scholarly journals Targeting ubiquitin-activating enzyme induces ER stress–mediated apoptosis in B-cell lymphoma cells

2019 ◽  
Vol 3 (1) ◽  
pp. 51-62 ◽  
Author(s):  
Scott Best ◽  
Taylor Hashiguchi ◽  
Adam Kittai ◽  
Nur Bruss ◽  
Cody Paiva ◽  
...  
Keyword(s):  
Er Stress ◽  
B Cell ◽  
Cell Lines ◽  
Cell Lymphoma ◽  
Proteasome Inhibitors ◽  
B Cell Lymphoma ◽  
Proteasome Inhibition ◽  
Cell Of Origin ◽  
Protein Ubiquitination

Abstract Alterations in the ubiquitin proteasome system (UPS) leave malignant cells in heightened cellular stress, making them susceptible to proteasome inhibition. However, given the limited efficacy of proteasome inhibitors in non-Hodgkin lymphoma (NHL), novel approaches to target the UPS are needed. Here, we show that TAK-243, the first small-molecule inhibitor of the ubiquitin activating enzyme (UAE) to enter clinical development, disrupts all ubiquitin signaling and global protein ubiquitination in diffuse large B-cell lymphoma (DLBCL) cells, thereby inducing endoplasmic reticulum (ER) stress and the unfolded protein response (UPR). Activation of the ER stress response protein kinase R (PKR)–like ER kinase and phosphorylation of eukaryotic translation initiator factor 2α led to upregulation of the proapoptotic molecule C/EBP homologous protein and cell death across a panel of DLBCL cell lines independent of cell of origin. Concurrently, targeting UAE led to accumulation of Cdt1, a replication licensing factor, leading to DNA rereplication, checkpoint activation, and cell cycle arrest. MYC oncoprotein sensitized DLBCL cells to UAE inhibition; engineered expression of MYC enhanced while genetic MYC knockdown protected from TAK-243–induced apoptosis. UAE inhibition demonstrated enhanced ER stress and UPR and increased potency compared with bortezomib in DLBCL cell lines. In vivo treatment with TAK-243 restricted the growth of xenografted DLBCL tumors, accompanied by reduced cell proliferation and apoptosis. Finally, primary patient-derived DLBCL cells, including those expressing aberrant MYC, demonstrated susceptibility to UAE inhibition. In sum, targeting UAE may hold promise as a novel therapeutic approach in NHL.

scholarly journals Repurposing dasatinib for diffuse large B cell lymphoma

2019 ◽  
Vol 116 (34) ◽  
pp. 16981-16986 ◽  
Author(s):  
Claudio Scuoppo ◽  
Jiguang Wang ◽  
Mirjana Persaud ◽  
Sandeep K. Mittan ◽  
Katia Basso ◽  
...  
Keyword(s):  
B Cell ◽  
Cell Lines ◽  
Kinase Inhibitor ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Multikinase Inhibitor ◽  
Large B Cell Lymphoma ◽  
Large B Cell

To repurpose compounds for diffuse large B cell lymphoma (DLBCL), we screened a library of drugs and other targeted compounds approved by the US Food and Drug Administration on 9 cell lines and validated the results on a panel of 32 genetically characterized DLBCL cell lines. Dasatinib, a multikinase inhibitor, was effective against 50% of DLBCL cell lines, as well as against in vivo xenografts. Dasatinib was more broadly active than the Bruton kinase inhibitor ibrutinib and overcame ibrutinib resistance. Tumors exhibiting dasatinib resistance were commonly characterized by activation of the PI3K pathway and loss of PTEN expression as a specific biomarker. PI3K suppression by mTORC2 inhibition synergized with dasatinib and abolished resistance in vitro and in vivo. These results provide a proof of concept for the repurposing approach in DLBCL, and point to dasatinib as an attractive strategy for further clinical development in lymphomas.

scholarly journals Establishment of B-Cell Lymphoma Cell Lines Persistently Infected with Hepatitis C Virus In Vivo and In Vitro: the Apoptotic Effects of Virus Infection

2003 ◽  
Vol 77 (3) ◽  
pp. 2134-2146 ◽  
Author(s):  
Vicky M.-H. Sung ◽  
Shigetaka Shimodaira ◽  
Alison L. Doughty ◽  
Gaston R. Picchio ◽  
Huong Can ◽  
...  
Keyword(s):  
Hepatitis C ◽  
B Cell ◽  
Cell Line ◽  
Cell Lines ◽  
Culture System ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Hcv Rna

