Primary Diffuse Large B-cell Lymphoma Samples Research Articles

Blocking the CD47-Sirpa Axis Enhances Tafasitamab-Mediated Phagocytosis

Introduction Around 40% of diffuse large B cell lymphoma (DLBCL) patients have refractory disease or experience relapse, thus highlighting the need for novel, more effective therapies (Coiffier et al). The immunotherapy Tafasitamab (MOR208) is an Fc-modified, humanized, monoclonal anti-CD19 antibody approved in combination with lenalidomide as a second line of therapy for R/R DLBCL. Tafasitamab mediates its therapeutic effect directly or via antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP). Macrophages represent a key component of the DLBCL microenvironment and are critical effectors of antibody therapies (Uchida et al). However, ADCP is reportedly counteracted by increased expression of the immune checkpoint molecule CD47 on lymphoma targets (Chao et al). In addition, the inhibitory receptor of CD47, signal regulatory protein alpha (SIRPα) is increased on macrophages from patients with follicular lymphoma who relapse or progress after frontline lenalidomide and rituximab (Marques-Piubelli et al). Aim of the study The current study investigates the impact of the CD47-SIRPα axis on tafasitamab-mediated phagocytosis and the potential of concomitant treatment with an anti-CD47 blocking antibody to improve its efficacy. Results We show that both, CD47 and its inhibitory receptor SIRPa were elevated in lymph node biopsies from DLBCL patients. CD47 expression was evaluated in 6 DLBCL patients and 5 healthy controls and SIRPα was evaluated in 9 patients and 7 controls. In fact, increased expression of SIRPa on macrophages correlated with decreased ADCP activity of tafasitamab, and CRISPR-mediated CD47 overexpression on lymphoma targets impaired tafasitamab-mediated phagocytosis, in vitro. Combination of tafasitamab and an anti-CD47 blocking antibody enhanced ADCP activity of in vitro generated macrophages. Furthermore, lymphoma cells with low CD19 expression were efficiently eliminated by the combination therapy. Importantly, the combination enhanced tafasitamab-mediated phagocytosis of primary DLBCL cells using primary, patient-derived lymphoma-associated macrophages (LAMs) as effectors, in an autologous setting. Conclusion Our results show increased expression of CD47-SIRPa axis in primary DLBCL samples and indicate that CD47 blockade can enhance the phagocytic potential of tumor-targeting immunotherapies such as tafasitamab. Our findings suggest that there is merit in exploring the clinical utility of combining tafasitamab with an anti-CD47 blocking antibody in lymphoma. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Fatty acid synthase and adenosine monophosphate-activated protein kinase regulate cell survival and drug sensitivity in diffuse large B-cell lymphoma

Fatty acid synthesis is crucial in supporting the survival and proliferation of multiple forms of cancer. The high metabolic demands of fatty acid synthesis are regulated by the AMP-activated kinase and activity of the fatty acid synthase enzyme. In this study, the roles of these enzymes in diffuse large B-cell lymphoma (DLBCL) were investigated by genetic knock-down and pharmacological activation of AMP-activated kinase by metformin, and selective inhibition of fatty acid synthase using the novel drug Fasnall. We observed distinct heterogeneity and adaptive plasticity of lipid metabolism in a panel of DLBCL cell lines and demonstrate the therapeutic potential of inhibiting fatty acid synthesis in a subset of DLBCL cells. The translational relevance of these in vitro data is supported by the strong correlation between AMP-activated protein kinase expression in primary DLBCL samples and disease relapse. Inhibition of fatty acid synthase with Fasnall may represent a therapeutic option for DLBCL that preferentially subverts to de novo fatty acid synthesis.

High frequency of inactivating tetraspanin CD37 mutations in diffuse large B-cell lymphoma at immune-privileged sites

AbstractTetraspanin CD37 is predominantly expressed on the cell surface of mature B lymphocytes and is currently being studied as novel therapeutic target for B-cell lymphoma. Recently, we demonstrated that loss of CD37 induces spontaneous B-cell lymphoma in Cd37-knockout mice and correlates with inferior survival in patients with diffuse large B-cell lymphoma (DLBCL). Here, CD37 mutation analysis was performed in a cohort of 137 primary DLBCL samples, including 44 primary immune-privileged site-associated DLBCL (IP-DLBCL) samples originating in the testis or central nervous system. CD37 mutations were exclusively identified in IP-DLBCL cases (10/44, 23%) but absent in non-IP-DLBCL cases. The aberrations included 10 missense mutations, 1 deletion, and 3 splice-site CD37 mutations. Modeling and functional analysis of CD37 missense mutations revealed loss of function by impaired CD37 protein expression at the plasma membrane of human lymphoma B cells. This study provides novel insight into the molecular pathogenesis of IP-DLBCL and indicates that anti-CD37 therapies will be more beneficial for DLBCL patients without CD37 mutations.

