GCB Diffuse Large B-cell Lymphoma Research Articles

Cerdulatinib, a novel dual SYK/JAK kinase inhibitor, has broad anti-tumor activity in both ABC and GCB types of diffuse large B cell lymphoma

B-cell receptor (BCR) and JAK/STAT pathways play critical roles in diffuse large B-cell lymphoma (DLBCL). Herein, we investigated the anti-lymphoma activity of cerdulatinib, a novel compound that dually targets SYK and JAK/STAT pathways. On a tissue microarray of 62 primary DLBCL tumors, 58% expressed either phosphorylated SYK or STAT3 or both. SYK and STAT3 are also phosphorylated in a panel of eleven DLBCL cell lines although ABC and GCB subtypes exhibited different JAK/STAT and BCR signaling profiles. In both ABC and GCB cell lines, cerdulatinib induced apoptosis that was associated with caspase-3 and PARP cleavage. The compound also blocked G1/S transition and caused cell cycle arrest, accompanied by inhibition of RB phosphorylation and down-regulation of cyclin E. Phosphorylation of BCR components and STAT3 was sensitive to cerdulatinib in both ABC and GCB cell lines under stimulated conditions. Importantly, JAK/STAT and BCR signaling can be blocked by cerdulatinib in primary GCB and non-GCB DLBCL tumor cells that were accompanied by cell death. Our work provides mechanistic insights into the actions of cerdulatinib, suggesting that the drug has a broad anti-tumor activity in both ABC and GCB DLBCL, at least in part by inhibiting SYK and JAK pathways.

Abstract 5308: Distinct expression, activity, regulation and gene expression signature of NF-κB subunit c-Rel and the prognostic impact of crosstalk between p53, p63 and c-Rel in different subsets of diffuse large B-cell lymphoma

Abstract Background: Diffuse large B-cell lymphoma (DLBCL) is a heterogeneous aggressive non-Hodgkin lymphoma and can be classified into GCB or ABC DLBCL. Aberrant activation of NF-κB has been associated with tumor proliferation and survival ABC-DLBCL. The “canonical” pathway of NF-κB activation involves activities of IκB kinase IKK2, which lead to degradation of NF-κB inhibitors IκBα/β/ϵ and nuclear translocation of p50/p65 and p50/c-Rel dimers from the cytoplasm. Objective: To investigate the common mechanisms for c-Rel activation and function, and to evaluate the prognostic significance of c-Rel in a large group of DLBCL patients (n = 460). Results: Nuclear c-Rel expression (c-Rel+) was observed in 29.6% of DLBCL patients and correlated with higher expression levels of p50 (P = 0.12), p52 (P = 0.724E-8), p65 (P = 0.073), RelB (P = 0.013). Coimmunoprecipitation analysis using nuclear extract purified from primary GCB and ABC cells showed that in addition to the known p50 to form dimers with c-Rel, p65, p52 and RelB could also form dimers with c-Rel in some cell lines. A unique function of c-Rel in terminating NF-κB as negative feedback of IKK2 activation was suggested by the significantly upregulated genes encoding IκBα/β/ϵ, A20 and TNIP1 at the mRNA level in either GCB- or ABC-DLBCL. The B-cell receptor signaling (BCR) appeared most relevant for c-Rel activation in GCB-DLBCL, with upreglated B-cell receptors (CD19, CD20, CD79A) and downstream LYN, SYK, CARD11, MALT1, BLNK, TAK1. The role of c-Rel in cytokine production was validated in our study cohort, showing significant correlation between c-Rel+ and mRNA levels of IL1, IL3, IL6, IL10, IL12, IL21 and chemokine receptor CXCR4, mostly in GCB-DLBCL. GEP analysis showed enrichment in cytokine, growth factors, inflammation, gene expression, metabolism, actin, collagen, cell morphology and adhesion. c-Rel did not correlate with prognosis in overall DLBCL. However, c-Rel+ patients with MUT-p53 had significantly worse survival compared with other MUT-p53 DLBCL patients, especially in in ABC-DLBCL, in which c-Rel+ was associated with upregulated MUT-TP53 transcription (P = 0.0087). Conversely, MUT-p53 expression was associated significantly with upregulated REL mRNA expression (P = 0.0021), predominantly in the ABC-DLBCL subtype (P = 0.0047). Furthermore, the lack of c-Rel prognostic impact is likely due to the antagonism of p63, whose expression was correlated with c-Rel nuclear expression, and significantly better OS and PFS in ABC-DLBCL. Conclusions: Unique function and upstream activation regulation of c-Rel was suggested by analysis in 460 DLBCLs. TP53 mutation status appears to a critical determinant for c-Rel prognostic impact. p63 is another tumor suppressor for the c-Rel oncogenic function. Citation Format: Zijun Y. Xu-Monette, Ken H. Young. Distinct expression, activity, regulation and gene expression signature of NF-κB subunit c-Rel and the prognostic impact of crosstalk between p53, p63 and c-Rel in different subsets of diffuse large B-cell lymphoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5308. doi:10.1158/1538-7445.AM2015-5308

Akt activation confers an inferior survival in patients with activated B-cell subtype of diffuse large B-cell lymphoma: a report from The International DLBCL Rituximab-CHOP Consortium Program

Context Akt plays an essential role in diverse cellular processes such as cell proliferation, cell growth and apoptosis. Dysregulation of Akt signaling has been shown to correlate with an inferior survival in patients with a number of solid tumors and some hematologic malignances. The prognostic significance of Akt expression and the gene expression profile of Akt activation in diffuse large B-cell lymphoma (DLBCL) have not been thoroughly assessed. Objective In this study, we investigated Akt activation and analyzed its prognostic importance in a large cohort of patients with de novo DLBCL treated with R-CHOP immunochemotherapy. We also characterized the gene expression profile of DLBCL with activated Akt. Patients The study group consisted of 493 DLBCL patients treated with R-CHOP. These cases were organized as a part of the International DLBCL R-CHOP Consortium Program Study, and diagnosed according to the WHO criteria. Patients with primary mediastinal large B-cell lymphoma, primary cutaneous DLBCL, primary central nervous system DLBCL, and DLBCLs transformed from a low-grade B-cell lymphoma or associated with HIV infection were excluded. Methods Immunohistochemical studies (IHC) were performed in formalin-fixed paraffin-embedded sections of tissue microarrays to evaluate the expression of phosphorylated (activated) Akt and other relevant markers. Genetic alterations were examined by FISH for MYC , BCL-2 , and BCL-6 translocations and sequencing for TP53 mutation. Gene expression profiling (GEP) was performed to characterize the gene expression profile in Akt-activated DLBCL. The cell-of-origin (COO) subtypes were determined by GEP (gold standard) and IHC. Results Phosphorylated (activated) Akt (pAkt) expression was detected in 98 of 493 (20%) DLBCL tumors including 49 of 253 (19%) GCB DLBCL and 49 of 240 (20%) ABC DLBCL. In the GCB subtype, MYC, BCL-2 and MYC/BCL-2 translocations were more frequently found in pAkt+ DLBCL compared with pAkt- DLBCL ( P =0.04, 0.007 and 0.03, respectively). In contrast, translocations of these genes were rare and detected equally in pAkt+ and pAkt- subgroups within the ABC subtype ( P =0.7, 0.9 and 0.9, respectively). In the GCB group, TP53 mutation frequency was similar between pAkt+ and pAkt- DLBCL ( P =0.45). In contrast, TP53 mutations were significantly less frequent in ABC group (5% versus 22%; P = 0.01). Akt activation predicted a poorer survival, but the negative prognostic impact of Akt activation was significant in the ABC (5-year survival rate: 44% in pAkt+ versus 65% in pAkt-; P =0.01; 5-year PFS: 40% in pAkt+ versus 59% in pAkt-; P =0.01), but not GCB subtype (5-year survival rate: 67% in pAkt+ versus 74% in pAkt-, P =0.31; 5-year PFS rate: 61% in pAkt+ versus 69% in pAkt-, P =0.27). The negative impact of Akt activation appeared to be independent of Myc/Bcl-2 expression and TP53 mutation. In multivariate analysis, pAkt expression was an independent predictor of poorer OS (HR=2.72, 95% CI, 1.50-4.98, P =0.001) and PFS (HR=2.16, 95% CI, 1.22-3.82, P =0.008) in patients with ABC wild type- TP53 . In addition, AKT1 mRNA expression conferred significantly poor survival in DLBCL. Gene expression profiling revealed a distinct high-risk signature characterized by increased expression of multiple pro-survival genes (6 of 16 up-regulated genes; 40%) and decreased expression of genes encoding tumor suppressors (5 of 20 down-regulated genes; 25%) in pAkt+ ABC DLBCL. Conclusions Akt is activated in DLBCL, is equally distributed in COO subtypes, and has prognostic significance in patients with ABC DLBCL. Given the promising anti-lymphoma activity of Akt inhibitors in clinical trials, these findings may have clinical implications for DLBCL patients. Akt activation may be useful for prognostic stratification and could serve as a biomarker to guide patient selection and predict response to targeted anti-Akt therapy. Disclosures No relevant conflicts of interest to declare.

