The Effect of Levetiracetam Treatment on Survival in Patients with Glioblastoma: A Systematic Review and Meta-Analysis.

Background:Levetiracetam (LEV) is an anti-epileptic drug (AED) that sensitizes glioblastoma (GBM) to temozolomide (TMZ) chemotherapy by inhibiting O6-methylguanine-DNA methyltransferase (MGMT) expression. Adding LEV to the standard of care (SOC) for GBM may improve TMZ efficacy. This study aimed to pool the existing evidence in the literature to quantify LEV’s effect on GBM survival and characterize its safety profile to determine whether incorporating LEV into the SOC is warranted.Method:A search of CINAHL, Embase, PubMed, and Web of Science from inception to May 2021 was performed to identify relevant articles. Hazard ratios (HR), median overall survival, and adverse events were pooled using random-effect models. Meta-regression, funnel plots, and the Newcastle-Ottawa Scale were utilized to identify sources of heterogeneity, bias, and statistical influence.Results:From 20 included studies, 5804 GBM patients underwent meta-analysis, of which 1923 (33%) were treated with LEV. Administration of LEV did not significantly improve survival in the entire patient population (HR=0.89, p=0.094). Significant heterogeneity was observed during pooling of HRs (I2=75%, p<0.01). Meta-regression determined that LEV treatment effect decreased with greater rates of MGMT methylation (RC=0.03, p=0.02) and increased with greater proportions of female patients (RC=-0.05, p=0.002). Concurrent LEV with the SOC for GBM did not increase odds of adverse events relative to other AEDs.Conclusions:Levetiracetam treatment may not be effective for all GBM patients. Instead, LEV may be better suited for treating specific molecular profiles of GBM. Further studies are necessary to identify optimal GBM candidates for LEV.

GammaTile® brachytherapy in the treatment of recurrent glioblastomas

Background GammaTile® (GT) is a recent U.S. Food and Drug Administration (FDA) cleared brachytherapy platform. Here, we report clinical outcomes for recurrent glioblastoma patients after GT treatment following maximal safe resection. Methods We prospectively followed twenty-two consecutive Isocitrate Dehydrogenase (IDH) wild-type glioblastoma patients (6 O6-Methylguanine-DNA methyltransferase methylated (MGMTm); sixteen MGMT unmethylated (MGMTu)) who underwent maximal safe resection of recurrent tumor followed by GT placement. Results The cohort consisted of 14 second and eight third recurrences. In terms of procedural safety, there was one 30-day re-admission (4.5%) for an incisional cerebrospinal fluid leak, which resolved with lumbar drainage. No other wound complications were observed. Six patients (27.2%) declined in Karnofsky Performance Score (KPS) after surgery due to worsening existing deficits. One patient suffered a new-onset seizure post-surgery (4.5%). There was one (4.5%) 30-day mortality from intracranial hemorrhage secondary to heparinization for an ischemic limb. The mean follow-up was 733 days (range 279-1775) from the time of initial diagnosis. Six-month local control (LC6) and twelve-month local control (LC12) were 86 and 81%, respectively. Median progression-free survival (PFS) was comparable for MGMTu and MGMTm patients (~8.0 months). Median overall survival (OS) was 20.0 months for the MGMTu patients and 37.4 months for MGMTm patients. These outcomes compared favorably to data in the published literature and an independent glioblastoma cohort of comparable patients without GT treatment. Conclusions This clinical experience supports GT brachytherapy as a treatment option in a multi-modality treatment strategy for recurrent glioblastomas.

Sulfonated Amphiphilic Poly(α)glutamate Amine—A Potential siRNA Nanocarrier for the Treatment of Both Chemo-Sensitive and Chemo-Resistant Glioblastoma Tumors

