Loss of NR4A1 Accelerates the Development of Aggressive Lymphomas in Myc Induced Cancerogenesis

Introduction and Aim:

We recently described a reduced NR4A1 and NR4A3 expression chronic lymphocytic leukemia, follicular lymphoma, and diffuse large B-cell lymphoma compared to normal controls. Our survival analysis of aggressive lymphomas revealed that low NR4A1 expression was associated with poor cancer specific survival. Over-expression of NR4A1 in lymphoma cell lines led to a significantly higher proportion of lymphoma cells undergoing apoptosis and abrogated tumor growth in xenografts¹. The aim of this study is to define the role of NR4A1 as a tumor suppressor in the development of lymphoid malignancies in vivo.

Methods:

To identify, whether the loss of NR4A1 has an impact in Myc driven lymphomagenesis we crossed EµMyc mice with NR4A1-/- mice and performed phenotypical analysis including measurement of tumor development, survival and immunophenotypic determination of the newly developed lymphomas by FACS analysis. To further investigate the impact of NR4A1 loss on the oncogenic potential of EµMyc lymphoma cells we isolated viable tumor cells (B220+ and 7AAD-) and cultured them for 72h with or without lipopolysaccharide (LPS) and determined the number of viable cells and their viability (B220 and 7AAD-staining by flow cytometry analysis) after 24h, 48h and 72h. Finally, expression levels of NR4A1, NR4A3 and Myc with or without NR4A1 loss were evaluated by using RT-qPCR.

Results:

EµMyc mice with NR4A1 loss (EµMyc NR4A1-/-, n=46) developed visible tumors significantly faster compared to EµMyc mice with NR4A1 (EµMyc NR4A1+/+, n=75) (median = 44 days for EµMyc NR4A1 -/- vs. 107 days for EµMyc NR4A1+/+; p<0.001). Additionally, EµMyc NR4A1-/- mice showed a significantly shorter life span (median survival = 77 days) compared to EµMyc NR4A1 +/+ mice (median survival = 156 days; p<0.001). By comparing the immunophenotype of the newly developed lymphoma between the two groups (EµMyc NR4A1+/+, n=17 and EµMyc NR4A1 /- , n=19), no significant difference was observed. Interestingly, EµMyc NR4A1-/- mice showed an increased frequency of strong CD93 expression (10 of 18, respectively, vs. 2 of 17 EµMyc NR4A1 +/+ mice, p=0.004). Since most of the EµMyc NR4A1-/- lymphoma were IgM negative (7 of 10) it might be speculated that NR4A1 loss leads to a more immature phenotype of the lymphoma. The number of viable B220+ lymphoma cells isolated from EµMyc NR4A1-/- mice was higher compared to B220+ lymphoma cells isolated from EµMyc NR4A1+/+ mice after 72h in culture with or without LPS (p=0.056; p=0,052). This was accompagnied by a higher in vitro proliferation rate as demonstrated by a higher percentage of BrdU positive cells of the EµMyc NR4A1-/- mice compared to B220+ EµMyc NR4A1+/+ cells with and without LPS stimulation (p= 0,064, p=0,038). Interestingly, we detected a 12 fold higher NR4A3 mRNA expression (p=0,038) in EµMyc NR4A1-/- tumors compared to EµMyc NR4A1+/+.

Conclusion: Our data demonstrate that NR4A1 possesses tumor suppressive properties and that loss of NR4A1 accelerates Myc driven lymphomagenesis. Furthermore, this study indicates that deletion of NR4A1 confers a more aggressive behavior and increases the oncogenic potential of EµMyc driven lymphoma cells.

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.