Lck Is a Crucial Anergy Regulator in CLL

NFAT is a group of highly phosphorylated transcription factors which can be found in the cytoplasm of resting cells. Following activation via dephosphorylation, they translocate to the nucleus where they regulate developmental and activation programs in various cell types. The tyrosine kinase Lck plays an essential role in T cell activation. Lck expression in CLL cells has previously been described, but its function remains largely elusive. With respect to prognosis CLL constitutes a very heterogeneous disease. While some patients exhibit an indolent course without requiring specific treatment for lengthy periods, others progress rapidly and demonstrate limited survival. Patients featuring aggressive disease show an enhanced responsiveness to stimulation of the B cell receptor (BCR) complex, whereas those with a more indolent course are characterized by an anergic phenotype with B cell unresponsiveness to IgM ligation, PRDM1 (BLIMP-1) depression and an essential lack of phosphotyrosine induction as well as calcium flux. In up to 10% of CLL patients a transformation to aggressive lymphoma (Richter´s syndrome) can be observed which is associated with a significantly worse treatment outcome. We have preciously shown in a mouse model and in primary human CLL samples that NFAT2 is a crucial anergy regulator in this disease and defined a genetic anergy signature consisting of Lck and the E3 ligases Cbl-b and Grail (Märklin et al., Nature Commun., in press). Here, we show that Lck is a direct NFAT2 target and substantially contributes to the induction of anergy in CLL cells.

Sequence analysis of the Lck gene locus revealed a potential NFAT binding site with the sequence TTTCATCAG in the Lck promotor. To biochemically define Lck as an unequivocal NFAT2 target, we performed chromatin immunoprecipitation (ChIP) assays in primary human CLL cells using a monoclonal antibody against NFAT2 and subsequent qRT-PCR. Our results demonstrate that NFAT2 binds to the CD40L gene locus which is a well-defined NFAT2 target gene in B cells and thus served as a positive control. We could further demonstrate that NFAT2 also binds to the Lck promotor and thus constitutes a direct NFAT2 target in human CLL cells. To assess the role of Lck in the induction of anergy in CLL, we made use of the Eµ-TCL1 transgenic mouse model. These mice exhibit TCL1 expression limited to the B cell lineage and develop a human-like CLL within 60 days. By mouse breeding with conventional Lck knockout mice (Lck^-/-), we generated animal cohorts with B cell specific TCL1 expression and concomitant deletion of Lck (n=10). Eµ-TCL1 transgenic mice with intact Lck expression served as controls (n=10). After development of manifest CLL, animals were sacrificed and CD5+CD19+ CLL cells were isolated from bone marrow and spleens for further analysis. While CLL cells from Eµ-TCL1 transgenic exhibited an anergic phenotype characterized by compromised calcium flux as well as low IgM and PRDM1 (BLIMP-1) expression, Lck-deficient CLL cells showed evidence of enhanced calcium mobilization upon BCR stimulation and significantly increased expression of sIgM and PRDM1 (BLIMP-1) as assessed by flow cytometry or qRT-PCR. Utilizing immunofluorescence-based colocalization assays we were further able to show that Lck is recruited to the BCR in CLL cells from TCL1 mice upon BCR stimulation while in TCL1 mice with NFAT2 deletion no Lck recruitment to the BCR could be detected.

Taken together, our data demonstrate that Lck is a direct NFAT2 target in anergic CLL cells and that its deletion contributes to the loss of the anergic phenotype which is generally associated with an indolent clinical course. Therefore our data identify Lck as a crucial regulator of the anergic phenotype in CLL.