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The de novo DNA methyltransferases Dnmt3a and Dnmt3b are of crucial importance in hematopoietic stem cells. Dnmt3b has recently been shown to play a role in genic methylation. To investigate how Dnmt3b-mediated DNA methylation affects leukemogenesis, we analyzed leukemia development under conditions of high and physiological methylation levels in a tetracycline-inducible knock-in mouse model. High expression of Dnmt3b slowed leukemia development in serial transplantations and impaired leukemia stem cell (LSC) function. Forced Dnmt3b expression induced widespread DNA hypermethylation inMyc-Bcl2-induced leukemias, preferentially at gene bodies.MLL-AF9-induced leukemogenesis showed much less pronounced DNA hypermethylation upon Dnmt3b expression. Nonetheless, leukemogenesis was delayed in both models with a shared core set of DNA hypermethylated regions and suppression of stem cell-related genes. Acute myeloid leukemia patients with high expression of Dnmt3b target genes showed inferior survival. Together, these findings indicate a critical role for Dnmt3b-mediated DNA methylation in leukemia development and maintenance of LSC function.

Chronic inflammation represents a major risk factor for tumor formation, but the underlying mechanisms have remained largely unknown. Epigenetic mechanisms can record the effects of environmental challenges on the genome level and could therefore play an important role in the pathogenesis of inflammation-associated tumors. Using single-base methylation maps and transcriptome analyses of a colitis-induced mouse colon cancer model, we identified a novel epigenetic program that is characterized by hypermethylation of DNA methylation valleys that are characterized by low CpG density and active chromatin marks. This program is conserved and functional in mouse intestinal adenomas and results in silencing of active intestinal genes that are involved in gastrointestinal homeostasis and injury response. Further analyses reveal that the program represents a prominent feature of human colorectal cancer and can be used to correctly classify colorectal cancer samples with high accuracy. Together, our results show that inflammatory signals establish a novel epigenetic program that silences a specific set of genes that contribute to inflammation-induced cellular transformation. Cancer Res; 75(10); 2120–30. ©2015 AACR.

Purpose Positron emission tomography (PET) with choline tracers has found widespread use for the diagnosis of prostate cancer (PC). However, choline metabolism is not increased in a considerable number of cases, whereas prostate-specific membrane antigen (PSMA) is overexpressed in most PCs. Therefore, a 68Ga-labelled PSMA ligand could be superior to choline tracers by obtaining a high contrast. The aim of this study was to compare such a novel tracer with standard choline-based PET/CT. Methods Thirty-seven patients with biochemical relapse of PC [mean prostate-specific antigen (PSA) 11.1 ± 24.1 ng/ml, range 0.01–116] were retrospectively analysed after 18F-fluoromethylcholine and 68Ga-PSMA PET/CT within a time window of 30 days. Radiotracer uptake that was visually considered as PC was semi-quantitatively analysed by measuring the maximum standardized uptake values (SUVmax) of the scans acquired 1 h after injection of 68Ga-PSMA complex solution (median 132 MBq, range 59–263 MBq) and 18F-fluoromethylcholine (median 237 MBq, range 114–374 MBq), respectively. In addition, tumour to background ratios were calculated. Results A total of 78 lesions characteristic for PC were detected in 32 patients using 68Ga-PSMA PET/CT and 56 lesions were detected in 26 patients using choline PET/CT. The higher detection rate in 68Ga-PSMA PET/CT was statistically significant (p = 0.04). In five patients no lesion was found with both methods. All lesions detected by 18F-fluoromethylcholine PET/CT were also seen by 68Ga-PSMA PET/CT. In 68Ga-PSMA PET/CT SUVmax was clearly (>10 %) higher in 62 of 78 lesions (79.1 %) and the tumour to background ratio was clearly (>10 %) higher in 74 of 78 lesions (94.9 %) when compared to 18F-fluoromethylcholine PET/CT. Conclusion 68Ga-PSMA PET/CT can detect lesions characteristic for PC with improved contrast when compared to standard 18F-fluoromethylcholine PET/CT, especially at low PSA levels. Electronic supplementary material The online version of this article (doi:10.1007/s00259-013-2525-5) contains supplementary material, which is available to authorized users.