Screening of key genes associated with R‑CHOP immunochemotherapy and construction of a prognostic risk model in diffuse large B‑cell lymphoma

Diffuse large B-cell lymphoma (DLBCL) is a common subtype of non-Hodgkin lymphoma, which is curable in the majority of patients treated with rituximab plus cyclophosphamide, doxorubicin, vincristine and prednisone (R-CHOP) immunochemotherapy. However, the therapeutic mechanism of R-CHOP has not been elucidated. The GSE32918 and GSE57611 datasets were retrieved from The Gene Expression Omnibus database. The differentially expressed genes (DEGs) associated with R-CHOP therapy were identified using limma. Combined with prognostic information in GSE32918, DEGs found to be significantly associated with prognosis were selected using univariate Cox regression analysis and a risk prediction model was constructed. Based on this model, the samples in the training set (GSE32918) were divided into high and low risk score groups according to the median risk score. A total of 801 DEGs were identified between the R-CHOP treated DLBCL and primary DLBCL samples, from this 116 prognosis-associated genes were selected. Using Cox proportional hazards model, an optimal combination of 12 genes [including calcium/calmodulin dependent protein kinase I (CAMK1), hippocalcin like 4 (HPCAL4) and ephrin A5 (EFNA5)] was selected, and the sample risk score prediction model was constructed and validated. The DEGs between high risk score and low risk score groups were significantly enriched in functions associated with ‘response to DNA damage stimulus’, and pathways including ‘cytokine-cytokine receptor interaction’ and ‘cell cycle’. The optimal combination of the 12 genes, including CAMK1, HPCAL4 and EFNA5, was found to be useful in predicting the prognosis of patients with DLBCL after R-CHOP treatment. Therefore, these genes may be affected by R-CHOP in DLBCL.

SHP1 Deficiency Is Responsible for the Constitutive Activation of the BCR Pathway in GCB DLBCL

Functional and transcriptional profiling studies have identified a subset of diffuse large B cell lymphoma (DLBCL) tumors that rely on B cell receptor (BCR) signals for proliferation and survival. These BCR-dependent tumors have been identified among both the ABC and GCB DLBCL subtype. However, differences have been observed between the two subtypes in terms of the mechanism of BCR pathway activation and activity of downstream signaling molecules. BCR-dependent ABC DLBCL tumors are characterized by high basal NF-kB and PI3K/AKT activity and the presence of gain-of-function mutations in the CD79A or CD79B subunit of the BCR that are responsible for the chronic activation of the BCR pathway. In contrast, BCR-dependent GCB DLBCL tumors have low baseline NF-kB activity and lack CD79A/CD79B mutations, suggesting a different mechanism of BCR pathway activation. In this study, we investigated whether activation of the BCR pathway in GCB DLBCL is potentially caused by deficiency of the phosphatase SHP1, which is an important negative regulator of the BCR pathway and has been reported to be downregulated in approximately 40% of primary DLBCL tumors. For this purpose, we first correlated SHP1 expression with the presence of phosphorylated SYK and BLNK in the GCB DLBCL cell lines OCI-Ly1, OCI-Ly7, OCI-Ly18, SU-DHL-4, SU-DHL-6, WSU-NHL, SU-DHL-8, Toledo, BJAB, OCI-Ly19, OCI-Ly4, and Farage. Immunoblotting analysis revealed that SHP1 is expressed in SU-DHL-8, Toledo, BJAB, OCI-Ly19, OCI-Ly4, and Farage but not in the remaining 6 cell lines. Expression of SHP1 inversely correlated with expression of phosphorylated SYK (pSYK-Y352) and BLNK (pBLNK-Y84), suggesting that these two BCR-proximal signaling molecules are activated because of SHP1 deficiency. To further evaluate this possibility, we re-expressed SHP1 in OCI-Ly1 and SU-DHL-4 cells by transient transfection with a plasmid or a lentiviral SHP1 expression vector. Both approaches resulted in a substantial reduction of pSYK-Y352 and pBLNK-Y84 levels, confirming that SHP1 deficiency leads to activation of the BCR pathway.To determine whether activation of the BCR pathway contributes to tumor cell survival, we investigated the effects of the SYK inhibitor R406 (active substance of the drug fostamatinib) on the viability of the 6 SHP1-positive and 6 SHP1-negative GCB DLBCL cell lines. At concentrations up to 2 μM, R406 displayed only minimal toxicity against each of the SHP1-positive and SHP1-negative GCB DLBCL cell lines (<20% apoptotic cells), with the exception of WSU-NHL (60% apoptotic cells). However, analysis of the effects of R406 on the expression of BCL-2 family proteins showed downregulation of the antiapoptotic protein MCL-1 and upregulation of the proapoptotic proteins BIM and HRK in all of the SHP1-negative but in none of the SHP1-positive GCB DLBCL cell lines. Such changes were also observed following siRNA-mediated knockdown of SYK or overexpression of SHP1, further suggesting that they are on-target effects of R406. In addition, treatment with the BTK inhibitor ibrutinib or siRNA-mediated knockdown of BTK also resulted in downregulation of MCL-1 and upregulation of BIM in these cell lines. Similar changes were observed in 3 of the 5 investigated primary DLBCL samples following treatment with ibrutinib or fostamatinib. Importantly, downregulation of MCL-1 by RNA interference or upregulation of BIM by mRNA transfection resulted in substantially increased sensitivity of these cell lines to the BCL-2 inhibitor venetoclax, suggesting that SYK or BTK inhibitors could potentiate the cytotoxic effects of this drug. To further evaluate this possibility, we calculated the combination index of the R406 + venetoclax or ibrutinib + venetoclax drug combination in the 12 GCB DLBCL cell lines. Significant synergistic activity was seen in all five BCL-2-positive/SHP1-negative GCB DLBCL cell lines with the R406 + venetoclax combination and in four of them with the ibrutinib + venetoclax combination. In contrast, synergistic activity was detected in only one of the 6 SHP1-positive GCB DLBCL cell lines and was not seen in the BCL-2-negative/SHP1-negative cell line OCI-Ly7. Collectively, these findings suggest that SHP1 deficiency is responsible for the constitutive activation of the BCR pathway in GCB DLBCL and identify SHP1 and BCL-2 as potential predictive markers for response to treatment with a venetoclax/BCR inhibitor combination. DisclosuresNo relevant conflicts of interest to declare.