Development of chemo-resistance is a major challenge in glioblastoma (GB) treatment. This phenomenon is often driven by increased activation of genes associated with DNA repair, such as the alkyl-removing enzyme O6-methylguanine-DNA methyltransferase (MGMT) in combination with overexpression of canonical genes related to cell proliferation and tumor progression, such as Polo-like kinase 1 (Plk1). Hereby, we attempt to sensitize resistant GB cells using our established amphiphilic poly(α)glutamate (APA): small interfering RNA (siRNA) polyplexes, targeting Plk1. Furthermore, we improved brain-targeting by decorating our nanocarrier with sulfonate groups. Our sulfonated nanocarrier showed superior selectivity towards P-selectin (SELP), a transmembrane glycoprotein overexpressed in GB and angiogenic brain endothelial cells. Self-assembled polyplexes of sulfonated APA and siPlk1 internalized into GB cells and into our unique 3-dimensional (3D) GB spheroids inducing specific gene silencing. Moreover, our RNAi nanotherapy efficiently reduced the cell viability of both chemo-sensitive and chemo-resistant GB cells. Our developed sulfonated amphiphilic poly(α)glutamate nanocarrier has the potential to target siRNA to GB brain tumors. Our findings may strengthen the therapeutic applications of siRNA for chemo-resistant GB tumors, or as a combination therapy for chemo-sensitive GB tumors.

KDM6B sensitizes PARthanatos via suppression of O6-methylguanine DNA methyltransferase repair and sustained checkpoint response

Poly(ADP-ribose) polymerase-1 (PARP-1) is a DNA damage sensor and contributes to both DNA repair and cell death processes. However, how PARP-1 signaling is regulated to switch its function from DNA repair to cell death remains largely unknown. Here, we found that PARP-1 plays a central role in alkylating agent-induced PARthanatic cancer cell death. Lysine demethylase 6B (KDM6B) was identified as a key cell death effector in PARthanatos. Knockout of KDM6B or loss of KDM6B demethylase activity conferred cancer cells resistance to PARthanatic cell death in response to alkylating agents. Mechanistically, KDM6B knockout suppressed methylation at the promoter of O6-methylguanine-DNA methyltransferase (MGMT) to enhance MGMT expression and its direct DNA repair function, thereby inhibiting DNA damage-evoked PARP-1 hyperactivation and subsequent cell death. Moreover, KDM6B knockout triggered sustained Chk1 phosphorylation and activated a second repair machinery to fix DNA damage evading from MGMT repair. Inhibition of MGMT or checkpoint response re-sensitized KDM6B deficient cells to PARthanatos induced by alkylating agents. These findings provide new molecular insights into epigenetic regulation of PARP-1 signaling mediating DNA repair or cell death and identify KDM6B as a biomarker for prediction of cancer cell vulnerability to alkylating agent treatment.

Early Initiation of Temozolomide Therapy May Improve Response in Aggressive Pituitary Adenomas

IntroductionAggressive pituitary adenomas (APAs) are, by definition, resistant to optimal multimodality therapy. The challenge lies in their early recognition and timely management. Temozolomide is increasingly being used in patients with APAs, but evidence supporting a favorable response with early initiation is lacking.MethodsThis was a single-center study of all patients with APAs who received at least 3 cycles of temozolomide (150–200 mg/m2). Their baseline clinico-biochemical and radiological profiles were recorded. Immunohistochemical evaluation for cell-cycle markers O6-methylguanine-DNA methyltransferase (MGMT), MutS homolog 2 (MSH2), MutS homolog 6 (MSH6), MutL homolog 1 (MLH1), and postmeiotic segregation increased 2 (PMS2) was performed, and h-scores (product of the number of positive cells and staining intensity) were calculated. Response was assessed in terms of radiological response using the RECIST criteria. Patients with controlled disease (≥30% reduction in tumor volume) were classified as responders.ResultsThe study comprised 35 patients (48.6% acromegaly, 37.1% prolactinomas, and 14.3% non-functioning pituitary adenomas). The median number of temozolomide (TMZ) cycles was 9 (IQR 6–14). Responders constituted 68.6% of the cohort and were more likely to have functional tumors, a lower percentage of MGMT-positive staining cells, and lower MGMT h-scores. There was a significantly longer lag period in the initiation of TMZ therapy in non-responders as compared with responders (median 36 vs. 15 months, p = 0.01). ROC-derived cutoffs of 31 months for the duration between diagnosis and TMZ initiation, low-to-intermediate MGMT positivity (40% tumor cells), and MGMT h-score of 80 all had a sensitivity exceeding 80% and a specificity exceeding 70% to predict response.ConclusionEarly initiation of TMZ therapy, functional tumors, and low MGMT h-score predict a favorable response to TMZ in APAs.

