CD49d Is Overexpressed in Trisomy 12 Chronic Lymphocytic Leukemia by an Epigenetic-Dependent Transcriptional Control

Abstract 929

CD49d is an adhesion molecule with a variable expression in chronic lymphocytic leukemia (CLL). Only one third of CLL display CD49d expression in a significant fraction of cells, the remaining cases either expressing very low levels or completely lacking its expression. The negative prognostic impact of CD49d in CLL and its key role in microenvironmental interactions have been broadly described by several studies, whereas information on the mechanisms regulating CD49d expression in CLL is still lacking.

Aiming to understand the genetic mechanisms underlying the different expression pattern of CD49d in CLL, CD49d expression was investigated by flow cytometry in the neoplastic component of 972 CLL patients, and correlated with cytogenetic profiles assessed by fluorescence in situ hybridization (FISH). Using the 30% positive cut-off value, 603 cases (62%) were CD49d-, whereas 369 cases (38%) were CD49d+. Correlation of CD49d expression with the major cytogenetic alterations (del13q14.3, trisomy 12, del11q22-q23-, del17p13.1), highlighted a very strong association between CD49d expression and the presence of trisomy 12 (p<0.0001). In particular, high CD49d expression was found in 93/113 cases (82.3%) with trisomy 12 alone, and 140/170 cases (82.3%) bearing trisomy 12 either alone or in the presence of other cytogenetic lesions. Notably, 50% (15/30) of CD49d- cases bearing trisomy 12 displayed a CD49d bimodal expression, with a CD49d positive population ranging from 10 to 28%. Even in the context of CD49d+ CLL, trisomy 12 CLL showed significantly higher CD49d mean fluorescence intensity (MFI) levels (MFI=2323±149, n=56) as compared to CLL cases with normal FISH (MFI=1530±118, n=56, p=0.0001). The association between trisomy 12 and CD49d expression was corroborated by results from FISH analysis in the flow cytometrically sorted (>99% purity) CD49d- and CD49d+ components from 3 CLL cases characterized by both CD49d bimodal expression (27%, 57%, and 75% of CD49d positive cells), and the presence of trisomy 12 in a fraction of the cells (25%, 42% and 63% respectively). In all cases, the CD49d- components almost completely lacked trisomy 12 (5%, 4%, and 0%), whereas the CD49d+ fractions displayed 70%, 62%, and 75% of trisomy 12, respectively (p<0.0001).

A gene expression profiling was performed comparing CD49d+/trisomy12 (n=5) versus CD49d-/normal FISH CLL (n=5). According to bioinformatics tools for global analysis of gene function, the “Chromatin modification” (p<0.0001), “Methyltransferase activity” (p=0.017), and “Histone deacetylase (HDAC) activity” (p<0.0001) categories were found among the top-ranked Gene Ontology categories selected as containing differentially expressed genes. These categories included the genes of lysine demethylases KDM5A (fold change=1.8, p=0.01) and KDM2B (fold change=1.6, p=0.007), and lysine methyltransferase SETD8 (fold change=1.4, p<0.001), all up-regulated in the trisomy 12 group and mapping on chromosome 12, and the methyltransferase DNMT3A, down-regulated in trisomy 12 CLL (fold change=-2.4, p=0.01). Collectively, these results point to an epigenetic regulation of CD49d expression in CLL. To address this point, DNA methylation was studied within the CpG island (68 CpGs) of the CD49d gene 5'UTR region (725 pb before the translation start codon ATG) in CD49d+/trisomy12 (n=10) and CD49d-/normal FISH (n=9) purified CLL cells, by means of bisulfite genomic sequencing (at least 10 clones per sample). The CD49d-/normal FISH group showed a significantly higher degree of methylation of the CD49d 5' UTR gene in comparison to the CD49d+/trisomy12 group (average amount of methylated CpGs=9.8% vs. 0.6%, p<0.0001). Focusing on a smaller region (214 bp before ATG) consisting in 24 CpGs, even higher methylation levels (average amount of methylated CpGs= 13.8%) were found in the CD49d-/normal FISH group.

Altogether, our results demonstrate that the overall down-regulated CD49d expression characterizing CLL is subjected to epigenetic control. Re-expression of CD49d may occur through a fine regulation of methyltransferase and chromatin modification processes, particularly active in trisomy 12 CLL, where CD49d expression may have a role in determining the clinical and biological features of this particular CLL subset.