DHL-4 Cells Research Articles

Inhibition of PIM Kinases in Diffuse Large B-Cell Lymphoma Cells Targets MYC-Dependent Transcriptional Program, Increases CD20 Expression and Augments the Efficacy of Anti-CD20 Antibodies

R-CHOP immunochemotherapy remains standard frontline therapy for newly diagnosed diffuse large B-cell lymphoma (DLBCL) patients. However, this therapy is ineffective in approximately 1/3 of patients, underscoring the need for better treatment modalities. Targeting DLBCL oncogenic drivers is a promising strategy to improve the treatment efficacy and outcome. Although MYC transcription factor is one of the key oncogenes in DLBCL development, direct MYC targeting strategies have been largely ineffective, highlighting the need for other, indirect approaches. For example, MYC expression is stabilized by PIM serine-threonine kinases, indicating that PIM inhibition might be a rational approach to indirectly target MYC. In this study, we assessed the PIM-MYC relationship and the consequences of PIM inhibition in DLBCL. We first evaluated the expression of PIM1-3 and MYC proteins in 57 DLBCL diagnostic sections by immunohistochemistry. In this series, 70.17% of specimens were positive for at least one PIM isoform and 84.22% cases were MYC-positive. 100% of cases with high MYC expression (MYC present in ≥30% of the cells, n=35) were PIM-positive, whereas 86,36% of cases with undetectable or low MYC expression (MYC detected in ≤20% of cells, n= 22) were PIM-negative (Fisher's exact test, p<0.0001). Since the coexpression of MYC and PIMs highlights the functional link between these proteins in DLBCLs, we evaluated the expression of PIM kinases in cell lines following siRNA-mediated MYC knockdown or treatment with MYC-MAX dimerization inhibitor, 10058F4. The genetic or chemical MYC inhibition markedly decreased PIM1-3 expression in six GCB and ABC cell lines. Likewise, knockdown of all three PIM isoforms decreased MYC levels, attenuated proliferation and induced apoptosis. Similarly, PIM blockade with SEL24/MEN1703, a novel pan-PIM/FLT3 inhibitor tested currently in clinical trial in AML patients and exhibiting favorable safety profile, decreased the expression of multiple MYC-dependent genes. To assess the MYC role in PIM inhibitor-mediated toxicity, we generated DHL4 cells expressing degradation-resistant MYC_T58A mutant. MYC_T58A expression partially protected cells from PIM inhibitor-induced proliferation arrest and apoptosis, indicating that the inhibitor's toxicity is at least partially mediated by MYC depletion. The MS4A1 gene, encoding CD20 surface antigen and rituximab target, is regulated by an upstream promoter containing potential MYC-binding sites (E-boxes). MYC association to these regions was confirmed in chromatin immunoprecipitation assays. As expected, in SEL24/MEN1703-treated cells, MYC occupancy at the MS4A1 promoter markedly decreased. To determine the consequences of MYC binding to the MS4A1 promoter, we assessed CD20 levels in a lymphoblastoid cell line carrying tetracycline-regulated (tet-off) MYC. MYC repression markedly elevated transcript and surface CD20 levels in a time-dependent manner, reaching 17.3-fold (transcript) and 3.82-fold (surface) inductions at 96 h. Consistently, the pan-PIM inhibitor decreased MYC expression in DHL4 and RAJI cells and resulted in increased surface CD20 levels up to 3.72-fold of baseline. In cells expressing the MYC_T58A mutant, PIM inhibition did not increase CD20 level, indicating that PIM kinases modulate CD20 surface expression via MYC. Importantly, PIM inhibitors increased CD20 levels also in primary, patient-derived DLBCL cells. These data suggest that indirect MYC targeting via PIM inhibition would lead to increased rituximab activity. Indeed, in PIM inhibitor-treated DHL4 and RAJI cells, rituximab triggered higher complement-dependent toxicity. Likewise, PIM inhibitor potentiated rituximab-dependent uptake of DHL4 and DHL6 cells by human monocyte-derived macrophages in antibody-dependent cellular phagocytosis assay. Taken together, we characterize a PIM-MYC regulatory circuit promoting DLBCL growth and resistance to anti-CD20 antibody. We also demonstrate that PIM inhibition exhibits pleiotropic effects that combine direct cytotoxicity with increased surface CD20 levels and increased susceptibility to anti-CD20 antibody-based therapies. Study supported by Foundation for Polish Science (POIR.04.04.00-00-5C84/17-00), Polish National Science Centre (2016/22/M/NZ5/00668 and 2017/26/D/NZ5/00561) and Ministry of Science and Higher Education in Poland (iONCO) grants. Disclosures Golas: Ryvu Therapeutics: Current Employment. Green:KDAc Therapeutics: Current equity holder in private company. Tomirotti:Menarini Ricerche: Current Employment. Brzózka:Ryvu Therapeutics: Current Employment. Juszczynski:Ryvu Therapeutics: Other: member of advisory board.

R-Nanochop Incorporating a TFR1-Targeted Doxorubicin Nanocarrier Is Superior to R-CHOP in a PDX Model of Diffuse Large B-Cell Lymphoma

