DNA Methylator Research Articles

Nuclear growth and import can be uncoupled.

What drives nuclear growth? Studying nuclei assembled in Xenopus egg extract and focusing on importin α/β-mediated nuclear import, we show that, while import is required for nuclear growth, nuclear growth and import can be uncoupled when chromatin structure is manipulated. Nuclei treated with micrococcal nuclease to fragment DNA grew slowly despite exhibiting little to no change in import rates. Nuclei assembled around axolotl chromatin with 20-fold more DNA than Xenopus grew larger but imported more slowly. Treating nuclei with reagents known to alter histone methylation or acetylation caused nuclei to grow less while still importing to a similar extent or to grow larger without significantly increasing import. Nuclear growth but not import was increased in live sea urchin embryos treated with the DNA methylator N-nitrosodimethylamine. These data suggest that nuclear import is not the primary driving force for nuclear growth. Instead, we observed that nuclear blebs expanded preferentially at sites of high chromatin density and lamin addition, whereas small Benzonase-treated nuclei lacking DNA exhibited reduced lamin incorporation into the nuclear envelope. In summary, we report experimental conditions where nuclear import is not sufficient to drive nuclear growth, hypothesizing that this uncoupling is a result of altered chromatin structure.

Abstract 5369: Tumorigenesis in Lynch syndrome: Somatic mutation profiles compared to sporadic counterparts

Abstract Lynch syndrome (LS) is the most prevalent cancer predisposition syndrome in which germline mutation in one of four DNA mismatch repair (MMR) genes MLH1, MSH2, MSH6, and PMS2 causes significantly increased lifetime risks of colorectal, endometrial, ovarian, and other cancers. Whether or not breast cancer is part of LS tumor spectrum is currently debated. Moreover, the reasons for organ selectivity in germline mutation carriers are unknown. We have reported that breast carcinoma from LS individuals resembles common breast carcinoma in many respects, but differs with respect to genomic instability (Lotsari et al. 2012, Cancer Res.). To extend these preliminary observations, we are now carrying out an in-depth investigation into the spectra of somatic mutations in LS and sporadic breast carcinomas. All available breast carcinomas among 300 LS families from the nation-wide Hereditary Colon Cancer Registry of Finland have been identified and the tumors investigated for MMR status, DNA methylator phenotype, mechanisms of two-allele inactivation for MMR genes, and somatic mutation status. For the latter, we use massive parallel sequencing as described in our recent publication (Porkka et al. 2017, Oncotarget). To define the unique vs. shared molecular features of LS breast tumorigenesis, profiles of somatic alterations in LS breast carcinomas are compared to those of established LS spectrum carcinomas (colorectal and ovarian) as well as sporadic breast carcinomas with sequence information available from large electronic data-sets. Of 14 LS breast carcinoma samples, 9 (64 %) were MMR-deficient and 5 (36 %) MMR-proficient. This contrasts with LS colorectal and ovarian carcinomas, all of which were MMR-deficient (p=0.0012). MMR-deficient LS breast carcinomas harbored an average of 783 non-synonymous mutations and MMR-proficient tumors 555 mutations (statistically non-significant). In contrast to colorectal and ovarian carcinomas, in which loss of heterozygosity (LOH) or somatic MMR gene mutation provided a second hit consistent with two-hit inactivation in 89 % (25/28) of the tumors, a detectable second hit (mainly LOH) was present in only 43 % (6/14) of breast carcinomas (p=0.0025). Genes harboring high-frequency mutations in at least one-third of tumors were regarded candidates for driver genes. There were 133 such genes in LS colorectal carcinomas, 10 in ovarian carcinomas, and 18 in breast carcinomas. Genes involved in epigenetic regulation were significantly enriched in all three tumor types. In addition, a significant enrichment of NOTCH signaling associated genes characterized LS breast carcinomas. This research is likely to shed light to the mechanisms of organ-specific cancer susceptibility in germline carriers of MMR gene mutations. Our results are expected to guide the surveillance and other clinical management of LS individuals with breast carcinoma. Citation Format: Noora Porkka, Alisa Olkinuora, Satu Mäki-Nevala, Samuli Eldfors, Päivi Peltomäki. Tumorigenesis in Lynch syndrome: Somatic mutation profiles compared to sporadic counterparts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5369.

Aberrant miRNA promoter methylation and EMT-involving miRNAs in breast cancer metastasis: Diagnosis and therapeutic implications.