66. Identification and Characterization of a B Cell Aptamer That Targets Diffuse Large B Cell Lymphoma (DLBCL) and Chronic Myelogenous Leukemia (CML)

Diffuse large B cell lymphoma (DLBCL) is a heterogenous group of non-Hodgkin's lymphoma (NHL) constituting ~40% of all NHL diagnoses in the United States of America. Alternative therapies for DLBCL are required, since only 30-50% of patients respond to current therapy regimens of either chemotherapeutic drugs (CHOP) alone or CHOP with Rituxan (R-CHOP). The high incidence of relapse/refractory lymphoma following treatment with R-CHOP and Rituxan's non-specific binding to normal CD20+ B cell demonstrates the clinical need for alternative therapies that specifically target malignant B cells. Majority of lymphoma patients suffer from the adverse side-effects associated with aggressive therapies like radiation and chemotherapy such as nausea, hair loss, and organ damage in severe cases.In recent years, aptamers or nucleic acid ligands have been explored as novel therapeutics, since aptamers are highly specific, not immunogenic, and amenable for conjugation of therapeutic molecules such as toxins, chemotherapeutic agents, and siRNAs. Aptamers, both antagonists and agonists, can be used to target receptors on cells with high affinity and specificity. We describe the identification and characterization of an RNA aptamer against a biomarker expressed on certain types of GCB diffuse large B cell lymphoma and chonic myelogenous leukemias (CML). Aptamer appears to be specific to cancerous B cells and does not interact with healthy B cells. We are currently conducting experiments to identify the unique biomarker expressed on these GCB DLBCL cells of origin BJAB, HT, and OCI-Ly1, and of CML origin. We have performed a proof of concept study conjugating saporin to a novel aptamer that specifically targets DLBCL specific B cells and does not bind to healthy B cells. We have developed an aptamer to deliver toxic agents and other cargoes to a subset of leukemia and lymphoma cells. This aptamer is engineered into a chimeric molecule possessing two levels of specificity: the binding domain (aptamer), and the agent domain (saporin) that specifically targets and kills cancerous cells in vitro.The creation of a novel in vivo targeted therapeutic platform could result in a significant advance over the existing technology because the aptamer-drug chimera can potentially lower the amount of drug required to achieve efficacy by specifically targeting circulating, malignant B cells. In addition, this novel DLBCL-specific therapeutic will have the advantage of being more potent and cause less nonspecific damage to surrounding tissue, which are the current side-effects associated with standard treatment of care for DLBCL. Hematological malignancies are optimal for toxin conjugated therapeutics because malignant lymphocytes are intravascular and will be accessible to an intravenously administered aptamer-drug toxin. Our goal is to develop a novel aptamer-toxin chimera therapeutic that specifically targets and eliminates diffuse large B cell lymphomas. Once developed, such aptamer-drug chimeras can be evaluated in animal models of DLBCL for safety and efficacy and then be translated into first-in-human clinical studies to treat patients with DLBCL.

CC-122 Has Potent Anti-Lymphoma Activity through Destruction of the Aiolos and Ikaros Transcription Factors and Induction of Interferon Response Pathways

Background: IMiDs® Immunomodulatory agent lenalidomide (Len) is active in a range of hematologic cancers including diffuse large B-cell lymphoma (DLBCL). The molecular target of Len is cereblon (CRBN), a substrate receptor of the cullin 4 RING E3 ubiquitin ligase complex. In a CRBN-dependent manner, Len promotes ubiquitination of lymphoid transcription factors Ikaros and Aiolos leading to their subsequent degradation. This was shown to contribute to Len’s anti-Multiple Myeloma and T-cell activation properties. CC-122 is a non-phthalimide analog of thalidomide and has potent anti-proliferative, immunomodulatory and anti-angiogenic activities giving it a potentially broader range of activity than Len. CC-122, a first-in-class pleiotropic pathway modifier (PPM®), is currently in a Phase I clinical trial for DLBCL, multiple myeloma, and solid tumors (ClinicalTrials.gov NCT01421524) and showing encouraging clinical activity. The present study investigated the mechanism of action of CC-122 in pre-clinical models of DLBCL and in clinical samples from the ongoing Phase I study.Results: CC-122 inhibited proliferation (IC50=0.01-1.5μΜ) and induced apoptosis (6.5-12 fold) in 4 ABC and 3 GCB DLBCL cell lines in a dose-dependent manner. CC-122 induced rapid degradation of Aiolos and Ikaros in a CRBN-dependent manner; an effect blocked by MLN4924, a small molecule inhibitor of Cullin-RING E3 ligase complexes. In vivo anti-tumor activity of CC-122 was demonstrated in 2 xenograft models of human DLBCL, and was associated with significant reduction in Aiolos and Ikaros expression (94% and 69%, respectively, p<0.001) in tumor tissue within 6 hrs of administration. To identify proteins and signaling pathways affected by CC-122, tandem-mass tag proteomics was performed on DLBCL cells treated with vehicle or CC-122. While Aiolos and Ikaros levels decreased in a time- and dose-dependent manner, Interferon Stimulated Genes (ISGs) including IRF7, DDX58, and IFIT3 were notably increased (3, 5 and 12.5 fold, respectively). ISG induction with 24 hr CC-122 treatment was further confirmed by Western analysis. Surprisingly, the induction of ISGs occurred in the absence of increased interferon α, β, or γ levels, which was confirmed by qPCR and ELISA. Furthermore, shRNA targeting Aiolos led to inhibition of Aiolos protein expression (2-3 fold) and an increase in the mRNA and protein levels of IRF7, DDX58 and IFIT3 (10, 14 and 22 fold in mRNA expression, respectively). These data indicate that the increase in ISGs is a direct consequence of Aiolos degradation by CC-122 and bypasses the need for interferon production. Exploration of additional downstream functional gene candidates of CC-122 and shAiolos activity revealed a decrease in protein levels of Myc (up to 3 fold decrease with shAiolos), a key driver of DLBCL proliferation and survival associated with poor survival. Importantly, reduction of Aiolos expression and the consequent changes in the molecular pathways described led to inhibition of proliferation (2-4 fold versus control shRNA) and increased apoptosis (3-10 fold vs control shRNA), which paralleled CC-122 effects in DLBCL cells lines. Of note, CC-122 induction of ISGs was comparable in both ABC and GCB lines, whereas Len did not induce ISGs in GCB lines.The relationship between Aiolos and Ikaros protein expression and DLBCL cell of origin (COO) was further explored in primary DLBCL tissues. A tissue microarray containing 90 DLBCL patient cases immunohistochemically stained for Aiolos, Ikaros and COO markers (Hans) demonstrated Aiolos and Ikaros expression across GCB and non-GCB subtypes, suggesting that CC-122 may have broad-spectrum activity independent of COO. In fact, Aiolos and Ikaros were degraded in lymph node biopsies from both GCB and non-GCB DLBCL patients administered CC-122. The predictive value of Aiolos and Ikaros protein levels for clinical response will be assessed in the Phase I study.Conclusion: Aiolos degradation-dependent upregulation of ISGs represents a novel mechanism for the activity of CC-122. This mechanism bypasses the interferon-signaling pathway and occurs across cell of origin subtypes, suggesting this is a common mechanism accounting for the broad-spectrum activity of CC-122. Understanding of this mechanism has potential to greatly enhance the clinical development of CC-122 in DLBCL by guiding patient selection strategies and rational combination therapies. DisclosuresHagner:Celgene Corp: Employment, Equity Ownership. Wang:Celgene Corp: Employment, Equity Ownership. Couto:Celgene Corporation: Employment, Equity Ownership. Breider:Celgene Corp: Employment. Fontanillo:Celgene Corp: Employment, Equity Ownership. Trotter:Celgene Corporation: Employment, Equity Ownership. Bjorklund:Celgene Corp: Employment, Equity Ownership. Havens:Celgene Corporation: Employment, Equity Ownership, Patents & Royalties. Raymon:Celgene Corporation: Employment, Equity Ownership. Narla:Celgene Corporation: Employment, Equity Ownership. Barnes:Celgene Corporation: Employment, Equity Ownership. Waldman:Celgene Corporation: Employment, Equity Ownership. Daniel:Celgene Corp: Employment, Equity Ownership. Klippel:Celgene Corp: Employment. Pourdehnad:Celgene: Employment, Equity Ownership. Thakurta:Celgene Corp: Employment, Equity Ownership. Chopra:Celgene Corporation: Employment, Equity Ownership. Gandhi:Celgene Corp: Employment, Equity Ownership.

