from resected glioblastoma tissue that is set up on the day of surgery, established within 7 days and viable for at least 3 weeks. High-density 3D cultures contain tumor and host cells, including microglia, and retain key histopathological characteristics of their parent tumors, including proliferative activity, expression of the marker GFAP, and presence of giant cells. This provides a proof-of-concept that 3D primary cultures may be useful to model tumor heterogeneity. Importantly, we show that high-density 3D cultures can be used to test chemotherapy response within a 2–3-week timeframe and are predictive of patient response to Temozolomide therapy. Thus, primary high-density 3D cultures could be a useful tool for brain cancer research and prediction of therapeutic resistance.

Human EGFRvIII chimeric antigen receptor T cells demonstrate favorable safety profile and curative responses in orthotopic glioblastoma.

Glioblastoma is a highly aggressive and fatal brain malignancy, and effective targeted therapies are required. The combination of standard treatments including surgery, chemotherapy and radiotherapy is not curative. Chimeric antigen receptor (CAR) T cells are known to cross the blood-brain barrier, mediating antitumor responses. A tumor-expressed deletion mutant of the epidermal growth factor receptor (EGFRvIII) is a robust CAR Tcell target in glioblastoma. Here, we show our de novo generated, high-affinity EGFRvIII-specific CAR; GCT02, demonstrating curative efficacy in human orthotopic glioblastoma models. The GCT02 binding epitope was predicted using Deep Mutational Scanning (DMS). GCT02 CAR T cell cytotoxicity was investigated in three glioblastoma models in vitro using the IncuCyte platform, and cytokine secretion with a cytometric bead array. GCT02 in vivo functionality was demonstrated in two NSG orthotopic glioblastoma models. The specificity profile was generated by measuring T cell degranulation in response to coculture with primary human healthy cells. The GCT02 binding location was predicted to be located at a shared region of EGFR and EGFRvIII; however, the in vitro functionality remained exquisitely EGFRvIII specific. A single CAR T cell infusion generated curative responses in two orthotopic models of human glioblastoma in NSG mice. The safety analysis further validated the specificity of GCT02 for mutant-expressing cells. This study demonstrates the preclinical functionality of a highly specific CAR targeting EGFRvIII on human cells. This CAR could be an effective treatment for glioblastoma and warrants future clinical investigation.

RTID-03. DSP-0390, AN ORAL EMOPAMIL BINDING PROTEIN (EBP) INHIBITOR, IN PATIENTS WITH RECURRENT HIGH-GRADE GLIOMA: A FIRST-IN-HUMAN, PHASE 1 STUDY

Abstract The brain's cells are dependent on their own biosynthesis of cholesterol as the blood-brain barrier prevents uptake from the circulation. In normal glial cells, regulation of cholesterol synthesis depends on cell density and is turned off when density exceeds a certain level. Gliomas maintain high levels of cholesterol synthesis genes to support abnormal growth. Upregulation of cholesterol synthesis genes is associated with decreased survival in patients with glioblastoma (GBM). Therefore, gliomas are potentially sensitive to cholesterol synthesis inhibition. DSP-0390, an investigational small molecule, inhibits EBP, an enzyme in one of the last, crucial steps of cholesterol biosynthesis. By inhibiting cholesterol synthesis, cytotoxicity can be induced selectively against hyperproliferative GBM cells. DSP-0390 has shown significant antitumor activity in orthotopic xenograft models of human GBM (data on file).DSP-0390 will be evaluated in a phase 1 study in patients with recurrent, high-grade glioma (NCT05023551). Key eligibility criteria: age >18 years; Karnofsky Performance Status score >70%; adequate renal, hepatic, and hematologic function. Patients must not have multifocal disease, leptomeningeal metastasis or extracranial metastasis, abnormal electrocardiograms, or significant cardiovascular disease. In Dose Escalation, 21-30 patients with World Health Organization (WHO) grade III or IV malignant glioma who progressed after >1 prior therapy will be enrolled. Dose level enrollment will be guided by a Bayesian Logistic Regression Model until identification of the maximum tolerated dose or recommended dose for expansion. Dose Expansion for clinical activity will enroll approximately 20-40 patients with WHO grade IV GBM who progressed after primary therapy and have measurable disease. Patients will receive oral DSP-0390 once daily. Study endpoints include safety (treatment-emergent adverse events [AEs], serious AEs, and dose-limiting toxicities), efficacy (6-month progression-free survival [PFS], objective response, PFS, duration of response, and 12-month overall survival), pharmacokinetics (PK), and pharmacodynamic biomarkers. This study is currently recruiting in the United States and Japan.

