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Purpose: To investigate the prognostic significance of programmed cell death ligand-1 (PD-L1) and phosphorylated ERK (p-ERK) and their interactions in T-cell lymphoma (TCL). Methods: The mRNA levels of PD-L1 and ERK in TCL samples were analyzed. Formalin-fixed paraffin-embedded tissues from 69 TCL patients were collected to detect the expression of PD-L1 and p-ERK by multiplexed immunofluorescence staining. The total PD-L1 and p-ERK was measured by western blotting, and membrane PD-L1 was determined using flow cytometry. Results: PD-L1 and ERK mRNA levels were significantly upregulated in TCL. The expression rates of PD-L1 and p-ERK were 52.2% and 27.5%, respectively. PD-L1 expression correlated with stage (R=0.304, P=0.011) and IPI score (R=0.313, P=0.009), and p-ERK expression correlated with stage (R=0.330, P=0.006) and IPI score (R=0.376, P=0.002). PD-L1 expression positively correlated with p-ERK expression (R=0.355, P=0.003). Patients with co-expression of PD-L1 and p-ERK had the worst overall survival (P=0.007). In three TCL cell lines with PD-L1 expression, we demonstrated that the expression of p-ERK was upregulated after stimulation with PD-1, suggesting that ERK signaling was activated. Conclusions: The PD-1/PD-L1 axis activates intracellular ERK signaling in tumor cells and that PD-L1, p-ERK or their combination are potential biomarkers for predicting the prognosis in TCL patients.

Background: Activated B-cell like diffuse large B-cell lymphoma (ABC-DLBCL) is an aggressive lymphoma characterized by constitutive NF-κB activation. Nevertheless, the role and mechanisms of miR-17~92 in contributing to the NF-κB activation in ABC-DLBCL are still elusive. Methods: The expression of miR-17~92 primary transcript (MIR17HG) and NF-κB target genes was determined using RNA-sequencing. The expression of miR-17~92 was performed using microarray analysis. Plasmids carrying conditional over-expression and loss-of-function of miR-17~92 were respectively constructed and dual-luciferase reporter assay was used to validate the target gene of miR-17~92. Immunoprecipitation and polyubiquitination were further used to the study of potential mechanisms.Results: Expression of MIR17HG was positively correlated with NF-κB activity, miR-17~92 activated the NF-κB signaling in ABC-DLBCL, and its over-expression promoted ABC-DLBCL cell growth, accelerated cell G1 to S phase transition and enhanced cell resistance to NF-κB inhibitor. Importantly, miR-17~92 promoted NF-κB activation through directly targeting multiple ubiquitin-editing regulators to lead to increase the K63-linked polyubiquitination and decrease the K48-linked polyubiquitination of RIP1 complex in ABC-DLBCL. We further found that miR-17~92 selectively activated IκB-α and NF-κB p65 but not NF-κB p52/p100, and high miR-17~92 expression was also associated with poorer outcome in ABC-DLBCL patients. Conclusions: Taken together, miR-17~92 selectively activate the canonical NF-κB signaling via targeting ubiquitin-editing regulators to lead to constitutively NF-κB activation and poorer outcome, which is an innovative function of miR-17~92 and previously unappreciated regulatory mechanism of NF-κB activation in ABC-DLBCL. Targeting miR-17~92 may thus provide a novel bio-therapeutic strategy for ABC-DLBCL patients.

Objective: To explore the effect of dysregulation of epigenetic regulator EZH1 and EZH2 on the proliferation in MCL and theunderlying mechanisms. Methods: In this study, we elucidated the role of EZH1 and EZH2 overexpression by immunohistochemistry and correlated themto clinical outcome in 41 MCL patients. Quantitative real-time PCR and Western blot were applied to confirm the level of EZH1and EZH2 in well-characterized MCL cell lines which were compared to those of naive B cells. Then we manipulated theexpression of EZH1 and EZH2 in MCL cells using CRISPR/Cas9 system to directly investigate their functional roles in MCL. Wealso evaluated the effect of two small molecule selective inhibitors, EPZ005687 and UNC1999, on MCL cell proliferation, cell cycledistribution and apoptosis in vitro. Finally, we performed RNA-sequencing (RNA-Seq) and Chromatin immunoprecipitation(ChIP) assay to further gain insight into the underlying molecular mechanisms. Results: We found that EZH2 protein is overexpressed in approximately half of this cohort of MCL cases. More importantly, theoverexpression of EZH2 is associated with poor OS in the patients. Nevertheless, simple EZH2 depletion in vitro has little impacton the viability of MCL cells, predominantly because of the consequent up-regulation of EZH1. Consistently, UNC1999, a dualEZH1/2 inhibitor, unlike the EZH2 selective inhibitor EPZ005687, exerts a potent inhibitory effect on MCL cells. Furthermore, wediscover CDKN1C and TP53INP1 as the two important cell cycle regulators, the expression of which are repressed by EZH1/2mediated epigenetic regulation and are restored by EZH1/2 dual inhibition. Conclusions: Our study suggests that EZH2 participates in the pathogenesis of MCL which may serve as a potential biomarker forprognosis prediction. The dual inhibition of EZH1/2 is a promising therapeutic strategy for MCL. Cite this article as: Li W, Bi C, Han Y, Tian T, Wang X, Bao H, et al.Targeting EZH1/2 induces cell cycle arrest and inhibits cell proliferationthrough reactivation of p57CDKN1C and TP53INP1 in mantle cell lymphoma.Cancer Biol Med. 2019; 16: 530-9. doi: 10.20892/j.issn.2095-3941.2018.0380