Akt Activation Confers an Inferior Survival in Patients with Activated B-Cell Subtype of Diffuse Large B-Cell Lymphoma: A Report from the International DLBCL Rituximab-CHOP Consortium Program

ContextAkt plays an essential role in diverse cellular processes such as cell proliferation, cell growth and apoptosis. Dysregulation of Akt signaling has been shown to correlate with an inferior survival in patients with a number of solid tumors and some hematologic malignances. The prognostic significance of Akt expression and the gene expression profile of Akt activation in diffuse large B-cell lymphoma (DLBCL) have not been thoroughly assessed.ObjectiveIn this study, we investigated Akt activation and analyzed its prognostic importance in a large cohort of patients with de novoDLBCL treated with R-CHOP immunochemotherapy. We also characterized the gene expression profile of DLBCL with activated Akt.PatientsThe study group consisted of 493 DLBCL patients treated with R-CHOP. These cases were organized as a part of the International DLBCL R-CHOP Consortium Program Study, and diagnosed according to the WHO criteria. Patients with primary mediastinal large B-cell lymphoma, primary cutaneous DLBCL, primary central nervous system DLBCL, and DLBCLs transformed from a low-grade B-cell lymphoma or associated with HIV infection were excluded.MethodsImmunohistochemical studies (IHC) were performed in formalin-fixed paraffin-embedded sections of tissue microarrays to evaluate the expression of phosphorylated (activated) Akt and other relevant markers. Genetic alterations were examined by FISH for MYC, BCL-2, and BCL-6 translocations and sequencing for TP53 mutation. Gene expression profiling (GEP) was performed to characterize the gene expression profile in Akt-activated DLBCL. The cell-of-origin (COO) subtypes were determined by GEP (gold standard) and IHC.ResultsPhosphorylated (activated) Akt (pAkt) expression was detected in 98 of 493 (20%) DLBCL tumors including 49 of 253 (19%) GCB DLBCL and 49 of 240 (20%) ABC DLBCL. In the GCB subtype, MYC, BCL-2 and MYC/BCL-2 translocations were more frequently found in pAkt+ DLBCL compared with pAkt- DLBCL (P=0.04, 0.007 and 0.03, respectively). In contrast, translocations of these genes were rare and detected equally in pAkt+ and pAkt- subgroups within the ABC subtype (P=0.7, 0.9 and 0.9, respectively). In the GCB group, TP53 mutation frequency was similar between pAkt+ and pAkt- DLBCL (P=0.45). In contrast, TP53 mutations were significantly less frequent in ABC group (5% versus 22%; P= 0.01). Akt activation predicted a poorer survival, but the negative prognostic impact of Akt activation was significant in the ABC (5-year survival rate: 44% in pAkt+ versus 65% in pAkt-; P=0.01; 5-year PFS: 40% in pAkt+ versus 59% in pAkt-; P=0.01), but not GCB subtype (5-year survival rate: 67% in pAkt+ versus 74% in pAkt-, P=0.31; 5-year PFS rate: 61% in pAkt+ versus 69% in pAkt-, P=0.27). The negative impact of Akt activation appeared to be independent of Myc/Bcl-2 expression and TP53 mutation. In multivariate analysis, pAkt expression was an independent predictor of poorer OS (HR=2.72, 95% CI, 1.50-4.98, P=0.001) and PFS (HR=2.16, 95% CI, 1.22-3.82, P=0.008) in patients with ABC wild type-TP53. In addition, AKT1 mRNA expression conferred significantly poor survival in DLBCL. Gene expression profiling revealed a distinct high-risk signature characterized by increased expression of multiple pro-survival genes (6 of 16 up-regulated genes; 40%) and decreased expression of genes encoding tumor suppressors (5 of 20 down-regulated genes; 25%) in pAkt+ ABC DLBCL.ConclusionsAkt is activated in DLBCL, is equally distributed in COO subtypes, and has prognostic significance in patients with ABC DLBCL. Given the promising anti-lymphoma activity of Akt inhibitors in clinical trials, these findings may have clinical implications for DLBCL patients. Akt activation may be useful for prognostic stratification and could serve as a biomarker to guide patient selection and predict response to targeted anti-Akt therapy. DisclosuresNo relevant conflicts of interest to declare.

Accurate Diagnosis of Aggressive B Cell Non-Hodgkin Lymphomas Using Gene Expression Profiling of Formalin-Fixed, Paraffin-Embedded Tissues

Accurate Diagnosis of Aggressive B Cell Non-Hodgkin Lymphomas Using Gene Expression Profiling of Formalin-Fixed, Paraffin-Embedded Tissues

Somatic Mutations in E3 Ubiquitin Ligase Deltex 1 Are Associated with Survival in Diffuse Large B-Cell Lymphoma

Diffuse large B-cell lymphoma (DLBCL) is a heterogeneous disease both biologically and clinically. Although a number of genetic alterations have been associated with the pathogenesis of DLBCL, survival association of most of the identified mutations remains to be shown. Here we have used exome and RNA sequencing (RNAseq) data to identify novel genetic events associated with treatment outcome after chemoimmunotherapy.Initial screen was performed with eight primary tumor samples and matched normal DNA using exome sequencing. The patients had clinically high risk DLBCL, and were treated in a Nordic phase II protocol with dose dense chemoimmunotherapy and CNS prophylaxis. After stringent filtering (VarScan2 p < 0.05), and removal of known germline variants, 967 somatic point mutations and indels in 561 different genes were identified. Of these, 436 were non-synonymous, and 27 were detected in more than one patient. These included both novel and several previously described DLBCL associated mutations, such as MYD88, CD79B, B2M and PTEN.To correlate the identified mutations with survival, we used RNAseq and survival data of 92 DLBCL samples from the Cancer Genome Characterization Initiative (CGCI) repository. The mutational status of the 436 genes identified in our screen was assessed with Bambino, which confirmed 405 of these genes also to be mutated in the CGCI set. Kaplan-Meier survival estimates per gene with at least one single nucleotide variant (SNV; with criteria of minimum 3 reads and 10% coverage and known polymorphisms filtered out) revealed 17 genes that were associated with overall survival (OS, p<0.05, minimum 10 samples mutated).We highlight mutations affecting Deltex1 (DTX1), which functions as an E3 ubiquitin ligase and targets Notch1 intracellular domain for degradation. In the study cohort, 14% of the patients had a non-synonymous DTX1 mutation. Mutations were equally distributed between GCB and ABC DLBCLs and low (0-2) and high (3-5) International Prognostic Index (IPI) risk groups. According to Kaplan-Meier analysis, the 5-year PFS rate for the patients with DTX1 mutations was significantly worse as compared to ones without mutations (46% vs 78%, p=0.010). The corresponding OS values were 54% and 85% (p=0.006). In multivariate analysis with IPI, DTX1 mutation status remained as an independent prognostic factor for both PFS (RR, 2.83; 95% CI, 1.17-6.85, p=0.021) and OS (RR, 3.51; 95% CI, 1.41-8.71, p=0.007). Mutational status was also found to correlate with the expression levels; the patients with mutations had lower DTX1 mRNA levels (p=0.006). When DTX1 mutations were analyzed according to different functional domains, 11 out of 16 (65%) were located in the first exon encoding the WWE 1 domain and a binding site for intracellular domain of Notch1. Interestingly, the patients with mutations in exon 1 had a significantly worse survival in comparison to other patients (Figure 1).Taken together, the results demonstrate that DTX1 mutations in DLBCL are an independent predictor of survival after chemoimmunotherapy, and mutations enriched in exon 1 identify cases with dismal prognosis. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

SYK and STAT3 Are Active in Diffuse Large B-Cell Lymphoma: Activity of Cerdulatinib, a Dual SYK/JAK Inhibitor