A Novel Method to Identify Epigenetic Subclones with Increased Fitness from Genomic DNA Methylation Data of Lymphoma and Leukemia Patients

DNA methylation has been functionally implicated in X-inactivation, genomic imprinting, and silencing of transposable elements. DNA methylation also has a complex regulatory relationship with gene expression. Canonically, methylation around the promoters of tumor-suppressor genes induces gene-silencing, thereby representing a hit in the two-hit hypothesis for the development of cancer. Unfortunately, profiling studies conducted to determine how aberrant methylation may contribute to cancer progression are confounded by heterogeneity in the original clinical sample. Thus, though studies in patients with Acute Myeloid Leukemia (AML), Diffuse Large B-cell Lymphoma (DLBCL), and Chronic Lymphocytic Leukemia (CLL) have found that variation in detected methylation values from patients at diagnosis correlates with prognosis following therapy, they do not address which subclonal methylation events contribute to cancer progression.To address this concern, we developed a novel computational method to deconvolve the bisulfite sequencing data from a sample into its major methylation profiles and their respective prevalence in the sample. Our method, based on a modified Hidden Markov Model, effectively models the autocorrelations found in methylation data and outperforms existing algorithms. Our method was validated across a wide range of mixture simulations, where bisulfite sequencing reads from various different cell types were subsampled to form test samples that could be deconvolved. We were able to accurately (98%) distinguish distinct methylation patterns corresponding to the expected underlying subpopulations, such as for CD14 and CD22 in mixtures of germinal center B-cells and monocytes and for CD4 and CD8A in mixtures of CD4+ T-lymphocytes and CD8+ T-lymphocytes. These patterns also recapitulated differentially methylated regions (DMRs) identified by an independent DMR-caller.Given that our method does not rely on cell-type specific parameters and is therefore robust to all samples, to further validate and demonstrate the applicability of our method, we conducted Agilent Methyl-Seq on 5 primary DLBCL samples procured by the Lymphoma Core at the Siteman Cancer Center. As a positive control, our method identified differential methylation profiles at loci expected to differ from underlying CD19+ and CD4+ cells, which comprise a large majority of each sample. Our method also identified distinct methylation profiles not found in reference profiles from normal cell-types, suggesting these methylation profiles may be specific to DLBCL. To further validate these findings, we used single-cell bisulfite-sequencing at ten loci to demonstrate that the methylation profiles predicted by our method from the original sample are found in individual cells. We found several methylation patterns that only existed in a subset of CD19+ cells, which may represent distinct epigenetic subclones of DLBCL.Using our novel computational method, we next profiled the subclonal epigenetic architecture of publicly available (dbGaP) paired samples from patients with AML (n=137) at diagnosis and following therapy. We were able to not only identify subclonal methylation profiles that were specific to cancer but also find profiles at higher prevalence in patients at relapse compared to diagnosis. These methylation profiles, which were enriched for genes in cancer pathways as seen by Gene Set Enrichment Analysis, may confer fitness advantages for a cancer subclone to expand. We are currently conducting additional analyses to characterize the epigenetic regulatory circuits that contribute to our observed increase in subclonal fitness. In summary, we have developed a robust method to identify subclonal methylation changes that may contribute to cancer progression and prognosis, as seen in AML, and may lead to new avenues for improving treatment for patients with leukemia or lymphoma. DisclosuresNo relevant conflicts of interest to declare.

