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BackgroundThe potential therapeutic efficacy of daratumumab in natural killer T-cell lymphoma (NKTL) was highlighted when its off-label usage produced sustained remission in a patient with highly refractory disease. This is corroborated recently by a phase II clinical trial which established that daratumumab monotherapy is well tolerated and displayed encouraging response in relapsed/refractory NKTL patients. However, little is known regarding the molecular factors central to the induction and regulation of the daratumumab-mediated antitumor response in NKTL.MethodsCD38 expression was studied via immunohistochemistry, multiplex immunofluorescence and correlated with clinical characteristics of the patient. The therapeutic efficacy of daratumumab was studied in vitro via CellTiter-Glo (CTG) assay, complement-dependent cytotoxicity (CDC), antibody-dependent cell cytotoxicity (ADCC), and in vivo, via a patient-derived xenograft mouse model of NKTL, both as a single agent and in combination with L-asparaginase. Signaling mechanisms were characterized via pharmacologic treatment, RNA silencing, flow cytometry and corroborated with public transcriptomic data of NKTL.ResultsEpstein-Barr virus-positive NKTL patients significantly express CD38 with half exhibiting high expression. Daratumumab effectively triggers Fc-mediated ADCC and CDC in a CD38-dependent manner. Importantly, daratumumab monotherapy and combination therapy with L-asparaginase significantly suppresses tumor progression in vivo. Ablation of complement inhibitory proteins (CIP) demonstrate that CD55 and CD59, not CD46, are critical for the induction of CDC. Notably, CD55 and CD59 expression were significantly elevated in the late stages of NKTL. Increasing the CD38:CIP ratio through sequential CIP knockdown, followed by CD38 upregulation via All-Trans Retinoic Acid treatment, potently augments complement-mediated lysis in cells previously resistant to daratumumab. The CD38:CIP ratio consistently demonstrates a statistically superior correlation to antitumor efficacy of daratumumab than CD38 or CIP expression alone.ConclusionThis study characterizes CD38 as an effective target for a subset of NKTL patients and the utilization of the CD38:CIP ratio as a more robust identifier for patient stratification and personalisation of treatment. Furthermore, elucidation of factors which sensitize the complement-mediated response provides an alternative approach toward optimizing therapeutic efficacy of daratumumab where CDC remains a known limiting factor. Altogether, these results propose a strategic rationale for further evaluation of single or combined daratumumab treatment in the clinic for NKTL.

RUNX3, runt-domain transcription factor, is a master regulator of gene expression in major developmental pathways. It acts as a tumor suppressor in many cancers but is oncogenic in certain tumors. We observed upregulation of RUNX3 mRNA and protein expression in nasal-type extranodal natural killer (NK)/T-cell lymphoma (NKTL) patient samples and NKTL cell lines compared to normal NK cells. RUNX3 silenced NKTL cells showed increased apoptosis and reduced cell proliferation. Potential binding sites for MYC were identified in the RUNX3 enhancer region. Chromatin immunoprecipitation-quantitative PCR revealed binding activity between MYC and RUNX3. Co-transfection of the MYC expression vector with RUNX3 enhancer reporter plasmid resulted in activation of RUNX3 enhancer indicating that MYC positively regulates RUNX3 transcription in NKTL cell lines. Treatment with a small-molecule MYC inhibitor (JQ1) caused significant downregulation of MYC and RUNX3, leading to apoptosis in NKTL cells. The growth inhibition resulting from depletion of MYC by JQ1 was rescued by ectopic MYC expression. In summary, our study identified RUNX3 overexpression in NKTL with functional oncogenic properties. We further delineate that MYC may be an important upstream driver of RUNX3 upregulation and since MYC is upregulated in NKTL, further study on the employment of MYC inhibition as a therapeutic strategy is warranted.

We performed gene expression profiling in Epstein-Barr virus (EBV)-associated T/natural killer (NK)-cell lymphoproliferative disorder in children and young adults (TNKLPDC) in order to understand the molecular pathways deregulated in this disease and compared it with nasal-type NK/T-cell lymphoma (NKTL). The molecular and phenotypic signature of TNKLPDC is similar to NKTL, with overexpression of p53, survivin and EZH2. Down-regulation of EZH2 in TNKLPDC cell lines led to an increase in apoptosis and decrease in tumor viability, suggesting that EZH2 may be important for the survival of TNKLPDC cells and hence potentially a useful therapeutic target. Notably, our gene expression profiling revealed a distinctive enrichment of stem cell related genes in TNKLPDC compared to NKTL. This was validated by a significantly higher expression of aldehyde dehydrogenase 1 (ALDH1) in TNKLPDC cell lines compared to NKTL cell lines. The novel discovery of cancer stem cell properties in TNKLPDC has potential therapeutic implications in this group of disorders.