Roles of the hydroxy group of tyrosine in crystal structures of Sulfurisphaera tokodaii O 6-methylguanine-DNA methyltransferase

O 6-Methylguanine-DNA methyltransferase (MGMT) removes cytotoxic O 6-alkyl adducts on the guanine base and protects the cell from genomic damage induced by alkylating agents. Although there are reports of computational studies on the activity of the enzyme with mutations at tyrosine residues, no studies concerning the crystal structure of its mutants have been found. In this study, the function of Tyr91 was investigated in detail by comparing the crystal structures of mutants and their complexes with substrate analogs. In this study, tyrosine, a conserved amino acid near the active-site loop in the C-terminal domain of Sulfurisphaera tokodaii MGMT (StoMGMT), was mutated to phenylalanine to produce a Y91F mutant, and the cysteine which is responsible for receiving the methyl group in the active site was mutated to a serine to produce a C120S mutant. A Y91F/C120S double-mutant StoMGMT was also created. The function of tyrosine is discussed based on the crystal structure of Y91F mutant StoMGMT. The crystal structures of StoMGMT were determined at resolutions of 1.13–2.60 Å. They showed no structural changes except in the mutated part. No electron density for deoxyguanosine or methyl groups was observed in the structure of Y91F mutant crystals immersed in O 6-methyl-2′-deoxyguanosine, nor was the group oxidized in wild-type StoMGMT. Therefore, the hydroxy group of Tyr91 may prevent the oxidant from entering the active site. This suggests that tyrosine, which is highly conserved at the N-terminus of the helix–turn–helix motif across species, protects the active site of MGMTs, which are deactivated after repairing only one alkyl adduct. Overall, the results may provide a basis for understanding the molecular mechanisms by which high levels of conserved amino acids play a role in ensuring the integrity of suicide enzymes, in addition to promoting their activity.

CBIO-02. IN VITRO TUMOR TREATING FIELDS (TTFields) REDUCE PROLIFERATION AND ALTER MLH1 EXPRESSION IN TEMOZOLOMIDE RESISTANT (TMZR) PATIENT-DERIVED GLIOBLASTOMA (GBM) CELLS

BACKGROUND TTFields therapy is approved as a monotherapy for the treatment of recurrent GBM, tumors that often have acquired resistance to TMZ. To examine TTFields efficacy in the context of TMZR in vitro, TTFields were applied to TMZR GBM cells to assess proliferation, clonogenicity, and changes in expression of O6-methylguanine-DNA methyltransferase (MGMT) that confers TMZ resistance and DNA Mismatch Repair Protein Mlh1 (MLH1) that may confer TMZ sensitivity. METHODS 15-037 GBM cells were isolated from a newly-diagnosed patient tumor (IDH-WT, unmethylated MGMT promoter. Cells were grown in DMEM/F12 media with 10% FBS and gentamicin. U-251 MG cells and 15-037 cells were incubated with 100 µM TMZ for three 5-day cycles to establish TMZR. TMZR cells were plated on coverslips (1×104cells/coverslip) and incubated overnight prior to TTFields application. TTFields were applied at 200 kHz (field intensity of ~1.6 V/cm) for 14 days. Then coverslips (n=3/group) were harvested, counted, and replated for clonogenic assay at 1000 cells/35mm2 well. Clonogenic assay plates were stained with crystal violet, imaged, and colonies counted. Additional coverslips (n=3/group) were harvested and MGMT and MLH1 expression were assessed by qRT-PCR with the delta-delta Ct method using GAPDH as housekeeping control. Cell counts and colony counts were compared with two-tailed t-test with significance at p&lt; 0.05. RESULTS In U-251 MG and 15-037 TMZR cells, TTFields application significantly reduced both cell counts and clonogenic assay colony counts compared to untreated controls (U-251 MG: 45.4% and 26.5%; 15-037: 96.2% and 60.9%, respectively, p&lt; 0.05). In both TMZR cell lines, MGMT expression was unchanged by TTFields, but MLH1 expression increased 2.2 fold in U-251 MG cells and 7.1 fold in 15-037 cells after TTFields application. CONCLUSIONS Not only are TMZR GBM cells sensitive to TTFields in vitro, but TTFields increased expression of MLH1 which may be able to reduce resistance to TMZ.