Non-Hodgkin Lymphoma (NHL) represents about 5 percent of all cancers diagnosed in the United States. While incidence of NHL has increased slightly over the past decade, death rates have been declining steadily. These declines in mortality can be attributed to improvements in treatment that are based on an increased understanding of the biology of the disease. Diffuse large B-cell lymphoma (DLBCL) accounts for ~30% of NHLs and greater than 80% of aggressive NHLs. Recent studies including large-scale genetic analyses have demonstrated the critical roles of the B-cell receptor’s (BCR) and JAK/STAT pathways in DLBCL. Herein, we investigated the anti-lymphoma activity of cerdulatinib (aka PRT062070), a novel compound that dually targets both SYK and JAK/STAT signaling pathways.To determine whether targeting both SYK and JAK/STAT is relevant in DLBCL, we examined the expression of p-SYK (pY525/526) and p-STAT3 (pY705) on a tissue microarray of 62 DLBCL primary tumors, including 41 GCB and 21 non-GCB cases. p-SYK expression was detected in 29 (47%) cases with a characteristic peri-membrane staining pattern. Of those 29 p-SYK positive cases, 17 were GCB type (17/41, 41%) and 12 were non-GCB type (12/21, 57%). p-STAT3 exhibits a characteristic nuclear staining pattern in DLBCL cases. A total of 26 (42%) stained positive for p-STAT3; 16 were GCB type (16/41, 39%) and 10 were non-GCB type (10/21, 48%). Interestingly, there are 19 cases (31%) with reactivity for both p-SYK and p-STAT3, among which, 11 were GCB type (27%) and 8 were non-GCB type (38%).SYK and STAT3 are also phosphorylated in a panel of nine DLBCL cell lines. Immunoblotting analyses showed that ABC and GCB subtypes of DLBCL cells appear to exhibit different JAK/STAT and BCR signaling profiles. For instance, p-AKT was highly expressed in GCB cells, whereas p-STAT3 was more strongly expressed in ABC cells. Overall, the DLBCL cells are more sensitive to the dual inhibitor than to the SYK-specific inhibitor alone. In both GCB and ABC cell lines, cerdulatinib induced apoptosis via down-regulation of MCL1 protein and PARP cleavage. The compound also blocked G1/S transition and caused cell cycle arrest through inhibition of RB phosphorylation and down-regulation of cyclin E. Further analyses of the cell signaling activities showed that STAT3 phosphorylation was sensitive to inhibition by cerdulatinib in ABC cell lines while phosphorylation of SYK, PLCg2, AKT and ERK was sensitive to inhibition by cerdulatinib in GCB cell lines. Importantly, JAK/STAT and BCR signaling can be blocked by cerdulatinib in GCB and non-GCB primary human DLBCL cells, which led to cell death of these cells. Our work provided mechanistic insights into the actions of SYK/JAK dual inhibitor cerdulatinib, suggesting that the drug may be a potent treatment of DLBCL with a broader anti-tumor activity in both ABC and GCB subtypes of the lymphoma. DisclosuresPandey:Portola Pharmaceuticals: Employment. Conley:Portola Pharmaceuticals: Employment. Coffey:Portola Pharmaceuticals: Employment.

The Impact of Immunophenotypic Subtypes and Treatment Regimens on Patient Outcomes in Monomorphic Post-Transplant Lymphoproliferative Disorders (Diffuse Large B-Cell Lymphoma)

Introduction: Post-Transplant Lymphoproliferative Disorders (PTLD) are a group of heterogeneous disorders that arise as a consequence of iatrogenic immunosuppression for solid organ or allogeneic bone marrow/stem cell transplantation. Though they all arise in a common clinical context, different types of PTLD differ with respect to their underlying biology, clinical presentation and treatment. The aims of this study were to: (1) Define the cell of origin (COO) of monomorphic Diffuse Large B-cell Lymphoma (DLBCL) PTLD and evaluate their impact on clinical presentation and survival and (2) assess the impact of different Rituximab containing treatment regimens on survival outcomes in monomorphic DLBCL PTLD patients.Methods: We conducted a retrospective review of our institutional databases to identify all the cases of monomorphic PTLD (DLBCL) diagnosed and treated at our medical center from 2000-2013. COO classification into germinal center B-cell like (GCB) and non-germinal center B-cell like (non-GCB) type was performed by immunohistochemistry using the Hans algorithm.Results: Cell of origin: 40 cases of monomorphic PTLD (DLBCL) were diagnosed during the study interval. Tissue material for COO subtyping was available for 25 patients. By immunohistochemistry 16/25 (64%) were non-GCB and 9/25 (36%) were GCB subtype, median age of presentation being 46 years (range 3-75) and 48 years (range 3-64), respectively. A trend towards EBV positivity (by in situ hybridization for EBV encoded RNA) was noted in the non-GCB group (75% vs. 33%) [p=0.09]. Non-GCB DLBCL PTLD presented earlier post-transplant at a median 1.5 years (range 0.2-15) vs. 3.9 years (range 0.7-17) for GCB cases. When comparing immunosuppressive therapy at the time of PTLD presentation, an association between Tacrolimus therapy and non-GCB phenotype was identified [p=0.03]. Non-GCB DLBCL PTLD demonstrated a trend towards higher rates of extra nodal involvement (88% vs. 44%) [p=0.06] and advanced stage disease (Stage III/IV 75% vs. 33%) [p=0.09]. No significant differences in organ transplanted, LDH, ECOG performance and IPI were observed. While acknowledging the heterogeneity of therapies administered, no significant differences in Progression Free Survival (PFS) (median PFS non-GCB = 17 months vs. GCB = 15 months [p=0.36]) and Overall Survival (OS) (median OS non-GCB = 33 months vs. GCB = 27 months [p=0.22]) were identified.Impact of Treatment: 35 adults (age≥18) were treated at our center. The four most common first line therapies administered were R-CHOP (14), R-EPOCH (7), Palliative Care (5) and Rituximab monotherapy (4). Five patients were given 4 other different therapies. In patients given Rituximab monotherapy, two patients presenting with stage I disease responded while two with stage IV disease progressed. When focusing on patients who commenced R-CHOP or R-EPOCH as their initial therapy, no significant differences in age, stage, LDH, extra nodal disease, ECOG performance status, IPI and immunosuppression therapy was identified between the groups. The complete response (CR) rate for R-CHOP was 50% vs. 71% for R-EPOCH. Primary refractory disease was present in 29% of patients receiving R-CHOP vs. 14% with R-EPOCH. Death during first line therapy occurred equally in both groups (14%). All four primary refractory disease patients in the R-CHOP arm died, while the one patient who was primary refractory to R-EPOCH is alive 3 years post autologous stem cell transplant. R-EPOCH demonstrated prolonged PFS (median PFS R-CHOP = 15 months vs. R-EPOCH not reached [p=0.049]) and prolonged OS (Figure 1) [p=0.036].Conclusions: (1) In monomorphic PTLD (DLBCL), the non-GCB subtype predominates and is associated with the use of Tacrolimus. It commonly presents with advanced stage disease and extra nodal involvement however, no difference in PFS and OS was noted when compared to GCB DLBCL PTLD. (2) R-EPOCH demonstrated prolonged PFS and OS when compared to R-CHOP. The survival differences reflect the higher rates of primary refractory disease in the R-CHOP group and the inability to salvage patients once they become relapse/refractory. Given the retrospective nature of our analysis, further studies in a larger cohort of patients are ongoing to validate these results. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Determining Cell-Of-Origin Subtypes In Diffuse Large B-Cell Lymphoma Using Gene Expression Profiling On Formalin-Fixed Paraffin-Embedded Tissue – An L.L.M.P.P. Project

