Genetic Loss of NFAT2 Induces Profound Acceleration of CLL in the TCL1 Mouse Model

Abstract 862

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. NFAT is inactivated and relocated to the cytoplasm by a network of several kinases. Although identified originally as a major transcriptional regulator in T cells, it is now clear that NFAT transcription factors also possess important roles in other cells of the hematopoietic system including dendritic cells, mast cells, megakaryocytes and B cells. Several recent studies have demonstrated that Calcineurin/NFAT signaling is involved in the pathogenesis of a wide array of hematological malignancies including diffuse large B cell lymphoma, CLL as well as Burkitt and Burkitt-like lymphomas.

Here, we analyzed the role of NFAT2 in the pathogenesis of B-CLL. For this purpose, we generated mice with a conditional NFAT2 knock out allele (NFAT2^fl/fl). In order to achieve NFAT2 deletion limited to the B cell lineage, we bred NFAT2^fl/fl mice to CD19-Cre mice, in which the Cre recombinase is expressed under the control of the B cell-specific CD19 promoter. To investigate the role of NFAT2 in the pathogenesis of CLL we made use of the Eμ-TCL1 transgenic mouse model in which the TCL1 oncogene is expressed under the control of the Eμ enhancer. 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. To analyze the role of NFAT2 in CLL, we generated mice (n=10) whose B cells exhibited a specific deletion of this transcription factor in addition to their transgenic expression of the TCL1 oncogene (TCL1 CD19-Cre NFAT2^fl/fl). TCL1 transgenic mice without an NFAT2 deletion served as controls (n=10).

Mice with NFAT2 knock out exhibited a significantly accelerated accumulation of CD5⁺CD19⁺ CLL cells as compared to control animals. Flow cytometric analysis at distinct time points showed a tremendous infiltration by CD5⁺ B cells in the peritoneal cavity, spleen, lymph nodes, liver and bone marrow which was significantly stronger in the NFAT2 ko cohort. Most of the CD5⁺ B cells in TCL1+NFAT2 ko mice showed high expression of ZAP70 and CD38, whereas TCL1 transgenic mice only demonstrated very few CD5⁺ B cells with concomitant expression of ZAP70 and CD38. At approximately 26 weeks of age, NFAT2 ko mice showed an approximately 40 fold increased lymphocyte count in the peripheral blood than their litter mate controls (1500/μL vs. 60000/μL). Splenomegaly and lymphatic adenopathy was also significantly increased in the NFAT ko population. Furthermore, NFAT2 ko mice showed a dramatically reduced median survival (200 vs. 325 days) and maximum survival (265 vs. 398 days) in comparison to regular TCL1 transgenic mice. To investigate the effects of an NFAT2 ko on proliferation and apoptosis of CD5⁺CD19⁺ CLL cells, we performed in vivo BrdU incorporation assays with subsequent flow cytometric analysis. Interestingly, we could show that CLL cells isolated from spleens, bone marrow and peripheral blood from mice with an NFAT ko at an age of approximately 7 months exhibited significantly higher rates of proliferation than control animals.

In summary, our data provide strong evidence that NFAT2 is a critical regulator of CD38 and ZAP70 expression and substantially controls cell cycle progression in CLL cells implicating Ca²⁺/NFAT signaling as a potential target for the treatment of this disease.