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Refractory/relapsed (R/R) diffuse large B-cell lymphoma (DLBCL) patients' failure of salvage chemotherapy had extremely worse prognoses. Herein, 14 R/R DLBCL patients failed to salvage chemotherapy were exposed to dual epigenetic agents (Chidamide 30 mg biw*2w and Decitabine 10 mg/m

Constitutive photomorphogenic 1 (COP1) is a p53-targeting E3 ubiquitin ligase that is downregulated by DNA damage through mechanisms that remain obscure. Here, we report that COP1 is not downregulated following DNA damage in 14-3-3σ null cells, implicating 14-3-3σ as a critical regulator in the response of COP1 to DNA damage. We also identified that 14-3-3σ, a p53 target gene product, interacted with COP1 and controlled COP1 protein stability after DNA damage. Mechanistic studies revealed that 14-3-3σ enhanced COP1 self-ubiquitination, thereby preventing COP1-mediated p53 ubiquitination, degradation, and transcriptional repression. In addition, we found that COP1 expression promoted cell proliferation, cell transformation, and tumor progression, manifesting its role in cancer promotion, whereas 14-3-3σ negatively regulated COP1 function and prevented tumor growth in a mouse xenograft model of human cancer. Immunohistochemical analysis of clinical breast and pancreatic cancer specimens demonstrated that COP1 protein levels were inversely correlated with 14-3-3σ protein levels. Together, our findings define a mechanism for posttranslational regulation of COP1 after DNA damage that can explain the correlation between COP1 overexpression and 14-3-3σ downregulation during tumorigenesis. Cancer Res; 71(3); 884–94. ©2010 AACR.

Supplementary Figures 1-3, Table 1 from 14-3-3σ Exerts Tumor-Suppressor Activity Mediated by Regulation of COP1 Stability

BACKGROUND The main aim of this study was to determine some simple but meaningful parameters that indicate immunochemotherapy-related interstitial lung disease (ILD) early in B-cell lymphoma and provide direction to hematologists. METHODS The clinical and laboratory characteristics, the treatments and outcomes of 21 B-cell lymphoma patients with ILD who underwent rituximab (RTX) -based immunochemotherapy were collected and retrospectively analyzed. RESULTS More cycles of immunochemotherapy and higher cumulative doses of RTX and doxorubicin hydrochloride liposome conferred a high risk of ILD. Compared to the baseline, patients had a significantly lower white blood cell count (WBC), absolute lymphocyte count (ALC), and albumin level (4.95×109 vs. 6.32×109, 0.71×109 vs. 1.61×109, 34.1 vs. 40.4 g/L; P

BACKGROUND: Chimeric antigen receptor T cell (CAR-T) therapy has achieved remarkable effects in refractory/relapsed (R/R) diffuse large B-cell lymphoma (DLBCL). However, many patients receiving CAR-T therapy still eventually die of disease recurrence or progression due to target antigen loss or exhaustion of CAR-T cells. Therefore, maintaining the efficacy of CAR-T has become a particular research focus. As lenalidomide can regulate T cell function, we conducted a study to evaluate the efficacy of lenalidomide maintenance after CAR-T therapy in R/R DLBCL patients. METHODS: Seven R/R DLBCL patients who received lenalidomide maintenance after CAR-T therapy and nine DLBCL patients that underwent CAR-T treatment alone were included. The clinical data of all subjects were collected to evaluate the efficacy of lenalidomide maintenance. In order to understand the possible mechanisms of lenalidomide in CAR-T therapy, CAR-T copies of peripheral blood were regularly detected by quantitative real-time polymerase chain reaction, and an in vitro test was also conducted. RESULTS: Overall survival (OS) was significantly prolonged in the lenalidomide maintenance group. Furthermore, one case responding to CAR-T therapy initially but suffering a relapse shortly achieved complete remission again after lenalidomide exposure, with an increase in the number of CAR-T copies detected. The in vitro test showed that lenalidomide could delay the exhaustion of CAR-T cells. CONCLUSIONS: Lenalidomide maintenance after CAR-T therapy is a safe and effective choice for R/R DLBCL patients. We confirmed that lenalidomide maintenance can improve patients’ OS, and the delayed exhaustion of CAR-T cells may contribute to this OS benefit.

Chimeric antigen receptor T-cell (CAR-T) therapy demonstrates impressive efficacy in relapsed/refractory (R/R) diffuse large B-cell lymphoma (DLBCL). However, CAR-T therapy-related severe cytokine release syndrome and neurological toxicity limit its clinical application in R/R DLBCL patients with high tumor burden. Here, we conducted a phase II clinical trial testing the efficacy and toxicities of CAR-T therapy in R/R non-Hodgkin lymphoma patients (NCT03196830). Among the enrolled patients, 10 R/R DLBCL patients with high tumor burden were analyzed. Before CAR-T therapy, 4 were treated with intensive combined chemotherapy (C-CAR-cohort), and 6 were exposed to radiotherapy (R-CAR-cohort). Patients in the R-CAR-T-cohort showed a higher overall response rate (100% vs. 25%, P=0.033) and less severe cytokine release syndrome (0% vs. 100%, P=0.0048) and neurotoxicity (0% vs. 75%, P=0.033) incidences than patients in the C-CAR-T-cohort. Furthermore, one case who responded to CAR-T therapy initially and who suffered a relapse shortly was exposed to radiation and achieved complete remission, with an increase in the number of CAR-T copies detected. This study demonstrates that radiotherapy is an optimal debulking regimen to managing R/R DLBCL patients before CAR-T therapy and a promising alternative salvage therapy for patients who suffer a relapse after CAR-T therapy by fuelling CAR-T copies.

Relapse is the major cause of mortality in patients with acute myeloid leukemia (AML) after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Effective preventive intervention in high-risk AML may be crucial. In this study, we investigated the clinical efficacy and safety of low dose decitabine (DAC) as part of a modified Busulfan-Cyclophosphamide (Bu-Cy) regimen for high-risk AML patients undergoing allo-HSCT to reduce relapse rate. Fifty-nine patients received DAC (20 mg/m2/d, i.v.) for 5 days, followed by modified Bu-Cy (DAC group). A matched-pair control (CON) group of 177 patients (matched 1:3) received modified Bu-Cy only. The differences were more substantial among patients with active disease: 2-year OS, 80.7% (DAC) versus 43.5% (CON), P = 0.011 and 2-year LFS, 64.9% (DAC) versus 39.2% (CON), P = 0.024. Median time to relapse was 8 months (DAC) versus 5 months (CON) for the entire groups and 6.5 months (DAC) versus 3.5 months (CON) for patients with active disease. In summary, our data indicated that the conditioning regimen containing low dose DAC may confer a survival advantage in high-risk AML patients with active disease undergoing allo-HSCT, and a prospective randomized trial is warranted to confirm these observations.

To investigate the prognosis of patients with adult B-cell acute lymphoblastic leukemia (B-ALL) with Ikaros family zinc-finger 1 (IKZF1) mutation and determine the role of allogeneic hematopoietic stem cell transplantation (allo-HSCT) in improving the clinical outcome, we detected the IKZF1 mutation and BCR-ABL fusion gene at diagnosis in the bone marrow of 164 adult patients with B-ALL, and analyzed the clinical data of these patients retrospectively. Our analysis showed that grade III-IV acute graft-versus-host disease and IKZF1 mutation in the transplantation group and age and IKZF1 mutation in the non-transplantation group were independent factors for poor prognosis by univariate and multivariate analyses.The 3-year overall survival (OS) and leukemia-free survival (LFS) rates were much lower in the IKZF1+/BCR-ABL+ subgroup than in the IKZF1+/BCR-ABL- and IKZF1-/BCR-ABL- subgroups in both the transplantation and non-transplantation groups. The 3-year OS and LFS rates were significantly higher in the transplantation group than in the non-transplantation group with IKZF1 mutation.The study demonstrated that IKZF1 mutation was an independent factor indicating the poor prognosis of adult B-ALL and much worse prognosis in the BCR-ABL+ subgroup in both non-transplantation and transplantation groups. However, allo-HSCT significantly improved the OS and LFS of patients and also their clinical outcomes.

