Concentrations Of Temozolomide Research Articles

MELATONIN ENHANCES TEMOZOLOMIDE-INDUCED APOPTOSIS IN GLIOBLASTOMA AND NEUROBLASTOMA CELLS.

The combination of temozolomide (TMZ) and paclitaxel (PTX) is the most commonly used chemotherapy regimen for glioblastoma, but there is no specific treatment for neuroblastoma due to the acquired multidrug resistance. Approximately half of treated glioblastoma patients develop resistance to TMZ and experience serious side effects. Melatonin (MEL), a multifunctional hormone long known for its antitumor effects, has a great advantage in combination cancer therapy thanks to its ability to affect tumors differently than normal cells. This study aims to evaluate the in vitro inhibitory effects of MEL in combination with TMZ on cancer cell viability and to elucidate the underlying mechanisms in the glioblastoma and neuroblastoma cell lines. C6 (Rattus norvegicus) and N1E-115 (Mus musculus) cancer cell lines and C8-D1A (mice) healthy cell lines were used. Cell proliferation was evaluated using the MTT test. IC50 values were determined by probit analysis. Two concentrations of TMZ (IC50 and 1/2 IC50) were used to induce cytotoxicity in the C6 and N1E-115 cell lines, both alone and in combination with PXT and MEL (all at IC50). The viable, dead, and apoptotic cells were determined by image-based cytometry using Annexin V/PI staining. The gene expression related to signaling pathways was assessed by the quantitative reverse transcription polymerase chain reaction (qRT-PCR), and key proteins were identified by the Western blot analysis. MTT assay showed that the combination of TMZ and MEL significantly reduces the viability of both glioblastoma and neuroblastoma cells compared to the vehicle-treated controls. Notably, MEL combined with 1/2 IC50 TMZ showed a significant death rate of cancer cells compared to controls and PTX. According to qRT-PCR data, the TMZ + MEL combination resulted in the upregulation of the genes of antioxidative enzymes (Sod1 and Sod2) and DNA repair genes (Mlh1, Exo1, and Rad18) in both cell lines. Moreover, the levels of Nfkb1 and Pik3cg were significantly reduced following the TMZ + MEL treatment. The combination of MEL with TMZ also enhanced the cell cycle arrest and increased the expression of p53 and pro-apoptotic proteins (Bax and caspase-3), while significantly decreasing the expression of anti-apoptotic protein Bcl-2. Our findings indicate that the combination of MEL with a low dose of TMZ may serve as an upstream inducer of apoptosis. This suggests the potential development of a novel selective therapeutic strategy as an alternative to TMZ for the treatment of both glioblastoma and neuroblastoma.

Kalata B1 Enhances Temozolomide Toxicity to Glioblastoma Cells.

Glioblastoma (GBM) is the most aggressive cancer originating in the brain, but unfortunately combination treatments with resection, radiation, and chemotherapy are relatively ineffective. Therefore, novel methods of adjuvant therapy are critically needed. Cyclotides are plant-derived circular peptides that chemosensitize drug-resistant breast cancer to doxorubicin. We analyzed naturally occurring and synthetic cyclotides (Cycloviolacin O3, Cycloviolacin O19, natural Kalata B1, synthetic Kalata B1, and Vitri E) alone and in co-exposure treatments with the drug temozolomide (TMZ) in human glioblastoma cells. The cyclotides were identified by UPLC-PDA and HPLC-UV. The synthetic Kalata B1 sequence was verified with orbitrap LC-MS, and structural confirmation was provided by NMR spectroscopy. The cyclotides displayed dose-dependent cytotoxicity (IC50 values 2.4-21.1 µM) both alone and as chemosensitizers of U-87 MG and T 98 cells to TMZ. In fact, a 16-fold lower concentration of TMZ (100 µM) was needed for significant cytotoxicity in U-87 MG cells co-exposed to synthetic Kalata B (0.5 µM). Similarly, a 15-fold lower concentration of TMZ (75 µM) was required for a significant reduction in cell viability in T 98 cells co-exposed to synthetic Kalata B1 (0.25 µM). Kalata B1 remained stable in human serum stability assays. The data support the assertion that cyclotides may chemosensitize glioblastoma cells to TMZ.

Acquired Temozolomide Resistance Instructs Patterns of Glioblastoma Behavior in Gelatin Hydrogels.

Acquired drug resistance in glioblastoma (GBM) presents a major clinical challenge and is a key factor contributing to abysmal prognosis, with less than 15 months median overall survival. Aggressive chemotherapy with the frontline therapeutic, temozolomide (TMZ), ultimately fails to kill residual highly invasive tumor cells after surgical resection and radiotherapy. Here, a 3D engineered model of acquired TMZ resistance is reported using two isogenically matched sets of GBM cell lines encapsulated in gelatin methacrylol hydrogels. Response of TMZ-resistant versus TMZ-sensitive GBM cell lines within the gelatin-based extracellular matrix platform is benchmarked and drug response at physiologically relevant TMZ concentrations is further validated. The changes in drug sensitivity, cell invasion, and matrix-remodeling cytokine production are shown as the result of acquired TMZ resistance. This platform lays the foundation for future investigations targeting key elements of the GBM tumor microenvironment to combat GBM's devastating impact by advancing the understanding of GBM progression and treatment response to guide the development of novel treatment strategies.

