Edgar Jost

Epigenetic alterations complement mutation of JAK2 tyrosine kinase in patients with BCR/ABL-negative myeloproliferative disorders

An acquired autoactivating mutation with a V617F amino-acid substitution in the JAK2 tyrosine kinase is frequently found in BCR/ABL-negative myeloproliferative disorders (MPD). Hypermethylation of CpG islands within gene promoter regions is associated with transcriptional inactivation and represents an important mechanism of gene silencing in the pathogenesis of hematopoietic malignancies. In this study, we determined the DNA methylation status of 13 cancer-related genes in the context of JAK2 mutations in 39 patients with MPD. Genes analyzed for hypermethylation were SOCS-1, SHP-1, E-cadherin, MGMT, TIMP-2, TIMP-3, p15, p16, p73, DAPK1, RASSF1A, RARβ2 and hMLH1. We found at least one hypermethylated gene in 15/39 MPD patient specimens, and in 6/39 samples aberrant methylation of the negative cytokine regulator SOCS-1 was present. The JAK2V617F mutation was found in 21/39 patients as determined by allele-specific polymerase chain reaction. Hypermethylation of SOCS-1 was observed in 3/21 patients with an autoactivating JAK2 mutation and in 3/18 patients with wild-type JAK2. Our results suggest that epigenetic inactivation of SOCS-1 may be a complementary mechanism to the JAK2V617F mutation in the pathogenesis of MPD that leads to dysregulation of JAK-STAT signal transduction and thus contributes to growth factor hypersensitivity.

Granzyme B-H22(scFv), a human immunotoxin targeting CD64 in acute myeloid leukemia of monocytic subtypes

Acute myeloid leukemia (AML) cells of subtypes M4 and M5 show enhanced expression of CD64 (FcγRI), the high-affinity receptor for IgG, which is normally expressed at high levels only on activated cells of the myeloid lineage. CD64 is therefore a prime target for the specific delivery of cytotoxic agents. A promising toxin candidate is granzyme B, a human serine protease originating from cytotoxic granules of CD8+ T lymphocytes and natural killer cells. After evaluating the sensitivity of the AML-related cell line U937 toward cytosolic granzyme B, we genetically fused granzyme B to H22, a humanized single-chain antibody fragment (scFv) specific for CD64, to obtain Gb-H22(scFv), a fusion protein lacking the immunogenic properties of nonhuman immunofusions. Gb-H22(scFv) was successfully expressed in human 293T cells, secreted, and purified from cell culture supernatants. The purified protein bound specifically to CD64+ U937 cells. Despite linkage to the binding domain, the proteolytic activity of functional Gb-H22(scFv) was identical to that of free granzyme B. Target cell-specific cytotoxicity was observed with a half-maximal inhibitory concentration (IC50) between 1.7 and 17 nmol/L. In addition, the induction of apoptosis in U937 cells was confirmed by Annexin A5 staining and the detection of activated caspase-3 in the cytosol. Finally, apoptosis was observed in primary CD64+ AML cells, whereas CD64− AML cells were unaffected. This is the first report of a completely human granzyme B-based immunotoxin directed against CD64, with activity against an AML-related cell line and primary AML cells. [Mol Cancer Ther 2008;7(9):2924–32]

Epigenetic inactivation of secreted Frizzled-related proteins in acute myeloid leukaemia

The Wnt signalling pathway has a key function in stem cell maintenance and differentiation of haematopoietic progenitors. Secreted Frizzled‐related protein genes (SFRPs), functioning as Wnt signalling antagonists, have been found to be downregulated by promoter hypermethylation in many tumours. To analyse epigenetic dysregulation of SFRPs in acute myeloid leukaemia (AML), we examined the promoter methylation status of SFRP1, ‐2, ‐4 and ‐5 in AML cell lines by methylation‐specific polymerase chain reaction (MSP). Aberrant CpG island methylation was found for all four SFRP genes. By real‐time reverse transcription‐PCR, corresponding transcriptional silencing for SFRP1 and ‐2 was demonstrated and treatment of cell lines with 5‐aza‐2′‐deoxycytidine resulted in re‐expression. The methylation status of the SFRP genes was analysed in 100 specimens obtained from AML patients at diagnosis. The frequencies of aberrant methylation among the patient samples were 29% for SFRP1, 19% for SFRP2, 0% for SFRP4 and 9% for SFRP5. For SFRP2, a correlation between promoter hypermethylation and transcriptional downregulation was found in primary AML samples. Among AML cases with a favourable karyotype, hypermethylation of SFRP genes was restricted to patients with core binding factor (CBF) leukaemia, and aberrant methylation of the SFRP2 promoter was an adverse risk factor for survival in CBF leukaemia.