MYC-driven lymphomas, especially those with concurrent MYC and BCL2 dysregulation, are currently a challenge in clinical practice due to rapid disease progression, resistance to standard chemotherapy, and high risk of refractory disease. MYC plays a central role by coordinating hyperactive protein synthesis with upregulated transcription in order to support rapid proliferation of tumor cells. Translation initiation inhibitor rocaglates have been identified as the most potent drugs in MYC-driven lymphomas as they efficiently inhibit MYC expression and tumor cell viability. We found that this class of compounds can overcome eIF4A abundance by stabilizing target mRNA-eIF4A interaction that directly prevents translation. Proteome-wide quantification demonstrated selective repression of multiple critical oncoproteins in addition to MYC in B-cell lymphoma including NEK2, MCL1, AURKA, PLK1, and several transcription factors that are generally considered undruggable. Finally, (-)-SDS-1-021, the most promising synthetic rocaglate, was confirmed to be highly potent as a single agent, and displayed significant synergy with the BCL2 inhibitor ABT199 in inhibiting tumor growth and survival in primary lymphoma cells in vitro and in patient-derived xenograft mouse models. Overall, our findings support the strategy of using rocaglates to target oncoprotein synthesis in MYC-driven lymphomas.

Inhibitors that target specific kinases or oncoproteins have become popular additions to or replacements for cytotoxic chemotherapies to treat many different types of cancer. However, many tumors lack a discernable target kinase and an amplified oncoprotein and/or rely on several cooperating mechanisms for progression. Thus, combinations of targeted therapies are essential for treating many cancers to avoid the rapid emergence of resistance. In this issue of the JCI, Ren et al. use an elegant kinase activity–profiling method and identify activity of the oncogene polo-like kinase-1 (PLK1) as an important driver of double-hit lymphoma (DHL), an aggressive subgroup of B cell lymphoma characterized by chromosomal translocations involving c-MYC and BCL2 or BCL6. Moreover, PLK1 activity was associated with MYC expression and poor prognosis in DHL patients. PLK1 inhibition with volasertib, alone and in combination with the BCL-2 inhibitor venetoclax, was efficacious in multiple DHL models, including mice harboring DHL patient–derived xenografts. Together, these data support PLK1 as a promising prognostic marker and therapeutic target for DHL.

Mechanistic target of rapamycin (mTOR) complex 1 is a central integrator of nutrient and growth factor inputs that controls cell growth in eukaryotes. The second generation of mTOR kinase inhibitors (TORKi), directly targeting the mTOR catalytic site, are more effective than rapamycin and its analogs in cancer treatment, particularly in inducing apoptosis. However, the mechanism underlying the cytotoxic effect of TORKi remains elusive. Herein, we demonstrate that TORKi-induced apoptosis is predominantly dependent on the loss of mTOR complex 1-mediated 4EBP activation. Knocking out RICTOR, a key component of mTOR complex 2, or inhibiting p70S6K has little effect on TORKi-induced apoptosis. Conversely, increasing the eIF4E:4EBP ratio by either overexpressing eIF4E or knocking out 4EBP1/2 protects lymphoma cells from TORKi-induced cytotoxicity. Furthermore, downregulation of MCL1 expression plays an important role in TORKi-induced apoptosis, whereas BCL-2 overexpression confers resistance to TORKi treatment. We further show that the therapeutic effect of TORKi in aggressive B-cell lymphomas can be predicted by BH3 profiling, and improved by combining it with pro-apoptotic drugs, especially BCL-2 inhibitors, both in vitro and in vivo. Taken together, the study herein provides mechanistic insight into TORKi cytotoxicity and identified a potential way to optimize its efficacy in the clinical treatment of aggressive B-cell lymphoma.

8035 Background: The goal of the North American Mantle Cell Lymphoma (MCL) Project is to evaluate the clinical, biological, and genomic markers that affect the outcome of patients with MCL. Methods: We have retrospectively studied the clinical and pathological features of 307/421 patients diagnosed with MCL between January 2000 to December 2012 from 23 institutions across North American. Results: The male to female ratio of MCL patients was 3.5:1, with a median age of 66 years (range: 24-106 years). Approximately 29% of patients (78/269) presented with B symptoms and 257 (257/307, 83.7%) patients had extranodal involvement at diagnosis. Median follow-up was 7.1 years (range, 0.03 to 16.6 years) with the five-year PFS and OS at 27.8%, and 54.4%, respectively. Univariate analysis revealed that the following factors were significantly associated with both inferior OS and PFS ( p< 0.05): older age (≥60 years), presence of B symptoms, advanced Ann Arbor stage, elevated LDH, low platelets (≤100K/ml), blastoid/pleomorphic cytology, Ki67 proliferation ≥30%, circulating tumor cells, no transplantation (vs. transplantation), and allogeneic (vs. autologous) stem cell transplantation. In addition, large tumor size (maximal diameter > 3cm), high WBC ( > 10×103/ml), CD5 or CD23 positivity, and a complex karyotype were associated with inferior OS ( p< 0.05). Multivariate Cox regression analysis showed age (≥ 60y; p= 0.0028, HR = 2.44, 95% CI: 1.36-4.38) and high LDH ( p= 0.0062, HR = 2.19, 95% CI: 1.25-3.84) were the two factors predicting the clinical outcome. MIPI-c, a commonly used prognostic scoring system which includes Ki67, stratified the 100 MCL cases into four group with distinct clinical outcomes ( p< 0.001). Using readily-available clinical and pathological variables, we developed a simple and robust scoring system, MIPI-P (pathology), which consisted of age (≥60 years), LDH (high), Ki67 index (≥30%), Ann Arbor stage (III/IV), and cytological type (blastoid/pleomorphic), each contributing one point. The MIPI-P system stratified 104 MCL cases into three distinct groups ( p< 0.001). Median survival for the different groups were: low grade (0-1 points): 11.8 years; intermediate grade (2-3 points): 4.9 years; and high grade (4-5 points): 1.6 years. We further validated this system in an independent cohort of 33 MCL cases and confirmed that the modified MIPI-P provided robust prognostic predication ( p= 0.014). Conclusions: The clinical and biologic characteristics of MCL can provide information assisting with the prognosis of patients with MCL.

