Survival Of Diffuse Large B-cell Lymphoma Cells Research Articles

Low NDRG2, regulated by the MYC/MIZ-1 complex and methylation, predicts poor outcomes in DLBCL patients.

Diffuse large B-cell lymphoma (DLBCL) represents the most common tumor in non-Hodgkin's lymphoma. N-Myc downstream-regulated gene 2 (NDRG2) is a tumor suppressor highly expressed in healthy tissues but downregulated in many cancers. Although cell proliferation-related metabolism rewiring has been well characterized, less is known about the mechanism of metabolic changes with DLBCL. Herein, we investigated the expressions of NDRG2, MYC and Myc-interacting zinc finger protein 1 (MIZ-1) in seven human lymphoma (mostly DLBCLs) cell lines. NDRG2 expression was inversely correlated with the expressions of MYC and MIZ-1. Further, we explored the regulatory mechanism and biological functions underlying the lymphomagenesis involving NDRG2, MYC and MIZ-1. MYC and MIZ-1 promoted DLBCL cell proliferation, while NDRG2 induced apoptosis in LY8 cells. Moreover, NDRG2 methylation was reversed by the 5-Aza-2'-deoxycytidine (5-Aza-CDR) treatment, triggering the downregulation of MYC and inhibiting DLBCL cell survival. MYC interacts with NDRG2 to regulate energy metabolism associated with mTOR. Remarkably, supporting the biological significance, the converse correlation between NDRG2 and MYC was observed in human DLBCL tumor tissues (R = -0.557). Bioinformatics analysis further validated the association among NDRG2, MYC, MIZ-1, mTOR, and related metabolism genes. Additionally, NDRG2 (P = 0.001) and MYC (P < 0.001) were identified as promising prognostic biomarkers in DLBCL patients through survival analysis. Together, our data demonstrate that the MYC/MIZ-1 complex interplays with NDRG2 to influence the proliferation and apoptosis of DLBCL cells and show the characterizations of NDRG2, MYC and MIZ-1 for metabolism features and prediction prognosis in DLBCL.

Ajania pacifica (Nakai) K. Bremer and Humphries Extract Limits MYC Expression to Induce Apoptosis in Diffuse Large B Cell Lymphoma.

The proto-oncogene MYC is frequently dysregulated in patients with diffuse large B-cell lymphoma (DLBCL) and plays a critical role in disease progression. To improve the clinical outcomes of patients with DLBCL, the development of strategies to target MYC is crucial. The use of medicinal plants for developing anticancer drugs has garnered considerable attention owing to their diverse mechanisms of action. In this study, 100 plant extracts of flora from the Republic of Korea were screened to search for novel agents with anti-DLBCL effects. Among them, Ajania pacifica (Nakai) K. Bremer and Humphries extract (APKH) efficiently suppressed the survival of DLBCL cells, while showing minimal toxicity toward normal murine bone marrow cells. APKH suppressed the expression of anti-apoptotic BCL2 family members, causing an imbalance between the pro-apoptotic and anti-apoptotic BCL2 members. This disrupted mitochondrial membrane potential, cytochrome c release, and pro-caspase-3 activation and eventually led to DLBCL cell death. Importantly, MYC expression was markedly downregulated by APKH and ectopic expression of MYC in DLBCL cells abolished the pro-apoptotic effects of APKH. These results demonstrate that APKH exerts anti-DLBCL effects by inhibiting MYC expression. Moreover, when combined with doxorubicin, an essential component of the CHOP regimen (cyclophosphamide, doxorubicin, vincristine, and prednisone), APKH synergistically enhanced the therapeutic effect of doxorubicin. This indicates that APKH may overcome drug resistance, which is common in patients with refractory/relapsed DLBCL. To identify compounds with anti-DLBCL activities in APKH, the chemical profile analysis of APKH was performed using UPLC-QTOF/MSe analysis and assessed for its anticancer activity. Based on the UPLC-QTOF/MSe chemical profiling, it is conceivable that APKH may serve as a novel agent targeting MYC and sensitizing drug-resistant DLBCL cells to CHOP chemotherapy. Further studies to elucidate how the compounds in APKH exert tumor-suppressive role in DLBCL are warranted.

STAT6 mutations enriched at diffuse large B-cell lymphoma relapse reshape the tumor microenvironment

Diffuse large B-cell lymphoma (DLBCL) relapses in approximately 40% of patients following frontline therapy. We reported that STAT6D419 mutations are enriched in relapsed/refractory DLBCL (rrDLBCL) samples, suggesting that JAK/STAT signaling plays a role in therapeutic resistance. We hypothesized that STAT6D419 mutations can improve DLBCL cell survival by reprogramming the microenvironment to sustain STAT6 activation. Thus, we investigated the role of STAT6D419 mutations on DLBCL cell growth and its microenvironment. We found that phospho-STAT6D419N was retained in the nucleus longer than phospho-STAT6WT following IL-4 stimulation, and STAT6D419N recognized a more restricted DNA-consensus sequence than STAT6WT. Upon IL-4 induction, STAT6D419N expression led to a higher magnitude of gene expression changes, but in a more selective list of gene targets compared with STATWT. The most significantly expressed genes induced by STAT6D419N were those implicated in survival, proliferation, migration, and chemotaxis, in particular CCL17. This chemokine, also known as TARC, attracts helper T-cells to the tumor microenvironment, especially in Hodgkin’s lymphoma. To this end, in DLBCL, phospho-STAT6+ rrDLBCL cells had a greater proportion of infiltrating CD4+ T-cells than phospho-STAT6− tumors. Our findings suggest that STAT6D419 mutations in DLBCL lead to cell autonomous changes, enhanced signaling, and altered composition of the tumor microenvironment.

