CBIO-28. IDENTIFYING THE ROLE OF MISMATCH REPAIR IN TEMOZOLOMIDE-INDUCED ATR ACTIVATION FOR MGMT-METHYLATED GLIOBLASTOMA MULTIFORME

The methylation status of the O6-methyl guanine methyltransferase (MGMT) gene promoter is a prognostic biomarker for treatment with the alkylator, temozolomide (TMZ) in many solid tumors including gliomas and colorectal cancers. It is well established that patients with a methylated MGMT promoter (MGMT-) who are treated with the TMZ have a better overall survival than patients with an unmethylated MGMT promoter (MGMT+). The enzyme produced by the MGMT gene is responsible for removing cytotoxic O6-methylguanine (O6-meG) lesions formed by TMZ. In the MGMT- setting, the O6-meG lesion activates the mismatch repair (MMR) pathway which functions to remove the damage. Published work from our group reported differential activation of the ataxia telangiectasia and RAD3 related protein (ATR) in MGMT- and MGMT+ glioblastoma multiforme (GBM) cells in response to TMZ treatment, as demonstrated through the phosphorylation of CHK1. Though it is known that MMR proteins are involved in ATR activation, the specific MMR proteins required for ATR activation by TMZ-induced alkyl lesions remain unknown in the MGMT- setting. Here, we demonstrate that specific mismatch repair proteins, including MSH2, MSH6, and PMS2 play a role in ATR activation in the presence of O6-meG lesions. We show that there is potent synergy with ATRi and TMZ in the MGMT- MMR- proficient GBM cell line, which is abrogated in an shMMR MGMT- GBM cell line. Additionally, we observe decreased levels of pCHK1 in the shMMR MGMT- setting compared to the MGMT- MMR-proficient cells, suggesting that MMR is integral in the activation of ATR upon TMZ treatment. Mechanistic understanding of how the MMR system is involved in ATR activation by TMZ can ultimately be exploited for therapeutic gain.

Species-specific mobile genetic elements in the gene of repair enzyme MGMT in new world monkeys

Aim.To analyze the distribution of species-specific mobile genetic elements (MGE) in orthologs of the MGMT gene in Platyrrhina. Methods. The homology between nucleotide sequences was determined by BLAST 2.6.1. The results of the search and identification of MGE were performed using the CENSOR program. Results. On the example of orthologs of the MGMT gene in New World monkeys, it has been shown that different species-specific MGE identified in their intron sequences may have different evolutionary chronologies. In the case of the element Alu2_TS, which originated in the Tarsiiformes representative, it was found that in evolutionarily close primates it undergoes deletion degradation, while fragments of the human-specific L1Hs element are found in the genomes of evolutionarily distant primates long before the formation and emergence of this retroelement. Conclusions. The chronology of evolutionary changes in the gene MGMT and its species-specific MGE can be of different nature and occur in parallele and independently. Keywords: Platyrrhina, MGMT gene, MGE, Alu2_TS, L1Hs.

Glioma Image Analysis to Accurately Classify Mgmt and Predict Drug Effectiveness

Glioblastoma multiforme is a deadly brain cancer with a median patient survival time of 18-24 months. A single biopsy cannot provide complete assessment of the tumor’s microenvironment, making personalized care limited. 50% of the patients do not respond to the anti-cancer drug Temozolomide(TMZ) because of the over-expression of MGMT gene. Epigenetic silencing of the MGMT gene by methylation results in decreased MGMT expression, resulting in increased sensitivity to TMZ, and longer survival. The purpose of this research is to use artificial intelligence (AI) to design a low cost methodology to determine the MGMT’s methylation status and suggest non- invasive treatment plan

Transcriptional Pausing and Activation at Exons-1 and -2, Respectively, Mediate the MGMT Gene Expression in Human Glioblastoma Cells

