183 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 cutoplasm by a network of several kinases including CK-1, GSK-3 and DYRK. 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. Here we have analyzed the role of NFAT2 in B cell development. Analysis of the role of this family member in hematopoiesis has been complicated by the fact that deletion of this gene is embryonic lethal around ED 13 because of defects in heart valve development. To circumvent this problem we generated mice with a conditional NFAT2 knock out allele (NFAT2fl/fl). In order to achieve NFAT2 deletion limited to the B cell lineage, we bred NFAT2fl/fl mice to CD19-Cre mice, in which the Cre recombinase is expressed under the control of the B cell-specific cd19 promoter. B cells from these mice were isolated using CD19-labeled magnetic beads and subjected to analysis by flow cytometry. While CD19+ splenocytes from conditional NFAT2 knock-out mice occurred in normal numbers, these cells showed significantly reduced expression of CD38 and ZAP70 upon stimulation with anti-IgM antibody as compared to CD19+ splenocytes from wild-type controls. The reduction of these proteins could also be detected in B cells isolated from the peripheral blood and from bone marrow and was confirmed by western blotting and quantitative RT-PCR. CD38 and ZAP70 are well characterized prognostic factors in chronic lymphocytic leukemia (CLL) and their increased expression has been shown to correlate with poor patient survival. Our data indicate that the expression of these markers is at least in part regulated by Ca2+/NFAT signaling and that deregulation of this pathway can contribute to their overexpression in disease. Next we analyzed bone marrow and peritoneal lavages from conditional NFAT knock-out mice by flow cytometry. While we found no significant differences in the abundance of B cell subpopulations in bone marrow, we detected an almost complete absence of CD5+CD43+ B1a cells in the peritoneal cavity, clearly demonstrating the requirement of NFAT2 in the development of this subclass. B1a cells are a phenotypically and functionally distinct population of B cells which are long-lived and typically express CD5, CD43 and high levels of surface IgM together with low surface IgD and CD45 (B220). A human B cell equivalent of the murine B1a cell has been suggested as the leukemic precursor cell in chronic lymphocytic leukemia (CLL). To further delineate the role of NFAT2 in the development of B1a cells we determined the abundance of B1 progenitor cells (B1P) in bone marrow and spleen by FACS analysis. In NFAT2 knock-out mice we observed a significant reduction of the frequency of B220- CD19+ CD93+ B1P cells in bone marrow (0.8% vs. 4.7%) and spleen (0.16% vs. 0.82%) demonstrating that NFAT2 is essential for normal development of this precursor cell population. In summary, our data provide strong evidence that NFAT2 is critical for the expression of CD38 and ZAP70 in B cells and substantially controls B1a cell homeostasis implicating Ca2+/NFAT signaling as a potential target for the treatment of CLL. Disclosures: No relevant conflicts of interest to declare.

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.

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. Expression of CD38 and ZAP70 define a subgroup of patients with enhanced responsiveness to stimulation of the B cell receptor (BCR) complex and more aggessive disease. In contrast, another subset of CLL patients with more indolent course is characterized by an anergic B cell phenotype refering 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 mice with a conditional NFAT2 knock out allele (NFAT2fl/fl). In order to achieve NFAT2 deletion limited to the B cell lineage, we bred NFAT2fl/fl mice to CD19-Cre mice. 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 wks 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 NFAT2fl/fl). TCL1 transgenic mice without an NFAT2 deletion served as controls (n=10). To identify novel NFAT2 target genes in CLL cells, we performed a comparative gene expression analysis on CLL cells with intact NFAT2 expression and on CLL cells with NFAT2 deletion using affymetrix microarrays. Mice with NFAT2 knock out exhibited a significantly more aggressive disease course with accelerated accumulation of CD5+CD19+ CLL cells and a significantly reduced life expectancy (200 vs. 325 days) as compared to control animals. Flow cytometric analysis at distinct time points showed a pronounced infiltration by CD5+ B cells of 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. 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 exhibited significantly higher rates of proliferation than control animals. To identify NFAT2 target genes resonsible for the observed alterations in the disease phenotype, we subsequently peformed a gene expression analysis with CD5+CD19+ CLL cells from TCL1+NFAT2 ko mice with CLL cells from TCL1+ mice serving as controls. Here, we detected a significantly altered expression of 22 genes associated with B cell anergy in the TCL1+NFAT2 ko cohort. The vast majority of these genes was expressed significantly less in the absence of NFAT2 with Lck, Pacsin1, Hspa14 and CD166 constituting the strongest hits with up to 10fold reduced gene expression. Downregulation of the identified target genes was subsequently confirmed using RT-PCR and Western Blotting. 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. In addition, we could show that NFAT2 controls the expression of several anergy-associated genes and that its absence prevents the acquisition of an anergic phenotype by the CLL cells correlating with a significantly more aggressive course of the disease. Taken together, our data demonstrate that NFAT2 plays an essential role in the pathogenesis of CLL and implicate this transcription factor as a potential target in its treatment. Disclosures: No relevant conflicts of interest to declare.

