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N. Martínez-Calle et al.
wide approach of DNA methylation analysis using the 450k array allowed us to interrogate regulatory regions outside traditional promoters and obtain a deeper insight into the aberrant DNA methylome of MF. Changes in DNA methylation levels are known to cooperate with the deposition of chromatin marks, particularly H3K4 methy- lation, to render the enhancers/promoters accessible/inac- cessible to the transcription machinery.35-37 Hence, the changes in DNA methylation observed in MF are expected to have an impact on the transcriptional profile of MF and potentially contribute to the MF malignant phenotype. Enhancer DNA methylation changes have been described to play a more prominent role in transcriptional regulation than promoter DNA methylation, governing processes such as hematopoietic differentiation and neoplastic trans- formation through the regulation of key transcription fac- tors and genes.12,13,27,37,38 Translated into the context of Philadelphia chromosome-negative myeloproliferative neoplasms, this evidence might support the involvement of aberrant enhancer DNA methylation in the abnormal pattern of differentiation leading to MF. Enhancer hyper- methylation has been reported in neutrophils,12 B cells,39 AML cells26 and myeloma13 adding evidence to dynamic enhancer DNA hypermethylation as a relevant regulatory mechanism of gene expression both in normal and neo- plastic hematopoietic cells.
Although the potential involvement of ZFP36L1 in myeloid differentiation has been described previously,40 our results suggest that epigenetic downregulation of ZFP36L1 might be a prominent event in the pathobiology of MF; more importantly, hypermethylation of an enhancer regulatory element represents a novel mecha- nism of disrupted gene expression in the context of MF and ZFP36L1. ZFP36L1 has been previously implicated in normal hematopoiesis41 and specifically associated with erythroid and myeloid differentiation,40 suggesting a pos- sible role of this gene in MF onset and progression. Moreover, ZFP36L1 is also known to mediate mRNA decay of genes relevant to cell proliferation, survival and differentiation such as CDK6, TNFα, BCL2, NOTCH1 and STAT5B.42,43 Interestingly, the enhancer region associated with this candidate gene was consistently hypermethylat- ed in the cohort of MF patients and was located in its intragenic region, presumably acting as a cis-regulatory element of ZFP36L1 transcription. The motif discovery experiments support our hypothesis of epigenetic deregu- lation of ZFP36L1, suggesting that MF samples with ZFP36L1 loss of expression experience upregulation of the gene’s putative targets. Consequently, when ZFP36L1 expression levels are restored with the lentiviral model,
SET-2 cells lose their malignant proliferative phenotype, strengthening the tumor suppressor role of this gene in MF. Taken together, these results link ZFP36L1 to the pathobiology of MF, ultimately resulting in transcriptome deregulation of genes relevant to cell proliferation, sur- vival and differentiation, as previously described.40,42,44,45
Conclusion
The DNA methylation landscape of patients with pri- mary MF or post-ET/post-PV MF is consistently different from that of healthy individuals. The absence of differ- ences between primary MF and post-ET/post-PV MF sug- gests that the changes seen in MF are founding epigenetic alterations occurring at the level of stem cells of this myeloproliferative neoplasm and maintained in differenti- ated myeloid cells. Aberrant DNA methylation in MF is predominantly located in enhancer regions and has a sig- nificant impact on the expression of their target genes. Combining DNA methylation and gene expression data, we identified ZFP36L1 as an attractive new possible ther- apeutic target that shows a decrease of gene expression mediated by enhancer hypermethylation. Our results also suggest a direct effect of ZFP36L1 downregulation on the gene expression profile of MF, through upregulation of mRNA harboring canonical sites with AU-rich elements. In vitro rescue of ZFP36L1 expression had an impact on cell proliferation and induced apoptosis in the SET-2 cell line, indicating a possible role of ZFP36L1 as a tumor suppres- sor gene in MF. Moreover, treatment with 5’-azacytidine further evidenced the plausibility of ZFP36L1 pharmaco- logical manipulation. Taken together, these results provide evidence of an unexplored therapeutic target in MF patients, which remains to be properly evaluated in the pre-clinical setting.
Acknowledgments
We particularly acknowledge the patients for their participa- tion and the Biobank of the University of Navarra for its collab- oration. We thank John J Murphy and Amor Alcaraz for provid- ing ZFP36L1 expression constructs. This research was funded by grants from Instituto de Salud Carlos III (ISCIII) PI14/01867, PI16/02024 and PI17/00701, TRASCAN (EPICA), CIBERONC (CB16/12/00489; co-financed with FEDER funds), RTICC (RD12/0036/0068) and the Departamento de Salud del Gobierno de Navarra 40/2016. NM is supported by a FEHH-Celgene research grant, MP was supported by a Sara Borrell fellowship CD12/00540 and RO was supported by the Ministerio de Ciencia, Innovación y Universidades of Spain, Subprograma de Formación de Profesorado Universitario (FPU) award number FPU14/04331.
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