Ubiquitin carboxyl-terminal hydrolase 1 (UCHL1) is a potential tumour suppressor in prostate cancer and is frequently silenced by promoter methylation

BackgroundWe have previously reported significant downregulation of ubiquitin carboxyl-terminal hydrolase 1 (UCHL1) in prostate cancer (PCa) compared to the surrounding benign tissue. UCHL1 plays an important role in ubiquitin system and different cellular processes such as cell proliferation and differentiation. We now show that the underlying mechanism of UCHL1 downregulation in PCa is linked to its promoter hypermethylation. Furthermore, we present evidences that UCHL1 expression can affect the behavior of prostate cancer cells in different ways.ResultsMethylation specific PCR analysis results showed a highly methylated promoter region for UCHL1 in 90% (18/20) of tumor tissue compared to 15% (3/20) of normal tissues from PCa patients. Pyrosequencing results confirmed a mean methylation of 41.4% in PCa whereas only 8.6% in normal tissues. To conduct functional analysis of UCHL1 in PCa, UCHL1 is overexpressed in LNCaP cells whose UCHL1 expression is normally suppressed by promoter methylation and found that UCHL1 has the ability to decrease the rate of cell proliferation and suppresses anchorage-independent growth of these cells. In further analysis, we found evidence that exogenous expression of UCHL1 suppress LNCaP cells growth probably via p53-mediated inhibition of Akt/PKB phosphorylation and also via accumulation of p27kip1 a cyclin dependant kinase inhibitor of cell cycle regulating proteins. Notably, we also observed that exogenous expression of UCHL1 induced a senescent phenotype that was detected by using the SA-ß-gal assay and might be due to increased p14ARF, p53, p27kip1 and decreased MDM2.ConclusionFrom these results, we propose that UCHL1 downregulation via promoter hypermethylation plays an important role in various molecular aspects of PCa biology, such as morphological diversification and regulation of proliferation.

A systematic comparison of quantitative high-resolution DNA methylation analysis and methylation-specific PCR.

Assessment of DNA methylation has become a critical factor for the identification, development and application of methylation based biomarkers. Here we describe a systematic comparison of a quantitative high-resolution mass spectrometry-based approach (MassARRAY), pyrosequencing and the broadly used methylation-specific PCR (MSP) technique analyzing clinically relevant epigenetically silenced genes in acute myeloid leukemia (AML). By MassARRAY and pyrosequencing, we identified significant DNA methylation differences at the ID4 gene promoter and in the 5′ region of members of the SFRP gene family in 62 AML patients compared with healthy controls. We found a good correlation between data obtained by MassARRAY and pyrosequencing (correlation coefficient R2 = 0.88). MSP-based assessment of the identical samples showed less pronounced differences between AML patients and controls. By direct comparison of MSP-derived and MassARRAY-based methylation data as well as pyrosequencing, we could determine overestimation of DNA methylation data by MSP. We found sequence-context dependent highly variable cut-off values of quantitative DNA methylation values serving as discriminator for the two MSP methylation categories. Moreover, good agreements between quantitative methods and MSP could not be achieved for all investigated loci. Significant correlation of the quantitative assessment but not of MSP-derived methylation data with clinically important characteristics in our patient cohort demonstrated clinical relevance of quantitative DNA methylation assessment. Taken together, while MSP is still the most commonly applied technique for DNA methylation assessment, our data highlight advantages of quantitative approaches for precise characterization and reliable biomarker use of aberrant DNA methylation in primary patient samples, particularly.

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.

