Base Excision Repair Glycosylase Activity Is Impaired in a Subgroup of Acute Myeloid Leukemia Resulting in Increased Levels of Oxidative Base Lesions

Abstract Base excision repair (BER) is the primary DNA repair mechanism dealing with oxidative base lesions. Oxidative DNA base lesions are the predominant type of DNA damage in mammalian cells. Deficiencies in glycosylases, the BER initiating enzymes, have been associated with increased genomic instability and increased frequencies of cancer. Here we investigated the role of oxidative BER in acute myeloid leukemia (AML). We determined oxidative BER activity in 99 primary AML blast cell samples, 34 CD34+ umbilical cord blood cell samples and 27 AML cell lines using the alkaline comet assay. Oxidative base lesion levels were determined in 10 AML cell lines using a modified version of the Comet assay with the bacterial enzymes Fpg and Endo III as well as using liquid chromatography-coupled tandem mass spectrometry (LC-MS/MS). Using nuclear protein extracts in an oligonucleotide incision assay we tested the enzymatic activity of oxidative glycosylases. Finally, mutational analysis, gene expression analysis and protein expression of oxidative glycosylases was used using Sanger sequencing, real time PCR and western blot of nuclear extracts, respectively. We found DNA strand incision of oxidatively damaged bases significantly impaired in primary AML cells as compared to UCB cells (p= 0.003) suggesting a deficiency in BER glycosylases. In addition, 5/27 AML cell lines showed impaired DNA strand incision activity. We hypothesized that BER deficient cells harbor an increased number of oxidative base lesions compared to BER proficient cells. Using a modified comet assay and LC-MS/MS we were able to show that increased numbers of unrepaired oxidative base lesions were indeed present in glycosylase deficient AML cells (comet assay: p= 0.0001; mass spec: p= 0.03). We then evaluated the activity of the predominant oxidative DNA glycosylase, OGG1, and found significantly decreased DNA strand incision activity in BER deficient cells as compared to proficient cells (p= 0.002) further supporting the fact that glycosylases are impaired in BER deficient cells. Determining causes of BER deficiency preliminary experiments showed significantly decreased expression of nuclear OGG1 protein in BER deficient cells but did not reveal novel non-synonymous mutations or a difference in gene expression. Taken together we found impaired BER glycosylases in a substantial number of primary AML samples and AML cell lines resulting in increased levels of potentially mutagenic oxidative DNA base lesions Disclosures No relevant conflicts of interest to declare.

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Base Excision Repair Deficiency in Acute Myeloid Leukemia.

Blood ◽

10.1182/blood.v114.22.1106.1106 ◽

2009 ◽

Vol 114 (22) ◽

pp. 1106-1106

Author(s):

Werner Olipitz ◽

Nicole Scheer ◽

Franz Quehenberger ◽

Karin Hiden ◽

Julia Rankl ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

Cell Lines ◽

Base Excision Repair ◽

Myeloid Leukemia ◽

Excision Repair ◽

Significant Proportion ◽

Hematopoietic Stem ◽

Single Strand Break ◽

Base Excision ◽

Acute Myeloid

Abstract Abstract 1106 Poster Board I-128 Base excision repair (BER) is the main DNA repair mechanism for single DNA base lesions resulting from oxidative stress, chemical damage or ionizing radiation. We investigated BER in acute myeloid leukemia (AML), a disorder characterized by widespread genomic instability. AML cell lines were treated with H2O2 and DNA damage induction and repair were monitored using the alkaline comet assay. Significantly reduced single strand break (SSB) formation - representing BER intermediates - was observed in 5/10 cell lines. Significantly reduced SSB formation was also demonstrated in 15/30 leukemic samples from patients with therapy-related AML, 13/35 with de novo AML and 14/26 with AML following a myelodysplastic syndrome but only in 1/31 CD34+ hematopoietic stem and progenitor cell specimens isolated from umbilical cord blood (P=.0000056). Reduced SSB formation was not due to differences in intracellular ROS concentrations or selection for a damage resistant subpopulation. To determine whether initial steps of BER were impaired, incision assays with oligonucleotides harboring either 7,8-dihydro-8-oxoguanine or the AP site analog furan were performed. Significantly diminished cleavage for both substrates was observed in cell lines that did not exhibit SSB formation upon H2O2 treatment. These data demonstrate that BER is functionally impaired in a significant proportion of myeloid cell lines and leukemic cells from patients with AML. Disclosures No relevant conflicts of interest to declare.

