Deleted In Lymphocytic Leukemia 1 Expression Research Articles

Survival‑related DLEU1 is associated with HPV infection status and serves as a biomarker in HPV‑infected cervical cancer.

Human papillomavirus (HPV) is the most common risk factor for the occurrence of cervical cancer (CC). In recent years, the important roles of long non-coding RNAs (lncRNAs) in CC have emerged, but studies on the relationship between lncRNAs and HPV-positive (HPV+) CC remain scarce. The present study aimed to investigate whether lncRNA deleted in lymphocytic leukemia 1 (DLEU1) is associated with HPV infection and explore the clinical significance of DLEU1 in HPV+ patients with CC. DLEU1 expression was detected by reverse transcription-quantitative PCR. The ability of DLEU1 to screen patients with CC from controls and differentiate individuals with different HPV infection status was evaluated by receiver operating characteristic analysis. The association of DLEU1 with the survival prognosis of patients with CC was assessed by Kaplan-Meier survival analysis and Cox regression analysis. The RNA Interactome Database was used to predict molecules interacting with DLEU1. The results indicated that DLEU1 expression was significantly upregulated in CC tissues and cell lines, particularly in those that were HPV+. In addition, DLEU1 had a high diagnostic value in discriminating patients with CC and differentiating between HPV+ and HPV- patients with CC, and had a certain ability to screen HPV+ controls. DLEU1 was correlated with HPV infection in CC patients. Furthermore, DLEU1 was indicated to be associated with survival prognosis in both total patients with CC and HPV+ patients with CC, and independently predict the prognosis of patients with CC. Most of the molecules interacting with DLEU1 were microRNAs. In conclusion, abnormal DLEU1 expression is associated with HPV infection and may serve as a diagnostic and prognostic biomarker for HPV+ patients with CC.

DLEU1 promotes oral squamous cell carcinoma progression by activating interferon-stimulated genes

Long noncoding RNAs (lncRNAs) are deeply involved in cancer development. We previously reported that DLEU1 (deleted in lymphocytic leukemia 1) is one of the lncRNAs overexpressed in oral squamous cell carcinoma (OSCC) cells, where it exhibits oncogenic activity. In the present study, we further clarified the molecular function of DLEU1 in the pathogenesis of OSCC. Chromatin immunoprecipitation-sequencing (ChIP-seq) analysis revealed that DLEU1 knockdown induced significant changes in the levels of histone H3 lysine 4 trimethylation (H3K4me3) and H3K27 acetylation (H3K27ac) in OSCC cells. Notably, DLEU1 knockdown suppressed levels of H3K4me3/ H3K27ac and expression of a number of interferon-stimulated genes (ISGs), including IFIT1, IFI6 and OAS1, while ectopic DLEU1 expression activated these genes. Western blot analysis and reporter assays suggested that DLEU1 upregulates ISGs through activation of JAK-STAT signaling in OSCC cells. Moreover, IFITM1, one of the ISGs induced by DLUE1, was frequently overexpressed in primary OSCC tumors, and its knockdown inhibited OSCC cell proliferation, migration and invasion. These findings suggest that DLEU1 exerts its oncogenic effects, at least in part, through activation of a series ISGs in OSCC cells.

Knockdown of lncRNA DLEU1 inhibits the tumorigenesis of oral squamous cell carcinoma via regulation of miR‑149‑5p/CDK6 axis.

Oral squamous cell carcinoma (OSCC) is a frequent malignant tumor worldwide. Long non-coding RNAs (lncRNAs) are known to play key roles in different types of cancer, including OSCC. It was previously reported that lncRNA deleted in lymphocytic leukemia 1 (DLEU1) is notably upregulated in OSCC; however, the role of DLEU1 in OSCC remains unclear. Gene and protein expression levels in OSCC cells were detected by reverse transcription-quantitative PCR and western blotting, respectively, in the present study. A Transwell assay was performed to measure cell migration and invasion. Flow cytometry was used to detect cell apoptosis, and the dual-luciferase reporter assay was applied to confirm the interaction between DLEU1, microRNA (miR)-149-5p and CDK6 in OSCC cells. DLEU1 expression was negatively associated with the survival rate of patients with OSCC. In addition, silencing of DLEU1 notably inhibited the proliferation of OSCC cells by inducing apoptosis. Meanwhile, DLEU1 directly bound to miR-149-5p, and CDK6 was found to be the direct target of miR-149-5p. Furthermore, DLEU1 knockdown-induced inhibition of OSCC cell proliferation was significantly reversed by the miR-149-5p antagomir. Knockdown of lncRNA DLEU1 reversed the proliferation of OSCC cells via regulation of the miR-149-5p/CDK6 axis. Thus, DLEU1 may serve as a novel target for treating OSCC.

