Diffuse Large B-cell Lymphoma Tumors Research Articles

SHP1 Loss Augments DLBCL Cellular Response to Ibrutinib: A Candidate Predictive Biomarker

BackgroundB-cell receptor (BCR) signaling plays a key role in B-cell non-Hodgkin lymphoma (NHL). Protein tyrosine kinases such as LYN, SYK and BTK are essential in the activation of BCR leading to B-cell survival, proliferation and differentiation. SHP1 is a cytosolic protein tyrosine phosphatase functioning as a negative regulator of the BCR signaling cascade. It dephosphorylates and counteracts the activities of the tyrosine kinases, including CD79A/CD79B, SYK and BLNK (Curr. Opin. Immunol.12, 307).Diffuse large B-cell lymphoma (DLBCL) is the most common type of aggressive NHL. DLBCL demonstrates heterogeneity in clinical presentation, genomic alterations and cell signaling. BCR signature has been identified in a subset of DLBCL (Monti Blood 105, 1851). Chronic active BCR plays a particular role in the subtype of activated B-cell like (ABC) DLBCL by cell-of-origin subclassification. Based on the success of ibrutinib in other B-cell NHL, clinical trials of ibrutinib in DLBCL was conducted and the initial results showed a 37% response rate in ABC-lymphomas vs 5% in GCB-DLBCL (Wilson Nat Med 21, 922). However, a large phase 3 clinical trial of ibrutinib-containing regimen in >800 non-GCB patients did not find any substantial difference in response between those received ibrutinib vs placebo. Thus, biomarker based on cell-of-origin did not seem to help stratify patients as expected (Younes JCO 27, 1285). New predictive biomarkers are needed to identify patients who likely benefit from ibrutinib or other BCR-directed therapeutic agents.We hypothesized that SHP1 may play a role in defining the level of the activity of the BCR pathway. Suppressed SHP1 expression in various types of NHL has been reported. We hypothesized that lack of SHP1 may contribute to the hyperactivity of BCR in DLBCL; and tumors with low SHP1 may respond better to ibrutinib. In this work, we set out to explore the relationship between SHP1 & ibrutinib sensitivity in DLBCL.Methods and ResultsWe first reviewed the published literature on SHP1 in NHL using a meta-analysis to provide a quantitative and thus unbiased analysis. Seventeen articles were selected based on criteria including inclusion of primary tumors with normal benign controls. The analysis of the SHP1 protein expression by immunohistochemistry (IHC) revealed that lack of SHP1 expression is found in 178 of 242 (73.6%) NHL tissues vs. 32 of 264 (12%) normal/benign controls. The odds ratio derived by the meta-analysis was 119.58 with a 95% confidence interval of 37.60-380.15, suggesting loss of SHP1 is strongly associated with NHL including DLBCL. Likewise, the analysis of the methylation studies revealed a strong association between SHP1 promoter hypermethylation and NHL (OR 69.46, 95% CI 12.32-391.74).To further understand the range of variability of SHP1 expression in subtypes of DLBCL tumors, we constructed a tissue microarray with 48 DLBCL tumors and 12 cell lines. By immunohistochemistry, we found a wide range of variability with regards to the expression levels and no substantial difference between the GCB and non-GCB subtypes.Using 12 cell lines together with resting B-cells from normal donors, we identified a strong reverse linear correlation between SHP1 protein expression and promoter methylation (r = 0.85, P=0.0002) suggesting that promoter hypermethylation may be responsible for reduced SHP1 expression in DLBCL cells.Using CRISPR, we knocked out SHP1 in both GCB and ABC cell lines. The phosphorylation of early BCR components, BTK, SYK and LYN, were increased in the SHP1 knockout clones compared to the wild type ones. The results demonstrate that SHP1 depletion results in increased proximal BCR signaling activity. Moreover, the knock out clones demonstrated increased sensitivity of ibrutinib. We further showed that re-introduction of SHP1 vector to the knockout clones via stable lentiviral transduction restored normal sensitivity of cells to ibrutinib.ConclusionsTogether, our results show that SHP1 expression is suppressed in a substantial fraction of DLBCL tumors regardless of cell-of-origin subclassifications. Using genetic approaches, we demonstrated that loss of SHP1 increases BCR signaling activity and sensitizes tumor cells to the inhibition by ibrutinib. Our results suggest that loss of SHP1 may be used as an alternative biomarker to cell-of-origin to identify patients who potentially benefit from ibrutinib treatment. DisclosuresWang: Incyte Research Institute: Ended employment in the past 24 months.

Novel Irreversible Menin Inhibitor, BMF-219, Shows Potent Single Agent Activity in Clinically Relevant DLBCL Cells

Novel Irreversible Menin Inhibitor, BMF-219, Shows Potent Single Agent Activity in Clinically Relevant DLBCL Cells

Immune Profiling of Diagnostic DLBCL Biopsies Dramatically Improves upon Cell-of-Origin Risk Stratification

Background:Diffuse large B cell lymphoma (DLBCL) is an aggressive malignancy of mature B cells. The disease has traditionally been subdivided into cell-of-origin (COO) subtypes - germinal centre B cell-like (GCB) or activated B cell-like (ABC) - as determined by expression profiling or immunohistochemistry of the tumor cells. However the role of the immune microenvironment, and how the tumor and immune system interact to influence patient outcomes, remains to be fully investigated.Methods:In this project, we used mass cytometry (CyTOF) to deeply profile both tumor cell phenotypes and the immune microenvironments, alongside ABC/GCB classification and mutation profiling, in a discovery cohort of 54 DLBCL cases. As well, a validation cohort of 129 DLBCL patients were immunologically profiled by high-dimensional conventional flow cytometry, and their immune profiles alongside ABC/GCB classification, mutation profiling, and RNAseq data, were correlated with patient outcomes as measured by progression-free survival (PFS).Results:Analysis of the CyTOF/discovery cohort demonstrated that DLBCL tumor cells are phenotypically unique to each patient, with a small number of samples displaying distinct sub-clonal structure, often distinguished by differential expression of immune-related proteins like MHC-II. ABC/GCB classifications could be recapitulated based on tumor cell phenotypes, demonstrating that while COO was a robust feature, a great deal of heterogeneity exists within these established subtypes.Immunological profiling of the CyTOF/discovery cohort revealed that DLBCL samples could be divided into three distinct groups which roughly correlated with abundances of naïve, activated, or terminally differentiated T cells, respectively. This profiling schema was extended to the validation cohort of 129 patients which in turn led to identification of a subset of patients with a very high risk of disease progression (5-year PFS; 30% high risk vs. 80% low risk, p<0.0001).This final classifier was based on a combination of ABC-DLBCL designation, combined with the presence of an immune microenvironment dominated by terminally differentiated (CD57+) T cells. We performed a limited series of functional studies using primary DLBCL biopsy samples to characterize further these CD57+ T cells as clonally restricted and incapable of responding to antigenic challenge. Interestingly, traditional immune markers of T cell exhaustion such as PD-1, TIM3, LAG3 and TIGIT were not correlated with patient outcomes.Conclusions:Overall, this study demonstrates the utility of immune profiling in risk stratification based on initial diagnostic biopsy material and highlights a subset of DLBCL patients who may benefit from immune-based therapies to rejuvenate the anti-tumor T cell response. We conclude that T cell senescence, rather than exhaustion, is the more relevant feature in DLBCL disease biology and highlights an alternate target for immunomodulatory therapy. [Display omitted] DisclosuresCraig: Bayer: Consultancy. Slack: Seagen: Consultancy, Honoraria. Scott: Abbvie: Consultancy; AstraZeneca: Consultancy; Celgene: Consultancy; NanoString Technologies: Patents & Royalties: Patent describing measuring the proliferation signature in MCL using gene expression profiling.; BC Cancer: Patents & Royalties: Patent describing assigning DLBCL COO by gene expression profiling–licensed to NanoString Technologies. Patent describing measuring the proliferation signature in MCL using gene expression profiling. ; Rich/Genentech: Research Funding; Janssen: Consultancy, Research Funding; Incyte: Consultancy. Steidl: Epizyme: Research Funding; Bayer: Consultancy; Curis Inc.: Consultancy; Seattle Genetics: Consultancy; AbbVie: Consultancy; Trillium Therapeutics: Research Funding; Bristol-Myers Squibb: Research Funding.

