Target In Diffuse Large B-cell Lymphoma Research Articles

Eukaryotic initiation factor 3a promotes the development of diffuse large B-cell lymphoma through regulating cell proliferation

BackgroundOne-third of diffuse large B-cell lymphoma (DLBCL) patients suffer relapse after standard treatment. Eukaryotic initiation factor 3a (eIF3a) is a key player in the initial stage of translation, which has been widely reported to be correlated with tumorigenesis and therapeutic response. This study aimed to explore the biological role of eIF3a, evaluate its prognostic and therapeutic potential in DLBCL.MethodsRNA-seq datasets from GEO database were utilized to detect the expression and prognostic role of eIF3a in DLBCL patients. Protein level of eIF3a was estimated by western blot and immunohistochemical. Next, DLBCL cells were transfected with lentiviral vector either eIF3a-knockdown or empty to assess the biological role of eIF3a. Then, samples were divided into 2 clusters based on eIF3a expression and differentially expressed genes (DEGs) were identified. Function enrichment and mutation analysis of DEGs were employed to detect potential biological roles. Moreover, we also applied pan-cancer and chemosensitivity analysis for deep exploration.ResultseIF3a expression was found to be higher in DLBCL than healthy controls, which was associated with worse prognosis. The expression of eIF3a protein was significantly increased in DLBCL cell lines compared with peripheral blood mononuclear cells (PBMCs) from healthy donors. eIF3a knockdown inhibited the proliferation of DLBCL cells and the expression of proliferation-related proteins and increase cell apoptosis rate. Besides, 114 DEGs were identified which had a close linkage to cell cycle and tumor immune. eIF3a and DEGs mutations were found to be correlated to chemosensitivity and vital signal pathways. Pan-cancer analysis demonstrated that high eIF3a expression was associated with worse prognosis in several tumors. Moreover, eIF3a expression was found to be related to chemosensitivity of several anti-tumor drugs in DLBCL, including Vincristine and Wee1 inhibitor.ConclusionsWe firstly revealed the high expression and prognostic role of eIF3a in DLBCL, and eIF3a might promote the development of DLBCL through regulating cell proliferation and apoptosis. eIF3a expression was related to immune profile and chemosensitivity in DLBCL. These results suggest that eIF3a could serve as a potential prognostic biomarker and therapeutic target in DLBCL.

PGD2 displays distinct effects in diffuse large B-cell lymphoma depending on different concentrations

Prostaglandin D2 (PGD2), an arachidonic acid metabolite, has been implicated in allergic responses, parasitic infection and tumor development. The biological functions and molecular mechanisms of PGD2 in diffuse large B-cell lymphoma (DLBCL) are still undefined. In this study, we firstly found the high concentration of serum PGD2 and low expression of PGD2 receptor CRTH2 in DLBCL, which were associated with clinical features and prognosis of DLBCL patients. Interestingly, different concentration of PGD2 displayed divergent effects on DLBCL progression. Low-concentration PGD2 promoted cell growth through binding to CRTH2 while high-concentration PGD2 inhibited it via regulating cell proliferation, apoptosis, cell cycle, and invasion. Besides, high-concentration PGD2 could induce ROS-mediated DNA damage and enhance the cytotoxicity of adriamycin, bendamustine and venetoclax. Furthermore, HDAC inhibitors, vorinostat (SAHA) and panobinostat (LBH589) regulated CRTH2 expression and PGD2 production, and CRTH2 inhibitor AZD1981 and high-concentration PGD2 enhanced their anti-tumor effects in DLBCL. Altogether, our findings demonstrated PGD2 and CRTH2 as novel prognostic biomarkers and therapeutic targets in DLBCL, and highlighted the potency of high-concentration PGD2 as a promising therapeutic strategy for DLBCL patients.

The Cyclin-G Associated Kinase (GAK) Is a Critical Dependency for Cell-Cycle Progression in Diffuse Large B-Cell Lymphoma Associated with Deregulation of the Retinoblastoma Tumor Suppressor

Background: Recurrent genomic alterations in specific cancers reveal tumor drivers that inform the development of rational therapeutic approaches. In diffuse large B-cell lymphoma (DLBCL), such oncoprotein-focused drug development has achieved only limited success. Phenotypic screening, less explored in DLBCL drug discovery, is an emerging alternative that can be leveraged especially through the application of machine-learning analyses to raw screening data. Under this paradigm, we reveal strong therapeutic potential in DLBCL of inhibiting the cyclin-G associated kinase (GAK). GAK participates in clathrin-mediated endocytosis and regulates mitotic-spindle alignment during cell division. It has been studied as an antiviral target that interrupts internalization at cell membranes but is minimally explored in cancer therapeutics and is a novel target in DLBCL. Methods and Results: We performed a first-of-its-kind screen of kinase inhibitors in DLBCL followed by machine learning-based analysis using our in-house target identification platform, idTRAX. The screen revealed GAK as a strong target for both activated B-cell (ABC) and germinal center B-cell (GCB) derived cell lines with complete sparing of peripheral-blood mononuclear cells (PBMCs), included as non-malignant controls. We analyzed gene-expression data available for 414 previously untreated DLBCL patients (Lenz cohort) and found tumors with high GAK expression had dramatically worse overall survival (OS) compared to those with low expression (hazard ratio=1.84 (95% conf. int. 1.33-2.53), p=0.0002). Mechanistically, we explored GAK's role in endocytosis but found no change in cell-surface B-cell receptor (BCR) expression after treatment with the GAK tool compound SGC-GAK-1. In addition, there was no evident perturbation of signaling pathways downstream of BCR by immunoblotting. We found instead that GAK inhibition in DLBCL results in profound disruption of progression through mitosis, potently triggering the spindle assembly checkpoint (SAC) and causing accumulation in G2 before onset of apoptosis. We explored whether GAK dependency is induced due to cell cycle deregulation. Analysis of gene-expression data in the Lenz cohort showed a striking correlation between high GAK expression and low expression of RB1, encoding the tumor suppressive master cell-cycle regulator retinoblastoma-associated protein (RB, p<0.0001). The ABC-DLBCL cell lines RIVA and U2932 completely lack RB expression and show high sensitivity and accumulation in G2 in response to SGC-GAK-1 treatment. Immunofluorescent confocal microscopy revealed profound disruption of mitotic spindles and shattering of chromosomes in these cells and, to a lesser extent, in RB-competent lines. SGC-GAK-1 is not optimized for use as a chemical probe in vivo, but target validation through shRNA knockdown in DLBCL patient-derived xenograft (PDX) models is underway and expected at time of presentation. Conclusions: GAK is a novel kinase therapeutic target in DLBCL revealed by our target identification platform, idTRAX. GAK's role in mitotic-spindle alignment represents a critical kinase dependency for DLBCL tumors, in particular those with cell-cycle deregulation due to loss of RB function. Our ongoing efforts to synthesize novel GAK inhibitors and reveal synergistic targets may reveal new therapeutic strategies for translation to clinical trials for DLBCL patients with unmet clinical needs.