Diffuse large B-cell lymphoma (DLBCL) comprises a third of non-Hodgkin lymphoma (NHL), with incidence steadily rising in what is already the most common hematologic malignancy. The backbone of potentially curative frontline chemoimmunotherapy combinations like R-CHOP is doxorubicin (Dox), still considered the most active anti-lymphoma agent. Relapsed or refractory (rel/ref) disease after frontline treatment carries persistently poor prognosis, even with recent advances in immunotherapy. Development and implementation of new therapeutic strategies remain urgent priorities to address unmet clinical needs in both high-risk previously untreated and rel/ref patients. We have studied transferrin receptor (TFR1) expression as a marker of high-risk DLBCL revealing significantly worse outcomes following frontline therapy associated with high expression in diagnostic samples. TFR1 is therefore a rational target for treatments aimed specifically at high-risk DLBCL. We used third-generation carbon-nitride dot (CND) nanocarriers we have developed conjugated to Dox and holo-transferrin (TF) to develop a chemotherapeutic-nanocarrier (NanoDox) designed to deliver Dox to TFR1-expressing tumors while sparing non-malignant tissues. Dox's mechanism is primarily through nuclear DNA damage or induction of reactive oxygen species (ROS) H202. We treated DLBCL cell lines with NanoDox or Dox for 24-hours and observed induction of nuclear DNA damage marker γ-H2Ax at drastically lower doses (10 nM NanoDox vs. 100 nM Dox). Terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay results yielded similar findings in BJAB cells, as observed under γ-H2Ax analysis, with TUNEL induction at significantly lower NanoDox concentrations. Treatment with NanoDox did not generate significant changes in ROS H202 when compared to Dox at 24-hours. Dox is known for a strong cytotoxic profile that extends past its terminal half-life of 24-36 hours. Therefore, we treated BJAB, Farage and DHL4 cells for 24-hours with a range of NanoDox and Dox doses, followed by washout and tracked cell viability for 6 additional days. We found as low as 10 nM NanoDox induced rapid and irreversible cytotoxicity, an effect only seen at much higher doses of Dox. These results combined with fluorescent confocal microscopy studies confirmed NanoDox works primarily through rapid nuclear Dox accumulation, causing DNA damage and apoptosis. Building on initial NanoDox dosing studies in NOD Scid Gamma (NSG) mice, we constructed R-nanoCHOP as an alternate to R-CHOP by replacing Dox with NanoDox at a working dose (WD) of 33.0 mg/kg. We next engrafted patient-derived xenograft (PDX) tumors derived from a previously untreated patient with germinal center B-cell (GCB) subtype DLBCL. We randomized NSG animals to two groups at tumor engraftment and treated with 21-day cycles of R-nanoCHOP (n=10) or R-CHOP (n=11). The treatments controlled tumor volume (TV) similarly, but R-nanoCHOP significantly improved overall survival associated with greatly reduced toxicities to host animals, which tolerated an average of 2 additional cycles of R-nanoCHOP. Studies with murine cells in vitro, including A20 B-cell lymphoma cells, confirmed these effects were not due to species differences, with human holo-TF alone and as part of NanoDox binding mouse and human TFR1 with equal affinity. This work establishes proof of principle in DLBCL for targeting TFR1, a specific marker of clinically high-risk disease. CND nanocarriers provide a flexible platform to exploit this, with our initial studies enhancing the safety profile of Dox, which remains the most active frontline DLBCL agent more than 50 years after it entered use. We are now optimizing NanoDox by substituting the holo-TF with an anti-TFR1 single chain variable fragment (scFv), decreasing size of the overall nanoconjugate by >50%. We believe dosing optimization from this change and additional optimizations to Dox delivery intracellularly will permit investigational new drug (IND) studies of absorption, distribution, metabolism and excretion (ADME). In sum, we provide compelling clinically relevant evidence for targeting TFR1 in DLBCL as a new therapeutic approach. Disclosures No relevant conflicts of interest to declare.

Downregulation of Deptor By MiR-155 Promotes Cell Survival through Activation of PI3K/AKT and NFkB Signaling in ABC-Type Diffuse Large B-Cell Lymphomas

IntroductionMiR-155 expression in DLBCLs is induced by NFkB and directly targets key regulators of B-cell maturation, motility and BCR signaling. In this study, we searched for new targets of miR-155 potentially involved in deregulation of BCR-PI3K/AKT and NFkB signaling in DLBCLs. We identified two new miR-155 targets: c-CBL (SYK ubiquitin E3 ligase) and DEPTOR (mTOR phosphatase). DEPTOR suppresses mTOR activity and in different tumors plays a role of either a tumor suppressor or an oncogene. Since the role of DEPTOR in DLCBLs cells has not been determined, we assessed the consequences of its inhibition in this malignancy.MethodsPredicted miR-155 targets were validated with 3'UTR luciferase reporter assays. MiR-155 expression was modulated through transfection with miR-155 mimic or miR-155 inhibitor. The DEPTOR silencing in DLBCL cell was achieved with retroviral shRNA vector. DEPTOR mRNA expression and survival of DLBCL patients was determined using publicly available microarray data (Lenz et al, 2008, GEO accession GSE10846). Immunohistochemical assessment of DEPTOR expression was performed in 76 newly diagnosed DLBCL patients with available GCB/non-GCB designations based on Hans algorithm.ResultsUsing miRNA target finding algorithms, we identified miR-155-matching sequences in 3’UTRs of two genes involved in SYK/PI3K/AKT pathway regulation: c-CBL and DEPTOR. MiR-155 suppressed luciferase activities of vectors containing 3’UTR fragments from c-CBL and DEPTOR genes with wild-type, but not mutant miR-155 seed sequence. To establish a link between miR-155 and c-CBL or DEPTOR, DLBCL cell lines were transfected with a control non-targeting miR or miR-155 mimic. Introduction of miR-155 resulted in decreased expression of c-CBL and DEPTOR, accompanied by a marked increase in phospho-AKT level. Inhibition of endogenous miR-155 in U2932 cell line by anti-miR-155 exhibited opposite effects. Using publicly available gene expression data (Lenz et al, 2008, GEO accession GSE10846), we found that miR-155 exhibited a reciprocal expression pattern with DEPTOR and c-CBL (r =-0.15; p=.002 and r=-0.189; p<.0001), indicating that miR-155 likely modulates expression of c-CBL and DEPTOR in primary tumors.Since the role of DEPTOR in DLBCL has not been previously addressed, we investigated its expression in a series of 76 newly diagnosed DLBCL patients by immunohistochemistry. Complete loss of DEPTOR expression was noted solely in non-GCB tumors (9 of 37; 24%). Additional 12 patients exhibited low expression of this protein. In contrast, none of the 39 GCB tumors stained negative for DEPTOR (Chi-square, p=.005). DEPTOR mRNA greater than mean in primary DLBCL biopsies was associated with longer overall survival (OS; P=0.016).To elucidate the function of DEPTOR in DLBCL cells, we silenced the expression of this protein with shRNA. Attenuated protein level of DEPTOR markedly enhanced AKT activity and promoted proliferation of DHL4 cells. DHL4 cells with silenced DEPTOR were also less prone to starvation- or SYK inhibition- induced apoptosis. Since AKT has been reported to stimulate activity of NFκB, we hypothesized that miR-155-induced decrease in DEPTOR expression would affect NFκB signaling. Consistent with this, inhibition of endogenous miR-155 in U2932 cells led to marked downregulation of NFĸB-controlled genes (CD40, BFL-1, RelB, IĸBα, A20, MIR155HG) and sensitized U2932 cells to ibrutinib, indicating that miR-155 amplifies NFĸB signaling in these cells.ConclusionsTaken together, our data underscore the role of miR-155 in the regulation of AKT and NFkB prosurvival signaling in DLBCL. By targeting multiple negative regulators of PI3K/AKT pathway, miR-155 creates a feed - forward loop leading to increased NFkB activity. We also show that DEPTOR protein expression is decreased or lost selectively in a large fraction of ABC-DLBCLs. Since DEPTOR modulates AKT activity and its silencing promotes proliferation of DLBCL cells, these data suggest that DEPTOR functions as a tumor suppressor in ABC-DLBCLs. DisclosuresProchorec-Sobieszek:Roche: Other: travel, accommodation. Warzocha:BMS: Consultancy, Honoraria; Novartis: Consultancy, Honoraria. Juszczynski:Selvita S.A.: Consultancy, Membership on an entity's Board of Directors or advisory committees.

MiR-17-92 represses PTPROt and PP2A phosphatases and amplifies tonic BCR signaling in DLBCL cells.