Abstract 7226: Synergy between ONC201 and temozolomide on ATF4 integrated stress response in glioblastoma

Abstract Glioblastoma multiforme (GBM) is the most common primary brain tumor in adults, with a median survival of 15 months. The standard of care involves surgical resection followed by chemoradiotherapy. Even with the full course of treatment, GBM is uncurable, and patients eventually relapse. Although temozolomide (TMZ) is the first-choice chemotherapeutic for GBM, tumor drug resistance limits the effects of the treatment. There is a growing need for new therapies with enhanced efficacy on GBM tumors. Dordaviprone (ONC201) is a TNF-related apoptosis inducing ligand (TRAIL)-inducing compound that is increasingly being studied for GBM treatment. This imipridone small molecule induces apoptosis by activating the integrated stress response, which involves transcription factors ATF4 and CHOP. Subsequently, these transcription factors upregulate proapoptotic protein TRAIL and its receptor, Death Receptor 5 (DR5). This study aims to investigate the combinatory effects of chemotherapeutics TMZ and ONC201 on GBM. Preliminary studies examined the cell viability of GBM cell line U251MG after dual treatment of 0 to 10uM of ONC201 and 0 to 200uM of TMZ using the CellTiter-Glo assay. Synergy was observed between the two drugs at higher concentrations of TMZ. This synergistic interaction was further characterized using western blots. When 0, 2, and 4uM of ONC201 were administered alone or in combination with 0, 60, and 120uM of TMZ, greater ATF4 expression was observed with the dual treatments than with the monotherapies. Subsequent western blots were conducted using three GBM cell lines (U251MG, T98G, and SNB19-GFP) at three time points (8, 24, and 48 hours). All cell lines were treated with 0 and 5uM of ONC201 alone or in combination with 0 and 200uM of TMZ. For all three cell lines, ATF4 expression increased in the combined presence of TMZ and ONC201. However, the effects of TMZ on ONC201-induced ATF4 expression were most pronounced in U251MG and least conclusive in T98G. Additionally, the time points in which this increase in ATF4 expression occurred varied among cell lines. CHOP expression was also probed and was seen to increase with the combination treatment in U251MG. These observations suggest a potential synergistic relationship between chemotherapeutics ONC201 and TMZ in the ATF4 integrated stress response pathway in GBM. By exploring the effects of this dual treatment on upstream and downstream factors in the pathway, we can identify, characterize, and potentially target the root cause of this synergy. Citation Format: Josephine Chen, Tyler J. Roady, Lanlan Zhou, Wafik S. El-Deiry. Synergy between ONC201 and temozolomide on ATF4 integrated stress response in glioblastoma [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 7226.

Abstract 3757: The development of MAGMAS as a potential therapeutic target in temozolomide-resistant glioma

Abstract Glioblastoma (GBM) is one of the most aggressive types of brain cancer and remains a significant challenge for drug development in oncology today. Temozolomide (TMZ) is the standard of care for GBM, and resistance to this drug is modulated partly by the DNA repair enzyme O6-methylguanine-DNA methyltransferase (MGMT). Clinical studies demonstrated that low MGMT expression in GBM enhances sensitivity to TMZ and prolongs survival for patients. However, recent clinical studies indicate MGMT is a discordant biomarker since the predictive value of MGMT methylation is relatively low and there is a lack of modalities to inhibit MGMT expression. Furthermore, glioma stem cells (GSC) are another contributing factor for resistance due to tumor heterogeneity, self-renewal capacity and activation of DNA repair responses to overcome the effect of radiation and TMZ treatment. These resistance mechanisms contribute to tumor recurrence and present major impediments for the development of effective treatments. MAGMAS, 13.8 kDa mitochondria protein, is a translocase of the inner mitochondrial membrane and regulates oxidative phosphorylation. Specifically, MAGMAS regulates the ATP stimulatory activity of DNAJC19 on mtHSP70 that drives the movement of peptides through the TIM23 complex into the mitochondrial matrix. Recent evidence suggests that MAGMAS can regulate oxidative phosphorylation via interactions with the respiratory complexes present in the inner mitochondrial membrane. Previously, our lab has shown MAGMAS overexpression in malignant glioma tissues of patients and murine intracranial xenografts. Additionally, pharmacological inhibition of MAGMAS with the small molecule BT9 promoted cytotoxicity and impaired respiratory functions in malignant glioma cells demonstrating that MAGMAS is a promising potential target for GBM patients. However, further studies are needed to investigate the role of MAGMAS across the spectrum from initial tumorigenesis to disease progression and to further determine how MAGMAS is implicated during the development of treatment resistance. This study is designed to investigate the role of MAGMAS in GSCs and their contribution to TMZ resistance. We observed temozolomide-resistant (TR) U251-TR and D54 MG-TR GBM cell lines expressed significantly higher levels of MAGMAS and several stem cell markers than their drug-sensitive (S) parental cell lines. Therefore, we hypothesized that silencing MAGMAS re-sensitizes TR GBM cell lines to TMZ. We found that MAGMAS knockdown in D54-MG and U251 S/TR cells in the presence of escalating TMZ concentrations, caused enhanced sensitivity to TMZ. Consistent with these results, inhibition of MAGMAS with the novel BT9 inhibitor enhances the TMZ sensitivity in the TMZ-resistant cell lines. Additional studies of patient-derived cell lines are being utilized further to validate MAGMAS as it is an attractive target in TMZ-resistant GBM patients. Citation Format: Jennifer D. Tran, Javier J. Lepe, Maria C. Kenney, Bhaskar C. Das, Daniela A. Bota. The development of MAGMAS as a potential therapeutic target in temozolomide-resistant glioma [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 3757.

