scholarly journals Involvement of Allele-Specific Methylation of Asparagine Synthetase Gene in Asparaginase Sensitivity of BCP-ALL

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 3966-3966
Author(s):  
Atsushi Watanabe ◽  
Takeshi Inukai ◽  
Minori Tamai ◽  
Tamao Shinohara ◽  
Shinpei Somazu ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Lines ◽  
Cpg Island ◽  
Methylation Status ◽  
Gene Expression Level ◽  
Expression Level ◽  
Ic50 Value ◽  
Allele Specific ◽  
Specific Restriction ◽  
Allele Specific Methylation

Abstract Asparaginase is one of the most important components for the treatment of ALL. ALL cells are supposed to be unable to synthesize adequate amounts of Asparagine (Asn), and, therefore, depend on extracellular source of Asn to survive. Asparaginase therapy induces the depletion of serum Asn by catalyzing the deamination of Asn and leads to cell death of ALL cells. Asparagine synthetase (ASNS) is an enzyme that produces Asn from Aspartic acid. Thus, silencing of the ASNS gene in ALL cells could be crucial for complete starving ALL cells of the Asn. Considering that the ASNS gene has a CpG island in its promotor, aberrant methylation of CpG island could be one of epigenetic mechanisms for silencing of ASNS gene in ALL cells. Previous qualitative analysis of ALL samples using methylation-specific restriction enzyme revealed frequent methylation of CpG island in the ASNS gene. However, associations of methylation status of ASNS gene with its expression level and sensitivity to asparaginase in ALL cells remain unknown. Moreover, little is known about mechanisms for leukemia-specific ASNS gene silencing by methylation. To shed light on these issues, we analyzed a large panel of BCP-ALL cell lines. We quantified ASNS gene expression level by real time RT-PCR in 79 BCP-ALL cell lines cultured in the presence or the absence of L-asparaginase (L-asp), and determined IC50 values of L-asp using alamar blue assay. In the majority of cell lines, although degree of the induction was highly variable, ASNS gene expression level was upregulated in the presence of L-asp. IC50 value of L-asp showed significant correlation with ASNS gene expression level cultured in the presence of L-asp (r=0.222, p=0.049) rather than that in the absence of L-asp (r=0.193, p=0.089). We next analyzed methylation status of the ASNS gene in 79 BCP-ALL cell lines by bisulfite PCR sequencing using a next-generation sequencer (NGS). Strong correlation was confirmed between mean % methylation by NGS and Sanger sequencing in representative cell lines. Of importance, mean % methylation in 79 BCP-ALL cell lines showed significant negative correlation with ASNS gene expression level cultured in the presence of L-asp (r=-0.482, p=6.73x10-6) and, subsequently, IC50 value of L-asp (r=-0.39, p=3.86x10-4). Unexpectedly, % methylation of 79 cell lines distributed in three clusters; 15 cell lines (19%) were highly methylated (>66%, median; 89%), 26 cell lines (32.9%) were moderately methylated (33-66%, median; 40%), and 38 cell lines (48.1%) were weakly methylated (<33%, median; 3.7%). In the majority of moderately methylated cell lines, histograms of % methylation in each read of NGS showed two peaks of high and low methylation, suggesting an allele-specific methylation. In the middle of CpG island, tandem repeat polymorphism of 14bp nucleotides is located adjacent to methylation-specific restriction enzyme site of Aor13HI. Of note, in 7 out of 8 moderately methylated cell lines with heterozygous tandem repeat genotype, only single PCR product was detectable when PCR was performed after Aor13HI treatment, whereas two PCR products derived from two- and three-repeat alleles was detectable when PCR was performed without treatment, indicating an allele-specific methylation. We next analyzed a possible one-allele-loss of the ASNS gene in highly methylated (>66%; 8 cell lines) and weakly methylated (<20%; 12 cell lines) cell lines. We directly sequenced genotype in a portion of introns 2 and 4 and exon 5 based on the imputated SNP genotypes, and confirmed heterozygous genotype in every cell lines at least in one of eight SNPs analyzed, demonstrating that loss-of-heterozygosity is not the mechanism for high or low methylation of the ASNS gene. Similar pattern of methylation was observed in 52 BCP-ALL samples. Taken together, these observations indicate that stepwise allele-specific methylation of ASNSgene is critically involved in the sensitivity to L-asp of BCP-ALL. Disclosures No relevant conflicts of interest to declare.

scholarly journals Association of allele-specific methylation of the ASNS gene with asparaginase sensitivity and prognosis in T-ALL

