PB1872 IS THERE A ROLE OF VITAMIN D IN THE PATHOPHYSIOLOGY OF CHRONIC LYMPHOCYTIC LEUKEMIA?
Authors: Gerousi M. , Kotta K. , Stavroyianni N. , Anagnostopoulos A. , Kotsianidis I. , Ntoufa S. , Stamatopoulos K. 
Published: June 2019

Background:

Calcitriol, the biologically active form of vitamin D, binds to the vitamin D receptor (VDR) which is translocated into the nucleus and regulates the transcription of target genes. Epidemiological studies have reported that low blood levels of vitamin D are associated with adverse disease outcome in chronic lymphocytic leukemia (CLL). However, very limited biological explanation is available for this observation.

Aims:

In this study, we investigated the potential pathophysiological implication of the VDR system in CLL.

Methods:

CD19+ primary CLL cells were negatively selected. VDR transcripts were detected by RT‐qPCR, VDR+ and CYP24A1+ cells were determined by Flow Cytometry (FC). CLL cell apoptosis was determined using FC (AnnexinV−/Pi− cells). CLL cells were cultured in vitro with calcitriol, anti‐IgM, CpG, and/or CD40 ligand, or co‐cultured with the HS‐5 mesenchymal cell line.

Results:

VDR mRNA expression was assessed in 75 CLL cases, revealing significantly higher (FD = 3.2, p < 0.01) levels in patients with unmutated IGHV genes (U‐CLL, n = 43) compared to patients with mutated IGHV genes (M‐CLL, n = 32). Interestingly, however, CLL cases showed remarkably reduced VDR mRNA levels (FD = 3.1, p < 0.0001) compared to tonsillar CD19+ B cells from healthy donors (n = 24). In order to examine whether the microenvironment of CLL cells affects VDR expression, we cultured U‐CLL cells (n = 8) for 14 hours under the following conditions (a) BcR stimulation with anti‐IgM; (b) TLR9 stimulation with CpG and (c) CD40 stimulation with CD40L. These treatments resulted in a statistically significant reduction of VDR mRNA expression compared to the unstimulated control (FD = 1.7, p < 0.01; FD = 2.9, p < 0.01; FD = 2, p < 0.05; for BcR, TLR9, CD40 respectively). Following, in order to activate the calcitriol/VDR system, we cultured CLL cells with calcitriol as well as a combination of anti‐IgM/CD40L/calcitriol for 24 hours and measured the protein expression of CYP24A1, a target gene of the activated VDR. The addition of calcitriol was found to increase the percentage of CYP24A1+ CLL cells compared to unstimulated control cells in both U‐CLL (n = 9;FD = 5.1, p < 0.01) and M‐CLL (n = 7; FD = 2.4, p < 0.05). Co‐stimulation with anti‐IgM/CD40L/calcitriol also induced CYP24A1 expression in U‐CLL (FD = 4.4, p < 0.01) and M‐CLL (FD = 2.1, p < 0.05) but to a lesser extent. Moreover, co‐culturing of CLL cells with the HS‐5 cells induced CYP24A1+ cells, while calcitriol showed additive effects. In contrast, no differences in VDR+ cells were observed under the conditions described above.

Summary/Conclusion:

We show that VDR is expressed at low levels in CLL cells compared to normal B cells, which can be further reduced by microenvironmental signals, indicating that the tumor microenvironment competes with VDR expression. However, calcitriol supplementation activates the VDR system suggesting that VDR is functional in CLL cells and can potentially induce the expression of target genes with antitumor activity.