Purpose Prostate-specific membrane antigen (PSMA) is a cell surface protein with high expression in prostate carcinoma (PC) cells. Recently, procedures have been developed to label PSMA ligands with 68Ga, 99mTc and 123/124/131I. Our initial experience with Glu-NH-CO-NH-Lys-(Ahx)-[68Ga(HBED-CC)](68Ga-PSMA) suggests that this novel tracer can detect PC relapses and metastases with high contrast. The aim of this study was to investigate its biodistribution in normal tissues and tumour lesions.

PET imaging with somatostatin receptor ligands, such as 68Ga-DOTATOC, is a well-established method for detection and target volume definition of meningiomas prior to radiotherapy. Since DOTATOC PET delivers a higher contrast between meningiomas and surrounding tissues than MRI, we conducted a retrospective analysis to compare the diagnostic accuracy of contrast-enhanced MRI (CE-MRI) with 68Ga-DOTATOC PET/CT in patients with cranial meningiomas prior to radiotherapy. Over a period of 6 years, 134 patients (20–82 years of age, 107 women and 27 men) underwent cranial CE-MRI and 68Ga-DOTATOC PET/CT. To compare the two methods, the lesions considered typical of meningiomas visually were counted and analysed with respect to their location and SUVmax. In the 134 patients investigated by both modalities, 190 meningiomas were detected by 68Ga-DOTATOC PET/CT and 171 by CE-MRI. With knowledge of the PET/CT data, the MRI scans were reinvestigated, which led to the detection of 4 of the 19 incidental meningiomas, resulting in an overall detection rate of 92 % of the meningioma lesions that were found by PET/CT. Ga-DOTATOC PET/CT demonstrated an improved sensitivity in meningioma detection when compared to CE-MRI. Tumours adjacent to the falx cerebri, located at the skull base or obscured by imaging artefacts or calcification are particularly difficult to detect by MRI. Therefore 68Ga-DOTATOC PET/CT may provide additional information in patients with uncertain or equivocal results on MRI or could help to confirm a diagnosis of meningioma based on MRI or could help to confirm MRI-based diagnosis of meningiomas in cases of biopsy limitations. It is possible that not only radiotherapy and surgical planning, but also follow-up strategies would benefit from this imaging modality.

Introduction: Colorectal adenomas are precursor lesions of colorectal cancers and represent clonal amplifications of single cells from colonic crypts. DNA methylation patterns specify cell-type identity during cellular differentiation and therefore provide novel opportunities for tumor subclassification and patient stratification. Methods: Infinium 450k and EPIC data from of non-malignant colorectal adenomas was applied to a consensus clustering algorithm. This defined epigenetic subtypes that were subsequently tested for clinico-pathological features, such as morphology and mutation status. Additionally, the subtypes were compared with Infinium 450k data from colonic crypt sections and also colorectal cancer samples provided by The Cancer Genome Atlas. Finally, clinical significance was addressed by testing for subtype specific overall survival rates. Results: The analysis of the adenoma dataset defined 3 distinct epigenetic subtypes of human premalignant lesions. These 3 subtypes also reflect 3 of 4 epigenetic subtypes identified in colorectal cancer. The fourth, cancer-specific subtype represents the previously known subclass defined by microsatellite instability phenotype and the CpG island methylator phenotype. Comparisons to Crypt section methylomes and to other reference methylomes revealed that the other 3 subtypes represent epigenetic programs reflecting various intestinal crypt cell differentiation stages. Patient survival correlated with the differentiation stages with a particular poor prognosis for patients with stem cell-related epigenetic signatures. Conclusion: Our results establish a novel and clinically relevant approach for colorectal adenoma and cancer classification and illustrate how differences in the cell-type of origin shape the tumor methylome. Citation Format: Felix Bormann, Manuel Rodríguez-Paredes, Felix Lasitschka, Dominic Edelmann, Axel Benner, Yehudit Bergman, Claudia R. Ball, Hanno Glimm, Heinz G. Linhart, Frank Lyko. Cell-of-origin differentiation stages define methylation-based subtypes of human colorectal adenomas and carcinomas [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 3305.

