Claudia Schubert

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.

Calreticulin-mutant proteins induce megakaryocytic signaling to transform hematopoietic cells and undergo accelerated degradation and Golgi-mediated secretion

BackgroundSomatic calreticulin (CALR), Janus kinase 2 (JAK2), and thrombopoietin receptor (MPL) mutations essentially show mutual exclusion in myeloproliferative neoplasms (MPN), suggesting that they activate common oncogenic pathways. Recent data have shown that MPL function is essential for CALR mutant-driven MPN. However, the exact role and the mechanisms of action of CALR mutants have not been fully elucidated.MethodsThe murine myeloid cell line 32D and human HL60 cells overexpressing the most frequent CALR type 1 and type 2 frameshift mutants were generated to analyze the first steps of cellular transformation, in the presence and absence of MPL expression. Furthermore, mutant CALR protein stability and secretion were examined using brefeldin A, MG132, spautin-1, and tunicamycin treatment.ResultsThe present study demonstrates that the expression of endogenous Mpl, CD41, and the key megakaryocytic transcription factor NF-E2 is stimulated by type 1 and type 2 CALR mutants, even in the absence of exogenous MPL. Mutant CALR expressing 32D cells spontaneously acquired cytokine independence, and this was associated with increased Mpl mRNA expression, CD41, and NF-E2 protein as well as constitutive activation of downstream signaling and response to JAK inhibitor treatment. Exogenous expression of MPL led to constitutive activation of STAT3 and 5, ERK1/2, and AKT, cytokine-independent growth, and reduction of apoptosis similar to the effects seen in the spontaneously outgrown cells. We observed low CALR-mutant protein amounts in cellular lysates of stably transduced cells, and this was due to accelerated protein degradation that occurred independently from the ubiquitin-proteasome system as well as autophagy. CALR-mutant degradation was attenuated by MPL expression. Interestingly, we found high levels of mutated CALR and loss of downstream signaling after blockage of the secretory pathway and protein glycosylation.ConclusionsThese findings demonstrate the potency of CALR mutants to drive expression of megakaryocytic differentiation markers such as NF-E2 and CD41 as well as Mpl. Furthermore, CALR mutants undergo accelerated protein degradation that involves the secretory pathway and/or protein glycosylation.

Epigenetic Biomarker to Support Classification into Pluripotent and Non-Pluripotent Cells

Quality control of human induced pluripotent stem cells (iPSCs) can be performed by several methods. These methods are usually relatively labor-intensive, difficult to standardize, or they do not facilitate reliable quantification. Here, we describe a biomarker to distinguish between pluripotent and non-pluripotent cells based on DNA methylation (DNAm) levels at only three specific CpG sites. Two of these CpG sites were selected by their discriminatory power in 258 DNAm profiles – they were either methylated in pluripotent or non-pluripotent cells. The difference between these two β-values provides an Epi-Pluri-Score that was validated on independent DNAm-datasets (264 pluripotent and 1,951 non-pluripotent samples) with 99.9% specificity and 98.9% sensitivity. This score was complemented by a third CpG within the gene POU5F1 (OCT4), which better demarcates early differentiation events. We established pyrosequencing assays for the three relevant CpG sites and thereby correctly classified DNA of 12 pluripotent cell lines and 31 non-pluripotent cell lines. Furthermore, DNAm changes at these three CpGs were tracked in the course of differentiation of iPSCs towards mesenchymal stromal cells. The Epi-Pluri-Score does not give information on lineage-specific differentiation potential, but it provides a simple, reliable, and robust biomarker to support high-throughput classification into either pluripotent or non-pluripotent cells.

OASIS/CREB3L1 is epigenetically silenced in human bladder cancer facilitating tumor cell spreading and migration in vitro

CREB3L1 has been recently proposed as a novel metastasis suppressor gene in breast cancer. Our current study highlights CREB3L1 expression, regulation, and function in bladder cancer. We demonstrate a significant downregulation of CREB3L1 mRNA expression (n = 64) in primary bladder cancer tissues caused by tumor-specific CREB3L1 promoter hypermethylation (n = 51). Based on pyrosequencing CREB3L1 methylation was shown to be potentially associated with a more aggressive phenotype of bladder cancer. These findings were verified by an independent public data set containing data from 184 bladder tumors. In addition, immunohistochemical evaluation showed that CREB3L1 protein expression is decreased in bladder cancer tissues as well. Interestingly, protein loss is predominately observed in the nuclei of aggressive tumor cells. Based on in vitro models we clearly show that CREB3L1 re-expression mediates suppression of tumor cell migration and colony growth of high grade and invasive bladder cancer cells. The candidate tumor suppressor and TGF-β signaling inhibitor HTRA3 was furthermore identified as putative target gene of CREB3L1 in both invasive J82 bladder cells and primary bladder tumors. Hence, our data provide for the first time evidence that the transcription factor CREB3L1 may have an important role as a putative tumor suppressor in bladder cancer.

