Combination Of Temozolomide Research Articles

CTNI-29. A PHASE 2 STUDY TO EVALUATE EFFICACY AND SAFETY OF ACT001 AND TEMOZOLOMIDE COMBINATION TREATMENT IN PATIENTS WITH RECURRENT GLIOBLASTOMA MULTIFORME

Abstract BACKGROUND Han Chinese patients with rGBM were randomly assigned to one of three cohorts in this phase 2 open label study: Arm A (ACT001, 400mg, BID), B (ACT001 and temozolomide combination) and C (TMZ or CCNU). Study endpoints include 1-year OS rate, PFS (by iRANO) and AE incidence. RESULTS 7, 6 and 6 patients were enrolled to Arms A, B and C respectively including one patient in Arm C with a mutant IDH1 (R132H). The median age was 52 years (range: 34-68) and male to female ratio 11/8. In addition to 7 SAEs, 189 TEAEs were reported in this interim analysis including 49 and 70 chemo-related AEs in Arm B and Arm C. Post enrollment MRI scans were performed in 6,6 and 4 patients in Arms A, B and C respectively. Despite the lack of objective response and difference in 1-year OS, a prolonged PFS lasting at least 12 months was noted in 3 patients including the patient with mutant IDH1 in Arm C (23 months and ongoing) and two patients in Cohort B (19 and 22 months, both ongoing) who were sensitive to TMZ in first line treatment as shown by at least 12-month interval between TMZ initiation and first relapse. Among 18 patients with wild type IDH, the prolonged stable disease was restricted to combination Arm whereas either the four patients with positive MGMT methylation or two patients with a similar history of sensitivity to first line TMZ treatment didn’t have such prolonged stable disease after being treated with TMZ or ACT001 alone during this study. CONCLUSIONS ACT001 restored sensitization of rGBM patients to TMZ treatment in sub-group of patients likely due to reduced DNA repair capacity by ACT001 treatment. Further study is warranted to evaluate the effect of ACT001 and TMZ combination in phase 2b study.

CTIM-05. RESULTS OF SAFETY LEAD IN OF PHASE II TRIAL OF COMBINATION OLAPARIB, TEMOZOLOMIDE, AND PEMBROLIZUMAB FOR PATIENTS WITH IDH-MUTANT GLIOMA

Abstract BACKGROUND Recurrent gliomas are universally fatal with few adequate treatment options. IDH-mutant gliomas may be susceptible to PARP inhibition, thus combination therapy using alkylating agents and checkpoint inhibition could be synergistic. METHODS This is an open label, phase II study of olaparib, temozolomide, and pembrolizumab for patients with recurrent IDH-mutant glioma, grade 2 or 3, with measurable enhancing disease per RANO. Patients received pembrolizumab 200mg IV on day 1 of each 21-day cycle. Oral olaparib 200mg twice daily and temozolomide 50mg/m2 daily were given days 1-7 of cycles 3-11. Here, we report the results of our safety lead in, consisting of the first 6 patients to complete cycle 3. RESULTS A total of seven patients were enrolled to the safety lead-in: 2 women; 5 patients with astrocytoma, 2 oligodendroglioma. Patients had a median age of 45 (range 29-63) and median KPS 90 (range 80-100). Median number of recurrences was 3.5 (range 1-5). All had received prior alkylating therapy and all but 1 patient had received prior RT. No patients were taking steroids at the time of enrollment. Six patients completed the dose-limiting toxicity (DLT) period without any DLTs. One patient experienced clinical progression on pembrolizumab monotherapy prior to the start of combination therapy and was thus replaced. No grade 3 or 4 toxicities were seen through the DLT period. The most common clinical toxicities at least possibly related to treatment were grade 1 and 2 fatigue (n = 4), nausea (n = 3) and constipation (n = 2). Hematological toxicities include grade 1 anemia (n = 2) and decreased platelet count (n = 1). CONCLUSIONS The combination of olaparib, temozolomide, and pembrolizumab was well tolerated in this cohort. Following completion of the safety lead-in, the trial was redesigned to minimize the length of pembrolizumab monotherapy and introduce combination therapy in cycle 1. Enrollment is ongoing. NCT05188508.

DDDR-29. TARGETING FGFR FUSIONS: A CASE OF CATEQUENTINIB (AL3818) AND TEMOZOLOMIDE COMBINATION THERAPY FOR RECURRENT MGMT METHYLATED TACC-FGFR FUSION POSITIVE GLIOBLASTOMA