Aberrant Translation Regulated By METTL1/WDR4-Mediated tRNA N 7-Methylguanosine Modification Drives DLBCL Progression

Cancer cells selectively promote translation of specific oncogenic transcripts to facilitate cancer survival and progression, but the underlying mechanisms are poorly understood. Here, we find that N 7 - methylguanosine (m 7G) tRNA modification and its methyltransferase complex components, METTL1 and WDR4, are significantly upregulated in diffuse large B-cell lymphoma (DLBCL) and associated with poor prognosis. We further reveal the critical role of METTL1/WDR4 in promoting DLBCL cell survival and progression using loss- and gain-of-function assays in vivo. Mechanistically, m 7G tRNA modification selectively regulates the translation of oncogenic transcripts, in m 7G-tRNA-decoded codon-frequency-dependent mechanisms. Moreover, using overexpression and knockout mouse models, we demonstrate the crucial oncogenic function of Mettl1-mediated m 7G tRNA modification in promoting DLBCL tumorigenesis and progression in vivo. Our study uncovers the important physiological function and mechanism of METTL1-mediated m 7G tRNA modification in the regulation of oncogenic mRNA translation and cancer progression.

Role of Autoantigen Induced-B-Cell Receptor Internalization and Its Inhibition for the Biology and Survival of Diffuse Large B-Cell Lymphoma (DLBCL) Cells

In normal B-cells, antigen binding by the B cell receptor (BCR) triggers rapid BCR clustering, initialization of signaling, and subsequent endocytosis of the BCR-antigen complex. Numerous studies showed that BCR signaling is continued as the BCR is trafficked intracellularly. Similarly, the internalized BCR in complex with TLR9 in DLBCL cells elicits crucial pro-survival signaling for DLBCL cells. Despite these observations, the role of endocytosis in generating a pathological signal in DLBCL, and a potential of endocytosis blockade as a strategy to inhibit BCR signaling, have not been defined. To address these questions, we generated DLBCL cell models with modified, ovoalbumin (OVA)-specific hypervariable regions (HVR) in BCR using the knock-in CRISPR-Cas9 methodology. This model allows to study BCR receptor internalization and the signaling that accompanies this process in highly controlled conditions. The change of BCR specificity led to inhibition of spontaneous (autoantigen-initiated) BCR internalization, observed as an increased surface BCR level, and lower proliferative potential of autoautigen-dependent U2932 and HBL1 cells, but not in cells in which autoantigens were not detected (LY19). Using the kinome analysis platform (PamGene technology), we identified kinases activated by BCR-autoantigen binding, including proximal mediators of BCR signaling, cyclin-dependent kinases, protein kinase C and S6 ribosomal kinases. Simultaneous analysis of the OVA-induced BCR internalization kinetics and signaling activation in the above-described models indicated that the activation of certain signaling cascades may emanate from the inside of the cell. Using the Proximity Ligation Assay (PLA) method, we identified the presence of BCR-TLR9-IkB complexes (activators of the NF-kB transcription factor), in the endosomal compartment of autoantigen-dependent DLBCL cells. Cells with OVA-specific BCR showed a drastically decreased number of BCR-TLR9-IkB complexes, which was associated with lower expression of NF-kB-dependent genes. To confirm the crucial role of the autoantigen-induced BCR endocytosis in sustaining the signaling and promoting DLBCL cells survival, we reanalyzed available data from the CRISPR-Cas9-Screen studies. This approach revealed the key role of genes involved in endocytosis, including dynamin-2, for DLBCL cell survival. Based on these observations, we generated cell models characterized by inducible expression of the dominant-negative (DN) form of dynamin-2, which inhibits dynamin-2-dependent endocytosis. Experiments performed using these models showed that BCR internalization is dependent on dynamin-2, and the induction of the DN-dynamin-2 form blocks internalization of the BCR receptor, BCR-TLR9-IkB complexes assembly and activation of BCR-initiated signaling pathways, including the transcription factor NF-kB, which markedly inhibited the growth of DLBCL cells. In addition, clinically available Dynamin-2 inhibitors (e.g. certain phenothiazine derivatives), also inhibited BCR endocytosis and were toxic to DLBCL cells. In summary, our observations demonstrate that autoantigen-induced endocytosis of the BCR receptor is the key mechanism supporting the BCR signal and ensuring the survival of DLBCL cells with autoantigen-specific BCRs. Furthermore, we show that endocytosis is a rational therapeutic target in this group of lymphoid malignancies.

The Alternative RelB NF-kB Subunit Exerts a Critical Survival Function upon Metabolic Stress in Diffuse Large B-Cell Lymphoma-Derived Cells.