Long non-coding RNA DLEUI promotes papillary thyroid carcinoma progression by sponging miR-421 and increasing ROCK1 expression.

We investigated the role of long non-coding RNA DLEU1 (deleted in lymphocytic leukemia 1) in the progression of papillary thyroid carcinoma (PTC). DLEU1 levels were higher in PTC cell lines (BHP5-16, TPC-1,8505C, and SW1736) and patient tissues (n=54) than in a human thyroid follicular epithelial cell line (Nthy-ori3-1) or adjacent normal thyroid tissues. High DLEU1 expression correlated positively with lymph node metastasis and advanced clinical stages in PTC patients. Bioinformatics, dual luciferase reporter, and RNA pulldown assays confirmed that DLEU1 directly binds to miR-421. Moreover, bioinformatics and dual luciferase reporter assays showed that miR-421 directly binds to the 3’untranslated region of the rho-related coiled-coil kinase 1 (ROCK1) in TPC-1 cells. PTC patient tissues and cell lines showed high ROCK1 mRNA and protein levels as well as low miR-421 levels. CCK-8, flow cytometry, wound healing, and Transwell invasion assays demonstrated that DLEU1 silencing decreases TPC-1 cell proliferation, survival and progression, but they can be rescued by miR-421 knockdown or ROCK1 overexpression. DLEU1 knockdown in TPC-1 cells decreased in vivo xenograft tumor size and weight compared to controls in nude mice. These findings demonstrate that DLEU1 promotes PTC progression by sponging miR-421 and increasing ROCK1 expression.

Exosomal-lncRNA DLEU1 Accelerates the Proliferation, Migration, and Invasion of Endometrial Carcinoma Cells by Regulating microRNA-E2F3.

PurposeLong non-coding RNAs (lncRNAs) may act as oncogenes in several cancers, including endometrial carcinoma (EC). The purpose of the current study is to investigate the regulatory mechanism of exosomal-lncRNA deleted in lymphocytic leukemia1 (DLEU1) on EC.MethodsThe expression levels of lncRNA DLEU1, microRNA-381-3p and E2F Transcription Factor 3 (E2F3) in EC tissues or cells were detected using quantitative reverse transcription–polymerase chain reaction (qRT-PCR). We then analysed the proliferation, migration, and invasion of EC cells by performing the MTT assay, wound healing assay, and transwell invasion assay, respectively. Identification of exosomes was detected using Western blot assay. The uptake of exosomes was detected by a confocal microscope. The effects of exosomes on EC cells were investigated by construction of cell co-culture system. The interactions among DLEU1, miR-381-3p and E2F3 were confirmed using the dual-luciferase reporter (DLR) assay.ResultsLncRNA DLEU1 expression was highly up-regulated in EC tissues and cells. Knockdown of DLEU1 inhibited the proliferation, migration, and invasion of EC cells. Exosomes could be uptaken by the ambient EC cells. MiR-381-3p was a target of DLEU1 and was negatively modulated by DLEU1. Overexpression of miR-381-3p suppressed the proliferation, migration, and invasion of EC cells. Additionally, E2F3 was the target gene of miR-381-3p and was negatively modulated by miR-381-3p. Upregulation of miR-381-3p and down-regulation of E2F3 reversed the promoting effect of exosomal DLEU1 on EC cells.ConclusionExosomal DLEU1 accelerates the development of EC by regulating the miR-381-3p/E2F3 axis, thus DLEU1 may act as a possible therapeutic target for treating EC.

LncRNA DLEU1/microRNA-300/RAB22A axis regulates migration and invasion of breast cancer cells.