The diffuse large B-cell lymphoma (DLBCL) cell-of-origin (COO) distinction into germinal center B cell (GCB) and activated B cell (ABC) subtypes, as molecularly described by our group, has profound biological, prognostic, and potential therapeutic implications. New therapeutic agents with selective activity in ABC and GCB DLBCL are under development. An accurate diagnostic assay is urgently needed to qualify patients for clinical trials using targeted agents and as a predictive biomarker. Although the subtypes were originally defined using gene expression profiling on snap-frozen tissues (frozen-GEP), it has become common practice to use less precise but relatively inexpensive and broadly applicable immunohistochemical (IHC) methods in formalin-fixed paraffin-embedded tissue (FFPET). We sought to create a robust, highly accurate molecular assay for COO distinction using new GEP techniques applicable to FFPET.Studies were performed on centrally reviewed DLBCL FFPET biopsies using cases that had “gold standard” COO assigned by frozen-GEP using Affymetrix U133 plus 2.0 microarrays. The training cohort consisted of 51 cases comprising 20 GCB, 19 ABC and 12 Unclassifiable (U) cases. An independent validation cohort, consisting of 68 cases (28 GCB, 30 ABC, 10 U) drawn from the validation cohort of Lenz et al (NEJM 2008) had the typical proportions of COO subtypes seen in DLBCL populations. Nucleic acids were extracted from 10um FFPET scrolls. Digital gene expression was performed on 200ng of RNA using NanoString technology (Seattle, WA). All FFPET GEP studies were performed in parallel at two independent sites (BC Cancer Agency, Vancouver, and NCI, Frederick, MD) using different FFPET scrolls to determine inter-site concordance and assess the robustness and portability of the assay. To assign COO by IHC, tissue microarrays were made using 0.6mm duplicate cores from 60/68 validation cohort cases, and stained for CD10, BCL6, MUM1, FOXP1, GCET1 and LMO2. Two hematopathologists independently assessed the proportion of tumor cells stained, with consensus on discordant cases reached with a third hematopathologist. For the validation studies, those producing and analyzing the GEP and IHC data were blinded to the “gold standard” COO.All 119 FFPET biopsies yielded sufficient RNA. A pilot study using the training cohort identified 20 genes (15 genes of interest and 5 house keeping genes) whose expression, measured using NanoString, would allow accurate replication of the COO assignment model of Lenz et al (NEJM 2008). NanoString was then used to quantitate these 20 genes in the training cohort, allowing the COO model to be optimized. Despite the age of the FFPET blocks (6-32 years old), 95% (49/51) of the training samples gave gene expression data of sufficient quality. The model, including coefficients, thresholds and QC parameters was then “locked” and applied to the independent validation cohort. Ninety-nine percent (67/68) of the samples from the validation cohort (5-12 years old) provided gene expression of adequate quality. Three cases did not give interpretable IHC results. When considering the “gold standard” ABC and GCB cases, the COO assignments by the NanoString assay at the NCI site were 93% concordant, with 5% labeled U and 1 ABC misclassified as GCB (see table). This 2% rate of misclassification of ABC and GCB cases compares favorably with the 9%, 6% and 17% rates for the interpretable cases from the Hans, Tally and Choi algorithms, respectively. Furthermore, the 98% concordance of COO assignment (95% if “gold standard” U cases are also included) between the NCI and BC Cancer Agency sites indicates that, in contrast to the IHC algorithms, the assay is reproducible.TableNanoString GEP assay - NCIHans algorithmTally algorithmChoi algorithmGCBUABCGCBNon-GCBGCBABCGCBABCFrozen GEPGCB2800210183192U721552864ABC1325422026620In summary, 119 well-characterized DLBCL cases from the LLMPP, previously subtyped by our published disease-defining algorithm using frozen-GEP, were used to develop a highly accurate and robust NanoString 20 gene assay, applicable to RNA from FFPET that is routinely obtained for diagnosis. This new assay shows excellent performance in archival FFPET, and the rapid turn-around time (<36 hours from FFPET block to result) will allow prospective implementation in future therapeutic trials and, ultimately, clinical practice. Disclosures:No relevant conflicts of interest to declare.

Accurate Classification Of GCB/ABC and MYC/BCL2 Diffuse Large B-Cell Lymphoma With a 14 Genes Expression Signature and a Simple and Robust RT-MLPA Assay

Diffuse large B-cell lymphoma (DLBCL) is the most common non-hodgkin lymphoma. It is subdivided into two molecular subtypes called germinal center B cell-like (GCB) and Activated B cell-like (ABC), deriving from different stages of B-cell differentiation. Recent studies have shown that ABC DLBCLs, which are associated with inferior survival, could benefit from new therapies targeting the NFkB pathway. Unfortunately, gene expression profiling methods to distinguish these lymphomas are not available in routine diagnosis and alternative immunohistochemical algorithms are often considered poorly reproducible. To circumvent these limitations we have developed a simple assay which allows a rapid and efficient classification of these tumours. By comparing gene expression data from the literature and from our own studies, we established a 10 genes expression signature which discriminates ABC from GCB cases (ABC: IRF4, FOXP1, IGHM, TNFRSF13B, CCND2; GCB: LMO2, MYBL1, BCL6, NEK6, TNFRSF9). These genes were incorporated into a Reverse Transcriptase Multiplex Ligation-dependant Probe Amplification assay (RT-MLPA), together with cMYC and BCL2, whose co-expression was recently shown to be prognostic in these diseases, and the CCND1 and MS4A1 (encoding CD20) genes used as controls (Figure 1A-B). We next applied this assay on a training series of 50 cases previously identified as belonging to the ABC or GCB subtypes by gene expression profiling using the Wright's algorithm to construct a Bayesian predictor. Within this first series, this predictor classified 44 cases within the expected subtypes, 5 were considered unclassified and only one ABC case was misclassified. The efficiency of this predictor was next verified on an independent validation series of 50 cases, where 46 cases were classified within the expected subtype, 3 were considered unclassified and again only one ABC case was misclassified. To test the prognostic strength of this assay we next included 85 additional cases, to reach a total of 185 DLBCLs (Figure 1C). 141 of these tumours, all treated between 2001 and 2011 at the centre H. Becquerel, Rouen, France, by a combination of Rituximab and chemotherapy, were retained for this analysis. Within this series, the RT-MLPA predictor identified 49 GCB and 64 ABC DLBCLs, and 28 cases remained unclassified. As expected, ABC cases were significantly associated with a worst event free survival (EFS)(P=0.015) and showed a trend toward a lower overall survival (OS)(P=0.060). Univariate analysis confirmed that the expression of several individual genes within this signature is significantly associated with poor prognosis (LMO2 low: OS P=0.005; BCL6 low: OS P=0.012; IRF4 high: OS P=0.008; TNFRSF13B high: OS P=0.004). We could also verify that the double MYC+/BCL2+ signature is significantly associated with a worst outcome (OS P=0.012) and identified a group of patients with particularly poor prognosis within the ABC subtype (OS P=0.004). Finally, we could demonstrate the robustness of our assay by classifying correctly 12 DLBCLs (6 ABC and 6 GCB) starting from RNA extracted from paraffin embedded (FFPE) biopsies. In conclusion, our RT-MLPA assay is a rapid (it can be achieved in less than one day, and we repeatedly tested up to 40 patients in parallel), cheap (the cost of one test is less than 5 dollars), and efficient gene expression profiling method to discriminate ABC from GCB DLBCLs and to obtain important information on others prognosis markers. It does not require specific equipments (only one conventional PCR module and one capillary genetic analyser), can be achieved from FFPE samples, and could easily be transferred in many routine diagnosis laboratories. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Dual SYK/JAK Inhibition Has a Broader Anti-Tumor Activity In Both ABC and GCB Types Of Diffuse Large B Cell Lymphoma

With increasing understanding of the genetic basis of diffuse large B cell lymphoma (DLBCL), more targeted therapies have been developed. However, most of these therapies have activities only against a subset of DLBCL, activated B-cell like subtype (ABC), in particular, where the BCR pathway is known to be chronically active. Specific single-molecule directed therapies, although effective, often induce resistance over long term treatment. Agents with broader activities are needed for the treatment of this heterogeneous disease. Since both the BCR and JAK/STAT pathways are strongly implicated in the pathogenesis of DLBCL, in the present study, we evaluated the anti-lymphoma activity of PRT062070 (PRT2070), a novel compound that dually targets both JAK and SYK signaling pathways. We analyzed a panel of DLBCL cells lines, of both ABC and germinal center B-cell like (GCB) subtypes, as well as DLBCL patient samples, for cellular and molecular events affected by PRT2070.Immunoblotting analyses showed that ABC and GCB subtype of DLBCL cells exhibit different JAK/STAT and BCR signaling profiles. For instance, AKT was highly expressed in GCB cells, whereas STAT3 was more strongly expressed in ABC cells. In GCB cell lines, PRT2070 blocked G1/S transition and caused cell cycle arrest. In contrast, in ABC cells, the drug induced apoptosis and down-regulated MCL1 protein. Furthermore, array of PRT2070-treated ABC subtype of DLBCL cells revealed that several STAT3 regulated cytokines and chemokines, including IL10, were down-regulated, confirming that PRT2070 affects ABC-DLBCL cells via JAK/STAT pathway. Genetic knockdown of both JAK and SYK reduced the growth and the survival of DLBCL cells more pronouncedly than knockdown of JAK or SYK alone, suggesting the anti-tumor activity of PRT2070 was mediated by dual inhibition of JAK and SYK signaling pathways. Importantly, JAK/STAT and BCR signaling can be blocked by PRT2070 in GCB and non-GCB primary human DLBCL cells, which led to death of these cells. Our work provided mechanistic insights into the actions of JAK/SYK dual inhibitor PRT2070 suggesting that the drug may be a potent treatment of DLBCL with a broader anti-tumor activity in both ABC and GCB subtypes of the lymphoma. Disclosures:Coffey:Portola Pharmaceuticals: Employment. Pandey:Portola Pharmaceuticals: Employment.