Breast cancer (BC) is the most prevalent cancer in women worldwide. Although extensive studies are ongoing concerning its intricate molecular mechanisms, development of novel therapies and more accurate diagnostic and prognostic approaches is still a challenge. Epithelial-mesenchymal transition (EMT) enables the invasion of metastatic cancer cells and has recently been highlighted in a Cancer Stem Cell (CSC) model of BC. Epigenetic events as well as miRNA expression are the master regulators of tumorigenesis and add a further layer to the complexity of BC pathogenesis. The miRNAs are related to epigenetic event and additionally affect epigenetic pathways. Recent evidence demonstrates that epigenetic mechanisms such as DNA methylation may control miRNA expression. Because each miRNA may regulate several target genes, dysregulation of miRNA caused by aberrant DNA methylation patterns of the locus may influence important downstream pathways. Furthermore, some miRNAs is believed to regulate important DNA methylator factors. Any disruption or modification of this intricate network can contribute to the disease process; thus, it is essential to understand these changes. Advancements in new sequencing technologies to detect DNA methylation patterns has provided the opportunity to determine differentially methylated regions (DMRs) of the miRNA locus and their effect on expression profiles to improve BC diagnosis and treatment. The current review examines the interplay of DNA methylation mechanisms and miRNA function in invasive tumorigenesis, specifically EMT and CSC of BC, to highlight its potential for advancements on BC etiology, diagnosis, and therapy.

Gene markers in brain tumors: what the epileptologist should know.

Gene markers or biomarkers can be used for diagnostic or prognostic purposes for all different types of complex disease, including brain tumors. Prognostic markers can be useful to explain differences not only in overall survival but also in response to treatment and for development of targeted therapies. Multiple genes with specific types of alterations have now been identified that are associated with improved response to chemotherapy and radiotherapy, such as O(6)-methylguanine methyltranferase (MGMT) or loss of chromosomes 1p and/or 19q. Other alterations have been identified that are associated with improved overall survival, such as mutations in isocitrate dehydrogenase 1 (IDH1) and/or isocitrate dehydrogenase 2 (IDH2) or having the glioma CpG island DNA methylator phenotype (G-CIMP). There are many biomarkers that may have relevance in brain tumor-associated epilepsy that do not respond to treatment. Given the rapidly changing landscape of high throughput "omics" technologies, there is significant potential for gaining further knowledge via integration of multiple different types of high genome-wide data. This knowledge can be translated into improved therapies and clinical outcomes for patients with brain tumors.

Mismatch Repair-dependent Iterative Excision at Irreparable O6-Methylguanine Lesions in Human Nuclear Extracts

The response of mammalian cells to Sn1 DNA methylators depends on functional MutSalpha and MutLalpha. Cells deficient in either of these activities are resistant to the cytotoxic effects of this class of chemotherapeutic drug. Because killing by Sn1 methylators has been attributed to O6-methylguanine (MeG), we have constructed nicked circular heteroduplexes that contain a single MeG-T mispair, and we have examined processing of these molecules by mismatch repair in nuclear extracts of human cells. Excision provoked by MeG-T is restricted to the incised heteroduplex strand, leading to removal of the MeG when it resides on this strand. However, when the MeG is located on the continuous strand, the heteroduplex is irreparable. MeG-T-dependent repair DNA synthesis is observed on both reparable and irreparable 3' and 5' heteroduplexes as judged by [32P]dAMP incorporation. Labeling with [alpha-32P]dATP followed by a cold dATP chase has demonstrated that newly synthesized DNA on irreparable molecules is subject to re-excision in a reaction that is MutLalpha-dependent, an effect attributable to the presence of MeG on the template strand. Processing of the irreparable 3' heteroduplex is also associated with incision of the discontinuous strand of a few percent of molecules near the thymidylate of the MeG-T base pair. These results provide the first direct evidence for mismatch repair-mediated iterative processing of DNA methylator damage, an effect that may be relevant to damage signaling events triggered by this class of chemotherapeutic agent.

An update on phase I study of dose-escalating imatinib mesylate plus standard-dosed temozolomide for the treatment of patients with malignant glioma

