DLEU1 siRNA Gene Knockdown IN BURKITT LYMPHOMA (bl) IS ASSOCIATED with A SIGNIFICANT Decrease IN Cyclophosphamide (CY) and/or Rituximab Induced Apoptosis: Mechanism of Drug Resistance in BL.

Background: Burkitt lymphoma (BL) represents approximately 40% of all childhood or adolescent NHL (Cairo et al., Blood, 2007). Following a multivariate analysis for known prognostic factors, we have identified a subgroup of BL patients with a 13q deletion who had a significantly poorer outcome despite aggressive short, intensive multiagent chemotherapy (Poirel/Cairo et al Leukemia 2009). More recently, we identified in a subset analysis that children with BL and a 13q14.3 deletion by FISH analysis have a significant inferior OS (Nelson/Cairo/Perkins/Sanger et al BJHaem, In Press). 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. In addition, DLEU1 is recognized to interact with c-Myc, histone acetylase (HTATIP), tumor antigen p53, histone-lysine N-methyltransferase (SETDB1), Tubulin beta-2C (TUBB2C), RASSF1A, and E3 ubiquitin-protein ligase (UBR1). We have previously reported that DLEU1 may in part function as a potential tumor suppressor gene. 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 gene array profiling studies, we also found that the expression levels of RASSF1, ERG, UBR1, and TUBB2C were significantly higher in BL than in DLBCL (Day/Cairo et al, AACR 2008). Objective: In this study, we sought to examine the percent of apoptosis induced by CY and/or rituximab in DLEU1 siRNA transfected BL cells. Methods: Ramos BL cell lines were transiently transfected (24 hrs) with DLEU1 siRNA as previously described (5'-AUACUUGGCAUGAAUGAACUUAUGU-3' and 3'-UAUGAACCGUACUUACUUGAAUACA-5') (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 mg/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. Results: 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<0.05 to negative control; 895 nM CY, 10.86±0.67% reduction, p<0.01; 8950 nM, 9.85±0.32% reduction, p<0.05. There was a similar significant reduction in rituximab induced apoptosis in the BL transfected DLEU1 siRNA vs mock control cells (4 mg/mL Rituximab: 25.45±2.55% reduction, p<0.01 to negative control; 40 mg/mL Rituximab, 18.31±5.13% reduction, p<0.04; 400 mg/mL Rituximab, 32.33±1.77% reduction, p<0.02). There was no additive effect when combining CY (895 nM) and Rituximab (4 mg/mL) in DLEU1 siRNA transfected BL cells: 17.96±3.35 vs 29.85±1.83 vs 31.74±2.44 (Figure). Conclusion: 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.