Suppression of Extracellular Vesicle CD47 Induces Systemic Anti-DLBCL Immunity

Purpose:Tumor cells evade the immune surveillance by up-regulating surface expression of CD47, which interacts with SIRPa on macrophages to elicit the immune checkpoint response. Anti-CD47 antibody (IBI188) has shown promise in treating tumors, including Diffuse large B cell lymphoma (DLBCL). The objective response rate among DLBCL was 73% (ClinicalTrials.gov number, NCT02953509.). The basis of differential therapeutic success between patients remains unknown. Extracellular vesicles (EVs) carry bioactive molecules that influence the immune system. CD47 has be found on surface of EVs. The purpose of this study is to explore whether EV CD47 contributes to immunosuppression and is associated with anti-CD47 response in DLBCL.Methods: 1.Kaplan-Meier curves and Cox regression models were used to analyze PFS and OS. The area under the ROC curve (AUC) was used to assess the predicted validity of the National Comprehensive Cancer Network-International Prognostic Index (NCCN-IPI) model and the NCCN-IPI+ CD47 model.2.Immunofluorescence experiment was used to detect the infiltration of M1 and M2 subtype macrophages in DLBCL tumor tissue. M1 and M2 macrophages were generated from peripheral blood mononuclear cells obtained from healthy donors. Immunoelectron microscopy, flow cytometry and western blot were performed to detect CD47 on the surface of the EVs derived from DLBCL cells. The activity of macrophage phagocytosis of DLBCL cells was detected by confocal-based phagocytosis assay and flow cytometry-based phagocytosis assay.3.In DLBCL xenografts, the IBI188 antibody was a macrophage immune checkpoint inhibitor blocking CD47 that induces tumor-cell phagocytosis.4.Peripheral blood mononuclear cells from DLBCL patients and healthy donors was collected for EVs purification and subsequent detection of human CD47 proteins by ELISA.Results 1.The elevated expression of CD47 in DLBCL is significantly correlated with poor PFS and OS in a univariate analysis, and is statistically significant after adjusting for the NCCN-IPI in the univariate and multivariate analysis (Figure 1A, B). AUC analysis with cross-validation showed that the combination of the CD47 signature and NCCN-IPI had a better prediction of PFS and OS than without the CD47 signature (Figure 1C). Thus, CD47 in DLBCL can be used as a biomarker of prognosis and developed the prognostic stratification of DLBCL patients.2.EV CD47 derived from DLBCL cells has the same membrane topology as the cell surface CD47, with its extracellular domain exposed on the surface of the EVs (Figure 1D-G). Moreover, EV CD47 could function similarly to cell-surface CD47 in the suppression of macrophage phagocytosis of DLBCL cells (Figure 1H, I). DLBCL cells that had higher levels of cellular CD47 protein packaged greater amounts of CD47 into EVs, and these EVs displayed an increased binding to SIRPa of macrophages, thus inhibiting macrophage-mediated phagocytosis of DLBCL cells (Figure 1J-O).3.Antibodies against human CD47 specifically identified human CD47 on the circulating exosomes from mice bearing DLBCL xenografts but not the control mice. The level of circulating EV CD47 positively correlated with tumor size. Injection of EVs derived from DLBCL cells promoted the growth of tumors, whereas pre-treatment of the EVs with anti-CD47 antibodies (IBI188), but not IgG isotype or CD63-blocking antibodies, inhibited the effect (Figure 1P, Q).4.The presence of CD47 in the EVs isolated from the plasma of DLBCL patients, and the level of CD47 on the circulating EVs was significantly higher in DLBCL patients than that in healthy donors (Figure 1R, S). The receiver ROC curve shows that the level of circulating EV CD47 could distinguish DLBCL patients from healthy donors.ConclusionOur studies suggest that the EV CD47-SIRPa interaction may represent a critical mechanism by which DLBCL cells escape immune-mediated clearance. Our study also raises the possibility that disrupting the interaction between the EV CD47 and macrophage SIRPa is a mechanism in the CD47-SIRPa blockade-based therapies. Moreover, high levels of circulating EV CD47 would follow and correlate positively with the phagocytic activity of macrophages, and reflect the presence of a successful anti-tumor immunity elicited by the anti-CD47 therapy. Together, these findings show that EV CD47 represents an unexplored therapeutic target, which could overcome resistance to current CD47 antibody approaches. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Using Machine Learning Methods to Explore the Role of Non-Coding RNAs in DLBCL

Diffuse large B-cell lymphoma (DLBCL) is a complex and aggressive malignancy. The standard-of-care chemo-immunotherapeutic regimen, which consists of R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone), leads to a complete response in most patients. Unfortunately, 30-40% of patients are either refractory to the current treatment regimen or experience disease relapse after complete response, and thus, these patients exhibit a dismal prognosis. Gene expression profiling has delineated two distinct molecular subtypes of DLBCL, the germinal center B-cell-like (GCB) subtype and the activated B-cell-like (ABC) subtype; 10 to 15% of cases are unclassifiable, these groups differ in survival, and could potentially direct therapy (Alizadeh, Nature 2000). A study by Schmitz et al have used whole exome and transcriptome of 574 DLBCL tumors to refine the abovementioned genetic subtypes, in attempt to improve prognostic capacity, focusing on protein coding genes (Schmitz, N Engl J Med. 2018) . Non-coding RNAs (ncRNAs) has been shown to be differentially expressed and clustered across different groups of DLBCL samples, suggesting that these ncRNAs may also participate in DLBCL pathogenesis (Shi, OncoTargets and therapy 2020) .In this study, we applied machine learning methods for classification of DLBCL genetic subgroups based on ncRNA expression, and proposing a clinical-genetic survival predictive model.Out of 1866 ncRNAs from the study by Schmitz et al, 377 were selected using information gain algorithm, and were used to classify 234 DLBCL tumors to the different genetic subgroups (ABC, GCB, Unclassified). Classification models were trained using K Nearest Neighbor (KNN) (K=5), decision tree, random forest and multilayer perceptron algorithms leading to a weighted area under the ROC curve of 0.895, 0.749, 0.924 and 0.965 respectively.Using the information gain algorithm, we identified 28 ncRNAs which have an information gain score of >0 in classifying patients to either having achieved survival of three years or not. Of these, seven ncRNAs were found to have a significant correlation to overall survival (OS) (p<0.05 for each) using cox regression survival analysis. In multivariate analysis, including age, gender, ECOG, IPI, genetic subgroups and these seven ncRNAs, we found only age and three ncRNAs (NR_026893, NR_002939, NR_002186) to be significantly associated with OS, figure 1A. We performed Kaplan Mayer analysis using these three genes as binary variables (medians were used for cutoff), dividing the cohort into three groups (all three ncRNA up regulated, one/two down regulated and all three ncRNAs down regulated) with robust difference in overall survival (median OS was not reached, 5.5 CI 95% (1.1-9.9) years 1.5 CI 95% (0.4-2.7) years, respectively), as presented in figure 1B.In conclusion, we detected novel diagnostic and prognostic ncRNAs biomarkers which potentially be able to inform clinical management for patients with DLBCL. Further studying of ncRNAs expression profile and cellular mechanisms could help improve our understanding of the disease and potentially identify new therapeutic targets and support the development of new therapies. [Display omitted] DisclosuresAvivi: Kite, a Gilead Company: Speakers Bureau; Novartis: Speakers Bureau. Cohen: Karophram: Membership on an entity's Board of Directors or advisory committees, Research Funding; GSK: Consultancy, Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees, Research Funding; Neopharm / promedico: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Functional Evaluation and Mechanism Study of Sting Inhibitor H-151 in Diffuse Large B-Cell Lymphoma