ABSTRACT Hepatitis C virus (HCV) is a major cause of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. Studies of HCV replication and pathogenesis have so far been hampered by the lack of an efficient tissue culture system for propagating HCV in vitro. Although HCV is primarily a hepatotropic virus, an increasing body of evidence suggests that HCV also replicates in extrahepatic tissues in natural infection. In this study, we established a B-cell line (SB) from an HCV-infected non-Hodgkin's B-cell lymphoma. HCV RNA and proteins were detectable by RNase protection assay and immunoblotting. The cell line continuously produces infectious HCV virions in culture. The virus particles produced from the culture had a buoyant density of 1.13 to 1.15 g/ml in sucrose and could infect primary human hepatocytes, peripheral blood mononuclear cells (PBMCs), and an established B-cell line (Raji cells) in vitro. The virus from SB cells belongs to genotype 2b. Single-stranded conformational polymorphism and sequence analysis of the viral RNA quasispecies indicated that the virus present in SB cells most likely originated from the patient's spleen and had an HCV RNA quasispecies pattern distinct from that in the serum. The virus production from the infected primary hepatocytes showed cyclic variations. In addition, we have succeeded in establishing several Epstein-Barr virus-immortalized B-cell lines from PBMCs of HCV-positive patients. Two of these cell lines are positive for HCV RNA as detected by reverse transcriptase PCR and for the nonstructural protein NS3 by immunofluorescence staining. These observations unequivocally establish that HCV infects B cells in vivo and in vitro. HCV-infected cell lines show significantly enhanced apoptosis. These B-cell lines provide a reproducible cell culture system for studying the complete replication cycle and biology of HCV infections.

Combination Anti-CD74 (Milatuzumab) and CD20 (Rituximab) Monoclonal Antibody Therapy Has in Vitro and in Vivo Activity in Mantle Cell Lymphoma

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 886-886 ◽  
Author(s):  
Lapo Alinari ◽  
Erin Hertlein ◽  
David M. Goldenberg ◽  
Rosa Lapalombella ◽  
Fengting Yan ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
B Cell ◽  
Mantle Cell Lymphoma ◽  
Cell Lines ◽  
Combination Treatment ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Preclinical Model ◽  
Mantle Cell

