Exogenous Overexpression Of DLEU1 Significantly Induces Programmed Cell Death and Inhibits Cell Proliferation In Burkitt Lymphoma (BL) and Sensitizes Rituximab (RTX) Resistant BL Cells To RTX Induced Apoptosis
Authors: Lee Sanghoon , Yin Changhong , Ayello Janet , O'Connell Timmy , van de Ven Carmella , Muniz Miguel , Barth Matthew J. , Cairo Mitchell S. 
Published: October 2019

Abstract

Background
Pediatric Burkitt Lymphoma (PBL) represents over 40% of Non-Hodgkin Lymphoma (NHL) in children and adolescents (Cairo et al, Blood, 2007; Miles/Cairo, BJH, 2012) and the prognosis of PBL has dramatically improved over the past 30 years through the introduction of short intensive multi-agent chemotherapy. (Cairo et al, JCO, 2012). However, the success of this therapeutic approach has been compromised by significant chemotherapy-induced toxicity, requiring the need to identify a less toxic targeted therapy. We previously identified, in a multivariate analysis, that children with Burkitt Lymphoma (BL) and a 13q deletion had a significantly poorer outcome and inferior overall survival (OS) despite aggressive short, intensive multi-agent chemotherapy (Poirel/Cairo et al, Leuk., 2009; Nelson/Cairo/Sanger et al, BJH, 2009). Deleted in Lymphocytic Leukemia 1 (DLEU1) is a BL classifier gene and a target of c-Myc on the chromosome 13q14.3 region (Dave et al, NEJM, 2006). DLEU1 encodes for proteins of 72 residues and 19 interacting proteins including c-Myc, TUBB2C, RASSF1A and UBR1. We have previously suggested that DLEU1 may function as a tumor suppressor gene in transient DLEU1 overexpressed BL cells (Lee/Cairo, ASH, 2012). However, functions and mechanism(s) following dysregulation of DLEU1 expression in BL are poorly understood.

Objectives
We proposed that DLEU1 may act as a tumor suppressor gene in PBL, and therefore investigated whether 1) overexpression of DLEU1 in BL cells results in changes in programmed cell death, cell proliferation and cell migration, and 2) sensitizes Rituximab resistant BL cells to apoptosis in PBL.

Methods
A stable Raji cell line with DLEU1 overexpression (DLEU1-OE) was established from previous reported (Lee/Cairo et al, ASH, 2012) under G418 selection and all cells including Rituximab (RTX) resistant Raji BL cells (2R) (Barth et al, BJH, 2012) were cultured in RPMI with 10% FBS. Total RNA was isolated by Trizol (Invitrogen) and cDNA was synthesized by cDNA Synthesis Kit (Quantas). Quantitative RT-PCR was performed by CFX96 Real-time (Bio-rad), and MTS and Caspase 3/7 assays (Promega) were employed for measurement of cell proliferation and apoptosis. Statistical significance was performed by one-tailed Student t-test. For classic scratch-wound healing assay, wounded cells were created by scratching cells at 72 hours post-transient transfection with DLEU1 and images of wound areas were taken at 0 and 48 hours after scratching. For transient experiments using 2R, DLEU1 transient overexpressing Raji and 2R cells were treated with Rituximab (20ug/ml) for 48hours and cell proliferation and apoptosis assay were measured as above.

Results
The expression of DLEU1 mRNA showed a significant increase in DLEU1 stable overexpressing Raji (DLEU1-OE) cells compared to mock control cells (9.0 fold increase, p<0.02). In comparing mRNA expression levels of DLEU1 network genes, there was significant decrease of RASSF1 and TUBB2C mRNA expression (1.2 fold reduction, p<0.02 and 1.3 fold reduction, p<0.03, respectively) in DLEU1-OE cells to mock control cells. DLEU1-OE cells showed a significant decrease in cell proliferation (15% reduction, p<0.03; 19% reduction, p<0.03; and 15% reduction, p<0.01 at 24, 48 and 72 hours, respectively) (Fig 1A). There was significant increase in Caspase 3/7 activity (10.2% increase, p<0.02; 9.7% increase, p<0.05 at 24 and 48 hours, respectively) (Fig 1B). The wounded cells dramatically healed slower than those of control in DLEU1 transient overexpressed 293 cells compared to mock control 293 cells (Fig 2). There were significant increase in Caspase 3/7 activity and significant decrease in cell proliferation in RTX (20ug/ml) treated transient DLEU1 overexpressed 2R (13% increase, p<0.05 and 5% reduction, p<0.02, respectively) compared to RTX treated transient DLEU1 overexpressed Raji. RTX treated transient DLEU1 overexpressed 2R showed significant decrease of expression anti-apoptotic genes, Bcl-xL and Bcl-2 mRNA (<15% reduction, p<0.04 and <20% reduction, p<0.02, respectively).

Conclusion
These results suggest that 1) overexpression of DLEU1 significantly inhibited cell proliferation, wound healing and induced apoptosis, and 2) we hypothesize that overexpression of DLEU1 in PBL may result in sensitizing BL to chemotherapy induced apoptosis and may result in a potential future therapeutic strategy.

Disclosures:
No relevant conflicts of interest to declare.