An estimated one-third of the world’s population is currently latently infected with Mycobacterium tuberculosis. Latent M. tuberculosis infection (LTBI) progresses into active tuberculosis (TB) disease in ~5 to 10% of infected individuals. Diagnostic and prognostic biomarkers to monitor disease progression are urgently needed to ensure better care for TB patients and to decrease the spread of TB. Biomarker development is primarily based on transcriptomics. Our understanding of biology combined with evolving technical advances in high-throughput techniques led us to investigate the possibility of additional platforms (epigenetics and proteomics) in the quest to (i) understand the biology of the TB host response and (ii) search for multiplatform biosignatures in TB. We engaged in a pilot study to interrogate the DNA methylome, transcriptome, and proteome in selected monocytes and granulocytes from TB patients and healthy LTBI participants. Our study provides first insights into the levels and sources of diversity in the epigenome and proteome among TB patients and LTBI controls, despite limitations due to small sample size. Functionally the differences between the infection phenotypes (LTBI versus active TB) observed in the different platforms were congruent, thereby suggesting regulation of function not only at the transcriptional level but also by DNA methylation and microRNA. Thus, our data argue for the development of a large-scale study of the DNA methylome, with particular attention to study design in accounting for variation based on gender, age, and cell type., IMPORTANCE DNA methylation modifies the transcriptional program of cells. We have focused on two major populations of leukocytes involved in immune response to infectious diseases, granulocytes and monocytes, both of which are professional phagocytes that engulf and kill bacteria. We have interrogated how DNA methylation, gene expression, and protein translation differ in these two cell populations between healthy individuals and patients suffering from TB. To better understand the underlying biologic mechanisms, we harnessed a statistical enrichment analysis, taking advantage of predefined and well-characterized gene sets. Not only were there clear differences on various levels between the two populations, but there were also differences between TB patients and healthy controls in the transcriptome, proteome, and, for the first time, DNA methylome in these cells. Our pilot study emphasizes the value of a large-scale study of the DNA methylome taking into account our findings.

Aberrant gene silencing accompanied by DNA methylation is associated with neoplastic progression in many tumors that also show global loss of DNA methylation. Using conditional inactivation of de novo methyltransferase Dnmt3b in Apc(Min/+) mice, we demonstrate that the loss of Dnmt3b has no impact on microadenoma formation, which is considered the earliest stage of intestinal tumor formation. Nevertheless, we observed a significant decrease in the formation of macroscopic colonic adenomas. Interestingly, many large adenomas showed regions with Dnmt3b inactivation, indicating that Dnmt3b is required for initial outgrowth of macroscopic adenomas but is not required for their maintenance. These results support a role for Dnmt3b in the transition stage between microadenoma formation and macroscopic colonic tumor growth and further suggest that Dnmt3b, and by extension de novo methylation, is not required for maintaining tumor growth after this transition stage has occurred.

Colon polyps represent precursor lesions of colon cancers and their malignant potential varies according to histological subtype. A rare subtype of colon polyps is the Peutz–Jeghers (PJ) polyp. PJ polyps mostly occur in the context of Peutz–Jeghers syndrome, which is characterized by the development of multiple polyps in the intestinal tract and hyperpigmentation of oral mucosa and lips. Peutz–Jeghers is an autosomal dominant disorder caused by pathogenic variants of the serine threonine kinase STK11. PJ polyps very rarely occur outside of the syndrome and are then referred to as solitary PJ polyps. Contrary to the situation in Peutz–Jeghers, the genetic basis and the malignant potential of solitary PJ polyps are currently unknown. Here we describe a detailed and comprehensive genetic profile of a solitary PJ polyp. Pathological examination revealed a high tissue homogeneity with >80% epithelial cells. Whole-genome sequencing failed to identify any clonal mutations but demonstrated a significant number of subclonal mutations. No somatic or germline mutations were found at the STK11 locus, suggesting that solitary PJ polyps are genetically distinct from Peutz–Jeghers polyps. In addition, methylome analysis revealed global hypomethylation and CpG island hypermethylation, two features that have been described as hallmarks of the colorectal cancer epigenome. These results provide an example of a premalignant lesion that is defined by epigenetic, rather than genetic changes. Furthermore, our findings support the notion that solitary PJ polyps constitute neoplastic tissue with malignant potential that should be removed for cancer prevention.