B-cell receptor (BCR) signaling plays a pivotal role in the pathogenesis of diffuse large B-cell lymphoma (DLBCL) and targeting the BCR pathway is a highly promising therapeutic strategy in this malignancy. The oncogenic microRNA miR-17-92 modulates multiple cellular processes such as survival, proliferation, apoptosis, angiogenesis, and BCR signaling. In the present study, we identified new targets of miR-17-92, PTPROt (protein phosphatase, receptor type O, truncated) and PP2A (protein phosphatase 2A) phosphatases, which regulate the activity of spleen tyrosine kinase (SYK) and AKT, critical components of BCR signal transduction in DLBCL cells. Introduction of miR-17-92 into DLBCL cells dampened the expression of the PTPROt and PP2A regulatory subunits PPP2R2A (protein phosphatase 2, regulatory subunit B, alpha) and PPP2R5E (protein phosphatase 2, regulatory subunit B, epsilon isoform) and increased the magnitude of SYK and AKT phosphorylation upon BCR ligation. Finally, we found that miR-17-92 expression modulates response to inhibitors of BCR signaling because downregulation of miR-17-92 increased SYK inhibitor-mediated toxicity in DLBCL cells. Our study reveals novel posttranscriptional regulatory pathways that contribute to the deregulation of BCR signaling and modulate SYK inhibitor activity in DLBCL.

A Novel Pan-PIM Kinase Inhibitor, SEL24-B489, Induces Apoptosis and Inhibits Proliferation of Diffuse Large B-Cell Lymphoma Cells through Inhibition of Protein Translation and Attenuation of Myc and NFkB Activity

Introduction. In 1/3of diffuse large B-cell lymphoma (DLBCL) patients current treatment options are ineffective, underscoring the need for more effective targeted therapies. Simultaneous genetic ablation of PIM kinases (PIM-1/2/3) or their pharmacological inhibition induce apoptosis in DLBCL cell lines, providing proof of concept that these proteins are promising targets in this disease. To better understand mechanisms of toxicity of PIM inhibition in DLBCL, we investigated biochemical and biological consequences of PIM inhibition using novel pan-PIM inhibitor (SEL24-B489) in a panel of DLBCL cell lines in in vivo xenograft model.Methods. Protein expression and phosphorylation status of PIM substrates was assessed by immunobloting. Proliferation and apoptosis were assessed by MTS assay and PI/AnnexinV staining. Gene expression profiling was performed on Illumina HT-12 v4 Chip. NFkB target genes expression was assessed by Real-Time Quantitative PCR (qPCR). For xenograft studies, 9-11 weeks old SCID/beige CB17 mice were subcutaneously injected with 5x106 U2932 cells suspended in 0.1 ml mixture of PBS with Matrigel. When tumor volumes reached ~100 mm3, mice were randomized into uniform groups and subjected to the compound administration (50 mg/kg daily, BID, per os). Tumor volume was monitored every second day.Results. The newly developed pan-PIM inhibitor (SEL24-B489) was toxic to DLBCL cell lines in low-micromolar or sub-micromolar concentrations (IC50: 0,29 to 1,17µM). To determine mechanisms underlying toxicity of PIM inhibition in DLBCL, we first assessed the activity of 4EBP1 and ribosomal protein S6, proteins involved in protein translation. PIM inhibitor rapidly and uniformly decreased 4EBP1 and S6 phosphorylation in all tested DLBCL cell lines (DHL4, DHL6, Ly1, Ly7, Karpas 422, Pfeiffer, Toledo, U2932, HBL-1). Since PIM kinases have been shown to increase stability and/or activity of NFκB-p65 (RelA) and Myc, we assessed the abundance/activity of these transcription factors upon PIM inhibitor treatment. PIM inhibition led to downregulation of Myc protein in GCB DLBCL cell lines. Since Myc and PIMs cooperate in augmenting transcription of active genes, we assessed global RNA levels upon PIM inhibitor treatment. The RNA abundance in SEL24-B489-treated cells decreased by 22-37%. The underlying mechanism of the decrease in cellular mRNA content involved global inhibition of PIM-dependent histone H3 serine 10 (H3S10) phosphorylation and decreased phoshorylation of RNA polymerase II (serine 2).To test whether PIM inhibition also attenuates specifically NFκB-dependent transcription in DLBCL cell lines, we assessed gene expression profiles of vehicle- or SEL24-B489-treated HBL-1, U2932 and DHL4 cells. Following PIM inhibition, we observed significant downregulation of multiple validated NFκB target genes in HBL-1 and U2932 cells, but not in DHL4 cells. We further confirmed decreased transcript abundance of 4 NFκB target genes (NFKBIA, CD40, TNFAIP 3 and MIR 155) by qPCR. Since NFκB target gene expression in ABC-DLBCL is essential for their survival and can be specifically blocked with the BTK inhibitor ibrutinib, we hypothesized that simultaneous targeting of PIMs and BTK would synergistically inhibit NFkB activity and cell survival. To test this hypothesis, we incubated the ABC-DLBCL cell lines with the ibrutinib, SEL24-B489, or their combination. We identified synergistic growth inhibitory effects of the drug combination in ABC-DLBCL cell lines, with combination index (CI) of 0.51 and 0.2 for HBL-1 and U2932 cells, respectively. Finally, we investigated the efficacy of SEL24-B489 in vivo in the murine xenograft model using U2932 cells. In contrast to progressive tumor growth in animals exposed to vehicle alone, we observed marked inhibition of tumor growth (>90%) in SEL24-B489-treated mice.Conclusions. In conclusion, a novel pan-PIM inhibitor SEL24-B489 induces apoptosis of DLBCL cell lines in low/sub-micromolar concentrations and exhibits activity in a xenograft model. The mechanisms of SEL24-B489 toxicity include blocking of protein translation, induction of Myc degradation, decrease of RNA transcription and attenuation of NFκB activity. Moreover, we show marked synergy between the PIM inhibitor and ibrutinib. Hence, these results provide new insights into mechanism of action of PIM inhibitors and rationale for targeting PIM activity in DLBCLs. DisclosuresCzardybon:Selvita S.A.: Employment. Galezowski:Selvita S.A.: Employment. Windak:Selvita S.A.: Employment. Golas:Selvita S.A.: Employment. Brzozka:Selvita S.A.: Employment. Juszczynski:Selvita S.A.: Other: member of Selvita Scientific Advisory Board.