The novel Bruton's tyrosine kinase inhibitor ibrutinib has demonstrated high response rates in B-cell lymphomas; however, a growing number of ibrutinib-treated patients relapse with resistance and fulminant progression. Using chemical proteomics and an organotypic cell-based drug screening assay, we determine the functional role of the tumour microenvironment (TME) in ibrutinib activity and acquired ibrutinib resistance. We demonstrate that MCL cells develop ibrutinib resistance through evolutionary processes driven by dynamic feedback between MCL cells and TME, leading to kinome adaptive reprogramming, bypassing the effect of ibrutinib and reciprocal activation of PI3K-AKT-mTOR and integrin-β1 signalling. Combinatorial disruption of B-cell receptor signalling and PI3K-AKT-mTOR axis leads to release of MCL cells from TME, reversal of drug resistance and enhanced anti-MCL activity in MCL patient samples and patient-derived xenograft models. This study unifies TME-mediated de novo and acquired drug resistance mechanisms and provides a novel combination therapeutic strategy against MCL and other B-cell malignancies., Ibrutinib has demonstrated high response rates in B-cell lymphomas but a lot of ibrutinib-treated patients relapse with resistance. This study unified TME-mediated de novo and acquired drug resistance through B-cell receptor signalling and PI3K-AKT-mTOR axis and provides a combination therapeutic strategy against B-cell malignancies.

Diffuse large B-cell lymphoma (DLBCL) is the most common non-Hodgkin lymphoma, whose main prognostic factor is closely related to germinal center B-cell-like subtype (GCB- DLBCL) or activated B-cell-like type (non-GCB-DLBCL). The most common type of primary central nervous system lymphoma is diffuse large B-cell type with poor prognosis and the reason is unclear. This study aims to stratify primary central nervous system diffuse large B-cell lymphoma (PCNS-DLBCL) according to the cell-of-origin (COO) and to investigate the multiple proteins expression of C-MYC, BCL-6, BCL-2, TP53, further to elucidate the reason why primary central nervous system diffuse large B-cell lymphoma possesses a poor clinical outcome as well. Nineteen cases of primary central nervous system DLBCL were stratified according to immunostaining algorithms of Hans, Choi and Meyer (Tally) and we investigated the multiple proteins expression of C-MYC, BCL-6, BCL-2, TP53. The Epstein-Barr virus and Borna disease virus infection were also detected. Among nineteen cases, most (15-17 cases) were assigned to the activated B-cell-like subtype, highly expression of C-MYC (15 cases, 78.9%), BCL-2 (10 cases, 52.6%), BCL-6 (15 cases, 78.9%). Unfortunately, two cases were positive for PD-L1 while PD-L2 was not expressed in any case. Two cases infected with BDV but no one infected with EBV. In conclusion, most primary central nervous system DLBCLs show an activated B-cell-like subtype characteristic and have multiple expressions of C-MYC, BCL-2, BCL-6 protein, these features might be significant factor to predict the outcome and guide treatment of PCNS-DLBCLs.

Summary Drug-tolerant "persister" tumor cells underlie emergence of drug-resistant clones and contribute to relapse and disease progression. Here we report that resistance to the BCL-2 targeting drug ABT-199 in models of mantle cell lymphoma and double-hit lymphoma evolves from outgrowth of persister clones displaying loss of 18q21 amplicons that harbor BCL2. Further, persister status is generated via adaptive super-enhancer remodeling that reprograms transcription and offers opportunities for overcoming ABT-199 resistance. Notably, pharmacoproteomic and pharmacogenomic screens revealed that persisters are vulnerable to inhibition of the transcriptional machinery and especially to inhibition of cyclin-dependent kinase 7 (CDK7), which is essential for the transcriptional reprogramming that drives and sustains ABT-199 resistance. Thus, transcription-targeting agents offer new approaches to disable drug resistance in B-cell lymphomas.