The transcription factor CCAAT enhancer binding protein α (C/EBPα) is expressed at high levels in liver and adipose tissue. Cell culture studies show that C/EBPα is sufficient to trigger differentiation of preadipocytes into mature adipocytes, suggesting a central role for C/EBPα in the development of adipose tissue. C/EBPα knockout mice die within 7–12 h after birth. Defective gluconeogenesis of the liver and subsequent hypoglycemia contribute to the early death of these animals. This short life span impairs investigation of the development of adipose tissue in these mice. To improve the survival of C/EBPα−/− animals, we generated a transgenic line that expresses C/EBPα under the control of the albumin enhancer/promoter. This line was bred into the knockout strain to generate animals that express C/EBPα in the liver but in no other tissue. The presence of the transgene improved survival of C/EBPα−/− animals almost 3-fold. Transgenic C/EBPα−/− animals at 7 days of age show an absence of s.c., perirenal, and epididymal white fat despite excess lipid substrate in the serum, whereas brown adipose tissue is somewhat hypertrophied and shows minimal biochemical alterations. Interestingly, mammary gland fat tissue is present and exhibits normal morphology. The absence of white adipose tissue in many depots in the presence of high serum lipid levels shows that C/EBPα is required for the in vivo development of this tissue. In contrast, brown adipose tissue differentiation is independent of C/EBPα expression. The presence of lipid in brown adipose tissue serves as an internal nutritional control, indicating that neither nutritional intake nor lipoprotein composition is likely responsible for the absence of white fat.

PET imaging with somatostatin receptor ligands, such as (68)Ga-DOTATOC, is a well-established method for detection and target volume definition of meningiomas prior to radiotherapy. Since DOTATOC PET delivers a higher contrast between meningiomas and surrounding tissues than MRI, we conducted a retrospective analysis to compare the diagnostic accuracy of contrast-enhanced MRI (CE-MRI) with (68)Ga-DOTATOC PET/CT in patients with cranial meningiomas prior to radiotherapy.Over a period of 6 years, 134 patients (20-82 years of age, 107 women and 27 men) underwent cranial CE-MRI and (68)Ga-DOTATOC PET/CT. To compare the two methods, the lesions considered typical of meningiomas visually were counted and analysed with respect to their location and SUVmax.In the 134 patients investigated by both modalities, 190 meningiomas were detected by (68)Ga-DOTATOC PET/CT and 171 by CE-MRI. With knowledge of the PET/CT data, the MRI scans were reinvestigated, which led to the detection of 4 of the 19 incidental meningiomas, resulting in an overall detection rate of 92 % of the meningioma lesions that were found by PET/CT.Ga-DOTATOC PET/CT demonstrated an improved sensitivity in meningioma detection when compared to CE-MRI. Tumours adjacent to the falx cerebri, located at the skull base or obscured by imaging artefacts or calcification are particularly difficult to detect by MRI. Therefore (68)Ga-DOTATOC PET/CT may provide additional information in patients with uncertain or equivocal results on MRI or could help to confirm a diagnosis of meningioma based on MRI or could help to confirm MRI-based diagnosis of meningiomas in cases of biopsy limitations. It is possible that not only radiotherapy and surgical planning, but also follow-up strategies would benefit from this imaging modality.

A healthy immune system needs to be highly plastic to cope with host defense and surveillance. What mechanisms provide this plasticity? Considering the threat of infectious diseases to a large part of the world's population, can these mechanisms possibly be of use in the ongoing battle against infectious diseases? Against the backdrop of the pandemic nature of tuberculosis, we discuss whether and how epigenetic mechanisms can shed light on our understanding of infectious disease, and if epigenetic marks can be employed to monitor latent infection, disease reactivation or treatment response.