Primary testicular lymphoma (PTL) is quite rare. Immunohistochemical analyses show that the majority of cases are diffuse large B-cell lymphoma accounting for about 1–7% of all testicular malignanc...

BACKGROUND: Autologous stem cell transplantation (ASCT) is the standard treatment for refractory/relapsed B cell non-Hodgkin’s lymphoma (R/R B-NHL), whereas chimeric antigen receptor T (CAR-T) therapy targeting CD19 is emerging as an alternative strategy. Here, we report a comparative analysis of the 2 strategies in a single center. METHODS: We performed a prospective, single-arm study of CAR-T therapy in 29 patients with R/R B-NHL and compared the outcomes with 27 contemporaneous patients who received ASCT. NHL was diagnosed by histopathologic assessments, and the safety and efficacy of treatments were compared. RESULTS: The CAR-T group exhibited better rates of complete response (CR) (48.0% vs. 20.8%, P = 0.046) and 1-year overall survival (OS) (74.4% vs. 44.5%, P = 0.044) compared with the ASCT group. Subpopulation analysis showed that patients with International Prognostic Index scores of at least 3 achieved a significantly higher objective response rate and CR rate in the CAR-T group than in the ASCT group (ORR 72.0% vs. 10.0%, P = 0.002, and CR 38.9% vs. 0%, P = 0.030, respectively). The most common severe adverse events in the CAR-T group were cytokine release syndrome, neurotoxicity, and infection compared with cytopenia, gastrointestinal toxicity, and infection in the ASCT group. Additionally, the incidence of nonhematologic severe adverse events was markedly lower in the CAR-T group than in the ASCT group (20.7% vs. 48.1%, P = 0.030). CONCLUSION: CAR-T therapy exhibited superior clinical outcomes in safety and efficacy over ASCT in patients with R/R B-NHL, suggesting that CAR-T may be a recommended alternative to ASCT. TRIAL REGISTRATION: ClinicalTrials.gov NCT03196830. FUNDING: Funding was supplied by UniCar Therapy, National Natural Science Foundation of China (81730003), National Science and Technology Major Project (2017ZX09304021), and Science Planning Project of Suzhou (sys2018049).

Tumor necrosis factor receptor-associated factor 6 (TRAF6), an (K63) E3-ligase, plays a role in many biological processes and its activity is relevant in diffuse large B cell lymphoma (DLBCL) biology. Although molecules that trigger TRAF6 activation have been defined, those that stabilize TRAF6 and/or enhance TRAF6 function remain largely unclear. We found that TRAF6 amplifies pAKT signaling in DLBCL. Moreover, TRAF6 activation and stabilization of its ubiquitination profile are facilitated by smoothened (SMO), signal transducer of canonical Hedgehog signaling. Here, we report that SMO is needed to facilitate and maintain TRAF6-dependent elevated pAKT levels, and that the SMO/TRAF6 axis contributes to doxorubicin resistance in DLBCL. Mechanistically, we found that SMO, through its C-terminal tail, stabilizes and protects TRAF6 from degradation, an effect mediated by ubiquitin-specific protease-8. Moreover, this functional link between SMO and TRAF6 is reflected in DLBCL patients where high expression of both molecules correlates with poor prognosis. In summary, our study reveals a novel cell survival mechanism in which SMO stabilizes and protects TRAF6 from degradation. The axis SMO/TRAF6/AKT is highly relevant in the biology of DLBCL and is involved in doxorubicin resistance.

Backgrounds: Refractory or relapsed(R/R) acute leukemia (AL) patients with TP53 alterations have a dismal outcome with current therapy. Recently, chimeric antigen receptor T cells (CAR-T) therapy has emerged as a novel immunotherapy modality in treating patients with R/R acute B lymphocytic leukemia (B-ALL) patients, with consistently high response rates and durable remissions and even cures across multiple studies. However, successful treatment of CAR-T therapy in R/R AL with TP53 alterations has been a challenge. Here, we conducted a phase II trial testing efficacy and toxicities of CAR-T therapy in R/R AL patients with TP53 alterations. Methods: The second generation CAR-T cells were successfully made from 9 patients' autologous lymphocytes and 2 donors' allogeneic lymphocytes and qualified tests were done before infusion to patients. CAR constructs used were humanized CAR targeting CD19, CD22 or tandem CD19 and CD22 according to the expression levels of each target on blasts. In this ongoing trial, 11 R/R AL patients with TP53 alterations (10 with TP53 mutations and 1 with TP53 deletions) including 2 relapsed after allo- allogeneic hematopoietic stem cell transplantation were included to evaluate the efficacy and safety of CAR-T therapy. Among the enrolled 11 R/R patients, 9 patients were diagnosed with B-ALL, 1 was diagnosed with acute myeloid leukemia (AML) and 1 was diagnosed with mixed phenotype acute leukemia (MPAL) (B-ALL and AML). After leukapheresis, all patients received fludarabine-based lymphodepletion chemotherapy with FLAG regimen (Fludarabine 30mg/m2 × 5d, Cytarabine 2g/m2 × 5d and Granulocyte-colony stimulating factor 300ug ×6d) in two cases, FC regimen (Fludarabine 30mg/m2 × 3d and Cyclophosphamide 300mg/m2 × 3d) in five cases, and FC+DAC regimen (Decitabine 50mg × 3d, Fludarabine 30mg/m2 × 3d and Cyclophosphamide 300mg/m2 × 3d) in four cases. Two days after chemotherapy, autologous/allogeneic CAR-T cells were infused within 2 to 4 days in a dose-escalation. Results: With a median (range) dose of 1×107/kg (0.1-1.5×107/kg) CAR-T cells, we observed a high response rate with 10/11 (90.9%) evaluated R/R patients achieved complete remission (CR) and 9/11 (81.8%) patients achieved molecular CR (mCR). Moreover, Most cases (8/11, 72.7%) only experienced mild to moderate cytokine release syndrome (CRS) and no neurological toxicity were observed. With a median follow-up of 10 months, 3 cases died of progressive disease including 1 case failed to response to CAR-T therapy, 1 case achieved CR and relapsed after CAR-T therapy and 1 case achieved mCR after CAR-T therapy and relapsed after the following allo-HSCT, and the left 8 cases still kept in mCR and alive. The one-year overall survival (OS) and leukemia-free survival (LFS) rates were 68.4% and 78% respectively. The 2-year cumulative incidence of relapse was 22.2%, and the non-relapse mortality rate was 0%. Further subgroup analysis showed that all 6 cases who received decitabine therapy (DAC group) achieved and maintained in mCR, while only 2 cases achieved and kept in mCR and 3 cases died from progressive disease in the cases without receiving decitabine therapy (Non-DAC group). The median OS and LFS for Non-DAC group was 10 and 16.375 months respectively, while the median OS and LFS for DAC group were unreached. Relative IL-6 and CRP levels increased dramatically in DAC group than that in Non-DAC group. However, there were no significant difference on treatment related toxicities between cases in DAC group and Non-DAC group. Conclusions: Our trial indicates that CAR-T therapy is a safe and powerful salvage approach to R/R AL patients with TP53 alterations and application of decitabine may fuel CAR-T therapy and reduce the relapse rate without increasing therapy related mortality incidences in this high risk population. Disclosures No relevant conflicts of interest to declare.

