Overexpression Of Deleted In Lymphocytic Leukemia 1 (DLEU1) Significantly Induces Programmed Cell Death and Inhibits Cell Proliferation In Primary Mediastinal B-Cell Lymphoma (PMBL): DLEU1 May Be a Tumor Suppressor Gene In a Subset Of Patients With PMBL

Primary Mediastinal large B-cell lymphoma (PMBL) is a rare form of Non Hodgkin Lymphoma (NHL) representing 2% of mature B-cell non-Hodgkin lymphoma in patients less than 18 years of age (Lones/Cairo et al., JCO, 2000; Burkhardt et al., BJH, 2005). PMBL has similar histo-pathological features along the biologic spectrum between Diffuse Large B-Cell Lymphoma (DLBCL) and classical HL (cHL) (Abramson et al., Blood, 2005). We have recently reported that a significantly decrease in EFS among children and adolescent PMBL patients compared with other stage III non-PMBL pediatric DLBCL patients following FAB/LMB96 therapy, suggesting that children and adolescent PMBL may be an inherently different B-NHL (Gerrard/Cairo et al., Blood, 2013). Nevertheless, the genetic mechanisms underlying the pathogenesis of PMBL remain unknown. Using high-resolution, microarray-based genomic techniques chromosomal losses at 13q14 was identified in 5 of the 37 (13%) PMBL tumor samples indicating localization of potential tumor suppressor genes at 13q that may be involved in the etiology of PMBL (Wessondorf, et al, Leuk., 2007). DLEU1 (Deleted in lymphocytic Leukemia 1) is a Burkitt lymphoma (BL) classifier gene (Dave et al., NEJM, 2006) and is located in the chromosome 13q14.3 region. DLEU1 interacts with 19 proteins including of c-Myc, RASSF1, TUBB2C and UBR1. DLEU1 has been implicated as a tumor suppressor in BL (Lee/Yin/Cairo et al., ASH, 2012) and in chronic lymphocytic leukemia (Garding et al., Plos Genet., 2013).

We hypothesize that DLEU1 may act as a tumor suppressor gene through induction of caspase-3/7 activity thereby inhibiting cell proliferation and down-regulation of constitutively activated signaling pathways in PMBL.

The full length of cDNA encoding DLEU1 was fused into pEGFP-N3 vector and pEGFP-DLEU1fusion construct (Lee/Yin/Cairo et al., ASH, 2012) was transfected into Karpas-1106P cells using Amaxa Nucleofector kit, followed by the manufacturer’s instruction. Forty-eight hours post transient transfection, total RNA was extracted and 1ug of total RNA was used for cDNA synthesized as above. For comparison of mRNA expression of network genes, quantitative RT-PCR was performed by CFX96 Real-time (Bio-rad). The expression of DLEU1 protein in DLEU1 transient transfected Karpas-1106P cells was confirmed both by western blotting analysis and under fluorescent microscope. In brief, the total lysates was prepared from DELU1 transfected cells at 48hours post-transfection, and membrane was hybridized with Rabbit polyclonal DLEU1 antibody (ProteinTech) on immune blotting. MTS (Promega) and Caspase 3/7 assay (Promega) were employed to examine the effects on cell proliferation and apoptosis. Statistical significance was determined by one tailed paired Student t-test.

The expression of DLEU1 mRNA in DLEU1 transiently transfected Karpas-1106P (DLEU1-Karpas) cells was significantly higher than empty vector alone transfected cell as mock control (78-fold increase, p<0.01). Comparing mRNA expression of DLEU1 network genes in DLEU1-Karpas cells to mock control cells, we observed a significant decrease in mRNA expression of the anti-apoptotic gene, Bcl-xL (73% reduction, p<0.01) and suppressors of cell signaling genes, SOCS1 and SOCS3 (52% reduction, p<0.05 and 44% reduction, p<0.01, respectively). There was a significant reduction in the mRNA expression of oncogenes, Pim-1 and c-Myc (72% reduction, p<0.04 and 54% reduction, p<0.03) respectively, in DLEU1-Karpas cells compared to mock control cells. Cell proliferation was significantly inhibited 18% (p<0.03) whereas Caspase 3/7 activity was significantly increased 1.5-fold (p<0.03) in DLEU1-Karpas cells at 48hours post transfection. Lastly, we found significant decreases of protein expression in phoshpho-Akt (67% reduction, p<0.01), phoshpho-ikBa (48% reduction, p<0.02), phoshpho-STAT3 (15% reduction, p<0.02) and c-Myc (25% reduction, p<0.01), respectively in DLEU1-Karpas cells.

We demonstrated that transient overexpression of DLEU1 1) significantly inhibits cell proliferation and induces programmed cell death, and 2) downregulates constitutively activated signaling pathways in PMBL, and therefore, DLEU1 may in part act as a tumor suppressor gene in a subset of patients with PMBL.

No relevant conflicts of interest to declare.