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Base Excision Repair Gene OGG1 Affects the Relapse Risk of Acute Myeloid Leukemia

Blood ◽

10.1182/blood.v128.22.2901.2901 ◽

2016 ◽

Vol 128 (22) ◽

pp. 2901-2901

Author(s):

Nanami Gotoh ◽

Takayuki Saitoh ◽

Noriyuki Takahashi ◽

Rumi Ino ◽

Yuya Kitamura ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

Multivariate Analysis ◽

Base Excision Repair ◽

Gene Polymorphisms ◽

Myeloid Leukemia ◽

Excision Repair ◽

Genetic Mutation ◽

Transversion Mutation ◽

Base Excision ◽

Acute Myeloid

Abstract Background: Approximately 80% of acute myeloid leukemia (AML) patients can achieve complete remission, but around half of them relapse within five years. Recent studies have shown that AML relapse is associated with additional genetic mutation calls gclonal evolutionh in leukemic cell population (Ding, et al. Nature. 2012 & Parkin B, et al. Blood. 2013). These studies suggested that cytotoxic chemotherapy damaged cellular DNA and caused genetic mutation. In fact, anthracycline can produce 8-oxoguanine (8-OG) through induction of oxidative stress. 8-OG is most common DNA damage, which cause G:C to T:A transversion mutation. It is reported that transversion mutation more frequently observed in relapsed AML than primary AML. Base excision repair (BER) plays important role to correct base lesion including 8-OG and suppress genetic mutation. Therefore, we hypothesized BER gene polymorphisms may affect the risk of AML relapse, and focused five major functional polymorphisms: OGG1 S326C, MUTYH Q324H, APE1 D148E, XRCC1 R194W and XRCC1 R399Q. Material & Method: Ninety-four consecutive adults with AML who had achieved their first complete remission were recruited (male: 52, female: 42, age: 15-83 years, median age: 55.7 years). To remove the bias of the group, we also evaluated the risk in patients of non-M3 and under 65 years old (male: 22, female: 19, age: 15-64 years, median age: 49.0 years) (trimming group). These patients treated on Japan Adult Leukemia Study Group (JALSG) treatment protocols consisted of daunorubicin or idarubicin plus cytarabine (JALSG AML95, AML97, AML201, AML209). Genotyping was performed by PCR-RFLP method. The X2-test was used to compare the distribution of genotype and allele frequencies in patients. Leukemia-free survival (LFS) was calculated using the Kaplan-Meier method. Survival curves were compared using the log-rank test. In multivariate analysis, a stepwise selection procedure was performed using the proportional hazards Cox model for LFS. The variables were chosen with reference to previous studies; age, sex, white blood cell count and lactate dehydrogenase at diagnosis, number of induction courses, stem cell transplantation, MRC classification and history of tumor. This study was approved by the Institutional Review Board of Gunma University Hospital. Results: The OGG1 S326C CC genotype was observed significantly more often in the relapsed group (28.9% vs. 8.9%, OR = 4.10, 95% CI = 1.35-12.70, p = 0.01). In trimming group, the CC genotype was also observed more frequently in the relapsed group (50.0% vs. 6.9%, OR = 13.5, 95% CI = 2.17-84.0, p = 0.002). In addition, the OGG1 S326C CC genotype experienced a shorter median LFS than those with a non-CC genotype (CC vs. non-CC = 27.0 months vs. not reached, p = 0.02) (Figure 1). This genotype was also associated with poor LFS in trimming group (CC vs. non-CC = 11.0 months vs. not reached, p < 0.001) (Figure 1). Furthermore, multivariate analysis of LFS revealed OGG1 S326C CC genotype as an independent prognostic factor (HR = 4.32, 95% CI = 1.70-11.0, p = 0.002), like age, number of induction courses, and MRC classification (Table 1). Other polymorphisms had no significant effect on the risk of relapse. Conclusion: We previously reported that mutations by 8-OG were more efficiently suppressed in OGG1-S326 transduced cells than in OGG1-C326 transduced cells. Therefore, we hypothesized that low OGG1 activity promotes relapse of AML. To the best of our knowledge, this is the first report to show an association between BER gene polymorphisms and the relapse of AML. Our data suggest that OGG1 S326C can be a prognostic factor for AML relapse. Disclosures No relevant conflicts of interest to declare.