1560 Background: Imatinib mesylate, a kinase inhibitor of the PDGF receptor has been shown to decrease tumor interstitial pressure resulting in enhanced delivery of cytotoxic therapy. Recent phase II trial demonstrated promising anti-glioma activity of imatinib mesylate in combination with chemotherapy, hydroxyurea. Methods: The current phase I study is designed to determine the maximum tolerated dose (MTD) and dose-limiting toxicity (DLT) of imatinib mesylate when combined with temozolomide, a DNA methylator with established efficacy against gliomas. Eligibility criteria include: histologically confirmed malignant glioma; age of at least 18 years; KPS of at least 60%; less than grade 2 intratumoral hemorrhage; adequate hepatic, renal, and bone marrow function and lack of prior failure or significant toxicity following treatment with either imatinib mesylate or temozolomide. Temozolomide is dosed at 200 mg/m2 on days 4–8 of each 28-day cycle. Imatinib mesylate is administered on days 1–8 of each cycle and the dose is escalated in successive cohorts of 3–6 patients via a standard “3+3” dose escalation design. Patients are stratified based on concurrent use of enzyme-inducing anticonvulsants (EIAC) and both strata are independently escalated. Results: To date 47 patients have been enrolled including 40 with GBM and 7 with anaplastic gliomas. Median age is 53.9 years (range 28 to 72); 66% are male and 51% are on EIAC. The MTD has yet to be defined for either stratum. To date DLT of ALT elevation has been observed in one patient from non-EIAC stratum. Two patients discontinued therapy due to toxicities with one asymptomatic intracerebral hemorrhage and one severe hematologic toxicity. Pharmacokinetic sampling has been performed in approximately half of the patients. One patient completed the study (12 cycles) with stable disease. Eleven patients remain on study with one partial response and three patients have undergone more than 10 cycles of therapy with stable disease. Twenty-eight patients (59%) have developed progressive disease and discontinued therapy. Conclusions: Combination of imatinib mesylate and temozolomide is safe and well tolerated. Further accrual and dose escalation are ongoing. [Table: see text]

Decitabine in Acute Myeloid Leukemia

Standard induction chemotherapy, depending on patient tolerability, is at present the treatment of choice in patients with acute myeloid leukaemia (AML) under the age of 75 years. Since AML is a disease primarily of the elderly, a large proportion of patients do not receive this therapy; therefore, novel treatment modalities are warranted. In addition, younger patients with poor-risk cytogenetics often have unsatisfactory results with conventional chemotherapy, so novel treatment options would represent a significant advancement in the management of this disease. The encouraging results of low-dose schedules of demethylating agents in myelodysplastic syndromes (MDS) have stimulated interest in the use of these agents in patients with AML. The DNA methylator phenotype of MDS and AML offers a biological rationale for treating these patients with doses of demethylating agents which act by reverting the hypermethylated state to an unmethylated state rather than by "classic" cytotoxicity mechanisms typically associated with higher doses. However, many issues surrounding this therapy need to be unravelled, including the role of different target genes that may become demethylated, and the potential use of combination therapy of hypomethylating agents with other drugs. This review will provide a summary of epigenetic processes in AML and the role of the demethylating agent 5-aza-2'-deoxycytidine (decitabine; Dacogen, MGI Pharma, Inc, Bloomington, MN).

A phase I dose escalation study of imatinib mesylate plus standard-dosed temozolomide in the treatment of patients with malignant glioma

1540 Imatinib mesylate (Gleevec), an inhibitor of the PDGF receptor tyrosine kinase with anti-angiogenic activity, has been shown to decrease tumor interstitial pressure resulting in enhanced delivery of cytotoxic therapy. The current phase I study is designed to determine the maximum tolerated dose (MTD) and dose-limiting toxicity (DLT) of imatinib mesylate when combined with temozolomide, a DNA methylator with established anti-glioma activity, in patients with malignant glioma. Eligibility criteria include: histologically confirmed malignant glioma; age at least 18 years; KPS of at least 60%; less than grade 2 intratumoral hemorrhage; adequate hepatic, renal, and bone marrow function and lack of prior failure or significant toxicity following treatment with either imatinib mesylate or temozolomide. Temozolomide is dosed at 200 mg/m2 on days 1–5 of each 28-day cycle. Imatinib mesylate is administered on days 1–8 of each cycle and the dose is escalated in successive cohorts of 3–6 patients via a standard ...

Genetics supersedes epigenetics in colon cancer phenotype

A CpG island DNA methylator phenotype has been postulated to explain silencing of the hMLH1 DNA mismatch repair gene in cancer of the microsatellite mutator phenotype. To evaluate this model, we analyzed methylation in CpG islands from six mutator and suppressor genes, and thirty random genomic sites, in a panel of colorectal cancers. Tumor-specific somatic hypermethylation was a widespread age-dependent process that followed a normal Gaussian distribution. Because there was no discontinuity in methylation rate, our results challenge the methylator phenotype hypothesis and its hypothetical pathological underlying defect. We also show that the mutator phenotype dominates over the gradual accumulation of DNA hypermethylation in determining the genotypic features that govern the phenotypic peculiarities of colon cancer of the mutator pathway.

Marked inactivation of O6-alkylguanine-DNA alkyltransferase activity with protracted temozolomide schedules.

