Abstract 4357: DLEU1 siRNA gene knockdown is associated with a significant reduction in cyclophosphamide (CY) and /or rituximab induced apoptosis in Burkitt lymphoma (BL): Implication of DLEU1 as a tumor suppressor gene
Authors: Nancy S. Day, Janet Ayello, Carmella van de Ven, Megan S. Lim, Sherrie L. Perkins, Warren G. Sanger, Lauren Harrison, Stanton C. Goldman, Mitchell S. Cairo
Journal: Cancer Research (2010)

Abstract Pediatric Burkitt lymphoma (PBL) represents approximately 40% of all childhood or adolescent NHL (Cairo et al., Blood, 2007). Patients with PBL and a 13q deletion, particularly 13q14.3, had significantly poorer outcome and inferior OS despite aggressive short, intensive multiagent chemotherapy (Poirel/Cairo et al Leukemia 2009; Nelson/Cairo/Perkins/Sanger et al BJHaem 2009). DLEU1, a gene within the Burkitt classifier genes as reported by Dave/Staudt et al. NEJM, 2006, is located within the region of 13q14.3. DLEU1 is recognized to interact with c-Myc, tumor antigen p53, Tubulin beta-2C (TUBB2C), RASSF1A, ERG, and E3 ubiquitin-protein ligase (UBR1). The expression levels of RASSF1, ERG, UBR1, and TUBB2C were significantly higher in BL than in DLBCL (Day/Cairo et al, AACR 2008). When DLEU1 is down regulated by a DLEU1 siRNA, the spontaneous apoptotic rate was decreased with concomitant significantly reduced levels of UBR1 and TUBB2C gene expression (Day/Cairo et al SIOP 2008). In this study, we sought to examine the percent of apoptosis induced by CY and/or rituximab in DLEU1 siRNA transfected BL cells. Ramos BL cell lines were transiently transfected (24 hrs) with DLEU1 siRNA as previously described (Day/Cairo SIOP 2008). Stealth RNAi whose GC content is similar to that of this DLEU1 siRNA was used as negative control. The siRNA transfected cells were then treated with CY (0, 89.5, 895, 8950 nM) and/or rituximab (0, 4, 40, 400 µg/mL) for additional 4 hrs. Cells were evaluated for percent apoptosis using Annexin V-FITC and Propidium Iodide followed by FACS using BD LSRII. Statistics was conducted by one-way ANOVA followed by Dunnett multiple comparisons test. There was a significant reduction in apoptosis in the CY treated BL transfected DLEU1 siRNA vs mock control cells (89.5 nM CY: 10.26±0.23% reduction, p&amp;lt;0.05 to negative control; 895 nM CY: 10.86±0.67% reduction, p&amp;lt;0.01; 8950 nM, 9.85±0.32% reduction, p&amp;lt;0.05). There was a similar significant reduction in rituximab induced apoptosis in the BL transfected DLEU1 siRNA vs mock control cells (4µg/mL rituximab: 25.45±2.55% reduction, p&amp;lt;0.01 to negative control; 40 µg/mL rituximab: 18.31±5.13% reduction, p&amp;lt;0.04; 400 µg/mL rituximab: 32.33±1.77% reduction, p&amp;lt;0.02). There was no additive effect when combining CY (895 nM) and rituximab (4 µg/mL) in DLEU1 siRNA transfected BL cells: 17.96±3.35 vs 29.85±1.83 vs 31.74±2.44. In summary, we have demonstrated that DLEU1 may in part regulate programmed cell death in BL. DLEU1 siRNA gene knockdown studies resulted in significantly less apoptosis in CY and rituximab treated BL cells. Deletion of 13q14.3, which contains DLEU1, in pediatric BL may confer a phenotype of drug resistance and predispose pediatric patients with BL to a significantly decreased EFS following intensive multiagent chemotherapy. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4357.