2021 ◽  
Author(s):  
Koshi Akahane ◽  
Shunsuke Kimura ◽  
Kunio Miyake ◽  
Atsushi Watanabe ◽  
Keiko Kagami ◽  
...  
Keyword(s):  
Gene Cluster ◽  
Cell Lines ◽  
Cpg Island ◽  
Lymphoblastic Leukemia ◽  
Methylation Status ◽  
Aberrant Methylation ◽  
Imprinted Gene ◽  
Allele Specific ◽  
Allele Specific Methylation

Asparaginase therapy is a key component of chemotherapy for T-cell acute lymphoblastic leukemia (T-ALL) patients. Asparaginase depletes serum asparagine by deamination into aspartic acid. Normal hematopoietic cells can survive due to asparagine synthetase (ASNS) activity, while leukemia cells are supposed to undergo apoptosis due to silencing of the ASNS gene. Since the ASNS gene has a typical CpG island in its promoter, its methylation status in T-ALL cells may be associated with asparaginase sensitivity. Thus, we investigated the significance of ASNS methylation status in asparaginase sensitivity of T-ALL cell lines and prognosis of childhood T-ALL. Sequencing of bisulfite PCR products using next-generation sequencing technology in 22 T-ALL cell lines revealed a stepwise allele-specific methylation of the ASNS gene, in association with an aberrant methylation of a 7q21 imprinted gene cluster. T-ALL cell lines with ASNS hypermethylation status showed significantly higher in vitro l-asparaginase sensitivity in association with insufficient asparaginase-induced upregulation of ASNS gene expression and lower basal ASNS protein expression. A comprehensive analysis of diagnostic samples from childhood T-ALL patients in Japanese cohorts (n = 77) revealed that methylation of the ASNS gene was associated with an aberrant methylation of the 7q21 imprinted gene cluster. In childhood T-ALL patients in Japanese cohorts (n = 75), ASNS hypomethylation status was significantly associated with poor therapeutic outcome, and all cases with poor prognostic SPI1 fusion exclusively showed ASNS hypomethylation status. These observations demonstrate that ASNS hypomethylation status is associated with asparaginase resistance and is a poor prognostic biomarker in childhood T-ALL.

scholarly journals Molecular Mechanism for Synergistic Anti-Leukemic Activity of Tyrosine Kinase Inhibitors and Glucocorticoids Against Ph+ALL

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1059-1059
Author(s):  
Meixian Huang ◽  
Takeshi Inukai ◽  
Keiko Kagami ◽  
Masako Abe ◽  
Tamao Shinohara ◽  
...  
Keyword(s):  
Gene Expression ◽  
Tyrosine Kinase ◽  
Cell Lines ◽  
Induction Therapy ◽  
Kinase Inhibitors ◽  
Mapk Pathway ◽  
Gene Expression Level ◽  
Expression Level ◽  
Imatinib Resistant ◽  
T315i Mutation