Abstract Mantle cell lymphoma (MCL) is an incurable B-cell malignancy and patients with this disease have limited therapeutic options. Despite the success of Rituximab in treatment of B-cell malignancies, its use as a single agent or in combination with chemotherapy in MCL has demonstrated modest activity; thus, novel strategies are needed. CD74 is an integral membrane protein expressed on malignant B cells and implicated in promoting survival and growth, making it an attractive therapeutic target. The humanized anti-CD74 monoclonal antibody (mAb), Milatuzumab, (Immunomedics) has shown promising preclinical activity against several human B-cell lymphoma cell lines, but has not been studied in MCL. Since Rituximab and Milatuzumab target distinct antigens lacking known association, we explored a combination strategy with these mAbs in MCL cell lines, patient samples, and in a preclinical model of MCL. Flow cytometric analysis shows that the MCL cell lines Mino and JeKo, and MCL patient tumor cells, express abundant surface CD74 compared to the CD74-negative cell line, Jurkat. Incubation of Mino and JeKo cells with immobilized (goat anti-human IgG) Milatuzumab (5 μg/ml) resulted in mitochondrial depolarization and significant induction of apoptosis determined by Annexin V/PI and flow cytometry (apoptosis at 8hr=38.3±0.85% and 25.4±2.6%; 24hr=73.6±3.47% and 36±3.57%; 48hr=84.9±3.91% and 50.4±4.17%, respectively, compared to Trastuzumab (control). Expression of surviving cells from anti-CD74-treated MCL cells consistently demonstrated marked induction of surface CD74 (MFI 762) compared to control (MFI 6.1). Incubation with immobilized Rituximab (10 μg/ml) resulted in 39.5±2.5% and 37.1±8.35% apoptotic events at 8hr, 58.8±3.14%, 41.2±8.27% at 24hr, and 40.1±1.3% and 45.6±3.25% at 48hr, respectively. Combination treatment of Mino and JeKo cells with Milatuzumab and Rituximab led to significant enhancement in cell death, with 77.6±3.95% and 79.6±2.62% apoptosis at 8hr in Jeko and Mino cells (P=0.0008 and P=0.00004 vs. Milatuzumab alone; P=0.00015 and P=0.001 vs. Rituximab alone); 90.4±3.53% and 76.6±4.3% at 24hr, respectively (P=0.0042 and P=0.0002 vs. Milatuzumab, P=0.0003 and P=0.0027 vs. Rituximab alone); 92.8±0.77% and 85.6±2.62% at 48hr, respectively (P= 0.026 and P=0.0002 vs. Milatuzumab alone, P=0.0000005 and P=0.00008 compared to Rituximab alone, respectively). To examine the in vivo activity of Rituximab and Milatuzumab, a preclinical model of human MCL using the SCID (cb17 scid/scid) mouse depleted of NK cells with TMβ1 mAb (anti-murine IL2Rb) was used. In this model, intravenous injection of 40×106 JeKo cells results in disseminated MCL 3–4 weeks after engraftment. The primary end-point was survival, defined as the time to develop cachexia/wasting syndrome or hind limb paralysis. Mice were treated starting at day 17 postengraftment with intraperitoneal Trastuzumab mAb control (300 μg qod), Milatuzumab (300 μg qod), Rituximab (300 μg qod), or a combination of Milatuzumab and Rituximab. The mean survival for the combination-treated group was 55 days (95%CI:41, upper limit not reached as study was terminated at day 70), compared to 33 days for Trastuzumab-treated mice (95% CI:31,34), 35.5 days for the Milatuzumab-treated mice (95% CI:33,37), and 45 days for the Rituximab-treated mice (95%CI:30,46). The combination treatment prolonged survival of this group compared to Trastuzumab control (P=0.001), Milatuzumab (P=0.0006) and Rituximab (P=0.098). No overt toxicity from Milatuzumab or the combination regimen was noted. A confirmatory study with a larger group of mice and detailed mechanistic studies are now underway. These preliminary results provide justification for further evaluation of Milatuzumab and Rituximab in combination in MCL.

Acadesine Inhibits Proliferation and Induces Apoptosis In Multiple Myeloma Cells, and Synergizes with Standard of Care Antimyeloma Agents

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 5023-5023
Author(s):  
Susana Hernández-García ◽  
Mercè de Frias ◽  
Clara Campàs ◽  
Bruno Paiva ◽  
Enrique M. Ocio ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
B Cell ◽  
Cell Lines ◽  
Plasma Cells ◽  
Cell Lymphoma ◽  
Hematologic Malignancy ◽  
B Cell Lymphoma ◽  
In Vivo Studies ◽  
Mutational Status

Abstract Abstract 5023 Multiple myeloma (MM) is a malignancy characterized by the accumulation of plasma cells. The disease represents the second most common hematologic malignancy and remains incurable, despite recent advances in its treatment. Therefore, studies to develop new therapies are still necessary, particularly in patients with bad prognostic factors, such as 17p deleted/p53 mutated patients. In this study we describe the preclinical activity of 5-Aminoimidazole-4-carboxamide-1–4-ribofuranoside (AICAR or acadesine) in multiple myeloma. Acadesine is an analog of AMP that is widely used as an activator of AMP-kinase (AMPK), a protein that regulates the responses of the cell to energy changes. Acadesine induces apoptosis in different cell types including CLL, mantle cell lymphoma (MCL) and splenic marginal zone B-cell lymphoma (SMZL) cells and tumor cell lines, without affecting primary T lymphocytes. Thus, acadesine is a promising drug for the treatment of B-cell neoplasms. A clinical phase I/II study of acadesine is currently being performed in CLL patients. We studied the effects of acadesine on the MTT metabolization of several multiple myeloma cell lines (MM1S, MM1R, RPMI-8266, RPMI-LR5, U266, U266-LR7, U266 Dox4, MM144, MGG, SJR, OPM-2, NCIH-929). Acadesine inhibited MM cell growth and induced apoptosis, with IC50 values in the micromolar range, and independently of the p53 mutational status. Cancer treatment, including myeloma, is generally based on combinations of drugs with different mechanisms of action. Thus, we studied the effect of acadesine in double combinations with drugs used in myeloma therapy, such as dexamethasone, melphalan, doxorubicin, bortezomib, and lenalidomide. Analyses of these data using the Chou and Talalay method indicated that acadesine was synergistic with dexamethasone (CI values of 0.60), and particularly with lenalidomide (CI values of 0.42). These promising results with double combinations promoted the investigation of triple combinations in the MM1S cell line. Triple combination of acadesine plus dexamethasone plus lenalidomide or bortezomib notably improved the efficacy of the respective double combinations, being the combination of acadesine plus lenalidomide plus dexamethasone especially efficient. Further studies to determinate the mechanism of action, and in vivo studies in MM1S xenograph are ongoing. Disclosures: de Frias: Advancell: Employment. Campàs:Advancell: Employment.

