A Novel Functional Polymorphism in the CCAAT/Enhancer Binding Protein (C/EBP), Epsilon (CEBPE) Gene Promoter Influences Acute Lymphoblastic Leukemia Risk Via Interaction with IKZF1

Purpose: We sought to identify and characterize the functional polymorphism in the CCAAT/enhancer binding protein (C/EBP), epsilon (CEBPE) gene, which is known to harbor a genetic risk modifier for childhood acute lymphoblastic leukemia (ALL).

Background: Association between ALL risk and polymorphic variation at the CEBPE gene, a zinc-finger hematopoietic and apoptotic regulator expressed exclusively in myeloid cells, was established in a genome-wide association study and replicated in other populations. The actual functional genetic variation that actually contributes the leukemia risk is not known. The discovery and functional characterization of the functional variant may lead to risk stratification, prevention, and/or therapeutic modalities.

Methods: Utilizing California Childhood Leukemia Study genome-wide SNP data from Hispanic subjects, we refine the genetic signal to a specific single nucleotide polymorphism (SNP) within the CEBPE gene promoter (rs2239635). This SNP was identified first using genetic imputation using 1000 Genomes data as a framework, and further validated in additional subjects using Taqman genotyping. Functional work to characterize the impact of the polymorphism was performed using prediction methods (database searches), chromatin immunoprecipitation assays from normal subjects heterozygous for rs2239635, and luciferase expression vectors carrying the SNP.

Results: The minor allele of rs2239635 conferred a 1.87-fold increased risk of B-cell ALL (95% CI = 1.51-2.30; P = 6.2x10^-9) and a 2.94-fold increased risk of high hyperdiploid B-cell ALL (95% CI = 2.12-4.06; P = 8.2x10^-11), but only a 1.51-fold increased risk of non-hyperdiploid B-cell ALL (95% CI = 1.11-2.04; P = 8.8x10^-3). Adjusting for rs2239635 attenuated all SNP associations in the region, including the original GWAS allele, strongly suggest that the GWAS signal in CCLS Hispanics is attributable to rs2239635. We also demonstrate, using published functional data and our own chromatin immunoprecipitation assays, that this SNP disrupts a canonical transcription factor binding site for Ikaros (IKZF1), an essential hematopoietic transcription factor. Using expression vectors, we found the polymorphic locus to have transcriptional repressive properties that are diminished in the presence of the ALL risk allele. Additionally, a heritable risk allele within IKZF1 interacts with rs2239635 in a multiplicative fashion (P = 0.02) in case-control association analyses.

Discussion: In sum, evidence for a functional CEBPE polymorphism links two hematopoietic developmental transcriptional factors and highlights feedback control between them. The minor, risk-associated variant of CEBPE may alter the ability of IKZF1 to repress CEBPE expression in precursor B-cells, causing lineage confusion and enhanced pre-B cell leukemia risk. This risk primarily impacts risk of pediatric ALL that exhibits the high hyperdiploid phenotype.