Abstract BACKGROUND There is no gold-standard for the management of recurrent glioblastoma (GBM). However, advances at the interface of molecular testing and rational drug design have opened avenues for personalized medicine. Here we describe the case of a 52-year-old patient with recurrent GBM (WHO grade 4) with known FGFR3-TACC3 fusion and MGMT promoter methylation by Caris molecular profiling. Initial treatment included radiotherapy with concomitant and adjuvant temozolomide. However, after 18 months off therapy, the patient became clinically and radiographically progressive with multifocal disease. Herein, we describe our experience with temozolomide and catequentinib (AL3818) in a single patient. METHODS We reviewed the available literature on targeting FGFR3-TACC3 fusion positive glioblastoma with catequentinib (1-[[[4-(4-fluoro-2-methyl-1H-indol-5-yloxy)-6-methoxyquinolin-7-Yl] oxy] methyl] cyclopropanamine dihydrochloride). Catequentinib is an oral small-molecule pan-kinase inhibitor with selective VEGFR2 inhibition, it is under investigation in multiple phase 2 and 3 clinical trials and can safely be administered concomitantly with temozolomide. We obtained single-patient Investigational New Drug (IND) approval and free drug was shipped directly from the manufacturer (Advenchen Laboratories; Jiangsu Chia-Tai Tianqing Pharmaceutical) to our institution’s investigational chemotherapy pharmacy. We administered catequentinib 12 mg by mouth for 14 days on and 7 days off of a 21-day cycle along with temozolomide 50mg/m2/day of a 28-day cycle with routine monitoring of laboratory parameters. RESULTS The combination of temozolomide and catequentinib was well-tolerated, resulted in clinical stability, and partial radiographic response at 3-month follow-up. CONCLUSION Catequentinib in combination with metronomic temozolomide was well tolerated in this single patient. This patient’s response shows the promise of intentional, patient-specific care at the intersection of molecular testing and rational drug design. The patient will continue therapy and updated clinical and radiographic outcomes will be presented.

Anti-Glioma Effects of Ligustilide or n-Butylphthalide on Their Own and the Synergistic Effects with Temozolomide via PI3K/Akt Signaling Pathway.

Ligustilide (LIG) and n-butylphthalide (NBP) have neuroprotective effects in cerebral ischemia; however, their roles in gliomas are not well-known.This study aimed to explore the anti-glioma effects of LIG and NBP individually and the synergistic effects of temozolomide (TMZ) via the PI3K/Akt Signaling Pathway. Cytotoxicity of LIG and NBP alone and in combination with TMZ in U251 cells was determined using the CCk-8. The effect of compounds alone or in combination on cell migration was detected using the wound healing assay, and the invasion was evaluated by transwell assays, respectively. Cell apoptosis was quantified by flow cytometry and the changed expressions of proteins were detected by Western blotting. The results showed that LIG and NBP significantly inhibited the growth of U251 cells at concentrations of 4-10 µg/mL and 1.5-6 µg/mL in a dose-dependent manner (p<0.05, p<0.01). The combination of 20 µg/mL TMZ with LIG in the concentration range of 4-10 µg/mL or with NBP of 0.5-6 µg/mlachieved synergistic effect towardsU251 cells. LIG and NBP, alone or in combination with TMZ, markedly inhibited cell invasion (p< 0.001) and enhanced apoptosis (p< 0.05). The combination of TMZ with LIG or NBP markedly inhibited cell migration (p< 0.001). Western blot analysis showed that LIG, NBP, and TMZ, alone and in combination, significantly decreased the expression of Bcl-2, p-PI3K, and p-Akt, and increased the expression of Bax. Both LIG and NBP exert anti-glioma effects on their own through the PI3K/Akt pathway and enhance TMZ-mediated anti-glioma efficiency via the same pathway.

Synergism of d-limonene and temozolomide on migratory and apoptotic behaviors of human glioblastoma cell lines.

Glioblastoma (GBM), which is a heterogeneous and aggressive type of brain tumor, is known for its poor survival outcomes. The treatment of GBM remains challenging primarily due to the drug resistance to the current standard therapeutic option, temozolomide (TMZ). Researchers are currently focusing on developing an appropriate alternative combinatorial therapeutic to enhance treatment outcomes. D-limonene (DL) is a monoterpene derived from citrus fruit. This study aims to assess the impact of combining DL with TMZ and explore its potential mechanism of action in U87MG and LN229 GBM cells. The effects of the combined treatment of DL and TMZ were assessed on various cellular aspects, including cell viability, anchorage-independent cell growth, and DNA damage. Furthermore, the influence of this combination on cell cycle progression, cell migration, and cell death was also investigated. The combination of DL+TMZ demonstrated a synergistic effect, resulting in reduced cell proliferation and suppressing the colony formation ability of a single cell. Treatment with DL and TMZ arrested the cells in G0/G1 phase. Furthermore, the DL+TMZ combination induced apoptosis by upregulating the expression of Bax, and Caspase (CASP)-3, while reducing the expression of the Bcl-2 gene in GBM cells. In addition, the combined treatment of DL+TMZ significantly decreased the expression of matrix metalloproteinase (MMP)-2 and MMP-9, expression, indicating inhibition of cell migration in GBM cells. In conclusion, the combination of DL and TMZ demonstrated a synergistic effect in reducing cell proliferation, suppressing colony formation, inducing apoptosis, and inhibiting cell migration in GBM cells. These findings suggest the potential of DL+TMZ combination therapy as an effective treatment for GBM.