Background: c-MYC (MYC) is commonly dysregulated in aggressive B cell lymphomas. MYC associated lymphoma, especially Double Hit lymphoma (DHL) and Double-Expression Lymphoma (DEL) which are characterized by MYC and BCL2 dual overexpression usually present with the inferior outcome as rapid disease progression and poor response to standard chemotherapy regimen. Nevertheless, MYC is considered as an "undruggable" target and targeting strategies such as suppressing MYC transcription by bromodomain (BRD)-4 inhibitors have been widely investigated in both preclinical models and clinical trials. However, increasing evidence has shown that lymphoma cells displayed a wide range of resistance to BRD-4 inhibition, due to transcription adaptation or kinome reprogramming. Hence, alternative approaches for suppressing MYC or its function are urgently needed. Strategies directed against oncoprotein translation may efficiently repress key oncoproteins regardless of the abundant MYC mRNA due to genetic aberrations and secondary transcription up-regulated by MYC. Rocaglate is a class of natural products derived from plants of the Aglaia genus that have been demonstrated to potently inhibit protein translation initiation via eIF4A. The use of rocaglates for anti-cancer treatment was limited due to the scarcity and instability of these natural products e.g. Silvestrol. Recent chemical modification and screening studies have unveiled a few synthetic rocaglates that are more potent than Silvestrol such as SDS-1-021-(−), which unlocked the potential use of rocaglates for clinical applications. Methods and Results: To probe effective reagents for MYC-driven lymphoma, a screening of 50 drugs targeting common oncogenic pathways and tumorigenic machinery was performed in two isogenic B-lymphoma lines. Several protein translation inhibitors, such as mTOR kinase inhibitors (TORKi), and eIF4A inhibitor Silvestrol were identified as the most potent drugs in all of the tested cells. Further, we found that Silvestrol but not TORKi efficiently repressed MYC protein translation in MYC-driven B lymphoma cells, whereas neither of them inhibited BCL2 expression. Moreover, we demonstrated that eIF4E knockdown or eIF4E/eIF4G disruptor Briciclib did not significantly affect MYC expression, whereas eIF4A inhibitor hippuristanol and rocaglates derivate SDS-1-021-(−) diminished MYC expression similar to that observed in Silvestrol treated cells. By using dual luciferase assay, we demonstrated that rocaglates had stronger cap-dependent and IRES translation inhibition than TORKi in lymphoma cells. Next, by native RNA immunoprecipitation and siRNA knocking down, we found that rocaglates repressed MYC translation not only via eIF4A1 but also via eIF4A2, however, with different underlying mechanisms. Furthermore, to explore the molecular targets of rocaglates treatment in B cell lymphoma, we performed TMT-Mass Spectrometry which identified multiple oncoproteins including NEK2, MYC, MCL1, TCF3, BCL6, PLK1, AURKA, and WEE1 were significantly down-regulated by SDS-1-021-(−) treatment. Finally, we demonstrated that SDS-1-021-(−) is highly potent as a single agent and synergized with ABT199 at a low dose (0.2mg/kg) in PDX models with DHL/DEL. Brief summary : Our pre-clinical study provided strong evidence that rocaglates but not TORKi efficiently suppress MYC protein translation because 1). Rocaglates exhibit strong inhibition on both Cap- and IRES-dependent translation, 2). Rocaglates decrease PLK1 and AURKA/B thus destabilizing MYC protein. The synthetic rocaglate SDS-1-021-(−) is a potent agent that exhibits significant synergistic killing effect with ABT199 on DHL/DEL cells in the pre-clinical animal study. Figure Figure. Disclosures Lunning: TG Therapeutics: Consultancy; AbbVie: Consultancy; Genentech: Consultancy; Astra-Zeneca: Consultancy; Genzyme: Consultancy; Celgene: Consultancy; Bayer: Consultancy; Gilead: Consultancy; Spectrum: Consultancy; Genentech: Consultancy; Seattle Genetics: Consultancy; Portola: Consultancy; Kite: Consultancy; Juno: Consultancy; Janssen: Consultancy; Verastem: Consultancy. Vose:Novartis: Honoraria, Research Funding; Epizyme: Honoraria; Incyte Corp.: Research Funding; Bristol Myers Squibb: Research Funding; Kite Pharma: Research Funding; Merck Sharp & Dohme Corp.: Research Funding; Abbvie: Honoraria; Legend Pharmaceuticals: Honoraria; Seattle Genetics, Inc.: Research Funding; Acerta Pharma: Research Funding; Celgene: Research Funding; Roche: Honoraria.

Background: c-MYC is a transcription factor that promotes oncogenesis by activating and repressing its target genes that control cell growth, metabolism, and proliferation. MYC is deregulated in a large proportion of aggressive B-cell lymphomas. A typical example is the Double-Hit Lymphoma (DHL) and Double-Expression Lymphoma (DEL) which present with a rapidly progressing clinical course, refractory to treatment, poor clinical outcome, and currently considered incurable. Nevertheless, MYC is considered as an "undruggable" target since it has no "active site" amenable to binding by conventional small molecule inhibitors. Moreover, MYC has a broad spectrum of functions in cell proliferation, survival, metabolism, and others, so direct inhibition would likely cause severe side effects. Besides direct inhibition, another practical strategy is to target druggable proteins that are essential for the viability of MYC-driven tumors, inducing MYC-dependent "synthetic lethality". The advantage of such approach is a capability of killing tumor cells discriminately, while leaving non-tumor cells intact or less influenced. This study is designed to identify such targets and explore practical novel strategies to treat MYC-driven lymphomas, especially DHL/DEL. Methods and Results: By integrating activity-based proteomic profiling and drug screens in isogenic MYC on/off lymphoma cells, we identified polo-like kinase-1 (PLK1) as an essential regulator of the MYC-dependent kinome in DHL/DEL. Notably, PLK1 was expressed at high levels in DHL, correlated with MYC expression and connoted poor outcome. Further, PLK1 is directly activated by MYC on transcriptional level and in turn, PLK1 signaling augmented MYC protein stability by promoting its phosphorylation and suppressing its degradation. Thus, MYC and PLK1 form a feed-forward circuit in lymphoma cells. Finally, both in vitro and in vivo studies demonstrated that inhibition of PLK1 triggered degradation of MYC and of the anti-apoptotic protein MCL1, and PLK1 inhibitors showed synergy with BCL-2 antagonists in blocking DHL/DEL cell growth, survival, and tumorigenicity. These data support that PLK1 is a promising therapeutic target in MYC-driven lymphomas. Brief summary: Functional pharmacoproteomics identified PLK1 as a therapeutic vulnerability for MYC-driven lymphoma, which was a synthetic lethal for DHL/DEL when targeted with BCL-2 inhibitors. Disclosures Vose: Roche: Honoraria; Merck Sharp & Dohme Corp.: Research Funding; Acerta Pharma: Research Funding; Seattle Genetics, Inc.: Research Funding; Novartis: Honoraria, Research Funding; Kite Pharma: Research Funding; Bristol Myers Squibb: Research Funding; Epizyme: Honoraria; Legend Pharmaceuticals: Honoraria; Abbvie: Honoraria; Celgene: Research Funding; Incyte Corp.: Research Funding.

The purpose of this study was to examine the incidence of Epstein-Barr virus (EBV)-positive diffuse large B-cell lymphoma of the elderly in a representative northern Chinese population. Overall, 212 cases of primary diffuse large B-cell lymphoma were analyzed for EBV-positive status by detection of EBV-encoded RNA using in situ hybridization. Immunophenotypic identity was verified by a streptavidin-biotin peroxidase detection system using the markers CD20, CD3, CD10, BCL6 and MUM1. The prevalence of EBV-positive diffuse large B-cell lymphoma in elderly northern Chinese patients was 3.8% (eight of 212). All eight cases were negative for CD10 and BCL6 immunostaining. The incidence is less frequent than that of southern China and other East-Asian countries. Patients tend to be older, present with a non-germinal center B-cell-like immunophenotype and have a poor outcome.