The best-understood mechanism by which EZH2 exerts its oncogenic function is through polycomb repressive complex 2 (PRC2)-mediated gene repression, which requires its histone methyltransferase activity. However, small-molecule inhibitors of EZH2 that selectively target its enzymatic activity turn out to be potent only for lymphoma cells with EZH2-activating mutation. Intriguingly, recent discoveries, including ours, have placed EZH2 into the category of transcriptional coactivators and thus raised the possibility of noncanonical signaling pathways. However, it remains unclear how EZH2 switches to this catalytic independent function. In the current study, using natural killer/T-cell lymphoma (NKTL) as a disease model, we found that phosphorylation of EZH2 by JAK3 promotes the dissociation of the PRC2 complex leading to decreased global H3K27me3 levels, while it switches EZH2 to a transcriptional activator, conferring higher proliferative capacity of the affected cells. Gene expression data analysis also suggests that the noncanonical function of EZH2 as a transcriptional activator upregulates a set of genes involved in DNA replication, cell cycle, biosynthesis, stemness, and invasiveness. Consistently, JAK3 inhibitor was able to significantly reduce the growth of NKTL cells, in an EZH2 phosphorylation-dependent manner, whereas various compounds recently developed to inhibit EZH2 methyltransferase activity have no such effect. Thus, pharmacological inhibition of JAK3 activity may provide a promising treatment option for NKTL through the novel mechanism of suppressing noncanonical EZH2 activity.

We performed a comprehensive genome-wide miRNA expression profiling of extranodal nasal-type natural killer/T-cell lymphoma (NKTL) using formalin-fixed paraffin-embedded tissue (n = 30) and NK cell lines (n = 6) compared with normal NK cells, with the objective of understanding the pathogenetic role of miRNA deregulation in NKTL. Compared with normal NK cells, differentially expressed miRNAs in NKTL are predominantly down-regulated. Re-expression of down-regulated miRNAs, such as miR-101, miR-26a, miR26b, miR-28-5, and miR-363, reduced the growth of the NK cell line and modulated the expression of their predicted target genes, suggesting the potential functional role of the deregulated miRNAs in the oncogenesis of NKTL. Taken together, the predicted targets whose expression is inversely correlated with the expression of deregulated miRNA in NKTL are significantly enriched for genes involved in cell cycle-related, p53, and MAPK signaling pathways. We also performed immunohistochemical validation for selected target proteins and found overexpression of MUM1, BLIMP1, and STMN1 in NKTL, and notably, a corresponding increase in MYC expression. Because MYC is known to cause repression of miRNA expression, it is possible that MYC activation in NKTL may contribute to the suppression of the miRNAs regulating MUM1, BLIMP1, and STMN1.

We performed comprehensive genome-wide gene expression profiling (GEP) of extranodal nasal-type natural killer/T-cell lymphoma (NKTL) using formalin-fixed, paraffin-embedded tissue (n = 9) and NK cell lines (n = 5) in comparison with normal NK cells, with the objective of understanding the oncogenic pathways involved in the pathogenesis of NKTL and to identify potential therapeutic targets. Pathway and network analysis of genes differentially expressed between NKTL and normal NK cells revealed significant enrichment for cell cycle-related genes and pathways, such as PLK1, CDK1, and Aurora-A. Furthermore, our results demonstrated a pro-proliferative and anti-apoptotic phenotype in NKTL characterized by activation of Myc and nuclear factor kappa B (NF-κB), and deregulation of p53. In corroboration with GEP findings, a significant percentage of NKTLs (n = 33) overexpressed c-Myc (45.4%), p53 (87.9%), and NF-κB p50 (67.7%) on immunohistochemistry using a tissue microarray containing 33 NKTL samples. Notably, overexpression of survivin was observed in 97% of cases. Based on our findings, we propose a model of NKTL pathogenesis where deregulation of p53 together with activation of Myc and NF-κB, possibly driven by EBV LMP-1, results in the cumulative up-regulation of survivin. Down-regulation of survivin with Terameprocol (EM-1421, a survivin inhibitor) results in reduced cell viability and increased apoptosis in tumour cells, suggesting that targeting survivin may be a potential novel therapeutic strategy in NKTL.