Aranose: Domestic original cytostatic agent for treatment of neuroendocrine tumors of all localizations

Aranose is an original cytostatic, synthesized in the Russian Cancer Research Center, belongs to the class of nitrosourea derivatives (this class of drugs also including streptozotocin). In preclinical trials, Aranose has shown its activity in neuroendocrine tumors (NETs). Prospective clinical studies have confirmed high efficacy and good tolerability of the drug in different lines of treatment of patients with NET G1 and G2. Median PFS while Aranose treatment in a single mode or in combination with capecitabine, doxorubicin and temozolomide did not differ significantly (15.3 vs 15.8, 15.3 and 17.9 months, respectively, p = 0.791). After updating the histological classification and highlighting the prognostically unfavorable subgroup of NET G3, a prospective single-center clinical study of Aranose in the first line of NET G3 therapy was conducted. The standard dosage regimen of the drug was used: 500 mg/m2 from the first to the third days, a cycle of 21 days. On average, patients received nine courses of Aranose chemotherapy, but in case of an increase in the radiological response treatment continued until disease progression or unacceptable toxicity. Median PFS in the Aranose group was 12 months, in the group of patients receiving capecitabine and oxaliplatin combination – 5 months, in capecitabine and temozolomide combination – 7 months, in the etoposide with cisplatin or carboplatin group only 4 months. The frequency of objective responses in the Aranose group was 37 % (10/27), no complete responses were recorded. Disease stabilization was achieved in 40.7 % (11/27), thus, the frequency of disease control was 77.7 % (21/27). Disease control was maintained after 6 months or more in 63 % of patients.

Development and characterization of a temozolomide-loaded nanoemulsion and the effect of ferrocene pre and co-treatments in glioblastoma cell models.

Glioblastoma is a severe brain tumor that requires aggressive treatment involving surgery, radiotherapy, and chemotherapy, offering a survival rate of only 15months. Fortunately, recent nanotechnology progress has enabled novel approaches and, alongside ferrocenes' unique properties of cytotoxicity, sensitization, and interaction with reactive oxygen species, have brought new possibilities to complement chemotherapy in nanocarrier systems, enhancing treatment results. In this work, we developed and characterized a temozolomide-loaded nanoemulsion and evaluated its cytotoxic potential in combination with ferrocene in the temozolomide-resistant T98G and temozolomide-sensitive U87 cell lines. The effects of the treatments were assessed through acute assays of cell viability, cell death, mitochondrial alterations, and a treatment protocol simulation based on different two-cycle regimens. Temozolomide nanoemulsion showed a z-average diameter of 173.37 ± 0.86nm and a zeta potential of -6.53 ± 1.13mV. Physicochemical characterization revealed that temozolomide is probably associated with nanoemulsion droplets instead of being entrapped within the nanostructure, allowing a rapid drug release. In combination with ferrocene, temozolomide nanoemulsion reduced glioblastoma cell viability in both acute and two-cycle regimen assays. The combined treatment approach also reversed T98G's temozolomide-resistant profile by altering the mitochondrial membrane potential of the cells, thus increasing reactive oxygen species generation, and ultimately inducing cell death. Altogether, our results indicate that using nanoemulsion containing temozolomide in combination with ferrocene is an effective approach to improve glioblastoma therapy outcomes.

Early Salvage Chemo-Immunotherapy with Irinotecan, Temozolomide and Naxitamab Plus GM-CSF (HITS) for Patients with Primary Refractory High-Risk Neuroblastoma Provide the Best Chance for Long-Term Outcomes.

Patients with high-risk neuroblastoma (HR-NB) who are unable to achieve a complete response (CR) to induction therapy have worse outcomes. We investigated the combination of humanized anti-GD2 mAb naxitamab (Hu3F8), irinotecan (I), temozolomide (T), and sargramostim (GM-CSF)-HITS-against primary resistant HR-NB. Eligibility criteria included having a measurable chemo-resistant disease at the end of induction (EOI) treatment. Patients were excluded if they had progressive disease (PD) during induction. Prior anti-GD2 mAb and/or I/T therapy was permitted. Each cycle, administered four weeks apart, comprised Irinotecan 50 mg/m2/day intravenously (IV) plus Temozolomide 150 mg/m2/day orally (days 1-5); naxitamab 2.25 mg/kg/day IV on days 2, 4, 8 and 10, (total 9 mg/kg or 270 mg/m2 per cycle), and GM-CSF 250 mg/m2/day subcutaneously was used (days 6-10). Toxicity was measured using CTCAE v4.0 and responses through the modified International Neuroblastoma Response Criteria (INRC). Thirty-four patients (median age at treatment initiation, 4.9 years) received 164 (median 4; 1-12) HITS cycles. Toxicities included myelosuppression and diarrhea, which was expected with I/T, and pain and hypertension, expected with naxitamab. Grade ≥3-related toxicities occurred in 29 (85%) of the 34 patients; treatment was outpatient. The best responses were CR = 29% (n = 10); PR = 3% (n = 1); SD = 53% (n = 18); PD = 5% (n = 5). For cohort 1 (early treatment), the best responses were CR = 47% (n = 8) and SD = 53% (n = 9). In cohort 2 (late treatment), the best responses were CR = 12% (n = 2); PR = 6% (n = 1); SD = 53% (n = 9); and PD = 29% (n = 5). Cohort 1 had a 3-year OS of 84.8% and EFS 54.4%, which are statistically significant improvements (EFS p = 0.0041 and OS p = 0.0037) compared to cohort 2. In conclusion, naxitamab-based chemo-immunotherapy is effective against primary chemo-resistant HR-NB, increasing long-term outcomes when administered early during the course of treatment.