Diffuse large B-cell lymphoma (DLBCL) is the most common non-Hodgkin lymphoma in adults and reveals distinct genetic and metabolic signatures. NF-κB transcription factor family is involved in diverse biological processes enabling tumor development and resistance to anticancer-therapy through activation of its two main pathways, the canonical and the alternative NF-κB pathways, the main actor of the latter being the RelB NF-kB subunit. RelB DNA binding activity is frequently activated in DLBCL patients and cell lines. RelB activation defines a new DLBCL subgroup with dismal outcome upon immunochemotherapy, and RelB confers DLBCL cell resistance to DNA damage. However, whether RelB can impact on DLBCL cell metabolism and survival upon metabolic stress is unknown. Here, we reveal that RelB controls DLBCL oxidative energetic metabolism. Accordingly, RelB inhibition reduce DLBCL mitochondrial ATP production, and sensitizes DLBCL cells to apoptosis induced by Metformin and L-asparaginase (®Kidrolase), two FDA approved antimetabolic drugs targeting mitochondrial metabolism. RelB also confers DLBCL cell resistance to glutamine deprivation, an essential amino acid that feeds the TCA cycle. Taken together, our findings uncover a new role for RelB in the regulation of DLBCL cell metabolism and DLBCL cell survival upon metabolic stress.

Cfi-400945 Induces Cytokinesis Failure By Activating Hippo Signaling Pathway in Diffuse Large B-Cell Lymphoma

Targeting mitosis has been found to be a therapeutic strategy for cancer since microtubule poisons have proved efficacy in a broad range of malignancies. Cytokinesis failure can increase chromosomal instability beyond a critical threshold that triggers tumor suppression. Thus, agents developed to block cytokinesis can stop proliferation of cancer cells. We previously demonstrated that the polo-like kinase 4 (PLK4) inhibitor, CFI-400945, triggers growth inhibition and apoptotic death in diffuse large B-cell lymphoma (DLBCL). Since PLK4 is involved in centrosome duplication for cell division, we aimed to explore the effects of CFI-400945 on cytokinesis in DLBCL cells. Here, we demonstrated that CFI-400945 induces cytokinesis failure in DLBCL. CFI-400945-treated cells led to binucleated/multinucleated cells (Fig. 1a). Previous study showed that the Hippo signaling pathway played a critical role in cytokinesis. Consistent with this finding, we found that CFI-400945 led to phosphorylation of LATS1 and YAP (Fig. 1b). Confocal microscopy confirmed the reduced nuclear YAP upon treatment of CFI-400945 (Fig. 1c). These results show that reduced nuclear YAP levels and activities contribute to growth inhibition by CFI-400945 in DLBCL cells. In addition, we further explored the synergy of CFI-400945 and doxorubicin (DOX) both in vitro and in vivo. A combined treatment of CFI-400945 and DOX significantly inhibited DLBCL cell survival and induced more apoptosis compared with CFI-400945 or DOX alone (Fig. 1d, Fig. 1e).In the mice xenograft model, the combination of CFI-400945 and DOX significantly delayed tumor progression compared to those treated with vehicle or DOX alone (Fig. 1f). Tumors from combination-treated mice displayed an increase in γ-H2AX staining and a reduction in Ki67 staining (Fig. 1g). Strikingly, tumor cells were generally larger, heterogeneous in size and frequently binucleated (Fig. 1h). Combined, our results indicate for the first time that CFI-400945 treatment induces cytokinesis failure and activation of Hippo signaling pathway, leading to vulnerability to mitotic catastrophe. Moreover, this study proves an attractive combination treatment that simultaneous targeting PLK4 with CFI-400945 could exacerbate mitotic defects to improve response of anthracycline-based chemotherapy in DLBCL. Figure 1 Disclosures No relevant conflicts of interest to declare.

Human MYD88L265P is insufficient by itself to drive neoplastic transformation in mature mouse B cells

AbstractMYD88L265P is the most common mutation in lymphoplasmacytic lymphoma/Waldenström macroglobulinemia (LPL/WM) and one of the most frequent in poor-prognosis subtypes of diffuse large B-cell lymphoma (DLBCL). Although inhibition of the mutated MYD88 pathway has an adverse impact on LPL/WM and DLBCL cell survival, its role in lymphoma initiation remains to be clarified. We show that in mice, human MYD88L265P promotes development of a non-clonal, low-grade B-cell lymphoproliferative disorder with several clinicopathologic features that resemble human LPL/WM, including expansion of lymphoplasmacytoid cells, increased serum immunoglobulin M (IgM) concentration, rouleaux formation, increased number of mast cells in the bone marrow, and proinflammatory signaling that progresses sporadically to clonal, high-grade DLBCL. Murine findings regarding differences in the pattern of MYD88 staining and immune infiltrates in the bone marrows of MYD88 wild-type (MYD88WT) and MYD88L265P mice are recapitulated in the human setting, which provides insight into LPL/WM pathogenesis. Furthermore, histologic transformation to DLBCL is associated with acquisition of secondary genetic lesions frequently seen in de novo human DLBCL as well as LPL/WM-transformed cases. These findings indicate that, although the MYD88L265P mutation might be indispensable for the LPL/WM phenotype, it is insufficient by itself to drive malignant transformation in B cells and relies on other, potentially targetable cooperating genetic events for full development of lymphoma.

