We established homogeneous sublines that contained two or three homozygous DNA loci with long (GATA) n tracts from polymorphic Ephestia laboratory strains. Crossbreeding analysis assigned these loci to two or three different chromosomes, respectively. A nonrelated and rather recently isolated strain contained at least three other poly(GATA) loci located on different chromosomes. Germline mutations, visible as restriction fragment size changes between parents and offspring or loss of a poly(GATA) tract, are relatively rare in some strains but unusually frequent in the ml strain. The mutations affect not only GATA repeats but also flanking sequences. In five mutations investigated by crossbreeding, the altered poly(GATA)-containing restriction fragments remained in their original linkage groups.Key words: Bkm DNA, microsatellite, simple sequence repeats.
Abstract Acute myeloid leukemia (AML) with the t(7;12)(q36;p13) translocation occurs only in very young children and has a poor clinical outcome. The expected oncofusion between breakpoint partners ( MNX1 and ETV6 ) has only been reported in a subset of cases. However, a universal feature is the strong transcript and protein expression of MNX1, a homeobox transcription factor that is normally not expressed in hematopoietic cells. Here, we map the translocation breakpoints on chromosomes 7 and 12 in affected patients to a region proximal to MNX1 and either introns 1 or 2 of ETV6 . The frequency of MNX1 overexpression in pediatric AML (n=1556, own and published data) is 2.4% and occurs predominantly in t(7;12)(q36;p13) AML. Chromatin interaction assays in a t(7;12)(q36;p13) iPSC cell line model unravel an enhancer-hijacking event that explains MNX1 overexpression in hematopoietic cells. Our data suggest that enhancer-hijacking is a more common and overlooked mechanism for structural rearrangement-mediated gene activation in AML. Key points Expression analysis of over 1500 pediatric AML samples demonstrates MNX1 expression as a universal feature of t(7;12)(q36;p13) AML as well as in rare cases without t(7;12)(q36;p13) MNX1 is activated by an enhancer-hijacking event in t(7;12)(q36;p13) AML and not, as previously postulated, by the creation of a MNX1 :: ETV6 oncofusion gene.
Topic: 3. Acute myeloid leukemia - Biology & Translational Research Background: Acute myeloid leukemia (AML) is an aggressive hematological malignancy resulting from a block in the differentiation of myeloid progenitors and activation of growth promoting genes. In clinics, hypomethylating agents (HMA) such as 5-azacitidine (AZA) or 2-deoxy-5-azacytidine (decitabine) are routinely used to treat patients with AML. Chromosomal rearrangements in AML can lead to aberrant activation of an oncogene by juxtaposition with an activating enhancer, a phenomenon known as enhancer hijacking. However, there is currently no therapeutic agent known to specifically target oncogenic enhancers in AML. Aims: To find an epigenetic inhibitor which abrogates enhancer hijacking leading to oncogenic overexpression of MNX1 (motor neuron and pancreas homeobox 1) in a subset of AML cases. Methods: 174 different epigenetic inhibitors were screened in the AML cell line GDM-1, which carries a translocation t(6;7) and overexpresses MNX1 due to hijacking of an enhancer from the MYB locus on chromosome 6. Candidate inhibitors were identified by cell viability assays. Western blot, real-time quantitative polymerase chain reaction (RT-qPCR) and microRNA (miRNA) sequencing were then applied to analyze changes of MNX1 and miRNA expression, respectively. Results: Top hits included compounds targeting DNMT, BET, KDM6A/6B, P300/CBP proteins and domains found in the SWI/SNF complex. Treatment with the HMA, decitabine (DAC), resulted in significant reduction of MNX1 expression at both RNA and protein level. DAC treatment induced activation of cancer testis antigens (CTAs), long terminal repeats (LTRs). Hypomethylation after DAC treatment will be confirmed through MiSeq of LINE-1. To investigate the mechanism of DAC-dependent MNX1 downregulation, we performed high throughput sequencing of microRNA (miRNA). However, we could not detect upregulated miRNAs upon DAC treatment precluding the contribution of miRNAs on MNX1 downregulation. Summary/Conclusion: We screened 174 epigenetic inhibitors in the AML cell line GDM-1 and showed that DAC treatment significantly reduces MNX1 expression, which is activated by enhancer hijacking. The contribution of activated miRNAs targeting MNX1 and DAC treatment mediated disruption of the topologically associated domain, which allows the enhancer-promoter interaction, are investigated as possible mechanisms for MNX1 expression. Further candidate compound validation will make use of a GDM-1 derivative control cell line without t(6;7) translocation but with ectopic MNX1 expression. Epigenetic inhibitor screening will be repeated in this isogenic cell line. The epigenetic inhibitor, which will not lead to reduction in cell viability of the isogenic line but result in a significant reduction in cell viability of wild type GDM1, will be identified as the inhibitor that disrupts the enhancer-promoter interactions driving oncogenic MNX1 overexpression. This inhibitor may offer clinical benefit in other MNX1-dependent AMLs with enhancer hijacking events.Keywords: Acute myeloid leukemia, decitabine, Epigenetic, Screening
Epigenetic patterns in a cell control the expression of genes and consequently determine the phenotype of a cell. Cancer cells possess altered epigenomes which include aberrant patterns of DNA methylation, histone tail modifications, nucleosome positioning and of the three-dimensional chromatin organization within a nucleus. These altered epigenetic patterns are potential useful biomarkers to detect cancer cells and to classify tumor types. In addition, the cancer epigenome dictates the response of a cancer cell to therapeutic intervention and, therefore its knowledge, will allow to predict response to different therapeutic approaches. Here we review the current state-of-the-art technologies that have been developed to decipher epigenetic patterns on the genomic level and discuss how these methods are potentially useful for precision oncology.
Non-coding RNAs are much more common than previously thought. However, for the vast majority of non-coding RNAs, the cellular function remains enigmatic. The two long non-coding RNA (lncRNA) genes DLEU1 and DLEU2 map to a critical region at chromosomal band 13q14.3 that is recurrently deleted in solid tumors and hematopoietic malignancies like chronic lymphocytic leukemia (CLL). While no point mutations have been found in the protein coding candidate genes at 13q14.3, they are deregulated in malignant cells, suggesting an epigenetic tumor suppressor mechanism. We therefore characterized the epigenetic makeup of 13q14.3 in CLL cells and found histone modifications by chromatin-immunoprecipitation (ChIP) that are associated with activated transcription and significant DNA-demethylation at the transcriptional start sites of DLEU1 and DLEU2 using 5 different semi-quantitative and quantitative methods (aPRIMES, BioCOBRA, MCIp, MassARRAY, and bisulfite sequencing). These epigenetic aberrations were correlated with transcriptional deregulation of the neighboring candidate tumor suppressor genes, suggesting a coregulation in cis of this gene cluster. We found that the 13q14.3 genes in addition to their previously known functions regulate NF-kB activity, which we could show after overexpression, siRNA–mediated knockdown, and dominant-negative mutant genes by using Western blots with previously undescribed antibodies, by a customized ELISA as well as by reporter assays. In addition, we performed an unbiased screen of 810 human miRNAs and identified the miR-15/16 family of genes at 13q14.3 as the strongest inducers of NF-kB activity. In summary, the tumor suppressor mechanism at 13q14.3 is a cluster of genes controlled by two lncRNA genes that are regulated by DNA-methylation and histone modifications and whose members all regulate NF-kB. Therefore, the tumor suppressor mechanism in 13q14.3 underlines the role both of epigenetic aberrations and of lncRNA genes in human tumorigenesis and is an example of colocalization of a functionally related gene cluster.