Chronic Lymphocytic Leukemia (CLL) is a hematological malignancy of mature B cells and constitutes the most common leukemia in adults. It is characterized by a progressive accumulation of clonal B cells, which coexpress CD19, CD23 and CD5. The clinical course of CLL can be predicted by serveral prognostic markers like CD38, ZAP70 and cytogenetic abnormalities. While the treatment of CLL has significantly improved during recent years, it remains an essentially incurable disease and the molecular events that lead to its development are still largely elusive. NFAT is a family of highly phosphorylated transcription factors residing in the cytoplasm of resting cells. Upon dephosphorylation NFAT proteins translocate to the nucleus where they orchestrate developmental and activation programs in diverse cell types. NFAT is inactivated by a network of several kinases. Several recent studies have demonstrated that Ca2+/NFAT signaling is involved in the pathogenesis of a wide array of different tumor types including pancreatic adenocarcinoma, breast cancer and Non Hodgkin´s lymphoma. In this study we investigated the significance of the Ca2+/NFAT signaling pathway in B-CLL. For this purpose, we analyzed CLL cell lines (MEC-1, JVM-3) as well as primary blood samples from patients with CLL (n=30). The analyzed patient population exhibited a representative distribution of age, sex, Binet stage, WBC count, cytogenetics and IGVH mutational status. We detected a profound overexpression of NFAT2 mRNA as well as NFAT2 protein in all CLL samples. Using qRT-PCR we found that CD19+CD5+ CLL cells exhibited an at least three fold overexpression of NFAT2 as compared to CD19+ B cells isolated from healthy donors. In one case, NFAT2 expression in CLL cells was 200 times higher than in the corresponding controls. This profound overexpression of NFAT2 in CLL cells could be confirmed on the protein level using Western Blotting and Immunocytochemistry. We could further demonstrate that even under resting conditions significant amounts of NFAT2 protein had translocated to the nucleus in CLL cells, whereas virtually all NFAT2 was in the cytoplasm in healthy B cells. NFAT2 nuclear translocation could be inhibited using pretreatment with Cyclosporin A demonstrating that this process was still calcineurin-dependent in CLL cells. We could further show that nuclear NFAT2 in CLL cells was able to bind DNA using electrophoretic mobility shift assays (EMSA). To assess the transcriptional activity of NFAT2 in human CLL we determined the expression of the apoptosis regulators OX40L, osteopontin and PD-L2, which we previously identified as NFAT2 target genes in a gene expression analysis with CD19+CD5+ CLL cells from TCL1 transgenic mice with intact NFAT2 and NFAT2 deletion, respectively. Interestingly, qRT-PCR revealed a tremendous reduction of all three target genes in the analyzed CLL samples as compared to control B cells from healthy donors. This is particularly remarkable, since in the TCL1 mouse model we observed a similar reduction of the expression of these genes in CLL cells with NFAT2 ablation. In summary, these results provide strong evidence that the Ca2+/NFAT signaling axis is constitutively activated in CD19+CD5+ CLL cells. Our data suggest that the profound overexpression of NFAT2 in CLL cells leads to its targeting to aberrant genetic loci different from its phsiological target genes resulting in a consecutive knock out phenotype with respect to the expression of the apoptosis regulators OX40, osteopontin and PD-L2 in CLL. Further investigation is therefore warranted to decipher the therapeutic potential of modulating the Ca2+/Calcineurin/NFAT signaling pathway in this disease. Disclosures: No relevant conflicts of interest to declare.

2486 Approximately twenty percent of acute lymphoblastic leukemias (ALL) in adults are positive for the Philadelphia chromosome t(9;22) upon cytogenetic examination and express a Bcr-abl fusion protein with constitutive tyrosine kinase activity as part of their pathogenesis. While the majority of these leukemias initially respond well to tyrosine kinase inhibition (TKI) with imatinib or dasatinib, most of them develop TKI-resistance over time and exhibit progressive disease to which most patients eventually succumb. NFAT is a family of highly phosphorylated transcription factors 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 proteins get rephosphorylated and inactivated by a network of multiple NFAT kinases including GSK-3, CK-1 and DYRK. Several recent studies have demonstrated that Calcineurin/NFAT signalling 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. In this study, we investigated the role of the Ca/NFAT signalling pathway in the development of TKI-resistance in Ph+ ALL. We analyzed several Ph+ ALL cell lines (SD-1, TOM-1, SUP-B15), which had been shown previously to be resistant to imatinib, as well as primary leukemia samples from patients with TKI-resistant disease (n=3). NFAT expression and aberrant nuclear translocation was assessed by Western Blotting and the NFAT2 (NFATc1) mRNA level was confirmed by quantitative RT-PCR. The cells were subsequently grown under optimized cell culture conditions in the presence or absence of TKI (0–1 μM imatinib), and cell viability was assessed using propidium iodide staining and flow cytometry. Proliferation assays were performed after 48–72 h using a luminescence assay measuring the amount of cellular ATP. All cell lines and primary leukemia samples showed marked expression and evidence of aberrant nuclear translocation of NFAT2 when analyzed by Western Blotting. Even maximum concentrations of imatinib had only minor effects on cellular proliferation documenting resistance to tyrosine kinase inhibition. To inhibit the calcineurin/NFAT signalling cascade as a potential therapeutic target, we treated the cells with the calcineurin inhibitor cyclosporin A (CsA) in addition to imatinib. The combined treatment showed a dramatic effect on ALL cell proliferation, resulting in almost complete cell death after 48 h. This effect could be reproduced using tacrolimus (FK506) instead of CsA indicating that it was specifically mediated through calcineurin inhibition and not by potential off target-activites of CsA. In summary, our data provide strong evidence that Calcineurin/NFAT signalling contributes to the proliferation of TKI-resistant Ph+ ALL cells and that pharmacological inhibition of NFAT signalling can break treatment resistance to TKI. Targeting Ca/NFAT signalling in combination with Bcr-abl inhibition might therefore be a novel option in the treatment of Ph+ ALL. Disclosures: No relevant conflicts of interest to declare.