To explore the possible role of deleted in lymphocytic leukemia 1 (DLEU1) in regulating the metastasis of breast cancer and its underlying mechanism. The expression levels of DLEU1 in 60 cases of breast cancer tissues and adjacent normal tissues were detected by quantitative Real Time-Polymerase Chain Reaction (qRT-PCR). Survival analysis and receiver operating characteristic (ROC) curves were introduced to analyze the correlation between DLEU1 expression and the clinical features of enrolled breast cancer patients. After altering expressions of DLEU1, RAB22A or microRNA-300 by plasmid transfection, the abilities of breast cancer cells to migrate and invade were evaluated by transwell assay. Western blot was conducted to detect protein level changes of epithelial-mesenchymal transition (EMT)-related genes regulated by DLEU1, RAB22A or microRNA-300. Dual-luciferase reporter gene assay was performed to detect the binding relation between microRNA-300 with DLEU1 and RAB22A. DLEU1 expression remains higher in breast cancer tissues than in normal adjacent tissues, and has a certain diagnostic value. The overall survival of breast cancer patients was negatively correlated with DLEU1 expression. Overexpression of DLEU1 or RAB22A, or microRNA-300 knockdown could enhance the migratory and invasive capacities, as well as increase EMT ofBT549 cells. On the contrary, knockdown of DLEU1 or RAB22A, or microRNA-300 overexpression in MCF-7 cells obtained the opposite trends of cellular behaviors. Dual-luciferase reporter gene assay confirmed that DLEU1 and RAB22A could bind to microRNA-300. Further verification showed that RAB22A expression was positively regulated by DLEU1, but negatively regulated by microRNA-300. Finally, we found that DLEU1 overexpression could reverse the inhibitory effects of microRNA-300 on proliferation, migration, and EMT of breast cancer cells. DLEU1 is highly expressed in breast cancer, which promotes migration, invasion, and EMT of breast cancer cells by targeting microRNA-300 to mediate RAB22A.

Long noncoding RNA DLEU1 aggravates glioma progression via the miR-421/MEF2D axis.

BackgroundLong noncoding RNA (lncRNA) deleted in lymphocytic leukemia 1 (DLEU1) was reported to be involved in the development and progression of multiple cancers. However, the accurate expression pattern, biological function and potential molecular mechanism of DLEU1 in glioma are not yet known. The present study investigated the role of DLEU in the development and progression of glioma, as well as the potential mechanism played by DLEU1 in glioma.Materials and methodsThe levels of DLEUI in glioma tissues and cell lines were examined using quantitative real-time PCR. The potential effects of DLEU1 on the proliferation, mobility, invasion and apoptosis of glioma cells were evaluated using corresponding in vitro experiments. The association between DLEU1 and microRNA (miR)-421 was also determined using luciferase reporter activity and RNA immunoprecipitation (RIP) assays.ResultsThe results revealed that DLEU1 was significantly upregulated in glioma tissues and cell lines. Increased DLEU1 was positively associated with the high-grade carcinoma (III–IV). Functional studies revealed that knockdown of DLEU1 expression by siRNA led to decreased proliferation, migration and invasion and increased apoptosis in human glioma cells. Furthermore, luciferase reporter activity and RIP assays confirmed that DLEUI could act as a competing endogenous RNA (ceRNA) for miR-421 that functioned as a tumor suppressor in glioma. Moreover, inhibition miR-421 partially restored the effect of DLEU1 knockdown on the glioma cells. DLEU1 could regulate myocyte enhancer factor 2D (MEF2D) expression, a known target of miR-421 in glioma cells.ConclusionTaken together, these findings suggested that DLEU1 regulated MEF2D expression to promote glioma progression by sponging miR-421 and that DLEU1 might be a potential therapeutic target for glioma.

Long non‑coding RNA DLEU1 promotes cell proliferation of glioblastoma multiforme.