The aberrant expression of microRNA-155 (miR-155), which has emerged as having a significant impact on the biological characteristics of lymphocytes, plays important roles in B-cell malignancies, such as diffuse large B-cell lymphoma (DLBCL). DLBCL is the most common non–Hodgkin's lymphoma in the adult population, accounting for approximately 40% of newly diagnosed non–Hodgkin's lymphoma cases globally. To determine the specific function of miR-155, a quantitative proteomics approach was applied to examine the inhibitory effects of miR-155 on protein synthesis in DLBCL cells. PIK3R1 (p85α), a negative regulator of the phosphatidylinositol 3-kinase (PI3K)–AKT pathway, was identified as a direct target of miR-155. A luciferase reporter was repressed through the direct interaction of miR-155 and the p85α 3′-untranslated region, and overexpression of miR-155 down-regulated both the transcription and translation of p85α. The PI3K-AKT signaling pathway was highly activated by the sustained overexpression of miR-155 in DHL16 cells, whereas knockdown of miR-155 in OCI-Ly3 cells diminished AKT activity. Taken together, our results reveal a novel target involved in miR-155 biological characteristics and provide a molecular link between the overexpression of miR-155 and the activation of PI3K-AKT in DLBCL.

The novel genetic information gained from genome-wide high throughput techniques has greatly improved our understanding of peripheral T-cell lymphoma (PTCL). PTCL consists of numerous distinct entities and is currently diagnosed using a combination of clinical and morphologic features and immunophenotyping together with limited molecular assays leading to an often fragmented, complicated diagnostic system. The diagnosis of many cases is challenging even for expert hematopathologists and more than a third of the cases cannot be further classified and thus put into the PTCL-NOS category. Gene expression profiling (GEP) has significantly improved the molecular classification of PTCLs and identified robust molecular signatures for common nodal subtypes of PTCL including angioimmunoblastic T-cell lymphoma (AITL), anaplastic T-cell lymphoma (ALCL), adult T-cell leukemia/lymphoma (ATLL) and extra-nodal NK/T cell lymphoma (ENKTL). These studies also led to identification of novel molecular subtypes with distinct prognosis, that otherwise could not be identified by conventional methods. Integration of massive sequencing strategies and gene expression has characterized driver genetic alterations in common subtypes like AITL, ALCL, ENKTL and other PTCLs. These studies have identified oncogenic pathways and genes affected in specific disease subtypes that can be potentially targeted by specific therapies. Novel treatment options with FDA approved drugs directed towards mutant IDH2, the NF-κB, JAK/STAT, or mTOR pathways illustrate the usefulness of genome-wide techniques to identify targets for therapy. In this review, we highlight recent advances in the molecular diagnosis and prognosis of PTCL using these genome-wide techniques.

Burkitt lymphoma (BL) is the most common non-Hodgkin lymphoma (NHL) in children. Although it accounts for only 1-5% of NHL in adults, approximately 60% of the BL cases diagnosed each year in western countries occur in patients >40 years of age. Although adult and pediatric BL cases are indistinguishable by molecular classification, pediatric patients have a significantly better outcome than adults. While translocation of MYC to the immunoglobulin heavy or light chain genes is characteristic of pediatric and adult BL, genetic differences may contribute to the superior clinical outcome of childhood cases. Therefore, we aimed to identify the spectrum of additional genetic abnormalities that occur in adult and pediatric BL. Copy number analysis, gene expression profiling (GEP), and targeted sequencing of ~400 genes known to be mutated in NHLs were performed on a cohort of molecularly defined BL samples. Copy number abnormalities (CNAs) were identified by the Affymetrix 250k NspI SNP array in 73 BL tumors (28 adult, 45 pediatric), and sequencing was performed on 52 BLs (21 adult, 31 pediatric). Pediatric cases had fewer CNAs than adults. The most common focal abnormality identified was a gain on 13q31.3 encompassing MIR17HG. It was more frequent in adult compared to pediatric cases (35% vs 16%, p=0.085) and was associated with increased expression of miR-17~92 cluster members; and among adults, patients with this gain trended towards worse overall survival, though the number of cases with available information was small. Gain of 8q was found in ~20% of adult cases, but in no pediatric cases. Surprisingly, cases with 8q gain had significantly lower MYC mRNA expression (p< 0.001) and lower protein expression. In cases with MYC gain 0/4 cases were positive for MYC protein expression by immunohistochemistry; in contrast,6/10 cases with no MYC gain were positive for MYC expression. This suggests that gain of 8q is driven by another gene in the region. Additional genetic alterations included gains of genomic loci encompassing MCL1 and MDM4 (1q21-24) and losses encompassing RB1, p53 and CDKN2A/CDKN2B. Pathway analysis of genes differentially expressed by CN status showed an enrichment of genes involved in cell cycle regulation, the p53 signaling pathway, and the ubiquitin proteasome pathway. The frequencies of mutations in commonly mutated genes including MYC, ID3, TP53, CCND3, DDX3X, ARID1A, and TCF3 were not significantly different in adult and pediatric BL. However, BCL2, (43%, p Gene expression studies suggested activated BCR signaling in BL cases with CN gain of miR-17~92. In vitro analysis of miR17~92 in BL cell lines (n=4) showed that functional loss of miR-17 ~ 92 expression using a miRNA sponge led to reduced proliferation. Treatment of BL cell lines with anti-IgM induced BCR activation in a time- and dosage-dependent manner, as estimated by increased phosphorylation of downstream mediators (SYK and BLNK). This activation was reduced upon loss of functional miR-17 ~ 92 expression in cell lines. Since miR-17~92 can directly inhibit proximal negative regulators of BCR signaling, treatment with the FDA-approved BTK inhibitor, Ibrutnib, further inhibited proliferation of BL cell lines carrying the miRNA sponge. The BCR signaling pathway is one example of how unique abnormalities in adult BL can provide possible targets for therapeutic intervention. Disclosures No relevant conflicts of interest to declare.