Diffuse Large B-Cell Lymphoma Remodels the Fibroblastic Reticular Network That Acquires Aberrant Immunosuppressive Capabilities; Implications for the Regulation of Anti-Tumor Immunity in the Immuno-Oncology Era

Immunotherapy has demonstrated potential to reactivate or transfer T cell immunity and regress tumors, offering hope to relapsed or refractory diffuse large B-cell lymphoma (DLBCL) patients. However, many DLBCL patients do not experience therapeutic benefit, likely owing to a lack of pre-existing anti-tumor immunity and/or poorly understood immunosuppressive mechanisms in the tumor microenvironment (TME). Understanding the different obstacles that cytotoxic T cells face in the DLBCL TME will help the development of novel therapeutic approaches to overcome them and optimize immunotherapy. Stroma-associated gene signatures reflecting fibroblast and immune cell activity as well as angiogenesis have been associated with outcome in DLBCL but the biology underlying these signatures has been understudied. Here we have examined beyond tumor ‘effacement’ and hypothesized that, rather than being sparse bystanders, lymph node stromal cells may be important players in driving immune suppression in lymphoma.Multiplex immunofluorescence (IF) confocal microscopy analysis of the major stromal cell subsets revealed a marked expansion and remodeling of podoplanin, PDPN+ fibroblastic reticular cells (FRCs) in DLBCL lymph node tissue TME biopsies across GCB and ABC subtypes (n=40) compared to reactive control tissues (n=10). FRC myofibroblasts were similarly remodeled in tumors from the transgenic mouse model of DLBCL (Iμ-HABCL6, n=10) compared to wild type littermates (n=5). These altered PDPNhigh, αSMAhigh FRC networks were interspersed within effaced lymph node tissues in close proximity to DLBCL tumor cells.To model the interactions between tumor cells and FRCs, we established 2D and 3D co-culture platforms that combined DLBCL cells (or non-malignant control B-cells) and PDPN+ FRCs derived from human or murine lymph node tissues. These quantitative assays have shown that tumor cells activate FRCs promoting their proliferation, increased expression of PDPN, marked elongation/stretching and subsequent reduced ability to contract 3D collagen matrix (non-contractile) (P<.01). Exposure to tumor cells caused uncoupling of PDPN from RHOA signaling and redistribution of PDPN into lipid rafts, permitting FRC stretching. FRCs purified from both human and murine tumor lymph nodes showed the same activated morphology and phenotype, demonstrating that our co-culture systems recapitulate in vivo findings. Screening experiments have shown that lymphoma-expressed membrane and soluble lymphotoxins (LTα1β2, LTα3) as well as TNFα significantly contribute to the remodeling of FRCs (P<.01). Co-culture assays have revealed evidence for mutualistic interaction as FRCs, that express BAFF, promote the survival of DLBCL cells in 3D matrix gels (P<.01). Expanded lymphoma PDPNhigh FRCs in situ co-expressed BAFF compared to a more restricted expression profile (marginal zone FRCs) in reactive lymph node tissues. Flow cytometry revealed that lymphoma FRCs exhibit a cancer-associated fibroblast (CAF)-like immunophenotype including upregulation of fibroblast activation protein (FAP) and αSMA, as well as immunomodulatory MHC class I, PD-L1 and PD-L2 molecules compared to control FRCs (P<.01).An important function of FRCs in regulating immunity is attracting and maintaining T cells by secreting chemokines and promoting their migration along the network. Functional assays revealed that T cells show significantly reduced chemotaxis as well motility (quantitative time-lapse movies) across 2D and 3D lymphoma FRC networks compared to control FRCs (P<.01). Multiplex IF analysis revealed reduced CCL19 and CCL21-expressing FRCs in DLBCL compared to the reactive control FRC network as well as low numbers of tumor-infiltrated CD8+ T cells, which localized closely with remodeled FRCs. We next determined whether lymphoma FRCs (CAFs) could negatively regulate T cell function. Triple culture autologous assays (murine and human) have shown that prior exposure of tumor-infiltrated CD8+ T cells to FRCs significantly decreased their cytolytic killing activity against tumor cells (P<.01).In conclusion our data indicate that DLBCL tumor cells convert FRCs into immunosuppressive CAFs, which exhibit altered immumomodulatory activities at different levels that we believe has important implications for the regulation of anti-tumor immunity as well as response to immunotherapy. DisclosuresVardi:Gilead: Research Funding; Janssen: Honoraria. Ramsay:Roche Glycart AG: Research Funding; MedImmune: Research Funding; Celgene Corporation: Research Funding.

Discovery of DNA G-Quadruplexes As a New Target for B-Cell Receptor Signaling Inhibition in Diffuse Large B-Cell Lymphoma