Preclinical efficacy and biological effects of the oral proteasome inhibitor ixazomib in diffuse large B-cell lymphoma

Despite advances in deciphering the molecular pathogenesis of diffuse large B-cell lymphoma (DLBCL), patients with relapsed/refractory disease, particularly those with adverse genetic features (e.g., mutated p53 or double hit lymphoma (DHL)) have very poor prognoses, and effective therapies are lacking. In this study we examined the preclinical efficacy and associated biological effects of the first oral proteasome inhibitor, ixazomib, in DLBCL in vitro and in vivo models. We demonstrated that ixazomib exhibited anti-tumor activities in 28 representative DLBCL cell lines, 10 primary DLBCL samples, and a DHL xenotransplant mouse model, at clinically achievable drug concentrations. Ixazomib sensitivity in DLBCL cells is correlated with immunoproteasomal activity; stimulating lymphoma cells with interferon gamma induced immunoproteasome activity and sensitized these cells to ixazomib. In addition, we showed that ixazomib induces apoptosis and the DNA damage response pathway, through activation of the checkpoint kinase 2 (CHK2). Hence, pharmacological inhibition of CHK2 enhances the anti-tumor activity of ixazomib in DLBCL cells. Our results indicate that ixazomib is an effective proteasome inhibitor active in DLBCL, including DHL, and its combination with a CHK2 inhibitor offers a potentially more robust therapeutic regimen for treatment-resistant DLBCL.

Comparative analysis of primary versus relapse/refractory DLBCL identifies shifts in mutation spectrum.

Current understanding of the mutation spectrum of relapsed/refractory (RR) tumors is limited. We performed whole exome sequencing (WES) on 47 diffuse large B cell lymphoma (DLBCL) tumors that persisted after R-CHOP treatment, 8 matched to primary biopsies. We compared genomic alterations from the RR cohort against two treatment-naïve DLBCL cohorts (n=112). While the overall number and types of mutations did not differ significantly, we identified frequency changes in DLBCL driver genes. The overall frequency of MYD88 mutant samples increased (12% to 19%), but we noted a decrease in p.L265P (8% to 4%) and increase in p.S219C mutations (2% to 6%). CARD11 p.D230N, PIM1 p.K115N and CD79B p.Y196C mutations were not observed in the RR cohort, although these mutations were prominent in the primary DLBCL samples. We observed an increase in BCL2 mutations (21% to 38% of samples), BCL2 amplifications (3% to 6% of samples) and CREBBP mutations (31% to 42% of samples) in the RR cohort, supported by acquisition of mutations in these genes in relapsed compared to diagnostic biopsies from the same patient. These increases may reflect the genetic characteristics of R-CHOP RR tumors expected to be enriched for during clinical trial enrollment. These findings hold significance for a number of emerging targeted therapies aligned to genetic targets and biomarkers in DLBCL, reinforcing the importance of time-of-treatment biomarker screening during DLBCL therapy selection.

MYC Protein-positive Diffuse Large B-Cell Lymphoma Features an Activated B-Cell Receptor Signal Pathway.

Components of the B-cell receptor (BCR) signaling pathway represent promising therapeutic targets in diffuse large B-cell lymphoma (DLBCL) and other B-cell malignancies. MYC, a transcriptional factor and oncoprotein, is overexpressed in a fraction of DLBCL and indicates poor prognosis and aggressive clinical course when treated with rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP). However, BCR signaling status in MYC-positive DLBCL cases and the potential efficacy of BCR signal inhibitors in treating this aggressive disease are unknown. To further elucidate the BCR signaling pathway in MYC-positive DLBCL, we analyzed the levels of BCR-associated genes according to MYC gene status, detected phosphorylated protein with primary DLBCL samples, and estimated the patient survival with MYC expression. In addition, we manipulated MYC gene expression and tested its effects on BCR signaling in vitro. We found that CD19, SYK, and BLK were highly expressed in DLBCL with MYC gene overexpression. MYC-positive DLBCL had higher levels of pSYK and pBLK, but only pSYK level correlated with patient survival. The in vitro studies demonstrated that overexpression of the MYC gene augmented BCR signaling, whereas MYC gene knockdown attenuated BCR signaling. Thus, MYC protein-positive DLBCL features highly activated BCR signaling and may represent a potential candidate for BCR inhibitor therapy.

FOXP2-positive diffuse large B-cell lymphomas exhibit a poor response to R-CHOP therapy and distinct biological signatures.

FOXP2 shares partially overlapping normal tissue expression and functionality with FOXP1; an established diffuse large B-cell lymphoma (DLBCL) oncogene and marker of poor prognosis. FOXP2 is expressed in the plasma cell malignancy multiple myeloma but has not been studied in DLBCL, where a poor prognosis activated B-cell (ABC)-like subtype display partially blocked plasma cell differentiation. FOXP2 protein expression was detected in ABC-DLBCL cell lines, and in primary DLBCL samples tumoral FOXP2 protein expression was detected in both germinal center B-cell-like (GCB) and non-GCB DLBCL. In biopsies from DLBCL patients treated with immunochemotherapy (R-CHOP), ≥ 20% nuclear tumoral FOXP2-positivity (n = 24/158) correlated with significantly inferior overall survival (OS: P = 0.0017) and progression-free survival (PFS: P = 0.0096). This remained significant in multivariate analysis against either the international prognostic index score or the non-GCB DLBCL phenotype (P < 0.05 for both OS and PFS). Expression of BLIMP1, a marker of plasmacytic differentiation that is commonly inactivated in ABC-DLBCL, did not correlate with patient outcome or FOXP2 expression in this series. Increased frequency of FOXP2 expression significantly correlated with FOXP1-positivity (P = 0.0187), and FOXP1 co-immunoprecipitated FOXP2 from ABC-DLBCL cells indicating that these proteins can co-localize in a multi-protein complex. FOXP2-positive DLBCL had reduced expression of HIP1R (P = 0.0348), which is directly repressed by FOXP1, and exhibited distinct patterns of gene expression. Specifically in ABC-DLBCL these were associated with lower expression of immune response and T-cell receptor signaling pathways. Further studies are warranted to investigate the potential functional cooperativity between FOXP1 and FOXP2 in repressing immune responses during the pathogenesis of high-risk DLBCL.