High expression of AP2M1 correlates with worse prognosis by regulating immune microenvironment and drug resistance to R-CHOP in diffuse large B cell lymphoma.

First-line treatment with R-CHOP has cured 50%-60% patients of diffuse large B cell lymphoma (DLBCL), and more than one-third patients will eventually progressed to relapsed/refractory disease with dismal outcomes. Adaptor Related Protein Complex 2 Subunit Mu 1 (AP2M1) is required for the activity of a vacuolar ATPase and may also play an important role in regulating the intracellular trafficking and function of CTLA-4 protein. Herein, using both public databases and our own tumor samples, we aimed to demonstrate the prognostic role of AP2M1 and the potential tumor-promoting mechanisms in DLBCL. Using public datasets of DLBCL from both GEO and TCGA databases, we analyzed the role of AP2M1 in mediating chemoresistance to R-CHOP and its correlation with various clinical parameters and prognosis. By using various R packages, we evaluated the role of AP2M1 on regulating tumor immune microenvironment. Moreover, tumor samples of DLBCL from Beijing TongRen Hospital were used to validate our findings by immunohistochemistry staining. Expression of AP2M1 was significantly increased in DLBCL, which was correlated with poor prognosis and a variety of clinical indicators. On the basis of enrichment analysis, it was found that AP2M1 may be related to intracellular receptor signaling pathway. Through immune analysis and drug prediction, we found that the expression of AP2M1 affected the immune environment and drug response of DLBCL, which further revealed the important role of AP2M1 in DLBCL. By analyzing 61 patients treated uniformly with R-CHOP regimen in our center, we validated the above findings that high expression of AP2M1 correlated with inferior survival outcomes and affected sensitivity to R-CHOP treatment. Expression of AP2M1 may affect the prognosis of DLBCL patients probably by affecting the immune environment and the responses to many drugs in treating DLBCL, indicating AP2M1 as a potential therapy target in DLBCL.

Abstract A12: Pervasive hypermutation of super-enhancer regions dysregulates oncogene expression in diffuse large B-cell lymphoma

Abstract Coding-genome sequencing efforts have identified several genes/pathways altered in Diffuse Large B-cell Lymphoma (DLBCL), including new potential therapeutic targets. However, the mutational contribution of the non-coding genome of DLBCL remains largely unexplored. To identify functionally relevant non-coding mutations targeting regulatory domains, we integrated enhancer (E)/super-enhancer (SE) identification, by ChIP-seq analysis of the hallmark H3K27 histone mark, with whole genome sequencing (WGS) and RNA-seq analysis in 29 DLBCL cell lines representative of the major DLBCL subtypes, along with germinal center (GC) B cells representing the normal counterpart of DLBCL. Following validation in 93 normal/tumor DLBCL biopsies, we found that active SEs are specifically hypermutated (≥3 somatic mutations/Kb) in 99% of DLBCLs, as compared to the same loci when not active as SE (in total, 159 hypermutated SEs, with at least 2/case). As evidence of oncogenic relevance, we have shown that the hypermutated SEs linked to the BCL6 and BCL2 proto-oncogenes prevent the binding and transcriptional downregulation by transcriptional repressors. Genetic correction of selected mutations using the CRISPR/Cas9 technology restored repressor DNA-binding, downregulated target gene expression, and led to the counter-selection of cells harboring corrected alleles, indicating oncogenic dependency from the SE mutations. A third recurrently hypermutated SE was the CXCR4-SE, affected in 19% of cases. CXCR4 encodes for a G-protein coupled chemokine receptor involved in cell migration within the GC, and is mutationally activated in 40% of Waldenström macrobulinemia (WM), a post-GC lymphoproliferative disease. We identified a mutational hotspot preventing the binding of the NR3C1 glucocorticoid receptor in 7.5% of DLBCL cases. Correction of the mutations in cell lines led to counter selection and reduced expression of CXCR4. Conversely, the introduction of a mutation disrupting NR3C1 binding in WT DLBCL cells increased CXCR4 expression. Together, these results are consistent with dependency of the mutant cells on the CXCR4-SE mutation, and confirm a direct link between SE mutations in this region and deregulated gene expression through escape from NR3C1-mediated suppression. In primary cases, mutations in the NR3C1 binding site of the CXCR4-SE were observed in the absence of NR3C1 coding mutations and correlated with increased CXCR4 transcript levels, as documented by RNA-seq. Together with the oncogenic role of CXCR4 in WM, these findings reveal this chemokine receptor as a potential oncogenic target in DLBCL. Collectively, these findings reveal a new layer of genetic alterations, which identifies novel mechanisms of dysregulation for known oncogenes, as well as new dysregulated genes and pathways, with implications for precision classification and therapeutic targeting of DLBCL. * Co-senior authors. Citation Format: Elodie Bal, Rahul Kumar, Mohammad Hadigol, Antony B. Holmes, Laura K. Hilton, Jui Wan Loh, Kostiantyn Dreval, Jasper Wong, Sofija Vlasevska, Clarissa Corinaldesi, Rajesh Kumar Soni Soni, Katia Basso, Ryan D. Morin, Hossein Khiabanian, Laura Pasqualucci, Riccardo Dalla-Favera. Pervasive hypermutation of super-enhancer regions dysregulates oncogene expression in diffuse large B-cell lymphoma [abstract]. In: Proceedings of the Third AACR International Meeting: Advances in Malignant Lymphoma: Maximizing the Basic-Translational Interface for Clinical Application; 2022 Jun 23-26; Boston, MA. Philadelphia (PA): AACR; Blood Cancer Discov 2022;3(5_Suppl):Abstract nr A12.