Background: Relapse remains the main cause of treatment failure post transplantation. Relapse prevention is an important strategy for acute myeloid leukemia (AML) patients. M ethods: We retrospectively analyzed the results of21 high risk AML patients who received a median number of 3 courses (range, 2 - 8) of decitabine (DAC) maintenance treatment (20mg/m2/d ×5d every 3 months for 1 year). Meanwhile, another 63 high risk AML patients without any prophylactic treatment after transplantation were included as a control group for 1:3 pair matched study. Results: With median follow-up of 23 months, 20 out of 21 (95.2%) patients maintained complete molecular remission (CMR) in DAC group, while 35 out of 63 (55.6%) patients maintained CMR in control group. Comparing with control group, the patients of DAC group had higher 3-year overall survival (OS) rates and 3-year leukemia free survival (LFS) rates (92.9% vs 51.8%, P =0.003; 94.1% vs 54.7%, P=0.002 respectively). Moreover, DAC maintenance was well tolerated in all patients and grade 3/4 or 4/4 hematological toxicities were observed in 11 of 21 (52.4%) patients. Conclusion: Our results suggested that DAC maintenance therapy was an effective and safe treatment option to prevent relapse after transplantation for high risk AML patients. Disclosures No relevant conflicts of interest to declare.

Background: CAR-T immunotherapy has shown remarkable promising results for relapsed/ refractory (R/R) acute lymphoblastic leukemia (ALL). However, CD19-targeted CAR T cell therapy for R/R ALL has a relapse rate of approximately 50% at 1 year after CART therapy, and the most common mechanism of relapse is due to CD19 antigen loss or decreased target expression on the surfaces of the lymphoblastic leukemia cells. Potential approaches to overcome this challenge include engineering CAR T cells to achieve multispecificity and to respond to lower expression levels of target antigens by dual target CAR T strategies. Objective: In order to evaluate the efficacy and safety of CD19/CD22 dual target CAR T cells for R/R ALL patients to determine if targeting multiple antigens can prevent treatment failure and improve response rates and durability of response. Method: A novel CAR T cells with tandem targeting CD19 and CD22 antigens and a third generation CAR construct of CD28 and OX40 co-stimulatory domains were administered to 23 patients with R/R ALL with a median age of 24(6-56)years. High-risk patients were enrolled in this cohort, including 10 with BCR-ABL+ALL (6 of 10 with T 315I mutation), 4 with TP53 mutation, 2 with Ph-like ALL and 3 with relapsed ALL post allo-HSCT. The patients received a single infusion of CAR T cells with dose escalation schedule:10%--30%--60%. The median infusion dose of CAR T cells was 1 (0.5- 2.5) ×107cells/kg. Results: The median follow-up was 9 (3.5-20) months. The day 28 CR/CRi rate was 100% with21/22 (95.5%) molecular remission. Six-month OS was 94.4% and RFS was 76.9%. One-year OS was 57.2% and RFS was 67.3%. One-year OS of high tumor burden (BM blast> 30%)(15/22) and low tumor burden(BM blast≤30%)(7/22) was 47.5% and 75% respectively (p>0.05). The 1-year OS of the CAR T- to- allo-HSCT group(14/22) was better than that of the non-transplant group(8/22) (OS 72.9% vs. 26.7%, p = 0.116). The cumulative relapse rate of 6 months and 1 year were 23.1% and 32.7% respectively. The overall incidence of CRS were 91% included 22.7%grade III CRS ,4.5% grade IV CRS and 0% severe CRES. Only one patient presented MAH syndrome and was cured with low dose dexamethasone. There was no CAR T-related death. Conclusions: The tandem CD19/CD22 dual target CAR T cells therapy is a safe and high efficacy treatment for R/R ALL patients. It is possible that multi-targeted CAR-T cell therapy may overcome this resistance mechanism and improve clinical outcomes. Disclosures No relevant conflicts of interest to declare.

Background: Chemo-resistance is a core challenge in successful treatment of diffuse large B cell lymphoma (DLBCL). It has been previously reported that DNA hypermethylation and histone deacetylation are two major epigenetic modifications contributing to chemo-resistance in multiple tumors. Moreover, combination of histone deacetylase inhibitors (HDI) and DNA methyltransferase inhibitors (DNMTI) resulted in synergistic anti-lymphoma effect toward refractory and/or relapsed (R/R) DLBCL cells in vitro and in vivo xenografts. R-GemOx (rituximab, gemcitabine, and oxaliplatin), as a first-line chemotherapy regimen for elderly primary DLBCL patients or a salvage chemotherapy regimen for R/R DLBCL patients not candidates for high-dose therapy, has shown high activity with a relative low toxicity profile. Herein, we therefore aimed to assess the efficacy, safety, and feasibility of the dual epigenetic agents plus R-GemOx regimen (CD-R-GemOx) as a salvage treatment in R/R DLBCL patients. Methods: 13 R/R DLBCL patients including 8 males and 5 females, diagnosed with R/R DLBCL on the basis of the 2008 World Health Organization guidelines, who had failed from previous salvage treatment were exposed to dual epigenetic agents (Chidamide 30mg biw and Decitabine 10mg/m2 qd d1-d5) and sequential R-GemOx(rituximab 375 mg/m² qd d6; gemcitabine 1 g/m² d7,d14; and oxaliplatin 100 mg/m² d8) for salvage chemotherapy. Median age of these patients was 56 (35-67) years old. Most (10/13) patients have advanced Ann Arbor stages. The cycle was repeated every 4 weeks. Clinical efficacy were assessed after two cycles. Results: All thirteen (100%) patients achieved disease control response. Ten (76.9%) patients achieved an overall response at the end of the treatment, with three (23.1%) achieving a complete response. Common grade 3-4 adverse events were haematological toxicities (thrombocytopenia in ten [76.9%] patients, anaemia in six [46.2%], and neutropenia in twelve [92.3%]) and gastrointestinal complications (nausea in four [30.8%] patients, vomiting in one [7.7%], diarrhoea in six [46.2%] and mucositis in six [46.2%]). Besides, 6/13 (46.2%) cases manifested with pyrexia; 3/13(23.8%) cases manifested with increase of ALT and hyperbilirubinemia. All above toxicities were reversible and recovered within 1 month after chemotherapy. No neurological toxicities and treatment-related deaths were observed. Conclusions: Our study indicates that combination of dual epigenetic agents and R-GemOx is a safe and promising salvage approach to managing R/R DLBCL patients and may serve as an optimal salvage treatment for bridging autologous transplantation. Disclosures No relevant conflicts of interest to declare.

Diffuse large B-cell lymphoma (DLBCL) is the most common lymphoid malignancy in adults. Aberrant activation of Hedgehog (Hh) and nuclear factor (NF)-κB pathways is ubiquitously observed and known to mediate tumor growth, survival, and chemoresistance in DLBCL. Here, we find that activation of Hh signaling is positively correlated with NF-κB pathway in DLBCL tumors, and that smoothened (SMO), the signal transducer subunit of Hh pathway, contributes to NF-κB activation through recruiting G protein subunits Gαi and Gα12 to activate PKCβ/CARMA1/TRAF6/NEMO signaling axis followed by assembling of the CARMA1/BCL10/MALT1/TRAF6 complex to SMO. Moreover, functional inhibition of SMO enhances the cytotoxic effects of NF-κB inhibitor. Altogether, our study reveals a noncanonical Hh signaling pathway in which SMO activates trimeric G proteins and CARMA1-associated signaling complex, leading to NF-κB activation. This signaling cascade contributes to the survival of DLBCL and may serve as a potential target for combination therapies in DLBCL.