Objective:Diffuse large B-cell lymphoma (DLBCL) is a subtype of non-Hodgkin's lymphoma with a higher incidence rate and is highly heterogeneous.The current first-line treatment effect is limited. After entering the relapse/refractory treatment, the second-line treatment lacks an efficient solution. Explore DLBCL Potential therapeutic targets are imperative. The cGAS/STING pathway can promote or inhibit tumorigenesis. The specific tumor type,genetic background,pathway activation level and microenvironment may determine the tumor's response to STING agonists or inhibitors. The disorder of cGAS/STING pathway function can lead to many diseases such as tumors and autoimmune diseases, but it has not been reported in DLBCL. This study found for the first time that STING is heterogeneously expressed in DLBCL and is related to tumor cell proliferation. The STING inhibitor H-151 can kill DLBCL tumor cells. At the same time, multi-omics methods are used to further screen the specific DLBCL population sensitive to H-151, provide a theoretical basis for the improvement of the prognostic model of DLBCL and the choice of targeted therapy drugs.MethodsImmunofluorescence staining to detect the expression of cGAS/STING on tissue microarrays in patients with DLBCL; Confocal microscopy imaging technology for localization and quantitative analysis of intracellular cGAS/STING expression; Whole-exome sequencing analysis of mutant genes related to drug sensitivity and pathway;The combination of quantitative proteomics and transcriptome sequencing to compare the biological characteristics of cell lines in different response groups, analyze gene expression differences and related activation pathways;Western Blotting to detect the expression of related proteins;CCK8 and flow cytometry were used to detect cell proliferation and apoptosis;NOD-SCID mice and Hu-PBMC mice were used to construct DLBCL tumor-bearing mouse models to explore the effect of STING inhibitors in vivo.ResultsThe cGAS/STING pathway-related proteins are expressed heterogeneously in real-world clinical DLBCL samples and DLBCL cell lines, and the expression of cGAS/STING pathway-related proteins is positively correlated with the proliferation level of DLBCL cell lines(Figure A). Based on this, we speculate the increased expression of cGAS/STING pathway-related proteins may potentially promote the proliferation of tumor cells. The STING inhibitor H-151 can inhibit the proliferation and induced apoptosis of some DLBCL tumor cells in a concentration-dependent manner.The IC50 of H-151 has a certain positive correlation with the expression of cGAS/STING pathway-related proteins, which suggests that the up-regulation of cGAS/STING pathway-related proteins may be potentially related to the resistance of DLBCL to H-151(Figure B).Whole transcriptome sequencing combined with single-cell transcriptome sequencing (scRNA-seq) analyzes potential subgroups related to H-151 sensitivity and related activation pathways(Figure C-E).. We also use whole exome sequencing to screen potential targets for assessing H-151 sensitivity We found that celllines with ITIH6, NPIPA5, NPY2R and TUBGCP3 mutations are more sensitive to the treatment of H-151, while cell lines with SH2B2 and LILRB5 mutations were less responsive to H-151,while the related gene mutations in the C-MYC pathway may be potentially related to the resistance of DLBCL cells to H-151, so as to screen potentially sensitive target populations for the effective use of H-151(Figure F).ConclusionThis study found that the cGAS/STING pathway is heterogeneously expressed in DLBCL and is related to tumor cell proliferation, and the STING inhibitor H-151 has anti-DLBCL effects. The anti-tumor mechanism of STING inhibitor H-151 was further explored by using multi-omics methods, and the sensitivity evaluation system of H-151 was established.Thus providing new ideas for precise and efficient treatment of DLBCL. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

RAS Mediates BET Inhibitor-Endued Repression of Lymphoma Migration and Prognosticates a Novel Proteomics-Based Subgroup of DLBCL through Its Negative Regulator IQGAP3.

Simple SummaryThe inhibitors of BET proteins represent a promising class of therapeutic agents that target the oncogenic activity of MYC and repress DLBCL cell migration, but the mechanism of such repression remains elusive. Herein, we found that BET inhibitor JQ1 abrogated the amoeboid movement of DLBCL cells through a small GTPase-driven mechanism, including both restrained RAS signaling and MYC-mediated suppression of GTP-RhoA activity. BET inhibition drastically increased the expression of a GTPase regulatory protein, the IQ motif containing GTPase activating protein 3 (IQGAP3), in DLBCL. Proteomics-based re-stratification identified a specific subgroup of DLBCL patients whose tumors harbored an enhanced PI3K activity and had an inferior survival, whereas a lower IQGAP3 expression level further portended a very dismal outcome for those patients. The inhibitors of both BET and RAS (through attenuated PI3K signaling) activities effectively ameliorated the outspread of in vivo DLBCL tumors, indicating the potential of their synergism in the treatment of specific DLBCL subtypes.Phenotypic heterogeneity and molecular diversity make diffuse large B-cell lymphoma (DLBCL) a challenging disease. We recently illustrated that amoeboid movement plays an indispensable role in DLBCL dissemination and inadvertently identified that the inhibitor of bromodomain and extra-terminal (BET) proteins JQ1 could repress DLBCL migration. To explore further, we dissected the impacts of BET inhibition in DLBCL. We found that JQ1 abrogated amoeboid movement of DLBCL cells through both restraining RAS signaling and suppressing MYC-mediated RhoA activity. We also demonstrated that BET inhibition resulted in the upregulation of a GTPase regulatory protein, the IQ motif containing GTPase activating protein 3 (IQGAP3). IQGAP3 similarly exhibited an inhibitory effect on RAS activity in DLBCL cells. Through barcoded mRNA/protein profiling in clinical samples, we identified a specific subgroup of DLBCL tumors with enhanced phosphatidylinositol-3-kinase (PI3K) activity, which led to an inferior survival in these patients. Strikingly, a lower IQGAP3 expression level further portended those with PI3K-activated DLBCL a very dismal outcome. The inhibition of BET and PI3K signaling activity led to effective suppression of DLBCL dissemination in vivo. Our study provides an important insight into the ongoing efforts of targeting BET proteins as a therapeutic approach for DLBCL.

The microenvironment of DLBCL is characterized by noncanonical macrophages recruited by tumor-derived CCL5

AbstractTissue invasion by tumor cells induces a host inflammatory response that variably impacts tumorigenesis. This has been well documented for tumor-associated macrophages (TAMs) that could play a pro/M2- or an anti/M1-tumoral function. TAMs frequently infiltrate diffuse large B-cell lymphoma (DLBCL), an aggressive neoplasm arising from germinal center–experienced B cells. However, the pathway leading to the presence of TAMs in DLBCL remains unknown, and their impact is unclear. Here, we show that some DLBCL tumor cells expressed the chemokine CCL5, enabling the differential recruitment of blood monocytes through their expression of CCR1 and CCR5. CCL5 expression by DLBCL was not related to molecular subtypes, and healthy tonsillar B cells did not produce this chemokine, implying a posttransformation event. A single-cell analysis revealed that most DLBCL TAMs had a noncanonical gene signature with the concomitant expression of M1 and M2 genes. The presence of noncanonical TAMs may explain the lack of impact of macrophages on DLBCL development reported in some survival studies.