Highlights from Recent Cancer Literature

Breaking Insights| August 16 2022 Highlights from Recent Cancer Literature Author & Article Information Online ISSN: 1538-7445 Print ISSN: 0008-5472 ©2022 American Association for Cancer Research2022American Association for Cancer Research Cancer Res (2022) 82 (16): 2809–2810. https://doi.org/10.1158/0008-5472.CAN-82-16-BI Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review Share Icon Share Facebook Twitter LinkedIn MailTo Tools Icon Tools Get Permissions Cite Icon Cite Search Site Article Versions Icon Versions Version of Record August 16 2022 Citation Highlights from Recent Cancer Literature. Cancer Res 15 August 2022; 82 (16): 2809–2810. https://doi.org/10.1158/0008-5472.CAN-82-16-BI Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search Search Dropdown Menu toolbar search search input Search input auto suggest Search Advanced Search Although recurrent genetic alterations in coding regions have been well characterized in diffuse large B-cell lymphoma (DLBCL), these alterations alone appear to be insufficient to drive lymphomagenesis. Bal and colleagues have now uncovered recurrent mutations in noncoding regions in super-enhancers, leading to loss of transcriptional repression of oncogenes, resulting in tumorigenesis. These recurrent mutations share an activation-induced cytidine deaminase signature implicating activation-induced cytidine deaminase as the mutational driver. CRISPR–Cas9-mediated reversion of these mutations led to a significant decrease in oncogene expression and prevented proliferation and survival of DLBCL cell lines. Expert Commentary: This study has uncovered a previously unrecognized role for activation-induced cytidine deaminase-mediated hypermutation of super-enhancers in lymphomagenesis and suggests potential new drug targets in DLBCL. Bal E, Kumar R, Hadigol M, Holmes AB, Hilton LK, Loh JW, et al. Super-enhancer hypermutation alters oncogene expression in B cell lymphoma. Nature; Published online July 6, 2022; doi: 10.1038/s41586-022-04906-8. Clear... You do not currently have access to this content.

PB2170: HIGH EXPRESSION OF AP2M1 CORRELATES WITH WORSE PROGNOSIS BY REGULATING IMMUNE MICROENVIRONMENT AND DRUG RESPONSE TO R-CHOP IN DLBCL

Background: DLBCL (diffuse large B cell lymphoma) is the most common subtype of non-Hodgkin lymphoma (NHL). After anthracycline-based chemotherapy, more than 70% of DLBCL patients can get complete response or partial response, but only 50% - 60% of patients can survive for a long time. Adaptor Related Protein Complex 2 Subunit Mu 1 (AP2M1) encodes a subunit of the hetero tetrameric coat assembly protein complex 2 (AP2), which required for the activity of a vacuolar ATPase and may also play an important role in regulating the intracellular trafficking and function of CTLA-4 protein. There are few reports on the relationship between DLBCL and AP2M1. Meanwhile, it is unclear whether it is related to the pathogenesis of DLBCL. Aims: Here, in this study, the relationship between DLBCL and AP2M1 and its effect in DLBCL chemotherapy were studied. Methods: Using public datasets of DLBCL from both GEO and TCGA databases, we analyzed the role of AP2M1 in mediating chemoresistance to R-CHOP and its correlation with various clinical parameters and prognosis. By using various R packages, we evaluated the role of AP2M1 on regulating tumor immune microenvironment. Moreover, FFPE samples of DLBCL from Beijing Tongren Hospital were used to valiate our findings. Results: We found that the expression of AP2M1 was significantly increased in DLBCL, which was correlated with poor prognosis and a variety of clinical indicators. On the basis of enrichment analysis, it was found that AP2M1 may be related to intracellular receptor signaling pathway. Through immune analysis and drug prediction, we found that the expression of AP2M1 affected the immune environment and drug response of DLBCL, which further revealed the important role of AP2M1 in DLBCL. Using FFPE DLBCL samples from patients treated in our center, we validated the above findings that high expression of AP2M1 correlated with inferior survival outcomes and affected sensitivity to R-CHOP treatment. Image:Summary/Conclusion: In this study, we found that the expression of AP2M1 could affect the prognosis of DLBCL patients by affecting the immune environment, and the expression of AP2M1 affected the response of many drugs in DLBCL treatment, indicating AP2M1 as a potential therapy target in DLBCL.

Identification of the estrogen receptor beta as a possible new tamoxifen-sensitive target in diffuse large B-cell lymphoma

Diffuse large B-cell lymphoma (DLBCL) is the most common lymphoma subtype. Despite the proven efficacy of combined immunochemotherapy (R-CHOP) in the majority of patients, ~40% of DLBCL patients do not respond or will relapse and consequently have a very poor prognosis. The development of targeted therapies has not improved patient survival, underscoring the need for new treatment approaches. Using an unbiased genome-wide CD20 guilt-by-association approach in more than 1800 DLBCL patients, we previously identified the estrogen receptor beta (ERβ) as a new target in DLBCL. Here, we demonstrate that ERβ is expressed at significantly higher levels in DLBCL compared to normal B cells, and ERβ plays a role in the protection against apoptosis in DLBCL. Targeting of the ERβ with the selective estrogen receptor modulator tamoxifen reduces cell viability in all tested DLBCL cell lines. Tamoxifen-induced cell death was significantly decreased in an ERβ knock-out cell line. The activity of tamoxifen was confirmed in a xenograft human lymphoma model, as tumor growth decreased, and survival significantly improved. Finally, tamoxifen-treated breast cancer (BC) patients showed a significantly reduced risk of 38% for DLBCL compared to BC patients who did not receive tamoxifen. Our findings provide a rationale to investigate tamoxifen, a hormonal drug with a good safety profile, in DLBCL patients.