Correlation of C-Reactive Protein with Survival and Radiographic Response to First-Line Platinum-Based Chemotherapy in Advanced Non-Small Cell Lung Cancer

Objective: This study was conducted to assess the prognostic role of regular measurements of C-reactive protein (CRP) in patients with advanced-stage non-small cell lung cancer (NSCLC) during platinum-based first-line therapy. Methods: A total of 210 patients were retrospectively analyzed regarding CRP values, infections, histological type, stage, performance status, gender, age, body mass index and survival. Additionally, in 88 of these patients, changes of CRP values were correlated with response to chemotherapy by radiographic imaging. Results: Elevation of CRP prior to the first cycle was an adverse prognostic factor for overall survival. Comparing CRP values before and after 2 cycles correlated with response and identified patient groups with a remarkable difference of overall survival (18.8 vs. 7.5 months). Normalization of CRP was associated with a low risk for progression, whereas patients with an increase of CRP values of more than 25% showed a progressive disease in most cases. Besides performance status, no correlation of CRP with other clinical data was found. Conclusions: Measurement of CRP before initiation and during a platinum-based chemotherapy can provide prognostic information for the individual patient with advanced NSCLC and is able to support or even replace assessment of response by radiographic imaging in defined situations.

Epigenetic dysregulation of secreted Frizzled-related proteins in multiple myeloma

We analysed the clinical impact of epigenetic dysregulation of the Wnt pathway in malignant plasma cell disorders. In multiple myeloma (MM) cell lines, aberrant promoter hypermethylation of the secreted Frizzled-related protein (SFRP) genes was a common event, and hypermethylation of SFRP1,-2 and -5 was associated with transcriptional silencing. Among 76 primary patient samples, the frequency of aberrant methylation was 35.5% for SFRP1, 52.6% for SFRP2, 1.3% for SFRP4 and 6.9% for SFRP5. Hypermethylation of SFRP1 and -2 genes was detected in monoclonal gammopathy of undetermined significance and all MM stages including plasma cell leukaemia (PCL), while SFRP5 methylation was restricted to advanced MM stages and PCL. Our data indicate that epigenetic silencing of Wnt antagonists is an early event in MM pathogenesis and that SFRP5 hypermethylation may play a role in disease progression.

Epimutations mimic genomic mutations of DNMT3A in acute myeloid leukemia

Mutations in the genetic sequence of the DNA de novo methyltransferase DNMT3A (DNA methyltransferase 3A) are found in many patients with acute myeloid leukemia (AML). They lead to dysfunction of DNMT3A protein and represent a marker for poor prognosis. Effects of genetic mutations can be mimicked by epigenetic modifications in the DNA methylation (DNAm) pattern. Using DNAm profiles of the Cancer Genome Atlas Research Network (TCGA), we identified aberrant hypermethylation at an internal promoter region of DNMT3A, which occurred in about 40% of AML patients. Bisulfite pyrosequencing assays designed for this genomic region validated hypermethylation specifically in a subset of our AML samples. High DNAm levels at this site are particularly observed in samples without genetic mutations in DNMT3A. Epimutations and mutations of DNMT3A were associated with related gene expression changes such as upregulation of the homeobox genes in HOXA and HOXB clusters. Furthermore, epimutations in DNMT3A were enriched in patients with poor or intermediate cytogenetic risk, and in patients with shorter event-free survival and overall survival (OS). Taken together, aberrant DNA hypermethylation within the DNMT3A gene, in analogy to DNMT3A mutations, is frequently observed in AML and both modifications seem to be useful for risk stratification or choice of therapeutic regimen.

In vivo efficacy of the recombinant anti-CD64 immunotoxin H22(scFv)-ETA' in a human acute myeloid leukemia xenograft tumor model.

Target‐specific acute myeloid leukemia (AML) immunotherapy requires selective cell‐surface antigens on AML blast cells. CD64 is a promising candidate antigen because it is abundantly expressed on monocytoid differentiated AML subtypes. In previous studies, a chemically linked full‐length anti‐CD64 immunotoxin based on ricin A showed promising results in several animal models, but further development has been hindered by its substantial, dose‐limiting off‐target effects. We recently constructed the recombinant immunotoxin H22(scFv)‐ETA′, comprising a truncated Pseudomonas exotoxin A (PE) and a humanized scFv antibody against CD64. This molecule was shown to kill CD64+ AML‐derived tumor cell lines and primary patient‐derived AML cells specifically, both in vitro and ex vivo. Here we describe the in vivo efficiency of H22(scFv)‐ETA′ in the U937/SCID mouse xenograft model for human AML, by providing immunohistochemical evidence for the elimination of human CD64+ tumor cells in mouse organs. H22(scFv)‐ETA′ showed potent antitumor activity against myeloid tumor cells and significantly prolonged the overall survival of AML xenograft animals. In conclusion, H22(scFv)‐ETA′ is efficacious against AML with monocytoid differentiation in vitro and in animal models in vivo, providing the basis for a novel therapeutic strategy for the treatment of AML patients.