Human cancer cells frequently have regions of their DNA hypermethylated, which results in transcriptional silencing of affected genes and promotion of tumor formation. However, it is still unknown whether cancer-associated aberrant DNA methylation is targeted to specific genomic regions, whether this methylation also occurs in noncancerous cells, and whether these epigenetic events are maintained in the absence of the initiating cause. Here we have addressed some of these issues by demonstrating that transgenic expression of DNA methyltransferase 3b (Dnmt3b) in the mouse colon initiates de novo DNA methylation of genes that are similar to genes that become methylated in human colon cancer. This is consistent with the notion that aberrant methylation in cancer may be attributable to targeting of specific sequences by Dnmt3b rather than to random methylation followed by clonal selection. We also showed that Dnmt3b-induced aberrant DNA methylation was maintained in regenerating tissue, even in the absence of continuous Dnmt3b expression. This supports the concept that transient stressors can cause permanent epigenetic changes in somatic stem cells and that these accumulate over the lifetime of an organism in analogy to DNA mutations.

Following publication of this article [1] the authors noticed that an incorrect probe reference was cited on page 3, 4, 5 and 6 ("UP #69, Roche Applied Science"). The correct probe that was used for the 1lox/2lox allele ratio analysis in the paper is as follows Probe for 1lox/2lox allele quantification: 5'-6-FAM-atAaCtTCgtatagCATaCattatac-BHQ-1 -3' (uppercase letters = LNA bases) Manufacturer: EUROGENTEC, Seraing, Belgium All other information and reaction conditions in the paper are correct as stated.

Cerebellar mutism (CM) is a rare and severe form of speech and language impairment, mostly diagnosed in children and adolescents and rarely reported in adults. We here review the literature and summarize all anatomical structures related to the pathogenesis of this rare syndrome. We also report two illustrative cases of CM following surgical treatment of Lhermitte-Duclos disease (LDD; dysplastic gangliocytoma) in two adult patients. LDD is a rare benign cerebellar tumor. Surgical excision appears to be the only effective treatment. However, surgery is hampered by the difficulty to distinguish between tumor and healthy cerebellar tissue, which may result in extensive resection and cause neurological deficits such as CM. A review of the literature and our two cases suggest that lesions or functional impairment of paravermian structures including dentate nuclei, vermis, lateral hemispheres, and cerebellocortical pathways contribute to the development of CM. However, there is no single anatomical structure identified to be associated with CM. It is unknown whether some diseases such as LDD carry a higher risk of postoperative CM than others. As illustrated by our two cases, although there are no special means, optimal preoperative diagnosis might contribute to the prevention of this syndrome. Despite the severity, CM carries a favorable prognosis and generally resolves within a few months.To conclude, we review the clinical signs and particularly the pathophysiological observations and anatomical structures affected in the development of postoperative CM and contribute two cases illustrating the pathogenesis, prognosis, and possible prevention of this syndrome, to focus that CM might also occur in adults even in association with rare tumors.

Epidemiologic studies have linked nutritional folate deficiency to an increased risk of cancer, but recent trials suggest that folate supplementation does not protect against tumor formation. Our aim was to analyze the genetic and epigenetic consequences of folate deficiency and to investigate whether impairment of the uracil base excision repair pathway can enhance its effects.Wild-type mice and those deficient in uracil DNA glycosylase (Ung(-/-)) were placed on a folate-deficient diet for 8 months. We measured tumor incidence in major organs, DNA mutation rates, DNA mutation spectra, local DNA methylation, and global DNA methylation in colon epithelial cells.The experimental diet increased plasma homocysteine (60%, P.001) and DNA uracil content (24%, P.05) but not tumor formation. Global DNA methylation was slightly decreased in splenocytes (9.1%) and small intestinal epithelial cells (4.2%), and significantly reduced in colon epithelial cells (7.2%, P.04). No gene-specific changes in methylation were detected at the mouse B1 element, the H19 DMR, or the Oct4 gene. By lambda CII assay and sequencing analysis of 730 mutants, we found that Ung(-/-) mice had a higher frequency of point mutations and increased C:G to T:A transitions at non-CpG sites. However, folate deficiency had no additional effect on the DNA mutation frequency or spectrum in Ung(-/-) or wild-type mice.Contradicting current concepts, these findings indicate that the effects of a low-folate diet on DNA methylation and point mutations are insufficient to promote tumor development, even in the presence of Ung deficiency.