A Novel Autochthonous Mouse Model Serves As a Preclinical Evaluation Platform and Explores Dual BTK and BCL2 Inhibition for Activated B Cell-like Diffuse Large B Cell Lymphoma

A Novel Autochthonous Mouse Model Serves As a Preclinical Evaluation Platform and Explores Dual BTK and BCL2 Inhibition for Activated B Cell-like Diffuse Large B Cell Lymphoma

Fascin Inhibition As a Novel Therapeeutic Strategy in Diffuse Large B-Cell Lymphoma

Fascin is an actin bundling protein used to form cellular projections (e.g. filipodia) involved in cell motility and invasion. Cancer cells often upregulate fascin, and in many cancer types this is associated with poor prognosis, likely due to increased invasive and metastatic potential. We have developed fascin inhibitors that prevent actin bundling and block breast cancer metastasis in mice. Here we explored the potential role of fascin inhibition in lymphoma. We have shown that fascin is upregulated in 44% of canine B-cell (n=62) and 57% of T-cell lymphoma cases (n=28), and that increased fascin expression is correlated with worse overall survival (p=0.03). B-cell lymphoma cases had a median survival time of 388 days while that of T-cell cases was 263 days. We have further shown that a fascin inhibitor, NP-G2-044, kills human and canine diffuse large B-cell lymphoma (DLBCL) cells in vitro, and in vivo in mice bearing canine DLBCL xenografts, with GI50s ranging from 5 to 200uM. Animal toxicology studies (performed by Charles River Laboratories) show no hematologic or other toxicity in dogs and rats at concentrations up to 1,000 mg/kg/day for 28 days; these doses are much higher than the doses needed to achieve concentrations that kill lymphoma in vivo . Therefore, we are planning clinical studies in pet dogs with lymphoma to establish proof of principle prior to a human clinical trial. Collectively, our results demonstrate that fascin is a potential new therapeutic target in DLBCL. DisclosuresHuang:Novita: Equity Ownership, Patents & Royalties: Cornell University and Novita Pharmaceutical has patient on the fascin described here. .

Expression and Prognostic Significance of PD-L2 in Diffuse Large B-Cell Lymphoma.

Recent studies suggest that programmed death ligand-2 (PD-L2) constitutes an important antitumor immune response. Here, we investigated the relationship between PD-L2 expression and clinicopathological features in diffuse large B-cell lymphoma (DLBCL). Immunohistochemistry showed that positive expression of PD-L2 was observed in 45 of 181 newly diagnosed patients, including 14 cases with expression exclusively on tumor cells (TCs) and 31 cases with the expression on both TCs and immune cells (ICs) in the tumor microenvironment (TME). In 21 recurrent patients, positive expression of PD-L2 was present in six cases, including two cases with expression exclusively on TCs, and four cases with the expression on both TCs and ICs in the TME. Patients with PD-L2 tumor proportion score (TPS) ≥1% exhibited a better ECOG performance status (PS) (ECOG PS score <2, P = 0.041), lower international prognostic index (IPI) score (P < 0.001), and early Ann Arbor stage (Ann Arbor stage I or II, P = 0.010). Similarly, patients with PD-L2 immune proportion score (IPS) ≥1% also exhibited a better ECOG PS (ECOG PS score < 2, P = 0.006) and lower IPI score (P = 0.001). Survival analysis showed that patients with PD-L2 TPS ≥1% exhibited prolonged overall survival (OS) and progression-free survival (PFS). However, survival analysis showed no prognostic significance based on expression of PD-L2 on ICs in the TME. TC PD-L2 expression was significantly associated with OS (P = 0.041) and PFS (P = 0.001). In the multivariate analysis, TC PD-L2 expression was an independent prognostic risk factor for PFS (P = 0.013), but not for OS (P = 0.249). Furthermore, we found that higher TC and IC PD-L2 expression was associated with higher objective response rate (ORR). Moreover, we demonstrated that the expression level of PD-L2 was positively correlated with the expression status of M1 macrophage markers CD86. Our findings highlight PD-L2 as a promising therapeutic target in DLBCL.

EGR1 Addiction in Diffuse Large B-cell Lymphoma.

Early growth response gene (EGR1) is a transcription factor known to be a downstream effector of B-cell receptor signaling and Janus kinase 1 (JAK1) signaling in diffuse large B-cell lymphoma (DLBCL). While EGR1 is characterized as a tumor suppressor in leukemia and multiple myeloma, the role of EGR1 in lymphoma is unknown. Here we demonstrate that EGR1 is a potential oncogene that promotes cell proliferation in DLBCL. IHC analysis revealed that EGR1 expression is elevated in DLBCL compared with normal lymphoid tissues and the level of EGR1 expression is higher in activated B cell-like subtype (ABC) than germinal center B cell-like subtype (GCB). EGR1 expression is required for the survival and proliferation of DLBCL cells. Genomic analyses demonstrated that EGR1 upregulates expression of MYC and E2F pathway genes through the CBP/p300/H3K27ac/BRD4 axis while repressing expression of the type I IFN pathway genes by interaction with the corepressor NAB2. Genetic and pharmacologic inhibition of EGR1 synergizes with the BRD4 inhibitor JQ1 or the type I IFN inducer lenalidomide in growth inhibition of ABC DLBCL both in cell cultures and xenograft mouse models. Therefore, targeting oncogenic EGR1 signaling represents a potential new targeted therapeutic strategy in DLBCL, especially for the more aggressive ABC DLBCL. IMPLICATIONS: The study characterizes EGR1 as a potential oncogene that promotes cell proliferation and defines EGR1 as a new molecular target in DLBCL, the most common non-Hodgkin lymphoma.

