Validation of epigenetic mechanisms regulating gene expression in canine B-cell lymphoma: An in vitro and in vivo approach

Abstract

Despite canine B-cell Lymphoma (BCL) representing the most common haematological tumour, epigenetic events driving development and progression are scarcely known. Recently, canine Diffuse Large BCL (DLBCL) DNA methylome by genome-wide CpG microarray has identified genes and pathways associated to pathogenesis. To validate data previously obtained by array analysis, the CLBL-1 cell line was used and the HOXD10, FGFR2, ITIH5 and RASAL3 genes were selected. CLBL-1 cells were treated with two hypomethylating drugs (HDs; IC50, 50% inhibitory concentration), i.e. azacytidine and decitabine (DEC), either alone or in combination with three histone deacetylase inhibitors (HDACis; IC20), i.e. valproic acid, trichostatin and vorinostat. Following the incubation with both HDs, an overall decrease of promoter methylation was highlighted, thus confirming target genes hypermethylation. The highest mRNA restoration was observed following the exposure to HDs combined with HDACis, and mostly with valproic acid. Contrasting results were only obtained for RASAL3. An in vivo confirmation was finally attempted treating Nod-Scid mice engrafted with CLBL-1 cells with DEC. Although DEC did not arrest tumour growth, target genes promoter methylation was significantly reduced in DEC-treated mice vs controls. Overall, this work demonstrates that CLBL-1 cell line represents a reliable in vitro model to validate the methylation-dependent silencing of key genes for BCL; moreover, it may be useful for xenograft models in mice, despite its aggressive behaviour. In future, functional studies will be performed to deepen the role of selected genes on BCL pathogenesis and progression, and their methylation-dependent mechanism of regulation.

Genome-wide DNA methylation analysis using MethylCap-seq in canine high-grade B-cell lymphoma

“…This finding is also consistent with another MethylCap‐seq study focusing on human pancreatic cancer 18 . Several hypermethylated genes have been reported in previous studies, including HOXD10 , FGFR2 , ITIH5 , RASAL3 , DAPK , and TFPI‐2 11–13,78 . In our study, HOXD10 hypermethylation, identified by array analysis in the previous study, is also discovered in the MEDIUM‐HIGH fraction, and was observed in four cHGBL cases, suggesting the reliability of these two methods for hypermethylation detection.…”

Hypermethylation-Mediated Silencing of CIDEA, MAL and PCDH17 Tumour Suppressor Genes in Canine DLBCL: From Multi-Omics Analyses to Mechanistic Studies

“…Overall, the combined bioinformatics and literature-based screenings allowed the selection of 21 putative TSGs, considered in the subsequent experimental filtering ( Figure 1 ). Among them, HOXD10 was previously identified and validated by the same research group [ 13 , 24 ]; further results related to HOXD10 will be published elsewhere.…”

“…The use of a multi-omics approach (DNA methylation, RNA-seq and αCGH) coupled with bioinformatics and literature-based screening allowed the detection of 21 putative TSGs with low expression profiles, hypermethylation status in the promoter region and no chromosome loss. One of these genes was HOXD10 , a gene previously identified and validated in cDLBCL [ 13 , 24 ].…”

“…The epigenetic modification that impedes TF recruitment is crucial for tumourigenesis since it has been reported that the inactivation of certain tumour suppressor genes (TSGs) occurs as a consequence of hypermethylation within the promoter regions [ 20 ]. Previous studies have demonstrated the aberrant methylation and silencing of TSGs in canine B- and T-cell lymphoma cell lines [ 21 , 22 , 23 , 24 ] as well as in cDLBCL samples [ 25 ].…”

Epigenetic Mechanisms in Canine Cancer

“…Both human and canine melanoma cells have hypermethylated DNA in the CpG islands of the microRNA-203 (61), a suppressor of growth in melanoma cells, as shown by bisulfite sequencing and MSP (62). Hypermethylation and epigenetic silencing has also be observed for several other important tumor suppressor genes such as tissue factor pathway inhibitor 2 (TFPI-2) (63), death-associated protein kinase (DAPK) (64), cyclin-dependent kinase inhibitor 2A (CDKN2A/p16), HOXD10, FGFR2, ITIH5, and RASAL3 in B-cell lymphoma (65)(66)(67). In addition, DAPK hypermethylation is associated with overall survival and considered a negative prognostic factor in canine high-grade B-cell lymphoma (68).…”