Complement-dependent cytotoxicity (CDC) is one of the major mechanisms mediating the anti-tumor efficacy of Daratumumab. We have previously demonstrated that a majority of Natural Killer/T- Cell Lymphoma (NKTL) patient samples express CD38 and Daratumumab is highly effective against NKTL cell lines expressing mid-high levels of CD38. In this report we show that subsequent testing in an NKTL mouse xenograft model confirms the potency of Daratumumab in vivo as evidenced by the inhibition in tumour progression and prolongation of mouse survival. When treatment was continued over a month, some tumors began to rapidly enlarge ('Resistant') while the rest remained similar or smaller ('Sensitive') than the tumour volume at the initiation of Daratumumab treatment. An mRNA analysis comparing 'Resistant' and 'Sensitive' tumors showed that while both tumours bore similar levels of CD38 expression, resistant tumours displayed an upregulation of complement inhibitory proteins (CIP), CD55 and CD59 but not CD46. This led us to hypothesize that CD59 and CD55 may play a critical role in Daratumumab-mediated CDC in NKTL. FACS analyses demonstrated that the number of membrane molecules of CD55 and CD59 appeared inversely correlated to Daratumumab-mediated CDC. A single CIP knockdown was first performed to delineate the role of each CIP. Silencing CD46 confirmed that it does not have any effect on CDC in NKTL. However, single knockdown of CD55 or CD59 was able to induce cytotoxicity in CDC-resistant cell line CD38midCD55hiCD59lo NKYS, and promote NKS1 CD38hiCD55hiCD59mid to further lysis. Both single and double knockdown of CD55 and CD59 could not enhance Daratumumab-induced CDC in CD38loCD55hiCD59hi HuT78 which recapitulates the importance of CD38 levels. Unexpectedly, the double knockdown did not sensitize CD38hiCD55hiCD59hi KMS12BM either. This led us to conjecture that it may be the ratio of CD38:CIPs which is predictive of response to Daratumumab than CD38 or CIPs alone. All-Trans Retinoic Acid (ATRA) binds the RARE element in CD38 gene leading to upregulation of mRNA and protein expression of CD38. We thus downregulated the expression of CIPs with siRNA followed by amplification of CD38 expression with ATRA in NKTL. This strategy resulted in a significant increase in the CD38:CIP ratio and induced almost a total lysis of NKS1 cells, as well as sensitised HuT78 to a massive amount of Daratumumab-mediated CDC. These experiments suggest that by increasing the CD38: CIP, ratio we can overcome resistance to Daratumumab-mediated CDC. To further statistically study this, a Spearman's rank correlation analyses was performed. The Spearman correlation coefficient shows that the number of surface molecules of CD38 positively correlates to CDC while that of CD55 displays an inverse correlation. CD46 and CD59 do not show any significant correlation. Notably, when correlating the CD38:CIP ratio instead to CDC, the CD38:CD46, CD38:CD55 and CD38:CD59 ratios always show a significant positive correlation coefficient. This suggests that the potential efficacy of Daratumumab can be predicted more accurately based on the ratio of CD38:CIP than any of the molecules alone. Detection of a low CD38:CIP ratio in patient samples could be a biomarker for potentially poorer response to Daratumumab treatment. Daratumumab-resistant NKTL cell lines are being developed in our lab and RNA sequencing comparing sensitive and resistance cells will be subsequently performed in order to gain further insights to mechanisms that may lead to resistance. Preliminary analyses on CD38 and CIP expression so far has shown that CD38 protein and mRNA expression are prominently downregulated in resistant cell lines while the level of CIPs remain similar or increased. The total outcomes of these studies will contribute valuable insights to clinical trials that currently involve Daratumumab treatment. Disclosures Zhou: Janssen R&D: Employment. Yang:Janssen R&D: Employment. Chng:Celgene: Consultancy, Honoraria, Other: Travel, accommodation, expenses, Research Funding; Takeda: Consultancy, Honoraria, Other: Travel, accommodation, expenses; Janssen: Consultancy, Honoraria, Other: Travel, accommodation, expenses, Research Funding; Aslan: Research Funding; Amgen: Consultancy, Honoraria, Other: Travel, accommodation, expenses; Merck: Research Funding.