Discovery of novel targets for diffuse large B-cell lymphoma

The newly emerging CD19-targeted Chimeric Antigen Receptor T-cells (CAR-T) therapy has revolutionized the treatment of advanced diffuse large B-cell lymphoma (DLBCL) and achieved a functional cure in some patients. However, a majority of patients suffer relapse. A major pathway for tumor relapse is the loss of the target antigen, CD19, on the cancer cell surface, essentially making them invisible to the therapy. Consequently, new targets are required for the continued treatment of relapsed patients. Facing this dilemma, we hypothesized that a systematic search of the human genome may reveal more novel targets of DLBCL with characteristics similar to CD19. These include high expression in DLBCL and other B-cell tumors, but minimal expression in normal tissues. To test this hypothesis, we used several well-annotated public genomic and proteomic databases and carried out in-depth screening to pinpoint optimal new targets for DLBCL. Our study uncovered several new targets that have not been extensively pursued in the public domain, such as CD79A and TNFRSF13C. Based on our refined criteria for target evaluation, we finally concluded that CD79A represents the most ideal novel target for DLBCL and potentially other B-cell tumor types. This target should be explored as the antigen of next-generation CAR-T therapy that has the potential to save and extend the lives of patients.

Targeting Glycosylated Ptgds Displays Anti-Tumor Activities in Diffuse Large B-Cell Lymphoma through Down-Regulation of Wnt Pathway

Introduction: Prostaglandin D2 Synthase (PTGDS), a member of lipocalin superfamily, plays a dual role in catalyzing the conversion of PGH2 to PGD2 and transporting lipophilic substances. PTGDS protein is with different degrees of glycosylation. AT56 is a selective, competitive, and highly bioavailable inhibitor of PTGDS. However, the function and mechanism of PTGDS in diffuse large B-cell lymphoma (DLBCL) remains ill defined. Herein, we aimed to investigate the functional significance of PTGDS and proposed a novel therapeutic strategy for DLBCL. Methods: Lymph node biopsies from 53 de novo DLBCL patients and 28 reactive hyperplasia cases, and peripheral blood mononuclear cells (PBMCs) from healthy volunteers were collected with informed consents. CD19+ B cells were purified by CD19+ magnetic microbeads. The expression levels of PTGDS in DLBCL cell lines and serum were detected by western blotting and ELISA, respectively. Lentivirus vectors were transfected to stably knockdown or overexpress PTGDS. After the treatment of Tunicamycin (Tun), an N-glycosylation inhibitor, western blotting and immunofluorescence were performed to validate the molecular weight and location of PTGDS protein. Results: We first evaluated the expression of PTGDS in DLBCL. Upregulation of PTGDS mRNA in DLBCL cells was identified based on Oncomine database (Fig.1A). Then, the expression level of PTGDS protein in tumor tissue (n=53) was validated to be higher in comparison with control group (Fig.1B). Furthermore, survival analysis revealed that PTGDS high expression was associated with reduced overall survival of DLBCL patients (Fig.1C). Moreover, high level of soluble PTGDS protein was detected in the serum of DLBCL patients (n=53, Fig.1D). High expression of PTGDS was also confirmed in DLBCL cells by western blotting (Fig.2A). The biological function of PTGDS in progression of DLBCL was further verified. Gene ontology and KEGG analysis revealed that PTGDS was enriched in cellular process and biological regulation (Fig.2B). After the treatment with rhPTGDS, increased proliferation of DLBCL cell was observed in a dose-dependent manner (Fig.2D), and overexpression of PTGDS also promoted cell growth (Fig.2E). Furthermore, knockdown of PTGDS (shPTGDS) significantly restrained cell proliferation (Fig.2F), promoted cell cycle arrest (Fig.2G) and cell apoptosis (Fig.2H). AT56 suppressed the proliferation of DLBCL cells in a dose- and time-dependent manner (Fig. 3A). Additionally, with the treatment of AT56, DLBCL cells exhibited induced G0/G1 phase arrest (Fig. 3B) and increased cell apoptosis (Fig. 3C). As Bendamustine and Adriamycin were found to decrease the mRNA level of PTGDS (Fig. 3D), we further observed that AT56 enhanced sensitivity to them in cell proliferation (Fig. 3E) and cell apoptosis (Fig. 3F). Next, we explored the underlying mechanism of PTGDS in DLBCL progression. The expression of Wnt pathway molecules, such as p-LRP6, β-catenin, p-GSK3-β, Lef-1, p-STAT3, were decreased with treatment of AT56 in dose-dependent manner (Fig. 4A). Besides, STAT3 inhibitor WP1066 was found to restore the proliferation promotion (Fig. 4B) caused by PTGDS overexpression. Moreover, we validated that Wnt pathway activator Wnt3a could restore the phenotype changes caused by AT56, including cell proliferation (Fig. 4C), cell apoptosis (Fig. 4D) and cell cycle (Fig. 4E). Glycosylation, a kind of post-translational modification, could significantly alter protein function and then cellular characteristics. Protein Blast analysis indicated the potential glycosylation sites (Fig. 5A) on PTGDS protein (Ser29, Asn51 and Asn78). Furthermore, Tunicamycin was used to inhibit the N-glycosylation of protein and molecular weight of PTGDS changed from 37kD to 21kD (Fig.5B). Besides, the deglycosylation could promote the translocation of PTGDS into the nucleus (Fig.5C-D), indicating the potential role of glycosylated PTGDS in DLBCL. Conclusions : Our investigations identified for the first time the aberrant overexpression of PTGDS in DLBCL, which was associated with poor prognosis. AT56 exerted anti-tumor effect via inhibiting Wnt pathway. Besides, PTGDS protein in DLBCL cells were highly glycosylated and deglycosylation promoted its translocation into nucleus, indicating the mechanism of PTGDS in DLBCL. Further investigation is warranted to substantiate PTGDS as a promising therapeutic target in DLBCL. Disclosures No relevant conflicts of interest to declare.

