Abstract MP244: Deciphering The Mechanism Of Elevation Of Mir34a And Its Attenuation Via Corm A1 And Subsequent Improvement In Mitochondrial Status In Atherogenic Mdms

Elevated levels of miR34a and its association with macrophage polarization is reported in atherosclerosis but the underlying mechanism that upregulates miR34a lacks clarity. Herein, the mechanism of miR34a elevation in atherogenic human monocyte derived macrophages (MdMs) and subsequent changes in mitochondria were monitored. Further, CO supplementation (via Carbon monoxide Releasing Molecule A1; CORM A1) to atherogenic (OxLDL treated) MdMs was used to achieve downregulation of miR34a. Herein, we also hypothesize that lowering of miR34a in atherogenic MdMs improves the cellular health and mitochondrial function. Transcriptional factors (P53, NF-κb), transcriptional inhibitors (Zeb1, snai1, stat3) and epigenetic modification (methylation) in promoter region of miR34a were evaluated. OxLDL treated MdMs recorded significant decrement in mRNA levels of the said transcription inhibitors whereas; the same were reversed in CORM A1 co-treated group. Further, hypomethylation was recorded in the promotor region of miR34a on oxLDL treatment but methylation status was reverted to the control levels following CORM A1 co-supplementation. The mRNA levels of transcription factors showed non-significant changes in all the experimental groups. In silico docking studies had shown that CO effectively binds to the DNA binding domains of p53 that possibly prevents subsequent binding to their respective miR34a promotor regions. Positive docking of miR34a to 3’UTR of SIRT-1 supported our observation on lowered SIRT-1 and PGC-1α levels in oxLDL group that were found to be restored in CORM A1 co-treated group. Poor indices of mitochondrial biogenesis (SIRT-1, PGC1α, Nrf-1, Drp1, Mito Tracker Red staining), function (ATP assay), mitochondrial membrane potential (JC-1) and mitochondrial antioxidants (SOD2 and TrxR2), cellular ROS (DCFDA) following oxLDL treatment was found to be restored by CORM A1 co-treatment. In conclusion, atherogenic elevation of miR34a is as a result of hypomethylation in its promotor region and lowered mRNA transcripts of its inhibitors (Zeb1, snai1, stat3). Further, lowering of miR34a by CORM A1, improves atherogenic status of MdMs as evidenced by an improved cellular and mitochondrial health.

PATH-28. EXTENT AND PROGNOSTIC VALUE OF MGMT PROMOTOR METHYLATION DEPEND ON IDH MUTATION AND 1p19q CO-DELETION IN GLIOMA WHO GRADE II

INTRODUCTION Methylation of the promotor region of the O6-methylguanin-DNA-methyltransferase (MGMT) gene is associated with increased survival in low- and high-grade glioma. It is unknown whether this association also applies to the 2016 WHO categories of glioma WHO grade II. MATERIAL AND METHODS We retrospectively searched the institutional database of the Center for Neuro-Oncology for patients with glioma WHO grade II. Patients were assigned to one of three groups according to the 2016 WHO classification system: 1. 1p19q co-deleted oligodendroglioma, IDH mutant; 2. 1p19q non-codeleted astrocytoma, IDH mutant; 3. 1p19q non-codeleted astrocytoma, IDH wild-type. MGMT methylation status was analysed using Sanger sequencing of the CpG sites 74-98 within the MGMT promotor region. The total number of methylated CpG sites was calculated for each patient. RESULTS 155 patients with glioma WHO grade II were encountered, including 81 1p19q co-deleted, IDH mutant oligodendrogliomas; 54 IDH mutant astrocytomas; and 20 IDH wild-type astrocytomas. The mean number of methylated CpG sites among oligodendrogliomas was significantly higher when compared to IDH mutant astrocytomas (18.9 ± 0.4 vs. 16.3 ± 0.6; p = 0.001). In turn, the number of methylated CpG sites among IDH mutant astrocytomas was higher when compared to IDH wild-type astrocytomas (16.3 ± 0.6 vs. 12.3 ± 1.9; p = 0.007). Median follow-up was estimated at 35 months. Median time to malignant progression was 87 months for all patients, and median overall survival was not reached. In the entire cohort, a larger number of methylated CpG sites was prognostic of overall survival and time to malignant progression. When analysed separately for the three WHO subgroups, a similar association was only retained in IDH wild-type astrocytoma. CONCLUSION In our series of WHO II gliomas, MGMT promotor methylation appeared strongly associated with 1p19q codeletion and IDH mutations. MGMT promotor methylation was only prognostic in IDH wild-type astrocytoma.

Transcriptional Regulation of the Outer Membrane Protein A in Acinetobacter baumannii

Acinetobacter baumannii is known for its virulence in severely ill, hospitalized patients and for exhibiting multidrug resistance. A. baumannii infection treatment poses a serious problem in clinical environments. The outer membrane protein A (OmpA) of the Acinetobacter genus is involved in bacterial virulence. Regulatory factors of OmpA in the post-transcriptional stage have been previously identified. However, the regulatory factors that act before the transcriptional stage remain unclear. We investigated the A1S_0316 gene that encodes a putative transcription factor for OmpA expression in A. baumannii. A1S_0316 was purified and examined using size-exclusion chromatography, which revealed that it forms an oligomer. The binding affinity of A1S_0316 to the OmpA promoter region was also examined. We compared the binding affinity to the OmpA promotor region between A1S_0316 and the AbH-NS protein. A1S_0316 showed higher binding affinity to the OmpA promotor region than did H-NS. We examined the regulatory effect of these proteins on OmpA expression in A. baumannii using real-time qPCR and various in vitro tools. Our results indicated that A1S_0316 acts as an anti-repressor on the promotor region of the OmpA gene by inhibiting the binding of the AbH-NS protein. This study was the first demonstration of the transcriptional regulation of OmpA expression.