Background EBV-associated T/NK-cell lymphoproliferative diseases (TNKLPD) is a rare spectrum of disease that occurs more commonly in Asia, and Central and South America. It commonly affects children and young adults and is an aggressive disease that is poorly understood with no known biologic markers that can predict prognosis. The systemic form of TNKLPD includes chronic active EBV infection of T/NK type, aggressive NK cell leukemia and systemic EBV + T-cell lymphoproliferative disease of childhood. Methods In this study, we analyse the clinicopathologic and genetic features of 22 cases of systemic TNKLPD in non-immunocompromised patients, including chronic active EBV infection of T/NK cell type and systemic EBV + T-cell lymphoproliferative disease of childhood. We also performed gene expression profiling in a subset of cases to identify markers that may be of prognostic relevance and validated our results using immunohistochemistry. Results The median age is 14.9 years and two of our 22 cases occurring in patients older than 30 years. Fifteen of 17 cases (88%) with adequate data were of T-cell origin. Eleven of 22 cases revealed polymorphic cellular infiltrate (P-group) while the rest showed monomorphic lymphoid infiltrate (M-group). We found a significant difference in survival between P-group vs M-group patients with median survival not yet reached in P-group, and 1 month in M-group (p = 0.0001), suggesting a role for morphology in predicting patient outcome. We also performed gene expression profiling in a subset of patients and compared the genes differentially expressed between P-group and M-group cases to identify markers of prognostic value. We identified cyclin E2 gene and protein to be differentially expressed between patients with good outcome (P-group, median expression 8%) and poor outcome (M-group, median expression 42%) (p = 0.0005). In addition, the upregulation of cyclin E2 protein in M-group cases correlated with a higher Ki67 proliferation rate (Pearson correlation r = 0.73, p = 0.0006) detected by immunohistochemistry. High cyclin E2 expression was also significantly associated with shorter survival (p = 0.0002). Conclusion Our data suggests the potential role of monomorphic morphology, high cyclin E2 and Ki67 expression as adverse prognostic factors for TNKLPD. Electronic supplementary material The online version of this article (doi:10.1186/s13023-014-0165-x) contains supplementary material, which is available to authorized users.

The role of enhancer of zeste homolog 2 (EZH2) in cancer is complex and may vary depending on the cellular context. We found that EZH2 is aberrantly overexpressed in the majority of natural killer/T-cell lymphoma (NKTL), an aggressive lymphoid malignancy with very poor prognosis. We show that EZH2 upregulation is mediated by MYC-induced repression of its regulatory micro RNAs and EZH2 exerts oncogenic properties in NKTL. Ectopic expression of EZH2 in both primary NK cells and NKTL cell lines leads to a significant growth advantage. Conversely, knock-down of EZH2 in NKTL cell lines results in cell growth inhibition. Intriguingly, ectopic EZH2 mutant deficient for histone methyltransferase activity is also able to confer growth advantage and rescue growth inhibition on endogenous EZH2 depletion in NKTL cells, indicating an oncogenic role of EZH2 independent of its gene-silencing activity. Mechanistically, we show that EZH2 directly promotes the transcription of cyclin D1 and this effect is independent of its enzymatic activity. Furthermore, depletion of EZH2 using a PRC2 inhibitor 3-deazaneplanocin A significantly inhibits growth of NK tumor cells. Therefore, our study uncovers an oncogenic role of EZH2 independent of its methyltransferase activity in NKTL and suggests that targeting EZH2 may have therapeutic usefulness in this lymphoma.