Neuropilin-1 enhances temozolomide resistance in glioblastoma via the STAT1/p53/p21 axis.

Glioblastoma (GBM) is the most prevalent primary intracranial tumor. Temozolomide (TMZ) is the first-line chemotherapy for GBM. Nonetheless, the development of TMZ resistance has become a main cause of treatment failure in GBM patients. Evidence suggests that neuropilin-1 (NRP-1) silencing can attenuate GBM cell resistance to TMZ. This study aims to determine potential mechanisms by which NRP-1 affects TMZ resistance in GBM. The parental U251 and LN229 GBM cells were exposed to increasing concentrations of TMZ to construct TMZ-resistant GBM cells (U251/TMZ, LN229/TMZ). BALB/c nude mice were injected with U251/TMZ cells to establish the xenograft mouse model. Functional experiments were carried out to examine NRP-1 functions. Western blotting and real-time quantitative polymerase chain reaction were used to evaluate molecular protein and mRNA expression, respectively. Immunohistochemical staining showed NRP-1 and STAT1 expression in mouse tumors. The results showed that NRP-1 was highly expressed in TMZ-resistant cells. Moreover, knocking down NRP-1 attenuated the TMZ resistance of U251/TMZ cells, while upregulating NRP-1 enhanced TMZ resistance of the parental cells. NRP-1 silencing elevated GBM cell sensitivity to TMZ in tumor-bearing mice. Depleting NRP-1 reduced STAT1, p53, and p21 expression in U251/TMZ cells. STAT1 depletion offset NRP-1 silencing evoked attenuation of GBM cell resistance to TMZ. Collectively, our study reveals that NRP-1 enhances TMZ resistance in GBM possibly by regulating the STAT1/p53/p21 axis.

CRISPR-Cas9 library screening combined with an exosome-targeted delivery system addresses tumorigenesis/TMZ resistance in the mesenchymal subtype of glioblastoma.

Rationale: The large-scale genomic analysis classifies glioblastoma (GBM) into three major subtypes, including classical (CL), proneural (PN), and mesenchymal (MES) subtypes. Each of these subtypes exhibits a varying degree of sensitivity to the temozolomide (TMZ) treatment, while the prognosis corresponds to the molecular and genetic characteristics of the tumor cell type. Tumors with MES features are predominantly characterized by the NF1 deletion/alteration, leading to sustained activation of the RAS and PI3K-AKT signaling pathways in GBM and tend to acquire drug resistance, resulting in the worst prognosis compared to other subtypes (PN and CL). Here, we used the CRISPR/Cas9 library screening technique to detect TMZ-related gene targets that might play roles in acquiring drug resistance, using overexpressed KRAS-G12C mutant GBM cell lines. The study identified a key therapeutic strategy to address the chemoresistance against the MES subtype of GBM. Methods: The CRISPR-Cas9 library screening was used to discover genes associated with TMZ resistance in the U87-KRAS (U87-MG which is overexpressed KRAS-G12C mutant) cells. The patient-derived GBM primary cell line TBD0220 was used for experimental validations in vivo and in vitro. Chromatin isolation by RNA purification (ChIRP) and chromatin immunoprecipitation (ChIP) assays were used to elucidate the silencing mechanism of tumor suppressor genes in the MES-GBM subtype. The small-molecule inhibitor EPIC-0412 was obtained through high-throughput screening. Transmission electron microscopy (TEM) was used to characterize the exosomes (Exos) secreted by GBM cells after TMZ treatment. Blood-derived Exos-based targeted delivery of siRNA, TMZ, and EPIC-0412 was optimized to tailor personalized therapy in vivo. Results: Using the genome-wide CRISPR-Cas9 library screening, we found that the ERBIN gene could be epigenetically regulated in the U87-KRAS cells. ERBIN overexpression inhibited the RAS signaling and downstream proliferation and invasion effects of GBM tumor cells. EPIC-0412 treatment inhibited tumor proliferation and EMT progression by upregulating the ERBIN expression both in vitro and in vivo. Genome-wide CRISPR-Cas9 screening also identified RASGRP1(Ras guanine nucleotide-releasing protein 1) and VPS28(Vacuolar protein sorting-associated protein 28) genes as synthetically lethal in response to TMZ treatment in the U87-KRAS cells. We found that RASGRP1 activated the RAS-mediated DDR pathway by promoting the RAS-GTP transformation. VPS28 promoted the Exos secretion and decreased intracellular TMZ concentration in GBM cells. The targeted Exos delivery system encapsulating drugs and siRNAs together showed a powerful therapeutic effect against GBM in vivo. Conclusions: We demonstrate a new mechanism by which ERBIN is epigenetically silenced by the RAS signaling in the MES subtype of GBM. Restoration of the ERBIN expression with EPIC-0412 significantly inhibits the RAS signaling downstream. RASGRP1 and VPS28 genes are associated with the promotion of TMZ resistance through RAS-GDP to RAS-GTP transformation and TMZ efflux, as well. A quadruple combination therapy based on a targeted Exos delivery system demonstrated significantly reduced tumor burden in vivo. Therefore, our study provides new insights and therapeutic approaches for regulating tumor progression and TMZ resistance in the MES-GBM subtype.