SNS01-T Exhibits Significant Anti-Tumoral Activity In Models Of Multiple Myeloma and Non-Hodgkins B Cell Lymphoma and Induces Cell Death In Malignant But Not Normal B Cells

SNS01-T is a novel nanoparticle that is designed to selectively initiate apoptosis in B-cell cancers such as multiple myeloma and non-Hodgkins B-cell lymphomas. SNS01-T comprises a plasmid DNA (pExp5A) encoding a pro-apoptotic form of the eukaryotic translation initiation factor 5A (eIF5A) containing a single-point mutation that prevents hypusination, an eIF5A siRNA that inhibits expression of the pro-survival hypusine-eIF5A protein, and a polymer that serves to assemble the nucleic acids into a nanoparticle. SNS01-T is currently being investigated in a multi-site, open-label Phase1b/2a dose escalation study in subjects with relapsed or refractory multiple myeloma (MM), mantle cell lymphoma (MCL), or diffuse large B cell lymphoma (DLBCL).SNS01-T has demonstrated activity in MM xenograft models as well as in B cell lymphoma models of MCL and DLBCL, when administered twice weekly at doses ≥ 0.18 mg(nucleic acid)/kg. In this study we compared the ability of SNS01-T to transfect, regulate eIF5A expression, and kill MM, DLBCL, and MCL cell lines. Furthermore, the activity of SNS01-T in normal B cells was investigated. A previous study using a KAS-6/1 MM xenograft model demonstrated that the eIF5A siRNA and plasmid pExp5A both have anti-tumoral activity in MM but had a greater impact on tumour growth when combined together as SNS01-T. This finding was confirmed in this study in a second MM model (RPMI 8226) as well as in a DLBCL xenograft model.To determine the efficiency of SNS01-T transfection into malignant or normal B cells, the pExp5A plasmid and eIF5A siRNA were labeled with FITC and DY547, respectively, packaged into nanoparticles using polyethylenimine polymer, and used to transfect cultured cells. FACS analysis was used to determine the percent of the cell population transfected with plasmid, siRNA, or both. RT-qPCR was used to assess biological activity of SNS01-T by quantifying the expression of eIF5AK50R mRNA transgene and endogenous eIF5A mRNA in a variety of B cell lines. The IC50 of SNS01-T in a panel of MM, MCL, and DLBCL cell lines was determined by XTT assay. SCID mice bearing either RPMI 8226 MM tumours or SuDHL6 GCB DLBCL tumours were treated with pExp5A plasmid (formulated with PEI and control siRNA), eIF5A siRNA (formulated with PEI and a control plasmid), or SNS01-T at 0.375 mg/kg twice per week by intravenous injection.SNS01-T was able to transfect MM, MCL, and DLBCL cell lines, although the proportion of cells transfected with both plasmid and siRNA was higher in MM cells. Transfection of SNS01-T resulted in expression of the transgene as well as a statistically significant reduction in expression of eIF5A mRNA compared to untreated controls for all three cell types. In contrast, normal B cells were found to take up fluorescently-labeled SNS01-T with reduced efficiency compared to RPMI 8226 MM cells. Futhermore, SNS01-T was observed to induce cell death in RPMI 8226 MM cells but not in normal B cells.In the RPMI 8226 xenograft model, treatment with either the pExp5A plasmid alone or eIF5A siRNA alone resulted in a 66 % reduction (p < 0.0001) or 44 % reduction (p < 0.05) in tumor volume compared to the control group at day 24 of the study. In contrast, treatment with SNS01-T, which contains both the pExp5A plasmid and the eIF5A siRNA, resulted in an 86 % (p < 0.0001) reduction in tumor volume. A similar result was observed in the SuDHL6 model with a 14 % reduction or 27 % reduction (p < 0.05) in tumor volume compared to the control group at day 20 of the study following treatment with pExp5A plasmid or eIF5A siRNA, respectively. In contrast, treatment with SNS01-T resulted in a 79 % (p < 0.0001) reduction in tumor volume.Collectively, these preclinical studies indicate that SNS01-T therapy has significant potential against MM, MCL, and DLBCL.▪ Disclosures:Taylor:Senesco Technologies: stock options Other. Dondero:Senesco Technologies: Employment. Thompson:Senesco Technologies: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Research Funding.

The Role Of Mir-155 In Controlling Key Growth and Survival Pathways In ABC-Subtype DLBCL

The incidence of diffuse large B-cell lymphoma (DLBCL) is rising, a situation that highlights the need for improved therapy with greater efficacy and fewer side effects. The fundamental clinical problem is that standard frontline combination chemotherapy for DLBCL (R-CHOP) achieves lasting remissions but is not curative. Development of a new, effective, low-toxicity frontline therapy against DLBCL that incorporates targeted agents is much needed. Constitutive activation of the NF-kB, NFAT, and STAT3 (NNS) pathways is a hallmark of DLBCL, particularly the activated B-cell–like (ABC) subtype. Although these pathways have been studied intensively recently, the precise mechanisms controlling these pathways in DLBCL is still uncertain. Elucidating the key mechanism(s) controlling these pathways should improve our understanding of the pathophysiology of DLBCL as well as the therapeutic strategy against this deadly disease. MicroRNAs (miRNA) are short 18-23 nucleotide long noncoding RNAs that posttranscriptionally regulate gene expression by binding to mRNA. Aberrant expression of miRNAs has been implicated in numerous diseases, including cancers. MiRNA-155 (miR- 155) has shown to be overexpressed in aggressive hematologic malignancies, including DLBCL. The oncogenic nature of this miRNA was confirmed in Em-miR-155 transgenic mice, where the mice develop aggressive pre B cell neoplasms within 3-4 weeks. However, the role miR-155 in the pathophysiology of DLBCL is still unclear. We performed gene expression profiling (GEP) in 24 representative DLBCL cell lines (17 GCB- and 7 ABC-subtype). Our data show that miR-155 expression is significantly higher in ABC vs. GCB DLBCL cell lines (p-value<0.0001). We then analyzed the expression levels of miR-155 in primary DLBCL cells using Oncomine (https://www.oncomine.org), a publicly available cDNA cancer microarray database. Analysis of a representative data set (from Lenz G. et al) indicated that miR-155 levels were 3.12-fold higher in primary ABC-DLBCL (167 cases) than in GCB-DLBCL (183 cases). Since ABC-DLBCL subtype has been shown to have active B-cell receptor (BCR) signaling mediating NNS pathways and previous studies have shown that activation of mir-155 in normal and malignant B cells is through BCR stimulation, we examined whether miR-155 is involved in these pathways in DLBCL. Our results indicated that inhibition of miR-155 by transiently transfecting an anti-miR-155 into ABC-DLBCL cell lines harboring high expression of miR-155, suppresses NNS activation, leading to cell growth inhibition as wells as reducing DLBCL clonogenic activity. Next, we examined whether blocking the BCR pathway, using a novel small molecule Btk inhibitor, ibrutinib, can lead to miR-155 inhibition. Ibrutinib at low drug concentrations can effectively inhibit miR-155 expression, leading to NNS inhibition. To test the efficacy and specificity of ibrutinib in DLBCL, we screened ibrutinib in 24 DLBCL cell lines using proliferation assays. Our data showed that ibrutinib preferentially suppress proliferation of most ABC-DLBCL cell lines, whereas most GCB DLBCL cell lines were not highly responsive with drug concentrations less than 10 uM. Our result indicated that miR-155 plays a critical role in the BCR-mediated NNS growth survival pathways in ABC-DLBCL subtype and represents a potential target for therapy in personalized medicine. Disclosures:No relevant conflicts of interest to declare.

Abstract A119: The BET-bromodomain inhibitor OTX015 shows synergism with several anticancer agents in preclinical models of diffuse large B-cell lymphoma (DLBCL).