Following acute-phase infection, activated T cells are terminated to achieve immune homeostasis, failure of which results in lymphoproliferative and autoimmune diseases. We report that sterile α- and heat armadillo-motif-containing protein (SARM), the most conserved Toll-like receptors adaptor, is proapoptotic during T-cell immune response. SARM expression is significantly reduced in natural killer (NK)/T lymphoma patients compared with healthy individuals, suggesting that decreased SARM supports NK/T-cell proliferation. T cells knocked down of SARM survived and proliferated more significantly compared with wild-type T cells following influenza infection in vivo. During activation of cytotoxic T cells, the SARM level fell before rising, correlating inversely with cell proliferation and subsequent T-cell clearance. SARM knockdown rescued T cells from both activation- and neglect-induced cell deaths. The mitochondria-localized SARM triggers intrinsic apoptosis by generating reactive oxygen species and depolarizing the mitochondrial potential. The proapoptotic function is attributable to the C-terminal sterile alpha motif and Toll/interleukin-1 receptor domains. Mechanistically, SARM mediates intrinsic apoptosis via B cell lymphoma-2 (Bcl-2) family members. SARM suppresses B cell lymphoma-extra large (Bcl-xL) and downregulates extracellular signal-regulated kinase phosphorylation, which are cell survival effectors. Overexpression of Bcl-xL and double knockout of Bcl-2 associated X protein and Bcl-2 homologous antagonist killer substantially reduced SARM-induced apoptosis. Collectively, we have shown how T-cell death following infection is mediated by SARM-induced intrinsic apoptosis, which is crucial for T-cell homeostasis., Singapore-MIT Alliance Computational and Systems Biology Flagship Project

We performed comprehensive genome-wide gene expression profiling (GEP) of extranodal nasal-type natural killer/T-cell lymphoma (NKTL) using formalin-fixed, paraffin-embedded tissue (n = 9) and NK cell lines (n = 5) in comparison with normal NK cells, with the objective of understanding the oncogenic pathways involved in the pathogenesis of NKTL and to identify potential therapeutic targets. Pathway and network analysis of genes differentially expressed between NKTL and normal NK cells revealed significant enrichment for cell cycle-related genes and pathways, such as PLK1, CDK1, and Aurora-A. Furthermore, our results demonstrated a pro-proliferative and anti-apoptotic phenotype in NKTL characterized by activation of Myc and nuclear factor kappa B (NF-κB), and deregulation of p53. In corroboration with GEP findings, a significant percentage of NKTLs (n = 33) overexpressed c-Myc (45.4%), p53 (87.9%), and NF-κB p50 (67.7%) on immunohistochemistry using a tissue microarray containing 33 NKTL samples. Notably, overexpression of survivin was observed in 97% of cases. Based on our findings, we propose a model of NKTL pathogenesis where deregulation of p53 together with activation of Myc and NF-κB, possibly driven by EBV LMP-1, results in the cumulative up-regulation of survivin. Down-regulation of survivin with Terameprocol (EM-1421, a survivin inhibitor) results in reduced cell viability and increased apoptosis in tumour cells, suggesting that targeting survivin may be a potential novel therapeutic strategy in NKTL.

Purpose: To characterize pregnane X receptor (PXR) polymorphic variants in healthy Asian populations [Chinese, Malay and Indian (n = 100 each)], and to investigate the association between PXR haplotypes and hepatic mRNA expression of PXR and its downstream target genes, CYP3A4 and ABCB1, as well as their influence on the clearance of doxorubicin in Asian breast cancer patients. Experimental Design: PXR genotyping was done by direct DNA sequencing, and PXR haplotypes and haplotype clusters were derived by expectation-maximization algorithm. Genotype-phenotype correlations were done using Mann-Whitney U test and Kruskal-Wallis test. Results: Significant interethnic variations were observed in PXR pharmacogenetics among the three Asian ethnic groups. The expression of PXR mRNA in liver tissues harboring the PXR*1B haplotype clusters was 4-fold lower compared with the non-PXR*1B (*1A + *1C) haplotype clusters [PXR*1B versus PXR*1A; P = 0.015; PXR*1B versus PXR*1C; P = 0.023]. PXR*1B-bearing liver tissues were associated with significantly lower expression of CYP3A4 (PXR*1B versus non-PXR*1B, P = 0.030) and ABCB1 (PXR*1B versus non-PXR*1B, P = 0.060) compared with non–PXR*1B-bearing liver tissues. Doxorubicin clearance in breast cancer patients harboring the PXR*1B haplotypes was significantly lower compared with patients carrying the non-PXR*1B haplotypes [PXR*1B versus non-PXR*1B, CL/BSA (L h−1 m−2): 20.84 (range, 8.68-29.24) versus 24.85 (range, 13.80-55.66), P = 0.022]. Conclusions: This study showed that PXR*1B was associated with reduced hepatic mRNA expression of PXR and its downstream targets, CYP3A4 and ABCB1. Genotype-phenotype correlates in breast cancer patients showed PXR*1B to be significantly associated with lower doxorubicin clearance, suggesting that PXR haplotype constitution could be important in influencing interindividual and interethnic variations in disposition of its putative drug substrates.