Targeting of BCL2 Family Proteins with ABT-199 and Homoharringtonine Reveals BCL2- and MCL1-Dependent Subgroups of Diffuse Large B-Cell Lymphoma.

To investigate the roles of BCL2, MCL1, and BCL-XL in the survival of diffuse large B-cell lymphoma (DLBCL). Immunohistochemical analysis of 105 primary DLBCL samples, and Western blot analysis of 18 DLBCL cell lines for the expression of BCL2, MCL1, and BCL-XL. Pharmacologic targeting of BCL2, MCL1, and BCL-XL with ABT-199, homoharringtonine (HHT), and ABT-737. Analysis of DLBCL clones with manipulated expressions of BCL2, MCL1, and BCL-XL. Immunoprecipitation of MCL1 complexes in selected DLBCL cell lines. Experimental therapy aimed at inhibition of BCL2 and MCL1 using ABT-199 and HHT, single agent, or in combination, in vitro and in vivo on primary cell-based murine xenograft models of DLBCL. By the pharmacologic targeting of BCL2, MCL1, and BCL-XL, we demonstrated that DLBCL can be divided into BCL2-dependent and MCL1-dependent subgroups with a less pronounced role left for BCL-XL. Derived DLBCL clones with manipulated expressions of BCL2, MCL1, and BCL-XL, as well as the immunoprecipitation experiments, which analyzed MCL1 protein complexes, confirmed these findings at the molecular level. We demonstrated that concurrent inhibition of BCL2 and MCL1 with ABT-199 and HHT induced significant synthetic lethality in most BCL2-expressing DLBCL cell lines. The marked cytotoxic synergy between ABT-199 and HHT was also confirmed in vivo using primary cell-based murine xenograft models of DLBCL. As homoharringtonine is a clinically approved antileukemia drug, and ABT-199 is in advanced phases of diverse clinical trials, our data might have direct implications for novel concepts of early clinical trials in patients with aggressive DLBCL.

VDJ-Seq: Deep Sequencing Analysis of Rearranged Immunoglobulin Heavy Chain Gene to Reveal Clonal Evolution Patterns of B Cell Lymphoma.

Understanding tumor clonality is critical to understanding the mechanisms involved in tumorigenesis and disease progression. In addition, understanding the clonal composition changes that occur within a tumor in response to certain micro-environment or treatments may lead to the design of more sophisticated and effective approaches to eradicate tumor cells. However, tracking tumor clonal sub-populations has been challenging due to the lack of distinguishable markers. To address this problem, a VDJ-seq protocol was created to trace the clonal evolution patterns of diffuse large B cell lymphoma (DLBCL) relapse by exploiting VDJ recombination and somatic hypermutation (SHM), two unique features of B cell lymphomas. In this protocol, Next-Generation sequencing (NGS) libraries with indexing potential were constructed from amplified rearranged immunoglobulin heavy chain (IgH) VDJ region from pairs of primary diagnosis and relapse DLBCL samples. On average more than half million VDJ sequences per sample were obtained after sequencing, which contain both VDJ rearrangement and SHM information. In addition, customized bioinformatics pipelines were developed to fully utilize sequence information for the characterization of IgH-VDJ repertoire within these samples. Furthermore, the pipeline allows the reconstruction and comparison of the clonal architecture of individual tumors, which enables the examination of the clonal heterogeneity within the diagnosis tumors and deduction of clonal evolution patterns between diagnosis and relapse tumor pairs. When applying this analysis to several diagnosis-relapse pairs, we uncovered key evidence that multiple distinctive tumor evolutionary patterns could lead to DLBCL relapse. Additionally, this approach can be expanded into other clinical aspects, such as identification of minimal residual disease, monitoring relapse progress and treatment response, and investigation of immune repertoires in non-lymphoma contexts.

Transcriptome sequencing reveals thousands of novel long non-coding RNAs in B cell lymphoma.