Background: The therapeutically important DNA repair gene O6-methylguanine DNA methyltransferase (MGMT) is silenced by promoter methylation in human brain cancers. The co-players/regulators associated with this process and the subsequent progression of MGMT gene transcription beyond the non-coding exon 1 are unknown. As a follow-up to our recent finding of a predicted second promoter mapped proximal to the exon 2 [Int. J. Mol. Sci.2021, 22(5), 2492], we addressed its significance in MGMT transcription. Methods: RT-PCR, RT q-PCR, and nuclear run-on transcription assays were performed to compare and contrast the transcription rates of exon 1 and exon 2 of the MGMT gene in glioblastoma cells. Results: Bioinformatic characterization of the predicted MGMT exon 2 promoter showed several consensus TATA box and INR motifs and the absence of CpG islands in contrast to the established TATA-less, CpG-rich, and GAF-bindable exon 1 promoter. RT-PCR showed very weak MGMT-E1 expression in MGMT-proficient SF188 and T98G GBM cells, compared to active transcription of MGMT-E2. In the MGMT-deficient SNB-19 cells, the expression of both exons remained weak. The RT q-PCR revealed that MGMT-E2 and MGMT-E5 expression was about 80- to 175-fold higher than that of E1 in SF188 and T98G cells. Nuclear run-on transcription assays using bromo-uridine immunocapture followed by RT q-PCR confirmed the exceptionally lower and higher transcription rates for MGMT-E1 and MGMT-E2, respectively. Conclusions: The results provide the first evidence for transcriptional pausing at the promoter 1- and non-coding exon 1 junction of the human MGMT gene and its activation/elongation through the protein-coding exons 2 through 5, possibly mediated by a second promoter. The findings offer novel insight into the regulation of MGMT transcription in glioma and other cancer types.

Effect of temozolomide combined with radiotherapy on survival and MGMT protein expression in recurrent malignant glioma patients

Purpose: To investigate the effect of temozolomide (TMZ) combined with radiotherapy (RT) on O-6- methylguanine-DNA methyltransferase (MGMT) protein and survival of recurrent malignant glioma patients. Methods: Ninety-two patients with malignant glioma in our hospital from January 2014 to January 2015 were assigned to study and control groups using the random table method. Subjects in the control group received radiotherapy (total dose in the range of 60 – 75 Gy), while those in the study group were given TMZ orally (75 mg/m2) daily in addition to radiotherapy, as well as TMZ at 150 – 200 mg/m2. After treatment, clinical effectiveness was compared for the two groups. Changes in methylation of MGMT gene were determined in the two groups. The patients were followed up for 3 years, and the degrees of survival and recurrence were recorded. Results: Total effectiveness of clinical treatment was markedly higher in the study group (76.09 %) than in the control group (45.65 %; p < 0.05). One month after radiotherapy, significant decrease in MGMT gene methylation was seen in patients in the study group, relative to control patients (p < 0.05). Patients in the study group had lower median recurrence but higher degree of survival in the 2nd and 3rd years, relative to control patients (p < 0.05). Conclusion: The combination of temozolomide and radiotherapy is more effective than radiotherapy in the treatment of recurrent malignant glioma. The combined treatment significantly inhibits tumor recurrence in patients, and improves their prognosis and standard of life.

MGMT gene polymorphisms in patients with severe hematological toxicity treated with temozolomide for adult diffuse gliomas: Results from a tertiary-care comprehensive cancer center.

e14032 Background: Polymorphisms in MGMT gene have been implicated in temozolomide (TMZ)-induced hematological toxicity in patients with adult diffuse gliomas. We aimed to investigate the association of three single nucleotide polymorphisms (SNPs) in the MGMT gene viz. L84F, I143V/K178R with severe hematological toxicity in patients of adult diffuse glioma treated with TMZ at an academic neuro-oncology unit of a tertiary-care comprehensive cancer centre from India. Methods: Thirty-three patients of adult diffuse glioma treated with multi-modality adjuvant therapy including TMZ who developed CTCAE V5.0 grade 2-4 hematological toxicity were included after written informed consent. Genomic DNA was extracted from peripheral blood mononuclear cells for SNP analysis. Correlation of MGMT SNP with patient demographics and hematological toxicity was assessed using the Chi-square and Fisher’s exact test. Results: Twenty-four patients (72.7%) developed grade 3-4 hematological toxicity with TMZ with a distinct female predilection. The variant T allele of L84F was expressed in 28.7% of the total 66 analyzed alleles which was markedly higher than previously reported in the general population of South Asian ancestry. The variant G allele of I43V/K178R was expressed in 9.3% (6/64) of 64 analyzed alleles. Persistent myelosuppression lasting beyond 2 months after cessation of TMZ correlated with I143V/K178R hetero/homozygous compared to wild type allele (p = 0.03). However, no significant correlation could be seen between any of the tested MGMT SNPs and grade of hematological toxicity. Conclusions: There is a higher prevalence of the L84F polymorphism in Indian patients with severe hematological toxicity than previously reported in the literature. The variant G allele of I143V/K178R is associated with prolonged and persistent myelosuppression induced by TMZ. A larger case-control is being planned to further elucidate the causal relationship between MGMT gene polymorphisms and TMZ-induced severe hematological toxicity.