Epigenetic Status Of CEBPA Promoter and Expression Of CEBPα Correlates With Molecular Genetic Subtype and CD2 Aberrant Expression In B Cell Precursor ALL
Authors: Slamova Lucie , Starkova Julia , Fiser Karel , Fronkova Eva , Reznickova Leona Rezkova , Zaliova Marketa Kubricanova , Polgarova Kamila , Stary Jan , Trka Jan , Hrusak Ondrej , Mejstrikova Ester 
Published: October 2019

Abstract
CCAAT/enhancer binding protein alpha (CEBPα) is one of the crucial transcription factors involved in hematopoietic differentiation and leukemogenesis. CEBPα promotes myeloid differentiation by up-regulation of lineage specific genes and by cell proliferation arrest. Epigenetic regulation of CEBPα expression through DNA methylation has been demonstrated in acute myeloid leukemia (AML) (Figueroa et al, Cancer Cell, 2010). However, only limited data are available regarding CEBPA promoter methylation and its expression in B cell precursor acute lymphoblastic leukemia (BCP-ALL).
Methylation status of CEBPA promoter (-295 to -593bp upstream of the transcription start site (TSS), 24 CpG dinucleotides) was analyzed by bisulfite sequencing. Five subgroups of BCP-ALLs were analyzed: MLL gene rearranged (n=5), hyperdiploid (n=6), mBCR-ABLpos(n=5), ETV6-RUNX1pos(n=6) and other BCP-ALLs (no hyperdiploidy, MLL gene rearrangement, BCR-ABL or ETV6-RUNX1 fusion gene (“BCP-others”, n=29)). CEBPA promoter was hypermethylated in MLL-rearranged, hyperdiploid and ETV6-RUNX1pos BCP-ALL (5/5, 6/6 and 4/6 respectively). Surprisingly CEBPA promoter was hypomethylated in all mBCR-ABLpos cases (5/5). In subgroup of other BCP-ALLs both hypermethylation (10/29) and hypomethylation of CEBPA promoter (19/29) were detected (Figure 1A).
In previous study we found association of CD2 (LFA-2) aberrant expression and switch to the monocytic lineage during the early phase of treatment in BCP-ALLs (Slamova et al, ASH 2012). We were interested if a possible link between hypomethylation of CEBPA promoter correlates with aberrant expression of CD2. There was a significant association between aberrant expression of CD2 antigen and hypomethylation in CEBPA promoter in BCP-others (Fisher exact test, p<0.0001). Interestingly, in the only hypomethylated ETV6-RUNX1pos case we found aberrant CD2 expression on blasts, which is exceptional in ETV6-RUNX1pos ALL.
We next asked whether methylation of CEBPA promoter correlates with CEBPα expression. It is generally accepted that promoter hypomethylation is often associated with increased expression of the relevant gene. Our data prove that in general, this holds true also for BCP-ALL. However, in two genetically defined subsets we observed either high expression despite hypermethylation (MLL-rearranged ALL) or low expression despite hypomethylation (mBCR-ABLpos ALL) (Figure 1B). In BCP-others hypomethylation of CEBPA promoter was significantly associated with upregulation of myeloid antigens (CD14 and/or CD33) and downregulation of B cell marker CD19 on blasts during the first weeks of the treatment (Fisher test, p=0.0009).

In summary
Methylation status of CEBPA promoter correlates with genetic subtypes of BCP-ALL. The notion that hypomethylation leads to overexpression was confirmed in majority of BCP-ALLs, while in mBCR-ABLpos and MLL gene rearranged BCP-ALL it did not follow this pattern. Hypomethylation of CEBPA promoter in BCP- others correlates with CD2 expression on blasts and increased CEBPα gene expression. During the early phase of the treatment in other BCP-ALLs with hypomethylated CEBPA promoter increase of myeloid and decrease of B lymphoid markers on blasts was observed.
Supported by: GACR P301/10/1877, GACR P304/12/2214, GAUK 914613, UNCE 204012, NT13462, NT12397- 4, project for conceptual development of research organization (Ministry of Health, CZ) 00064203, the FACS Aria instrument was supported by EU-Prague project CZ.2.16/3.1.00/24022

Disclosures:
No relevant conflicts of interest to declare.