Extensive gene expression profiling and RNA interference studies revealed the frequently chemo-resistant activated B-cell-like (ABC) subtype of diffuse large B-cell lymphoma (DLBCL) relies on constitutive B-cell receptor (BCR) signaling. As such, the clinical importance of BCR signaling inhibition is well appreciated and thus far led to the development of kinase and protease inhibitors. However, this therapeutic approach fails to achieve complete, sustained responses in DLBCL patients because of inherent resistance due to additional genetic lesions in other components of the BCR pathway or acquired kinase mutations. The emerging field of DNA secondary structures support that guanine (G)-rich stretches of DNA capable of adopting G-quadruplex (G4) motifs act as transcription regulatory units, or switches, that can turn gene expression on or off. Targeting G4s is likely to overcome activating kinase mutations by limiting the amount of gene available for translation into protein. Here, we explore a drug discovery effort based on targeting G4 within BCR genes critical for ABC DLBCL cell survival, CD79A, CD79B, CARD11, and MYD88.We first interrogated the BCR-related genes within the hg19 human reference genome for G-rich DNA using a G4 algorithm and discovered each of the four genes contain G4 forming sequences near promoter regions. These G4 elements formed stable G4 structures as determined by circular dichroism (CD) spectroscopy, the standard for visualizing macromolecule secondary structure formation. Melting curves are also generated from CD spectroscopy to determine the thermal stability of a given structure. The CD79A, CD79B, CARD11, and MYD88 G-rich sequences displayed classic, stable G4 structure spectra consisting of negative minima absorption peaks at 240-265 nm and a positive maximum at 260-295 nm with melting temperatures ranging from 62 to 95 °C.We then developed a high-throughput screening assay based on fluorescence resonance energy transfer (FRET) to identify G4 interactive compounds from the NCI Diversity Set IV library (1584 compounds) that uniquely interact with each of the BCR G4 sequences. This screen used the BCR G4 sequences as molecular bait where the 5´-end and 3´-end of the oligomers were labeled with a FAM- and a TAMRA-fluorophore, respectively, such that G4 formation leads to an increase in fluorescence emission (Figure 1). The initial FRET screen tested compounds at a 1:5 molecular ratio of probe to compound and measured the change in fluorescence relative to probe alone. Overall, the screen resulted in a ~1% “hit” rate for each BCR target, except for CD79B, which yielded a lower percent of interactive compounds (0.3%). Seven compounds, which included ellipticine, quinoline, and daunomycin derivatives, were identified to selectively target the CARD11 (n=3), MYD88 (n=3), or CD79A (n=1) G4s relative to other G4, single-stranded, and double-stranded DNA. Of note, all five compounds found to interact with the CD79B G4 also altered FRET of the other BCR G4 sequences. Subsequent FRET validation and CD analyses where each of the BCR sequences was incubated with increasing concentrations of candidate compounds demonstrated dose-dependent effects on G4 structure formation, particularly stabilization of the CARD11 G4 with compound NCI 9037 that resulted in a 300% FRET increase and an 8 °C shift in melting temperature at a 1:10 ratio.This study identifies DNA G4 as a new class of molecular targets for inhibiting an important oncogenic pathway. Discovery of selective compounds in addition to those with “pan” interaction, suggests the CARD11, MYD88, and CD79A G4 have unique folding patterns whereas the CD79B G4 may exhibit more common structural features. These compounds will be used as molecular tools to provide further insight into the structures and mechanisms in which G4 regulate gene transcription. In establishing a high-throughput screen, we discovered compounds for which preclinical development is ongoing and includes evaluation of the effects on BCR target gene and protein expression, inhibition of downstream BCR signaling, and consequent ABC DLBCL tumor growth and survival. This treatment strategy has high potential for leading to a breakthrough in effectively targeting the constitutively active molecules and greatly impacting the clinical management of patients with BCR-dependent DLBCL. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

CXCR7 expression in diffuse large B-cell lymphoma identifies a subgroup of CXCR4+ patients with good prognosis.

The CXCR4/CXCL12 axis has been extensively associated with different types of cancer correlating with higher aggressiveness and metastasis. In diffuse large B-cell lymphoma (DLBCL), the expression of the chemokine receptor CXCR4 is involved in the dissemination of malignant B cells and is a marker of poor prognosis. CXCR7 is a chemokine receptor that binds to the same ligand as CXCR4 and regulates de CXCR4-CXCL12 axis. These findings together with the report of CXCR7 prognostic value in several tumor types, led us to evaluate the expression of CXCR7 in diffuse large B-cell lymphoma biopsies. Here, we describe that CXCR7 receptor is an independent prognostic factor that associates with good clinical outcome. Moreover, the expression of CXCR7 associates with increased survival in CXCR4+ but not in CXCR4- DLBCL patients. Thus, the combined immunohistochemical evaluation of both CXCR7 and CXCR4 expression in DLBCL biopsies may improve their prognostic value as single markers. Finally, we show that CXCR7 overexpression in vitro is able to diminish DLBCL cell survival and increase their sensitivity to antitumor drugs. Hence, further studies on the CXCR7 receptor may establish its role in DLBCL and the molecular mechanisms that modulate CXCR4 activity.

Targeting nucleolin for better survival in diffuse large B-cell lymphoma.

Anthracyclines have been a cornerstone in the cure of diffuse large B-cell lymphoma (DLBCL) and other hematological cancers. The ability of anthracyclines to eliminate DLBCL depends on the presence of topoisomerase-II-alpha (TopIIA), a DNA repair enzyme complex. We identified nucleolin as a novel binding partner of TopIIA. Abrogation of nucleolin sensitized DLBCL cells to TopIIA targeting agents (doxorubicin/etoposide). Silencing nucleolin and challenging DLBCL cells with doxorubicin enhanced the phosphorylation of H2AX (γH2AX-marker of DNA damage) and allowed DNA fragmentation. Reconstitution of nucleolin expression in nucleolin-knockdown DLBCL cells prevented TopIIA targeting agent-induced apoptosis. Nucleolin binding to TopIIA was mapped to RNA-binding domain 3 of nucleolin, and this interaction was essential for blocking DNA damage and apoptosis. Nucleolin silencing decreased TopIIA decatenation activity, but enhanced formation of TopIIA-DNA cleavable complexes in the presence of etoposide. Moreover, combining nucleolin inhibitors: aptamer AS1411 or nucant N6L with doxorubicin reduced DLBCL cell survival. These findings are of clinical importance because low nucleolin levels versus high nucleolin levels in DLBCL predicted 90-month estimated survival of 70% versus 12% (P<0.0001) of patients treated with R-CHOP-based therapy.