Abstract B027: Synergy between ONC201 and temozolomide on ATF4 integrated stress response in glioblastoma

Abstract Glioblastoma multiforme (GBM) is the most common primary brain tumor in adults with a median survival time of 15 months. The standard of care involves surgical resection followed by chemoradiotherapy. Even with the full course of treatment, GBM is uncurable, and patients eventually relapse. Although temozolomide (TMZ) is the first-choice chemotherapeutic for GBM, tumor drug resistance limits the effects of the treatment. There is a growing need for new therapies with enhanced efficacy on GBM tumors. ONC201 is a TNF-related apoptosis-inducing ligand (TRAIL)-inducing compound that is increasingly being studied for GBM treatment. This small molecule induces apoptosis by activating the integrated stress response, which involves transcription factors ATF4 and CHOP. Subsequently, these transcription factors upregulate proapoptotic protein TRAIL and its receptor, Death Receptor 5 (DR5). This study aims to investigate the combinatory effects of chemotherapeutics ONC201 and TMZ on GBM. Preliminary studies examined the cell viability of GBM cell line U251MG using ONC201 concentrations up to 40uM and TMZ concentrations up to 200uM. Additionally, western blots performed with 0, 2, and 4uM of ONC201 in combination with 0 and 120uM of TMZ demonstrated greater ATF4 expression when both drugs were administered concurrently than when either drug was administered alone. Subsequent western blots were conducted using three GBM cell lines (U251MG, T98G, and SNB19-GFP) at three time points (8, 24, and 48 hours). All cell lines were treated with 0 and 4uM of ONC201 in combination with 0 and 120uM of TMZ. At all three time points and for all three cell lines, ATF4 expression increased in the combined presence of TMZ and ONC201. However, the effects of TMZ on ONC201-induced ATF4 expression were most pronounced in U251MG. These observations suggest synergy between chemotherapeutics ONC201 and TMZ in the ATF4 integrated stress response pathway in GBM. By exploring the effects of this dual treatment on upstream and downstream factors in the integrated stress response pathway, we can identify, characterize, and potentially target the root cause of this synergy. Citation Format: Josephine Chen, Andrew George, Lanlan Zhou, Wafik El-Deiry. Synergy between ONC201 and temozolomide on ATF4 integrated stress response in glioblastoma [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B027.

DDEL-13. CILIA INHIBITORS SYNERGIZE WITH TEMOZOLOMIDE TO DRAMATICALLY IMPROVE SURVIVAL IN ORTHOTOPIC MURINE MODELS OF GLIOBLASTOMA

Abstract Patients with malignant brain tumors such as glioblastoma face a poor prognosis with a median survival of less than two years. Recent glymphatic studies demonstrate the interconnectivity between the fluid in the brain parenchymal and ventricular spaces. We reasoned enhancing the accumulation of a drug in the cerebral spinal fluid (CSF) of the ventricular space will also enhance the accumulation of the drug in the parenchyma where a GBM resides. To improve drug accumulation in the CSF, we modulated the motility of ependymal cell cilia. Ependymal cells coat the ventricles and display motile cilia that mechanically propel CSF through the ventricular space. Impairing ciliary motion could slow down CSF turnover without affecting the overall amount of CSF in the brain and central nervous system. To test these hypotheses, we identified FDA-approved therapeutics that are known to impair airway cilia as a “side effect.” Seven candidate drugs were administered into the ventricle of mice bearing orthotopic GBM in combination with systemic temozolomide. Five of the seven cilia-inhibiting drugs significantly improved overall survival when combined with temozolomide compared to controls. The lead candidate, lidocaine, demonstrated a synergistic relationship with temozolomide, and 100% of animals treated with the combination survived to the study endpoint tumor free. We demonstrate lidocaine can impair cilia motility in vitro and in vivo. Combining intraventricular lidocaine with temozolomide is well tolerated and results in a 2.8-fold increase in brain temozolomide concentration per dose compared to controls. Finally, we validate these data in two human PDX orthotopic xenograft GBM murine models, including an un-methylated MGMT GBM that became sensitive to temozolomide with the combination treatment. These studies offer a new concept for treating malignant brain tumors and potentially a scheme to improve the treatment of additional encephalopathies in the future.

Coixendide efficacy in combination with temozolomide in glioblastoma and transcriptome analysis of the mechanism

The purpose of this study was to explore the role of coixendide (Coix) combine with temozolomide (TMZ) in the treatment of Glioblastoma (GBM) and explore its possible mechanism. CCK-8 was used to determine the inhibitory rate of Coix group, TMZ group and drug combination group on GBM cells, and the combination index (CI) was calculated to determine whether they had synergistic effect. Then RNA was extracted from each group, transcriptome sequencing was performed, and differentially expressed genes (DEGs) were identified. The possible mechanism was analyzed by GO enrichment analysis and KEGG enrichment analysis. The CI of Coix and TMZ indicating a synergistic effect when TMZ concentration is 0.1 mg/ml and Coix concentration is 2 mg/ml. Transcriptome sequencing analysis showed that interferon (IFN) related genes were down-regulated by Coix and up-regulated by TMZ and combined drugs, however, the up-regulation induced by combined drugs was less than that of TMZ. Besides IFN related genes, cholesterol metabolism pathway were also been regulated. Coix and TMZ have synergistic effects in the treatment of GBM at certain doses. RNA-Seq results suggested that the abnormal on genetic materials caused by DNA damage induced by TMZ treatment can be sensed by IFN related genes and activates antiviral IFN signaling, causing the activation of repairing mechanism and drug resistance. Coix inhibits IFN related genes, thereby inhibits drug resistance of TMZ. In addition, the activation of ferroptosis and the regulation of DEGs in cholesterol metabolism pathway were also contributed to the synergistic effects of Coix and TMZ.