Hedgehog (Hh) signaling pathway is activated in diffuse large B-cell lymphoma (DLBCL). Genetic abnormalities that explain activation of Hh signaling in DLBCL are unknown. We investigate the presence of amplifications of Hh genes that might result in activation of this pathway in DLBCL. Our data showed few extra copies of GLI1 and SMO due to chromosomal aneuploidies in a subset of DLBCL cell lines. We also showed that pharmacologic inhibition of PI3K/AKT and NF-κB pathways resulted in decreased expression of GLI1 and Hh ligands. In conclusion, our data support the hypothesis that aberrant activation of Hh signaling in DLBCL mainly results from integration of deregulated oncogenic signaling inputs converging into Hh signaling.

Radiotherapy is the primary treatment for nasopharyngeal carcinoma (NPC), but radioresistance severely reduces NPC radiocurability. Here, we have established a radio-resistant NPC cell line, CNE-2R, and investigate the role of miRNAs in radioresistance. The miRNAs microarray assay reveals that miRNAs are differentially expressed between CNE-2R and its parental cell line CNE-2. We find that miR-205 is elevated in CNE-2R. A target prediction algorithm suggests that miR-205 regulates expression of PTEN, a tumor-suppressor. Introducing miR-205 into CNE-2 cells suppresses PTEN protein expression, followed by activation of AKT, increased number of foci formation and reduction of cell apoptosis post-irradiation. On the other hand, knocking down miR-205 in CNE-2R cells compromises the inhibition of PTEN and increases cell apoptosis. Significantly, immunohistochemistry studies demonstrate that PTEN is downregulated at late stages of NPC, and that miR-205 is significantly elevated followed the radiotherapy. Our data conclude that miR-205 contributes to radioresistance of NPC by directly targeting PTEN. Both miR-205 and PTEN are potential predictive biomarkers for radiosensitivity of NPC and may serve as targets for achieve successful radiotherapy in NPC.

Successful treatment of diffuse large B-cell lymphoma (DLBCL) is frequently hindered by the development of resistance to conventional chemotherapy resulting in disease relapse and high mortality. High expression of antiapoptotic and/or drug transporter proteins induced by oncogenic signaling pathways has been implicated in the development of chemoresistance in cancer. Previously, our studies showed that high expression of adenosine triphosphate-binding cassette drug transporter ABCG2 in DLBCL correlated inversely with disease- and failure-free survival. In this study, we have implicated activated hedgehog (Hh) signaling pathway as a key factor behind high ABCG2 expression in DLBCL through direct upregulation of ABCG2 gene transcription. We have identified a single binding site for GLI transcription factors in the ABCG2 promoter and established its functionality using luciferase reporter, site-directed mutagenesis and chromatin-immunoprecipitation assays. Furthermore, in DLBCL tumor samples, significantly high ABCG2 and GLI1 levels were found in DLBCL tumors with lymph node involvement in comparison with DLBCL tumor cells collected from pleural and/or peritoneal effusions. This suggests a role for the stromal microenvironment in maintaining high levels of ABCG2 and GLI1. Accordingly, in vitro co-culture of DLBCL cells with HS-5 stromal cells increased ABCG2 mRNA and protein levels by paracrine activation of Hh signaling. In addition to ABCG2, co-culture of DLBCL cells with HS-5 cells also resulted in increase expression of the antiapoptotic proteins BCL2, BCL-xL and BCL2A1 and in induced chemotolerance to doxorubicin and methotrexate, drugs routinely used for the treatment of DLBCL. Similarly, activation of Hh signaling in DLBCL cell lines with recombinant Shh N-terminal peptide resulted in increased expression of BCL2 and ABCG2 associated with increased chemotolerance. Finally, functional inhibition of ABCG2 drug efflux activity with fumitremorgin C or inhibition of Hh signaling with cyclopamine-KAAD abrogated the stroma-induced chemotolerance suggesting that targeting ABCG2 and Hh signaling may have therapeutic value in overcoming chemoresistance in DLBCL.

Background: Chemoresistance is a core challenge in successful treatment of diffuse large B cell lymphoma (DLBCL). Chimeric antigen receptor T cells (CAR-T) therapy, as a novel immunotherapy modality, demonstrates impressive efficacy and durable remissions in relapsed/refractory(R/R) B-cell malignancies including DLBCL. However, CAR-T therapy is also associated with potentially fatal toxicities, including cytokine release syndrome (CRS) and neurological toxicities which limit the clinical application of CAR-T therapy in R/R DLBCL with high tumor burden. How to improve the prognosis of this population is urgently underway. Here, we conducted a trial testing efficacy and toxicities of CAR-T therapy in R/R lymphoma patients (This study is registered at www.clinicaltrials.gov as NCT03196830). Methods:Human T cells were collected from autologous/allogenous peripheral blood mononuclear cells (PBMC) . CD3+T cells were separated from PBMS of patients or donors by degradable anti-CD3 magnetic microbeads followed by 24h stimulation with CD3 and CD28 monoclonal antibody (5ug/ml). Then CD3+T cells were transduced with lentivirus particles incorporated with a humanized CAR construct targeting CD19, CD20 and CD22 and further expanded for 7 to 10 days in vitro. The transfection efficacy, ratio of CD4+ versus CD8+, antitumor activities, pathogen detection as well as cytokines releasing of CAR-T cells were evaluated before infusion to patients. In this ongoing trial, 14 patients were diagnosed as R/R DLBCL with high tumor burden. Two debulking conditioning regimens including intensive combined chemotherapy or radiation (40 gray in 20 fractions) were carried out before CAR-T therapy followed by sequential FC regimen (cyclophosphamide 300mg/m2 and fludarabine 30mg/m2 X 3d) for lymphodepletion chemotherapy. Two days after FC regimen, autologous/allogenous CAR-T cells targeting CD19 and CD20 or CD22 provided by the unicar-therapy bio-medicine technology co.(Shanghai, China) at average dose of 1-2×107 cells for each target per kilogram(kg) were infused within 3 days. Results: 14 R/R DLBCL patients with high tumor burden were enrolled in the study. Patients ranged from 39 to 66 years of age had received two to four prior lines of therapy. Out of the 14 patients, 7 patients received intensive second-line chemotherapy (median age, 46 years) and 7 received radiotherapy (median age, 63years) to debulking tumor after leukapheresis and during CAR-T cells preparation. All Adverse Events occurring within 30 days of CAR-T cells infusion were graded and reported for the 14(100%) treated patients. All patients experienced CAR-T-related CRS of any grade with severe CRS (grade 3/4/5) reported in all patients of chemotherapy cohort experienced and mild CRS (grade 1/2) reported in all patients of radiation cohort. 4 patients (57.1%) in chemotherapy cohort manifested with neurological toxicity while no patient in radiation cohort manifested with neurological toxicity. Comparable hematological toxicity as well as non-hematological and non-neurological toxicity were observed in two groups. Four of seven (57.1%) patients achieved an objective response after CAR-T infusion, with three of seven (42.9%) achieving a CR in chemotherapy cohort, while seven of seven (100%) achieved an objective response after CAR-T therapy, with four of seven (57.1%) achieving a CR in radiation cohort. Conclusions:Our clinical trial indicates that compared to intensive second-line chemotherapy, radiation is a better approach to enhancing efficacy and decreasing toxicity of CAR-T therapy in R/R DLBCL patients with high tumor burden. Radiotherapy in combination with CAR-T cell infusion may be the optimal alternative treatment model of R/R DLBCL with high tumor burden. Disclosures No relevant conflicts of interest to declare.