Identification of the Adapter Molecule MTSS1 as a Potential Oncogene-Specific Tumor Suppressor in Acute Myeloid Leukemia

The adapter protein metastasis suppressor 1 (MTSS1) is implicated as a tumor suppressor or tumor promoter, depending on the type of solid cancer. Here, we identified Mtss1 expression to be increased in AML subsets with favorable outcome, while suppressed in high risk AML patients. High expression of MTSS1 predicted better clinical outcome of patients with normal-karyotype AML. Mechanistically, MTSS1 expression was negatively regulated by FLT3-ITD signaling but enhanced by the AML1-ETO fusion protein. DNMT3B, a negative regulator of MTSS1, showed strong binding to the MTSS1 promoter in PML-RARA positive but not AML1-ETO positive cells, suggesting that AML1-ETO leads to derepression of MTSS1. Pharmacological treatment of AML cell lines carrying the FLT3-ITD mutation with the specific FLT3 inhibitor PKC-412 caused upregulation of MTSS1. Moreover, treatment of acute promyelocytic cells (APL) with all-trans retinoic acid (ATRA) increased MTSS1 mRNA levels. Taken together, our findings suggest that MTSS1 might have a context-dependent function and could act as a tumor suppressor, which is pharmacologically targetable in AML patients.

Epigenetic dysregulation of secreted frizzled-related proteins in myeloproliferative neoplasms complements the JAK2V617F-mutation.

BackgroundSecreted frizzled-related proteins (SFRPs) are antagonists of the Wnt signaling pathway, which plays a central role in stem cell maintenance and differentiation of stem cells and hematopoietic progenitors. Epigenetic downregulation of SFRPs by promoter hypermethylation has been described to be involved in the pathogenesis of hematopoietic malignancies. There is an association between aberrant Wnt signaling and the established cancer stem cell concept. In contrast to BCR-ABL1-positive chronic myeloid leukemia CML, BCR-ABL1-negative myeloproliferative neoplasms (Ph-MPN) are characterized by the frequent occurrence of an autoactivating mutation in the JAK2 tyrosine kinase (JAK2V617F) or other mutations in the JAK-STAT pathway. However, pathogenetic mechanisms of JAK2 mutated or unmutated Ph-MPN remain not completely understood. We determined the promoter methylation status of SFRP-1, -2, -4, and -5 in 57 MPN patient samples by methylation-specific polymerase chain reaction (PCR) (MSP). JAK2V617F was assessed by allele-specific PCR.ResultsAberrant methylation among primary MPN samples was 4% for SFRP-1, 25% for SFRP-2, 2% for SFRP-4, and 0% for SFRP-5. Hypermethylation of SFRP-2, which was the most frequently hypermethylated gene in our study, could not be correlated to any specific MPN subtype. However, we detected a significant correlation between SFRP-2 methylation and presence of a JAK2V617F mutation (P = 0.008). None of the 10 CML samples showed any SFRP-methylation.ConclusionsOur data indicate that epigenetic dysregulation of the Wnt signaling pathway is a common event in MPN with aberrant methylation of at least one SFRP being detected in 25% of the primary patient samples and in 30% if only accounting for Ph-MPN. A significant correlation between SFRP-2 methylation and presence of JAK2V617F in our data supports the hypothesis that epigenetic dysregulation may be a complementary mechanism to genetic aberrations. Aberrant methylation of crucial stem cell maintenance genes seems to contribute to disease pathogenesis in Ph-MPN.

Dissecting Genomic Aberrations in Myeloproliferative Neoplasms by Multiplex-PCR and Next Generation Sequencing

In order to assess the feasibility of amplicon-based parallel next generation sequencing (NGS) for the diagnosis of myeloproliferative neoplasms (MPN), we investigated multiplex-PCR of 212 amplicons covering genomic mutational hotspots in 48 cancer-related genes. Samples from 64 patients with MPN and five controls as well as seven (myeloid) cell lines were analyzed. Healthy donor and reactive erythrocytosis samples showed several frequent single-nucleotide polymorphisms (SNPs) but no known pathogenic mutation. Sequencing of the cell lines confirmed the presence of the known mutations. In the patient samples, JAK2 V617F was present in all PV, 4 of 10 ET, and 16 of 19 MF patients. The JAK2 V617F allele burden was different in the three groups (ET, 33+/-22%; PV 48+/-28% and MF 68+/- 29%). Further analysis detected both previously described and undescribed mutations (i.e., G12V NRAS, IDH1 R132H, E255G ABL, and V125G IDH1 mutations). One patient with lymphoid BC/Ph+ ALL who harbored a T315I ABL mutation and was treated with ponatinib was found to have developed a newly acquired V216M TP53 mutation (12% of transcripts) when becoming resistant to ponatinib. Ponatinib led to a decrease of ABL T315I positive transcripts from 47% before ponatinib treatment to 16% at the time of ponatinib resistance in this patient, suggesting that both TP53 and ABL mutations were present in the same clone and that the newly acquired TP53 mutation might have caused ponatinib resistance in this patient. In conclusion, amplicon-sequencing-based NGS allows simultaneous analysis of multiple MPN associated genes for diagnosis and during treatment and measurement of the mutant allele burden.