Increased methylation of CpG islands and silencing of affected target genes is frequently found in human cancer; however, in vivo the question of causality has only been addressed by loss-of-function studies. To directly evaluate the role and mechanism of de novo methylation in tumor development, we overexpressed the de novo DNA methyltransferases Dnmt3a1 and Dnmt3b1 in ApcMin/+ mice. We found that Dnmt3b1 enhanced the number of colon tumors in ApcMin/+ mice approximately twofold and increased the average size of colonic microadenomas, whereas Dnmt3a1 had no effect. The overexpression of Dnmt3b1 caused loss of imprinting and increased expression of Igf2 as well as methylation and transcriptional silencing of the tumor suppressor genes Sfrp2, Sfrp4, and Sfrp5. Importantly, we found that Dnmt3b1 but not Dnmt3a1 efficiently methylates the same set of genes in tumors and in nontumor tissues, demonstrating that de novo methyltransferases can initiate methylation and silencing of specific genes in phenotypically normal cells. This suggests that DNA methylation patterns in cancer are the result of specific targeting of at least some tumor suppressor genes rather than of random, stochastic methylation followed by clonal selection due to a proliferative advantage caused by tumor suppressor gene silencing.

Although several lines of evidence suggest the involvement of the Wnt pathway in the development of gastric cancers, the functional significance of the pathway in gastric carcinogenesis is still poorly defined. To examine the role of the Apc/β-catenin signaling pathway in the development of gastric cancers, we investigated the gastric mucosa of the ApcMin/+ mouse, which is a murine model for familial adenomatous polyposis, carrying a germ-line mutation at codon 850 of Apc. We found that aged ApcMin/+ mice spontaneously develop multiple tumors in the stomach, which are accompanied by loss of heterozygosity of Apc. Such tumors consisted of adenomatous glands with strong nuclear accumulation of β-catenin. Even a single adenomatous gland already showed nuclear accumulation of β-catenin, suggesting that Apc/β-catenin pathway is an initiating event in gastric tumorigenesis in ApcMin/+ mice. Myc and cyclin D1 expressions, which are transcriptional targets of β-catenin/Tcf, increased in the adenomatous lesions. Furthermore, β-catenin/Tcf reporter transgenic mice with ApcMin allele showed higher levels of the transcriptional activity of β-catenin/Tcf in the gastric tumors. We also treated ApcMin/+ and wild-type mice with N-methyl-N-nitrosourea (MNU), an alkylating agent that induces adenomas and adenocarcinomas in the stomach. Consequently, MNU-treated ApcMin/+ mice significantly enhanced the tumor development in comparison with ApcMin/+ mice or MNU-treated wild-type mice. Several gastric tumors in MNU-treated ApcMin/+ mice showed invasion into the submucosal layer. These results indicate that the Apc/β-catenin pathway may play an important role in at least subset of gastric carcinomas. In addition, ApcMin/+ mice combined with MNU could be a useful short-term model to investigate multistage carcinogenesis in the stomach. [Cancer Res 2007;67(9):4079–87]

Genome-wide DNA hypomethylation and concomitant promoter-specific tumor suppressor gene hypermethylation are among the most common molecular alterations in human neoplasia. Consistent with the notion that both promoter hypermethylation and genome-wide hypomethylation are functionally important in tumorigenesis, genetic and/or pharmacologic reduction of DNA methylation levels results in suppression or promotion of tumor incidence, respectively, depending on the tumor cell type. For instance, DNA hypomethylation promotes tumors that rely predominantly on loss of heterozygosity (LOH) or chromosomal instability mechanisms, whereas loss of DNA methylation suppresses tumors that rely on epigenetic silencing. Mutational and epigenetic silencing events in Wnt pathway genes have been identified in human colon tumors. We used Apc Min/+ mice to investigate the effect of hypomethylation on intestinal and liver tumor formation. Intestinal carcinogenesis in Apc Min/+ mice occurs in two stages, with the formation of microadenomas leading to the development of macroscopic polyps. Using Dnmt1 hypomorphic alleles to reduce genomic methylation, we observed elevated incidence of microadenomas that were associated with LOH at Apc . In contrast, the incidence and growth of macroscopic intestinal tumors in the same animals was strongly suppressed. In contrast to the overall inhibition of intestinal tumorigenesis in hypomethylated Apc Min/+ mice, hypomethylation caused development of multifocal liver tumors accompanied by Apc LOH. These findings support the notion of a dual role for DNA hypomethylation in suppressing later stages of intestinal tumorigenesis, but promoting early lesions in the colon and liver through an LOH mechanism.