FOXO1 Activation Is an Effector of SYK and AKT Inhibition in Tonic BCR Signal-Dependent Diffuse Large B-Cell Lymphomas

Introduction: In normal B lymphocytes, B-cell receptor (BCR)-induced activation of PI3K-AKT kinases and subsequent inactivation of FOXO1 is a critical pro-survival component of tonic BCR signaling. In murine models, conditional deletion of FOXO1 protected quiescent peripheral B cells from apoptosis mediated by inducible loss of the BCR, demonstrating that PI3K-AKT-FOXO1 axis plays a central role in B-cell homeostasis. Disruption of the BCR signaling by SYK inhibitor leads also to the apoptosis of BCR-dependent DLBCLs, at least in part via a mechanism involving decreased activity of PI3K/AKT axis. Herein, we investigated the role of FOXO1 in toxicity of BCR pathway/SYK inhibition in human BCR-dependent lymphomas.Methods: BCR-dependent DLBCL cell lines (DHL4, DHL6, Ly1, Ly7) were incubated with SYK inhibitor R406 (4 µM) and AKT phosphorylation, FOXO1 activation and transcriptional activity were assessed by phospho-specific flow cytometry, Western Blot and qPCR. Toxicity of active FOXO1 was assessed by transducing DLBCL cells with a constitutively nuclear FOXO1 mutant. FOXO1 knock-down in DLBCL cell was achieved with shRNA. DREAM cleavage and HRK expression were assessed by Western Blot and qPCR, respectively, in the absence or presence of caspase inhibitors. DLBCL cell viability and apoptosis after incubation with SYK and/or AKT inhibitors R406 and MK2206, were assessed by MTS assay and Annexin V/PI staining. Drug interactions were quantitated by CompuSyn software. The expression of p-SYK and FOXO1 in DLBCL samples was assessed by immunohistochemistry (IHC) in 60 DLBCL patients.Results: Since FOXO1 is a major effector of tonic BCR signaling, we assessed the activity of FOXO1 in DLBCL cells after SYK inhibition. In all tested cell lines, AKT and FOXO1 phosphorylations decreased after incubation with SYK inhibitor. Diminished FOXO1 phosphorylation resulted in its nuclear relocalization and induction of FOXO1-dependent expression of p27, BIM, TRAIL and GADD45A. To assess whether the increased activity of FOXO1 is sufficient to induce apoptosis of DLBCL cells, we transduced DHL4 cells with a constitutively nuclear and transcriptionally active FOXO1-3A mutant. The mutant induced G1/S cell cycle arrest and triggered apoptosis, whereas wild-type FOXO1 did not change proliferation or cellular viability, demonstrating that FOXO1 activation is sufficient to induce apoptosis of DLBCL cells. Next, we assessed the toxicity of SYK inhibitor in cells lacking FOXO1. Cells with depleted FOXO1 exhibited 70% lower sensitivity to SYK inhibitor than control cells (p<0.0001). Since in previous studies expression of the proapoptotic member of BCL2 family, HRK, was required for SYK-inhibitor induced cell death in DLBCL cells, we determined the role of FOXO1 activation in HRK expression. HRK expression was dramatically increased in SYK-inhibitor treated control cells, but not in FOXO1-deficient cells. Consistent with this, SYK inhibitor triggered cleavage of HRK transcriptional repressor DREAM only in control cells, but not in FOXO1-depleted cells. HRK induction was blocked by caspase inhibitors. Since AKT is major kinase regulating both FOXO1 activity and HRK induction, we assessed the combined effects of the AKT inhibitor MK-2206 with R406 and found markedly synergistic toxicity (combination index [CI] 0.5-0.8). Combination of inhibitors in FOXO1-depleted cells did not trigger cell death, highlighting the critical effector role of FOXO1. FOXO1 expression was present in 80% of primary DLBCL samples and correlated with SYK activity (p=0.009). High levels of FOXO1 protein expression were associated with longer OS (log rank, p=0.04).Conclusions: Taken together, our results demonstrate that targeting the BCR signaling at multiple levels is a rational therapeutic strategy. Proapoptotic activity of FOXO1 is an important effector of SYK and AKT inhibition in DLBCLs and its expression is required for SYK-and AKT inhibitor-induced toxicity. The underlying mechanism linking FOXO1 activation and cell death involves caspase-dependent cleavage of transcriptional repressor DREAM and subsequent induction of a proapoptotic BCL2 family member, HRK. DisclosuresNo relevant conflicts of interest to declare.

Abstract 4205: Mass-spectroscopy phosphorylation profiling identified differential activation of signaling molecules via spleen tyrosine kinase in B-cell receptor signaling and stromal cell stimulation in DLBCL and CLL cells that are inhibited by GS-9973

Abstract Dysregulation of protein kinase activity leading to aberrant or excessive downstream signaling is widely believed to underlay the pathogenesis of multiple cancers. The purpose of this study was to identify the spleen tyrosine kinase (SYK)-dependent phosphoproteins that were activated in a diffuse large B-cell lymphoma (DLBCL) cell line, DHL4, and primary chronic lymphocytic leukemia (CLL) cells, upon either B-cell receptor (BCR) activation, stromal cell contact, or in a resting state and demonstrate inhibition with a selective SYK inhibitor, GS-9973. A commercial PTMScan Direct procedure from Cell Signaling Technology was utilized in this assessment. Cell lysates generated from resting, αIgG treatment or HS-5 human stromal cell line contact were digested with trypsin and the resultant peptides were enriched by immunoprecipitation with either an anti-phosphotyrosine or anti-phosphoserine/threonine antibody cocktail, and subjected to mass spectrometric analysis to identify and quantify phosphorylated peptides. The phosphorylated peptide profile was then compared to a similarly treated lysate after incubation with 1 μM GS-9973. Protein phosphorylations that were significantly changed by &amp;lt; or &amp;gt; 2.5-fold with GS-9973 treatment were identified and confirmed, where possible, by western blot of DHL-4 lysates or in primary CLL samples with antibodies specific for the identified sites. PTMScan Direct analysis showed overlapping, but not equivalent, patterns of phosphorylated peptides upon αIgG or stromal cell activation. A shared set of GS-9973 inhibited phosphoproteins were identified including pSyk, pAkt, pMek, pErk, pJnk2, pPKCδ, and pS6 kinase. Western blot analysis validated 13 out of 14 proteins as being modulated by 1 μM GS-9973 treatment. Furthermore, dose-response inhibition was demonstrated for pAkt-T473, pBCR-Y177, pBlnk-Y84, pMek-S217/221, and pErk-T202/Y204 with EC50s ranging from 23 to 50 nM. These data support the concept that Syk inhibition can block multiple signaling pathways known to be important in malignant B-cells function. Furthermore, identification of these sites elucidates possible pharmacodynamic signatures of GS-9973 inhibition that may be followed in clinical investigations of GS-9973 in oncology. Citation Format: Julie A. Di Paolo, Astrid Clarke, Hong Rong, Emma Rousseau. Mass-spectroscopy phosphorylation profiling identified differential activation of signaling molecules via spleen tyrosine kinase in B-cell receptor signaling and stromal cell stimulation in DLBCL and CLL cells that are inhibited by GS-9973. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4205. doi:10.1158/1538-7445.AM2014-4205

Cytotoxic Effect of Bendamustine Combined with Kinase Inhibitors in Hematologic Cell Lines