Correlation between molar activity, injection mass and uptake of the PARP targeting radiotracer [18F]olaparib in mouse models of glioma

PurposeRadiopharmaceuticals targeting poly(ADP-ribose) polymerase (PARP) have emerged as promising agents for cancer diagnosis and therapy. PARP enzymes are expressed in both cancerous and normal tissue. Hence, the injected mass, molar activity and potential pharmacological effects are important considerations for the use of radiolabelled PARP inhibitors for diagnostic and radionuclide therapeutic applications. Here, we performed a systematic evaluation by varying the molar activity of [18F]olaparib and the injected mass of [TotalF]olaparib to investigate the effects on tumour and normal tissue uptake in two subcutaneous human glioblastoma xenograft models.Methods[18F]Olaparib uptake was evaluated in the human glioblastoma models: in vitro on U251MG and U87MG cell lines, and in vivo on tumour xenograft-bearing mice, after administration of [TotalF]olaparib (varying injected mass: 0.04–8.0 µg, and molar activity: 1–320 GBq/μmol).ResultsSelective uptake of [18F]olaparib was demonstrated in both models. Tumour uptake was found to be dependent on the injected mass of [TotalF]olaparib (µg) but not the molar activity. An injected mass of 1 μg resulted in the highest tumour uptake (up to 6.9 ± 1.3%ID/g), independent of the molar activity. In comparison, both the lower and higher injected masses of [TotalF]olaparib resulted in lower relative tumour uptake (%ID/g; P < 0.05). Ex vivo analysis of U87MG xenograft sections showed that the heterogeneity in [18F]olaparib intratumoural uptake correlated with PARP1 expression. Substantial upregulation of PARP1-3 expression was observed after administration of [TotalF]olaparib (> 0.5 µg).ConclusionOur findings show that the injected mass of [TotalF]olaparib has significant effects on tumour uptake. Moderate injected masses of PARP inhibitor-derived radiopharmaceuticals may lead to improved relative tumour uptake and tumour-to-background ratio for cancer diagnosis and radionuclide therapy.

Preclinical Efficacy and Toxicology Evaluation of RAC1 Inhibitor 1A-116 in Human Glioblastoma Models.

Simple SummaryMalignant gliomas are the most common primary central nervous system tumors in adults. Currently, this disease is associated with poor prognosis and is virtually incurable. There is a need to find novel targets and treatments to improve patient survival. This study shows the preclinical evaluation of 1A-116, a Rac1 inhibitor that showed in vitro antitumor activity on glioma cells. We also evaluated 1A-116 in vivo, showing a favorable toxicological profile and antitumor efficacy in an intracranial mouse tumor model. Altogether, our study provides important evidence of 1A-116 as a signal transduction-based precision therapy for glioma and also increases the evidence of Rac1 as a key molecular target in cancer.Malignant gliomas are the most common primary central nervous system tumor in adults. Despite current therapeutics, these tumors are associated with poor prognosis and a median survival of 16 to 19 months. This highlights the need for innovative treatments for this incurable disease. Rac1 has long been associated with tumor progression and plays a key role in glioma’s infiltrative and invasive nature. The aim of this study is to evaluate the 1A-116 molecule, a Rac1 inhibitor, as targeted therapy for this aggressive disease. We found that targeting Rac1 inhibits cell proliferation and cell cycle progression using different in vitro human glioblastoma models. Additionally, we evaluated 1A-116 in vivo, showing a favorable toxicological profile. Using in silico tools, 1A-116 is also predicted to penetrate the blood–brain barrier and present a favorable metabolic fate. In line with these results, 1A-116 i.p daily treatment resulted in a dose-dependent antitumor effect in an orthotopic IDH-wt glioma model. Altogether, our study provides a strong potential for clinical translation of 1A-116 as a signal transduction-based precision therapy for glioma and also increases the evidence of Rac1 as a key molecular target.