DNA methyltransferases (DNMT) play an important role in regulation of DNA methylation and mutations of DNMT3A are frequently found in AML. In previous studies using a tetracycline-inducible DNMT3B mouse model, we could show that overexpression of DNMT3B affected leukemia initiation and maintenance upon retroviral transduction and serial transplantation of hematopoietic stem and progenitor cells with MSCV-MLL-AF9-GFP and MSCV-cmyc-bcl2-mcherry oncogenic vectors, respectively. Sublethally irradiated recipient mice of DNMT3B overexpressing MLL-AF9 and cmyc/bcl2 leukemic cells developed leukemia with a prolonged latency when compared to recipients of wildtype cells. We performed serial transplantation assays of MLL-AF9 leukemic stem cells, which were sorted for high expression of ckit. The life-prolonging effect of DNMT3B expression was stem cell-specific, as the potential to initiate leukemia was maintained upon serial retransplantation and recipients of DNMT3B overexpressing leukemic stem cells also died significantly later in secondary (p Taken together, these findings demonstrate that DNMT3B exerts its anti-leukemic effect mainly via induction of aberrant DNA methylation in hematopoietic and leukemic stem cells, thereby changing expression patterns of genes known to be important for stem cell function. The identification of differentially expressed DNMT3B target genes could help to find promising targets for new therapeutic strategies in AML. Disclosures: No relevant conflicts of interest to declare.

Abstract 398 DNA methyltransferases (DNMT) play an important role in regulation of DNA methylation and mutations of DNMT3A are frequently found in AML. We analyzed the effects of DNMT3B overexpression on leukemogenesis using a tetracycline-inducible DNMT3B mouse model. To analyze the impact of DNMT3B overexpression on leukemogenesis, we retrovirally co- transduced lineage-negative bone marrow cells of wildtype and DNMT3B transgenic mice with an MSCV- cMyc-bcl2 or MSCV-MLL-AF9-hCD4 and an MSCV- tTA- GFP expressing vector. Using these conditions, doxycycline suppressed DNMT3B expression whereas absence of doxycycline led to overexpression of DNMT3B on the mRNA and protein level. To verify that expression of DNMT3b in this mouse model was able to induce DNA methylation, we performed Reduced Representation Bisulfite Sequencing (RRBS) on cMyc-Bcl2 leukemic bone marrow cells. Analysis of the global methylation levels revealed a strong hypermethylation in DNMT3b overexpressing bone marrow cells. Differentially methylated regions (DMR) were defined as those regions with at least 30% difference in methylation levels. DMRs were mainly located in promoter regions and first introns and the heatmap of these DMRs confirms distinct hypermethylation in DNMT3b transgenic cells whereas very few regions become hypomethylated in transgenic cells when compared to wildtype cells. This specific methylation pattern could be responsible for altered gene expression and consecutively altered (stem) cell characteristics. To analyze the influence of changed DNA methylation levels induced by expression of DNMT3b on leukemogenesis, sorted GFP+cells were transplanted into sublethally irradiated wildtype recipients. Mice with and without DNMT3B expression developed leukemia with splenomegaly and a tendency of delayed leukemogenesis in DNMT3B overexpressing mice. Mice with cMyc-Bcl2- and MLL-AF9-induced leukemia showed a clear myeloid phenotype with enhanced expression of the stem cell marker c-kit on MLL-AF9 leukemic cells. To consider the leukemogenic capacity, we transplanted the leukemic blasts into secondary recipients. cMyc-Bcl2 induced leukemia of both, wildtype and DNMT3b recipients was transplantable and lethal. DNMT3B tg leukemic cells were severely impaired in leukemia development in secondary recipients. Secondary recipients of cMyc-Bcl2 induced leukemic DNMT3B cells died significantly later (p= 0.04). As differential methylation can also have an effect on the responsiveness to chemotherapy, spleen cells from MLL-AF9 leukemic mice were treated with different cytotoxic and demethylating drugs to see if DNMT3b induced hypermethylation confers a resistance or increased sensitivity to chemotherapeutic treatment. DNMT3b overexpressing cells were more sensitive to treatment with Azacytidine and Decitabine. Taken together, these findings demonstrate that expression of DNMT3b in a doxycycline-regulatable mouse model induces widespread DNA hypermethylation, whereas a few small regions become hypomethylated when compared to wildtype cells. This specific methylation pattern is associated with delayed leukemogenesis and DNMT3b expression significantly impaired leukemia maintenance. In addition, response to chemotherapeutic treatment is altered by DNMT3b induced hypermethylation which underlines the importance of epigenetic regulatory mechanisms as promising targets for new therapeutic strategies in AML. Disclosures: Müller-Tidow: Celgene: Lecture fees Other; Deutsche Forschungsgemeinschaft: Research Funding.