Systematic Analysis of Competing Endogenous RNA Networks in Diffuse Large B-Cell Lymphoma and Hodgkin's Lymphoma.

Lymphoma is a systemic malignancy, originating from the lymphatic system, which accounts for 3 to 4% of all tumors. There are two major subtypes of lymphoma, namely, diffuse large B-cell lymphoma (DLBCL) and Hodgkin’s lymphoma (HL). Elucidation of the pathogenesis of these two lymphoma types is crucial for the identification of potential therapeutic targets. Compared with the corresponding knowledge of other diseases, the understanding of the regulatory networks involved in DLBCL and HL is relatively deficient. To address this, we comprehensively analyzed the mRNAs, lncRNAs, and miRNAs that were differentially expressed between normal and tumor samples of DLBCL and HL. In addition, functional enrichment analysis of the differentially expressed mRNAs was performed. We constructed two specific ceRNA networks of DLBCL and HL. The pathways enriched by dysregulated mRNAs in DLBCL and HL were mainly involved in immune responses, transcription process, and metabolism process. The ceRNA network analysis revealed that 45 ceRNAs were shared between the two ceRNA networks, including five pivotal lncRNAs (MALAT1, CTBP1-AS, THUMPD3-AS, PSMA3-AS1, and NUTM2A-AS1). In addition, we proposed a DLBCL survival risk model based on a DLBCL-specific network constructed by Lasso regression analysis. The model, which is based on eight mRNAs, exhibited excellent performance in regard to predicting outcomes in DLBCL patients, with a p value of 0.0017 and AUC of 0.9783. In summary, although the molecular mechanisms underlying tumorigenesis in DLBCL and HL were quite different, the same pivotal lncRNAs acted as key regulators. Our findings identify novel potential prognostic and therapeutic targets for DLBCL and HL.

Targeting Polo-like Kinase 4 for Therapeutic Benefit in the Patients with Diffuse Large B-Cell Lymphoma

Abstract: Polo-like kinase 4 (PLK4) is a serine/threonine kinase that plays a critical role in regulating centriole duplication. PLK4 overexpression causes centriole amplification and genomic instability, which is the hallmark of cancer. Thus, PLK4 has been suggested as a tumor promoter role in solid malignancies. CFI-400945 is a highly selective small-molecule inhibitor of PLK4. CFI-400945 treatment results in centriole duplication defects, which leads to mitotic catastrophe and cell death. The antineoplastic effects of CFI-400945 have been reported in many cancers and this inhibitor has entered Phase I clinical trials for solid tumors. However, the role of PLK4 and cellular effects of CFI-40045 in hematological malignancies remains elusive. In the present study, we found that PLK4 expression is up-regulated at both transcriptional and translational levels in diffuse large B-cell lymphoma (DLBCL) cell lines (Fig. 1a, Fig. 1b). DLBCL patients displayed high protein levels of PLK4 as visualized by immunohistochemistry (Fig. 1c). In vitro, treatment of DLBCL cell lines with CFI-400945 induced a decrease in cell proliferation and an increase in cell apoptosis and a block at the G2-M transition in a dose-dependent manner (Fig. 1d, Fig. 1e, Fig. 1f). Furthermore, we found that the antineoplastic effects of CFI-400945 in DLBCL cell lines were due to the increased DNA damage, as observed by increased accumulation of γH2AX through phosphorylation of CHK1/CHK2 (Fig. 1g). Since PLK4 inhibition affects DNA damage, we next aimed to explore the effects of PLK4 expression on sensitivity of DNA damage-inducing drugs (e.g., doxorubicin). We found that silencing of PLK4 via shRNA improve the anti-tumor effects of doxorubicin in DLBCL cell lines (Fig. 1h, Fig. 1i). PLK4 inhibition in combination with doxorubicin significantly increased phosphorylation of H2AX compared to cells treated with doxorubicin alone (Fig. 1j). In summary, our data indicates that PLK4 is aberrantly expressed in DLBCL cell lines and tissues. Targeting PLK4 with the selective inhibitor CFI-400945 suppresses cell proliferation and induces apoptotic deaths and causes DNA damage in vitro. Our findings established PLK4 as a potential therapeutic target in DLBCL. Silencing of PLK4 expression induces accumulation of DNA damage and enhances the cytotoxic effects of doxorubicin. Thus, the levels of PLK4 may serve as a determinant of doxorubicin sensitivity and a predictive biomarker of the patients with DLBCL treated with R-CHOP based therapy. Figure 1 Disclosures No relevant conflicts of interest to declare.

Combination of DPX-Survivac, Low Dose Cyclophosphamide, and Pembrolizumab in Recurrent/Refractory DLBCL: The Spirel Study