Relationship between Plasma Cell Density and Clinico-histopathological Characteristics of DLBCL

Diffuse Large B Cell Lymphoma (DLBCL) is a malignancy derived from the malignant transformation of B cells from the germinal center. This type of non-Hodgkin's lymphoma is the most common and represents about 30-40% of all cases. The tumor microenvironment of B-cell lymphoma plays an important role in the regulation of tumor cell survival and proliferation. Plasma cells in the tumor microenvironment have an important role in the formation of the anti-tumor immune response, even in low numbers, these cells can produce large amounts of cytokines and antibodies. The purpose of this study was to determine the relationship between plasma cell density and clinico-histopathological characteristics of DLBCL patients. A cross-sectional study was conducted. The samples of this study were paraffin blocks of DLBCL patients referred to Anatomic Pathology Departement Faculty of Medicine Universitas Sriwijaya/RSUP Dr. Mohammad Hoesin Palembang. From 50 cases during the 2018-2020 period, 32 samples were selected. Plasma cells were morphologically identified on HE slides. Statistical analysis was performed using the chi-square test. The distribution of DLBCL cases found more in the age group &lt;60 years (56,3%), male gender (68,8%), extranodal tumor location (56,3%). Centroblastic variants were higher (78,1%) than others, and the non-GCB subtype (78,1%) was higher than the GCB. There is no significant relationship between plasma cell density and the clinico-histopathological characteristics of DLBCL age, sex, tumor location, variant, and subtype.

Characteristics of PD-L1 expression in tumor cells and tumor microenvironment of DLBCL with MYD88 L265P mutation

Objective: To study the effect of MYD88 L265P mutation on the expression of PD-L1 in tumor cells and tumor microenvironment in diffuse large B-cell lymphoma (DLBCL), and to provide theoretical basis for immunotherapy for patients. Methods: Multiplex ligation-dependent probe amplification (MLPA) was used to detect the frequency of MYD88 L265P mutation in 72 cases of DLBCL diagnosed by pathologists in Cancer Hospital of Chinese Academy of Medical Sciences from August 2008 to May 2010. Expression of PD-L1 in tumor cells and tumor microenvironment in all samples was evaluated using PD-L1 (22C3) and PD-L1 (SP142) with Ventana automatic immunohistochemical (IHC) platform. The relationship between MYD88 L265P mutation and the expression of PD-L1 in DLBCL tumor cells and tumor microenvironment was assessed. Results: Of the 72 cases of DLBCL, MYD88 L265P mutation was detected in 15 (20.8%) cases. Nine cases with JAK2 amplification were excluded, and the remaining 63 cases of DLBCL were divided into MYD88 L265P mutant group (n=14) and MYD88 L265P wild-type group (n=49). IHC results showed that among the 14 cases of MYD88 L265P mutant groups, PD-L1 (22C3) was positive in 7 cases (7/14) of tumor cells and PD-L1 (SP142) was positive in 4 cases (4/14) of tumor microenvironment. Among the 49 cases of MYD88 L265P wild-type group, 9 cases (18.4%) were positive for PD-L1 (22C3) in tumor cells, and 38 cases (77.6%) were positive for PD-L1(SP142) in tumor microenvironment. In addition, among the 16 cases with PD-L1(22C3) expression in tumor cells, only 2 of the 7 cases with MYD88 L265P mutation were positive for PD-L1 (SP142) in tumor microenvironment. All 9 cases with wild-type MYD88 L265P were positive for PD-L1 (SP142) in tumor microenvironment. Statistical analysis showed that the expression level of PD-L1 (22C3) in tumor cells in the MYD88 L265P mutant group was significantly higher than that in the MYD88 L265P wild-type group (P=0.017). The expression level of PD-L1 (SP142) in tumor microenvironment in the MYD88 L265P mutant group was significantly lower than that in the MYD88 L265P wild-type group (P=0.001). Conclusions: MYD88 L265P mutation may play an important role in the regulation of PD-L1 expression in DLBCL tumor cells and tumor microenvironment. Further studies will provide a theoretical basis for immunotherapy of DLBCL patients with MYD88 L265P mutation.

Selective Inhibition of HDAC1 and HDAC3 Inhibits Survival of Diffuse Large Cell Lymphoma Via Epigenetic Modulation of Death Associated Protein (DAPK)

Histone deacetylases (HDACs) are key players of epigenetic regulation of gene expression and are therefore attractive targets for cancer therapy. Many epigenetic drugs including HDAC inhibitors (HDACi) have shown anti-tumor activity in several types of cancers. Recently SAHA and belinostat Pan-HDACi have been FDA-approved for the treatment of T cell lymphoma. However, the capacity of Pan-HDACi for distinguishing different classes and isoforms of HDACs is limited, and may produce profound side effects. Therefore, it is important to identify cancer-relevant HDACs to promote selective HDAC inhibition for therapy in other malignancies such as diffuse large B cell lymphoma (DLBCL), the most aggressive non-Hodgkin lymphoma. Recent studies have shown that HDAC1 (HDACs class I) are commonly overexpressed in multiple cancer types and contribute to poor overall survival. However, the role of HDAC3 is not well known. The goal of this study was to determine the mechanistic role of HDAC3 and HDAC1 in DLBCL.Immunohistochemistry performed on DLBCL tumors (n=29) demonstrate 42% positivity for HDAC3 and 100% positivity for HDAC1. Ectopically expressed HDAC3 led to increased thymidine uptake, whereas HDAC3 knockdown using siRNA led to a significant decrease in thymidine uptake (p<0.001) suggesting that HDAC3 could be necessary for DLBCL cell growth. Moreover, inhibition of HDAC3 using a selective pharmacological inhibitor RGFP966 suppressed cell proliferation in a dose dependent manner in several DLBCL cell lines (SUDHL6, OCILY1, OCILy10 and OCILy3). However, the effect of RGFP966 on cell survival as determined by annexin V/ propidium iodide was underwhelming. We sought to target both HDAC1 and HDAC3 using the pharmacological inhibitor RG283 and assessed biological effect of dual inhibition. Interestingly, dual inhibition of HDAC1 and HDAC3 with RG2833 significantly suppressed both cell proliferation (<0.0001) and survival (p<0.001) in most of the DLBCL cell lines tested. As expected, treatment with RG2833 increased acetylation of H3K9, H3K14, H3K27 and H4K5 histone marks and led to P21 upregulation in DLBCL cells. Interestingly, exogenously overexpressed HDAC3 but not HDAC1 increased sensitivity of DLBCL cells to Pan-HDAC inhibitors vorinostat and belinostat, which suggest that endogenous HDAC3 in DLBCL cells may exist in a protein complex that may evade targeting by Pan-HDACi. In order to identify a potential mechanism of action for dual HDAC3 and HDAC1 inhibition, we have focused on tumor suppressor gene DAPK (Death associated protein), which has been previously shown to be silenced in DLBCL patients via DNA methylation. DAPK is a serine/threonine kinase and possesses pro-apoptotic and tumor-suppressive functions. We have confirmed by RT-PCR that DAPK was suppressed/silenced in DLBCL cell lines (n=8) as compared to CD19+ (n=3) normal B cells. HDAC inhibition alone or in combination with DNA methyltransferase inhibitors (DNMTi) can induce apoptosis by modulating expression of tumor suppressor genes. We next asked whether HDAC3 and HDAC1 dual inhibition through RG2833 alone or its combination with DNMTi, azacytidine (AZA) will be able to restore expression of DAPK in DLBCL cells. Quantitative RT-PCR analysis in OCILy3 and OCILY10 cells demonstrated that treatment with RG2833 alone modestly increased DAPK expression; however, co-treatment with RG283 and AZA led to marked increase in DAPK expression. As expected, the combination of RG2833 and Aza had significantly greater pro-apoptotic effect in DLBCL cells than either drug alone suggesting that DAPK expression may be an important determinant of response to HDAC1/HDAC3 dual inhibitor in DLBCL cells.In summary, our study provides first evidence of antitumor activity of dual HDAC3 and HDAC1 targeted agent therapy in DLBCL patients. Moreover, we have identified epigenetic modulation of DAPK as potential modulator of cellular response to HDAC1/HDAC3 dual inhibition. This study suggests for further clinical trials and/or development of selective inhibitors of HDAC3 or HDAC1/HDAC3 in DLBCL patients. DisclosuresNo relevant conflicts of interest to declare.