Characterization and comparison of human glioblastoma models

Glioblastoma (GBM) is one of the deadliest cancers. Treatment options are limited, and median patient survival is only several months. Translation of new therapies is hindered by a lack of GBM models that fully recapitulate disease heterogeneity. Here, we characterize two human GBM models (U87-luc2, U251-RedFLuc). In vitro, both cell lines express similar levels of luciferase and show comparable sensitivity to temozolomide and lapatinib exposure. In vivo, however, the two GBM models recapitulate different aspects of the disease. U87-luc2 cells quickly grow into large, well-demarcated tumors; U251-RedFLuc cells form small, highly invasive tumors. Using a new method to assess GBM invasiveness based on detecting tumor-specific anti-luciferase staining in brain slices, we found that U251-RedFLuc cells are more invasive than U87-luc2 cells. Lastly, we determined expression levels of ABC transporters in both models. Our findings indicate that U87-luc2 and U251-RedFLuc GBM models recapitulate different aspects of GBM heterogeneity that need to be considered in preclinical research.

Purple sweet potato delphinidin-3-rutin represses glioma proliferation by inducing miR-20b-5p/Atg7-dependent cytostatic autophagy

Glioma is the most common primary malignant intracranial tumor. Owing to highly aggressive invasiveness and metastatic properties, the prognosis of this disease remains poor even with surgery, radiotherapy, and chemotherapy. Rutin is a glycoside natural flavonoid that modulates microglia inflammatory profile and improves anti-glioma activity. Here, a glycoside flavonoid was extracted and named purple sweet potato delphinidin-3-rutin (PSPD3R). In an experiment using the subcutaneous xenograft model of human glioblastoma (GBM) and alamar blue assay, we found that PSPD3R suppressed the glioma proliferation both in vitro and in vivo. Flow cytometry assay and transmission electron microscopy observation revealed that PSPD3R stimulated glioma cell autophagy and apoptosis. High-throughput microRNA (miRNA) sequencing showed that PSPD3R substantially affected the miRNA expression of U251 cells. Acridine orange staining and immunoblotting indicated that PSPD3R regulated autophagy via Akt/Creb/miR-20b-5p in glioma cells. Luciferase reporter assays showed that autophagy-related gene 7 (Atg7) mRNA was the target gene of miR-20b-5p. The downregulation of miR-20b-5p inhibited glioma proliferation in vivo. In summary, PSPD3R regulated autophagy in glioma via the Akt/Creb/miR-20b-5p/Atg7 axis. This work unraveled the molecular mechanism of PSPD3R-induced autophagy in glioma and revealed its potential as a therapeutic agent for glioma treatment.

Immune landscape of a genetically engineered murine model of glioma compared with human glioma.

Novel therapeutic strategies targeting glioblastoma (GBM) often fail in the clinic, partly because preclinical models in which hypotheses are being tested do not recapitulate human disease. To address this challenge, we took advantage of our previously developed spontaneous Qk/Trp53/Pten (QPP) triple-knockout model of human GBM, comparing the immune microenvironment of QPP mice with that of patient-derived tumors to determine whether this model provides opportunity for gaining insights into tumor physiopathology and preclinical evaluation of therapeutic agents. Immune profiling analyses and single-cell sequencing of implanted and spontaneous tumors from QPP mice and from patients with glioma revealed intratumoral immune components that were predominantly myeloid cells (e.g., monocytes, macrophages, and microglia), with minor populations of T, B, and NK cells. When comparing spontaneous and implanted mouse samples, we found more neutrophils and T and NK cells in the implanted model. Neutrophils and T and NK cells were increased in abundance in samples derived from human high-grade glioma compared with those derived from low-grade glioma. Overall, our data demonstrate that our implanted and spontaneous QPP models recapitulate the immunosuppressive myeloid-dominant nature of the tumor microenvironment of human gliomas. Our model provides a suitable tool for investigating the complex immune compartment of gliomas.

