ALBERT

Description: Abstract 1730 Poster Board I-756 The sonic hedgehog (SHH) pathway, associated with proliferative stem cell niches of many organs, is frequently deregulated in diverse cancers. We have previously shown that the SHH pathway augments the survival of tumor cells by inducing antiapoptotic molecules including bcl-2 and provides resistance to a number of conventional cancer therapies. Aberrant activation of the SHH pathway has been associated with activation and maintenance of lymphoid malignancies. Additionally, our recent data indicates that p63, a p53 family member and an important marker of stem cells has multiple interacting nodes with the SHH pathway. Based on these observations, we hypothesized i) that there is crosstalk between the SHH and p53/p63 networks in cells and, ii) inhibition of the SHH pathway with simultaneous activation of the p53 pathway would result in increased cell death in cancer cell lines of lymphoid origin. Using SDS-PAGE followed by immunoblotting and RT-PCR we surveyed a panel of 18 leukemia/lymphoma cell lines for components of the SHH pathway and the p53/p63/p73 network. Robust SHH pathway expression was observed in 15 of the 18 cell lines examined. Interestingly, in p53 deficient cell lines there was an increase in p63/p73 expression as compared to cell lines with wild type (WT) p53. A set of the previously analyzed diffuse large B- cell lymphoma (DLBCL) cell lines were selected and were representative of both p53 deficient and WT cell lines and also the activated B-like DLBCL (ABC) and germinal center B-like DLBCL (GCB) subgroups. These cells were treated with cyclopamine, an inhibitor of the SHH pathway and/or nutlin, an HDM2 antagonist. Cell viability (MTS assay) was measured using both compounds at various drug concentrations and time points. In addition we also investigated the effects of the drugs individually and in combination on components of the p53/p63 and SHH axes and their targets. Our findings suggest that treatment of p53 (WT) cell lines with a combination of nutlin and cyclopamine results in reduced cell survival than treatment with either drug alone and at lower drug concentrations and that the p53 status of the cell line may be more important in determining therapeutic response to the selected compounds. In addition the p53/p63 pathway may have a novel role in regulation of specific components of the SHH pathway in cells of lymphoid origin. In conclusion, these observations provide proof of concept that a combinatorial therapeutic approach, targeting both the p53 and SHH axes would provide a more robust and favorable response in large B cell lymphomas. Acknowledgments This work was supported in part by the The Mehta Family Foundation Disclosures No relevant conflicts of interest to declare.

Description: Context and objective Approximately 5-10% of diffuse large B-cell lymphoma (DLBCL) patients carry MYC gene translocations (MYC-translocation+) with a poor prognosis after standard chemotherapy. MYC-translocation+ DLBCL patients carrying BCL2 translocations (MYC+/BCL2+ double-hit lymphoma) have a worse survival. The efficacy of adjuvant radiotherapy in this setting is unknown. The purpose of this study is to evaluate the efficacy of radiotherapy as a part of the therapeutic regimen for patients with MYC-translocation+ DLBCL. Patients and methods From the International DLBCL R-CHOP consortium program, we selected 581 patients with de novo DLBCL treated with standard R-CHOP immunochemotherapy (diagnosed and treated from 2000 to 2010). We excluded patients with transformed DLBCL, primary mediastinal, cutaneous, testicular or central nervous system large B-cell lymphomas, patients with HIV infection, and patients not treated with R-CHOP. The median follow-up was 54.9 months. Fluorescence in situ hybridization assessing MYC was performed for all the patients (n=581) and results were correlated with available clinical data to identify clinicopathologic features associated with MYC translocation, and to evaluate the prognostic significance of MYC translocations regarding overall survival (OS, from the time of diagnosis to death from any cause) and progression-free survival (PFS, from the time of diagnosis to relapse or death from any cause). In the MYC-translocation+ DLBCL group, 38 patients received chemotherapy alone and 21 patients received chemotherapy with adjuvant radiation therapy. The clinicopathologic features and survival of MYC-translocation+ DLBCL patients treated with (n=21) and without radiotherapy (n=38) after immunochemotherapy were compared to in order to evaluate the radiotherapy efficacy and other confounding factors. Results MYC translocations were detected in 59 DLBCL patients (10.2%). Patients with MYC-translocation+ DLBCL more often had bulky tumors, involvement of multiple extranodal sites, and poorer OS (hazard ratio [HR]: 2.0, 95% confidence interval [CI]: 1.20 - 3.35, P= .0083) and PFS (HR: 1.96, 95% CI: 1.22 - 3.13, P= .0005) independent of the International Prognostic Index score. Poor survival was primarily attributable to patients with MYC+/BCL2+ double-hit DLBCL who were predominantly of germinal center B-cell subtype. Among MYC-translocation+ DLBCL patients, a better survival was achieved in patients who received radiotherapy (for OS, HR: .32, 95% CI: .15 - .71, P= .0049; for PFS, HR: .35, 95% CI: .17 - .73, P= .0043). Conversely, radiotherapy abolished the adverse impact of MYC translocations. In addition, radiotherapy was associated with better survival in the subset of patients with MYC+/BCL2+ double-hit lymphoma (P = .017 for OS, and P = .05 for PFS). However, owing to the common use of radiotherapy as consolidation therapy, the favorable prognoses in the group of patients who received radiotherapy could also be attributed to limited-stage disease and more frequent complete remission (CR) after first-line treatment and therefore these factors confound interpretation of the data. To address these issues, we evaluated radiotherapy efficacy in separate patient groups: patients who achieved CR, non-CR (PR/SD/PD) patients, and patient with stage I/II, or stage III/IV disease. The efficacy of radiotherapy appeared more apparent in patients with advanced disease who did not achieve CR after first-line chemotherapy. Multivariate analysis after adjustment for stage and IPI score validated that radiotherapy significantly improved OS (HR: .28, 95% CI: .10 - .81, P= .018) and PFS (HR: .32, 95% CI: .13 - .80, P= .015) of MYC-translocation+ DLBCL patients. Conclusions The presence of MYC translocations in DLBCL is an important biomarker that facilitates prognostic prediction and treatment stratification independent of the IPI score. For chemoresistant MYC-translocation+ DLBCL patients, radiotherapy seems to be an effective adjuvant regimen likely due to the higher frequency of extranodal involvement and bulky disease in MYC-translocation+ DLBCL patients. Our results provide a rationale for larger scale studies to assess the potential role of radiotherapy in the management of MYC-translocation+ DLBCL patients, particularly patients with MYC+/BCL2+double-hit DLBCL. Disclosures: Winter: Millenium: Research Funding; Novartis : Research Funding; Pfizer (Wyeth): Research Funding; Seattle Genetics: Research Funding; Spectrum: Research Funding; Janssen (Pharmacyclics): Research Funding; Spectrum (Allos): Consultancy; Sanofi Aventis: Consultancy; Tgen: Consultancy; AMBIT Biosciences (Spouse): Research Funding; Celgene (Spouse): DSMB, DSMB Other, Research Funding; Ariad Pharmaceuticals (Spouse): Research Funding; Novartis (Spouse): Consultancy, Research Funding; Amgen (Spouse): Consultancy, Research Funding; Astellas (Spouse): Research Funding; Caremark/CVS: Consultancy; Pfizer (Spouse): Consultancy; Sanofi Aventis (Spouse): DSMB, DSMB Other; Bristol Myers Squibb (Spouse): DSMB, DSMB Other; UptoDate, Inc.(Spouse): Patents & Royalties.