Constitutively Active STAT6 Represses BCL6 in Primary Mediastinal B Cell Lymphoma.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2417-2417
Author(s):  
Olga Ritz ◽  
Jochen K Lennerz ◽  
Karolin Rommel ◽  
Karola Dorsch ◽  
Elena Kelsch ◽  
...  
Keyword(s):  
B Cell ◽  
Cell Lines ◽  
Conflicts Of Interest ◽  
Cell Lymphoma ◽  
Ectopic Expression ◽  
B Cell Lymphoma ◽  
Proximal Promoter ◽  
Constitutively Active

Abstract Abstract 2417 Primary mediastinal B-cell lymphoma (PMBL) is a subtype of diffuse large B-cell lymphoma (DLBCL) that affects predominantly young women (Swerdlow et al. 2008). Despite improvements due to addition of rituximab, which has become state of the art treatment, 20% of PMBL patients succumb to disease progression or relapse. Notably, here are currently no registered trials that are actively recruiting PMBL-patients and a better understanding of the underlying pathobiology may identify novel therapeutic targets and provide an alternative to dose escalation (Steidl and Gascoyne 2011). BCL6 is a key germinal center B-cell transcription factor that suppresses genes involved in lymphocyte activation, differentiation, cell cycle arrest and DNA damage response gene. BCL6 is aberrantly expressed in certain DLBCL subgroups and BCL6 overexpression is sufficient for lymphomagenesis in mice (Cattoretti et al. 2005). In cellular- and murine DLBCL models, targeting of BCL6 via retroinverted BCL6 peptid inhibitor (RI-BPI) appears effective (Polo et al. 2004; Cerchietti et al. 2010). In conjunction with the relatively restricted expression pattern of BCL6, these data collectively suggest BCL6 as a candidate for targeted therapy in BCL6-positive lymphomas. Despite substantial work on BCL6 in lymphomas, the function of BCL6 in PMBL is unknown. To address the BCL6 function in PMBL, we performed BCL6 depletion by siRNA in all three available PMBL cell lines: K1106, U-2940 and MedB-1. We found that BCL6 acts pro-proliferative and anti-apoptotic; however, PMBL models were only partially dependent on and not addicted to BCL6. Given that BCL6 expression in all PMBL cell lines is variable with a notable fraction of BCL6-negative cells, we argued that increasing the fraction of BCL6-positive cells might increase the level of BCL6-dependence. Since IL-4/STAT6 signaling upregulates BCL6 in mouse lymphocytes (Schroder et al. 2002), we treated PMBL cell lines with IL-4 (or IL-13) and, as expected, observed increased phosphorylated (p)STAT6 levels. Surprisingly, the pSTAT6 increase was not associated with higher – but with drastically lower BCL6 protein levels. Moreover, in untreated cells, co-localization studies for pSTAT6- and BCL6 demonstrated staining in mutually exclusive subsets of cells (Figure 1A), suggesting negative interaction between BCL6 and pSTAT6. Other STAT family members were already shown to participate in the transcriptional regulation of BCL6. Thus, we examined binding of STAT6 to the proximal promoter of BCL6 in all PMBL cell lines using shift assay and chromatin immunoprecipitation. We found that STAT6 can bind all five GAS binding sites within the BCL6 promoter in vitro and in all PMBL cell lines STAT6 was bound to proximal BCL6 promoter in vivo. Furthermore, transient STAT6 depletion by siRNA and/or ectopic expression of constitutively active STAT6 confirms that pSTAT6 is sufficient for transcriptional repression of BCL6. Co-localization studies in primary patient samples demonstrated mutually exclusive BCL6/pSTAT6 distribution as a visual hallmark of the repression mechanism (Figure 1B, C). Thus, our data demonstrate for the first time that constitutively active STAT6 transcriptionally represses BCL6 in PMBL. In conjunction with functional data, the delineated repression mechanism may prevent addiction to one single oncogenic pathway (i.e. BCL6) in PMBL. Figure 1. Mutually exclusive distribution of BCL6 and pSTAT6 in PMBL Figure 1. Mutually exclusive distribution of BCL6 and pSTAT6 in PMBL Disclosures: No relevant conflicts of interest to declare.