Abstract 349: Pharmacokinetics- and pharmacodynamics-based evaluation of quisinostat as a radiosensitizer in preclinical models of human glioblastoma

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

DSP-0390, an oral emopamil binding protein (EBP) inhibitor, in patients with recurrent high-grade glioma: A first-in-human, phase 1 study.

TPS2077 Background: The brain’s cells are fully dependent on their own de novo biosynthesis of cholesterol as the blood-brain barrier prevents its uptake from the circulation. In normal glial cells, proper regulation of cholesterol synthesis depends on its cell density and is turned off when the cell density exceeds a certain level. On the other hand, gliomas maintain high levels of cholesterol synthesis to support abnormal growth under any condition. Upregulation of cholesterol synthesis genes is associated with decreased survival in patients with glioblastoma (GBM). Therefore, gliomas are potentially sensitive to cholesterol synthesis inhibition. DSP-0390, an investigational small molecule, is an inhibitor of EBP, an enzyme in one of the last and crucial steps of cholesterol biosynthesis. By inhibiting de novo cholesterol synthesis, cytotoxicity can be induced more selectively against hyperproliferative GBM cells. DSP-0390 has shown significant antitumor activity in orthotopic xenograft models of human GBM (data on file). Methods: DSP-0390 will be evaluated in a phase 1 study in patients with recurrent, high-grade glioma (NCT05023551). Key eligibility criteria: age ≥18 years; Karnofsky Performance Status score ≥70%; and adequate renal, hepatic, and hematologic function. Patients must not have multifocal disease, leptomeningeal metastasis or extracranial metastasis, abnormal electrocardiograms, or significant cardiovascular disease. In Dose Escalation, 21–30 patients with World Health Organization (WHO) grade III or IV malignant glioma who progressed after ≥1 prior therapy will be enrolled. Dose level enrollment will be guided by a Bayesian Logistic Regression Model until identification of the maximum tolerated dose or recommended dose for expansion. Dose Expansion for clinical activity will enroll approximately 20–40 patients with WHO grade IV GBM who progressed after primary therapy and have measurable disease. Patients will receive oral DSP-0390 once daily. Study endpoints include safety (treatment-emergent adverse events [AEs], serious AEs, and dose-limiting toxicities), efficacy (6-month progression-free survival [PFS], objective response, PFS, duration of response, and 12-month overall survival), pharmacokinetics (PK), and pharmacodynamic biomarkers. This study is currently recruiting in the United States and Japan. Clinical trial information: NCT05023551.

The effects of CHF6467, a new mutated form of NGF, on cell models of human glioblastoma. A comparison with wild-type NGF

CHF6467 is a mutated form of human recombinant nerve growth factor (NGF). The mutation selectively disrupts the binding of NGF to its p75NTR receptor while maintaining the affinity toward TrkA receptor. Because of such different profile of receptor interaction, CHF6467 maintains unaltered the neurotrophic and neuroprotective properties of wild-type NGF but shows reduced algogenic activity. In this study, we investigated the effects of CHF6467 on mortality, proliferation, cell-damage and migration in three human glioblastoma cell lines (U87MG, T98G, LN18), and in the rat astrocytoma C6 cells. Both CHF6467 and wild-type NGF, given in the range 1-50 ng/ml, did not modify cell proliferation, metabolism and migration, as well as the number of live/dead cells. The present in vitro data are predictive of a lack of tumorigenic activity by both wild-type NGF and CHF6467 on these cell types in vivo, and warrant for CHF6467 further clinical development.

The Botanical Drug PBI-05204, a Supercritical CO2 Extract of Nerium Oleander, Is Synergistic With Radiotherapy in Models of Human Glioblastoma.