Modulating Wnt/β-Catenin Signaling Pathway on U251 and T98G Glioblastoma Cell Lines Using a Combination of Paclitaxel and Temozolomide, A Molecular Docking Simulations and Gene Expression Study.

One of the most lethal cancers, glioblastoma (GBM), affects 14.5% of all central nervous system (CNS) tumors. Patients diagnosed with GBM have a meager median overall survival (OS) of 15 months. Extensive genetic analysis has shown that many dysregulated pathways, including the Wnt/β-catenin signaling system, contribute to the pathogenicity of GBM. Paclitaxel (PTX) and temozolomide (TMZ) are recognized to have therapeutic potential in several types of cancer, including GBM. This work aimed to examine the impact of PTX and TMZ on the human glioma cell lines U251 and T98G using molecular docking simulations and gene expression profiles in the Wnt/β-catenin signaling pathway. Standard procedure for Molecular Docking simulation, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cytotoxicity assay, and Flow Cytometry assay was used. Genes implicated in the Wnt/β-catenin signaling pathway, including Dvl, Axin, APC, β-catenin, and glycogen synthase kinase3-β (GSK3β), were subjected to real-time PCR. The estimated parameters for targets revealed that the average binding energy and inhibition constant (Ki) for the DVL, β-Catenin, and GSK3β, when targeted by PTX, were - 5.01 kcal/mol, - 5.4 kcal/mol, and - 9.06 kcal/mol, respectively. This energy range was - 6.34 kcal/mol for DVL, - 5.52 kcal/mol for β-Catenin, and - 5.66 kcal/mol for GSK3β as a result of TMZ's inhibitory actions. Gene expression analyses indicated that PTX and PTX/TMZ suppressed GSK3β (p < 0.05). GSK3β from the Wnt/β-catenin signaling pathway was significantly targeted by PTX alone, and adding TMZ to PTX may improve the efficacy of glioblastoma treatment. In addition, the GSK3β gene may help GBM therapy strategies as a potential PTX target.

Germ line variant GFI1-36N affects DNA repair and sensitizes AML cells to DNA damage and repair therapy

AbstractGrowth factor independence 1 (GFI1) is a DNA-binding transcription factor and a key regulator of hematopoiesis. GFI1-36N is a germ line variant, causing a change of serine (S) to asparagine (N) at position 36. We previously reported that the GFI1-36N allele has a prevalence of 10% to 15% among patients with acute myeloid leukemia (AML) and 5% to 7% among healthy Caucasians and promotes the development of this disease. Using a multiomics approach, we show here that GFI1-36N expression is associated with increased frequencies of chromosomal aberrations, mutational burden, and mutational signatures in both murine and human AML and impedes homologous recombination (HR)–directed DNA repair in leukemic cells. GFI1-36N exhibits impaired binding to N-Myc downstream-regulated gene 1 (Ndrg1) regulatory elements, causing decreased NDRG1 levels, which leads to a reduction of O6-methylguanine-DNA-methyltransferase (MGMT) expression levels, as illustrated by both transcriptome and proteome analyses. Targeting MGMT via temozolomide, a DNA alkylating drug, and HR via olaparib, a poly-ADP ribose polymerase 1 inhibitor, caused synthetic lethality in human and murine AML samples expressing GFI1-36N, whereas the effects were insignificant in nonmalignant GFI1-36S or GFI1-36N cells. In addition, mice that received transplantation with GFI1-36N leukemic cells treated with a combination of temozolomide and olaparib had significantly longer AML-free survival than mice that received transplantation with GFI1-36S leukemic cells. This suggests that reduced MGMT expression leaves GFI1-36N leukemic cells particularly vulnerable to DNA damage initiating chemotherapeutics. Our data provide critical insights into novel options to treat patients with AML carrying the GFI1-36N variant.