Abstract 3446 Acute myeloid leukaemia (AML) is a malignant disease with poor prognosis, which is, among other biological features, characterized by epigenetic changes including alterations in DNA methylation. DNA methyltransferases (DNMT) play an important role in regulation of DNA methylation and mutations of DNMT3A are frequently found in AML. We analyzed the effects of DNMT overexpression on leukemogenesis using an inducible DNMT3B mouse model (Linhart et al., 2007). To analyse the impact of DNMT3B overexpression on leukemia we retrovirally co-transduced lineage-negative bone marrow cells of wildtype and DNMT3Btg mice with a MSCV-cMyc-bcl2 and a MSCV-tTA-GFP containing vector. Under these conditions, doxycycline suppressed DNMT3B expression whereas absence of doxycycline led to overexpression of DNMT3B on the mRNA and protein level. DNMT3B overexpression was not toxic since colony formation in vitro did not differ between DNMT3B expressing and physiologically expressing cells. To analyze leukemogenesis, 5 × 104 sorted GFP-positive cells were transplanted into sublethally irradiated wildtype recipients. Both recipients of transduced wildtype cells and recipients of transduced DNMT3Btg cells developed leukemia with a tendency of delayed leukemogenesis in DNMT3B overexpressing mice. GFP positive leukemic cells were sorted and doxycycline regulated DNMT3B expression was verified by Western blot analysis in vitro. To determine the repopulation capacity of the leukemic cells we performed transplantation of GFP-positive primary leukemia cells into secondary wildtype recipients. Leukemia of both, wildtype and DNMT3B-overexpressing donors was transplantable and lethal. However, DNMT3Btg leukemic cells were severely impaired in leukemia development in secondary recipients. Secondary recipients of leukemic DNMT3Btg cells died significantly later (p= 0.02). Taken together, these findings demonstrate that DNMT3B expression impairs leukemia maintenance. Loss of DNMT activity might contribute to the pool size of leukemia initiating cells. Disclosures: Krug: Boehringer Ingelheim: Research Funding.

Background Conditional gene deletion using Cre-lox recombination is frequently used in mouse genetics; however recombination is frequently incomplete, resulting in a mixture of cells containing the functional (2lox) allele and the truncated (1lox) allele. Conventional analysis of 1lox/2lox allele ratios using Southern Blotting is time consuming, requires relatively large amounts of DNA and has a low sensitivity. We therefore evaluated the utility of Real-Time PCR to measure 1lox/2lox allele ratios. Results We show that SYBR Green based Real-Time PCR analysis of 1lox/2lox allele ratios can generate erroneous peaks in the melting curve that are possibly caused by alternate hybridization products promoted by the palindromic loxP sequence motif. Since abnormal melting curves frequently contribute to dismissal of SYBR Green based data, we developed a convenient method with improved specificity that avoids such erroneous signals. Our data show that probe based Real-Time PCR, using a universal probe directed against the loxP site, can accurately detect small differences in 1lox/2lox allele ratios. We also validated this method in Fabpl4× at -132-Cre transgenic mice, measuring 1lox/2lox allele ratios that are in agreement with published data. Our Real-Time PCR protocol requires the use of one probe only for all reactions. Also the universal probe established in our assay is generally applicable to any experiment analyzing Cre-lox recombination efficiency, such that only primer sequences have to be adapted. Conclusions Our data show that 1lox/2lox allele ratios are detected with high accuracy and high sensitivity with Real-Time PCR analysis using a probe directed against the loxP site. Due to the generally applicable probe the assay is conveniently adapted to all models of Cre-lox mediated gene deletion.