CBIO-15. LOSS OF WILLIAMS SYNDROME TRANSCRIPTION FACTOR (WSTF) LEADS TO IMPROVED TEMOZOLOMIDE SENSITIVITY IN HUMAN GLIOBLASTOMA CELLS IRRESPECTIVE OF MGMT EXPRESSION

BACKGROUND The prognosis for glioblastoma multiforme (GBM) patients is poor with a median survival of approximately 15 months. The DNA repair protein O 6-methylguanine-DNA methyltransferase (MGMT) counteracts the effects of temozolomide (TMZ) chemotherapy and is thus associated with poor outcome in GBM patients. Williams Syndrome Transcription Factor (WSTF) has been suggested to regulate the DNA damage response pathway (DDR) in both an indirect (through chromatin remodeling) and direct manner (by phosphorylating H2AX at Tyr142). However, whether WSTF has any role in the development of resistance against chemotherapy through its functions in the DDR in GBMs, is so far unknown. In this study, we investigated whether a loss of WSTF sensitizes different MGMT-proficient and -deficient GBM cell lines to TMZ treatment. METHODS We generated WSTF knockout clones from both MGMT-proficient (LN18, T98G) and -deficient GBM cell lines (U-251) using CRISPR/Cas9 gene-editing technology with lentiviral vectors. The PCR-based screening results combined with the T7 endonuclease mismatch assay for bi-allelic monoclonal knockouts were verified via sequencing and immunoblotting to identify candidate knockout clones. Colony formation assays were performed to determine the survival ability in response to TMZ treatment. Statistical analysis was performed using two-way ANOVA. RESULTS WSTF knockout clones showed a significant decrease in colony formation after TMZ-treatment compared to the corresponding control groups (non-target single guide RNA) (LN18: Clone 59 vs control: p= 0.0456, T98G: All three studied clones vs control: p&lt; 0.0001, U-251: Clone 7/35.1/70.2 vs control: p&lt; 0.0001/p= 0.0107/p= 0.0119). CONCLUSION WSTF is an important factor in both MGMT de- and proficient GBM cell lines for response against TMZ chemotherapy. The loss of WSTF leads to a significantly increased TMZ sensitivity in clinically relevant concentrations for all the studied cell lines. Ongoing studies are investigating the underlying mechanisms and potential alterations in the DDR pathway caused by WSTF loss.

RADT-15. FIRST EXPERIENCE WITH MAXIMAL SAFE RESECTION AND GAMMATILE BRACHYTHERAPY AS TREATMENT FOR RECURRENT GLIOBLASTOMA

INTRODUCTION Gammatile (GT) is a recently FDA-cleared brachytherapy platform with 131Cs seeds imbedded into a resorbable collagen carrier for surgically targeted radiation delivery. We report the first experience for recurrent glioblastoma patients who underwent GT treatment following surgical resection. METHODS Twenty-two consecutive patients with 23 isocitrate dehydrogenase (IDH) wild-type glioblastomas (14 second; eight third recurrence) who underwent intra-operative MRI/5-ALA guided maximal safe resection followed by GT placement were prospectively followed. There were 6 methylguanine-DNA-methyltransferase promoter methylated (MGMTm) and 17 unmethylated (MGMTu) glioblastomas. RESULTS The median hospital stay was one day (range:1-15 days). There was one 30-day readmission (4.5%) for a cerebrospinal fluid leak from the incision site, which resolved with lumbar drainage. There were no other wound complications. One patient (4.5%) suffered new post-operative seizure. Eight patients experienced worsened neurological deficit (8/22 or 36%). While all deficits improved by the 30-day follow-up, 7 of these 8 patients suffered KPS decline due to persistent deficits. There was one 30-day mortality (4.5%) from intracranial hemorrhage secondary to heparinization for an ischemic limb. The median follow-up after GT placement for the remaining 21 patients was 296 days (range:111-931 days). Six months local control (LC) was achieved in ~75% of the patients irrespective of MGMT status. Median overall survival (OS) was 715 days for the MGMTu patients, and not reached (&gt;1000 days) for MGMTm patients. These outcomes compared favorably to the published literature (LC: 3-49%; OS MGMTu: 135-285 days; OS MGMTm: 174-564 days) and an age, KPS, extent of resection matched glioblastoma cohort who underwent maximal safe resection without GT at our institution (LC: 52%; OS MGMTu: 462 days; OS MGMTm: 821 days; p=0.0089 and p=0.0271, respectively when compared to the GT treated patients). CONCLUSION This clinical experience supports the safety and efficacy of GT brachytherapy as a treatment option for recurrent glioblastomas.