Abstract The tyrosine kinase inhibitors (TKIs) have dramatically altered the management of patients with Philadelphia chromosome-positive ALL (Ph+ALL). In earlier trials of imatinib monotherapy for relapsed or refractory patients with lymphoid blast crisis of CML (CML-LC) and Ph+ALL, complete hematologic remission (CHR) was achieved only in 20%, and relapses occurred in most of the patients within several months. Thereafter, combination induction therapy of imatinib with conventional chemotherapeutic agents profoundly improved the therapeutic outcome in the patients with Ph+ALL. In elderly patients, to reduce the therapy-related toxicities, combination induction therapy of imatinib with glucocorticoids (GCs) followed by imatinib monotherapy was performed. Surprisingly, CHR was obtained in all of the patients with this simple therapy, and median survival from diagnosis was 20 months (Blood 2007). Similarly, induction therapy of dasatinib, a second-generation TKI, combined with GCs achieved CHR in all of the newly diagnosed Ph+ALL patients (Blood 2011). These clinical findings indicate a synergistic anti-leukemic activity of TKIs with GCs in Ph+ALL, but its underlying molecular mechanisms remain totally unknown. Thus, we analyzed synergistic effects of TKIs and dexamethasone (Dex) in a panel of leukemic cell lines derived from Ph+ALL. Indeed, in the presence of 0.5μM of imatinib, IC50 values of Dex were approximately 3-8 times lower than those in the absence of imatinib in the most of Dex-sensitive Ph+ALL cell lines. Since gene expression level of GC receptor (GR; NR3C1) was associated with Dex-sensitivity in Ph+ALL cell lines, we next analyzed the effects of imatinib on gene expression level of NR3C1. Of note, NR3C1 gene expression level was significantly upregulated in the presence of 0.5 μM of imatinib approximately 1.5-7-fold in all of 14 Ph+ALL cell lines except for SK9, which was an imatinib-resistant cell line having a T315I mutation of BCR-ABL, whereas it was unchanged in all of 11 Ph-negative ALL cell lines. Induction of GR was also confirmed by immuno-blotting in representative Ph+ALL cell lines. Moreover, treatment with either dasatinib or nilotinib clearly upregulated the NR3C1 gene expression in the representative Ph+ALL cell lines. Of importance, although gene expression level of NR3C1 was significantly upregulated in the presence of imatinib in the imatinib-sensitive Ph+ALL cell lines, SU-Ph2 and TCCY, it was unchanged in their imatinib-resistant sublines, SU/SR and TCCY/SR, respectively, in which T315I mutation was acquired after the culture with increasing concentrations of imatinib, indicating that upregulation of GR by TKIs in Ph+ALL cell lines was mediated by an inactivation of BCR-ABL. To further verify the downstream pathway of BCR-ABL that is critically involved in the TKI-induced GR upregulation, we treated imatinib-sensitive SU-Ph2 and its imatinib-resistant subline SU/SR with specific inhitors of PI3K (GDC0941, LY294002, and AS606240), JAK2(SD1029), and MAPK(UO126). Among five agents, only UO126 effectively upregulated the NR3C1 gene expression both in SU-Ph2 and in SU/SR, suggesting that TKIs upregulate GR in Ph+ALL cell lines mainly through an inactivation of the MAPK pathway. Previous reports revealed that three promoters, 1A, 1B, and 1C, are mainly involved in the NR3C1 gene expression in ALL cells. We therefore performed real time RT-PCR analysis of the NR3C1 gene using three sets of primers that are specific for exons 1A3, 1B, or 1C. The strongest induction by an imatinib-treatment was observed in the 1A promoter in Ph+ALL cell lines. Finally, since BIM, one of BH3-only pro-apoptotic members of BCL2 family, has been reported to be critically involved in the anti-leukemic activities of both TKIs and GCs, we analyzed BIM expression. Synergistic induction of BIM was confirmed both in mRNA and protein expression levels by a simultaneously treatment of Ph+ALL cell lines with imatinib and Dex. Taken together, these observations in Ph+ALL cell lines indicate that TKIs induce GR expression in Ph+ALL mainly through the MAPK pathway and the 1A promoter of NR3C1 gene by inactivating BCR-ABL and subsequently exert a synergistic anti-leukemic activity with GCs through the induction of BIM. Disclosures No relevant conflicts of interest to declare.

SET (TAF-IB) Gene Is Highly Expressed in Acute Leukemia Patients without Associated with Methylation Status.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4308-4308
Author(s):  
Sema Sirma ◽  
Cumhur G. Ekmekçi ◽  
Ayten Kandilci ◽  
Gerard Grosveld ◽  
Ugur Ozbek
Keyword(s):  
Gene Expression ◽  
Bone Marrow ◽  
Acute Leukemia ◽  
Gene Silencing ◽  
Lymphoblastic Leukemia ◽  
Methylation Status ◽  
Prognostic Significance ◽  
Gene Expression Level ◽  
Expression Level ◽  
Metastasis Suppressor