Temozolomide, an oral DNA methylator that inactivates the DNA repair enzyme O6-alkylguanine-DNA alkyltransferase (AGAT), has demonstrated anticancer activity on protracted schedules. Protracted schedules may lead to an ‘autoenhancement’ of temozolomide's inherent cytotoxic potential by cumulative reduction of the cell's capacity for AGAT-mediated DNA repair and resistance. This study was undertaken to characterise AGAT inactivation and regeneration in the peripheral blood mononuclear cells (PBMCs) of patients treated on two protracted temozolomide schedules. O6-alkyl guanine-DNA alkyltransferase activity was measured in the PBMCs of patients treated on two phase I protracted temozolomide studies. Patients were treated daily for either 7 days every 2 weeks (Schedule A) or 21 days every 4 weeks (Schedule B). The effects of various temozolomide doses (75–175 mg m−2), treatment duration (7–21 days), and temozolomide plasma levels on AGAT inactivation and regeneration, as well as the relation between AGAT inactivation and toxicity, were studied. O6-alkyl guanine-DNA alkyltransferase activity in PBMCs was measured serially in 52 patients. Marked inactivation of AGAT occurred following 7 days of temozolomide treatment, with mean AGAT activity decreasing by 72% (P<0.0001). Similarly, mean AGAT activity decreased by 63 and 73% after 14 and 21 days of treatment, respectively (P<0.001 for both comparisons). O6-alkyl guanine-DNA alkyltransferase inactivation was greater after 7 days of treatment with higher doses of temozolomide than lower doses and remained markedly reduced 7 days post-treatment. However, AGAT inactivation following temozolomide treatment for 14 and 21 days was similar at all doses. On the continuous 21-day schedule, AGAT inactivation was significantly greater in patients who experienced severe thrombocytopenia than those who did not (90.3±5.5 vs 72.5±16.1%, P<0.045). In Conclusion, protracted administration of temozolomide, even at relatively low daily doses, leads to significant and prolonged depletion of AGAT activity, which may enhance the antitumour activity of the agent.

A new resistance mechanism to cisplatin?

BACKGROUND alignant melanoma is an increasingly common cancer with the death rate in the United States doubling in the last 35 years; in 1995 alone there were 32 000 new cases and 7200 deaths. While the majority of patients presenting with early stage (stage 1) malignant melanoma can be cured by surgery, the prognosis when the disease has already spread to the regional lymph nodes is much worse.A key reason for this poor prognosis (with only 20‐25% of patients surviving 5 years post-diagnosis) is the lack of response of melanoma to chemotherapy. While hundreds of drugs have been tested against the disease, the most active single agent remains the DNA methylator, dacarbazine (DTIC). Dacarbazine typically produces response rates of only 15 to 20%, with responses lasting on average 3 to 6 months. 1

DNA methylator and mismatch repair phenotypes are not mutually exclusive in colorectal cancer cell lines.

A potential link between DNA repair and de novo methylation of exogenous sequences in colorectal cancer cell lines suggested that cells deficient in mismatch repair (MMR-) had an increased ability to silence the introduced virus promoter by DNA methylation due to the presence of a methylator phenotype (MET+) (Lengauer et al., 1997a). We explored this relationship in more detail and found that although there was a clear difference in the abilities of MMR+ cells to express the viral promoter compared to their MMR- counterparts, this difference was not consistently explained by levels of methylation in the viral promoter. Furthermore, we were unable to distinguish differences between the levels of methylation of six endogenous known CpG islands or 100 random DNA fragments containing CCGG sites within the cells. No consistent differences between the abilities of the cells to methylate the CpG island in exon 2 of the p16 gene were observed after transient demethylation by 5-aza-2'-deoxycytidine nor in the levels of expression of three human methyltransferase enzymes. Our results do not therefore support the existence of mutually exclusive DNA methylation (MET) and DNA repair (MMR) phenotypes. Oncogene (2000) 19, 943 - 952.

HMutSalpha- and hMutLalpha-dependent phosphorylation of p53 in response to DNA methylator damage.

hMSH2.hMSH6 heterodimer (hMutSalpha) and hMLH1.hPMS2 complex (hMutLalpha) have been implicated in the cytotoxic response of mammalian cells to a number of DNA-damaging compounds, including methylating agents that produce O(6)-methylguanine (O(6)MeG) adducts. This study demonstrates that O(6)MeG lesions, in which the damaged base is paired with either T or C, are subject to excision repair in a reaction that depends on a functional mismatch repair system. Furthermore, treatment of human cells with the S(N)1 DNA methylators N-methyl-N-nitrosourea or N-methyl-N'-nitro-N-nitrosoguanidine results in p53 phosphorylation on serine residues 15 and 392, and these phosphorylation events depend on the presence of functional hMutSalpha and hMutLalpha. Coupled with the previous demonstration that O(6)MeG.T and O(6)MeG.C pairs are recognized by hMutSalpha, these results implicate action of the mismatch repair system in the initial step of a damage-signaling cascade that can lead to cell-cycle checkpoint activation or cell death in response to DNA methylator damage.