Abstract 3498 Nasal-type Natural Killer/T-cell lymphoma (NKTL) is an aggressive lymphoid malignancy associated with very poor survival. A better understanding of the molecular abnormalities underlying this disease will lead to a better therapy. We recently performed whole genome gene expression studies and identify a number genes that are differentially expressed in NKTL as well as pathways which are activated in NKTL. EZH2, one of the genes identified in our study to be aberrantly over-expressed in NKTL, is a H3K27-specific histone methyltransferase and a component of the polycomb repressive complex 2 (PRC2), which plays a key role in the epigenetic maintenance of repressive chromatin mark. To the best of our knowledge, the mechanism of EZH2 overexpression in NKTL has not yet been described. In this study, we showed that EZH2 overexpression in NKTL can be attributed to a deregulated MYC-miRNA-EZH2 regulatory axis where MYC activation represses miRNAs that normally downregulate EZH2. He functionally demonstrated this relationship using NKTL cell lines while also demonstrating the correlation between MYC activation and EZH2 expression in clinical samples through the analysis of gene expression data as well as histological detection of nuclear MYC and EZH2 protein using a tissue microarray containing 35 NKTL clinical samples using immunohistochemistry. We then investigated the functional role of EZH2 in NKTL. We showed that ectopic overexpression of EZH2 in both primary NK cells and NK cell lines led to a significant growth advantage. Conversely, knock-down of EZH2 in NK cell lines resulted in growth inhibition of tumor cells. Intriguingly, ectopic EZH2 mutant deficient for histone methyltransferase activity was also able to confer growth advantage and rescue the growth inhibition upon endogenous EZH2 depletion in NKTL cells, indicating an oncogenic role of EZH2 independent of its gene silencing activity. Indeed, EZH2 expression in clinical NKTL samples is associated with higher Ki67 staining implying a role in driving NKTL proliferation. We further demonstrated that EZH2 directly binds to the gene promoter of Cyclin D1 and EZH2 promotes the transcription of Cyclin D1 independent of its enzymatic activity. Consistent with its potential oncogenic role, depletion of EZH2 using an inhibitor called DZNep also induced significant growth inhibition in NKTL cells. Taken together, our study demonstrates an unconventional role of EZH2 in promoting oncogenic growth in NKTL and provides novel insights into the oncogenic function of EZH2 in human cancers. The pro-proliferative properties of EZH2 in NKTL support the rationale for using of EZH2 inhibitors in the treatment of NKTL. However, it is important to note that in some tumor, EZH2 may be mediating its oncogenic functions through non-enzymatic mechanism. This has critical implications on the choice of specific inhibitors of its enzymatic function or compounds that can deplete EZH2 as the most appropriate therapeutic approach. Since targeting of EZH2 is an active area of drug development at present, there is great potential for the development of better treatment modalities and this is especially important for aggressive cancers, such as NKTL, for which no effective curative treatment is currently available. Disclosures: No relevant conflicts of interest to declare.