Lung cancer is a leading cause of morbidity and mortality worldwide. Cyclooxygenase-2 (COX-2) expression is upregulated in lung carcinomas and is considered an attractive therapeutic target. In this study, the effect of curcumin and curcumin analogues on COX-2 expression induced by phorbol 12-myristate 13-acetate (PMA) were investigated. We found that a novel curcumin analogue (GL63) inhibited PMA-induced COX-2 mRNA and protein levels in H460 cells to a greater degree than curcumin. To understand the molecular mechanisms governing COX-2 regulation, the effect on COX-2 mRNA degradation was examined; we found that GL63 significantly decreased COX-2 mRNA stability by reducing cytoplasmic localization and protein abundance of human antigen R (HuR). The 3'-untranslated region (3'-UTR) report gene assay also showed GL63 substantially reduced the 3'-UTR green fluorescent protein values, indicating that the destabilizing effect on COX-2 mRNA may be couple with the posttranscriptional inhibition of COX-2. Taken together, our results provide evidence that the novel curcumin analogue can effectively inhibit PMA-induced COX-2 expression in H460 cells, a mechanism associated with COX-2 mRNA stability and post-transcriptional regulation.

Tumor necrosis factor receptor-associated factor 6 (TRAF6), an (K63) E3-ligase, plays a crucial role in many biological processes and its activity is relevant in the biology of multiple cancers including diffuse large B cell lymphoma (DLBCL). Although molecules that trigger TRAF6 activation have been defined, those that stabilize TRAF6 levels and/or enhance TRAF6 function remain largely unclear. Previously, we found that activation of smoothened (SMO) with recombinant Hedgehog (Hh) ligand increased the binding between SMO with TRAF6, as well as TRAF6 protein levels (Blood 2013; 121:4718-28). In addition, transient overexpression of SMO resulted in increased K63-Ub of both TRAF6 and NEMO indicating stabilization of these proteins resulting in NF-kB activation. This is relevant, as more recently we found that TRAF6 amplifies pAKT signaling in DLBCL and that TRAF6 is the dominant E3 ligase for the K63-Ub of AKT in DLBCL. Moreover, TRAF6 recruitment to the cell membrane, and stabilization of its ubiquitination profile are facilitated by SMO. SMO is a member of the Frizzled-class G-protein-coupled receptor (GPCRs) and is traditionally known for its role as signal transducer in canonical Hedgehog (Hh) signaling. These observations prompted us to investigate whether the ability of SMO to increase TRAF6 levels is limited to ligand induced signaling, whether it contributes to chemoresistance in DLBCL cells, and whether SMO directly participates in controlling TRAF6 levels. To confirm the regulatory role of SMO in the TRAF6/AKT axis in DLBCL cells (HBL1 and HT) and further outline the nature of the underlying regulation, we measured the impact of activation of the Hh pathway with recombinant Shh ligand on TRAF6 levels, with and without SMO knockdown or recombinant SMO overexpression. Canonical Hh signaling results in the activation of the GLI1 transcription factor and the subsequent elevation of GLI1 mRNA levels is an established indicator of activation of the Hh pathway. However, neither SMO activation nor the knockdown of GLI1 had a significant impact on TRAF6 mRNA levels. These findings indicate that TRAF6 is not transcriptionally regulated by SMO signaling through GLI1 (canonical Hh signaling). In contrast, overexpression of SMO or siRNA knockdown of SMO resulted in an increase or decrease of TRAF6 protein levels, respectively. Consistent with the decrease of AKT activation (pAKT T308 and S473) after TRAF6 knockdown, the increase in TRAF6 levels that follows SMO overexpression resulted in an increase in the levels of AKT phosphorylation. Altogether, these observations suggest a post-translational regulation of TRAF6 by SMO. Indeed, stable knockdown of SMO dramatically reduces the half-life of TRAF6 in both HBL1 and HT cells in the presence of cyclohexamide. Furthermore, overexpression of SMO increases K63-Ub of both TRAF6 and AKT. In contrast, the SMO induced decrease in K48-Ub occurred only for TRAF6 but not for AKT. These data link the SMO-stimulated activation of TRAF6 to the enhancement of AKT signaling and protection of TRAF6 from proteasomal degradation. Mechanistically, we found that SMO, through its C-terminal tail, stabilizes TRAF6 and protects TRAF6 from proteosomal degradation, an effect mediated by ubiquitin-specific protease-8 (USP8). Importantly, this functional link between SMO and TRAF6 is reflected in DLBCL patient samples where high expression of both molecules correlates with poor prognosis. Resistance to DXR is a serious challenge in the treatment of DLBCL, and activated AKT is known to contribute to DXR resistance in multiple cancers including DLBCL. We evaluated whether SMO and TRAF6 support resistance to DXR in DLBCL cell lines. We exposed HT and HBL1 cells as well as their counterparts with stable knockdown of TRAF6 or SMO to DXR for 96hrs. Cell viability after exposure to DXR was determined by an Annexin V and PI staining assay. Silencing SMO or TRAF6 dramatically decreased cell survival after treatment with DXR. In summary, we report that SMO is needed to facilitate and maintain TRAF6-dependent elevated pAKT levels in DLBCL cell lines of germinal (GC) and non-GC subtypes, and that the SMO/TRAF6 axis contributes to DXR resistance in DLBCL. Our study reveals a novel and potential central cell survival signaling mechanism in which SMO stabilizes and protects TRAF6 from proteosomal degradation. Disclosures Lossos: Affimed: Research Funding.

Aberrant activation of Hedgehog signaling has been described in a growing number of cancers, including malignant lymphomas. Here, we report that canonical Hedgehog signaling modulates the transcriptional expression of AKT genes and that AKT1 is a direct transcriptional target of GLI1. We identified two putative binding sites for GLI1 in the AKT1 promoter region and confirmed their functionality using chromatin immunoprecipitation, luciferase reporter, and site-directed mutagenesis assays. Moreover, we provide evidence that GLI1 contributes to the survival of diffuse large B-cell lymphoma (DLBCL) cells and that this effect occurs in part through promotion of the transcription of AKT genes. This finding is of interest as constitutive activation of AKT has been described in DLBCL, but causative factors that explain AKT expression in this lymphoma type are not completely known. In summary, we demonstrated the existence of a novel cross-talk at the transcriptional level between Hedgehog signaling and AKT with biological significance in DLBCL.

Previously, we have demonstrated that inhibition of Hedgehog pathway induces predominantly apoptosis in diffuse large B-cell lymphoma (DLBCL) cell lines of activated B-cell (ABC) type but predominantly cell cycle arrest in those of germinal center (GC). Here, we explored the possibility of overcoming the resistance to apoptosis to SMO inhibitors in five DLBCL cells of GC type using the combination of the SMO inhibitor HhAntag (Genentech Inc) with the BH3 mimetic ABT-737 (Abbott Laboratories). As controls we have used two DLBCL of ABC type (OCI-LY10 and OCI-LY3). Combinatorial treatments were performed with increasing concentrations of the HhAntag with low doses (equal or less than the IC20) of ABT-737. MTS assays were used to detect changes in cell viability and Annexin-V and PARP1 cleavage assays were used to detect apoptosis. Combining low doses of ABT-737 with increasing concentrations of HhAntag in GC DLBCL cell lines resulted in significantly increase of apoptosis in comparison to treatments with the SMO inhibitor alone. We concluded that in GC DLBCL cell lines, in contrast to those of ABC type, functional inhibition of BCL2 family members is usually needed to overcome the resistance to apoptosis to SMO inhibitors. These findings provide a rationale to explore the use of SMO and BCL2 inhibitors as adjuvant therapy for treatment of DLBCL of GC type.