Purpose We previously reported that constitutive STAT3 activation is a prominent feature of the activated B-cell subtype of diffuse large B-cell lymphomas (ABC-DLBCL). In this study, we investigated whether STAT3 activation can risk stratify patients with DLBCL. Patients and Methods By an immunohistochemical method, we investigated phosphotyrosine STAT3 (PY-STAT3) expression from 185 patients with DLBCL treated with R-CHOP (rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone). Cell line-based siRNA experiments were also performed to generate an 11-gene, PY-STAT3 activation signature, which was used to study a previously published cohort of 222 patients with DLBCL. The STAT3 activation status determined by these two methods and by STAT3 mRNA levels were then correlated with survival. Results PY-STAT3 was detected in 37% of DLBCL and enriched in ABC-DLBCL cases (P = .03). PY-STAT3 positivity significantly correlated with poor overall survival (OS; P = .01) and event-free survival (EFS; P = .006). Similar observations were made for high levels of STAT3 mRNA. In multivariable analysis, PY-STAT3 status (P = .02), International Prognostic Index (P = .02), and BCL2 expression (P = .046) were independent prognosticators of OS in this cohort. Among the cell-of-origin subgroups, PY-STAT3 was associated with poor EFS among non–germinal center B-cell DLBCL cases only (P = .027). Similarly, the 11-gene STAT3 activation signature correlated with poor survival in the entire DLBCL cohort (OS, P < .001; EFS, P < .001) as well as the ABC-DLBCL subgroup (OS, P = .029; EFS, P = .025). Conclusion STAT3 activation correlated with poor survival in patients with DLBCL treated with R-CHOP, especially those with tumors of the ABC-DLBCL subtype.

The activated B-cell-like (ABC) subtype of diffuse large B-cell lymphoma (DLBCL) is a very aggressive lymphoma characterized by constitutive NF-kB activation, but whether miRNAs dysfunction contributes to this event, and their exact function and mechanism remain unclear. Starting from an integrative screening strategy, we revealed that there were some interactions between the NF-kB signaling and miR-17~92 cluster, which was essential for B-cell development and commonly gained and/or overexpressed in ABC-DLBCL. Several important NF-kB negative regulators including TNFAIP3 (A20), CYLD and Rnf11 were predicted and validated to be the direct targets of miR-17~92. Conditional knock-down of miR-17~92 using sponge could suppress NF-kB activity and elevate the A20, CYLD and Rnf11 expression in 293T cells. Furthermore, we demonstrated that enforced overexpression of miR-17~92 could also decrease the A20, CYLD and Rnf11 expression in ABC-DLBCL cells. Conditional overexpression of miR-17~92 could promote ABC-DLBCL cells growth, accelerate the cells G1/G0 phase to S phase transition, and suppress NF-kB inhibitor-induced apoptosis. Conversely, conditional knock-down of miR-17~92 could inhibit ABC-DLBCL cells growth and sensitize the cells to NF-kB inhibitor-induced apoptosis. The miR-17~92 could induce the IkB-a and NF-kB p65 phosphorylation, leading to the NF-kB activation and aberrant expression of NF-kB transcriptional target genes. However, miR-17~92 did not regulate the NF-kB p52/p100 phosphorylation. Overexpression of miR-17~92 enhanced K63-linked ubiquitination and reduced K48-linked ubiquitination of the TNFa receptor 1 complex including RIP1. Importantly, we found that high expression level of miR-17~92 was associated with poorer survival in ABC-DLBCL patients. Our results uncovered a novel mechanism for the canonical but not the non-canonical transcription factor NF-kB pathway by modulation of miR-17~92 in ABC-DLBCL, and suggested that targeting the miR-17~92 might be novel bio-therapeutic strategies, which could be single-agent or combined with NF-kB inhibitor treatment, for ABC-DLBCL patients. Figure 1. Inhibition of miR-17~92 blocks the activity of NF-kB in HER293T cells: (A) The schematic representation of reporter constructs involved in assay. (B) HEK293T cells were co-transfected with Dul-Luciferase reporter constructs and the miR-17~92 sponge plasmid. TNF-a stimulation or without stimulation for 18 h. Figure 1. Inhibition of miR-17~92 blocks the activity of NF-kB in HER293T cells: (A) The schematic representation of reporter constructs involved in assay. (B) HEK293T cells were co-transfected with Dul-Luciferase reporter constructs and the miR-17~92 sponge plasmid. TNF-a stimulation or without stimulation for 18 h. Figure 2. miR-17~92 directly regulates A20, CYLD and Rnf11 in ABC-DLBCL cells. (A) Fluorescence images of tranduced ABC-DLBCL cells. (B) Expression of sponge or miR-17~92 in tranduced ABC-DLBCL cells. (C) Inhibition of miR-17~92 increase the expression of A20, CYLD and Rnf11. Overexpression of miR-17~92 reduce the expression of A20, CYLD and Rnf11 in ABC-DLBCL cells. Figure 2. miR-17~92 directly regulates A20, CYLD and Rnf11 in ABC-DLBCL cells. (A) Fluorescence images of tranduced ABC-DLBCL cells. (B) Expression of sponge or miR-17~92 in tranduced ABC-DLBCL cells. (C) Inhibition of miR-17~92 increase the expression of A20, CYLD and Rnf11. Overexpression of miR-17~92 reduce the expression of A20, CYLD and Rnf11 in ABC-DLBCL cells. Figure 3. miR-17~92 modulate mediate NF-kB activity in ABC-DLBCL. (A) Immunoblot analysis of IkB-a, NF-kB p65, NF-kB p100/p52 and their phosphorylation. (B) Heat-map display of quantitative real-time RT-PCR measurements of six independent NF-kB transcriptional targets show significantly lower expression in sponge expressing cells and higher expression in miR-17~92 expressing cells. Figure 3. miR-17~92 modulate mediate NF-kB activity in ABC-DLBCL. (A) Immunoblot analysis of IkB-a, NF-kB p65, NF-kB p100/p52 and their phosphorylation. (B) Heat-map display of quantitative real-time RT-PCR measurements of six independent NF-kB transcriptional targets show significantly lower expression in sponge expressing cells and higher expression in miR-17~92 expressing cells. Disclosures No relevant conflicts of interest to declare.

