NFAT2 Regulates Anergy Induction in CLL through Lck
Authors: Märklin Melanie , Heitmann Jonas S. , Poljak Alexandra , Bugl Stefanie , Kopp Hans-Georg , Haap Michael , Kanz Lothar , Rao Anjana , Wirths Stefan , Müller Martin R. 
Published: October 2019

Abstract
NFAT is a family of highly phosphorylated proteins residing in the cytoplasm of resting cells. Upon dephosphorylation by calcineurin, NFAT proteins translocate to the nucleus where they orchestrate developmental and activation programs in diverse cell types. CLL is a clonal disorder of mature B cells characterized by the expression of CD19, CD23 and CD5. With respect to prognosis, it constitutes a heterogeneous disease with some patients exhibiting an indolent course for many years and others progressing rapidly and requiring early treatment. A defined subgroup of patients shows enhanced responsiveness to stimulation of the B cell receptor (BCR) complex and more aggressive disease. In contrast, another subset of CLL patients with more indolent course is characterized by an anergic B cell phenotype referring to B cell unresponsiveness to IgM ligation and essential lack of phosphotyrosine induction and calcium flux. Here, we analyzed the role of NFAT2 in the pathogenesis of B-CLL and in anergy induction in CLL cells.
For this purpose, we generated conditional CD19-Cre NFAT2 knock out mice, which exhibit NFAT2 deletion limited to the B cell lineage. To investigate the role of NFAT2 in the pathogenesis of CLL, we used the Eµ-TCL1 transgenic mouse model. TCL1 transgenic mice develop a human-like CLL at the age of approximately 14 weeks to which the animals eventually succumb at an average age of 10 months. We generated TCL1+NFAT2 ko mice with TCL1 transgenic mice without an NFAT2 deletion serving as controls. To identify novel NFAT2 target genes in CLL cells, we also performed a comparative gene expression analysis on CLL cells with intact NFAT2 expression and on CLL cells with NFAT2 deletion using affymetrix microarrays. In order to asses the anergic phenotype in CLL cells and the role of NFAT2 in its induction, we performed Ca2+ mobilization assays using a flow cytometric approach and performed Western Blots for multiple downstream signaling molecules.
Mice with NFAT2 ko exhibited a significantly more aggressive disease course with accelerated accumulation of CD5+CD19+ CLL cells in different organs, significantly higher proliferation rates and a dramatically reduced life expectancy (200 vs. 325 days) as compared to TCL1 control animals. To identify NFAT2 target genes responsible for the observed alterations in the disease phenotype, we subsequently performed a gene expression analysis with CLL cells from both leukemic cohorts. Here, we detected a substantially altered expression profile of genes associated with B cell anergy in the TCL1+NFAT2 ko mice. The vast majority of these genes was expressed significantly less in the absence of NFAT2 with Lck, Pacsin1 and the E3 ligase Cbl representing the biggest hits.
To further delineate the anergic phenotype and the role of NFAT2 in its induction, we subsequently performed Ca2+ mobilization assays. While anergic CLL cells from TCL1 mice exhibited an unresponsive phenotype with respect to Ca2+ flux upon IgM ligation, TCL1+NFAT2 ko mice showed an entirely normal capacity to mobilize intracellular Ca2+. Furthermore, IgM stimulation did not activate normal phosphotyrosine induction (phosphorylation of AKT and ERK kinases) in TCL1 mice while NAFT2-deficient CLL cells exhibited an unremarkable activation pattern with respect to AKT and ERK as assessed by Western Blotting. NFAT2-deficient CLL cells on the contrary exhibited compromised activation of the anergy regulator Lck as assessed by Y394 phosphorylation. Bypassing the BCR by antigen-independent stimulation with CD40 and LPS demonstrated slightly increased proliferation in anergic TCL1 CLL cells while NFAT2-deficient CLL cells exhibited massive proliferation.
In summary, our data provide strong evidence that genetic loss of NFAT2 leads to more aggressive disease in CLL which is associated with the loss of the anergic phenotype. We could show that NFAT2 controls the expression of several important anergy-associated genes and identified Lck as a critical target of NFAT2 in this context. Taken together, our data demonstrate that the NFAT2-Lck axis plays an essential role in the pathogenesis of CLL and implicate it as a potential target in its treatment.

Disclosures
No relevant conflicts of interest to declare.