Ccytosporone-B Induces NR4A1 Meditated Apoptosis In Aggressive Lymphoma Cells

Abstract 467

NR4A1 (also called Nur77, TR3, or NGFI-B) and NR4A3 (Nor-1 or Minor) are two of three members of the orphan nuclear hormone receptor (NR) family referred to as Nur77 family. For both NRs no physiological ligand has been identified. These orphan NRs have been implicated in cell cycle regulation, apoptosis, inflammation, and more recently in carcinogenesis. Their function as critical tumour suppressors genes is demonstrated by the observation that the NR4A1 and NR4A3 double knock out mouse develops rapidly acute myeloid leukemia involving decreased expression of the AP-1 transcription factors JunB and c-Jun and defective extrinsic apoptotic (Fas-L and TRAIL) signalling and their reduced expression in leukemic blasts from human AML patients.

NR4A1 and NR4A3 expression analyses in lymphoid malignancies revealed a down regulation of both NRs in all lymphoma cell lines analysed (Ly 1, 3, 4, 7, 8, Karpas 422 and Sc-1), in two of eight “low grade lymphoma” entities (B-CLL and FL II) and in three of five “high grade lymphoma” entities (DLBCL, FL III, MBL) and in ALLs compared to normal controls. Further expression analyses of genes involved in extrinsic (Trail and Fas-L) and intrinsic (Bim and Puma) apoptotic pathways resulted in a uniform regulation (significant positive correlation) of all four apoptotic genes to NR4A1 and NR4A3 expression suggesting that NR4A1 and NR4A3 act as putative tumour suppressor in a substantial number of lymphoma entities and that their abrogation leads to a defective extrinsic and intrinsic apoptotic signalling (1).

To investigate whether the reduction of NR4A1 and NR4A3 expression is caused by epigenetic (hypermethylation of CpG islands) or genetic (mutations) aberrations, the regulatory region and coding sequence of both NRs were subjected to a comprehensive methylational and mutational analyses in primary aggressive lymphoma samples by methylation specific PCR and direct sequencing. Whereas no hypermethylation of CpG islands in the regulatory region was detected, a total of four mutations were found in four of 17 aggressive lymphoma samples (1 of 5 FLIII, 2 of 6 DLBCL-GCB and 1 of 6 DLBCL-NGCB). One mutation in the group of the DLBCL-GCB led to an amino acid exchange.

To further delineate the function of NR4A1 in aggressive lymphomas, we treated three lymphoma cell lines (Karpas422, SC-1 and Ly8) with Cytosporone B (Csn-B), a natural occurring ligand of NR4A1 known to induce NR4A1 expression (2). Treatment with Csn-B led to an induction of NR4A1 expression accompanied by a marked inhibition of lymphoma cell growth in a dose dependent manner in all three lymphoma cell lines. To investigate whether Csn-B induced growth inhibition is mediated by apoptosis we performed various apoptosis assays, and cell cycle analyses in SC-1 and Ly8 cell lines. After 6h 1.5 ×10⁻⁴ M Csn-B treatment cleaved caspase 3 was detected in 91% of Sc-1 cells and in 77% of Ly8 cells as compared to 3% and 23% in untreated controls. Similarly, in the Annexin V assay Csn-B displayed a robust pro-apoptotic activity in both cell lines within 10 h (52 % in SC-1 and 67 % in Ly8 vs 2 % and 14 %) of treatment. By flow cytometry a significant higher sub-G1 DNA content (26 % in SC-1 cells and 45% in Ly8 vs 2% and 9%) could be detected. Induction of apoptosis was accompanied by a significant rise in NR4A1 expression.

Our data suggest that NR4A1 is down regulated in a substantial fraction of aggressive lymphomas and that Csn-B induces NR4A1 mediated apoptosis in lymphoma cells. Hence, regulation of NR4A1 by Csn-B is a promising new target for the development of new drugs for lymphoma therapy.

1. Deutsch AJA, Beham-Schmid C, Linkesch W, Neumeister P. The Nuclear Orphan Receptors NR4A1 and NR4A3 as Putative Tumour Suppressor Genes in Aggressive Non-Hodgkin's Lymphomas. ASH Annual Meeting Abstracts. 2009 November 20, 2009;114(22):2967.

2. Zhan Y, Du X, Chen H, Liu J, Zhao B, Huang D, et al. Cytosporone B is an agonist for nuclear orphan receptor Nur77. Nat Chem Biol. 2008 Sep;4(9):548-56.