Association of the reduced expression of NR4A1 and NR4A3 with poor survival in patients with aggressive lymphomas.

Abstract

e18525 Background: NR4A1 and NR4A3 belong to the orphan nuclear receptors. Their function as critical tumour suppressor genes was demonstrated by the rapid development of acute myeloid leukemia (AML) in NR4A1 and NR4A3 double knock-out mice and by their reduced expression in leukemic blasts from human AML patients. The aim of our study is to comprehensively study and functionally characterize NR4A1 and NR4A3 expression in Non-Hodgkin's Lymphomas (NHL). Methods: NR4A1 and NR4A3 expression were determined on mRNA- and protein levels in most lymphoid malignancies of B cell type. Additionally direct sequencing, methylation specific PCR and microRNA analyses of potential interacting NR4A partners were performed. For functional characterization NR4A1was over expressed in a Sc-1 lymphoma cell line by using an inducible lentiviral construct, and treated with Cytosporone-B (Csn-B), a non physiological NR4A1 ligand, followed by apoptotic assays (cleaved caspase 3, Sub-G1 peak determination, and the Annexin V staining). Results: We found a more than 50% reduction of both, NR4A1 and NR4A3, in B-CLL (71%) and Follicular Lymphoma (70%), and more pronounced in diffuse large B cell lymphoma (DLBCL) (74%) compared to normal controls. Mutational analyses in selected cases revealed four of 16 aggressive lymphoma samples mutated (one misssense- and three silent mutations (=25%)). Induction of NR4A1 expression in the transduced Sc-1 lymphoma cell line led to apoptosis in a significantly higher proportion of induced cells compared to their uninduced controls. Additionally, Csn-B treatment of an immortalized B cell line and three lymphoma cell lines caused NR4A1 mediated apoptosis. In a patient cohort of 83 DLBCL, NR4A1 and NR4A3 were more than 50% down-regulated in 88% and 74% of patients, respectively. A low expression of NR4A1 was significantly associated with non-germinal center B-cell like subtype (p<0.001) and with poor overall survival (p=0.042, HR=2.2, CI=1.01-4.9). Conclusions: Our data indicates that NR4A1 has pro-apoptotic functions and that Csn-B induces NR4A1 mediated apoptosis in lymphoma cells. Hence, regulation of NR4A1 by Csn-B is a promising target for the development of new therapeutic drugs.