Glioblastoma multiforme (GBM) is the most common as well as one of the most malignant types of brain cancer. Despite progress in development of novel therapies for the treatment of GBM, it remains largely incurable with a poor prognosis and a very low life expectancy. Recent studies have shown that oleandrin, a unique cardiac glycoside from Nerium oleander, as well as a defined extract (PBI-05204) that contains this molecule, inhibit growth of human glioblastoma, and modulate glioblastoma patient-derived stem cell-renewal properties. Here we demonstrate that PBI-05204 treatment leads to an increase in vitro in the sensitivity of GBM cells to radiation in which the main mechanisms are the transition from autophagy to apoptosis, enhanced DNA damage and reduced DNA repair after radiotherapy (RT) administration. The combination of PBI-05204 with RT was associated with reduced tumor progression evidenced by both subcutaneous as well as orthotopic implanted GBM tumors. Collectively, these results reveal that PBI-05204 enhances antitumor activity of RT in preclinical/murine models of human GBM. Given the fact that PBI-05204 has already been examined in Phase I and II clinical trials for cancer patients, its efficacy when combined with standard-of-care radiotherapy regimens in GBM should be explored.

SS-4 is a highly selective small molecule inhibitor of STAT3 tyrosine phosphorylation that potently inhibits GBM tumorigenesis in vitro and in vivo

Glioblastoma (GBM) is a highly aggressive cancer with a dismal prognosis. Constitutively active STAT3 has a causal role in GBM progression and is associated with poor patient survival. We rationally designed a novel small molecule, SS-4, by computational modeling to specifically interact with STAT3. SS-4 strongly and selectively inhibited STAT3 tyrosine (Y)-705 phosphorylation in MT330 and LN229 GBM cells and inhibited their proliferation and induced apoptosis with an IC50 of ∼100 nM. The antiproliferative and apoptotic actions of SS-4 were Y-705 phosphorylation dependent, as evidenced by its lack of effects on STAT3 knockout (STAT3KO) cells or STAT3KO cells that overexpressed a phospho-Y705 deficient (STAT3Y705F) mutant, and the recovery of effects when wild-type STAT3 or a phospho-serine (S)727 deficient mutant was expressed in STAT3KO cells. SS-4 increased the expression of STAT3 repressed genes, while decreasing the expression of STAT3 promoted genes. Importantly, SS-4 markedly reduced the growth of GBM intracranial tumor xenografts. These data together identify SS-4 as a potent STAT3 inhibitor that selectively blocks Y705-phosphorylation, induces apoptosis, and inhibits growth of human GBM models in vitro and in vivo.

Ionizing Radiation-Induced GDF15 Promotes Angiogenesis in Human Glioblastoma Models by Promoting VEGFA Expression Through p-MAPK1/SP1 Signaling.

Glioblastoma multiforme (GBM), the most aggressive cancer type that has a poor prognosis, is characterized by enhanced and aberrant angiogenesis. In addition to surgical resection and chemotherapy, radiotherapy is commonly used to treat GBM. However, radiation-induced angiogenesis in GBM remains unexplored. This study examined the role of radiation-induced growth/differentiation factor-15 (GDF15) in regulating tumor angiogenesis by promoting intercellular cross-talk between brain endothelial cells (ECs) and glioblastoma cells. Radiation promoted GDF15 secretion from human brain microvascular endothelial cells (HBMVECs). Subsequently, GDF15 activated the transcriptional promoter VEGFA in the human glioblastoma cell line U373 through p-MAPK1/SP1 signaling. Upregulation of vascular endothelial growth factor (VEGF) expression in U373 cells resulted in the activation of angiogenic activity in HBMVECs via KDR phosphorylation. Wound healing, tube formation, and invasion assay results revealed that the conditioned medium of recombinant human GDF15 (rhGDF15)-stimulated U373 cell cultures promoted the angiogenic activity of HBMVECs. In the HBMVEC-U373 cell co-culture, GDF15 knockdown mitigated radiation-induced VEGFA upregulation in U373 cells and enhanced angiogenic activity of HBMVECs. Moreover, injecting rhGDF15-stimulated U373 cells into orthotopic brain tumors in mice promoted angiogenesis in the tumors. Thus, radiation-induced GDF15 is essential for the cross-talk between ECs and GBM cells and promotes angiogenesis. These findings indicate that GDF15 is a putative therapeutic target for patients with GBM undergoing radio-chemotherapy.