Recommended phase 2 dose (RP2D) for abemaciclib in combination with irinotecan (IRN) and temozolomide (TMZ) in pediatric patients with relapsed/refractory solid tumors (JPCS Part A).

10039 Background: There remains an unmet need to improve outcomes for pediatric patients (pts) with relapsed/refractory solid tumors. Abemaciclib is an orally administered, selective cyclin-dependent kinase (CDK) 4 & 6 inhibitor approved for breast cancer. Based upon preclinical data and the role of CDK4 & 6 in multiple pediatric solid tumor types, we hypothesized that abemaciclib in combination with IRN and TMZ would demonstrate clinical activity to support further pediatric development. This Phase 1b study aimed to determine the RP2D of abemaciclib in combination with IRN and TMZ in this pt population. Methods: This dose escalation study was part A of study JPCS (NCT04238819), and enrolled pts ≤18 years, weighing ≥10 kg, with a body-surface area ≥0.5 m2, and with relapsed/refractory solid tumors, including CNS tumors, that progressed on standard treatment or experienced disease recurrence. IRN (50 mg/m2) and TMZ (100 mg/m2) were administered on days 1-5 of 21-day cycles and remain the same for all dose levels, while abemaciclib was tested on four dose levels: 55, 70, 90, and 115 mg/m2 twice daily (BID). The starting dose was 70 mg/m2 BID for abemaciclib. The maximum tolerated dose was defined as the highest dose level at which < 33% of pts experienced a dose-limiting toxicity (DLT) during Cycle 1. The primary objective, determination of the abemaciclib RP2D, was defined based on the totality of safety, tolerability, and pharmacokinetic (PK) results, and confirmed in an expansion cohort. Secondary objectives included safety and response assessments. Results: At the data cut-off of 13 June 2022, a total of 20 pts received abemaciclib at 70 mg/m2 (n = 7) or 55 mg/m2 (n = 13). The most frequent diagnoses (≥3 pts) were medulloblastoma (n = 4), malignant glioma (n = 3), and rhabdomyosarcoma (n = 3). At the starting dose of 70 mg/m2, DLTs were experienced by 3 pts (neutropenia [n = 3; 43%]; gamma glutamyltransferase increased [n = 1; 14%]). The dose was de-escalated to 55 mg/m2, at which one pt (8%) experienced a DLT (thrombocytopenia). High grade adverse events (AEs) were mostly hematological. Discontinuations due to AEs occurred in 2 pts (29%) at 70 mg/m2 and 0 pts at 55 mg/m2. Five pts (71%) at 70 mg/m2 and 4 pts (31%) at 55 mg/m2 achieved a best overall response of stable disease; 1 pt (8%) at 55 mg/m2 achieved a complete response (rhabdomyosarcoma). No partial responses were observed. PK concentrations of abemaciclib at 55 mg/m2 achieved average plasma abemaciclib concentrations of ~150 ng/mL and the abemaciclib Ctrough concentrations appeared reasonably constant over time at ~100 ng/mL. PK concentrations of IRN and TMZ were as expected. Conclusions: The RP2D of abemaciclib is 55mg/m2 BID in combination with IRN and TMZ. This combination appears tolerable with plasma concentrations within the range associated with efficacy in adult studies. Clinical trial information: NCT04238819 .

PIK3CB as a potential target to regulate chemosensitivity in glioblastoma.

e14051 Background: Glioblastoma (GBM) is the most common primary central nervous system malignancy but exhibits universally poor outcomes in part due to chemoresistance. Temozolomide (TMZ) can significantly improve overall survival, but resistance mechanisms limit its consistent efficacy. The phosphoinositide 3 kinase (PI3K) pathway is involved in multiple TMZ resistance mechanisms and is highly druggable; however, pan-inhibition of its four grossly homologous but functionally distinct catalytic subunits (PIK3CA/B/D/G encode p110α/β/δ/γ, respectively) was unsuccessful clinically. Therefore, by understanding how the individual PI3K subunits regulate TMZ sensitivity, a more selective treatment strategy can be developed to combat chemoresistance. Methods: Differential expression of PI3K subunit genes was assessed using RNA sequencing (RNAseq) results obtained from public databases (e.g., DepMap, The Cancer Tissue Genome Atlas) for GBM cell lines and patient tumors expressing high or low O6-methylguanine-DNA-methyltransferase (MGMT), a DNA repair enzyme highly implicated in TMZ resistance. PI3K protein levels were assessed using immunohistochemistry of gliomas available at The Human Protein Atlas (THPA). PI3K gene expression was correlated with PI3K pathway activation, quantified as protein kinase B phosphorylation (pAKT). PI3K mRNAs and pAKT were then correlated with the half maximal inhibitory concentrations (IC50s) of TMZ in GBM cell lines to evaluate for a relationship between PI3K gene expression, PI3K pathway activity, and TMZ sensitivity. Finally, hazard ratios were used to compare PI3K gene expression in patient tumors with survival and response to chemotherapy to determine if expression was predictive of prognosis and/or chemosensitivity. Results: PIK3CB was the only PI3K gene enriched in both GBM cell lines and tumors, demonstrating significantly higher expression than all other PI3K genes in GBM (P < 0.0001), independent of MGMT levels. PIK3CB expression remained the highest even after controlling for GBM risk factors such as tumor recurrence, isocitrate dehydrogenase (IDH) mutations, molecular subtypes, or patient gender/age differences. Levels of the p110β protein were also significantly higher than those of other PI3K catalytic subunits (P < 0.001). PIK3CB expression was positively correlated with pAKT levels (P < 0.05), but only in cell lines and tumors with low MGMT activity. Both PIK3CB expression and pAKT levels correlated positively with TMZ IC50s (P < 0.05) in MGMT-deficient GBMs, suggesting an inverse relationship between PIK3CB and TMZ sensitivity. Finally, better prognosis and significant survival benefit with chemotherapy were both associated with lower levels of PIK3CB in MGMT-deficient patients. Conclusions: PIK3CB may selectively regulate chemoresistance in GBM, suggesting a role for p110β selective inhibition to improve chemosensitivity.