Rational Targeting of Cellular Cholesterol in Diffuse Large B-Cell Lymphoma (DLBCL) Enabled By Functional Lipoprotein Nanoparticles and the B Cell Receptor Inhibitor Ibrutinib: A Therapeutic Strategy Dependent on Cell of Origin (COO)

Introduction: Malignant cells, including lymphoma cells, have altered metabolism and, in some cases, an increased demand for cholesterol and cholesteryl esters. It is important to identify novel, rational treatments based on biology, and cellular cholesterol metabolism as a potential target for cancer is an innovative, exciting approach. Toward this end, we focused on DLBCL as a model because of differences in cholesterol biosynthesis driven by B-cell receptor (BCR) signaling in germinal center (GC) versus activated B-cell (ABC) DLBCL. To specifically target cellular cholesterol homeostasis, we employed bio-inspired high-density lipoprotein-like nanoparticles (HDL NP) that can generally reduce cellular cholesterol by targeting and blocking cholesterol and cholesteryl ester uptake through binding to the high-affinity HDL receptor, scavenger receptor type B-1 (SCARB1). We hypothesized that by inhibiting BCR signaling, and consequently cholesterol biosynthesis with the BTK inhibitor ibrutinib, we could overcome inherent resistance in cholesterol replete ABC DLBCL cell lines in vitro and in a xenograft model.Methods: HDL NPs were synthesized by surface-functionalizing a gold nanoparticle core as previously described (Thaxton et al JACS 2009) with the HDL defining apolipoprotein A-I and phospholipids, with the resultant HDL NPs having similar size, surface composition, charge and function as natural HDLs. However, owing to the gold nanoparticle core, HDL NPs are not an appreciable source of cholesterol or cholesteryl esters. GC DLBCL (SUDHL4) and ABC DBLCL (TMD8, HBL-1) cell lines were treated with HDL NPs as monotherapy or in combination with BCR signaling inhibitors (e.g. ibrutinib), and assayed for total cholesterol content and cell viability. Quantitative PCR was conducted to measure changes in expression of cholesterol biosynthesis genes. TMD8 tumor xenografts were initiated in SCID-beige mice, and following implantation, the mice were treated with vehicle, HDL NPs, ibrutinib, or a combination of the two treatments.Results: As we have previously reported (Yang et al PNAS 2013), GC DLBCL are exquisitely sensitive to HDL NP as monotherapy while ABC DLBCL are less sensitive. Here, our data demonstrate that HDL NPs, as a monotherapy, statistically significantly reduced total cellular cholesterol levels in SUDHL4 cells (7.96 ± 1.47 µg cholesterol/ 104 cells for 0nM HDL NP vs. 2.07 ± 2.07 µg cholesterol/ 104 cells for 100nM HDL NP; p=0.0007), resulting in profound cell death (HDL NP IC50 = 2nM). Further, HDL NP-induced cholesterol depletion resulted in upregulation of numerous cholesterol biosynthesis genes in SUDHL4 cells, including HMG-CoA synthase 1, 7-dehydrocholesterol reductase, lanosterol synthase, and others. In ABC DLBCL cells, the enhanced BCR signaling and resultant de novo cholesterol synthesis drastically diminishes the ability of HDL NPs to reduce cellular cholesterol (TMD8: 12.80 ± 5.08 µg cholesterol/ 104 cells for 0nM HDL NP vs. 16.35 ± 1.17 µg cholesterol/ 104 cells for 100nM HDL NP, p=0.84; HBL-1: 29.16 ± 5.96 µg cholesterol/ 104 cells for 0nM HDL NP vs. 23.51 ± 5.65 µg cholesterol/ 104 cells for 100nM HDL NP, p=0.75) and induce cell death (TMD8 HDL NP IC50 = 69nM; HBL-1 HDL NP IC50 = 75nM). Additionally, HDL NPs had little to no effect on the expression of the cholesterol biosynthesis genes in the ABC cell lines. To reduce cholesterol biosynthesis, which depends on BCR signaling, we combined HDL NPs with the BCR signaling inhibitor ibrutinib. We found that HDL NP synergize with ibrutinib in the ABC derived TMD8 cell line in vitro and in a xenograft model. Correspondingly, HDL NPs and ibrutinib reduced the total cellular cholesterol content of TMD8 and HBL-1 cells when administered together, but had no effect when given alone. Thus, by targeting both cellular cholesterol uptake and BCR-associated de novo cholesterol synthesis we achieved synergistic cellular cholesterol reduction and subsequent cell death, both in vitro and in the TMD8 in vivo tumor xenograft model.Conclusion: These results in DLBCL lymphoma cell lines and tumor xenografts demonstrate that depletion of cellular cholesterol, in a specifically targeted manner dependent on the HDL receptor SCARB1 and differential BCR signaling in ABC vs GCB cells, is a powerful mechanism to induce apoptosis in lymphoma and opens a new paradigm in lymphoma biology and therapy. DisclosuresThaxton:Aurasense: Other: The HDL NPs technology was licensed by Northwestern University to a biotech company (Aurasense) founded by C. Shad Thaxton . Gordon:Janssen: Other: Data Monitoring Committee.

Development and Characterization of the Novel Orally Bioavailable EZH1/2 Dual Inhibitor DS-3201

Polycomb repressive complex 2 (PRC2) methylates histone H3 lysine 27 (H3K27) and represses gene expression to regulate cell proliferation and differentiation. Enhancer of zeste homolog 2 (EZH2) or its close homolog EZH1 function as catalytic subunits of PRC2, so there are two PRC2 complexes containing either EZH2 or EZH1. Many reports suggest that inhibiting PRC2 activity through EZH2 knockdown gives rise to anti-cancer effect against hematological and solid cancers.To date, some EZH2 selective inhibitors have demonstrated anti-cancer potential in preclinical and clinical studies. In spite of this promise, there is concern that the efficacy anticipated with EZH2 selective inhibitors may be compromised due to compensation by EZH1 activity.Herein, we describe the novel, orally bioavailable EZH1/2 dual inhibitor DS-3201. This compound strongly and selectively inhibited the methyltransferase activity of both EZH1 and EZH2 with an IC50 value of 10 nM and 6.0 nM, respectively in cell-free enzymatic assays. This compound also suppressed trimethylation of H3K27 in cells (IC50: 0.55 nM) more potently than EZH2 selective inhibitors.In order to identify the anti-cancer spectrum of DS-3201, we tested its in vitro growth inhibition activity against a panel of cancer cell lines including a variety of hematological cancers. Among the panel, hematological cell lines showed particular sensitivity to DS-3201, with GI50 values less than 100 nM, including 1) acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) cell lines harboring fusion genes, such as MLL-AF9, MLL-AF4, or AML1-ETO, 2) diffuse large B-cell lymphoma (DLBCL) cell lines having EZH2 gain-of-function (GOF) mutations, 3) most of the peripheral T-cell lymphoma and multiple myeloma cell lines tested. Our data thus shows this compound has broader anti-cancer activity than EZH2 selective inhibitors, especially in hematology cancers. We also evaluated the in vivo anti-tumor efficacy of DS-3201 against KARPAS-422, a DLBCL tumor xenograft with an EZH2 GOF mutation. A 100 mg/kg once daily oral administration of DS-3201 caused nearly complete tumor regression without any body weight loss and 25 mg/kg administration also showed clear tumor growth delay.Based on these results, we initiated clinical studies of DS-3201 for patients with lymphoma (Clinical trial information: NCT02732275) and for those with AML and ALL (NCT03110354). DisclosuresHonma:Daiichi Sankyo Co., Ltd.: Employment. Adachi:Daiichi Sankyo Co., Ltd.: Employment. Kanno:Asubio Pharma Co., Ltd.: Employment. Watanabe:Daiichi Sankyo Co., Ltd.: Employment. Hirasawa:Daiichi Sankyo Co., Ltd.: Employment. Nosaka:Daiichi Sankyo Co., Ltd.: Employment. Shiroishi:Daiichi Sankyo Co., Ltd.: Employment. Takizawa:Daiichi Sankyo RD Novare Co., Ltd.: Employment. Yasumatsu:Daiichi Sankyo RD Novare Co., Ltd.: Employment. Horiuchi:Daiichi Sankyo Co., Ltd.: Employment. Nakao:Daiichi Sankyo Co., Ltd.: Employment. Suzuki:Daiichi Sankyo Co., Ltd.: Employment. Yamasaki:Daiichi Sankyo Co., Ltd.: Employment. Nakajima:Daiichi Sankyo Co., Ltd.: Employment. Hayakawa:Daiichi Sankyo Co., Ltd.: Employment. Yamazaki:Daiichi Sankyo Co., Ltd.: Employment. Araki:Daiichi Sankyo: Employment. Fujiwara:Daiichi Sankyo Co., Ltd.: Employment.