Constitutive photomorphogenic 1 (COP1) is a p53-targeting E3 ubiquitin ligase that is downregulated by DNA damage through mechanisms that remain obscure. Here, we report that COP1 is not downregulated following DNA damage in 14-3-3σ null cells, implicating 14-3-3σ as a critical regulator in the response of COP1 to DNA damage. We also identified that 14-3-3σ, a p53 target gene product, interacted with COP1 and controlled COP1 protein stability after DNA damage. Mechanistic studies revealed that 14-3-3σ enhanced COP1 self-ubiquitination, thereby preventing COP1-mediated p53 ubiquitination, degradation, and transcriptional repression. In addition, we found that COP1 expression promoted cell proliferation, cell transformation, and tumor progression, manifesting its role in cancer promotion, whereas 14-3-3σ negatively regulated COP1 function and prevented tumor growth in a mouse xenograft model of human cancer. Immunohistochemical analysis of clinical breast and pancreatic cancer specimens demonstrated that COP1 protein levels were inversely correlated with 14-3-3σ protein levels. Together, our findings define a mechanism for posttranslational regulation of COP1 after DNA damage that can explain the correlation between COP1 overexpression and 14-3-3σ downregulation during tumorigenesis. Cancer Res; 71(3); 884–94. ©2010 AACR.

The mammalian constitutive photomorphogenesis 9 (COP9) signalosome (CSN), a protein complex involved in embryonic development, is implicated in cell cycle regulation and the DNA damage response. Its role in tumor development, however, remains unclear. Here, we have shown that the COP9 subunit 6 (CSN6) gene is amplified in human breast cancer specimens, and the CSN6 protein is upregulated in human breast and thyroid tumors. CSN6 expression positively correlated with expression of murine double minute 2 (MDM2), a potent negative regulator of the p53 tumor suppressor. Expression of CSN6 appeared to prevent MDM2 autoubiquitination at lysine 364, resulting in stabilization of MDM2 and degradation of p53. Mice in which Csn6 was deleted died early in embryogenesis (E7.5). Embryos lacking both Csn6 and p53 survived to later in embryonic development (E10.5), which suggests that loss of p53 could partially rescue the effect of loss of Csn6. Mice heterozygous for Csn6 were sensitized to γ-irradiation-induced, p53-dependent apoptosis in both the thymus and the developing CNS. These mice were also less susceptible than wild-type mice to γ-irradiation-induced tumorigenesis. These results suggest that loss of CSN6 enhances p53-mediated tumor suppression in vivo and that CSN6 plays an important role in regulating DNA damage-associated apoptosis and tumorigenesis through control of the MDM2-p53 signaling pathway.

Background: Constitutive activation of NF-κB signaling is a hallmark of DLBCL. Activation of NF-κB is a multifactorial process resulting from oncogenic mutations (CARD11, MYD88…), chromosomal abnormalities, chronic activation of B-cell receptor signaling (BCR) as well as stimuli from the microenvironment. Chronic activation of BCR is not only the result of gene mutations (e.g.CD79B) but also is the result of stimuli generated from the lymphoma microenvironment. We previously found that smoothened (SMO), transducer of hedgehog (Hh) signaling, enhanced NF-κB activation in lymphoma cells, independently of the presence of oncogenic mutations (Changju et al., Blood 2013). Hh ligands are provided by the lymphoma microenvironment (e.g. stromal cells) to activate SMO in the lymphoma cells. Activated SMO, recruits and activates trimeric-G-coupled proteins to activate PKCβ/CARMA1/TRAF6/NEMO axis, followed by assembling of the CARMA1/BCL10/MALT1/TRAF6 complex to SMO resulting in NF-κB activation. Now, we reveal an additional mechanism by which SMO further contributes to the activation of NF-κB in DLBCL. Summary of results: We explored if recombinant Hh ligand can activate NF-κB signaling in the presence of the BTK inhibitor (PCI-32765). Hh stimulation resulted in phosphorylation of PLCg2 (downstream of BTK) and partially rescued the inhibitory effect of the BTK inhibitor on phosphorylation of PLCg2 suggesting a crosstalk between SMO and BCR receptor signaling. We identified that SMO forms a complex with TRAF-6 and PLCg2 using immunoprecipitation and Duolink in situ proximity ligation assays. We found that SMO stabilizes and protects TRAF-6 from proteosomal degradation. SMO-dependent TRAF6 stabilization is mediated by the ubiquitin-specific protease-8 (USP-8) by removing ubiquitin from K48-linked lysine of TRAF6. Conclusions: Collectively, our data reveals multilayer crosstalk between Hh and BCR/NF-κB pathways and provide significant insights into how SMO contributes to chemotolerance and progression of DLBCL. We expect that our results will delineate novel molecular mechanisms involved in the pathobiology of DLBCL that may serve as therapeutic targets. Disclosures Vega: Seatle Genetics: Honoraria; NIH: Research Funding.

Hedgehog (Hh) signaling plays an important role in the pathobiology of B-cell malignancies including diffuse large B cell lymphoma (DLBCL). The GLI family members that include GLI1, GLI2 and GLI3 are Hh signaling transcription factors. While most research has focused on smoothened (SMO), Hh transducer receptor subunit, many lines of evidence indicate that other signaling pathways can activate GLI transcription factors. GLI1 is a zinc finger transcription factor and established oncogene. GLI1 expression increases in response to Hh signaling activation and potentiates the transcriptional output of Hh signaling. We previously demonstrated that the canonical Hh ligand-PTCH1-SMO-GLI axis is functional and plays an important role in cell proliferation, survival and chemotolerance in DLBCL (Leukemia 2010 and Oncogene 2011). Moreover, using stable GLI1 knockdown DLBCL cell lines we found that GLI1 contributes to cell survival and proliferation of DLBCL (J Biol Chem 2013). However, our understanding of the mechanisms controlling the transcriptional activity of GLI1 is limited. To identify regulatory components that participate in the transcriptional regulation of GLI1, we explored GLI1 putative interacting proteins by liquid chromatography tandem mass spectrometry following immuno-precipitation (IP) of endogenous GLI1. We found that the NF-κB kinase, IKKβ, is one of the proteins associated with GLI1. This finding was of interest as we found a positive correlation between NF-kB activity and Hh signaling in DLBCL (Blood 2013). To validate the functional interaction between endogenous GLI1 with IKKβ in-vivo, lysates of 293T cells were IP with GLI1 or control antibody and subjected to immunoblot analysis with antibodies recognizing IKKβ subunit and SuFu (known partner of GLI1) respectively. IKKβ and SuFu were co-immunoprecipitated with GLI1. To confirm these findings, 293T cells were transiently co-transfected with plasmids carrying GLI1 and FLAG-tagged IKKα and IKKβ wild-type or kinase dead (K44A) forms. Both forms (IKKα and IKKβ) were detected in the immunoprecipitated GLI1 protein complex, however only transfection of IKKβ resulted in abundance of GLI1 protein suggesting a functional role of IKKβ kinase in the regulation of GLI1 protein levels. The association (and nuclear co-localization) between IKKβ and GLI1 was confirmed in DLBCL cell lines and 293T cells by immunofluorescence studies. To investigate the role of IKKβ in the transcriptional activity of GLI1, we knocked down IKKβ gene in HBL1 (cells with CD79B mutation) cells using two independent IKKβ shRNAs. Silencing IKKβ resulted in a decrease of IKKβ expression associated with decreased levels of GLI1 and GLI1 downstrean targets (PTCH1, CCND1 and BCL2). To better understand the functional role of IKKβ kinase activity in the transcriptional regulation of GLI1 target genes, we explore the IKKβ dependent GLI1 phosphorylation sites. We co-transfected full length GLI1 and IKKβ plasmids in 293T cells and IKKβ-GLI1 complex were purified using anti-GLI1 antibody. Peptides resulting from digestion of GLI1 with several different proteases were analyzed by nanospray ion trap mass spectrometry. Seven phosphorylation sites in GLI1 C-terminal residues were identified during analysis of trypsin-digested GLI1 by mass spectrometry. These data suggest that IKKβ phosphorylates GLI1 and plays an important role in the regulation of GLI1 transcriptional activity. Because activation of NF-κB is a frequent event in DLBCL, our study will help to understand the cross talk between NF-κB and Hh signaling pathways and will open potential new avenues for targeted therapy in DLBCL. Disclosures: Vega: NIH: Research Funding; Leukemia & Lymphoma Society: Research Funding.