Unsaturated free fatty acid emulsion infusion into carotid artery enhances drug delivery to the rat brain.

To determine whether the blood-brain barrier (BBB) opens to enhance drug delivery during the acute stage of unsaturated fat embolism. We infused oleic, linoleic, and linolenic acid emulsions through the right common carotid artery of rats, followed by trypan blue for gross and lanthanum for electron microscopic (EM) examination. Doxorubicin and temozolomide were also administered, and then the rats were euthanized at 30min, 1h, and 2h. Trypan blue hue was analyzed to semiquantitatively measure BBB opening. Desorption electrospray ionization-mass spectrometry (DESI-MS) imaging was used to evaluate drug delivery. Trypan blue staining observed in each group 30min after emulsion infusion increased at 1h and decreased after 2h in the oleic acid group. The linoleic and linolenic acid groups showed weak staining over time. The hue and trypan blue analysis results were corroborative. EM showed tight junction opening, whereas DESI-MS imaging showed increased doxorubicin and temozolomide signal intensities in ipsilateral hemispheres of all three groups. We demonstrated that oleic, linoleic, and linolenic acid emulsions opened the BBB, promoting drug delivery to the brain. Hue analysis and DESI-MS imaging are appropriate for analysis of doxorubicin and temozolomide concentrations in brain tissue.

Expert opinion on translational research for advanced glioblastoma treatment

Malignant gliomas are known to be one of the most difficult diseases to diagnose and treat because of the infiltrative growth pattern, rapid progression, and poor prognosis. Many antitumor drugs are not ideal for the treatment of gliomas due to the blood-brain barrier. Temozolomide (TMZ) is a DNA alkylating agent that can cross the blood-brain barrier. As the only first-line chemotherapeutic drug for malignant gliomas at present, TMZ is widely utilized to provide a survival benefit; however, some patients are inherently insensitive to TMZ. In addition, patients could develop acquired resistance during TMZ treatment, which limits antitumor efficacy. To clarify the mechanism underlying TMZ resistance, numerous studies have provided multilevel solutions, such as improving the effective concentration of TMZ in tumors and developing novel small molecule drugs. This review discusses the in-depth mechanisms underlying TMZ drug resistance, thus aiming to provide possibilities for the establishment of personalized therapeutic strategies against malignant gliomas and the accelerated development and transformation of new targeted drugs.

Abstract 2788: Extracellular vesicles as a pharmacodynamic reporter in glioblastoma

Abstract Glioblastoma (GBM), a lethal astrocytic brain tumor, has limited available treatment options. A major hindrance to the progress of new therapies is the selectivity of the blood brain barrier (BBB) which limits drug access to the tumor. Further, there are currently no minimally-invasive tests that can quickly and accurately determine whether novel therapeutics have crossed the BBB during clinical trials. Therefore, development of a non-invasive test that can identify drug delivery and target engagement in GBM represents an opportunity to enhance knowledge during early phase trials of a new compound’s pharmacological suitability and potential efficacy. This study proposes that cargo contained in GBM-derived extracellular vesicles (EVs), which are small membrane-bound particles released from cells, may serve as one such pharmacodynamic reporter. We analyzed RNA-seq data for EVs isolated from two GBM patient-derived xenograft models grown as short-term in vitro cultures, GBM10 and GBM120, following treatment with either temozolomide (TMZ) or pevonedistat (MLN). We assessed whether EV RNA payloads vary with different drug concentrations (EC50 vs EC80) and at different time points (4hr vs 24hr) post treatment. In the samples treated with MLN, a few long non-coding RNAs are commonly upregulated across the different time points and two models. Under different TMZ concentrations, differentially expressed genes are overrepresented in biological processes such as cytoplasmic translation and cellular macromolecule biosynthesis. We also found a negative correlation between the TMZ concentration and Y RNA expression level, which are functionally involved with DNA replication. Additionally, using CIRI2, we predicted the amount of circular RNAs in the EV cargo; we found that the ratio of circular RNAs to their linear counterparts (CLR) varies under different treatments. In GBM10, the percentage of circRNA with CLR > 1 decreases with increasing drug effect. Thus, our preliminary results indicate that EV cargo post-treatment may be a novel pharmacodynamic reporter in GBM. To apply such an assessment to patients, we have simultaneously pursued the development of an immunoprecipitation (IP) approach to enrich GBM-derived EVs that can then undergo cargo profiling. Potential biomarkers were screened across isolated GBM EVs from in vitro supernatant by western blot and ExoView analysis. B7H3 has emerged as a lead candidate for GBM-EV enrichment. To validate the ability of EV cargo to reflect drug distribution across the BBB in patients, current efforts are focused on applying IP enrichment of GBM-EVs in longitudinally-acquired cerebrospinal fluid samples from patients during their disease course. Overall, we anticipate that the results of this study will lead to development of a clinical test that reflects BBB penetration and tumor response, which will likely aid in novel drug development efforts. Citation Format: Valerie DeLuca, Nanyun Tang, Yue Hao, Anjali Raju, Charles Shaffer, Cecile Riviere-Cazaux, Terry C. Burns, Jann Sarkaria, Ian Parney, Patrick Pirrotte, Kendall Van Keuren-Jensen, Michael E. Berens. Extracellular vesicles as a pharmacodynamic reporter in glioblastoma [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 2788.