Upregulation of Alternatively Glycosylated Smoothened (SMO) Is Involved in Doxorubicin (DXR) Resistance in Diffuse Large B-Cell Lymphoma

▪Diffuse large B-cell lymphoma (DLBCL) is the most common lymphoid malignancy in adults. Chemoresistance remains the most urgent clinical challenge in the management of DLBCL patients. The salvage therapy options for patients with relapsed disease are poor (responses rates less than 20%) and survival is usually short. Therefore, the development of less toxic yet effective therapeutic approaches for DLBCL patients will address a significant immediate medical need. Smoothened (SMO), a Frizzled-class G-protein-coupled-receptor (GPCR), is activated by cholesterol and regulated indirectly by Hedgehog (Hh) ligands through the release of PTCH1-mediated suppression. SMO contains an N-terminal extracellular segment with the cysteine-rich domain (CRD), a heptahelical transmembrane domain (TMD), and a C-terminal intracellular segment. Although SMO localizes in cilia to function as Hh signal transducer, our data support that SMO is also a constituent of lipid rafts, and plays a fundamental role in lipid raft organization in DLBCL. SMO in doxorubicin (DXR) resistant cell lines shows increased glycosylation of its extracellular segments and drug resistance is sensitive to modulation of cellular cholesterol levels, both features associated with protein localization to lipid raft and lipid raft composition, a poorly understood aspect in drug resistance. To improve our current therapeutic approaches for lymphoma patients we need to increase our understanding of the cellular mechanisms that contribute to tumor progression and drug resistance.We established a DXR-resistant cell line (SUDHL4) through long-term exposure to escalating doses of DXR (DXR-resistant cell line: virtually no cell death up to a DXR concentration of 120 nM; parental cell line DXR-IC50: 40nM). We observed that DXR-resistant SUDHL4 cells were also resistant to escalating doses of vincristine in comparison with the parental (sensitive) cells. Studies on the response of DXR resistant lymphoma cells to DXR confirmed high levels of DNA damage in resistant cells (measured by phosphorylation of histone H2AX) confirming that DXR resistance was not due to increase drug efflux. In addition, in this experimental model, both parental and DXR-resistant lymphoma cells have mutated TP53 (missense mutation p.V157A) showing that TP53 deficiency was not a factor in inducing DXR resistance.Several lines of evidence support a direct role of SMO in the DXR-resistance observed in DLBCL cells. First, DXR-resistant lymphoma cells in comparison with the parental cells showed a strong increase in SMO mRNA, but not its canonical downstream target of signaling, the transcription factor GLI1. Concordantly, SMO protein levels were substantially elevated in DXR-resistant cells (as detected by immunofluorescence and western blot). Moreover, upregulation of the expression of SMO was detected by immunohistochemistry in 4 of 5 relapsed DLBCL tumors in comparison with the matched tumors at the time of the initial diagnosis and before chemotherapy treatment. Second, we found that SMO is localized in the lipid rafts and mediates the assembly of a TRAF6 E3 ligase-dependent machinery that enhances AKT signaling. Additionally, we observed that SMO is highly glycosylated in DXR-resistant SUDHL4 cells but not in the parental cell line; previously, it has been found that SMO has 4 potential N-glycosilation sites that are dispensable for canonical Hh signaling but needed to induce a non-canonical SMO signal through Gα1. Finally, depletion of SMO using shRNA and a lentiviral-tetracycline inducible system and pharmacologically inhibition of SMO using the classic SMO inhibitor (cyclopamine-KAAD) or a cholesterol synthesis inhibitor (lovastatin) re-established sensitivity to DXR in DXR-resistant cells. Interestingly, lovastatin did not modify DXR sensitivity in the parental cell line. In summary, this study suggests a novel function of SMO in the development of DXR-resistance in DLBCL and opens new opportunities to regain or enhance DXR sensitivity through targeting this novel mechanism. DisclosuresNo relevant conflicts of interest to declare.

Functional Investigation of the Gene NFKBIZ and the Impact of 3'UTR Mutations in Diffuse Large B-Cell Lymphoma

Introduction:Diffuse large B-cell lymphoma (DLBCL) represents the most frequent non-Hodgkin lymphoma, accounting for over 30% of cases. The two main subtypes of DLBCL, germinal center B-cell-like (GCB) and activated B-cell-like (ABC), have distinct clinical outcomes, unique gene expression profiles and a growing list of defining genetic features. Recurrent somatic mutations affecting genes such as MYD88, CD79A/B and TNFAIP3 have been shown to constitutively activate NF-κB signaling, either directly or indirectly, in ABC DLBCL. Amplification of the NFKBIZ locus has been observed in ~10% of ABC DLBCL cases however, no other mutations affecting this locus have been previously described. Although some alterations leading to NF-κB activation have been identified and characterized, there remain ABC cases with NF-κB activation and no clear genetic explanation. Through a meta-analysis of published and in-house exome and genome sequence data for recurrent mutations affecting non-coding regions, we found NFKBIZ had the strongest enrichment for mutations within the 3' untranslated region (UTR). The majority of these mutations affect a highly conserved region of the mRNA that is predicted to form multiple stem-loops or more complex secondary structures (Figure 1A). The endoribonuclease Regnase-1, encoded by the gene ZC3H12A, targets mRNAs with conserved secondary structures in the 3'UTR such as those predicted to form in NFKBIZ . Mutations in the NFKBIZ 3'UTR potentially abrogate this interaction with Regnase-1.Aims and Methods:Based on the observed mutation pattern, we hypothesized that NFKBIZ 3'UTR mutations are more significantly enriched in ABC DLBCL and lead to increased activation of the NF-κB signaling pathway. We aimed to (1) determine the frequencies of mutations in these two genes in DLBCL, specifically within each sub-type in our cohort and (2) investigate the effects of NFKBIZ 3'UTR mutations on mRNA and protein expression in activating the NF-κB signaling pathway in B-cells. We analyzed RNA-seq data and performed targeted sequencing on these lines and 334 primary DLBCL tumors and 8 DLBCL cell lines. We assessed mRNA abundance and the allelic abundance of mutant and wild-type NFKBIZ using RNA-seq reads and a custom “wild-type negative” or “drop-off” droplet digital PCR (ddPCR) assay. The selected cell lines represent those naturally NFKBIZ mutant (amplification or containing a 3'UTR deletion), MYD88 mutant, or wild-type for both. These lines were used to investigate NFKBIZ mRNA and IκB-ζ protein expression.Results:Targeted sequencing and copy number analysis of 316 cases with available RNA-seq data uncovered 66 DLBCLs (21% of cases) containing any NFKBIZ mutation, 34 (10.8%) of which were 3'UTR mutations, and 19 DLBCLs with a mutation in ZC3H12A (6% of cases) . A substantial fraction of the NFKBIZ mutations were confirmed somatic by sequencing matched constitutional DNA. NFKBIZ 3'UTR mutations were mutually exclusive with MYD88 mutations and largely segregated from those affecting ZC3H12A (Figure 1B). In aggregate, mutations in either of these novel genes are present in 22.6% of all DLBCLs and 30.8% of ABC cases. The enrichment for these mutations in ABC cases was highly significant (OR 4.1, 95% CI 2.04-8.48). Results from our ddPCR experiments revealed allelic imbalance of NFKBIZ mRNA in mutant cell lines, specifically a relative increase of the mutant transcript. Patient RNA samples harboring NFKBIZ mutations (small deletions and SNVs) showed similar results. Western blot experiments confirmed increased levels of IκB-ζ protein consistent with increased mRNA in all NFKBIZ and MYD88 mutant cell lines (Figure 1C).Conclusion:Our current work has shown promising results indicating that NFKBIZ may be an underappreciated driver gene in ABC DLBCL. We have demonstrated that mutations affecting the 3'UTR of the NFKBIZ gene are significantly enriched in the ABC sub-type. These mutations cause deregulation of the mRNA and lead to over-expression of the IκB-ζ protein which activates the NF-κB signaling pathway. Uncovering new ways in which the NF-κB pathway can be dysregulated in ABC DLBCL will allow for better treatments to be developed and more accurate stratification of patients into the clinical trials that will be the most beneficial to their specific set of mutations. [Display omitted] DisclosuresConnors:Bristol-Myers Squibb: Research Funding; Genentech: Research Funding; Merck: Research Funding; Lilly: Research Funding; Cephalon: Research Funding; F Hoffmann-La Roche: Research Funding; Janssen: Research Funding; Bayer Healthcare: Research Funding; Seattle Genetics: Research Funding; Takeda: Research Funding; NanoString Technologies, Amgen, Bayer, BMS, Cephalon, Roche, Genentech, Janssen, Lilly, Merck, Seattle Genetics, Takeda,: Research Funding; Amgen: Research Funding; NanoString Technologies: Research Funding. Scott:Janssen: Consultancy, Honoraria; Celgene: Consultancy, Honoraria; BCCA: Patents & Royalties: Patent describing molecular subtyping of DLBCL licensed to NanoString Technologies. Patent describing measurement of the proliferation signature in MCL.. Steidl:Affimed Therapeutics: Consultancy; Juno Therapeutics: Consultancy. Morin:Epizyme, Inc: Consultancy.