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The Polymorphisms Of Base Excision Repair Genes Influence The Cytogenetic Risk Factors In Acute Myeloid Leukemia

Blood ◽

10.1182/blood.v122.21.1355.1355 ◽

2013 ◽

Vol 122 (21) ◽

pp. 1355-1355

Author(s):

Atsushi Iwasaki ◽

Takayuki Saitoh ◽

Yasuhiro Nitta ◽

Batchimeg Norjimaa ◽

Chiharu Omiya ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

Base Excision Repair ◽

Myeloid Leukemia ◽

Excision Repair ◽

Control Group ◽

Healthy Controls ◽

Base Excision ◽

And Control ◽

Repair Genes ◽

Acute Myeloid

Abstract Background Base excision repair (BER) systems have important role for repairing oxidative DNA damage, and known to influence the carcinogenesis and the response to anti-cancer treatments. Although few studies have shown that several DNA repair genes are associated with an increased risk of leukemia, the clinical significance of BER polymorphisms in acute myeloid leukemia (AML) patients remains unclassified. The aim of this study was to evaluate the impact of polymorphisms in genes encoding four main proteins of BER system: OGG1 Ser326Cys, MUTYH Gln324His, APE1 Asp148Glu, and XRCC1 Arg399Gln, and on the risk of AML. Methods Between December 1991 and May 2013, 99 patients (male/female 55/44, median age 58 years, range 15-86 years) diagnosed as AML and 192 healthy controls were included in this study. Cytogenetic subgroups were classified as good, intermediate, and adverse risk according to NCCN guidelines. Genomic DNA was isolated from peripheral blood using the DNA extraction kit. Genotyping was determined by the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique. Genotype and allele frequencies were compared between patients group and control group by using χ2-test. Probability values <0.05 were considered statistically significant. All patients and healthy controls received written information about the study. This study was approved by the Institutional Research Board of Gunma University Hospital. Results The APE1 Asp/Asp genotype increases the risk of AML (OR 2.30, 95% CI 1.41-3.77, p<0.001), whereas APE1 Glu/Glu genotype reduces the risk of AML (OR 0.34, 95% CI 0.14-0.80, p<0.05). In contrast, there were no significant differences in the genotype frequencies OGG1 Ser326Cys, MUTYH Gln324His, and XRCC1 Arg399Gln between AML patients and control group. Next we compared the frequency of cytogenetic abnormalities according to BER polymorphism. The AML patients with OGG1 Ser/Ser genotype increased the frequencies of (15;17) type (p<0.05) and good risk group. Moreover, the AML patients with MUTYH His/His genotype increased the frequencies of complex type (p<0.02) and reduced the frequencies of t(8;21) type. Conclusions According to our data, BER gene polymorphisms may affect the carcinogenesis and the cytogenetic risk of AML. Disclosures: No relevant conflicts of interest to declare.

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