The SCLtTAxBCR-ABL transgenic mouse model closely reflects the differential effects of dasatinib on normal and malignant hematopoiesis in chronic phase-CML patients

The second generation tyrosine kinase inhibitor (TKI) dasatinib is a clinically approved drug for chronic myeloid leukemia (CML) as well as Ph+ acute lymphoblastic leukemia. In addition to its antileukemic effects, dasatinib was shown to impact on normal hematopoiesis and cells of the immune system. Due to the fact that the murine in vivo studies so far have not been performed in a chronic-phase CML model under steady-state conditions, our aim was to study the hematopoietic effects of dasatinib (20 mg/kg p.o.) in BCR-ABL expressing SCLtTAxBCR-ABL double transgenic (dtg) mice. Dasatinib robustly antagonized the CML phenotype in vivo in our transgenic mouse model, and this effect included both mature and immature cell populations. However, similar to patients with CML, the fraction of LinnegSca-1+KIT+CD48negCD150+ hematopoietic stem cells was not reduced by dasatinib treatment, suggesting that these cells are not oncogene-addicted. Moreover, we observed differential effects of dasatinib in these animals as compared to wild-type (wt) animals: while granulocytes were significantly reduced in dtg animals, they were increased in wt mice. And Ter119+ erythrocytic and B220+ B cells were increased in dtg mice but decreased in wt mice. Finally, while dasatinib induced a shift from CD49b/NK1.1 positive NK cells from the bone marrow to the spleen in wt animals, there was no change in dtg mice. In conclusion, the present mouse model provides a useful tool to study mechanisms of TKI resistance and dasatinib-associated beneficial effects and adverse events.

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

Abstract 4872: Synergistic induction of cell death and apoptosis by the histone deacetylase inhibitor vorinostat and the demethylating agent 5-aza-2′-deoxycytidine in multiple myeloma

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Introduction: Hypermethylation of CpG islands in the promoter region of genes is an epigenetic modification associated with transcriptional silencing. In addition, acetylation of core histones is necessary for the maintenance of transcriptional activity of genes. DNA methylation and histone deacetylation are reversible and can be influenced by DNA methyltransferase (DNMT) inhibitors such as 5-aza-2′-deoxycytidine (DAC) and histone deacetylase (HDAC) inhibitors such as suberoylanilide hydroxamic acid (SAHA, vorinostat), respectively. Material and Methods: To investigate the in vitro effects of SAHA on the multiple myeloma (MM) cell lines U266, LP-1, RPMI-8226 or OPM-2 and assess possible interactions with DNMT inhibitors, cells were first incubated with DAC at a final concentration of 0.1 or 0.2 μM for 72 hours. After exposure to DAC, cells were incubated for 72 or 96 hours with SAHA at final concentrations between 0.1 and 20 μM. The toxic effects of the treatment were assessed by MTT assay. Induction of apoptosis was analyzed by flow cytometry with annexin V-binding. In addition, expression of the epigenetically silenced tumor suppressor genes SFRP-2 and DAB2 was determined by real-time RT-PCR. Results: In LP-1 and U-266 cells, no relevant enhancement of the cytotoxic effect of SAHA was observed after previous exposure to DAC. In contrast, in OPM-2 and RPMI-8226 cells, a significant increase in cytotoxicity of SAHA was observed, when the cells were first incubated with DAC with a decrease of the IC50 from 6.5 μM to 2.43 μM and 10.37 μM to 4.5 μM, respectively. After sequential exposure to DAC and SAHA for 72 hours each, no change in the apoptotic cell fraction was observed for the cell lines OPM-2 and RPMI-8226. However, for U266 and LP-1, a significant increase in apoptotic cells was observed after incubation with SAHA, when the cells were previously exposed to DAC with an increase in the apoptotic cell fraction from 39.5 % to 55.4 % and 2.5 % to 14.4 %, respectively. Corresponding transcriptional silencing for SFRP-2 and DAB2 was demonstrated in untreated cells, and exposure of cell lines to DAC and SAHA resulted in reexpression. A synergism for the induction of reexpression of these genes was observed when cells were incubated with DAC and SAHA sequentially. Discussion: After treatment with SAHA, we observed a dose-dependent induction of cell death and apoptosis as assessed by MTT and annexin V assay, respectively. In the different MM cell lines, we observed a synergism between SAHA and DAC for cytotoxic effects and induction of apoptosis. A synergism was also observed for the reexpression of epigenetically silenced genes after exposure to DAC and SAHA. These data can be considered as a basis for further in vitro studies and preclinical models with SAHA in combination with demethylating agents in order to improve therapeutic options in MM patients. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4872.