Diffuse large B-cell lymphoma (DLBCL) is the most common lymphoid malignancy in adults. Aberrant activation of Hedgehog (Hh) and NF-κB pathways is ubiquitously observed and known to mediate tumor growth, survival and chemo-resistance in DLBCL. Previously, we found that activation of Hh signaling is positively correlated with NF-κB pathway in DLBCL tumors and that modulating the activity of smoothened (SMO), the signal transducer subunit of Hh pathway, but not GLI1, transcriptional factor of Hh signaling, resulted in modulation of NF-kB pathway activation. Hereby, we investigate the molecular mechanisms that link SMO to NF-kB pathway. To elucidate whether there was interaction between G protein superfamily and SMO, we immunoprecipitated representative members of the Gα subfamilies (Gαi, Gα16, Gαq, Gαs and Gα12), and found that SMO was associated with Gαi, Gαq, and Gα12 in two DLBCL cell lines. Stimulation of SMO with Shh recombinant increased the recruitment of Gαi and Gα12 to SMO and also increased the GTPase activity of Gαi and Gα12. We also found that the activation of SMO with Shh in DLBCL cell lines was associated with increased total activity of PKC, phosphorylation of PKCβ1 and -2, and recruitment of CARMA1-MALT1-BCL10-TRAF6 to SMO receptor complex supporting that G proteins are involved in the transmission of the signal between SMO and NF-κB. Opposite results were seen when SMO was inhibited or silenced. As polyubiquitination of TRAF6 and NEMO (IKKγ) at lysine 63 (K63) are also important events in propagating NF-κB signaling, we examined the effect of SMO overexpression on K63-linked polyubiquitination of TRAF6 and NEMO. Transient overexpressing SMO resulted in increased polyubiquitination of TRAF6 and NEMO, supporting activation of both proteins by SMO. Moreover, functional inhibition of SMO (cyclopamine-KAAD) enhances the cytotoxic effect of NF-κB inhibitor (BAY-11-7082). Altogether, our study reveal a non-canonical Hh signaling pathway, in which SMO activates trimeric G proteins and CARMA1-associated signaling complex, leading to NF-κB activation. This signaling cascade contributes to the survival of DLBCL, and may serve as potential targets for combination therapies in DLBCL. Citation Format: Changju Qu, Yadong Liu, Kranthi Kunkalla, Rajesh Singh, Marzenna Blonska, Nitin Agarwal, Xin Lin, Francisco Vega. Trimeric G protein-CARMA1 axis links smoothened to NF-κB activation in diffuse large B-cell lymphoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4114. doi:10.1158/1538-7445.AM2013-4114

Abstract 2399 Activation of the Hedgehog (Hh)/glioma-associated oncogene (GLI) pathway has been found in a growing number of malignancies. We have provided evidence that canonical Hh signaling is required for cell survival and proliferation of DLBCL cell lines. To confirm the pathogenic role of GLI1 in DLBCL, we established GLI1 knock down DLBCL cell lines (OCI-Ly19, HBL-1 and BJAB) using a lentiviral shRNA system and performed cell viability and apoptosis assays. Cell viability assays demonstrated that GLI1 knockdown DLBCL cells experienced a statistically significantly decrease in the number of viable cells in comparison with control cells harboring scramble shRNA. To examine whether decreases number of cell viability in GLI1 knock down cells were due to apoptosis, we performed annexin V and PI assays. We observed marked increase of apoptosis in GLI1 knock down DLBCL cells versus controls (2.5 fold increase for OCI-Ly10, and 5 fold for HBL1 and BJAB). To investigate the mechanism by which GLI1 regulates tumorigenesis and cell survival, we searched for whole genome GLI1-target genes in DLBCL cells using CHIP sequencing technique and identified AKT genes as potential targets of GLI1. Using pharmacological and silencing approaches, we observed that Hh signaling modulates the expression of AKT genes in DLBCL cells. We further identified two putative binding sites for GLI1 in the AKT1 promoter region and confirmed their functionality using chromatin immunoprecipitation, luciferase reporter and site-directed mutagenesis assays. To investigate whether there is any correlation between AKT1 and GLI1 mRNA expression in human DLBCL tumors, we performed quantitative real-time PCR analyses in 17 frozen DLBCL specimens including apharesis samples from pleural effusions. The real time PCR analysis revealed a strong Spearmen correlation coefficient (R2=0.9) between GLI1 and AKT1 mRNA expression. In summary, we provide evidence of the role of GLI1 in the pathobiology of DLBCL and demonstrated a cross talk, at the transcriptional level, between Hh signaling and AKT in DLBCL. A link between these 2 pathways at the trasncriptional level was not previoulsy documented. This finding is of clinical interest as AKT has a key role in lymphoma cell survival and constitutive activation of AKT has been described in DLBCL. Disclosures: No relevant conflicts of interest to declare.