MicroRNA-26a/cyclin-dependent kinase 5 axis controls proliferation, apoptosis and in vivo tumor growth of diffuse large B-cell lymphoma cell lines

Diffuse large B-cell lymphoma (DLBCL) is the most frequent type of non-Hodgkin lymphoma. Despite a favorable therapeutic response to first-line chemo-immunotherapy, still 30–40% of patients is refractory, or relapse after this treatment. Thus, alternative strategies must be sought. Previous studies have indicated that cyclin-dependent kinase 5 (CDK5), a serine/threonine protein kinase, is involved in tumor development and progression, and it may represent a potential therapeutic target. However, its role in modulating DLBCL growth and progression remains largely unexplored. In this study, we show that CDK5 and its activator, cyclin-dependent kinase 5 activator 1 (CDK5R1 or p35), are overexpressed in DLBCL cell lines and that signal transducer and activator of transcription 3 (STAT3) phosphorylation and activity is dependent on CDK5 expression in DLBCL. Using public data sets, we also demonstrate that patients with DLBCL show a higher expression of CDK5 compared with healthy individuals. By using loss-of-function approaches, we demonstrate that CDK5’s activity regulates proliferation and survival of DLBCL cells. MicroRNAs (miRNAs or miRs) are small noncoding RNAs that negatively regulating gene expression and are involved in cancer initiation and progression. We identify miR-26a as direct regulator of p35 expression and CDK5 activity. We show that miR-26a expression is lower in DLBCL cell lines compared to B lymphocytes and that its ectopic expression leads to a drastic reduction of DLBCL tumor growth in vivo and decreased proliferation, cell-cycle progression, and survival in vitro. Remarkably, concomitant overexpression of a 3′-UTR-truncated form of p35 promoted tumor growth in vivo and cell proliferation, cell-cycle progression, and cell survival in vitro. In conclusion, these results demonstrate an important role for miR-26a and CDK5 together in the survival and growth of DLBCL cells, suggesting the existence of potential novel therapeutic targets for the treatment of DLBCL.

Tenovin-6 inhibits proliferation and survival of diffuse large B-cell lymphoma cells by blocking autophagy.

Diffuse large B-cell lymphoma (DLBCL) is one of the most aggressive non-Hodgkin lymphomas. It is curable but one-third of cases are refractory to therapy or relapse after initial response highlighting the urgent need for developing novel therapeutic approaches. Targeting sirtuins, particularly SIRT1 by genetic approaches or using pharmaceutical inhibitor tenovin-6, has shown promising therapeutic potential in various hematopoietic malignancies. However, it remains unknown whether these approaches are effective for DLBCL. In this study, we have found that tenovin-6 potently inhibits the proliferation and survival of DLBCL cells. Surprisingly, specific knockdown of SIRT1/2/3 has no effect on DLBCL. Mechanistically, tenovin-6 increases the level of microtubule-associated protein 1 light chain 3B (LC3B)-II in a SIRT1/2/3- and p53-independent manner in DLBCL cell lines. Tenovin-6-mediated increase of LC3B-II is through inhibition of classical autophagy pathway. Furthermore, inhibition of the autophagy pathway by using other inhibitors or by knocking down key genes in the pathway impairs cell proliferation and survival of DLBCL cells. These results indicate that targeting the autophagic pathway could be a novel therapeutic strategy for DLBCL and that precaution should be taken to interpret data where tenovin-6 was used as an inhibitor of sirtuins.

MYD88, CD79B, and CD79A Gene Mutations in CD5-Positive Diffuse Large B-Cell Lymphoma