Introduction: Recurrent/refractory diffuse large B cell lymphoma (DLBCL) remains an area of unmet clinical need. Although autologous stem cell transplant (ASCT), and more recently chimeric antigen receptor (CAR) T cell therapy, have made a significant impact, many patients are ineligible for these treatments due to advanced age, comorbidities, or financial or geographical limitations, or they have relapsed following these treatments. Survivin, a novel target in DLBCL, is overexpressed in approximately 60% of untreated DLBCL with higher rates in relapsed disease. DPX-Survivac, a T cell activating therapy, elicits a strong and prolonged immune response against tumors expressing survivin. DPX-Survivac is combined with pembrolizumab, an anti-PD-1 checkpoint inhibitor, and intermittent low dose cyclophosphamide (CPA), used for an immunomodulatory effect. Pre-clinical studies have demonstrated increased efficacy of this DPX-based drug combination in controlling of tumor growth in murine tumor models, resulting in improved survival (Weir et al. J Immunother Cancer 2016). Methods: "SPiReL" is a Phase 2 non-randomized, open label, efficacy and safety study. Subjects with recurrent/refractory DLBCL with confirmed survivin expression are eligible for participation. Participants must also be ineligible for curative therapy. Study treatment includes administering two doses of 0.5 mL of DPX-Survivac 3 weeks apart followed by up to six 0.1 mL doses every 8 weeks. Intermittent low dose cyclophosphamide is administered orally at 50 mg twice daily for 7 days followed by 7 days off. Pembrolizumab 200 mg is administered every 3 weeks. Study participants continue active therapy for up to one year or until disease progression, whichever occurs first. The primary objective is to document the response rate to this treatment combination using modified Cheson criteria. Secondary objectives include duration of response and safety. Exploratory endpoints include T cell response, tumor immune cell infiltration, and gene expression analysis. Enrollment is ongoing with a goal of up to 25 subjects in this national, multi-center study. Trial design is shown below the text. Results: At the time of data cut-off, 23 subjects have been screened and 12 have been enrolled. The demographics of enrolled subjects include: median age is 75.5 years (50-82), with 3 male subjects participating. The median number of prior therapies is 2.5 (1-6), with 4 subjects having previously undergone ASCT. The median time from diagnosis to screening is 31 months (8-151). Of the 12 enrolled subjects, 3 were not evaluable for clinical efficacy due to early disease progression. One subject has not yet reached the first time point for assessment. In the Per Protocol analysis of 8 evaluable subjects ("PP", N = 8); as compared to a Full Analysis Set ("FAS", N = 11); 7 of 8 subjects (PP = 87.5%) demonstrated clinical benefit, including 6 with tumor regressions (FAS = 63.6%); 2 subjects (PP = 25%) achieved a complete response, 1 of whom has completed the 1 year study period (FAS = 18.2%); 3 subjects (PP = 37.5%) had a partial response (FAS = 27.3%); and 3 (PP = 37.5%) had stable disease remaining on study for 4, 6 and 8 months (FAS = 27.3%). This treatment combination is well-tolerated with mostly grade 1 and 2 adverse events reported; a single grade 3 maculopapular rash and 1 case of grade 4 cytopenia has been observed. Of the 23 screened subjects, survivin expression analysis was performed on 18 subjects. All of the 18 samples analyzed were survivin positive, with a range of 60%-100% of lymphoma cells expressing survivin. Preliminary analysis of peripheral blood T cell responses to survivin by ELISPOT assay shows 2 of 4 subjects had strong survivin-specific T cell responses. Both of these subjects had clinical responses. Summary: DPX-Survivac, in combination with pembrolizumab and low dose cyclophosphamide, demonstrates clinical activity and a favorable safety profile in recurrent/refractory DLBCL with 87.5% of evaluable subjects deriving clinical benefit, including 2 complete responses (25%) and 3 partial responses (37.5%) to date. Enrollment is continuing to further explore the synergistic effect of this combination in this population. Figure Disclosures Berinstein: Merck: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees. Stewart:Merck: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees. OffLabel Disclosure: Keytruda (pemroblizumab) is approved for use to treat Melanoma, Metastatic, Non-Small Cell Lung Cancer, Head and Neck Cancer, Hodgkin's Lymphoma, Urothelial Carcinoma, Gastric Cancer, Cervical Cancer, Hepatocellular Carcinoma, Merkel Cell Carcinoma, Renal Cell Carcinoma, and Small Cell Lung Cancer. In this trial, it is being used to treat Diffuse Large B-Cell Lymphoma.

Abstract 3436: PRMT5 upregulation and its oncogenic cooperation with PI3K/AKT signaling in diffuse large B cell lymphoma

Abstract Protein arginine methyltransferase PRMT5, which regulates gene expression by symmetric dimethylation of histones and non-histone target proteins, is overexpressed and plays a pathogenic role in many cancers. A growing literature demonstrates a critical role of PRMT5 in tumorigenesis. PRMT5 expression is upregulated in various cancers, including diffuse large B cell lymphoma (DLBCL), the most common, aggressive form of non-Hodgkin lymphoma. PRMT5 upregulation is associated with Epstein-Barr virus (EBV) infection. Given that less than 10% of DLBCL is EBV-positive, the molecular mechanisms of PRMT5 overexpression in DLBCL are still largely unknown. Here, we demonstrated increased expression of PRMT5 in DLBCL cell lines and in a tissue microarray of 104 DLBCL cases when compared with normal naïve B cells. Notably, the level of PRMT5 expression was also elevated in germinal center B cells, which experience antigen stimulation and give rise to DLBCL. In addition, PRMT5 expression in these DLBCL cases correlated with BTK expression, a key component of the B cell antigen receptor (BCR) signaling pathway and an effective drug target in DLBCL. We dissected the mechanisms of differential BCR signaling in regulating PRMT5 expression. Using the CRISPR/Cas9 genome editing technology or by specific pharmacological inhibitors, we discovered that two main BCR downstream signaling pathways, NF-kB and PI3K/AKT, contribute to PRMT5 overexpression. Specifically, the NF-kB member p65 and MYC, a downstream target of the PI3K/AKT signaling pathway, induced PRMT5 transcription. More importantly, inhibition of PRMT5 by its sgRNA or its specific inhibitor was lethal to all DLBCL cell lines tested but not to normal B cells. The antitumor effect of PRMT5 sgRNA or its inhibitor was further revealed in two DLBCL xenograft mouse models as well as in patient derived xenografts (PDX). Further RNA-Seq and biochemical analysis demonstrated that PRMT5 promotes cell cycle progression and activates PI3K-AKT signaling, suggesting a positive feedback regulatory mechanism to enhance cell survival and proliferation. To intervene in the positive feedback regulatory mechanism, we used the PRMT5 inhibitor GSK3326595 and the AKT inhibitor AZD5363, both of which are currently used in clinical trials for solid cancers. Indeed, our in vitro analysis in DLBCL cell lines demonstrated their synergistic cytotoxicity. We will further test the synergism of these two inhibitors in xenograft mouse models as well as in primary cancer cells. Taken together, our study elucidates the mechanisms of PRMT5 upregulation and also demonstrates an oncogenic role for PRMT5 in DLBCL, suggesting targeting PRMT5 by a specific inhibitor or co-targeting with an AKT inhibitor as a potential therapeutic strategy for DLBCL patients. Citation Format: Lixin Rui. PRMT5 upregulation and its oncogenic cooperation with PI3K/AKT signaling in diffuse large B cell lymphoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3436.