Glioblastoma multiforme (GBM) is the most common malignant tumor with high morbidity and mortality. This study investigated the role of long non‑coding RNAs (lncRNAs) in glioblastomagenesis progression. Using the GSE2223 and GSE59612 datasets, and RNA sequencing data of GBM from The Cancer Genome Atlas, differentially expressed (DE) genes including DE messenger RNAs (DEmRNAs) and DElncRNAs between GBM and normal controls were identified. Based on the competing endogenous RNA hypothesis, DElncRNA‑micro RNA (miRNA)‑DEmRNA interactions were obtained by target gene prediction. Gene Ontology (GO) and Kyoto Encyclopedia of Gene and Genomes pathway analysis of DEmRNAs in the DElncRNA‑miRNA‑DEmRNA network was performed. Expression and function analyses of DElncRNAs were performed by reverse transcription‑polymerase chain reaction (RT‑PCR) and an established viability assay, respectively. In total, 712 DE genes were identified. Significant upregulation of lncRNA deleted in lymphocytic leukemia 1 (DLEU1) was revealed in GBM and a number of other types of cancer. DLEU1 interacted with 315 miRNAs and 105 DEmRNAs. The DEmRNAs were mainly enriched in tumorigenesis‑associated GO terms (angiogenesis, positive regulation of cell proliferation, positive regulation of fibroblast apoptotic processes and regulation of neutrophil migration) and pathways (Hippo signaling pathway, cancer pathways, and Wnt signaling pathway). Correlation analysis revealed that mRNA TNF receptor associated factor 4 (TRAF4) was associated with DLEU1 expression. RT‑PCR demonstrated that the expression levels of DLEU1 and TRAF4 were increased in GBM tissues. Small interfering RNA demonstrated that silencing DLEU1 downregulated TRAF4. The viability of GBM cells was significantly decreased following RNA interference with DLEU1 and TRAF4 production. The results demonstrate that DLEU1 and TRAF4 is highly expressed in GBM tissues and promotes proliferation of GBM cells. It may act as a competing endogenous RNA and influence tumorigenesis of GBM.

Long noncoding RNA DLEU1 aggravates pancreatic ductal adenocarcinoma carcinogenesis via the miR-381/CXCR4 axis.

Recent evidence has highlighted that long noncoding RNAs (lncRNA) are associated with many diseases, particularly cancer. However, current understanding of the lncRNA deleted in lymphocytic leukemia 1 (DLEU1) in pancreatic ductal adenocarcinoma (PDAC) remains limited. Our studies indicated that the DLEU1 expression level was upregulated in PDAC tissue samples compared with adjacent normal tissue. Moreover, the aberrant overexpression of DLEU1 indicated poor prognosis of patients with PDAC. Loss-of-function experiments revealed that DLEU1 knockdown inhibited the proliferation, migration, and invasion of PDAC cells in vitro and decreased tumor growth in vivo. Bioinformatics analysis predicted that miR-381 potentially targeted the DLEU1 3'-untranslated region (UTR), suggesting an interaction between miR-381 and DLEU1. Furthermore, miR-381 also targeted the chemokine receptor-4 (CXCR4) messenger RNA 3'-UTR, which was validated by luciferase reporter assay. Taken together, our study demonstrated the oncogenic role of DLEU1 in clinical PDAC specimens and cellular experiments, showing the potential involvement of DLEU1/miR-381/CXCR4 pathway. These results provide novel insight into PDAC tumorigenesis.

Screening for long noncoding RNAs associated with oral squamous cell carcinoma reveals the potentially oncogenic actions of DLEU1

Recent studies have shown that long noncoding RNAs (lncRNAs) have pivotal roles in human malignancies, although their significance in oral squamous cell carcinoma (OSCC) is not fully understood. In the present study, we identified lncRNAs functionally associated with OSCC. By analyzing RNA-seq datasets obtained from primary head and neck squamous cell carcinoma (HNSCC), we identified 15 lncRNAs aberrantly expressed in cancer tissues. We then validated their expression in 18 OSCC cell lines using qRT-PCR and identified 6 lncRNAs frequently overexpressed in OSCC. Among those, we found that knocking down DLEU1 (deleted in lymphocytic leukemia 1) strongly suppressed OSCC cell proliferation. DLEU1 knockdown also suppressed migration, invasion, and xenograft formation by OSCC cells, which is suggestive of its oncogenic functionality. Microarray analysis revealed that DLEU1 knockdown significantly affects expression of a number of cancer-related genes in OSCC cells, including HAS3, CD44, and TP63, suggesting that DLEU1 regulates HA-CD44 signaling. Expression of DLEU1 was elevated in 71% of primary OSCC tissues, and high DLEU1 expression was associated with shorter overall survival of HNSCC patients. These data suggest that elevated DLEU1 expression contributes to OSCC development, and that DLEU1 may be a useful therapeutic target in OSCC.