Abstract 4912Bendamustine, a kind of alkylating agent, has demonstrated a favorable anti-tumor activity both alone and in combination with rituximab in low grade B cell lymphoma. Because the kinase cascades, including B-cell receptor signaling, are necessary for the proliferation and survival of the lymphoma cells, the kinase inhibitors have been developed as molecular target agents of malignant lymphomas. We investigated the cytotoxic effect of bendamustine, both as a single agent and in combination with these new kinase inhibitors in human lymphoid cell lines. We used three B-cell lines, BALL-1 (acute lymphoblastic leukemia), SKLY16 (B-cell lymphoma) and DHL4 (diffuse large B cell lymphoma), and T-cell line THP-6 in this study. These cells were treated with bendamustine alone or combined with enzastaurin (a PKC-β inhibitor), CAL-101 (a p110δ PI3K inhibitor), PCI-32765 (a Btk inhibitor) or R406 (a Syk inhibitor) for 48 hours. Drug-induced cytotoxicity was evaluated using the MTT assay. Cell viability in each experiment was normalized using untreated controls. The treatment with bendamustine alone decreased cell viability in a dose-dependent manner on these three B-cell lines. In contrast, T-cell line THP-6 showed the resistance to bendamusitine. For the combination study, cells were exposed to bendamustine (10μM) and one of these kinase inhibitors at various concentrations simultaneously for 48 hours. CAL-101 and PCI-32765 did not enhance the cytotoxic effect on all of B-cell lines. In BALL-1 and SKLY16 cells, the combination with enzastaurin or R406 resulted in a higher cytotoxic activity than that induced by bendamustine alone. In DHL4 cells, the treatment combined with R406 inhibited cell growth effectively, but not with enzastaurin. To evaluate whether these drug combinations are synergistic, a combination index (CI) was calculated and normalized isobolograms were constructed from non-constant ratio drug combinations using Calcusyn software. The CI values were less than 0. 7 in SKLY16 cells treated with bendamustine and enzastaurin, indicating that these produced synergistic cytotoxic effects in cell line-dependent manner. Our results show that enzastaurin might potentiate the cytotoxic activity of bendamustine in vitro and be a good candidate for the combination with bendamustine. Disclosures:No relevant conflicts of interest to declare.

Abstract 3651: Methylseleninic acid (MSA) is an HDAC inhibitor and suppresses VEGF production in lymphoma cell lines

Abstract Background: Serum selenium (Se) concentration at presentation is prognostic of outcome in patients with aggressive B-cell lymphoma (Last et al., JCO, 2003). We have shown that the Se compound MSA enhanced the efficacy of chemotherapeutic agents at non-cytotoxic concentrations in lymphoma cell lines (Juliger et al., Can Res, 2007). Others have reported that Se enhanced the anti-tumour activity of cytotoxic agents, but reduced toxicity to normal tissue in a xenograft model (Cao et al., Clin Can Res, 2004). In addition, Se has been reported to have antiangiogenic properties including the reduction of tumour microvessel density (Wang et al., Int J Can, 2008). We have therefore investigated the effect of MSA on histone deacetylases (HDACs) and HIF1α, the latter being in part regulated by HDAC activity. Methods: Lymphoma cell lines (RL, SUD4, DHL4) and peripheral blood mononuclear cells (PBMCs) were exposed to MSA in concentration and time-course experiments in normoxic and hypoxic conditions (1% O2). Changes in protein expression were determined by western blotting, HDAC activity measured using an HDAC fluorimetric assay (Biomol), and VEGF levels in cellular supernatants determined using an electrochemiluminescence assay (Meso Scale Discovery). Results: In lymphoma cell lines, exposure to MSA concentrations ≥5μM for 24 hours resulted in acetylation of histone H3 and induction of p21 protein. Acetylation of α-tubulin (mediated by HDAC6, a class II HDAC) occurred to a lesser extent. MSA did not inhibit HDAC activity in an isolated enzyme assay using HeLa cell nuclear extract. However, in whole cells pre-incubated with MSA HDAC activity was inhibited in a concentration-dependent manner. In DHL4 cells, exposure to 10μM and 30μM MSA for 2 hours inhibited HDAC activity by 34% (p=0.01 cf control) and 50% (p=0.02) respectively. 24 hour exposure to MSA concentrations ≥5μM markedly increased histone H3 acetylation in PBMCs. These results suggest that a cellular metabolite of MSA inhibits HDAC activity in intact cells. The effect of MSA on HIF1α expression and VEGF secretion was then investigated. In DHL4 cells, exposure to hypoxia for 24 hours induced HIF1α expression (1.8 fold), an effect not seen in the presence of 20µM MSA. In addition, hypoxia increased VEGF levels to 157% of basal levels, but this was reduced to 43% by 20μM MSA (p=0.007). Conclusions: At clinically relevant concentrations, a cellular metabolite of MSA inhibits HDAC activity and suppresses HIFα induction and VEGF secretion. This effect may account for some of the observed pre-clinical activity with Se species, particularly in animal models. The mechanism of HDAC inhibition is now being investigated. In addition, histone H3 acetylation in PBMCs may serve as a biomarker of Se activity in the clinic. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3651.

Chronic Lymphocytic Leukemia B-Cells Lack or Express Markedly Reduced Levels of the Phosphatase PHLPP, Which Regulates B-Cell Receptor Induced AKT Activation.

The PI3K/AKT pathway plays a central role in regulating cellular growth and survival. This pathway is activated by signals derived from various receptors and is tightly regulated through the action of several phosphatases, including SHIP and PTEN, which hydrolyze the PI3K product PIP3, and the recently identified PHLPP, which directly dephosphorylates AKT. Hyperactivation of the PI3K/AKT pathway has been implicated in the pathogenesis of many types of cancer, including chronic lymphocytic leukemia (CLL) and B-cell lymphoma. In addition, gene expression profiling and real-time RT/PCR analysis have recently shown differential expression of PHLPP mRNA in CLL subsets classified according to the presence of the 13q14 abnormality, with many CLL cases demonstrating absent PHLPP expression altogether. These findings prompted us to compare the levels of PHLPP expression in primary CLL B-cells (n=17) with normal tonsillar B-cells (n=4) and various lymphoma cell lines, including the diffuse large B-cell lymphomas (DLBCL) DHL-4, DHL-6, DHL-8, DHL-10, WSU, Toledo, Ly1, Ly3, Ly7 and Ly18, the Burkitt's lymphoma BJAB and the prolymphocytic leukemia MEC1. Immunoblotting analysis revealed abundant and uniform expression of PHLPP in normal B-cells and in 7 out of 12 investigated lymphoma cell lines. Higher levels were observed in the BJAB, Ly1 and Ly18 cell lines, whereas PHLPP was undetectable in the DLBCL cell lines WSU and Toledo. Remarkably, PHLPP was either not expressed or was expressed at markedly reduced levels in all of the investigated CLL samples, with levels of expression ranging from 0 to 10% of the levels in normal B-cells. In contrast, the levels of expression of the phosphatase SHIP were relatively similar between CLL and normal B-cells. To determine what are the consequences of reduced PHLPP expression on signaling through AKT in malignant B-lymphocytes, we downregulated PHLPP in BJAB and DHL-4 cells by RNA interference. A significant reduction in the levels of PHLPP was achieved in both cell lines, which amounted to 20–40% of the levels in cells transfected with the control siRNA. Immunoblotting analysis of protein extracts from cells transfected with PHLPP and control siRNA did not show a difference in AKT phosphorylation on Ser473 and Thr308, indicating that a reduction in PHLPP expression is not sufficient to augment basal AKT activity. To determine the effects of PHLPP downregulation on agonist-induced AKT activation, we investigated phosphorylation on Ser473 and Thr308 in BJAB and DHL-4 cells stimulated through the B-cell receptor. In both cell lines downregulation of PHLPP resulted in more than a 50% increase in BCR-induced AKT phosphorylation. In contrast, phosphorylation of other signaling molecules that are also activated by BCR crosslinking, such as PLCγ2 and ERK, appeared unaffected by PHLPP downregulation. These data confirm the functional relevance of PHLPP in AKT regulation in B-lymphoid cells and implicate reduced or absent PHLPP expression in CLL B-cells as a potential determinant of BCR-induced AKT signaling in CLL.