INTRODUCTIONDiffuse large B-cell lymphoma (DLBCL) is regarded as a clinicopathologically heterogeneous group of lymphomas and is classified as activated B-cell-like (ABC) DLBCL or germinal center B-cell-like (GCB) DLBCL based on the cell-of-origin (COO). Nuclear factor-kappa B (NF-kB) activation is indispensable for ABC DLBCL cell survival. Genes in the NF-kB signaling pathway, such as myeloid differentiation factor 88 (MYD88) and CD79B, are mutated in 20-40% of ABC DLBCLs. CD5 is expressed in approximately 10% of DLBCLs. CD5-positive (CD5+) diffuse large B-cell lymphoma (DLBCL) is characterized by frequent central nervous system (CNS) relapse and a predominant activated B-cell-like nature. Primary DLBCL in sanctuary sites (DLBCL-SS) also shows these features, and more than 70% of the patients exhibit MYD88 (L265P) and CD79B mutations. This study aimed to ascertain the frequencies of MYD88 (L265P), CD79B, and CD79A mutations in CD5+ DLBCL and to determine whether CD5+DLBCL shows the same features as DLBCL-SS in terms of gene mutations.PATIENTS AND METHODSThis study included 40 patients with CD5+DLBCL. All the patients were diagnosed with DLBCL, not otherwise specified, according to the 2008 WHO classification between 1993 and 2014 at Mie University Hospital. CD5 expression in tumor cells was analyzed by immunohistochemistry using frozen sections or by flow cytometry. Mutation analysis was performed by direct sequencing.RESULTSDirect sequencing was successful with samples from all 40 patients with CD5+ DLBCL. The median age was 64 years (range: 15 to 91 years). The COO classification was determined by gene expression profiling (GEP) in 29 patients. In the other 11 patients, the COO was identified by immunohistochemistry according to Hans' criteria. Thirty-six (90%) cases were confirmed as ABC/non-germinal center DLBCL. Two patients fulfilled the criteria for primary testicular DLBCL. One patient had systemic CD5+ DLBCL with bone and brain involvement. None of the other patients had CNS involvement at diagnosis. The MYD88 L265P mutation was identified in 13 (33%) of the cases, and no other MYD88 mutations were found. CD79B mutations were detected in 15 (38%) cases, and 10 of these cases overlapped with the MYD88-mutated group. One of the two patients with testicular involvement had double mutations. The other had MYD88 mutation alone. One patient with brain involvement had double mutations. Only one case (3%) had a CD79A mutation as well as CD79B and MYD88 mutations. The incidence of MYD88 L265P, CD79B, and CD79A mutations in ABC/non-GCB DLBCLs was 13/36 (36%), 14/36 (39%), and 1/36 (3%), respectively. Of the 15 cases with a CD79B mutation, 14 had missense mutations in an immunoreceptor tyrosine-based activation motif (ITAM) domain; all of these mutations occurred in the first tyrosine of the ITAM (Y196H [7/14], Y196S [4/14], Y196N [2/14], and Y196C [1/14]), and two cases showed double mutations (Y196H/H225Y and Y196S/K219R). One case had a 13-base deletion in exon 5 before the first tyrosine of the ITAM. MYD88 and CD79B mutations were associated with localized disease (P = 0.038 and P = 0.003, respectively). Clinical information on first-line treatment was available for 38 patients. Before 2002, anthracycline-containing chemotherapies without rituximab were selected as first-line treatment. Since 2002, CHOP chemotherapy with rituximab (R-CHOP) has predominated. For this reason, 18 patients who were treated with R-CHOP were included in the survival analysis in this study. The COO of these 18 patients was confirmed as ABC/non-GCB DLBCL. Two patients with primary testicular lymphoma and one with CD5+ DLBCL with brain involvement were not included in this cohort of 18 patients. With a median follow-up of 7.1 years, there was no significant difference in overall survival based on MYD88 mutation status (P = 0.98) or CD79B mutation status (P= 0.69).CONCLUSIONSTo the best of our knowledge, this is the first study toreport the frequency of MYD88, CD79B, and CD79A mutations in the largest cohort of CD5+ DLBCLpatients. The incidence of MYD88 and CD79B mutations in CD5+ DLBCL is lower than that in DLBCL-SS, suggesting that CD5+DLBCL is not the same disease as DLBCL-SS. DisclosuresTakeuchi:Chugai: Honoraria. Yamaguchi:Chugai: Honoraria; Eisai: Honoraria; Takeda: Honoraria; Kyowa-Hakko Kirin: Honoraria; Zenyaku: Honoraria. Miyazaki:Eisai: Honoraria; Kyowa Kirin: Honoraria; Chugai: Honoraria. Tawara:Astellas: Honoraria. Katayama:Bristol-Myers Squibb Japan: Honoraria; Alexion Pharmaceuticals: Honoraria; Eisai: Honoraria; Taisho Toyama Pharma: Honoraria; Nippon Shinyaku: Honoraria; Kyowa Hakko Kirin: Honoraria, Research Funding; Astellas: Honoraria, Research Funding; Dainippon Sumitomo Pharma: Honoraria; Chugai: Honoraria, Research Funding; Takeda: Honoraria; Shire: Honoraria; Daiichi Sankyo: Honoraria; Shionogi: Honoraria; Celgene: Honoraria; Pfizer: Honoraria.

ATF3, a new player in DLBCL cell survival

In this issue of Blood, Juilland and colleagues reveal the expression pattern and the role of different members of the activating transcription factor (ATF) family in survival of diffuse large B-cell lymphoma (DLBCL) cells.

Inhibition of COP9-signalosome (CSN) deneddylating activity and tumor growth of diffuse large B-cell lymphomas by doxycycline

In searching for small-molecule compounds that inhibit proliferation and survival of diffuse large B-cell lymphoma (DLBCL) cells and may, therefore, be exploited as potential therapeutic agents for this disease, we identified the commonly used and well-tolerated antibiotic doxycycline as a strong candidate. Here, we demonstrate that doxycycline inhibits the growth of DLBCL cells both in vitro and in mouse xenograft models. In addition, we show that doxycycline accumulates in DLBCL cells to high concentrations and affects multiple signaling pathways that are crucial for lymphomagenesis. Our data reveal the deneddylating activity of COP-9 signalosome (CSN) as a novel target of doxycycline and suggest that doxycycline may exert its effects in DLBCL cells in part through a CSN5-HSP90 pathway. Consistently, knockdown of CSN5 exhibited similar effects as doxycycline treatment on DLBCL cell survival and HSP90 chaperone function. In addition to DLBCL cells, doxycycline inhibited growth of several other types of non-Hodgkin lymphoma cells in vitro. Together, our results suggest that doxycycline may represent a promising therapeutic agent for DLBCL and other non-Hodgkin lymphomas subtypes.