scholarly journals CD30 Promotes Proliferation Via ERK 1/2 and p38 MAPK Signal Pathways in ABC-Diffuse Large B Cell Lymphoma

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 4337-4337
Author(s):  
Weimin Huang ◽  
Xiaolei Wei ◽  
Yongqiang Wei ◽  
Ru Feng
Keyword(s):  
B Cell ◽  
P38 Mapk ◽  
Cell Lines ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
In Vivo Study ◽  
Signal Pathways ◽  
Large B Cell Lymphoma ◽  
Large B Cell

Abstract Background CD30 was expressed in about 40% diffuse large B cell lymphoma (DLBCL), epically in activated B cell subtype DLBCL(ABC-DLBCL) and implied superior outcome. however, the underlying mechanisms remains unclear. Methods Three ABC-DLBCL cell lines (SuDHL2, TMD8, HBL1) were used for construction of stably CD30 expression cell lines. The proliferation of these cell lines was measured by MTT assay. Cell cycles were detected by Flow Cytometry after staining with PI. Expression of CyclinD1, p27, CDK2, ERK, phosphorylation-ERK, p38, phosphorylation p38 was detected by Western Blot. Nude mice were used in vivo study. Results With CD30 overexpression, the proliferation and division of SuDHL2, TMD8 and HBL1 cell lines were promoted. CyclilnD1, CDK2 were upregulated and p27 was downregulated after of CD30 overexpression. Furthermore, CD30 was knock down by treated CD30 shRNA in CD30 overexpression cell lines. CD30 KD could reverse the the proliferation induced by CD30 expression. CD30 overexpression increased the phosphorylation of ERK and p38 in ABC-DLBCL cells. In vivo study, CD30 overexpression was correlated with cell proliferation and division. Conclusion Taken together, our date showed CD30 promotes proliferation and division by activating ERK 1/2 and p38 MAPK signal pathways, and targeting CD30 may be a novel therapeutic approach for improving the effectiveness of chemotherapy in ABC-DLBCLs. Key words: CD30, ERK 1/2, p38 MAPK, ABC- diffuse large B cell lymphoma Disclosures No relevant conflicts of interest to declare.

scholarly journals Identification of Anti-Lymphoma Biomarkers of Response to the Anti-CD37 Antibody Drug Conjugate (ADC) IMGN529

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 4187-4187 ◽  
Author(s):  
Eugenio Gaudio ◽  
Chiara Tarantelli ◽  
Alberto Arribas ◽  
Luciano Cascione ◽  
Ivo Kwee ◽  
...  
Keyword(s):  
T Cell ◽  
B Cell ◽  
Cell Lines ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
T Cell Lymphoma ◽  
Lymphoma Cell ◽  
Cell Of Origin ◽  
Antibody Drug Conjugate ◽  
Induced Apoptosis