Generation of multidrug resistant lymphoma cell lines stably expressing P-glycoprotein

The objective of this study was to generate new P-glycoprotein (P-gp)-expressing multidrug resistant (MDR) cell lines by drug selection. Since our previous studies have been carried out with cells infected with a P-gp-containing vector, it was important to confirm our findings in cells generated by drug selection. In this report, we describe three B-lymphoma cell lines which became drug-resistant by stepwise exposure to vincristine (VCR): Raji cells resistant to 18 nM VCR (R18V), Namalwa cells resistant to 21 nM VCR (N21V) and DHL-4 cells resistant to 12 nM VCR (DHL-4/12V). Cells overexpressed P-gp and continued to express CD19, CD20 and CD22, all of which are targets for monoclonal antibody (MAb) therapy. The P-gp pump in these new cells was functional as determined by the efflux of Rhodamine 123 and DIOC2, and the three cell lines were resistant to several chemotherapeutic drugs. We further determined that their P-gp phenotype was stable in xenografted SCID mice and that the tumors were also resistant to chemotherapy. We will now use these new MDR cells to determine whether monoclonal antibodies against CD19 and -20 can reverse P-gp, as we previously demonstrated using Namalwa cells infected with a human mdr1 gene-containing retrovirus.

Methylseleninic Acid (MSA) Decreases DNA Synthesis, Induces Cell Death, and Sensitizes Both Rituximab Sensitive and Rituximab-Resistant Non-Hodgkin's Lymphoma Cell Lines to the Anti-Tumor Activity of Cisplatin and Gemcitabine.

Selenium (Se) is an essential trace element with antioxidant properties. Examination of the dietary intake of Se in more than 25 countries found an inverse correlation with total age-adjusted cancer mortality. In addition, other investigator had shown that low serum levels of Se correlate with an adverse outcome in patients with diffuse large B-cell lymphoma (DLBCL) undergoing anthracycline-base chemotherapy. Various Se compounds had been developed and are undergoing preclinical testing such as MSA or Sodium selenite (SS). SS has been shown to decrease PKCg activity and induce apoptosis in murine lymphomas. In an attempt to further evaluate the role of Se in human lymphomas we studied the effects of MSA in a panel of rituximab-sensitive (RSCL) and rituximab-resistant cell lines (RRCL). Resistant clones were generated by chronic exposure of Raji, RL or DHL-4 cells to escalating doses of rituximab with (4RH) or without (2R) human complement. Functional assays were performed to demonstrate decrease in rituximab sensitivity on RRCL. In addition, resistance to chemotherapy (CDDP, doxorubicin, vincristine, etc.) agents was demonstrated in RRCL. Lymphoma cells were exposed in vitro to MSA (0 to 5mM) with or without CDDP (0 to 10mM) or gemcitabine (0 to 10mM). Following a 72 hour-period of MSA-drug exposure, changes in mitochondrial potential were determined by alamar blue reduction measured at 30 minutes intervals over a 48 hr period. In addition, changes in DNA synthesis and cell proliferation following drug exposure were performed using standard [3H]-thymidine incorporation assays. MSA induced dose-dependent cell death and a decrease in DNA synthesis in all the cell lines tested (both RSCL and RRCL). Up to a 50% reduction in mitochondrial potential and cell viability was observed in RRCL or RSCL exposed to 5mM of MSA. Incubation of RSCL and RRCL to 5mM of MSA induced synergistic cytotoxic and anti-proliferative effects when combined with CDDP or gemcitabine. Our data demonstrates that MSA is active against various RSCL and RRCL and potentiates the anti-tumor effects of chemotherapy agents. Our findings strongly suggest that MSA added to systemic chemotherapy (i.e. combination of gemcitabine and cisplatin) may result in a novel and potent salvage regimen against relapsed/refractory B-cell lymphomas.

GX15-070 and Bortezomib Induce Up-Regulation of BH3 Single Domain Pro-Apoptotic Proteins Puma and Noxa and Is Associated with Synergistic Anti-Tumor Activity in Rituximab-Sensitive, Rituximab-Resistant Cell Lines (RSCL and RRCL), and Primary Lymphoma Patient Specimens.

The interactions between the members of the BH3 domain family of proteins play an important role in the development, progression, and prognosis in various subtypes of B-cell lymphomas. Therapies that selectively favor a pro-apoptotic environment are attractive strategies to overcome chemotherapy resistance in B-cell lymphomas. We previously reported that by targeting Bcl-2 family proteins with either Bcl-2 anti-sense oligonucleotides or GX15-070, a novel pan-inhibitor of the Bcl-2 family members, improved rituximab and/or chemotherapy activity in vitro and/or in vivo (Ramanarayanan J, et al, BJH 2004; 127:519–30 and Hernandez-Ilizaliturri F, et al, Blood 2006; 108:2502a/2523a). Recently, investigators have demonstrated that the proteasome is an important regulator of various members of Bcl-2 family proteins. In our efforts to increase the therapeutic options for B-cell lymphoma patients we studied the biological effects of GX15-070 in combination with the proteosome inhibitor bortezomib in a panel of rituximab-sensitive (RSCL) and rituximab-resistant cell lines (RRCL). Resistant clones were generated by chronic exposure of Raji, RL, or DHL-4 cells to escalating doses of rituximab with (4RH) or without (2R) human complement. In addition, we utilized lymphoma cells isolated from patients with treatment-naïve or refractory/relapsed diffuse large B-cell lymphomas (DLBCL). NHL cells were exposed in vitro to escalating doses of GX15-070 (0, 2, 5, 10 and 20μM) and/or Bortezomib (0, 2, 10 and 20nM) for 24 and 48 hrs. Changes in the expression of BH3 domain Bcl-2 proteins were studied by Western blot (i.e. Bcl-2, Mcl-1, Bcl-XL, Bad, Bid, Bax, Bak, Puma, and Noxa). NHL cell lines and DLBCL cells isolated from patients were exposed to different concentrations of GX15-070 (0 to 20μM) with or without Bortezomib (0 to 20nM). Cell viability was determined by the Cell Titer-Glow luminescent assay, and DNA synthesis was evaluated by standard [3H]-Thymidine incorporation assays at 24 and 48 hrs. Statistical differences were analyzed by Chi-square test. In vitro exposure of RRCL and RSCL to GX15-070 resulted in a significant up-regulation of Puma while in vitro exposure of the same cells to bortezomib led to a dose- and time-dependent up-regulation of Noxa and Bak. In vitro exposure of RRCL, RSCL, and primary lymphoma specimens to GX15-070 and bortezomib resulted in significant synergistic activity compared to controls. In summary, deregulation of apoptosis by BH3 inhibition with GX15-070 and bortezomib:1.induces the expression of BH3 single domain proteins Puma and Noxa;2.results in cell death and antiproliferation not only in RSCL and RRCL, but also from “treatment-refractory” primary DLBCL patient samples.Our findings strongly suggest that GX15-070 added to bortezomib may result in a novel and potent therapeutic strategy against aggressive B-cell lymphomas.