The Novel Calcium Release-Activated Calcium (CRAC) Channel Inhibitor RP4010 Exerts Potent Antitumor Effects in NOD/SCID/IL2Rg-/- Mice with Diffuse Large B Cell Lymphoma (DLBCL) Cell Line Xenografts

INTRODUCTION: Calcium ions (Ca2+) act as second messengers in cell signaling triggering various cellular processes, including gene transcription, secretion, cell proliferation, migration, and apoptosis. Store-operated Ca2+ entry (SOCE) is activated by Ca2+ release from the endoplasmic reticulum. Two genes are responsible for SOCE activity: Stromal interaction molecule 1 (STIM1), an ER Ca2+ sensor that detects store depletion and Orai1, the pore-forming subunit of Ca2+ release-activated Ca2+ (CRAC) channel. Downregulation of Orai1, and consequently SOCE, protects the cells from diverse apoptosis-inducing pathways. As emerging evidence points to the involvement of aberrant CRAC channel activity in human diseases including Diffuse Large B Cell Lymphoma (DLBCL), we hypothesize that the CRAC channel inhibitorRP4010 might represent a unique therapeutic opportunity for DLBCL patients. RP4010 is a potent inhibitor of CRAC channel activity (IC50=60 nM) with demonstrated efficacy across a range of cancer cell lines in vitro . This study aimed at investigating the activity of RP4010 in preclinical DLBCL models.METHODS: The DLBCL cell lineswith GCB phenotype (SUDHL-4, SUDHL-6, SUDHL-16, and SUDHL-10) and ABC phenotype (SUDHL-8, RCK-8, RIVA, and SUDHL-2) were used to investigate in vitro the effects of RP4010 on cell growth and cell death and gene expression. The antitumor efficacy and of RP4010 was also analyzed in NOD/SCID/IL2Rg-/-mice bearing DLBCL cell line xenografts.RESULTS: Incubating GCB-DLBCL and ABC-DLBCL cell lines for up to 72 hours with increasing doses of RP4010 (1.25 - 10 µM)resulted in a significant dose- and time-dependent decrease in cell proliferation. For all cell lines, the peak of the cytostatic effect was detected upon incubation with 10 µM of RP4010 for 72 hours, when an average 80% decrease in cell proliferation over vehicle-treated controls (P ≤ 0.0001) was detected. RP4010 treatment resulted in a significant time- and dose-dependent increase in cell death, as assessed by AnnexinV/PI staining, in GCB-DLBCL cell lines and in two out of four ABC-DLBCL cell lines (SUDHL-8 and SUDHL-2), whereas modest or no effects were detected in the RCK-8 and RIVA ABC-DLBCL cell lines. Exposure to 5 µM RP4010 for 72 hours significantly increased cell death over vehicle-treated controls in SUDHL-4 (90 ± 4% vs. 11 ± 2%), SUDHL-6 (39 ± 4% vs. 16 ± 3%), SUDHL-16 (70 ± 5% vs. 18 ± 4%), SUDHL-10 (49 ± 5% vs. 15 ± 1%), SUDHL-8 (78 ± 4 vs. 8 ± 1%) and SUDHL-2 (57 ± 5 vs. 26 ± 4%) cell lines (P ≤ 0.0001). Interestingly, RP4010 (1.25 - 10 µM) failed to induce any cytotoxic effects on the in vitro growth of hematopoietic colony-forming cells from healthy volunteers. While no activation of caspase-8, -9, -3, or PARP were observed in ABC-DLBCL cell lines treated with RP4010, a potent caspase cleavage was observed upon RP4010 treatment in GCB-DLBCL cell lines. Pretreating cells with the pan-caspase inhibitor Z-VAD-fmk abrogated apoptosis only in GCB-DLBCL cell lines, but not in the ABC-DLBCL cell lines, supporting that RP4010 induces both caspase-dependent and caspase-independent cell death. Gene expression analysis revealed a strong correlation between Orai1 expression and sensitivity to RP4010-induced cell death. Thus, Orai1 has a pivotal role in the establishment of an apoptosis-resistant phenotype in ABC-DLBCL cells. Finally, in vivo experiments were conducted to investigate the antitumor activity of RP4010. Treatment of NOD/SCID/IL2Rg-/-mice bearing DLBCL tumor nodules with increasing dose of RP4010 (30, 60, 90 mg/Kg body weight, PO, 5 days/3 weeks) resulted in a dose-dependent inhibition of tumor growth (TGI) of SUDHL-8 [TGI = 35%, 74%, and 83% at 30, 60, and 90 mg/kg, respectively], RIVA and SUDHL-6[TGI = 66%, 52% at 90 mg/kg, respectively], compared to vehicle-treated controls. No mice experienced any apparent treatment-related toxicity. Despite the marked antitumor activity observed in DLBCL xenografts, RP4010 treatment did not induce any tumor necrosis in healthy tissues (including lungs, livers, spleens, and kidneys), suggesting that RP4010-induced growth inhibition was tumor restricted.CONCLUSIONS: RP4010 is associated with a potent anti-lymphoma activity, suggesting its therapeutic potential in DLBCL. A Phase 1/1b study in Relapsed/Refractory Non-Hodgkin Lymphoma (NHL) patients is currently ongoing in the US (ClinicalTrials.gov Identifier: NCT03119467). DisclosuresViswanadha:Incozen Therapeutics: Employment. Vakkalanka:Rhizen Pharmaceuticals S A: Employment, Equity Ownership. Santoro:Merck: Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees. Carlo-Stella:Boehringer Ingelheim: Consultancy, Honoraria; ADC Therapeutics: Research Funding; Rhizen Pharmaceuticals: Research Funding.