Abstract 2637 Background: Extranodal nasal-type Natural Killer/T-cell lymphoma (NKTL) is a relatively rare but aggressive type of non-Hodgkins lymphoma that is more prevalent in Asia. The outcome of patients with disseminated stage is universally fatal. Progress in therapy has been slow and is based on combination of chemotherapy. MicroRNA are short non-coding RNA sequences that could regulate the expression of a large number of genes by inhibiting translation or leading to mRNA degradation. It has been implicated in tumorigenesis and has prognostic value across a wide range of malignancies including haematologic malignancies. We performed a comprehensive genome-wide miRNA expression profiling (MEP) of NKTL to identify deregulated miRNA and their potential role in NKTL biology. Method: MEP was performed using the Agilent human miRNA Microarray V2 (Agilent Technologies, Santa Clara, CA) on formalin fixed paraffin-embedded tissue (FFPE) (n=30) and NK cell lines (n=6) in comparison with normal NK cells. Differential expressed miRNA were identified using fold change and Significance Analysis of Microarray (SAM). Some of the differentially expressed miRNA were validated using quantitative polymerase chain reaction (q-PCR). The functional relevance of candidate miRNAs are assessed using miRNA mimics or inhibitors, and observing for apoptosis and growth arrest in the cell lines. Potential targets of candidate miRNAs are identified using predictive algorithms and significant negative correlation with gene expression data. The strongest candidate target genes are further verified using luciferase assay and q-PCR. miRNA and target gene relationship was further confirmed in the patients samples using immunohistochemistry for the protein expression on tissue microarray of NKTL. Results: Compared to normal NK cells, differentially expressed miRNAs in NKTL are predominantly downregulated. Re-expression of downregulated miRNAs, such as mir-101, mir-26a, mir26b, mir-28-5 and mir-363, reduced the growth of NK cell line and modulated the expression of their predicted target genes, suggesting the potential functional role of the deregulated miRNAs in the oncogenesis of NKTL. Taken together, the predicted targets whose expression are inversely correlated with the expression of deregulated miRNA in NKTL are significant enriched for genes involved in cell cycle-related, p53 and MAPK signaling pathways. We validated and confirmed the regulation of STMN1, and BLIMP1 by miR-101 and miR-30b respectively. In addition, miR-101, miR26a and miR-26b also affect the expression of BCL2 and IGF-1. We also performed immunohistochemical validation for selected target proteins and found over-expression of MUM1, BLIMP1 and STMN1 in NKTL, and notably, a corresponding increase in MYC expression. Conclusion: miRNA are dysregulated in NKTL. Since MYC is known to cause repression of miRNA expression, it is possible that MYC activation in NKTL as we have shown previously may contribute to the suppression of the miRNAs. These suppressed miRNA in turn lead to increase and aberrant expression of proteins and pathways of biological relevance to NKTL including cell cycle related genes, genes involved in p53 and MAPK signaling pathways as well as MUM1, BLIMP1 and STMN1. Reintroduction of these suppressed miRNA lead to death of NKTL cells and may be a potential therapeutic strategy. Disclosures: No relevant conflicts of interest to declare.

Abstract 3931 Poster Board III-867 Extranodal nasal-type Natural Killer/T-cell lymphoma (NKTCL) is a distinct clinicopathologic entity most commonly affecting Asians and Central and South Americans, and characterized by a clonal proliferation of NK or T cells with a cytotoxic phenotype. There is a strong association with Epstein-Barr Virus. The tumor is aggressive with patient usually surviving short duration even with chemotherapy. We performed the first comprehensive genome-wide gene expression profiling (GEP) of extranodal nasal-type Natural Killer/T-cell lymphoma (NKTCL) using formalin-fixed paraffin embedded (FFPE) tissue (n=25) and NK cell lines (n=5) and compared the results to the GEP of normal NK cells using the Illumina DASL whole genome array, with the objective of understanding the oncogenic pathways involved in the pathogenesis of NKTCL and to identify potential therapeutic targets. Quantitative-PCR validation of the GEP findings revealed over-expression of candidate genes BIRC5 (survivin), EZH2 and STMN1 in NK cell lines compared to normal NK cells, consistent with the GEP data. We then extracted a list of genes that are differentially expressed between NKTCL and normal NK cells and tissue controls. We than subjected this list of genes to pathway and network analysis using Metacore. This revealed a significant enrichment for cell cycle related genes and pathways. Furthermore, the network analysis results demonstrated a pro-proliferative and anti-apoptotic phenotype in NKTCL characterized by activation of Myc and nuclear factor kappa B (NF-KB), and deregulation of p53. This was further corroborated using immunohistochemistry on tissue microarray of NKTCL (n=33, including all cases with GEP performed). We observed a significant percentage of NKTCL showing overexpression for c-Myc (45.4%), p53 (87.9%) and NF-KB p50 (67.7%) on immunohistochemistry. Notably, overexpression of survivin was observed in 97% of the cases. Based on our findings, we propose a model of NKTCL pathogenesis where deregulation of p53 together with activation of MYC and NF-KB, possibly driven by EBV LMP-1, result in the cumulative upregulation of survivin. When KHYG and NKYS cell lines were treated with a compound IDR E804 which inhibited survivin, there is significant inhibition of cell growth as assess by MTS assay and induction of apoptosis as measured using Annexin V staining by flow cytometry. This suggests that compounds inhibiting survivin may be a potentially useful novel therapeutic approach in NKTCL. Disclosures: No relevant conflicts of interest to declare.