Temozolomide-based sonodynamic therapy induces immunogenic cell death in glioma

BackgroundIn our previous study, we found for the first time that temozolomide (TMZ), the first-line chemotherapeutic agent for glioblastoma (GBM), can generate a large amount of reactive oxygen species (ROS) under ultrasound irradiation. Sonodynamic therapy (SDT) using TMZ as the sonosensitizer produced more potent antitumor effects than TMZ alone. Here, we further evaluate the effects of TMZ-based SDT on subcellular structures and investigate the immunogenic cell death (ICD)-inducing capability of TMZ-based SDT. MethodsThe sonotoxic effects of TMZ were explored in LN229 and GL261 glioma cells. The morphology of endoplasmic reticulum and mitochondria was observed by transmission electron microscopy. The nuclear DNA damage was represented by γ-H2AX staining. Bone marrow-derived dendritic cells (BMDCs) were employed to assess ICD-inducing capability of TMZ-based SDT. A cyclic arginine-glycine-aspartic (c(RGDyC))-modified nanoliposome drug delivery platform was used to improve the tumor targeting of SDT. ResultsTMZ-based SDT had a greater inhibitory effect on glioma cells than TMZ alone. Transmission electron microscopy revealed that TMZ-based SDT caused endoplasmic reticulum dilation and mitochondrial swelling. In addition, endoplasmic reticulum stress response (ERSR), nuclear DNA damage and mitochondrial permeability transition pore (mPTP) opening were promoted in TMZ-based SDT group. Most importantly, we found that TMZ-based SDT could promote the “danger signals” produced by glioma cells and induce the maturation and activation of BMDCs, which was associated with the mitochondrial DNA released into the cytoplasm in glioma cells. In vivo experiments showed that TMZ-based SDT could remodel glioma immune microenvironment and provoke durable and powerful anti-tumor immune responses. What's more, the engineered nanoliposome vector of TMZ conferred SDT tumor targeting, providing an option for safer clinical application of TMZ in combination with SDT in the future. ConclusionsTMZ-based SDT was capable of triggering ICD in glioma. The discovery of TMZ as a sonosensitizer have shown great promise in the treatment of GBM.

P10.27.A THE INFLUENCE OF DEXAMETHASONE ON TOTAL DNA METHYLATION LEVEL IN GLIOBLASTOMA CELL LINES

Abstract BACKGROUND Dexamethasone (DEX) is a first-line antiedematic drug for glioblastoma (GBM) patients. There is also some evidence it also inhibits GBM cell proliferation and migration, thus impacting patients’ outcomes. However, other studies show that the use of DEX is associated with poor treatment outcomes in GBM patients. The exact mechanism of DEX action is unclear, and no definitive conclusion has yet been reached on its benefits in neurooncological treatment. 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 DEX 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 DEX, and a combination of DEX and with TMZ. RESULTS We adjusted the DEX doses to the ones achieved in the central nervous system during treatment. We observed dose-dependent increase in total DNA methylation in all cell lines. However, the exposition of GBM cells to the combination of DEX and TMZ caused an adverse synergistic effect resulting in DNA demethylation in high doses of both drugs. In lower concentrations of both drugs DEX kept the increased DNA methylation and attenuated the demethylating effect of TMZ. CONCLUSION Total DNA methylation changes in glioma cell lines under DEX treatment suggest the new mechanism of that drug action and promote clinical implications for adjusting DEX and TMZ therapy in GBM patients. Our results show the potential and possible obstacles of the combined therapy of TMZ with DEX. Our experiments show that combined therapy with both drugs leads to total DNA hypomethylation in high doses of both drugs. Therefore the conclusion would be to adjust DEX administration during TMZ chemotherapy.

P17.14.B PHASE I TRIAL OF DNX-2401 ONCOLYTIC ADENOVIRUS COMBINED WITH A SHORT COURSE OF DOSE-DENSE TEMOZOLOMIDE FOR RECURRENT GLIOBLASTOMA

Abstract BACKGROUND Oncolytic virotherapy is emerging as a novel therapeutic strategy for glioblastoma (GBM). DNX-2401, an oncolytic adenovirus with selective replication and enhanced infectivity in tumor cells, showed anti-tumor effect in a previous clinical trial for recurrent GBM. The combination of DNX-2401 and Temozolomide (TMZ) has shown a synergistic anti-glioma effect in preclinical models. MATERIAL AND METHODS We conducted a phase I open-label single-center study to investigate the safety and efficacy of this therapeutic combination in patients with first recurrence of GBM. Following tumor biopsy or resection, 3x10e10 viral particles of DNX-2401 were injected intratumorally or into the post-resection cavity, respectively. Approximately 2 weeks after DNX-2401 injection, patients were treated with up to two 28-day cycles of oral 150 mg/m2/day TMZ in a 7 days on/7 days off schedule. RESULTS Thirty-one patients (13 [42%] female, 18 [58%] male) with confirmed recurrent GBM were enrolled and treated with DNX-2401 followed by a median of 1.5 cycles of TMZ (range, 0-2). Median age was 54 years (range, 25-78) and median KPS was 70 (range 70-90). Surgery consisted of biopsy in 14, partial resection in 15 and complete resection in 2 patients. IDH1/2 mutation was confirmed in 1 patient. MGMTP was methylated in 9 and unmethylated in 21 patients (1 patient, unknown). Best response according to RANO criteria was partial response in 1 patient, stable disease in 21 subjects and progressive disease in the remainder. Median PFS was 51 days (95% CI, 43-81). Median OS was 282 days (95% CI 202-344) with 6-month and 12-month OS rates of 72.5 (95% CI 52.4-85.2) and 27.5 % (95% CI 12.4-45.0), respectively. Four patients survived more than 18 months with 2 long-term survivors exceeding 4.5 years. Treatment was generally well tolerated. The safety profile was as expected for subjects treated with TMZ alone. Except one subject who experienced grade 2 brain edema possibly related to DNX-2401, no additional toxicity was considered attributable to DNX-2401. The most frequent reported adverse events were lymphopenia (54.8%), asthenia (48.4%), constipation (41.9%), headache (29%) and pyrexia (22.6%). Ongoing correlative analyses evaluating immune response are ongoing. CONCLUSION Our data suggest that treatment with DNX-2401 followed by a short-course of dose-dense TMZ is feasible, can be safely administered with expected adverse events related to chemotherapy, and appears to render a clinical benefit in a subset of patients with recurrent GBM. Further assessment of this therapeutic approach is warranted.

