Downregulation of DLEU1 Significantly Shortened Survival in a Rituximab-Treated DLEU1 Knockout Human Burkitt Lymphoma (BL) Xenograft NSG Mouse Model: DLEU1 May Act As a Tumor Suppressor Gene in Pediatric BL

BACKGROUND: Burkitt lymphoma (BL) is the most common histological subtype of non-Hodgkin lymphoma (NHL) in children and adolescents (Cairo et al., Blood, 2007; Miles/Cairo, BJH, 2012). We have previously identified secondary chromosomal aberrations in 70% of pediatric BL patients (PBL) with a C-MYC gene rearrangement (Poirel/Cairo et al., Leukemia, 2009). Specifically, we identified significantly inferior event free survival (EFS) and overall survival in children and adolescents with a specific loss of the 13q14.3 locus (Poirel/Cairo et al., Leukemia, 2009; Nelson/Cairo/Sanger et al., Br. J. Haematol., 2009). In a multivariate analysis controlling for stage, lactate dehydrogenase levels, country of treatment, and group classification, children with BL who had a 13q deletion had significantly poorer EFS compared to the remainder of patients treated with the same French-American-British (FAB) chemotherapy regimen (Poirel/Cairo et al., Leukemia, 2009). Deleted in lymphocytic leukemia 1 (DLEU1) is a BL classifier gene in the 13q14.3 region (Dave et al., NEJM, 2006) and interacts with C-MYC, Tubulin beta-2C chain (TUBB2C), E3 ubiquitin-protein ligase (UBR1), cellular tumor antigen p53, and Ras association (RalGDS/AF-6) domain family member 1 (RASSF1) (Stelzl et al., Cell, 2005). The sequence-specific Transcription Activator-Like Effector Nucleases (TALENs) have been developed for targeted genome editing in in vitro and in vivo experiments with high efficiency as a new therapeutic tool (Sander et al., Nat. Biotechnol., 2011). We have previously reported a down-regulated DLEU1 mRNA expression significantly associated with an increase in BL proliferation in vitro(Lee/Cairo et al., AACR, 2015). We hypothesize that DLEU1 may have function as a tumor suppressor gene; however, the role of DLEU1 regulating programmed cell death in BL are poorly understood.

OBJECTIVES: We hypothesize that DLEU1 may act as a tumor suppressor gene in BL and whether down-regulation of DLEU1 expression results in changes in BL survival following targeted immunotherapy.

METHODS: TALENs induced DLEU1 knockout Raji cells were previously generated (Lee/Cairo et al., ASH, 2013) and DLEU1 knockout cells were stably transfected with a firefly luciferase expression plasmid (ffluc-zeo), kindly provided by Laurence Cooper MD, PhD. Four- to six- week-old female NSG (NOD.Cg-Prkdc^scid Il2rg^tm1Wjl/SzJ) mice from The Jackson Laboratory were irradiated (2.5 Gy), and then mice were subcutaneously injected with 1x10⁶ ffluc-zeo tumor cells. Tumor burden and tumor progression were monitored by bioluminescence imaging system using the Xenogen IVIS-200 (Caliper Life Sciences). Mice were treated with either PBS, IgG isotype control, rituximab (30 mg/kg) or cyclophosphamide (25 mg/kg) and in combination by intraperitoneal (i.p) injection at 7 day intervals. Survival rates were analyzed by the Kaplan-Meier method and differences evaluated by log-rank test using the Prism Version 6.0 software.

RESULTS: There were significant increases of luciferase signal intensity in DLEU1 knockout mice (DLEU1-KO) compared to that in wild type (WT) mice on day 31 of rituximab (p<0.05), cyclophosphamide (p<0.05) or rituximab and cyclophosphamide (p<0.01) combined treatment. Consistent with these initial findings in tumor growth, we found that rituximab-treated DLEU1-KO mice (n=12 per group) had a significantly shortened survival time with a median of 42 days compared to that of WT mice (n=12 per group) (52 days, p<0.005) (Figure 1A). For the rituximab and cyclophosphamide combination treated group, WT mice (n=12 per group) had a significantly extended survival time compared to the DLEU1-KO group (55.5 days vs48 days, p<0.05) (Figure 1B). There were no significant differences in survival between WT and DLEU1-KO mice with PBS and IgG isotope treatment.

CONCLUSIONS: The down-regulation of DLEU1 expression significantly decreased the survival rate in DLEU1-KO xenografted NSG mice following rituximab and in combination with cyclophosphamide treatment. Therefore, the down-regulation of DLEU1 expression in BL may in part result in immunotherapy resistance and may result in a consideration of alternative therapeutic strategies.