Abstract Relapsed/refractory DLBCL is still the cause of death in a portion of patients, hence novel active therapies are needed. BET Bromodomain inhibitors have shown promising preclinical activity in hematological and solid tumors and are currently investigated in early clinical trials. We have shown preclinical single-agent activity of the BET Bromodomain OTX015 on mature lymphoid tumors, including DLBCL (AACR 2012; ICML 2013). Here, we report on OTX015 combinations with other anticancer compounds in DLBCL cell lines. Methods: 3 germinal center B cell (GCB) DLBCL and 2 activated B cell (ABC) DLBCL cell lines were exposed to increasing doses of OTX015 alone or in combination with increasing doses of other drugs. The MTT assay was performed after 72 hours of exposure. Synergy was assessed by Chou-Talalay combination index (CI) with the Synergy R package: CI&amp;lt;0.3, strong synergism; 0.3-0.9, synergism; 0.9-1.1, additive effect. Baseline gene expression profiles (GEP) were obtained in 38 lymphoma cell lines, including 22 DLBCL with Illumina HumanHT-12 v4 Expression BeadChip. GEP before and after OTX015 treatment were done in 3 DLBCL cell lines, too. The relationship between GEP and IC50 values was assessed by Pearson correlation. LIMMA was used for differential expression analysis, followed by Benjamini-Hochberg multiple test correction, and GSEA to test for enrichment of functionally-related genes. Results: Strong synergism was observed with OTX015 combined with the mTOR inhibitor everolimus (median CI, 0.11; range 0.1-0.17) and with the BTK-inhibitor ibrutinib in ABC-cells (CI=0.04; 0.02-0.1). A synergistic effect was estimated for OTX015 in combinations with the class I and II HDAC-inhibitor vorinostat (CI=0.45; 0.31-0.56), the anti-CD20 moAb rituximab (CI=0.47; 0.37-0.54), the hypomethylating agent decitabine (CI=0.62; 0.56-0.66), and the immunomodulant lenalidomide (CI=0.66; 0.59-0.72). OTX015 combinations with the class I HDAC inhibitor romidepsin (CI=1.08; 1-1.22) and with the chemotherapy agents bendamustine (CI=0.92; 0.83-1.1) and doxorubicin (CI=0.83; 0.71-0.96) presented a moderate additive effect. A stronger synergism was observed in ABC than in GCB DLBCL cells for ibrutinib (P&amp;lt;0.0001), lenalidomide (P=0.0001), and rituximab (P=0.007). Data mining of GEP obtained at baseline across 38 lymphoma cell lines with known OTX015 IC50s and GEP changes observed after OTX015 exposure indicated the relevance of genes involved in MYD88/JAK/STAT pathway and glucose metabolism as possible explanations of the observed synergism of OTX015 with targeted agents, such as ibrutinib and everolimus. Conclusions: Several targeted agents appeared to synergize with OTX015: additional studies are warranted to clarify the potential clinical role of BET bromodomain inhibitors. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A119. Citation Format: Eugenio Gaudio, Ivo Kwee, Elena Bernasconi, Andrea Rinaldi, Monica Testoni, Michela Boi, Anastasios Stathis, Eugenia Riveiro, Kay Noel, Emanuele Zucca, Francesco Bertoni. The BET-bromodomain inhibitor OTX015 shows synergism with several anticancer agents in preclinical models of diffuse large B-cell lymphoma (DLBCL). [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A119.

IκB-ζ controls the constitutive NF-κB target gene network and survival of ABC DLBCL

AbstractConstitutive activation of the nuclear factor-κ B (NF-κB) pathway is a hallmark of the activated B-cell-like (ABC) subtype of diffuse large B-cell lymphoma (DLBCL). Recurrent mutations of NF-κB regulators that cause constitutive activity of this oncogenic pathway have been identified. However, it remains unclear how specific target genes are regulated. We identified the atypical nuclear IκB protein IκB-ζ to be upregulated in ABC compared with germinal center B-cell–like (GCB) DLBCL primary patient samples. Knockdown of IκB-ζ by RNA interference was toxic to ABC but not to GCB DLBCL cell lines. Gene expression profiling after IκB-ζ knockdown demonstrated a significant downregulation of a large number of known NF-κB target genes, indicating an essential role of IκB-ζ in regulating a specific set of NF-κB target genes. To further investigate how IκB-ζ mediates NF-κB activity, we performed immunoprecipitations and detected a physical interaction of IκB-ζ with both p50 and p52 NF-κB subunits, indicating that IκB-ζ interacts with components of both the canonical and the noncanonical NF-κB pathway in ABC DLBCL. Collectively, our data demonstrate that IκB-ζ is essential for nuclear NF-κB activity in ABC DLBCL, and thus might represent a promising molecular target for future therapies.

MiR-181a Is a Master Regulator of the Nuclear Factor-κB Signaling Pathway in Diffuse Large B Cell Lymphoma

AbstractAbstract 417MicroRNAs (miRNAs) exhibit differential expression in cancer and can be used as prognostic biomarkers. MiR-181a expression is reported to be associated with survival and outcome in acute myeloid and chronic lymphocytic leukemia patients. We demonstrated that miR-181a levels are independently associated with improved survival of diffuse large B cell lymphoma (DLBCL) patients treated with R-CHOP (Rituximab, Cyclophosphamide, Adriamycin, Oncovin, Prednisolone). However, the mechanism underlying this observation and the function of miR-181a in DLBCL pathogenesis are unknown.MiR-181a was expressed at higher levels in centroblasts compared to naïve and memory B cells, and at significantly higher levels in GCB-like compared to ABC-like DLBCL cell lines (p=0.017). These observations suggested that miR-181a may differentially target critical signaling pathways in GCB and ABC DLBCL. NF-kB serves a critical role in ABC DLBCL survival. Utilizing 3 miRNA target prediction algorithms, multiple NF-κB signaling pathway transcripts harbored putative miR-181a binding sites. Consequently, we tested the effect of miR-181a on CARD11, IBKα, p105/p50, and C-Rel expression in DLBCL cell lines (HBL1, VAL). Compared with a scrambled miRNA control, miR-181a expression decreased protein and mRNA levels of these targets. To confirm the effect was direct, we fused the 3′-UTR sequences of CARD11, IBKα, p105 and C-Rel, each containing miR-181a putative binding sites, to a luciferase reporter gene. Co-transfecting miR-181a with the corresponding constructs, we demonstrated that all the constructs had significantly repressed luciferase activity compared with a non-targeting control. The effect was specific, since miR-181a did not affect luciferase activity of CARD11, IBKα, p105 and C-Rel reporter constructs with mutated binding sites. Using an NF-κB luciferase reporter assay, we next demonstrated that compared to a scrambled control, miR-181a significantly decreased NF-κB reporter activity in DLBCL cell lines (VAL, SUDHL6, OCILY7, OCILY19, HBL1, RCK8). MiR-181a also decreased NF-κB reporter activity induced by anti-IgM and TNFα stimulation. Concordantly, anti-miR-181a increased endogenous p105/p50 and C-Rel protein levels. Because ubiquitinated-IKKγ drives NF-κB signaling, we tested the effect of miR-181a in TNFα-stimulated 293T cells on ubiquitinated-IKKγ. MiR-181a decreased levels of ubiquitinated-IKKγ, corroborating the observed inhibitory effects on NF-κB signaling. We reasoned that NF-κB signaling repression should coincide with a decrease in endogenous transcription activity from NF-κB promoters. Indeed, miR-181a decreased mRNA expression levels of NF-κB target genes (BCL2, IRF4, IL-6, IKBa, FN1, PIM1, BLR1, CCL3, CFLAR, FCER2, TP53) as measured by qRT-PCR in miR-181a-transfected HBL1 cells. Because miR-181a directly targets p105/p50 and REL proteins, we postulated that this may be one of the main mechanisms of NF-κB signaling repression. Indeed, an electrophoresis mobility shift assay along with super-shifts analyses showed a decrease in the p105/p50 protein in HeLa nuclear extracts. To examine the biological significance of differential miR-181a expression between GCB- and ABC-like DLBCL and elucidate its potential role in DLBCL pathogenesis, we next assessed cell death (Annexin V, 7AAD) and cell proliferation (BrdU, 7AAD) in GCB (SUDHL4, OCILY7, OCILY19, VAL) and ABC (HBL1, OCILY10, RCK8, U2932) DLBCL cell lines transfected with GFP labeled precursor miR-181a. MiR-181a expression significantly increased cell death and apoptosis of ABC versus GCB DLBCL (p=0.006). This was associated with a more pronounced G1 phase growth arrest in the ABC DLBCL cells.Our studies demonstrate that miR-181a is a master regulator of canonical NF-kB signaling by regulating the expression of multiple components of this pathway, an effect that may underlie the distinct prognosis of DLBCL with different miR-181a expression levels. Furthermore, miR-181a down regulation may contribute to the pathogenesis of ABC DLBCL. Disclosures:No relevant conflicts of interest to declare.