P10.18.B JUGLONE AS A NOVEL EPIGENETIC DRUG FOR GLIOBLASTOMA

Abstract BACKGROUND Glioblastoma (GBM) is the most common and aggressive primary brain tumor and one of the human malignancies with the highest mortality. Standard approaches for GBM, including, gross total resection, radiotherapy, and chemotherapy, cannot destroy all the cancer cells and, despite advances in its treatment, the prognosis for GBM remains poor. The, until now, the most successful chemotherapy with temozolomide (TMZ) for brain gliomas is not fully effective, and therefore new therapeutic strategies for GBM are needed. We found that juglone (J), which exhibits cytotoxic, anti-proliferative, and anti-invasive effects on various cells could be a promising agent for GBM therapy. MATERIAL AND METHODS We analyzed the effects of juglone alone and in combination with temozolomide on glioblastoma cell lines. In addition to the analysis of cell viability and cell cycle, we looked at the epigenetic effects of these compounds on cancer cells. We estimated the total DNA methylation in T98G, U138, U118, and HaCaT lines after treatment with different J and J/TMZ concentrations and incubation time using TLC separation of radioactively labeled nucleotides. The combination treatment with J and TMZ was evaluated with total DNA methylation analysis and 8-oxo-dG (oxidative stress marker) estimation using EC detection. RESULTS We noticed concentration and time-dependent changes in total DNA methylation. The highest increase was observed for the T98G cell line at the incubation time of 24hrs. Normal cell line (HaCaT) did not present significant changes. TMZ alone induced a lowering of DNA methylation, but juglone diminished that effect. J induces minute oxidative stress in low concentrations, but is quite high and rapid at concentrations over 50uM, concomitantly with none/slight decrease in DNA methylation. However, the combination of J/TMZ in increasing concentrations diminished the oxidative stress effect. CONCLUSION Our results strongly suggest that a combination of juglone and temozolomide can be applied for better GBM treatment. At certain incubation times and concentrations, juglone increases DNA methylation, which is a positive therapeutic effect in oncogenesis. Small doses of J keep a low level of 8-oxo-dG with an increase in DNA methylation. The combination of J/TMZ in high concentrations of both drugs decreases the oxidative stress effect of chemotherapy. The study was supported by OPUS 19 (2020/37/B/NZ5/03249) grant from the National Science Center, Poland.

OS09.2.A REGORAFENIB IN COMBINATION WITH TEMOZOLOMIDE WITH OR WITHOUT RADIOTHERAPY IN PATIENTS WITH NEWLY DIAGNOSED MGMT-METHYLATED, IDH WILDTYPE GLIOBLASTOMA: A PHASE I DOSE-FINDING STUDY (REGOMA-2, EUDRA CT N° 2021-001604-15)

Abstract BACKGROUND Glioblastoma (GBM) is associated with the activation of multiple signaling pathways, including the receptor tyrosine kinases, VEGFR, FGFR and PDGFR. The potential response to anti-angiogenic agents makes GBM an attractive target for regorafenib (REG). Antitumor activity of REG in recurrent GBM patients was shown in the phase II REGOMA trial. Interestingly, in “in vivo” studies, beneficial effects were observed with the combination of REG and temozolomide (TMZ) in subcutaneous xenografts. MATERIAL AND METHODS This phase I open-label multicentric study in progress is evaluating the addition of REG to standard of care treatment with TMZ as adjuvant therapy (cohort A), and in combination with TMZ + radiotherapy (RT) as concomitant therapy (cohort B), in newly diagnosed MGMT-methylated, IDH wildtype GBM patients. Primary objectives are: to determine the safety and tolerability of REG and to establish the maximum tolerated dose (MTD) of REG in these two cohorts. Secondary objectives are: the assessment of pharmacokinetics of REG, TMZ, and possible pharmacokinetic interactions between TMZ and REG in cohort A; the assessment of preliminary antitumor activity; the evaluation of quality of life during the treatment. The dose escalation is being explored according to a “3 + 3” design, starting from cohort A and following 3 dose levels of REG [80 mg, 120 mg and 160 mg]. After finding the MTD in cohort A, the cohort B dose escalation will start, exploring escalating oral doses of REG in combination with concomitant TMZ + RT (60 Gy/30 fractions). In the adjuvant phase of cohort B, REG will be given with TMZ at MTD shown in the prior adjuvant phase dose escalation.The DLT (dose-limiting toxicity) evaluation period will be two cycles from the start of cycle 1 of adjuvant phase (in cohort A) and from day 1 to the last day of the concomitant phase (in cohort B). Recruitment started in May 2022; Level 1 in cohort A (3 patients) has been completed without DLT. Approximately 36 subjects will be enrolled.