BackgroundGene profiling of diffuse large B cell lymphoma (DLBCL) has revealed broad gene expression deregulation compared to normal B cells. While many studies have interrogated well known and annotated genes in DLBCL, none have yet performed a systematic analysis to uncover novel unannotated long non-coding RNAs (lncRNA) in DLBCL. In this study we sought to uncover these lncRNAs by examining RNA-seq data from primary DLBCL tumors and performed supporting analysis to identify potential role of these lncRNAs in DLBCL.MethodsWe performed a systematic analysis of novel lncRNAs from the poly-adenylated transcriptome of 116 primary DLBCL samples. RNA-seq data were processed using de novo transcript assembly pipeline to discover novel lncRNAs in DLBCL. Systematic functional, mutational, cross-species, and co-expression analyses using numerous bioinformatics tools and statistical analysis were performed to characterize these novel lncRNAs.ResultsWe identified 2,632 novel, multi-exonic lncRNAs expressed in more than one tumor, two-thirds of which are not expressed in normal B cells. Long read single molecule sequencing supports the splicing structure of many of these lncRNAs. More than one-third of novel lncRNAs are differentially expressed between the two major DLBCL subtypes, ABC and GCB. Novel lncRNAs are enriched at DLBCL super-enhancers, with a fraction of them conserved between human and dog lymphomas. We see transposable elements (TE) overlap in the exonic regions; particularly significant in the last exon of the novel lncRNAs suggest potential usage of cryptic TE polyadenylation signals. We identified highly co-expressed protein coding genes for at least 88 % of the novel lncRNAs. Functional enrichment analysis of co-expressed genes predicts a potential function for about half of novel lncRNAs. Finally, systematic structural analysis of candidate point mutations (SNVs) suggests that such mutations frequently stabilize lncRNA structures instead of destabilizing them.ConclusionsDiscovery of these 2,632 novel lncRNAs in DLBCL significantly expands the lymphoma transcriptome and our analysis identifies potential roles of these lncRNAs in lymphomagenesis and/or tumor maintenance. For further studies, these novel lncRNAs also provide an abundant source of new targets for antisense oligonucleotide pharmacology, including shared targets between human and dog lymphomas.Electronic supplementary materialThe online version of this article (doi:10.1186/s13073-015-0230-7) contains supplementary material, which is available to authorized users.

MCL1 Targeting Agent Homoharringtonine Exerts Strong Cytotoxicity Towards Diffuse Large B-Cell Lymphoma (DLBCL) Cells and Synergizes with BCL2 Targeting Agent ABT199 in Eliminating BCL2-Positive DLBCL Cells

Introduction:Diffuse large B-cell lymphoma (DLBCL) represents the most prevalent type of B-cell non-Hodgkin lymphomas (B-NHL) in the Western hemisphere. While BCL2 gene deregulation was repeatedly associated with poor prognosis, the role of MCL1 in the biology of DLBCL remains largely unknown. ABT199 is a highly-selective inhibitor of BCL2 protein currently evaluated in clinical trials. Homoharringtonine (HHT) is a plant alkaloid and as a semisynthetic compound (omacetaxine) it was approved for the treatment of relapsed chronic myelogenous leukemia (CML). Anti-tumor activity of HHT includes downregulation of the anti-apoptotic protein MCL1.Aim:The aim of the project was to evaluate the preclinical anti-lymphoma efficacy of BCL2 and MCL1-targeting agents ABT199 and HHT in DLBCL.Methods:Immunophenotype of primary DLBCL samples was determined by immunohistochemistry (IHC) using the Hans algorithm. Sensitivity of DLBCL cell lines to ABT199 and HHT was determined by Annexin V-based apoptotic assay and WST8-based cell proliferation assay. DLBCL clones with downregulation of selected anti-apoptotic proteins were derived using pLKO1-based lentiviral particles containing shRNAs against BCL2, BCL-XL and MCL1. For upregulation, BCL2, BCL-XL and MCL1 were cloned in the lentiviral expression vector pCDHNeo and the prepared lentiviral particles were used for the transduction of DLBCL cell lines.Results:We analyzed molecular mechanisms of cytotoxic activity of HHT in 7 DLBCL cell lines, and confirmed decreased expression of MCL1 protein in all cases. By semi-quantitative protein expression analysis (western blot or IHC) we demonstrated that BCL-XL and MCL1 were detectable in all DLBCL cell lines (n=18) and primary samples (n=114, GCB=51, ABC=63), while BCL2 was not detectable in 6 out of 18 DLBCL cell lines and 32 out of 114 primary DLBCL samples. 8 out of 12 BCL2-positive DLBCL cell lines were sensitive to 1 microM ABT199 (i.e. did not survive 1 microM ABT199 by standard proliferation assay). In contrary, 6 out of 6 BCL2-negative DLBCL cell lines were resistant to 1 microM ABT199. 11 out of 12 BCL2-positive DLBCL cell lines were sensitive to 30 nM HHT (considered a steady-state plasma level in CML patients treated with HHT). 5 out of 6 BCL2-negative DLBCL cell lines were sensitive to 30 nM HHT. Significant drug synergism between ≤1 microM ABT199 and ≤ 30 nM HHT was observed in 8 out of 12 BCL2-positive, but only in 1 out of 6 BCL2-negative DLBCL cell lines.We demonstrated that high expression of BCL2 positively correlated with sensitivity to ABT-199, irrespective of expression levels of BCL-XL and MCL1. Expression levels of BCL2 and BCL-XL negatively correlated with sensitivity to HHT. Expression level of MCL1 did not correlate with sensitivity to HHT.Both targeted downregulation and transgenic overexpression of BCL-XL in selected DLBCL cell lines confirmed that the expression of BCL-XL negatively correlates with sensitivity to HHT (but not to ABT199). While increase in sensitivity to HHT was observed in 3 out of 3 DLBCL cell lines with targeted knock-down of BCL2, increase in sensitivity to ABT199 was observed only in 1 out of these 3 DLBCL cell lines. Targeted knockdown of MCL1 was associated with increased sensitivity to HHT in 1 out of 2 DLBCL cell lines, but with no change of sensitivity to ABT199.Conclusions:HHT is a promising anti-DLBCL agent in both BCL2-positive and BCL2-negative cases. ABT199, as a single-agent or in combination with HHT, effectively eliminates BCL2-positive DLBCL cells. Based on the observed data two biological categories of DLBCL might be assumed: BCL2-dependent (ABT199-sensitive, HHT-sensitive) and MCL1-dependent (ABT199-resistant, HHT-sensitive) DLBCL.Grant support: IGA-MZ: NT13201-4/2012, GACR14-19590S, UNCE 204021, SVV-2013-266509, PRVOUK P24/LF1/3, GA-UK 1270214 DisclosuresNo relevant conflicts of interest to declare.