The effects of DLEU1 gene expression in Burkitt lymphoma (BL): potential mechanism of chemoimmunotherapy resistance in BL.

Following a multivariant analysis we demonstrated that children and adolescents with Burkitt lymphoma (BL) and a 13q14.3 deletion have a significant decrease in event free survival (EFS) despite identical short intensive multi-agent chemotherapy. However, how this deletion in the 13q14.3 region is associated with a significant decrease in EFS in children and adolescents with BL is largely unknown. The gene Deleted in Lymphocytic Leukemia 1 (DLEU1) is located in the region of 13q14.3. Here, we report that DLEU1 expression is implicated in the regulation of BL programmed cell death, cell proliferation, and expression of apoptotic genes in transcription activator-like effector nuclease (TALEN)s-induced DLEU1 knockdown and DLEU1 overexpressing BL cell lines. Furthermore, NSG mice xenografted with DLEU1 knockdown BL cells had significantly shortened survival (p < 0.05 and p < 0.005), whereas those xenografted with DLEU1 overexpressing BL cells had significantly improved survival (p < 0.05 and p < 0.0001), following treatment with rituximab and/or cyclophosphamide. These data suggest that DLEU1 may in part function as a tumor suppressor gene and confer chemoimmunotherapy resistance in children and adolescents with BL.

Abstract 3838: Upregulation of DLEU1 significantly prolongs 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

Abstract 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., BJHaem 2012). We identified that pediatric BL patients with chromosome 13q deletion, particularly 13q14.3, had significantly poorer outcome and inferior overall survival 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 in the 13q14.3 region (Dave et al., NEJM 2006) and interacts with C-MYC, TUBB2C and UBR1 (Stelzl et al., Cell 2005). We have previously reported a regulated DLEU1 expression significantly associated with changes in BL proliferation in vitro (Lee/Cairo et al., AACR 2015). We hypothesize that DLEU1 may have function as a tumor suppressor gene; however, functions and mechanisms underlying DLEU1 expression in the BL are poorly understood. OBJECTIVES: We hypothesize that DLEU1 may act as a tumor suppressor gene in BL and therefore examined whether up-regulation of DLEU1 results in changes in survival following targeted immunotherapy. METHODS: DLEU1 stably overexpressing Raji BL cells (Lee/Cairo et al., ASH 2013) were transfected with a firefly luciferase expression plasmid, kindly provided by Laurence Cooper MD, PhD. Four- to six- week-old female NSG (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ) mice from The Jackson Lab. Tumor burden and tumor progression were monitored by bioluminescence imaging system. Mice were treated with either rituximab (30 mg/kg) or cyclophosphamide (25 mg/kg) by intraperitoneal injection at 7 day intervals. Survival rates were analyzed by the Kaplan-Meier method using the Prism Version 6.0 software. RESULTS: We observed that DLEU1 stably overexpressing Raji BL cells xenografted (GFP-DLEU1) mice had significantly extended survival compared to GFP control mice following treatment rituximab (59 vs 51 days, p&amp;lt;0.05, n = 8), cyclophosphamide (50.5 vs 37.5 days, p&amp;lt;0.0001, n = 12) and in combination treatment (60.5 vs 53 days, p&amp;lt;0.05, n = 12) whereas there were no significant differences in survival between WT and DLEU1-KO mice with PBS treatment. We observed that there were no significant differences of luciferase intensity in between GFP and GFP-DLEU1 mice following rituximab, cyclophosphamide or in combination treatment. CONCLUSIONS: We demonstrated that the upregulation of DLEU1 expression significantly improved the survival in DLEU1 overexpressing BL cells xenografted NSG mice following rituximab, cyclophosphamide and in combination treatment. Therefore, the up-regulation of DLEU1 expression in BL may in part result in sensitizing to chemoimmunotherapy induced survival and may result in a consideration of potential therapeutic strategies Citation Format: Sanghoon Lee, Changhong Yin, Tishi Shah, Janet Ayello, Erin Morris, Carmella van de Ven, Mitchell S. Cairo. Upregulation of DLEU1 significantly prolongs 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. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3838.