Abstract SET gene, also known as TAF-I beta, was originally identified as a component of the SET-CAN fusion gene, which results from t(9;9) translocation, in a patient with acute undifferentiated leukemia (AUL). SET gene encodes a nuclear phosphoprotein that ubiquitously expressed. There is an accumulating data that suggest a role for SET in gene silencing either through prevention of histone acetylation as a subunit of inhibitor of acetyltransferases (INHAT) complex or through inhibition of DNA demethylation. SET also inhibits the activity of protein phosphatase 2A, which involves in regulation of cell proliferation and differentiation, and blocks DNase activity of the tumor metastasis suppressor NM23-H1. Taken together, available data suggest that SET might play a role in tumorogenesis via tumor suppressor gene silencing or inhibition of apoptosis. In this study, we investigated SET gene expression level in bone marrow samples of 77 patients with acute leukemia (57 acute lymphoblastic leukemia (ALL) and 26 acute myeloid leukemia (AML)) and 5 control bone marrow samples from healthy volunteers using quantitative real-time RT-PCR. The ALL patient ages ranged 10months – 17 years, with a median of 6 years and the AML patient ages ranged 1–72 years, with a median of 18 years. For determination of the prognostic significance of SET gene expression in ALL patients, the association between patient’s clinical characteristics and the SET gene expression level was assessed usind the Pearson’s chi-square test or Fisher’s Exact test. Overall survival (OS) in 48 patients and relapse-free survival (RFS) in 37 patients with ALL at 6 years (median follow-up 35 months, range 1–79 months) were analyzed according to Kaplan-Meier method. We also studied methylation of various genes (p15, p16, p73, SOCS1, RAR beta, E-Cadherin, GSTP1, DAP-Kinaz, ER and 5-HIC) using methylation spesific PCR and COBRA analysis in AML samples to examine whether high SET gene expression play a role in tumorogenesis via gene silencing. Here we demonstrate that 54.4% of ALL patients and 53.8% of AML patients show two fold and higher SET expression level compare to control samples (p=0.005 and p=0.016 respectively). There were no significant association between SET gene expression level and age, peripheral WBC count, sex, FAB group and immunophenotype (P=0.823, P=0.182, P=1.00, P=0.132, P=0.751) in ALL. The OS was not significantly different between high (83.10% ± 6.92%) and low SET expressed patients (51.14% ± 18.73%) (log-rank=3.36, P=0.067) and the probability of RFS was not significantly different between high (81.20% ± 7.60%) and low SET expressed patients (80.00% ± 17.89%) with ALL (log-rank=0.01, P= 0.92). There was no statisticall association between methylation index and SET gene expression level (P=0.618). Our data suggest that high level of SET expression may play an important role in leukemogenesis. Further analyses are required to determine prognostic significance of SET gene expression different types of leukemia.

scholarly journals A Possible Role of Angiogenin in Intergenerational Transmission of Diet-Induced Metabolic Stress

2021 ◽  
Vol 5 (Supplement_2) ◽  
pp. 508-508
Author(s):  
Eunbi Lee ◽  
Seo Yoon Choi ◽  
Jihye Park ◽  
Anders M Lindroth ◽  
Yoon Jung Park
Keyword(s):  
Gene Expression ◽  
High Fat Diet ◽  
Methylation Status ◽  
Metabolic Stress ◽  
Gene Expression Level ◽  
Expression Level ◽  
Trna Modification ◽  
High Fat ◽  
Compound C ◽  
Mtor Activity

Abstract Objectives Epigenetic is one of the possible mechanisms of transmit parental metabolic stress to offspring. tRNA modification or fragmentation is a promising candidate. We investigated the molecular mechanism of intergeneration transmission of diet-induced metabolic stress. Methods Male mice fed with control diet (CD) or high-fat diet (HFD) for 9 weeks. Body weight, amount of food intake and organ weights were measured. Metabolism-related proteins and gene expression were measured in testis and liver. tRNA methylation in sperm was investigated by bisulfite conversion-based sequencing. We analyzed publicly available omics data to profile changes of high-fat diet feeding. To explain mTOR activity linked to angiogenin (ANG) gene expression, we treated compound C (CC) or rapamycin (RP) in cell lines. Results We showed mTOR negative regulators were down regulated in HFD from analysis of transcriptome data from liver and sperm. Consistently, analysis on sperm metabolome data revealed that free amino acid level and tRNA amino-acyl biosynthesis pathway was up-regulated in HFD. Increased a level of phospho-mTOR protein was confirmed in testis, but not phospho-AMPK protein. Next, we measured tRNA modification-related gene expression levels in testis and liver. Expression of Dnmt2 and NSun2 related in tRNA methylation was elevated in HFD in testis and liver. However, expression of ANG related with tRNA cleavage was only increased in testis. In addition, methylation status in sperm tRNA-Asp-GTC was no different between diets. The data suggested that tRNA cleavage with ANG, rather than tRNA methylation process, was more likely involved in transmit transgenerational effect to offspring. Finally, we investigated changing mTOR activity could affect to Angiogenin gene expression level. Treatment with CC showed increased Angiogenin gene expression level, but RP treatment showed no change. Conclusions Our data suggested that diet-induced alteration of mTOR activity led to upregulation of Angiogenin expression in sperm, which might be a key of transgenerational mechanism. Funding Sources This study was supported by the National Research Foundation of Korea the Korean National Cancer Center. EL is grateful for financial support from Hyundai Motor Chung Mong-Koo Foundation and BK21 FOUR (Fostering Outstanding Universities for Research).

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