Dysregulation of autophagy in human follicular lymphoma is independent of overexpression of BCL-2

Overexpression of the anti-apoptotic protein BCL-2 is characteristic of human follicular lymphoma (FL) and some cases of diffuse large B cell lymphoma (DLBCL). We aimed to determine autophagy status in primary FL and DLBCL samples and the BCL-2+/BCL-2- lymphoma cell lines using both autophagy PCR array and tissue microarray (TMA). A greater number of autophagy machinery genes were up-regulated in the BCL-2+ Su-DHL4 cell line compared with BCL-2- Su-DHL8 cells, at both the basal level and in response to autophagic stress. The autophagy-related gene expression profiles were determined in purified and unpurified malignant human lymph node biopsies. Seven autophagy machinery genes were up-regulated in purified FL B-cells compared with reactive B-cells. Only 2 autophagy machinery genes were up-regulated in DLBCL B-cells. In unpurified tissue biopsies, 20 of 46 genes in FL and 2 of 5 genes in DLBCL with increased expression were autophagy machinery genes. Expression of autophagy substrates p62 and LC3 were determined by TMAs. FL samples showed significantly decreased levels of both p62 and LC3 compared with reactive and DLBCL, indicative of an increased autophagy activity in FL. In summary, these results demonstrate that FL showed increased basal autophagy activity, regardless of overexpression of BCL-2 in this disease.

A microRNA-mediated regulatory loop modulates NOTCH and MYC oncogenic signals in B- and T-cell malignancies.

Growing evidence suggests that microRNAs facilitate the cross-talk between transcriptional modules and signal transduction pathways. MYC and NOTCH1 contribute to the pathogenesis of lymphoid malignancies. NOTCH induces MYC, connecting two signaling programs that enhance oncogenicity. Here we show that this relationship is bidirectional and that MYC, via a microRNA intermediary, modulates NOTCH. MicroRNA-30a, a member of family of microRNAs that are transcriptionally suppressed by MYC, directly binds to and inhibits NOTCH1 and NOTCH2 expression. Using a murine model and genetically modified human cell lines, we confirmed that microRNA-30a influences NOTCH expression in a MYC-dependent fashion. In turn, through genetic modulation, we demonstrated that intracellular NOTCH1 and NOTCH2, by inducing MYC, suppressed microRNA-30a. Conversely, pharmacological inhibition of NOTCH decreased MYC expression, and ultimately de-repressedmicroRNA-30a. Examination of genetic models of gain and loss of microRNA-30a in diffuse large B-cell lymphoma (DLBCL) and T-acute lymphoblastic leukemia (T-ALL) cells suggested a tumor suppressive role for this microRNA. Finally, the activity of the microRNA-30a-NOTCH-MYC loop was validated in primary DLBCL and T-ALL samples. These data define the presence of a microRNA-mediated regulatory circuitry that may modulate the oncogenic signals originating from NOTCH and MYC.

Low expression of microRNA-146b-5p and microRNA-320d predicts poor outcome of large B-cell lymphoma treated with cyclophosphamide, doxorubicin, vincristine, and prednisone.

