NR4A3 Possesses Tumor Suppressive Properties like NR4A1 By Regulating Pro-Apoptotic Genes in Aggressive Lymphomas and Serves As a Potential Drug Target for Lymphoma Therapy

Recently, we described a significant down-regulation of NR4A1 (Nur77) and NR4A3 (Nor-1)-two members of the orphan nuclear receptors acting together as critical tumor suppressor genes in acute myeloid leukemia- in aggressive lymphoma¹. NR4A1 over-expression proved its pro-apoptotic function in aggressive lymphoma cells and its lymphoma suppressive properties in vivo was demonstrated in a xenograft mouse model. Since the role of NR4A3 in aggressive lymphomas and the effects of NR4A3 inducing agents on lymphoma cells are unknown, we aimed to investigate NR4A3 function and the effects of a NR4A3 inducing agent in aggressive lymphoma cells. For functional characterization, NR4A3 was over-expressed in a SuDHL4 lymphoma cell line by using an inducible lentiviral construct followed by various apoptotic assays. Induction of NR4A3 expression led to a significantly higher proportion of induced SuDHL4 cells undergoing apoptosis as demonstrated by DNA cleavage, Annexin V staining and increased caspase 3-7 activity suggesting a functional redundancy to NR4A1 in aggressive lymphoma. To test the tumor suppressor function of NR4A3 in vivo, the stably transduced SuDHL4 lymphoma cell line was further investigated in the NOD scid gamma (NSG) mouse model. Induction of NR4A3 in SuDHL4 abrogated tumor growth in the NSG mice, in contrast to vector control- and uninduced SuDHL4 cells, which formed massive lymphoid tumors. Additionally, four aggressive lymphoma cell lines (Karpas-422 and SuDHL4 as GCB- cell line, RI-1 and U2932 as ABC-cell lines) were treated with a NR4A3 inducing agent, named Thapsigargin followed by cell growth (MTS) and apoptotic (Annexin V staining and Caspase 3-7 activity assay) assays. Treatment with Thapsigargin induced NR4A3 expression accompanied with induction of apoptosis of all four lymphoma cell lines detected by increased percentage of Annexin V positive cell and increased caspase 3/7 activity. Inhibition of NR4A3 by siRNA reduced the apoptotic effects of Thapsigargin. To further compare the transcriptional activity as nuclear receptor of NR4A3 to NR4A1, both receptors were separately over-expressed in our four aggressive lymphoma cell lines followed by mRNA expression analysis of intrinsic (Bad, BIK, BID, BMF, Noxa, BAK, Bax, Puma, Bim, Bcl-2, Bcl-X and Mcl-1) and extrinsic (FasL, Fas, Trail, DR4 and DR5) apoptotic genes. mRNA expression analysis of apoptotic genes in aggressive lymphoma cells demonstrated that NR4A1 and NR4A3 over-expression induced Trail, Bim, Puma, BIK, BID and BAK in a similar pattern. Our data suggest that NR4A3 has a pro-apoptotic function in aggressive lymphoma and define that NR4A3 together with the functionally redundant NR4A1 as novel tumor suppressor involved in aggressive lymphoma development. Hence, NR4A3 and its inducing agents are promising novel targets for drug development in lymphoma therapy.

1. Deutsch AJ, Rinner B, Wenzl K, et al. NR4A1-mediated apoptosis suppresses lymphomagenesis and is associated with a favorable cancer specific survival in patients with aggressive B-cell lymphomas. Blood. 2014.