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-PrkdcscidIl2rgtm1Wjl/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 vs 48 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. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Abstract 2077: DLEU1 significantly alters programmed cell death in chemoimmunotherapy-treated TALENs-induced DLEU1 knockout and DLEU1 overexpressing Burkitt Lymphoma (BL): DLEU1 may act as a tumor suppressor gene in pediatric BL

Abstract BACKGROUND: Burkitt Lymphoma (BL) is the most common histological subtype of childhood and adolescent Non Hodgkin Lymphoma (Cairo et al, Blood 2007; Miles/Cairo, BJH 2012). Pediatric patients with BL with a chromosome 13q deletion, particularly 13q14.3, had significantly poorer outcome and inferior overall survival despite aggressive short, intensive multi-agent chemotherapy (Poirel/Cairo et al, Leukemia 2009; Nelson/Cairo/Sanger et al, BJH 2009). Deleted in Lymphocytic Leukemia 1 (DLEU1) is a BL classifier gene in the 13q14.3 (Dave et al, NEJM 2006) and it has been implicated in regulating programmed cell death in patients. Sequence-specific Transcription Activator-Like Effector Nucleases (TALENs) have been developed for precision targeted genome editing in in vitro and in vivo (Sander et al, Nat Bio 2011). OBJECTIVES: We hypothesize that DLEU1 may act as a tumor suppressor gene and therefore investigated whether DLEU1 expression results in changes in gene expression profiles, cell viability, apoptosis induced by chemoimmunotherapy regimens in DLEU1 knockout and/or DLEU1 overexpressing Raji BL cells. METHODS: TALEN-mediated DLEU1 knockout (DLEU1-KO) and DLEU1 overexpressing Raji BL cells (DLEU1-OE) were treated with Rituximab (RTX) and/or Cyclophosphamide (CTX). MTS, Caspase assay, qRT-PCR, western blot, and gene expression profiling using Affymetrix HG-133+ 2.0 arrays were performed. RESULTS: A significant decrease of apoptosis in DLEU1-KO (32.5±11.8%, p&amp;lt;.05) and a significant increase in DLEU1-OE (9.7±5.5%, p&amp;lt;.05) were observed vs control. A total 2,501 differentially expressed genes were identified by gene expression profiling (&amp;gt;2 fold, p&amp;lt;.05) and 79.8% of genes including signaling pathways and anti-apoptosis related genes including ATK1, ikBa and Bcl-2 were upregulated in DLEU1-KO.. There was a significant decrease in apoptosis with RTX, CTX and in combination (0.85±0.05, 0.76±0.09, and 0.65±0.17, p&amp;lt;.05, respectively) in DLEU1-KO whereas a significant increase with RTX (11.5%, p&amp;lt;.05), CTX (13.9%, p&amp;lt;.05) in DLEU1-OE. A significant increase in cell proliferation was observed with RTX, CTX and combination (1.14±0.06, 1.1±0.06, and 1.09±0.02, p&amp;lt;.05) in DLEU1-KO whereas a significant decrease with RTX, CTX and in combination (0.92±0.01, 0.89±0.04, and 0.87±0.04, p&amp;lt;.05, respectively) in DLEU1-OE vs control. CONCLUSIONS: We demonstrate that 1) DLEU1 knockout resulted in significant changes of ikBa and Akt1 expression, inhibition of apoptosis and increase of viability in RTX and CTX treatment; 2) DLEU1 overexpression significantly inhibited cell viability and increased apoptosis in RTX and CTX treatment. We hypothesize that deletion of DLEU1 in pediatric BL may in part result in chemotherapy resistance secondarily to a loss of a tumor suppressor gene. Citation Format: Sanghoon Lee, Changhong Yin, Janet Ayello, Carmella van de Ven, Mitchell S. Cairo. DLEU1 significantly alters programmed cell death in chemoimmunotherapy-treated TALENs-induced DLEU1 knockout and DLEU1 overexpressing Burkitt Lymphoma (BL): DLEU1 may act as a tumor suppressor gene in pediatric BL. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2077. doi:10.1158/1538-7445.AM2015-2077

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

BackgroundPediatric 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. ObjectivesWe 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. MethodsA 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. ResultsThe 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). [Display omitted] [Display omitted] ConclusionThese 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.