Mechanism of NLRC5 in diffuse large B-cell lymphoma

Diffuse large B-cell lymphoma (DLBCL) is the most common malignant lymphoma in China, which mostly starting from the lymph nodes. The studies find that the expression of NLRC5, member of nucleotide-binding oligomerization domain-like receptor (NLR) family, is evident in the lymphoid cells, which is likely to be an important basis for lymphocyte tumorigenesis. NLRC5 can block the nuclear factor-κB (NF-κB), a core component of signal transduction pathway, and affect the development of tumor. While continuing activation of NF-κB is a necessary condition for DLBCL cell survival. Therefore, NLRC5 is likely to inhibit excessive inflammation, thereby inhibiting the function of DLBCL, which promises to be a new target for immunotherapy treatment of DLBCL. Key words: Lymphoma, large B-cell, diffuse; NF-kappaB; Genes, MHC class Ⅰ; NLRC5

The Lipid Addiction of Diffuse Large B-Cell Lymphoma (DLBCL) and Potential Treatment Strategies with Novel Fatty Acid Synthase (FASN) Small Molecule Inhibitors

Background: Fatty acid (FA) metabolism is altered in several cancers through increased de novo synthesis of lipids via up-regulation fatty acid synthase (FASN) and increased utilization of lipids via β-oxidation. We investigated the dependence of DLBCL survival on FA metabolism. In addition, we examined novel FASN inhibitors TVB3567 and TVB3166 in comparison with cerulenin for the effects on cell survival and PI3K and MAPK-related biological pathways associated with tumor-related FA metabolism in DLBCL.Methods: FASN inhibitors, TVB3567 and TVB3166 (3V Biosciences, CA), cerulenin (FASN inhibitor), orlistat (anti-lipoprotein lipase (LPL) and FASN), PI3K/mTOR, and MEK small molecule inhibitors were studied in OCI-LY3, OCI-LY19, SUDHL4, SUDHL6, and SUDHL10 DLBCL cell lines for the effects of FA inhibition on lipid metabolism, cell signaling, and cell death. The effects of FASN inhibition on global gene expression profile (GEP) were also determined with Affymetrix Human 2.0 ST Genechip with Gene set enrichment analysis (GSEA). We also utilized short hairpin RNA interference (shRNA) to study interactions between FASN and PI3K/MAPK signaling. Finally, AutoDock Vina software (autodock.scripps.edu) was utilized to analyze drug target (FASN enzyme) binding affinity and assist in the design of FASN inhibitors with higher target binding affinity.Results: DLBCL cell lines OCI-LY3, SUDHL4, and SUDHL6 grown in the presence of lipoprotein-depleted serum showed exquisite sensitivity to lipid deprivation resulting in near complete cytotoxicity by MTT. Lipid deprivation-induced apoptotic cell death, detected as cleaved caspase 3 and PARP and Annexin-V/PI positivity, in these cells. Further, these effects were completely rescued by Very Low Density Lipoprotein (VLDL) supplementation to growth medium in SUDHL4 confirming the high lipid-dependency on cell survival in DLBCL. Treatment with pharmacological inhibitors of FASN (ie, TVB3567, TVB3166, cerulenin, or orlistat) resulted in a dose- and time-dependent reduction in cell viability in all DLBCL cell lines. Ingenuity Pathway Analysis (IPA) from GEP with cerulenin-treated OCI-LY3 showed prominent suppression of CD40, TNF, and NFκB dependent inflammatory responses as well as activation of apoptosis as predominant biological activities including significant down-regulation of genes involved in Krebs cycle and p38 MAPK pathways. Interestingly, upstream regulation by IPA predicted activation of MEK/ERK and MYC-dependent functions; and in OCI-LY3 with shRNA knock down of FASN, we observed constitutive activation of ERK as detected with increased phosphorylation by western blot. Activation of MEK/ERK and MYC is expected in part owing to metabolic stress induced by FASN inhibition. Considering the impact of MEK/ERK pathways on lipid metabolism, we next investigated the impact of MEK/ERK on FA metabolism. FASN was significantly decreased following MEK or ERK shRNA in OCILY-3 and SUDHL10 cells. Similarly, pharmacological inhibition of MEK or PI3K/mTOR (using novel small molecule agents AZD6244 (selumetinib) or BEZ235, respectively) resulted in marked down-regulation of FASN expression. Based on these results, FASN inhibition appears to a promising therapeutic target for the treatment of DLBCL, however attaining clinical efficacy with existing compounds require the effective drug concentration to be within the nanomolar range. Thus, we utilized AutoDock to determine drug docking enzyme inhibition constant (ki). We identified high ki values of 33μM and 180μM for Cerulenin and Orilstat, respectively. Therefore, we have developed/constructed modified novel and potent anti-FA compounds with ki <1μM that are currently being investigated.Conclusions: Collectively, we demonstrated that DLBCL cell survival is highly dependent on FA metabolism and that targeting lipid metabolism may be harnessed as a potential therapeutic strategy. We also showed that MEK/ERK-dependent mechanisms are intimately involved in promoting lipid addiction in DLBCL cells. Further investigation is warranted to delineate the mechanisms through which MEK/ERK regulate FASN expression and to determine in vivo implications of FASN inhibition on DLBCL tumor growth. In addition, continued development, design, and enhancement are needed to construct the most optimal anti-FA therapeutic agents. DisclosuresLansigan:Teva Pharmaceuticals: Research Funding; Spectrum Pharmaceuticals: Research Funding.

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

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