The Marine Natural Compound Elatol Is a Novel Protein Translation Inhibitor with Potent Activity Against Aggressive Non-Hodgkin Lymphomas

Prognosis remains poor for patients with relapsed or refractory diffuse large B-cell lymphoma (DLBCL) and other aggressive non-Hodgkin lymphomas. Targeted small-molecule signaling inhibitors have been poorly active in this setting due to a heterogeneous pathogenesis in which multiple different but overlapping signals may drive lymphomagenesis. Activation of cap-dependent protein translation is a convergence point of multiple lymphoma drivers, including B-cell receptor (BCR) signaling pathways. Cap-dependent protein translation begins with formation of the eIF4F complex, a heterotrimeric complex comprised of the cap-binding protein eIF4E, the scaffold protein eIF4G, and the ATP-hydrolyzing RNA helicase eIF4A, which is required for expression of many oncogenes, including the common lymphoma driver MYC. Despite evidence that disruption of eIF4F function is toxic to lymphoma cell lines and sensitizes mouse models of lymphoma to chemotherapy, eIF4A inhibitors have not progressed to clinical evaluation. We designed a screen to identify novel eIF4A inhibitors by measuring ATP hydrolysis and screened a collection of ~1,200 natural compounds and purified extracts. The marine natural product elatol was the top hit in our screen, showing selectivity for eIF4A over other DEAD-box family helicases and a panel of 97 common kinases. Elatol inhibits eIF4A's helicase activity in cell-free in vitro assays and binds to the target at a 2:1 ratio through interaction with two key lysine residues that fold together in its RNA-binding groove. It also induced apoptosis in a broad range of cancer cell lines from the NCI-60 (average LD50= 1.9 μM; LD50<1μM in 33/60) cancer cell line and CCLE panels, but our collection of non-Hodgkin lymphoma lines showed the greatest sensitivity as a group (average LD50= 1.3 μM; 9/12 <1μM; range 213-5749nM). In DLBCL cell lines, Elatol treatment reduced global protein translation measured through polysome profiling and O-propargyl puromycin incorporation as well as cap-dependent translation measured by luciferase reporter and western blotting. Elatol also blocked translational activation downstream of BCR stimulation in primary chronic lymphocytic leukemia (CLL) cells. The effects of elatol are similar to the rocaglate silvestrol, which inhibits eIF4A though through a different mechanism, but elatol requires higher concentrations to achieve these effects. Elatol's reduced in vitro potency, however, is off-set by a maximum tolerated in vivo dose to mice of 65mg/kg, compared to silvestrol's typical dosing at 0.2-05 mg/kg. Elatol significantly reduced the growth of DLBCL tumors in a xenograft model. Mechanistically, elatol and silvestrol both clearly function downstream of mTOR for translational inhibition, but unlike silvestrol, elatol causes strong and rapid induction of the stress response transcription factor ATF4. Assessment of drug activity in murine embryonal fibroblasts lacking ATF4 and those with an unphosphorylatable mutant allele of its upstream activator eIF2α shows elatol's anti-lymphoma activities are not dependent on ATF4 induction. In addition, an inhibitor of the eIF2α kinase PERK and other evidence demonstrate elatol does not produce an unfolded protein response, but the reason for ATF4 induction is not yet clear and additional eIF2α kinases are under investigation. The novel mechanism of inhibition of ATP hydrolysis may be driving these unique observations compared to other eIF4A inhibitors, but unexpected off targets are also under evaluation. In sum we report a new natural inhibitor of protein translation with potent anti-lymphoma activities. Elatol provides a novel starting point for development of eIF4A inhibitors in cancer therapeutics. DisclosuresNo relevant conflicts of interest to declare.

PET-CT Invisible Diffuse Large B-Cell Lymphoma Features a Silent B-Cell Receptor Signaling

Background:18F-fluorodeoxyglucose (FDG) positron emission tomography with computed tomography (PET-CT) has been widely used in lymphoma patients for diagnosis, staging, assessment of response and surveillance. And the standardized uptake value (SUV), a commonly used indicator in PET-CT scan, can quantitatively reflect the glucose uptake and glycolysis of the tumor. Diffuse large B-cell lymphoma (DLBCL), one of the most common subtypes of aggressive lymphomas, is supposed to exhibit high standardized uptake value (SUV) in 18F-FDG PET-CT due to high rate of glucose (18F-FDG) uptake and high level of glycolysis. Nevertheless, PET-CT “invisible” DLBCL cases have also been described, which featuring a relatively low 18F-FDG uptake denoted by low SUV. Herein, we analyzed this subgroup of under-estimated disease.Methods: Nineteen cases each with PET-CT “invisible” (PET-CTi) and PET-CT “visible” (PET-CTv) de novo DLBCL were submitted for an immunohistochemical detection for the expression level of B-cell receptor (BCR) signaling-related molecules. Nine xenografts derived from BCR-dependent and BCR-independent DLBCL cell lines were further submitted for a small animal 18F-FDG PET-CT scanning and the following immunohistochemical detection for BCR signaling-related molecules.Results: The immunohistochemical expression level of pSYK (pY525/526), a key effector molecule for BCR signaling, was significantly lower in PET-CTi DLBCL tumor tissues compared with that in PET-CTv DLBCL cases (P <0.01). With regard to in vivo PET-CT experiment, the xenografts derived from BCR-independent DLBCL cell lines exhibited remarkable lower SUV level than those derived from BCR-dependent cell lines (P < 0.01). And BCR-independent DLBCL xenografts featured relatively lower expression level of pSYK compared with those BCR-dependent ones (P <0.01).Conclusions:18F-FDG PET-CT negative DLBCL features a silent BCR signaling and 18F-FDG PET-CT may be of use in evaluating BCR signaling status. DisclosuresNo relevant conflicts of interest to declare.

A Phase I, Open Label, Multicenter Trial of Oral Azacitidine (CC-486) Plus R-CHOP in Patients with High-Risk, Previously Untreated Diffuse Large B-Cell Lymphoma, Grade 3B Follicular Lymphoma, or Transformed Lymphoma

A Phase I, Open Label, Multicenter Trial of Oral Azacitidine (CC-486) Plus R-CHOP in Patients with High-Risk, Previously Untreated Diffuse Large B-Cell Lymphoma, Grade 3B Follicular Lymphoma, or Transformed Lymphoma

Conditional expression of HGAL leads to the development of diffuse large B-cell lymphoma in mice

Diffuse large B-cell lymphomas (DLBCLs) are clinically and genetically heterogeneous tumors. Deregulation of diverse biological processes specific to B cells, such as B-cell receptor (BCR) signaling and motility regulation, contribute to lymphomagenesis. Human germinal center associated lymphoma (HGAL) is a B-cell–specific adaptor protein controlling BCR signaling and B lymphocyte motility. In normal B cells, it is expressed in germinal center (GC) B lymphocytes and promptly downregulated upon further differentiation. The majority of DLBCL tumors, primarily GC B-cell types, but also activated types, express HGAL. To investigate the consequences of constitutive expression of HGAL in vivo, we generated mice that conditionally express human HGAL at different stages of hematopoietic development using 3 restricted Cre-mediated approaches to initiate expression of HGAL in hematopoietic stem cells, pro-B cells, or GC B cells. Following immune stimulation, we observed larger GCs in mice in which HGAL expression was initiated in GC B cells. All 3 mouse strains developed DLBCL at a frequency of 12% to 30% starting at age 13 months, leading to shorter survival. Immunohistochemical studies showed that all analyzed tumors were of the GC B-cell type. Exon sequencing revealed mutations reported in human DLBCL. Our data demonstrate that constitutive enforced expression of HGAL leads to DLBCL development.