Abstract 1298 Aberrant activation of hedgehog (Hh) and NF-kB pathways contribute to tumor cell growth, survival and chemotolerance in diffuse large B-cell lymphoma (DLBCL). Previously, we documented a functional crosstalk between hedgehog (Hh) and NF-kB pathways that contribute to tumor cell growth and survival in diffuse large B-cell lymphoma (DLBCL). However, the molecular mechanisms that link Hh with NF-kB pathway have not been defined. Based on that smoothened (SMO) has been associated with heterotrimeric G protein members of the Gα i family and established as a GPCRs-like protein, we hypothesize that GPCRs-related mechanisms such as the protein kinase C (PKC)-CARMA1-BCL10/MALT1/TRAF6 axis may contribute to SMO-dependent activation of NF-kB. First, we confirmed that SMO contributes to the activity of total PKC in 293T and DLBCL cells. Transiently or stably silencing of SMO resulted in decreased total PKC activity in comparison with the controls, while overexpression of SMO resulted in increased activity of PKC. Cyclopamine-KAAD or recombinant Shh N-terminal peptide resulted in decrease or increase of the total activity of PKC, respectively. As PKC isoforms β-1 and β-2 are the major isoforms expressed in B-lymphocytes that mediates NF-kB activation induced by activation of the B-cell receptor, we assessed the activation status of these two isoforms in response to changes of SMO activity. Inhibiting SMO with cyclopamine-KAAD or silencing SMO by siRNA decreased the phosphorylation status of PKCβ-1 and −2. In contrast, activating SMO with Shh N-terminal peptide increased the phosphorylation of PKCβ-1 and −2. Next, we assessed if SMO can modulated the activity of CARMA1. CARMA1 is a scaffold protein that serves to integrate the upstream signal of PKCs with downstream effectors in hematopoietic cells. We found that cyclopamine-KAAD or silencing SMO by siRNA decreased the phosphorylation status (inactivation) of CARMA1 and that activation of SMO with Shh N-terminal peptide increased the phosphorylation (activation) of CARMA 1. Because the polyubiquitination of TRAF6 and NEMO (IKKg) at lysine 63 (K63) are important events in propagating NF-kB signaling we examined the effect of overexpressing SMO on K63 polyubiquitination of TRAF6 and NEMO. Overexpression of SMO resulted in increased polyubiquitination of TRAF6 and NEMO at K63 supporting NF-kB pathway activation. TRAF6 is involved in activation of TAK1 and the IKK complex, resulting in translocation of NF-kB to the nucleus and activation of NF-kB. Silencing SMO by shRNA decreased total levels of TRAF6 that was associated with faster proteosomal degradation of TRAF6 in 293T cells. Inhibition of SMO with cyclopamine-KAAD also decreased the total levels of TRAF6 in DLBCL cells. In addition, overexpression of SMO (including a constitutively active mutated SMO) in 293T cells caused increase of TRAF6 expression by decreased polyubiquitination of TRAF6 at lysine 48 (K48) that targets TRAF6 for proteosomal degradation. In summary, altogether, these findings support that at least one mechanism by which SMO contributes to modulate activation of NF-kB is through the activation of the axis PKCβ/CARMA1/TRAF6/NEMO and stabilization and blocking degradation of TRAF6. Disclosures: No relevant conflicts of interest to declare.

Abstract 3483 Activated Hedgehog (HH) signaling was identified in our laboratory to contribute to cell survival, proliferation and chemotolerance of diffuse large B-cell lymphoma (DLBCL) (Leukemia 2010;24:1025 & Oncogene 2011, in press). The HH receptor complex is integrated by two main proteins, smoothened (SMO) and patched-1 (PTCH1). SMO is a seven-transmembrane G protein-coupled receptor that transduces HH signal to the cytoplasm and has glioma-associated oncogene homologue (GLI) proteins as major signaling transcription effectors. PTCH1 is a 12 transmembrane protein that inhibits SMO in the absence of HH ligands. Here, we investigated potential cross talk between SMO with the activation status of PI3K/AKT and NF-kB, two relevant oncogenic pathways in DLBCL. Using a small interfering (si)RNA approach and DLBCL cell lines of germinal center (GC) and activated B-cell (ABC) type we found that the expression levels of SMO modulate the activation status of AKT and canonical NF-KB pathways in DLBCL cells of GC type and mainly AKT in those of ABC type. In DLBCL cells of GC cell type, silencing SMO resulted in decrease of the phosphorylation levels of ser473p-AKT and ser536p-P65 and silencing PTCH1 resulted in increase of the phosphorylation levels of both proteins. The same silencing experimental approach in DLBCL cells of ABC type resulted in similar modulation in the activation status of the AKT but not, or to less extent, in the activation of NF-kB pathway. The modulation of the activation status of the NF-KB pathway was also confirmed using protein nuclear extracts and DNA binding ELISA assays. In cells of both DLBCL subtypes, silencing of the SMO transcriptional effector GLI1 showed no changes in the activation status of both pathways. The modulation in the activation status of AKT and NF-KB was also detected using SMO inhibitors, cyclopamine-KAAD and HhAntag (Genentech Inc) or activators, purmorphamine and recombinant HH protein. Combinatorial treatments with increasing concentrations of SMO inhibitors (cyclopamine and HhAntag [1.6, 3.2 and 4.8 μM]) with minimal lethal doses of Ly294002 (PI3K inhibitor) or BAY-11 (NF-KB inhibitor) were also performed. Using cell viability, and apoptosis (Annexin-V) assays, we found that combined treatments of PI3K or NF-KB inhibitors with a SMO inhibitor resulted in an additive/synergistic decrease of cell viability and increase of apoptosis in comparison to the treatments with SMO inhibitors alone. Taken together, our data shed novel light on the contribution of SMO on the activation of PI3K/AKT and NF-kB pathways in DLBCL. Our data also provide a rationale to use SMO inhibitors in combination with inhibitors of other oncogenic pathways such as PI3K/AKT and/or NF-KB for the treatment of patients with DLBCL. Disclosures: No relevant conflicts of interest to declare.

Abstract 2479 Generally in cancers, high expression of anti-apoptotic proteins induced by oncogenic signaling pathways is a major cause for treatment failure. Previously, we have demonstrated that Hedgehog (Hh) signaling is activated and functional in diffuse large B-cell lymphoma (DLBCL) and that its inhibition, using the Smoothened (SMO) inhibitor cyclopamine-KAAD, induces apoptosis in cell lines of DLBCL of activated B-cell (ABC) subtype and cell cycle arrest (G1-phase) in those of germinal center (GC) subtype. Induction of apoptosis in ABC-DLBCL was due to decrease of BCL2 expression (protein and mRNA levels), a direct target of Hh signaling (Leukemia 2010;24:1025–36). We also found that BCL2 is upregulated in the tumor cells in the presence of stroma and that Hh inhibition abrogates this upregulation (Oncogene 2011, in press). Here, we explored the possibility of overcoming resistance to apoptosis-induced by Hh inhibition in GC-DLBCL using a combination of the SMO inhibitor “Hh antagonist” (Genentech Inc) with the BH3 mimetic ABT-737 (Abbot laboratories) in a panel of 5 GC type DLBCL cell lines, 4 of them with the t(14:18)(q32;q21) (DOHH2, SUDHL4, Toledo and OCI-LY19) and one without the translocation (HT). ABT-737 is a functional inhibitor of three major anti-apoptotic proteins of the BCL2 family (BCL2, BCL-xL and BCL-W). Combinatorial treatments were performed with increasing concentrations of the Hh antagonist (2.5, 5.0 and 7.5 μM) and with variable but minimal lethal doses of ABT-737 (inducing cell death less than 20%). Using cell viability, apoptosis (annexin-V) and cell cycle based assays, we found that the combined treatments resulted in a additive/synergistic decrease of cell viability and increase of apoptosis in comparison to the treatment with the Hh antagonist alone. In contrast to ABC type DLBCL cell lines, in DLBCL cells with t(14;18), Hh antagonist did not result in decrease expression of BCL2, BCL-xL and BCL-W indicating that the additive effect of ABT-737 was due to their functional inhibition and not by suppressing expression levels of these proteins. On the other hand, treatments with the Hh antagonist resulted in a concentration dependent decrease of BCL2 protein levels in HT cells. In summary, our data confirm that in the presence the t(14;18), inhibition of SMO does not result in decrease of BCL2 expression. In these cases, concomitant inhibition of BCL2 function and SMO is needed to induce apoptosis. Combined inhibition of Hh signaling and BCL2 function may have a therapeutic value for lymphomas with the t(14;18)(q32;q21). Disclosures: No relevant conflicts of interest to declare.