Description: Abstract 2406 The death receptor Fas has a key role in mediating homeostasis, elimination of defective cells and more recently promotion of cancer. Many effective anti-cancer therapies depend on Fas-mediated apoptosis to eradicate tumor cells and ineffective Fas- apoptotic signaling is a basis for primary as well as acquired resistance to chemotherapy. We hypothesized that Fas is subjected to direct regulation and inhibition of Fas attained by cancer cells and may explain the emergence of chemoresistance. To screen for potential binding modulators of Fas, we analyzed lymphoma cells for Fas binding proteins. We first purified Fas associated proteins by using activating CH-11 antibody bound to intact BJAB cells. After immunoprecipitation, any remaining Fas, considered activation–resistant, was subjected to the second immunoprecipitation with Fas antibody B-10 followed by liquid chromatography and tandem mass spectroscopy. This purification scheme identified high scoring peptides derived from nucleolin, a nuclear protein known to be overexpressed in cancer. Nucleolin is selectively expressed on the surfaces of cancer cells and blood vessels undergoing angiogenesis. In a cell culture system, we confirmed binding of nucleolin to Fas and the presence of nucleolin-Fas complexes on the surface of lymphoma cells by surface biotin labeling. Using deletion mutants of nucleolin, we identified RNA binding domain 4 and glycine/arginine rich region to be required for the binding to Fas. BJAB cells with partially knockdown (KD) nucleolin showed significantly higher rates of apoptosis in response to stimulation with CH-11 and FasL when compared to nontarget KD controls. Importantly, the lower levels of nucleolin in knockdown cells did not affect total and surface Fas expression. Nucleolin present on the cell surface prevented binding of FasL and CH-11 to the receptor and thus provides a mechanism for blocking activation of Fas apoptosis. To examine the role of nucleon in vivo, we transfected mice with nucleolin-expressing plasmids using the hydrodynamic transfection method. The mice overexpressing nucleolin showed significantly higher survival rates than vector control transfected mice (P=.01) after a challenge with a lethal dose of agonistic anti-Fas antibody. We next examined the expression of nucleolin in human lymphomas. Cell lines derived from lymphomas of different histological types consistently expressed nucleolin protein. We found nucleolin expressed on the surface of cells in over 20 primary lymphoma isolates, whereas peripheral blood lymphocytes showed low or undetectable levels. Lymphoma tissue microarray staining showed a correlation between nucleolin and Ki-67 expression. Whether nucleolin expression also correlates with adverse clinical features in lymphoma is currently under evaluation. Taken together, we show here that the known cancer associated protein nucleolin is overexpressed on surface of lymphoma cells where it binds to Fas receptor and blocks Fas signaling and apoptosis. We expect that further analysis of nucleolin properties will reveal how Fas-nucleolin interaction can be targeted to enhance killing of cancer cells leading eventually to cell surface nucleolin targeting therapy. Disclosures: Fayad: Roche: Research Funding.