Alterations in Metabolism and Mitochondrial Respiration Are Associated with Response to Targeted Inhibition of WNT Signaling in Lymphomas

Abstract 778 BackgroundWnt signaling is a highly conserved pathway with diverse roles in proliferation, self-renewal and differentiation in multiple tissue types. Aberrant Wnt signaling and deletions/mutations of Wnt regulatory genes have been found in a variety of malignancies including lymphoma. The factors which govern this pathway and the precise role of Wnt signaling in mediating tumor proliferation are unknown. We previously identified recurrent mutations in Wnt pathway genes including APC and WIF1 in diffuse large B cell lymphoma (DLBCL) using exome sequencing and thus sought to investigate if targeting of Wnt with molecular agents would be a viable treatment approach in NHL. MethodsAlamar blue proliferation assays were used to determine IC50 values for 2 small molecule inhibitors of Wnt signaling: ICG001 (a selective inhibitor of TCF/LEF1 mediated transcription) and salinomycin (an ionophore which has been shown to target Wnt) in a panel of 65 cell lines comprising subtypes of lymphoma (including 7 ABC DLBCL, 16 GCB DLBCL, 10 Burkitt lymphoma, 5 mantle cell lymphoma and 5 primary effusion lymphoma cell lines) and leukemia. Both molecules inhibited proliferation of cell lines in the screening panel at physiologically achievable concentrations (IC50 range for salinomycin 0.11–6.23 μM, IC50 range for ICG001 0.96–28.9 μM). IC50s values for salinomycin and ICG001 correlated well (Pearson r=0.56) suggesting a common mechanism of action between the 2 agents. Furthermore, we found lower IC50 values in primary effusion lymphoma (PEL) cell lines (0.35 μM vs. 1.37 μM for other cell line histologies, p=0.01 for salinomycin and 3.25 μM vs. 6.22 μM, p=0.13 for ICG001), which is a lymphoma subtype associated with aberrant Wnt signaling. We did not note significant differences in cytoxicity among other subsets. Wnt activity was confirmed using TOPFLASH luciferase assays and correlated with sensitivity to ICG001 and salinomycin.We then used gene expression profiles derived from Affymetrix Gene 1.0 arrays from each cell line and a Cox proportional hazards model to identify genes associated with sensitivity or resistance to both agents. A gene list consisting of 367 genes associated with either sensitivity or resistance at p<0.05 for both agents was derived from this analysis. Hierarchical clustering was used to define 2 groups of cell lines based upon these genes and analyzed with gene set enrichment (GSEA). GSEA comparisons of sensitive and resistance groups derived from this method revealed that genes associated with mitochondrial pathways were highly associated with response to both ICG001 and salinomycin (FDR=0.036, p=0.03).Specifically, expression of genes involved in glycolysis and mitochondrial respiration including FBG1, CS, UBQRB5, OXA1L, LDHA and ATP5B was higher in ICG001 and salinomycin-sensitive cell lines. Conversely, we noted higher IC50s in cell lines with higher expression of mitochondrial genes having opposing functions, including PC and CPT1A. This overall suggests that tumor cells utilizing glycolytic and oxidative pathways are sensitive to Wnt inhibition. Remarkably, the GCB DLBCL cell line Pfeiffer also was noted to contain an inactivating mutation in APC and had the lowest noted IC50 among DLBCL cell lines. ConclusionsThe Wnt pathway appears an important oncologic driver in at least a subset of NHLs and we found ICG001 and salinomycin have significant in vitro activity in NHLs. These agents may be most useful in subsets of tumors with high mitochondrial mass and/or oxidative phosphorylation. This work serves as a starting point to characterize the importance of the Wnt pathway in NHL and identifies inhibition of this pathway as a therapeutic opportunity in lymphomas. Disclosures:No relevant conflicts of interest to declare.

Enhancer Profiling Reveals SOX9 As a Novel Transcription Regulator of B Cell Activation and DLBCL Transformation

Abstract 527Antigen stimulation of naïve B cells (NBC) induces differentiation with a phenotype characterized by robust proliferation and genomic instability tolerance to enable activated germinal center B cells (GCB) to undergo immunoglobulin affinity maturation. Aberrant genetic events resulting from this process lead to malignant transformation and diffuse large B cell lymphoma (DLBCL). Phenotypic progression from quiescent NBC to activated GCB and malignant DLBCL involves major shifts in gene expression. Recent studies suggest that enhancers play a key role in mediating cell type-specific gene regulation. We therefore postulated that enhancers are involved in dictating the gene expression programs that govern normal and malignant B cell phenotypes; and systematic discovery of enhancers coupled with bioinformatic analysis would uncover key enhancer-binding transcription factors (TFs) that regulate these cell states.To test this hypothesis, we performed ChIP-seq on enhancer histone marks, i.e. H3K4me2, H3K27Ac, and H3K4me3, in primary NBC and GCB, and in DLBCL cell lines in biological replicates. We defined enhancers by the criterion of H3K4me2hiH3K4me3low. We observed a striking pattern of enhancer re-organization between cell types. First, we found a larger number of enhancers in primary B-cells (∼20,000) than in DLBCL (∼12,000). Second, we confirmed that enhancers are cell type-specific. For example, 11,492 out of 20,173 NBC enhancers were lost during transition to GCB (loss of H3K4me2 enrichment), while 13,088 new enhancers were gained in GCB. A similar phenomenon was also observed in DLBCL when compared to either NBC or GCB. This re-organization of enhancers suggests that cells may have dynamic gene regulatory programs during differentiation or malignant transformation. To discover TFs that act through enhancers, we used bioinformatic analyses, including FIRE and MEME, to search for TF consensus binding sequences within enhancers. Over-represented DNA motifs included motifs of SPI1, RUNX1, STAT3, RELA and SOX9, etc. SOX9 motif was significantly enriched in GCB specific enhancers (p=3.07e-15). SOX9 belongs to the SOX family TFs and plays an important role in cartilage development, sex determination, and intestinal differentiation but has not been implicated in B cell development.To investigate the role of SOX9 in B cell activation and malignant transformation, we first examined the expression of SOX9 in these cells. RNA-seq performed on human tonsilar NBC and GCB showed more than 20-fold increase of SOX9 mRNA in GCB as compared to NBC (6.75±0.80 vs 0.29±0.14, RPKM, p=0.0002). In addition, SOX9 expression was maintained in plasma B cells (2.88±0.49, RPKM). To understand how SOX9 regulates transcriptional programming in GCB, we performed SOX9 ChIP-seq in GCB to look for its targets. We found that SOX9 binds to 1,668 upstream distal enhancer regions (-5 to -100 kb of TSS) associated with 963 genes. These target genes were significantly enriched in many important pathways including cell cycle regulation (CCND2, CDC25B, CDK1), transcription regulation (BCOR, NCOR2), epigenetic regulation (BMI1, DNMT3A, MLL2, SUZ12, TET3), and MAPK signaling (MAP2K3, MAP3K7) (p<0.001). One of the SOX9 targets is PRMD1, a TF that controls the transition from GCB to plasma cells, suggesting that SOX9 may be involved in B cell terminal differentiation. To our surprise, we did not detect SOX9 mRNA in 10 out of 12 DLBCL cell lines by RNA-seq. Moreover, SOX9 was not expressed in the majority of primary malignant non-Hodgkin’s lymphoma cases studied by IHC in the Human Protein Atlas project. To examine whether reduced SOX9 expression could induce malignant transformation, we used shRNA to knockdown Sox9 in mouse BCL1 lymphoma cells and subjected them to colony forming assay in semi-solid methylcellulose. Knockdown of Sox9 increased BCL1 colony forming ability by 50% as compared to scramble, suggesting that loss of SOX9 expression maybe important for lymphomagenesis.In summary, we identified a novel germinal center TF, SOX9, by examining enrichment of TF motifs within enhancer regions uncovered by ChIP-seq. Our current data suggest that SOX9 may play an important role in germinal center reaction and subsequent terminal differentiation by regulating key factors, such as PRDM1, and that loss of SOX9 may contribute to DLBCL malignant transformation by potentially blocking the terminal differentiation of mature GCB. Disclosures:No relevant conflicts of interest to declare.