PATH-04. INFLUENCE OF INDIVIDUAL CpG METHYLATION STATUS OF THE MGMT PROMOTOR ON OUTCOME IN ADULT PATIENTS WITH GLIOBLASTOMA MULTIFORME RECEIVING ALKYLATING AGENT TREATMENT

BACKGROUND Methylation of O-6-methylguanine-DNA-methyltransferase (MGMT) promotor causes gene silencing and has been associated with a favourable prognosis in patients with glioblastoma multiforme (GBM) receiving alkylating chemotherapy. However, analysis of MGMT promotor methylation is usually reported as a cut-off depending on the results of the correspondent CpG-site testing. This approach disregards a possible heterogeneity concerning the methylation status within the individual CpG-sites and its possible association with prognosis in GBM patients. The current study aimed at elucidating the association between methylation of CpG-sites 74–98 within the MGMT promotor region and outcome in GBM patients receiving alkylating agents. METHODS Individual methylation status of 230 patients with histologically proven GBM following concomitant radio-chemotherapy with TMZ after stereotactic biopsy or open tumor resection was assessed by the Sanger-sequencing approach. Methylation of CpG-sites 74–98 within the MGMT promotor region was defined according to a ratio of cytosine/thymine peak > 50%. The total number of methylated CpG-sites was correlated with outcome using proportional hazards models. In a subset of 34 patients, a correlation between individual CpG-site methylation and MGMT mRNA-expression was performed. RESULTS Median progression-free (PFS) and overall survival (OS) were 7.8 and 14.6months, respectively. The cumulative total number of methylated loci within the CpG-sites 74–98 was strongly associated with both PFS and OS and retained its prognostic influence on outcome in multivariate models (p< 0.001). Furthermore, a linear coherence between the total number of methylated CpG-sites 74–98 and survival parameters could be observed. Moreover, low number of methylated CpG-sites was observed in tumor specimen with a high mRNA-expression and vice versa (Spearman-correlation-coefficient: -0.62). CONCLUSION Our data suggest a strong linear coherence between outcome and the total number of methylated CpG-sites 74–98, thus an up-front analysis of the individual GpC-site methylation status prior to initiation of alkylating chemotherapy might help improving treatment response in GBM patients.

P14.02 Influence of individual CpG methylation status on outcome in adult patients with glioblastoma multiforme receiving alkylating agent treatment

Background: Methylation of the O-6-methylguanine-DNA methyltransferase (MGMT) promotor causes gene silencing and has been associated with a favourable prognosis in patients with glioblastoma multiforme (GBM) receiving alkylating chemotherapy. However, analysis of MGMT promotor methylation is usually reported as a cut-off depending on the results of the correspondent CpG site testing. This approach disregards a possible heterogeneity concerning the methylation status within the individual CpG sites and its possible association with prognosis in GBM patients. The current study aimed at elucidating the association between methylation of CpG sites 74–98 within the MGMT promotor region and outcome in GBM patients receiving alkylating agents. Material and Methods: Individual methylation status of 230 patients with histologically proven GBM following concomitant radio-chemotherapy with TMZ after stereotactic biopsy or open tumor resection (OTR) was assessed by the Sanger sequencing (Sseq) approach. Methylation of CpG sites 74–98 within the MGMT promotor region was defined according to a ratio of cytosine /thymine peak >50%. The total number of methylated CpG sites as well as clinical factors such as age, Karnofsky Performance Score (KPS) and mode of surgical procedure were correlated with outcome using proportional hazards models. In a subset of 34 patients, a correlation between individual CpG methylation and MGMT mRNA expression was performed. Results: Median progression-free (PFS) and overall survival (OS) were 7.8 and 14.6 months, respectively. Alongside younger age, KPS> 80 and OTR, the cumulative total number of methylated loci within the CpG sites 74–98 was strongly associated with both PFS and OS and retained its prognostic influence on outcome in multivariate models (p <0.001). Furthermore, a linear coherence between the total number of methylated CpG sites 74–98 and survival parameters could be observed. Moreover, low number of methylated CpG sites was observed in tumor specimen with a high mRNA expression and vice versa (Spearman correlation coefficient: -0.62). Conclusion: In contrast to the concept of dichotomizing the MGMT promotor status into ‘methylated’ and ‘non-methylated’, our approach shows a clear heterogeneity within the methylation status of the CpG sites 74–98 within the GBM tumor specimens. Our data suggest a strong correlation between outcome and the total number of methylated CpG sites, thus an up-front analysis of the individual GpC site methylation status prior to initiation of alkylating chemotherapy might help to improve treatment response in GBM patients.