T-cell dysfunction in the glioblastoma microenvironment is mediated by myeloid cells releasing interleukin-10

Despite recent advances in cancer immunotherapy, certain tumor types, such as Glioblastomas, are highly resistant due to their tumor microenvironment disabling the anti-tumor immune response. Here we show, by applying an in-silico multidimensional model integrating spatially resolved and single-cell gene expression data of 45,615 immune cells from 12 tumor samples, that a subset of Interleukin-10-releasing HMOX1+ myeloid cells, spatially localizing to mesenchymal-like tumor regions, drive T-cell exhaustion and thus contribute to the immunosuppressive tumor microenvironment. These findings are validated using a human ex-vivo neocortical glioblastoma model inoculated with patient derived peripheral T-cells to simulate the immune compartment. This model recapitulates the dysfunctional transformation of tumor infiltrating T-cells. Inhibition of the JAK/STAT pathway rescues T-cell functionality both in our model and in-vivo, providing further evidence of IL-10 release being an important driving force of tumor immune escape. Our results thus show that integrative modelling of single cell and spatial transcriptomics data is a valuable tool to interrogate the tumor immune microenvironment and might contribute to the development of successful immunotherapies.

ATX-101, a Peptide Targeting PCNA, Has Antitumor Efficacy Alone or in Combination with Radiotherapy in Murine Models of Human Glioblastoma.

Simple SummaryPCNA is an interesting target for cancertreatment due to its essential activities in DNA replication and repair and its recently discovered regulatory roles in cellular signaling. Here, we demonstrate that ATX-101, a peptide targeting PCNA, has antitumor effects as a single agent and radiosensitizing properties in glioblastoma multiforme models.Cell proliferation requires the orchestrated actions of a myriad of proteins regulating DNA replication, DNA repair and damage tolerance, and cell cycle. Proliferating cell nuclear antigen (PCNA) is a master regulator which interacts with multiple proteins functioning in these processes, and this makes PCNA an attractive target in anticancer therapies. Here, we show that a cell-penetrating peptide containing the AlkB homolog 2 PCNA-interacting motif (APIM), ATX-101, has antitumor activity in a panel of human glioblastoma multiforme (GBM) cell lines and patient-derived glioma-initiating cells (GICs). Their sensitivity to ATX-101 was not related to cellular levels of PCNA, or p53, PTEN, or MGMT status. However, ATX-101 reduced Akt/mTOR and DNA-PKcs signaling, and a correlation between high Akt activation and sensitivity for ATX-101 was found. ATX-101 increased the levels of γH2AX, DNA fragmentation, and apoptosis when combined with radiotherapy (RT). In line with the in vitro results, ATX-101 strongly reduced tumor growth in two subcutaneous xenografts and two orthotopic GBM models, both as a single agent and in combination with RT. The ability of ATX-101 to sensitize cells to RT is promising for further development of this compound for use in GBM.

"Smart" nanoscalpel for microsurgery of glial tumors of the human brain

We studied the effectiveness of magnetomechanical therapy in the treatment of brain glial tumors using magnetic nanodiscs functionalized with DNA aptamers to human brain tumor glial cells. • Materials and methods. The formation of a model of human glioblastoma was carried out by intracranial injection of tumor cells of glioblastoma obtained from a patient with glioblastoma. Antitumor therapy was carried out using nanodiscs modified with the Gli233 aptamer. The growth of the glial tumor was monitored using NMR tomography. • Results and discussion. Therapy of a glial tumor during 4 sessions of magnetomechanical therapy using a "smart" nanoscalpel in MF (10Hz, 100Oe) led to a significant reduction in its size, while glial tumors in mice that were treated with nanodiscs modified with nonspecific aptamers continued to increase in size. • Conclusion. Microsurgery using three-layer magnetic nanodisks with a quasi-dipole structure (Au/Ni/Au) modified with the specific for glial cells Gli233 aptamer (“smart” nanoscalpel) is effective for the treatment of human glial tumors in the brain.