Regulation of CD38 by IRF4 in chronic lymphocytic leukemia

Abstract

Genome‐wide association analysis identified rs872071, a common variant in the 3’UTR of IRF4, as tagging a risk allele for chronic lymphocytic leukaemia (CLL). The risk allele is significantly associated with expression of CD38, a poor prognostic marker in CLL. IRF4 is a transcription factor with pleiotropic roles in the regulation of B cell development, and interrogation of the CD38 gene identified a number of putative binding sites for IRF4, suggesting that CD38 may be transcriptionally regulated by IRF4. Chromatin immunoprecipitation (ChIP) demonstrated significant IRF4‐CD38 binding at two composite ETS/IRF consensus element (EICE) sites in SU‐DHL‐6 and MEC‐1 mature B cell lines. Furthermore, there was evidence of IRF4‐CD38 binding at these EICE sites in primary CLL lymphocytes in some, but not all, cases. ChIP studies using markers of histone methylation suggested increased transcriptional activation at the IRF4‐CD38 binding sites in MEC‐1 cells with IRF4 knockdown. In contrast, transcriptional activity at these sites appeared to be reduced in SU‐DHL‐6 cells with IRF4 knockdown. Co‐culture of primary CLL lymphocytes on a CD40L‐expressing monolayer led to an upregulation of IRF4 expression, but no consistent effect on CD38 expression. In addition, ChIP studies using markers of histone methylation were suggestive of reduced transcriptional activity at the IRF4‐CD38 binding site after CD40L co‐culture, suggesting that IRF4 may be a negative regulator of CD38 in CLL. Taken together, the evidence indicates that IRF4 binds to the CD38 locus. However, direct experimental evidence for an effect on CD38 expression is lacking. It is necessary to consider that the interaction between IRF4 and CD38 is unlikely to be a linear signal transduction pathway. Indeed the prevailing evidence regarding the function of IRF4 in B cells suggests a complex signalling network involving numerous other transcription factors and cytokines. Furthermore, IRF4 has been suggested as a putative therapeutic target in haematological malignancies including myeloma. Transient IRF4 knockdown using RNA ii interference (RNAi) techniques was tolerated by SU‐DHL‐6, MEC‐1 and lymphoblastoid TK6 cell lines, though cell proliferation in TK6 and SU‐DHL‐6 was significantly impaired. Long‐term stable knockdown was also tolerated in TK6 cells, but not in MEC‐1 cells, suggesting an essential role for IRF4 in maintenance of MEC‐1 cells. Targeted knockdown of IRF4 also sensitised TK6 B cells and MEC‐1 CLL cells to the growth inhibitory effects of fludarabine, a nucleoside analogue used in the treatment of CLL. These findings indicate that IRF4 is necessary to the maintenance of MEC‐1 cells, which represent a CLL cell line model. A greater understanding of the role of IRF4 in the development and maintenance of CLL may indicate new therapeutic targets in CLL. Indeed, a recently developed novel mouse model of CLL has indicated that deficient IRF4 expression predisposes to the development of CLL. However, the role of IRF4 in the maintenance of CLL cells is yet to be determined. The heterogeneous nature of IRF4 expression in primary CLL lymphocytes determined here, and its potentially pleiotropic role in the transcriptional regulation of CD38 in B cells at different stages of differentiation, suggest that if IRF4 is a key player in the maintenance of the leukaemic clone, its role may be context or patient specific, and dependent on other signalling components or somatic genetic abnormalities.