E2A Transcriptionally Regulates CD38 Expression In Chronic Lymphocytic Leukemia

Abstract 3599

CD38 is a cell surface molecule endowed with enzymatic and receptor functions. As an enzyme, CD38 is part of a large family of nucleotide-metabolizing ecto-enzymes (NMEs) involved in the catabolism of extra-cellular nucleotides. Its activity results in the dismantling of NAD and the generation of Ca²⁺ mobilizing compounds. CD38 also bind CD31, a non substrate ligand, expressed by endothelial and stromal cells. In the neoplastic context, CD38/CD31 interactions lead to increased proliferation, survival and chemotaxis. CD38 is a negative prognostic marker for chronic lymphocytic leukemia (CLL) patients and its expression is higher in BM than in peripheral blood cells. Furthermore, the exposure of CLL cells to specific combination of activatory signals (e.g., IL-2, IL-4, CD40L) results in an up-regulation of the molecule, that relies on de novo gene transcription. The human CD38 gene has a genetic single-nucleotide polymorphism (SNP), with a C→G variation in a putative E-box located in the regulatory region. E proteins have the ability to bind to E-box elements and to activate gene transcription. The frequency of the CD38 rare allele (G) has been recently reported to be higher in a subset of CLL patients characterized by clinical and molecular markers of poor prognosis. The aim of this work is to test whether CD38 is a target of E2A at least in CLL cells and if the SNP may affect CD38 gene transcription, influencing the binding affinity of the transcription factor.

E2A expression was analyzed in a large cohort of CLL patients (n=72) and in normal B cells. Results indicate that the transcription factor was expressed by the majority of CLL samples, but at higher level in CD38⁺ ones. Moreover, it was absent in circulating B cells and splenocytes. A positive correlation between the presence of E2A in the nucleus and the surface expression of CD38 in G carrier patients was found. These results suggested that E2A is i) directly associated with CD38 expression and that ii) the binding of the transcription factor is influenced by CD38 genotype. Chromatin immunoprecipitation experiments indicated that E2A directly interacts with the CD38 regulatory region. Furthermore, the binding was stronger in the presence of the G allele. Silencing of E2A resulted in a significant reduction of CD38 surface expression, formally linking these two molecules and confirming the working hypothesis. A direct functional interplay between E2A and CD38 was obtained by mimicking in vitro conditions known to induce CD38 expression through de novo gene transcription. Exposure of CLL cells to TLR-9 ligands and IL-2, both inducers of CD38 expression, resulted in the up-regulation of the molecule. The effect was primarily conditioned by the presence of E2A and then by the G allele.

The results obtained in the present work indicate that E2A and CD38 expression are functionally linked in a common pathway and the activity of E-protein is a necessary element for an efficient induction of CD38 transcription.