Although diffuse large B-cell lymphoma (DLBCL) encompasses a biologically and clinically diverse set of diseases, increasing evidence has pointed to an important role of microRNAs (miRs) in the pathogenesis of DLBCL. We report here that low expression of miR-146b-5p and miR-320d is associated with poor prognosis of DLBCL patients treated with the standard cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP) regimen and that this is related to the inhibitory effect of these miRs on DLBCL cell proliferation. Analysis of a retrospective cohort of 106 primary nodal DLBCL samples from patients who were treated with CHOP showed that, when the median survival period (40.8 months) was used as the cutoff point, miR-146b-5p and miR-320d were expressed at lower levels in DLBCLs with poor prognosis. Indeed, whereas low expression of miR-146b-5p was correlated with reduced progression-free survival, low expression of miR-320d was associated with decreases in both progression-free survival and overall survival. Moreover, miR-146b-5p and miR-320d were expressed at significantly lower levels in DLBCLs with the MYC t(8;14) translocation. Functional studies demonstrated that overexpression of miR-146b-5p or miR-320d inhibited DLBCL cell proliferation, wheareas knockdown of miR-146b-5p or miR-320d promoted proliferation of DLBCL cells. Taken together, these results suggest that low expression of miR-146b-5p and miR-320d may be predictive of compromised responses of a subset of DLBCL patients to treatment with the CHOP regimen and that restoration of these miRs may be useful to improve the therapeutic efficacy of CHOP.

Smurf2 suppresses B-cell proliferation and lymphomagenesis by mediating ubiquitination and degradation of YY1

About half of patients with diffuse large B-cell lymphoma (DLBCL) do not respond to or relapse soon after the standard chemotherapy, indicating a critical need to better understand the specific pathways perturbed in DLBCL for developing effective therapeutic approaches. Mice deficient in the E3 ubiquitin ligase Smurf2 spontaneously develop B-cell lymphomas that resemble human DLBCL with molecular features of germinal center or post-germinal center B cells. Here we show that Smurf2 mediates ubiquitination and degradation of YY1, a key germinal center transcription factor. Smurf2 deficiency enhances YY1-mediated transactivation of c-Myc and B-cell proliferation. Furthermore, Smurf2 expression is significantly decreased in primary human DLBCL samples, and low levels of Smurf2 expression correlate with inferior survival in DLBCL patients. The Smurf2-YY1-c-Myc regulatory axis represents a novel pathway perturbed in DLBCL that suppresses B-cell proliferation and lymphomagenesis, suggesting pharmaceutical targeting of Smurf2 as a new therapeutic paradigm for DLBCL.

Smurf2 mediates ubiquitination and degradation of YY1 to suppress B-cell proliferation and lymphomagenesis (P2181)

Abstract Current treatment of diffuse large B-cell lymphoma (DLBCL), the most common type of non-Hodgkin’s lymphoma, has improved patient survival significantly. However, 40-50% of DLBCL patients still die from this disease. There is a critical need to better understand the specific molecular pathways that are perturbed in DLBCL for the development of new therapeutic approaches. Using a mouse model deficient in the E3 ubiquitin ligase Smurf2, we show that Smurf2 deficiency increases the susceptibility of these mice to spontaneous B-cell lymphomagenesis. Lymphomas developed in Smurf2-deficient mice resemble human DLBCL with molecular features of germinal center B cells. We discover that Smurf2 mediates ubiquitination and degradation of YY1, a key germinal center transcription factor. Importantly, loss of Smurf2 enhances YY1-mediated transactivation of c-Myc and B-cell proliferation. Furthermore, we find that expression of Smurf2 is significantly decreased in primary human DLBCL samples compared to normal B cells. Importantly, low levels of Smurf2 expression correlate with poor survival prognosis in DLBCL patients. The Smurf2-YY1-c-Myc regulatory axis represents a novel pathway perturbed in DLBCL that suppresses B-cell proliferation and lymphomagenesis, and identifies Smurf2 as a new therapeutic paradigm for DLBCL.

Abstract 1994: Smurf2 mediates ubiquitination and degradation of YY1 to suppress B-cell proliferation and lymphomagenesis.

Abstract Current treatment of diffuse large B-cell lymphoma (DLBCL), the most common type of non-Hodgkin's lymphoma, has improved patient survival significantly. However, 40-50% of DLBCL patients still die from this disease. There is a critical need to better understand the specific molecular pathways that are perturbed in DLBCL for the development of new and effective therapeutic approaches. We show that mice deficient in the E3 ubiquitin ligase Smurf2 spontaneously develop B-cell lymphomas that resemble human DLBCL with molecular features of germinal center B cells. We discover that Smurf2 mediates ubiquitination and degradation of YY1, a key germinal center transcription factor. Importantly, Smurf2 deficiency enhances YY1-mediated transactivation of c-Myc and B-cell proliferation. Furthermore, the expression of Smurf2 is significantly decreased in primary human DLBCL samples compared to normal B cells, and low levels of Smurf2 expression correlate with poor survival prognosis in DLBCL patients. The Smurf2-YY1-c-Myc regulatory axis represents a novel pathway perturbed in DLBCL that suppresses B-cell proliferation and lymphomagenesis, suggesting Smurf2 as a new therapeutic paradigm for DLBCL. Citation Format: Charusheila Ramkumar, Hang Cui, Yahui Kong, Stephen Jones, Rachel Gerstein, Hong Zhang. Smurf2 mediates ubiquitination and degradation of YY1 to suppress B-cell proliferation and lymphomagenesis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1994. doi:10.1158/1538-7445.AM2013-1994 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.