P10.06.B APPLYING TUMOR TREATING FIELDS (TTFIELDS) FOR ENHANCEMENT OF TEMOZOLOMIDE AND LOMUSTINE EFFICACY IN GLIOBLASTOMA CELL LINES

Abstract BACKGROUND Tumor Treating Fields (TTFields) are electric fields that disrupt cellular processes critical for cancer cell viability and tumor progression, ultimately leading to cell death. TTFields therapy concurrent with maintenance temozolomide (TMZ) is approved for treatment of newly-diagnosed glioblastoma (GBM). Recently, benefit was demonstrated in patients with O6-methylguanine DNA methyltransferase (MGMT) promoter methylation for the combination of TMZ with lomustine (CCNU). The addition of adjuvant TTFields to this treatment regimen further improved patient outcomes, leading to a CE mark. The current study aimed to elucidate the mechanism underlying the benefit of this treatment protocol by utilizing GBM cell lines with different MGMT expression status. MATERIAL AND METHODS Human GBM cell lines with different MGMT promoter methylation status [LN-18 (unmethylated MGMT promoter), U-118 MG (partially methylated MGMT promoter), and U-87 MG and LN-229 (methylated MGMT promoter)] were examined for MGMT expression levels. Next, the cells were treated with TTFields (intensity of 0.83 V/cm RMS; frequency of 200 kHz) using the inovitro system. Cell count and colony formation were employed to detect the effectiveness of TTFields concomitant with TMZ and/or CCNU. Western blot allowed the detection of expression levels of DNA repair proteins in control versus TTFields-treated cells. RESULTS The cell lines displayed comparable responses to TTFields and CCNU, while they differed in sensitivity to TMZ according to MGMT expression status. TTFields concomitant with TMZ displayed an additive effect, irrespective of MGMT expression levels. The effect of concomitant TTFields with CCNU was additive in MGMT-expressing cell lines, whereas it trended toward synergy in MGMT-non-expressing cell lines. Expression of proteins from the Fanconi Anemia (FA)-BRCA pathway for DNA repair was downregulated following TTFields application. TTFields concurrent with both TMZ and CCNU provided higher efficacy relative to TMZ plus CCNU or TTFields apart, with additivity in MGMT-expressing cell lines and a tendency toward synergy in MGMT-non-expressing cell lines. CONCLUSION The results support the clinical benefit demonstrated for concomitant TTFields with TMZ plus CCNU, showing that the efficacy of TMZ and CCNU may be enhanced by TTFields, with synergism seen for the latter case in cells not expressing MGMT. The BRCAness state induced by TTFields rationalizes this, as in the absence of MGMT DNA damage induced by CCNU requires the FA-BRCA pathway for repair.

Characterization of the Intracellular Acidity Regulation of Brain Tumor Cells and Consequences for Therapeutic Optimization of Temozolomide.

A well-known feature of tumor cells is high glycolytic activity, leading to acidification of the tumor microenvironment through extensive lactate production. This acidosis promotes processes such as metastasis, aggressiveness, and invasiveness, which have been associated with a worse clinical prognosis. Moreover, the function and expression of transporters involved in regulation of intracellular pH might be altered. In this study, the capacity of tumor cells to regulate their intracellular pH when exposed to a range of pH from very acidic to basic was characterized in two glioma cell lines (F98 and U87) using a new recently published method of fluorescence imaging. Our results show that the regulation of acidity in tumors is not the same for the two investigated cell lines; U87 cells are able to reduce their intracellular acidity, whereas F98 cells do not exhibit this property. On the other hand, F98 cells show a higher level of resistance to acidity than U87 cells. Intracellular regulation of acidity appears to be highly cell-dependent, with different mechanisms activated to preserve cell integrity and function. This characterization was performed on 2D monolayer cultures and 3D spheroids. Spatial heterogeneities were exhibited in 3D, suggesting a spatially modulated regulation in this context. Based on the corpus of knowledge available in the literature, we propose plausible mechanisms to interpret our results, together with some new lines of investigation to validate our hypotheses. Our results might have implications on therapy, since the activity of temozolomide is highly pH-dependent. We show that the drug efficiency can be enhanced, depending on the cell type, by manipulating the extracellular pH. Therefore, personalized treatment involving a combination of temozolomide and pH-regulating agents can be considered.

EPIC-1042 as a potent PTRF/Cavin1-caveolin-1 interaction inhibitor to induce PARP1 autophagic degradation and suppress temozolomide efflux for glioblastoma.