The Proteasome Inhibitor Bortezomib Acts Independently of p53 and Induces Cell Death via Apoptosis and Mitotic Catastrophe in B-Cell Lymphoma Cell Lines

Bortezomib is a proteasome inhibitor with proven efficacy in multiple myeloma and non-Hodgkin's lymphoma. This study reports the effects of bortezomib in B-cell lymphoma cell lines with differing sensitivity to bortezomib to investigate factors that influence sensitivity. Bortezomib induced a time- and concentration-dependent reduction in cell viability in five lymphoma cell lines, with EC(50) values ranging from 6 nmol/L (DHL-7 cells) to 25 nmol/L (DHL-4 cells) after 72 h. Bortezomib cytotoxicity was independent of p53 function, as all cell lines exhibited mutations by sequence analysis. The difference in sensitivity was not explained by proteasome or nuclear factor-kappaB (NF-kappaB) inhibition as these were similar in the most and least sensitive cells. NF-kappaB inhibition was less marked than that of a specific NF-kappaB inhibitor, Bay 11-7082. Cell cycle analysis showed a marked G(2)-arrested population in the least sensitive DHL-4 line only, an effect that was not present with Bay 11-7082 treatment. Conversely, in DHL-7 cells, bortezomib treatment resulted in cells moving into an aberrant mitosis, indicative of mitotic catastrophe that may contribute to increased sensitivity to bortezomib. These studies show that although bortezomib treatment had similar effects on apoptotic and NF-kappaB signaling pathways in these cell lines, different cell cycle effects were observed and induction of a further mechanism of cell death, mitotic catastrophe, was observed in the more sensitive cell line, which may provide some pointers to the difference in sensitivity between cell lines. An improved understanding of how DHL-7 cells abrogate the G(2)-M cell cycle checkpoint may help identify targets to increase the efficacy of bortezomib.

Investigation of the selenium species distribution in a human B-cell lymphoma line by HPLC- and GC-ICP-MS in combination with HPLC-ESIMS/MS and GC-TOFMS after incubation with methylseleninic acid

A methodology using the on-line combination of micro-HPLC with ICP-MS and electrospray ionisation (ESI) MS/MS, GC-ICP-MS and GC-TOFMS is proposed for investigation of the Se species distribution in lysates and headspace of human lymphoma cell lines after incubation with methylseleninic acid (MSA), which is known as a precursor of methylselenol, a metabolite with anti-cancer properties. The HPLC-ICP-MS results showed significant differences in speciation results of control (untreated) and Se-exposed DHL-4 cells, revealing the presence of three Se compounds in the cell extracts after 10 min incubation with MSA. These compounds were preliminarily identified as Se-methylselenocysteine (SeMC), selenomethionine (SeMet) and gamma-glutamyl-methylselenocysteine (γ-glutamyl-SeMC) by HPLC-ICP-MS on the basis of retention matching with standards. Quantification of Se-compounds in cell lysates was performed by reversed-phase HPLC coupled directly to ICP-MS. Calibration was by the standard additions technique, using peak area measurements of the chromatographic signals by monitoring the 82Se signal. Verification of the presence of SeMC, the major Se compound in the cell extracts, was achieved by on-line HPLC-ESIMS/MS in selected reaction monitoring (SRM) mode. Total selenium concentrations in cell lysates were determined by flow injection analysis coupled with ICP-MS. The capabilities of solid phase micro-extraction (SPME)-GC in combination with ICP-MS and GC-TOFMS were investigated for studying the distribution of volatile Se species in the headspace of the lymphoma DHL-4 cell cultures. A cryogenic oven cooling (COC)-GC system, which led to a significant improvement in the chromatographic performance (retention time, peak shape and consequently, chromatographic selectivity) of compounds with a low boiling point such as dimethylselenide (CH3SeCH3) in comparison with conventional GC was employed for the first time. A number of volatile Se species could be detected by GC-ICP-MS, from which dimethylselenide and dimethyldiselenide (CH3SeSeCH3) could be identified by COC-GC-TOFMS in the headspace of DHL-4 cell lines exposed to methylseleninic acid.

Targeting BH3-Domain Anti-Apoptotic Proteins with GX15-070 Decreases DNA Synthesis, Induces Cell Death and Sensitizes Rituximab-Sensitive and Resistant Non-Hodgkin's Lymphoma Cell Lines to the Anti-Tumor Activity of Chemotherapy Agents.

GX15-070 is a potent pan-bcl-2 inhibitor with known activity against chronic lymphocytic leukemia (CLL) cells and mantle cell lymphoma (MCL) cell lines, currently undergoing phase I testing. Bcl-2 over-expression is associated with chemotherapy resistance and correlates with a poor clinical outcome in diffuse large B-cell lymphoma. Recently, we demonstrated that acquisition of rituximab resistance is associated with deregulation of BH3-domain pro- and anti-apoptotic proteins. This leads to concomitant resistance to multiple chemotherapy agents. Targeting BH3-domain anti-apoptotic proteins with GX15-070 is an attractive strategy to potentially overcome acquired biologic and/or chemotherapy resistance. To this end we studied the effects of GX15-070 in a panel of rituximab-sensitive (RSCL) and rituximab-resistant cell lines (RRCL). Resistant clones were generated by chronic exposure of Raji, RL or DHL-4 cells to escalating doses of rituximab with (4RH) or without (2R) human complement. Functional assays (e.g. ADCC and CMC assays) demonstrated a significant decrease in rituximab sensitivity in RRCL. In addition, resistance to chemotherapy agents (CDDP, doxorubicin, vincristine, etc.) was demonstrated in RRCL. Lymphoma cells were exposed in vitro to GX15-070 (0 to 20mM) with or without CDDP (0 to 10mM) or Doxorubicin (0 to 1mM). Following a 24 and a 48 hour period of drug exposure: induction of apoptosis and the percentage of viability was determined by flow cytometric analysis, Western blotting and trypan-blue staining techniques. In addition, changes in DNA synthesis and cell proliferation following drug exposure were performed using standard [3H]-thymidine incorporation assays. GX15-070 induced a dose-dependent cell death and decrease in DNA synthesis in all the cell lines tested (both RSCL and RRCL). Up to 75% of cell death was observed in all cell lines exposed to 20mM of GX15-070. Anti-tumor activity was seen even at the lowest tested dose of GX15-070 (2mM, 20–25% death cells). Incubation of RSCL and RRCL with 2 or 5mM of GX15-070 induced synergistic cytotoxic and anti-proliferative effects when combined with CDDP and doxorubicin. The IC50 of CDPP and doxorubicin was decrease ten-fold by the concomitant in vitro exposure to GX15-070 in all cell lines tested, including RRCL. Our data demonstrates that GX15-070 is active against both RSCL and RRCL and augments the anti-tumor activity of chemotherapy agents. These results strongly suggest that GX15-070 added to systemic chemotherapy is a potentially valuable and novel therapeutic strategy in the treatment of B-cell NHL.