Temozolomide protects against the progression of glioblastoma via SOX4 downregulation by inhibiting the LINC00470-mediated transcription factor EGR2.

Temozolomide is extensively applied in chemotherapy for glioblastoma with unclear exact action mechanisms. This article seeks to address the potential molecular mechanisms in temozolomide therapy for glioblastoma involving LINC00470. Bioinformatics analysis was conducted to predict the potential mechanism of LINC00470 in glioblastoma, which was validated by dual-luciferase reporter, RIP, ChIP, and RNA pull-down assays. LINC00470 expression and the predicted downstream transcription factor early growth response 2 (EGR2) were detected in the collected brain tissues from glioblastoma patients. Following temozolomide treatment and/or gain- and loss-of-function approaches in glioblastoma cells, cell viability, invasion, migration, cycle distribution, angiogenesis, autophagy, and apoptosis were measured. In addition, the expression of mesenchymal surface marker proteins was assessed by western blot. Tumor xenograft in nude mice was conducted for in vivo validation. Mechanistic analysis and bioinformatics analysis revealed that LINC00470 transcriptionally activated SRY-related high-mobility-group box 4 (SOX4) through the transcription factor EGR2. LINC00470 and EGR2 were highly expressed in brain tissues of glioblastoma patients. LINC00470 and EGR2 mRNA expression gradually decreased with increasing concentrations of temozolomide in glioblastoma cells, and SOX4 expression was reduced in cells by temozolomide and LINC00470 knockdown. Temozolomide treatment induced cell cycle arrest, diminished cell viability, migration, invasion, and angiogenesis, and increased apoptosis and autophagy in glioblastoma, which was counteracted by overexpressing LINC00470 or SOX4 but was further promoted by LINC00470 knockdown. Temozolomide restrained glioblastoma growth and angiogenesis in vivo, while LINC00470 or SOX4 overexpression nullified but LINC00470 knockdown further facilitated these trends. Conclusively, temozolomide repressed glioblastoma progression by repressing the LINC00470/EGR2/SOX4 axis.

Mechanism of NURP1 in temozolomide resistance in hypoxia-treated glioma cells via the KDM3A/TFEB axis.

Temozolomide (TMZ) resistance is a major obstacle in glioma treatment. Nuclear protein-1 (NUPR1) is a regulator of glioma progression. This study investigated the mechanism of NUPR1 in TMZ resistance in hypoxia-treated glioma cells and its mechanism in modulating autophagy. We treated TMZ-resistant cells U251-TMZ and T98G-TMZ to normoxia or hypoxia and silenced NUPR1 in hypoxia-treated U251-TMZ and T98G-TMZ cells to assess cell viability, proliferation, apoptosis, LC3-II/LC3-I and p62 expressions, and autophagic flux under different concentrations of TMZ. We found that hypoxia upregulated NUPR1 expression and autophagy while NUPR1 silencing suppressed hypoxia-induced TMZ resistance and autophagy in glioma cells. We also investigated the interaction between NUPR1 and lysine demethylase 3A (KDM3A), as well as the enrichments of KDM3A and H3 lysine 9 dimethylation (H3K9me2) in the transcription factor EB (TFEB) promoter region. Our results suggest that hypoxia-induced NUPR1 promotes TFEB transcription by binding to KDM3A and reducing H3K9me2 levels, thereby augmenting glioma cell autophagy and TMZ resistance. Moreover, the overexpression of KDM3A or TFEB promoted glioma cell autophagy. In a xenograft tumor model, silencing NUPR1 suppressed TMZ resistance in glioma cells in vivo. Overall, our findings highlight a mechanism by which NUPR1 enhances glioma cell autophagy and TMZ resistance via the KDM3A/TFEB axis.

Hsa_circ_0043949 reinforces temozolomide resistance via upregulating oncogene ITGA1 axis in glioblastoma.

Temozolomide (TMZ) resistance limits its use in glioblastoma (GBM). Exosomes can carry circular RNAs (circRNAs) to regulate chemoresistance. To date, the role of exosomal hsa_circ_0043949 (circ_0043949) in GBM resistance to TMZ is unclear. Relative expression of circ_0043949 in clinical samples, GBM cell lines, and exosomes was detected by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The half-maximal inhibitory concentration (IC50) of TMZ, cell proliferation, apoptosis, invasion, and migration were analyzed via MTT, EdU, flow cytometry, transwell, and wound-healing assays. Relative protein levels were evaluated by western blotting. Target relationship was predicted by bioinformatics analysis and validated by dual-luciferase reporter and RNA pull-down assays. Exosomes were isolated by ultracentrifugation and verified by transmission electron microscopy, nanoparticle tracking analysis (NTA), and western blotting. The effect of exosomal circ_0043949 on TMZ resistance was validated by xenograft assay. Higher expression of circ_0043949 was gained in TMZ-resistant GBM samples and cells. Inhibition of circ_0043949 reduced TMZ resistance via decreasing IC50 of TMZ, repressing proliferation, invasion, migration, and inducing apoptosis in TMZ-resistant GBM cells. Circ_0043949 mediated integrinalpha1 (ITGA1) expression via function as a miR-876-3p sponge. Circ_0043949 was also upregulated in TMZ-resistant GBM cells-derived exosomes, and exosomal circ_0043949 increased the resistance of TMZ-resistant GBM cells to TMZ in xenograft models. TMZ-resistant GBM cells-derived exosomal circ_0043949 promoted TMZ resistance via upregulating ITGA1 expression via sequestering miR-876-3p, offering a potential target for the treatment of TMZ resistance in GBM.