Abstract 2055: Differential expression of TP73 isoforms modulates cell survival and therapeutic response in diffuse large B-cell lymphoma

Abstract Rearrangements of the chromosome locus 1p36 with ensuing deletion or disruption of TP73, one of the most distally located putative tumor suppressor genes, is frequent in non-Hodgkin lymphoma (NHL) and confers inferior prognosis. TP73 shares homology to TP53 and is capable of transactivating p53 target genes. Earlier reports in normal B-cells and lymphoma animal models suggest an important role of p73 in B-cell lymphomas, however, the biological significance of p73 isoforms in Diffuse large B-cell lymphoma (DLBCL) is not clear. Diffuse large B-cell lymphoma is the most common form of NHL, and although long-term remission is achieved with current therapies, relapse occurs in almost one-third of the patients. Previously, we have demonstrated that the differential expression of p73 isoforms (TAp73 and ΔNp73) correlate with proliferation and apoptosis in DLBCL. In the present study, we examined whether modulating the expression of these p73 isoforms in DLBCL cells regulates cell survival, proliferation, and response to the chemotherapeutic agent, doxorubicin. We performed in vitro studies in DLBCL cells and modulated the expression of the two opposing p73 isoforms (TAp73 and ΔNp73) using expression vectors and siRNA in DHL16 (GCB subtype) and OCI-Ly3 (ABC subtype) cells. Under normal conditions TAp73-transfected cells showed comparable growth to control vector-transfected cells; however, the TAp73-transfected cells were more susceptible to serum deprivation as well as doxorubicin. In contrast, ΔNp73-transfected cells had accelerated growth when compared with control vector-transfected cells under normal conditions and were more resistant to serum deprivation and doxorubicin treatment. Subsequently, we analyzed the p73 direct transcriptional pro-apoptotic targets in cells transfected with TAp73 and ΔNp73 and vector control. Both the common p53 and p73 targets (PUMA and NOXA) as well as the exclusively p73 targetsGRAMD4 and BIM, were up-regulated in the TAp73-transfected cells compared to control cells. A reduction of PUMA, BIM, and GRAMD4 was observed in the ΔNp73-transfected cells as compared to control cells. Furthermore, we observed that p73 knockdown using p73 siRNA altered the growth and the response to doxorubicin in both DLBCL cell lines. Together, these data demonstrate the significant biological role of p73 isoforms in DLBCL pathogenesis and therapeutic response. Our findings illustrate that modulation of TP73 isoforms induces apoptosis and expression of pro-apoptotic targets, thus establishing p73 as a potential therapeutic target in DLBCL. Citation Format: Hesham M. Hassan, Michelle L. Varney, Sugandha Saxena, Rakesh K. Singh, Bhavana J. Dave. Differential expression of TP73 isoforms modulates cell survival and therapeutic response in diffuse large B-cell lymphoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2055.

Abstract 3041: CD72 as a potential new therapeutic target in diffuse large B-cell lymphoma (DLBCL) and mantle cell lymphoma (MCL)

Abstract Cluster of differentiation 72 (CD72) is a 45 kDa type II transmembrane glycoprotein expressed primarily on the surface of B cells. In a search for a novel lymphoma specific target, public datasets were leveraged to assess mRNA expression. We investigated CD72 mRNA expression in cell lines (data from CCLE), patients (data from TCGA) and normal tissues (data from GTEx). The absence of CD72 in normal tissue and the presence in B cells and non-Hodgkin lymphomas (B-NHL) made CD72 a potential target for the treatment of B-NHL, mainly DLBCL and MCL. CD72 expression on the cell surface was tested by FACS in 15 DLBCL and 5 MCL cell lines. Of these, 12 DLBCL and 4 MCL were positive for CD72, while 3 DLBCL and 1 MCL showed no CD72 expression. In all cell lines tested, the cell surface expression of CD72 correlated with mRNA expression. To evaluate the potential of CD72 as an ADC target, 4 cell lines were treated in vitro with three commercial monoclonal anti-CD72 primary antibodies (clones J4-117, 3F3 and BU40), and MMAF-conjugated secondary antibody. The cell lines used were RL (DLBCL, CD72+), SCC-3 (DLBCL, CD72-), Jeko-1 and Mino (both MCL, CD72+). The cell growth inhibition after 96 hrs of treatment for for all control conditions; anti-CD72 primary antibody, IgG isotype, MMAF secondary antibody alone or combined with IgG isotype; tested in all cell lines, CD72+ or CD72- ranged from -3 to +4%. Conversely, anti-CD72 primary antibodies plus MMAF secondary antibody had marked growth inhibition effect ranged from 24 to 80% on the three CD72+ cell lines and no effect on cell growth in CD72- cell line, suggesting internalization of CD72 and growth inhibition dependent on the presence of CD72 on the cell surface. This is the first study demonstrating a potential role for CD72 as a novel target for DLBCL and MCL. These data suggest that CD72 may be a candidate for an ADC approach in DLBCL and MCL. Cell line% growth inhibition anti-CD72 + MMAF 2°Anti-CD72 clone J4-117Anti-CD72 clone 3F3Anti-CD72 clone BU40SCC-3 (CD72-)-31-5RL (CD72+)503330Jeko-1 (CD72+)433024Mino (CD72+)778066 Citation Format: Erika Von Euw, Eileen Taschereau, Da Ming Ou, Monica Mead, Sarah Larson, Dennis Slamon. CD72 as a potential new therapeutic target in diffuse large B-cell lymphoma (DLBCL) and mantle cell lymphoma (MCL) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3041.