The immunoglobulin heavy-chain gene 3′ enhancers deregulate bcl-2 promoter usage in t(14;18) lymphoma cells

In t(14;18) lymphomas, bcl-2 is juxtaposed to the immunoglobulin heavy-chain gene (IgH), resulting in increased bcl-2 transcription and resistance to apoptosis. Regulatory elements of both the bcl-2 promoter and the IgH enhancers are believed to play a role in the increased expression of bcl-2 in t(14;18) lymphoma cells. In addition, transcription of the translocated bcl-2 allele is deregulated with activation of the normally minor bcl-2 P2 promoter. The mechanisms involved in the promoter shift from P1 to P2 are not known. We found that the murine IgH 3' enhancers increased bcl-2 P2 promoter activity in an episomal model of the translocation, and IgH enhancer region HS12 had the greatest effect. Quantitative chromatin immunoprecipitation (ChIP) assays revealed that localized histone H3 hyperacetylation of the P2 promoter was observed on the translocated allele in t(14;18) DHL-4 cells and also on the stably transfected bcl-2 promoter-IgH enhancer episomal construct. Analysis of the HS12 enhancer region revealed that a previously identified nuclear factor-kappaB (NF-kappaB) site and a previously uncharacterized downstream Cdx site, both of which are conserved in the human and murine IgH enhancers, were important for its enhancer activity and promoter activation. ChIP assays showed that C/EBPbeta bound to the HS12 Cdx site in vivo, and mutation of this site abrogated the binding of C/EBPbeta. Reduced expression of C/EBPbeta by transfection of small interfering RNA or interference with NF-kappaB activity decreased transcription from the bcl-2 promoters. These results demonstrate that the IgH 3' enhancers, particularly HS12, are important for the deregulation of bcl-2 promoter usage in t(14;18) lymphomas.

Differentiation of tumour and inflammation: characterisation of [methyl-3H]methionine (MET) and O-(2-[18F]fluoroethyl)-L-tyrosine (FET) uptake in human tumour and inflammatory cells

Previous studies suggest that radiolabelled amino acids could be superior to FDG in differentiating tumour and inflammation. Therefore the aim of this study was to investigate the uptake of FET and MET in human tumour and inflammatory cells and to investigate their uptake kinetics. For uptake studies, cells were incubated with 370 kBq FET or 3.7 kBq MET for 15 min. Kinetic studies were performed at variable concentrations of FET and MET. Competitive inhibition studies were done with BCH, MeAIB and L: -serine. All inflammatory cells incorporated more MET than the tumour cells. The uptake of FET, in contrast, was significantly lower in all inflammatory cells than in the tumour cells. In tumour cells the uptake of MET was about five times the uptake of FET. The competitive inhibitors reduced uptake of both tracers to 20-40% in tumour cells and to 70% in inflammatory cells. Kinetic studies showed that MET and FET transport was saturable in all cells except macrophages and followed a Michaelis-Menten kinetic. Highest capacity (V (max)) and affinity (K (m)) for the uptake of MET was observed in granulocytes. Capacity and affinity for FET uptake were highest in the DHL-4 cells. In contrast to MET, FET accumulated to a significantly greater extent in tumour cells than in inflammatory cells. The marked differences between tumour and inflammatory cells concerning FET and MET uptake suggest that FET and MET are substrates of different subtypes of the L system.

Molecular characterization of PS-341 (bortezomib) resistance: implications for overcoming resistance using lysophosphatidic acid acyltransferase (LPAAT)-β inhibitors

PS-341 (bortezomib, Velcadetrade mark) is a promising novel agent for treatment of advanced multiple myeloma (MM); however, 65% of patients with relapsed refractory disease in a phase II study do not respond to PS-341. We have previously shown that lysophosphatidic acid acyltransferase (LPAAT)-beta inhibitor CT-32615 triggers caspase-dependent apoptosis, and can overcome resistance to conventional therapeutics (i.e., dexamethasone, doxorubicin, melphalan) in MM cells. In this study, we therefore determined whether CT-32615 could also overcome resistance to PS-341. We first characterized molecular mechanisms of resistance to PS-341 in DHL-4 cells. DHL-4 cells express low levels of caspase-3 and caspase-8; furthermore, no cleavage in caspase-8, caspase-9, caspase-3, poly ADP-ribose polymerase (PARP), or DNA fragmentation factor 45 was triggered by PS-341 treatment. We have previously shown that PS-341 treatment triggers phosphorylation of c-Jun NH(2)-terminal kinase (JNK), which subsequently induces caspase-dependent apoptosis; conversely, JNK inhibition blocks PS-341-induced apoptosis. We here show that phosphorylation of SEK-1, JNK, and c-Jun are not induced by PS-341 treatment, suggesting that PS-341 does not trigger a stress response in DHL-4 cells. Importantly, CT-32615 inhibits growth of DHL-4 cells in a time- and dose-dependent fashion: a transient G2/M cell cycle arrest induced by CT-32615 is mediated via downregulation of cdc25c and cdc2. CT-32615 triggered swelling and lysis of DHL-4 cells, without caspase/PARP cleavage or TUNEL-positivity, suggesting a necrotic response. Our studies therefore demonstrate that LPAAT-beta inhibitor CT-32615 triggers necrosis, even in PS-341-resistant DHL-4 cells, providing the framework for its evaluation to overcome clinical PS-341 resistance and improve patient outcome.

Interferon (IFN)-β induces apoptotic cell death in DHL-4 diffuse large B cell lymphoma cells through tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)

The effect of interferon (IFN)-α, β and γ on the growth of DHL-4 diffuse large B cell lymphoma cells was studied. IFN-β significantly inhibited the cell growth, and the effect was stronger than that of IFN-α. IFN-γ did not inhibit the cell growth because of lack of IFN-γ receptors. IFN-β caused apoptotic cell death which was accompanied by DNA fragmentation, caspase 3 activation and annexin V binding. IFN-β lead to the expression of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) mRNA. Anti-TRAIL antibody significantly prevented IFN-β-induced apoptosis. It was suggested that IFN-β might cause apoptosis in DHL-4 cells through TRAIL.