Selenoprotein GPX1 is a prognostic and chemotherapy-related biomarker for brain lower grade glioma

ObjectiveGlutathione peroxidase 1 (GPX1) is a major selenoprotein in most animal tissues, primarily expressed in the cytoplasm and mitochondria of cells and peroxidase structures of certain cells. GPX1 expression is highly correlated with carcinogenesis and disease progression. The goal of the study was to determine the association between GPX1 expression and tumor therapy, and to identify GPX1 prognostic value in various malignancies. MethodsThe Cancer Genome Atlas (TCGA), Genotype-Tissue Expression (GTEx), and Human Protein Atlas (HPA) databases were used to detect the levels of GPX1 expression in human tumor tissues and normal tissues. Indeed, correlations between GPX1 and tumor purity, tumor mutation burden (TMB), microsatellite instability (MSI), and DNA mismatch repair genes (MMRs) were explored using the TCGA cohort. Functional and enrichment analyses were performed by the GeneMANIA database and Gene Set Enrichment Analysis (GSEA), respectively. Cox regression models and Kaplan – Meier curves were used to screen for independent risk factors and estimate brain lower-grade glioma (LGG) survival probability. The Chinese Glioma Genome Atlas (CGGA) database was used to determine whether GPX1 had a race-specific effect on overall survival (OS) in LGG. The cross-interaction between GPX1 and chemoradiotherapy on LGG OS was determined by Kaplan – Meier curves. Logistic regression models of multiplicative interactions were constructed. Furthermore, the relationship between GPX1 and LGG treatment regimens was also explored through the Genomics of Drug Sensitivity in Cancer (GDSC) database. ResultsGPX1 was highly expressed in various tumors, GPX1 overexpression was significantly correlated with the poor prognosis of LGG. GPX1 was found to be an independent predictive factor for LGG in both univariate and multivariate Cox models. The nomogram showed a high predictive accuracy (C-index: 0.804, 95% CI: 0.74–0.86). In addition, GPX1 was significantly associated with TMB, MSI, and MMRs in diverse cancers. GPX1 was involved in IL6/JAK/STAT3, inflammatory response, and apoptosis signaling pathways. Besides, non-radiotherapy, chemotherapy, and low GPX1 expression were important factors affecting the better prognosis of LGG. GPX1 acted as a tumor promoter, which has taken the worst effect on LGG survival, but a multiplicative interaction of GPX1*chemoradiotherapy may improve the poor clinical outcome. GPX1 was negatively correlated with the half inhibition concentration (IC50) of temozolomide (TMZ) (Spearman = −0.44, P = 4.52 ×10−26). ConclusionIn LGG patients, high GPX1 expression was linked to a shorter OS. The interaction between GPX1 and chemoradiotherapy exhibits a beneficial clinical effect and chemotherapy was recommended for LGG patients, especially for those with high GPX1 expression. Besides, high GPX1 expression can predict TMZ sensitivity in LGG, providing potential evidence for chemotherapy. On the whole, this study presents a wealth of biological as well as clinical significance for the roles of GPX1 in human tumors, particularly in LGG.

P02.09.B Valproic acid changes total DNA methylation level and influences temozolomide effect in glioblastoma cell lines

Abstract Background Valproic acid (VPA) is a first-line antiepileptic drug for glioblastoma (GBM) patients. There is also some evidence it improves the clinical outcome in those patients. However, the exact mechanism of VPA action is vague. Temozolomide (TMZ) is a first-line chemotherapeutic in GBM. Epigenetics offers a connection between genetic and environmental factors that influence the development of the disease. The best-characterized epigenetic mark is 5-methylcytosine (m5C) in DNA. The aim of that project is to show the effects of VPA administration on the total DNA methylation level. Material and Methods Using the nucleotide post-labeling method, we analyzed the total amount of m5C in DNA of GBM (T98G, U118, U138), cancer (HeLa) and normal (HaCaT) cell lines treated with VPA, and a combination of VPA and with TMZ. Results We adjusted the VPA doses to the ones achieved in the central nervous system during treatment. We observed dose-dependent changes in the total DNA methylation in neoplastic cell lines and the lack of such effect in a normal cell line. VPA alone produced a clear dose-dependent increase in total DNA methylation in GBM cell lines and scarce in the non-neoplastic cell line. In GBM cell lines, TMZ decreased the level of m5C. However, the exposition of GBM cells to the combination of VPA and TMZ caused an adverse synergistic effect resulting in DNA demethylation. The highest loss of m5C was observed at the highest concentration of TMZ (100 μM) and VPA (350 μM), after 3 h of incubation in the T98G cell line. Conclusion Total DNA methylation changes in glioma cell lines under VPA treatment suggest the new mechanism of that drug action and promote clinical implications for adjusting VPA and TMZ therapy in GBM patients. Our results show the potential and possible obstacles of the combined therapy of TMZ with VPA. Our experiments show that combined therapy with both drugs leads to total DNA hypomethylation. Therefore the conclusion would be to stop VPA administration during TMZ chemotherapy temporarily.