Abstract Background IMGN529 is an antibody drug conjugate (ADC) consisting of an anti-CD37 antibody with direct anti-tumor activity conjugated via a thioether linker to the cytotoxic maytansinoid antimicrotubule agent DM1. IMGN529 has shown pre-clinical (Deckert et al, Blood 2013) and clinical activity in lymphoma (Stathis et al, ASH 2014; NCT01534715). Here, we assessed the anti-tumor activity of IMGN529 on a large panel of B cell and T cell human lymphomas to identify potential biomarkers of response. Methods Fifty-four lymphoma cell lines [diffuse large B cell lymphoma (DLBCL), n.=27; mantle cell lymphoma (MCL), n.=10; anaplastic large T-cell lymphoma, n.=5; marginal zone lymphomas, n=6, others, n=6] were exposed to increasing doses of IMGN529 or to the unconjugated DM1 for 72h. Cell proliferation was measured using the MTT. Apoptosis induction was defined by at least 1.5-fold increase in caspase 3/7 signal activation with respect to controls using the Promega ApoTox-Glo Triplex Assay. CD37 surface expression was assessed by cytofluorimetry. Gene expression profiling (GEP) was done with the Illumina HumanHT-12 Expression BeadChips on untreated cell lines followed by GSEA (NES > |2|, P<0.05, FDR<0.25) and limma t-test (FC> |1.2|; P< 0.05; top 200 up and top 200 down). Results. The IMGN529 median IC50 in the 54 cell lines was 780pM (95%C.I., 263pm-11.45nM). Activity was stronger (P<0.001) in B cell lymphoma cell lines (n= 46; median IC50=450pM; 95%C.I., 150-800pM) than in T cell lymphoma cell lines (n=8; median IC50=22.5nM; 95%C.I., 14-40nM). The median IC50 for DM1 was 30pM (C.I.95%, 20-40pM) with no differences between B and T cell lymphoma origin. IMGN529 induced apoptosis in 33/54 (61%) lymphoma cell lines. Surface CD37 expression was higher in cell lines derived from B than from T cells (P< 0.0001): IMGN529 IC50 values, but not of DM1, were negatively correlated with surface CD37 expression across all cell lines (R=-0.39; P= 0.018), but not within the individual B or T cell subgroups. Among B cell lines, DLBCL cell of origin, TP53 status or the presence of BCL2 translocation did not affect the sensitivity to IMGN529, while IC50s were higher in the presence of MYC translocation (P= 0.043). No association was seen between IMGN529-induced apoptosis or the sensitivity to DM1 with DLBCL cell of origin, TP53 status or the presence of BCL2 or MYC translocations. We then compared the baseline gene expression profiling of DLBCL cell lines that were highly sensitive to IMGN529 (IC50< 800pM; "S") versus less sensitive/resistant DLBCL cell lines (IC50>10nM, "R"), separately for germinal center B cell type (GCB) (S, n=11; R, n=8) and for activated B cell like (ABC) (S, n=4; R, n=3). In both DLBCL groups, MYC targets, genes involved in unfolded protein response, glycolysis and DNA repair were enriched in transcripts more expressed in R than S cell lines. Transcripts associated with low sensitivity included CD44, VIM, ANXA2, BCL2, ANXA2P1, HSP90B1, NFKBIZ, CDK6, BIRC5 in GCB and HSPA1B, HSP90AA1, CADM1, CD86, TUBB2A, TUBG1, NOTCH1 in ABC cell lines. HEBP1, PHB, PSME3, RNU6-15, RPL13 were more expressed in both GCB and ABC R. Genes involved in PI3K/AKT/mTOR, hypoxia, INF-gamma, TNFA signaling via NFKB and in complement were more expressed in S than in R cell lines. Genes associated with sensitivity to IMGN529 comprised: CD37 (IMGN529 target), CD79A, CHI3L2, FAM117B, LPAR5, NFATC1, PTPN22, RBM38, SGPP1, SLC6A16 in both GCB and ABC cell lines; BASP1, CXCR5, BIK, LY86, TLR10, CD86, LCK, CD22, PTPN22, BCL6, PIK3IP1, CDKN2A in GCB; AFF3, PIM1, MGMT, PDE4B, NFKBIE, SYK, FOXO1in ABC. Conclusions. IMGN529 showed a very strong anti-tumoral activity in pre-clinical lymphoma models. High expression of CD37 and mostly genes involved in BCR signalling were associated with sensitivity to IMGN529. Conversely, the presence of MYC translocation, a high expression of MYC targets and of genes known to be involved in drug resistance (BCL2, BIRC5, CDK6, heat-shock proteins, annexins, proteasome and tubulin components) appeared to negatively affect the response to the ADC but also represent therapeutic targets for novel combinations to be explored. Disclosures Rossi: Gilead: Honoraria, Research Funding; Abbvie: Honoraria; Janseen: Honoraria. Sloss:Immunogen Inc: Employment.

scholarly journals A Novel FBXO45-Gef-H1 Axis Controls Oncogenic Signaling in B-Cell Lymphoma

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 711-711
Author(s):  
Anagh Anant Sahasrabuddhe ◽  
Xiaofei Chen ◽  
Kaiyu Ma ◽  
Rui Wu ◽  
Richa Kapoor ◽  
...  
Keyword(s):  
B Cell ◽  
Cell Lines ◽  
Germinal Center ◽  
De Novo ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Lymphoma Cell ◽  
In Vivo Studies