Temozolomide (TMZ) treatment efficacy in glioblastoma is determined by various mechanisms such as TMZ efflux, autophagy, base excision repair (BER) pathway, and the level of O6-methylguanine-DNA methyltransferase (MGMT). Here, we reported a novel small-molecular inhibitor (SMI) EPIC-1042 (C20H28N6) with the potential to decrease TMZ efflux and promote PARP1 degradation via autolysosomes in the early stage. EPIC-1042 was obtained from receptor-based virtual screening. Co-immunoprecipitation and pull-down assays were applied to verify the blocking effect of EPIC-1042. Western blotting, co-immunoprecipitation, and immunofluorescence were used to elucidate the underlying mechanisms of EPIC-1042. In vivo experiments were performed to verify the efficacy of EPIC-1042 in sensitizing glioblastoma cells to TMZ. EPIC-1042 physically interrupted the interaction of PTRF/Cavin1 and caveolin-1, leading to reduced secretion of small extracellular vesicles (sEVs) to decrease TMZ efflux. It also induced PARP1 autophagic degradation via increased p62 expression that more p62 bound to PARP1 and specially promoted PARP1 translocation into autolysosomes for degradation in the early stage. Moreover, EPIC-1042 inhibited autophagy flux at last. The application of EPIC-1042 enhanced TMZ efficacy in glioblastoma in vivo. EPIC-1042 reinforced the effect of TMZ by preventing TMZ efflux, inducing PARP1 degradation via autolysosomes to perturb the BER pathway and recruitment of MGMT, and inhibiting autophagy flux in the later stage. Therefore, this study provided a novel therapeutic strategy using the combination of TMZ with EPIC-1042 for glioblastoma treatment.

Comparative analysis of α-pinene alone and combined with temozolomide in human glioblastoma cells

α-Pinene (PEN) is a phyto compound present in terpene plants. In traditional medicine, PEN has been used for its anti-inflammatory, pain-relieving, and bronchodilator properties. The effect of PEN in combination with temozolomide (TMZ) in glioblastoma multiforme (GBM) cells has been evaluated. The action of the PEN + TMZ combination on cell migration, soft-agar, and cell death was determined in LN229 and U87MG human glioblastoma cells. In combination, PEN with TMZ showed a synergistic inhibitory effect in the GBM cells. The PEN + TMZ treatment showed a higher fluorescent intensity and reduced the percentage of wound area closure compared to the compound alone. The compounds in combination also resulted in a reduction in single-cell colony formation. To conclude, the study showed that plant-derived PEN enhanced the effectiveness of standard chemotherapeutic, TMZ, in LN229 and U87MG cells.

Targeting sphingolipid metabolism with the sphingosine kinase inhibitor SKI-II overcomes hypoxia-induced chemotherapy resistance in glioblastoma cells: effects on cell death, self-renewal, and invasion

BackgroundGlioblastoma patients commonly develop resistance to temozolomide chemotherapy. Hypoxia, which supports chemotherapy resistance, favors the expansion of glioblastoma stem cells (GSC), contributing to tumor relapse. Because of a deregulated sphingolipid metabolism, glioblastoma tissues contain high levels of the pro-survival sphingosine-1-phosphate and low levels of the pro-apoptotic ceramide. The latter can be metabolized to sphingosine-1-phosphate by sphingosine kinase (SK) 1 that is overexpressed in glioblastoma. The small molecule SKI-II inhibits SK and dihydroceramide desaturase 1, which converts dihydroceramide to ceramide. We previously reported that SKI-II combined with temozolomide induces caspase-dependent cell death, preceded by dihydrosphingolipids accumulation and autophagy in normoxia. In the present study, we investigated the effects of a low-dose combination of temozolomide and SKI-II under normoxia and hypoxia in glioblastoma cells and patient-derived GCSs.MethodsDrug synergism was analyzed with the Chou-Talalay Combination Index method. Dose–effect curves of each drug were determined with the Sulforhodamine B colorimetric assay. Cell death mechanisms and autophagy were analyzed by immunofluorescence, flow cytometry and western blot; sphingolipid metabolism alterations by mass spectrometry and gene expression analysis. GSCs self-renewal capacity was determined using extreme limiting dilution assays and invasion of glioblastoma cells using a 3D spheroid model.ResultsTemozolomide resistance of glioblastoma cells was increased under hypoxia. However, combination of temozolomide (48 µM) with SKI-II (2.66 µM) synergistically inhibited glioblastoma cell growth and potentiated glioblastoma cell death relative to single treatments under hypoxia. This low-dose combination did not induce dihydrosphingolipids accumulation, but a decrease in ceramide and its metabolites. It induced oxidative and endoplasmic reticulum stress and triggered caspase-independent cell death. It impaired the self-renewal capacity of temozolomide-resistant GSCs, especially under hypoxia. Furthermore, it decreased invasion of glioblastoma cell spheroids.ConclusionsThis in vitro study provides novel insights on the links between sphingolipid metabolism and invasion, a hallmark of cancer, and cancer stem cells, key drivers of cancer. It demonstrates the therapeutic potential of approaches that combine modulation of sphingolipid metabolism with first-line agent temozolomide in overcoming tumor growth and relapse by reducing hypoxia-induced resistance to chemotherapy and by targeting both differentiated and stem glioblastoma cells.