NFATc1 Is Transcriptionally Activated in Chronic Lymphocytic Leukemia (CLL) By Promotor DNA-Hypomethylation Which Correlates with in-Vitro Vulnerability to Calcineurin Inhibitors

Chronic lymphocytic leukemia (CLL), the most frequent adult leukemia in Western countries, is characterized by progressive accumulation of mature, monoclonal B lymphocytes in blood, bone marrow, and lymphoid tissues. In the pathogenesis and treatment of CLL, B cell receptor (BCR) signaling plays a crucial role, and aberrations in downstream pathways that become activated in CLL need to be better defined. One downstream target of BCR signaling is NFATc1, a transcription factor with a high oncogenic and transforming potential. Employing a genome-wide comparative DNA methylation analysis the NFATc1 5’ region was identified to be DNA hypomethylated in CLL patient samples. The pilot series comprised CD19+ B-cells from 13 CLL patients without previous treatment (9 with mutated IGHV using a 98% similarity cut-off) and CD19+ B-cells from 6 age-matched healthy donors. DNA methylation was relatively quantified using methyl-CpG immunoprecipitation (MCIp) on custom oligonucleotide promoter arrays covering -8kbp to +2.5kbp of all human transcription start sites. In total 1944 genes showed significant DNA hypomethylation in CLL cells compared to non-malignant B-cells (p<0.05), with NFATc1 ranking at position 36. DNA hypomethylation of the NFATc1 promoter was validated in peripheral blood CD19+ B-cells from 76 CLL patients without previous treatment compared to 14 additional age-matched healthy donors using mass spectrometry based quantitative high resolution measurement of DNA methylation (MASSARRAY). These findings suggest activation of the NFATc1 gene in CLL cells. In fact, NFATc1 mRNA was significantly upregulated in CLL samples compared to non-malignant B-cells, and expression correlated highly significantly with DNA methylation (r=-0.66, p<0.001). Inhibition of NFATc1 in primary CLL cells from patients (n=5) using calcineurin inhibitors like CsA and FK-506 induced significant apoptosis in primary CLL cells in-vitro even when protected by co-culture with bone-marrow stromal cells, suggesting a dependence of CLL cells on the activity of NFATc1. For a mechanistic insight into NFATc1 signal transduction in primary CLL cells and to allow modeling of the signaling network, a panel of phospho-specific antibodies is currently used in flow cytometry. This allows to monitor multiple components of signaling pathways in parallel with high time resolution to understand the impact of NFATc1 upregulation on the cellular signaling networks. The approach will shed further light on how CLL cell survival is regulated how this pathway can be targeted in CLL.