BACKGROUND: Interleukin-22 (IL-22) was recently discovered as a T-cell derived cytokine that belongs to the IL-10 gene family. It has been shown that IL-22 can promote cell proliferation and activate several oncogenic pathways including STAT3, MAPK and AKT signaling. IL-22 exerts its function by binding to its heterodimeric receptor complex which is composed of IL-22 receptor (R) 1 and IL-10R2. IL-22R1 is restricted and barely detected in normal immune cells including B and T cells. In the current study, we sought to determine whether IL-22 plays a part in the development of diffuse large B-cell lymphoma (DLBCL). METHODS: Plasma IL-22 level was measured in newly diagnosed patients with DLBCL and normal individuals by ELISA assay. We also assessed the expression of IL-22R1 in FFPE sample and DLBCL cell lines (TMD8, OCI-LY3, SUDHL16, OCI-LY1) by immunohistochemistry and immunofluorescence staining respectively. Immunoblotting was performed to determine changes of signaling pathways in cell lines upon the treatment with IL-22 recombinant protein. RESULTS: The plasma IL-22 concentration was significantly increased in newly diagnosed DLBCL patients (n=12) compared with normal individuals (n=26, P= 0.0177). IL-22R1 protein was expressed in all of the four DLBCL cell lines, while not all but some of the primary tumor samples expressed it on membranes of tumor cells. Using immunoblotting, we demonstrated a substantial increase of pSTAT3 level with the treatment of IL-22 in ABC-DLBCL cell lines TMD8 and OCI-LY3, while no pSTAT3 was detectable in GCB-DLBCL cell lines SUDHL16 and OCI-LY1. Interestingly, upon IL-22 stimulation, the p-ERK1/2 level was increased shortly and quickly returned to the normal level in TMD8 cells, and steadily increased in LY3 cells during 30 min of treatment. However, there was no change about p-ERK1/2 in SUDHL16 and OCI-LY1 cells after IL-22 treatment. CONCLUSIONS: Our current study has shown the aberrant expression of IL-22R1 in DLBCL. And recombinant IL-22 protein increased the activation of STAT3 and ERK1/2 in ABC rather than GCB-DLBCL cell lines. For the first time, we suggested the importance of IL-22 and its receptor IL-22R1 pathway in ABC-DLBCL, which warrants further studies. Figure 1. The expression of IL-22R1 in DLBCL cell lines was detected by using immunofluorescence staining. Disclosures No relevant conflicts of interest to declare.

Mechanistic target of rapamycin complex 1 (mTORC1) is a central integrator of nutrient and growth factor inputs that controls cell growth in all eukaryotes. Rapamycin and its analogs (rapalogs) have been approved for the treatment of relapsed mantle cell lymphoma. A large proportion of aggressive B-cell lymphoma patients, however, respond poorly to rapalogs. The second generation of mTOR inhibitors function as ATP-competitive inhibitors (TORi), directly targeting the mTOR catalytic site. TORis have been proven to be more effective than rapalogs in cancer treatment. However, the mechanism underlying the cytotoxic effect of TORis in aggressive B-cell lymphomas remains unclear. In this study, we demonstrated that TORi-induced apoptosis is predominantly dependent on loss of mTORC1-mediated 4EBP phosphorylation. Knocking out Rictor, a key component of mTORC2, or inhibiting p70S6K has little effect on TORi-induced apoptosis. In contrast, increasing the EIF4E:4EBP ratio by either overexpressing EIF4E or knocking out 4EBP1/2 protected lymphoma cells from TORi-induced cytotoxicity. Furthermore, down-regulation of MCL1 and BCL-XL expression plays an important role in TORi-induced apoptosis whereas BCL-2, in cells with high expression, confers resistance to TORi treatment. Based on the mechanism study, we demonstrated that BH3 profiling, primarily NOXA and HRK stimulation, can effectively predict the cytotoxicity of the TORi in lymphoma cells. Also, in combination with pro-apoptotic drugs, especially BCL-2 inhibitors, the TORi exerted powerful anti-tumor effects both in vitro and in vivo. Taken together, this study provides mechanistic insight in TORi treatment in aggressive B-cell lymphoma and identified a mean to predict and improve its effectiveness clinically. Disclosures No relevant conflicts of interest to declare.

mTORC1 (mammalian target of rapamycin complex 1) plays a central role in integrating nutrient and growth factor inputs to control cell growth in all eukaryotes and is commonly deregulated in human cancers. Inhibition of mTORC1 is a promising strategy in lymphoma therapy. However, only a few drugs, such as rapamycin and its analogs (rapalogs), have been approved for treatment in a limited number of cancer types, due to their incomplete and nonspecific inhibition of mTORC1 as well as their limited effects toward the 4EBP1 pathway. 4EBP1 pathway regulates protein translation which is considered to be crucial in cancer cell survival and proliferation. In this study, we used the glycolysis inhibitor 2-deoxyglucose (2-DG) together with the mitochondrial respiratory inhibitor metformin to treat aggressive B cell lymphoma cells in vitro and in vivo. We found that the combined treatment inhibited mTORC1 and its major downstream targets, including 4EBP1. As a result, combined treatment significantly inhibited tumor cell growth and survival by the inhibition of 5’ cap-dependent translation involving lymphoma associated oncogenes such as MCL-1, BCL-XL and Cyclin D1. Moreover, the combination of 2-DG and metformin suppressed tumor growth in B cell lymphoma xenograft mouse models. Although the combined treatment dramatically decreased cellular ATP levels, mTORC1 inhibition was independent of AMPK activity but instead resulted from inhibitory effects on Rag-GTPases, which are upstream activators of mTORC1. Given that both 2-DG and metformin have been used in clinical diagnosis or treatment for decades, the combination of the two drugs hold promise as a new strategy to treat aggressive B cell lymphoma. Disclosures: No relevant conflicts of interest to declare.