Abstract Introduction: Diffuse large B cell lymphoma (DLBCL) is the most common form of malignant lymphoma and may arise de novo, or through transformation from a pre-existing low-grade B cell lymphoma such as follicular lymphoma (FL). However, the post-translational mechanisms and deregulated pathways underlying the pathogenesis of disease evolution are not fully understood. Methods: We employed integrated functional and structural genomics and mass spectrometry (MS)-driven proteomics which implicated a possible novel tumor suppressor role for a conserved E3 ubiquitin ligase FBXO45 in DLBCL pathogenesis. We generated conditional knockout mice targeting loss of Fbxo45 in germinal center (GC) B-cells using the Cg1-Cre-loxP system and an assortment of CRISPR-mediated knockouts of FBXO45 in B cell lymphoma cells (FL518, BJAB, U2932). We engineered B cell lines (BJAB, U2932) to inducibly express FLAG-tagged FBXO45 to identify candidate substrates of FBXO45 using liquid chromatography-tandem MS. In vitro biochemical and in vivo studies using a variety of genetically-modified lines in xenograft studies in immunodeficient mice were performed to validate observations from proteogenomic studies. Whole genome sequencing (WGS) and genomic copy number studies were interrogated to investigate structural alterations targeting FBXO45 in primary human lymphoma samples. Results: Conditional targeting of Fbxo45 in GCB-cells in transgenic mice resulted in abnormal germinal center formation with increased number and size of germinal centers. Strikingly, targeted deletion of Fbxo45 in GCB-cells resulted in spontaneous B cell lymphomas with (22/22);100%) penetrance and none of the wild-type (WT) littermates (0/20; 0%) developed lymphoma at 24 months. Macroscopic examination revealed large tumor masses, splenomegaly, and lymphadenopathy at different anatomic locations including ileocecal junction, mesenteric, retroperitoneal and cervical lymph nodes and thymus. Next generation sequencing of immunoglobulin heavy chain genes revealed monoclonal or oligoclonal B cell populations. Using proteomic analysis of affinity-purified FBXO45-immunocomplexes and differential whole proteome analysis from GCB-cells of Fbxo45 wt/wt vs Fbxo45 fl/fl mice, we discovered that FBXO45 targets the RHO guanine exchange factor GEF-H1 for ubiquitin-mediated proteasomal degradation. FBXO45 exclusively interacts with GEF H1 among 8 F-box proteins investigated and silencing of FBXO45 using three independent shRNA and CRISPR-Cas9-mediated knockouts in B-cell lymphoma cell lines promotes RHOA and MAPK activation, B cell growth and enhances proliferation. GEF-H1 is stabilized by FBXO45 depletion and GEF-H1 ubiquitination by FBXO45 requires phosphorylation of GEF-H1. Importantly, FBXO45 depletion and expression of a GEF-H1 mutant that is unable to bind FBXO45 results in GEF-H1 stabilization, promotes hyperactivated RHO and MAPK signaling and B-cell oncogenicity in vitro and in vivo. Notably, this phenotype is reverted by co-silencing of GEF-H1. Inducible ectopic expression of FBXO45 triggers accelerated turnover of GEF H1 and decreased RHOA signaling. Genomic analyses revealed recurrent loss targeting FBXO45 in transformed DLBCL (25%), de novo DLBCL (6.6%) and FL (2.3%). In keeping with our observation of prolonged hyperactivation of pERK1/2 consequent to FBXO45 ablation, in vitro and in vivo studies using B-cell lymphoma cell lines and xenografts demonstrated increased sensitivity to pharmacologic blockade with the MAP2K1/2 (ERK1/2) inhibitor Trametinib. Conclusions: Our findings define a novel FBXO45-GEF-H1-MAPK signalling axis, which plays an important role in DLBCL pathogenesis. Our studies carry implications for potential exploitation of this pathway for targeted therapies. Disclosures Siebert: AstraZeneca: Speakers Bureau. Lim: EUSA Pharma: Honoraria.

Exosome Mediated Wnt-Signaling Augments Side Population In Aggressive Lymphoma

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3010-3010
Author(s):  
Raphael Koch ◽  
Martin Demant ◽  
Thiha Aung ◽  
Annemarie Guentsch ◽  
Nina Diering ◽  
...  
Keyword(s):  
Wnt Signaling ◽  
B Cell ