Oliver Galm

A DNA methylation fingerprint of 1628 human samples

Most of the studies characterizing DNA methylation patterns have been restricted to particular genomic loci in a limited number of human samples and pathological conditions. Herein, we present a compromise between an extremely comprehensive study of a human sample population with an intermediate level of resolution of CpGs at the genomic level. We obtained a DNA methylation fingerprint of 1628 human samples in which we interrogated 1505 CpG sites. The DNA methylation patterns revealed show this epigenetic mark to be critical in tissue-type definition and stemness, particularly around transcription start sites that are not within a CpG island. For disease, the generated DNA methylation fingerprints show that, during tumorigenesis, human cancer cells underwent a progressive gain of promoter CpG-island hypermethylation and a loss of CpG methylation in non-CpG-island promoters. Although transformed cells are those in which DNA methylation disruption is more obvious, we observed that other common human diseases, such as neurological and autoimmune disorders, had their own distinct DNA methylation profiles. Most importantly, we provide proof of principle that the DNA methylation fingerprints obtained might be useful for translational purposes by showing that we are able to identify the tumor type origin of cancers of unknown primary origin (CUPs). Thus, the DNA methylation patterns identified across the largest spectrum of samples, tissues, and diseases reported to date constitute a baseline for developing higher-resolution DNA methylation maps and provide important clues concerning the contribution of CpG methylation to tissue identity and its changes in the most prevalent human diseases.

Targeted restoration of down-regulated DAPK2 tumor suppressor activity induces apoptosis in Hodgkin lymphoma cells.

Death-associated protein kinase 2 (DAPK2) is a calcium/calmodulin-regulated proapoptotic serine/threonine kinase that acts as a tumor suppressor. Here we show that DAPK2 is down-regulated in Hodgkin lymphoma-derived tumor cell lines and that promoter-region hypermethylation is one mechanism for DAPK2 inactivation. To determine whether selective reconstitution of DAPK2 catalytic activity in these cells could induce apoptosis, we created a fusion protein comprising a human CD30 ligand conjugated to a human DAPK2 calmodulin-deletion mutant. Thus, recombinant immunokinase DAPK2′-CD30L has a constitutive kinase activity with enhanced proapoptotic function. We show that this immunokinase fusion protein inhibits cell proliferation and induces apoptotic cell death specifically in CD30+/DAPK2-negative tumor cell lines. This proof-of–concept study provides the first demonstration of therapeutic strategies based on the restoration of a defective, tumor-suppressing kinase activity by a novel class of recombinant immunotherapeutics.

Methylation-Specific Polymerase Chain Reaction

Methylation-specific polymerase chain reaction (MSP) is a method that can rapidly assess the methylation status of virtually any group of CpG sites within a CpG island, independent of the use of methylation-sensitive restriction enzymes. This assay entails the initial modification of DNA by sodium bisulfite, converting all unmethylated cytosines to uracils but leaving the methylated cytosines unchanged, followed by subsequent amplification with primers specific for methylated vs unmethylated DNA. The great sensitivity of this technique allows qualitative methylation analysis from DNA obtained not only from fresh frozen tissues, peripheral blood, bone marrow, or body fluids but also from paraffin-embedded samples. It is a rapid and cost-effective method that does not require radioactive reagents and can be used for the analysis of a large number of clinical samples.

Inactivation of the tissue inhibitor of metalloproteinases-2 gene by promoter hypermethylation in lymphoid malignancies

The tissue inhibitor of metalloproteinases-2 (TIMP-2) is known to antagonize matrix metalloproteinase activity and to suppress tumor growth, angiogenesis, invasion and metastasis. We analysed the methylation status of the CpG island in the TIMP-2 promoter region by methylation-specific polymerase chain reaction (MSP) in hematopoietic cell lines. TIMP-2 promoter hypermethylation in the lymphoma cell line Raji and the leukemia cell line KG1a was associated with transcriptional repression. Treatment with the demethylating agent 5-aza-2′-deoxycytidine resulted in TIMP-2 upregulation in both cell lines. TIMP-2 was expressed in the cell lines HL60, U266 and XG1, which carry an unmethylated promoter region. MSP analysis of primary patient samples revealed aberrant methylation of TIMP-2 in 33/90 (36.7%) cases of non-Hodgkins lymphoma (NHL), but not in normal peripheral blood lymphocytes as well as in nonmalignant bone marrow and lymph nodes. The frequency of TIMP-2 methylation was slightly higher in aggressive NHL subtypes compared to those with an indolent subtype (38.6 versus 33.3%). In contrast, TIMP-2 was not hypermethylated in any of the 40 cases of acute myelogenous leukemia examined. We conclude that promoter hypermethylation of TIMP-2 is a novel epigenetic event in the pathogenesis of lymphoid malignancies and may contribute to a more aggressive NHL phenotype.

EHA Scientific Workshop Report: The Role of Epigenetics in Hematological Malignancies

The clinical application of targeting the epigenome in cancer cells through demethylation and histone acetylation is an exiting novel pharmacologic concept in the treatment of malignant diseases. The manipulation of gene expression through epigenetic modifications represents a new strategy in targeted anti-cancer therapy. A workshop was held in Cannes, France in February 2007 to discuss the latest findings in basic and clinical research regarding the role of epigenetics in haematological malignancies. Fundamental aspects of DNA methylation, the histone code, chromatin remodelling and transcriptional regulatory mechanisms were discussed. Recent technological advances are now allowing scientists to gain further insight in the altered epigenome during tumorigenesis and will provide numerous opportunities for translational research. The aim of this article is to summarize the major topics discussed at the workshop by leading experts in the field and highlight conclusions for future basic, translational and clinical research.

Beyond Genetics-The Emerging Role of Epigenetic Changes in Hematopoietic Malignancies

The term epigenetic refers to a heritable change in gene expression that is mediated by mechanisms other than alterations in the primary nucleotide sequence. DNA methylation at cytosine bases that are located 5′ to guanosine within a CpG dinucleotide is the main epigenetic modification in humans. Patterns of DNA methylation are profoundly deranged in human cancer and comprise genome-wide losses as well as regional gains in DNA methylation. Hypermethylation of CpG islands within gene promoter regions is associated with transcriptional inactivation and represents, in addition to genetic aberrations, an important mechanism of gene silencing in the pathogenesis of hematopoietic malignancies. This epigenetic phenomenon acts as an alternative to mutations and deletions to disrupt tumor suppressor gene function.A large number of genes involving fundamental cellular pathways may be affected in virtually all types of human cancer by aberrant CpG island methylation in association with transcriptional silencing. Altered methylation patterns can be used as biomarkers for cancer detection, assessment of prognosis, and prediction of response to antitumor treatment. Furthermore, clinical trials using epigenetically targeted therapies have yielded promising results for acute and chronic leukemias as well as for myelodysplastic syndromes.The exploration of our growing knowledge about epigenetic aberrations may help develop novel strategies for the diagnosis and treatment of hematopoietic malignancies in the future.

Synergism between rViscumin and cisplatin is not dependent on ERCC-1 expression.

Interactions between recombinant mistletoe lectin (rViscumin) and anticancer drugs were investigated in vitro. rViscumin enhanced the cytotoxic effects of vincristine, mafosfamide, idarubicin and cisplatin in the human leukemia cell lines K562 and KG1a. In human marrow progenitor cells, rViscumin inhibited colony formation and did not exert any protective activity against cisplatin-induced inhibition of clonogenicity. Quantitative real-time reverse transcription polymerase chain reaction analysis revealed that cisplatin treatment of K562 cells resulted in a 1.9-fold increase in mRNA expression of the nucleotide excision repair gene ERCC-1. This upregulation was not prevented when cells were post-incubated with rViscumin. Our study provides evidence that rViscumin is capable of enhancing cytotoxicity of anticancer agents in vitro. This synergism appears to be independent of transcriptional activity of DNA repair relevant genes.

Ungewöhnliche Ursache einer Erythrozytose bei einer 23-jährigen Erstgebärenden in der 22. Schwangerschaftswoche

CASE REPORT A 23-year-old pregnant woman presented with erythrocytosis and a spuriously elevated HbA1c. Family history revealed that her father has been treated with phlebotomies for the last 2 years because of erythrocytosis of unknown cause. An examination of the family members demonstrated that the patient and her father were carriers of the hemoglobin (Hb) variant Hb Andrew-Minneapolis. DISCUSSION Hb Andrew-Minneapolis belongs to a group of hemoglobin variants with a high oxygen affinity resulting in compensatory erythrocytosis. The carriers of such hemoglobin variants are usually clinically asymptomatic, exercise tolerance appears unimpaired and there is no higher incidence of cardiovascular diseases. There is no clear-cut evidence that a maternal hemoglobinopathy with high oxygen affinity is accompanied by negative consequences for the fetus or a higher abortion rate. CONCLUSION Hemoglobinopathies with a high oxygen affinity are a rare but important differential diagnosis of polycythemia. Under these circumstances erythrocytosis has to be accepted as the primary mode of compensation and does not require treatment, as long as blood viscosity is kept within tolerable limits. An excessively elevated or lowered HbA1c without a history or symptoms of diabetes should lead to further investigations concerning the possibility of hemoglobinopathy.ZusammenfassungFallbeschreibung: Eine 23-jährige Erstgebärende in der 22. Schwangerschaftswoche wurde wegen einer Erythrozytose und eines unplausibel erhöhten HbA1c-Wertes (glykiertes Hämoglobin) internistisch vorgestellt. Familienanamnestisch ließ sich eruieren, dass ihr Vater seit zwei Jahren aufgrund einer Erythrozytose unklarer Ätiologie durch regelmäßige Aderlässe behandelt werde. Die weiterführende Familienuntersuchung erbrachte bei der Patientin und ihrem Vater eine Hämoglobinopathie vom Typ Hämoglobin (Hb) Andrew-Minneapolis. Diskussion: Die Hämoglobinvariante vom Typ Hb Andrew-Minneapolis ist in die Grupp der Hämoglobinopathien mit stark erhöhter Sauerstoffaffinität und konsekutiver kompensatorischer Erythrozytose einzuordnen. Die betroffenen Patienten sind in der Regel beschwerdefrei und normal körperlich belastbar; eine erhöhte Inzidenz von kardiovaskulären Erkrankungen konnte bisher hicht beobachtet werden. Es gibt keine sicheren Hinweise, dass eine Hämoglobinanomalie der Mutter mit deutlich erhöhter Sauerstoffaffinität mit negativen Auswirkungen auf den Fetus oder einer erhöhten Komplikationsrate unter der Geburt vergesellschaftet ist. Schlussfolgerung: Hämoglobinopathien mit deutlich erhöhter Sauerstoffaffinität sind eine seltene, aber wichtige Differentialdiagnose der Erythrozytose, deren Klärung diagnostische Irrwege für Patient und Arzt vermeidbar macht. Für die prognostische Beurteilung ist bedeutsam, dass diese Erythrozytose bei den betroffenen Patienten in weiteren Grenzen als kompensatorisch zu akzeptieren ist. Ein außergewöhnlich erhöhter oder erniedrigter HbA1c-Wert ohne Vorliegen einer diabetischen Befundkonstellation sollte an eine Hämoglobinopathie denken lassen.AbstractCase Report: A 23-year-old pregnant woman presented with erythrocytosis and a spuriously elevated HbA1c. Family history revealed that her father has been treated with phlebotomies for the last 2 years because of erythrocytosis of unknown cause. An examination of the family members demonstrated that the patient and her father were carriers of the hemoglobin (Hb) variant Hb Andrew-Minneapolis. Discussion: Hb Andrew-Minneapolis belongs to a group of hemoglobin variants with a high oxygen affinity resulting in compensatory erythrocytosis. The carriers of such hemoglobin variants are usually clinically asymptomatic, exercise tolerance appears unimpaired and there is no higher incidence of cardiovascular diseases. There is no clear-cut evidence that a maternal hemoglobinopathy with high oxygen affinity is accompanied by negative consequences for the fetus or a higher abortion rate. Conclusion: Hemoglobinopathies with a high oxygen affinity are a rare but important differential diagnosis of polycythemia. Under these circumstances erythrocytosis has to be accepted as the primary mode of compensation and does not require treatment, as long as blood viscosity is kept within tolerable limits. An excessively elevated or lowered HbA1c without a history or symptoms of diabetes should lead to further investigations concerning the possibility of hemoglobinopathy.

Differential Role of gp130-Dependent STAT and Ras Signalling for Haematopoiesis Following Bone-Marrow Transplantation

Introduction Bone marrow transplantation (BMT) is a complex process regulated by different cytokines and growth factors. The pleiotropic cytokine IL-6 (Interleukin-6) and related cytokines of the same family acting on the common signal transducer gp130 are known to play a key role in bone marrow (BM) engraftment. In contrast, the exact signalling events that control IL-6/gp130-driven haematopoietic stem cell development during BMT remain unresolved. Methods Conditional gp130 knockout and knockin mice were used to delete gp130 expression (gp130ΔMx), or to selectively disrupt gp130-dependent Ras (gp130ΔMxRas) or STAT signalling (gp130ΔMxSTAT) in BM cells. BM derived from the respective strains was transplanted into irradiated wildtype hosts and repopulation of various haematopoietic lineages was monitored by flow cytometry. Results BM derived from gp130 deficient donor mice (gp130ΔMx) displayed a delayed engraftment, as evidenced by reduced total white blood cells (WBC), marked thrombocytopenia and anaemia in the early phase after BMT. Lineage analysis unravelled a restricted development of CD4(+) and CD8(+) T-cells, CD19(+) B-cells and CD11b(+) myeloid cells after transplantation of gp130-deficient BM grafts. To further delineate the two major gp130-induced signalling cascades, Ras-MAPK and STAT1/3-signalling respectively, we used gp130ΔMxRas and gp130ΔMxSTAT donor BM. BMT of gp130ΔMxSTAT cells significantly impaired engraftment of CD4(+), CD8(+), CD19(+) and CD11b(+) cells, whereas gp130ΔMxRas BM displayed a selective impairment in early thrombopoiesis. Importantly, gp130-STAT1/3 signalling deficiency in BM grafts severely impaired survival of transplanted mice, thus demonstrating a pivotal role for this pathway in BM graft survival and function. Conclusion Our data unravel a vital function of IL-6/gp130-STAT1/3 signals for BM engraftment and haematopoiesis, as well as for host survival after transplantation. STAT1/3 and ras-dependent pathways thereby exert distinct functions on individual bone-marrow-lineages.

Abstract 3142: Hypermethylation of the tissue inhibitor of metalloproteinases-2 gene in multiple myeloma

Multiple myeloma (MM) is a B-cell neoplasm that is characterized by the accumulation of malignant plasma cells in the bone marrow (BM) as well as abundant BM angiogenesis. Aberrant methylation of CpG islands near gene promoter regions is the most widely studied epigenetic abnormality in human malignancies and is associated with loss of gene function. There is increasing evidence for a role of the tissue inhibitor of metalloproteinases 2 (TIMP-2) gene as a tumor suppressor. Overexpression of TIMP-2 may result in decreased invasive potential, suppression of tumor growth and vascularization as well as inhibition of angiogenesis. We could previously show that TIMP-2 may become hypermethylated in association with transcriptional silencing in indolent and aggressive non-Hodgkin9s lymphomas. Recently, downregulation of TIMP-2 was also reported in MM, and this is thought to contribute to increased BM angiogenesis. In this study, we determined the methylation status of the promoter-associated CpG island of TIMP-2 in primary MM samples and investigated correlations between TIMP-2 methylation and clinical parameters. Methylation of the promoter-associated CpG island of TIMP-2 was analyzed by methylation-specific polymerase chain reaction (MSP) in samples from 84 MM patients (median age 65 years [range 40-94]; 48 males, 36 females; 79 BM samples, 5 peripheral blood samples). Overall survival curves were plotted according to the method of Kaplan and Meier and compared using the log-rank test. Correlations between variables were analyzed using the Fisher9s exact two-sided test and independent t-test, respectively. MSP analysis revealed aberrant methylation of the TIMP-2 promoter region in 4/84 MM patient samples. We found an association of TIMP-2 hypermethylation with plasma cell leukemia (p Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3142. doi:1538-7445.AM2012-3142