Abstract 5118 Mantle cell lymphoma (MCL) is an aggressive hematological malignancy with a median survival ranging between 3 and 5 years. Novel therapeutic strategies are urgently needed to improve the outcome. Mammalian target of rapamycin (mTOR) pathway which plays a central role in controlling cell growth, proliferation and metabolism has been shown to be deregulated in MCL. mTOR inhibitors, such as rapamycin and its analogues, have been approved for treatment of relapsed/refractory MCL. However, the molecular mechanism of mTOR activation in MCL has yet to be defined. MiRNA (miR)-17∼92 is a cluster of six miRNAs which are frequently overexpressed in MCL cases and overexpression of the miR-17∼92 cluster in MCL predicts poor prognosis. Our previous study demonstrated that miR-17∼92 activated the PI3K/AKT pathway by directly targeting PTEN and PHLPP2; and knock-down of miR-17∼92 expression inhibited MCL tumor growth in a xenograft/SCID MCL mouse model. In the present study, we further demonstrated that knock-down miR-17∼92 decreased the cell size, similar effect as seen in cells treated with mTORC1 inhibitors. Knockdown of miR-17∼92 expression also decreased the glycolysis, protein synthesis and glucose uptake in MCL cells. We found that knockdown miR-17∼92 activated AMPK signaling as demonstrated by increased phosphorylation of AMPK at Thr172, especially under low glucose condition. Activated AMPK further phosphorylated TSC2 and Raptor at S1387 and S792, respectively, thereby inhibiting mTORC1 signaling as evidenced by decreased phosphorylation of RPS6 and 4E-BP1. Using TargetScan and other prediction algorithms, we found that several factors in the LKB1/AMPK/mTOR pathway, such as LKB1, CAB39, PRKAA1 and TSC1, are predicted the targets of miR-17∼92. In this study, we validated these factors as direct targets of the miR-17∼92 by 3'UTR luciferase assays using reporter plasmids containing the 3'UTR of the targets or the 3'UTR with mutations in the predicted miRNA binding sites. The protein levels of these targets decreased in MCL cells with the miR-17∼92 overexpression. Conversely, the levels of these factors were increased upon knockdown of miR-17∼92 cluster. Our results indicate that overexpression of miR-17∼92 in MCL plays important role in mTORC1 activation by inactivating the LKB1/AMPK signaling, in addition to its effect on the PI3K/AKT pathway activation. Disclosures: No relevant conflicts of interest to declare.

We have previously reported aberrant activation of the signal transducer and activator of transcription 3 (STAT3) pathway promotes cell proliferation and survival in the activated B-cell-like (ABC) subtype, but not in the germinal center B-cell-like (GCB) subtype of diffuse large B-cell lymphoma (DLBCL). Herein, we investigated the effects of small molecular inhibitors of STAT3 in sensitizing DLBCL cell to chemotherapy drugs. ABC-DLBCL lines Ly3 and Pfeiffer were more resistant to vincristine-induced cell death than GCB-DLBCL cells Ly1 and Ly7 were. In ABC-DLBCL cells, constitutive activation of STAT3 provided survival signal and inhibited vincristine-induced apoptosis, while inhibition of STAT3 signaling abrogated IL-6 secretion and promoted cell apoptosis. STAT3 inhibitor and chemotherapy drugs synergistically induced cell apoptosis, with increased caspases 3/7 activity and PARP cleavage, in ABC-DLBCL cells but not in the GCB-DLBCL cells. The effect was correlated with reduced expression of MCL1 in these cells. To investigate the prognostic significance of STAT3 activation in DLBCL patients, we evaluated the levels of phospho-Tyr705-STAT3 (PY-STAT3) expression by immunohistochemistry in a cohort of 309 DLBCL patients treated with R-CHOP regimen. The ABC-DLBCL subgroup had significantly higher number of PY-STAT3 positive cases compared to the GCB subgroup (59.4% vs 36.2%; P = 0.003). Constitutive activation of STAT3 pathway has a significant impact on the overall-survival (OS) and event-free-survival (EFS) in the entire DLBCL cohort (OS, P = 0.010; EFS, P = 0.006) and in the ABC subgroup (OS, P = 0.062; EFS, P=0.016). Moreover, we constructed a subgroup of genes representing STAT3 activation from the gene expression profiling data of STAT3-siRNA treated ABC cell lines and primary DLBCL samples. Attributions of these genes include repression with the STAT3 knockdown in DLBCL cell lines, overexpression in the PY-STAT3-positive DLBCL tumors, and containing one or more STAT3 binding motif within their promoter regions. Eleven genes were obtained when trained this gene subgroup with patient survival through a semi-supervised algorithm. Averaged expression of the eleven genes predicted the OS (P < 0.001) and EFS (P < 0.001) in the entire DLBCL cohort, as well as in the ABC-DLBCL subgroup (OS, P = 0.029; EFS, P = 0.025). In conclusion, our study demonstrated that STAT3 activation is a significant prognostic factor in DLBCL and suggested that STAT3 may be a new therapeutic target in this aggressive malignancy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5568. doi:1538-7445.AM2012-5568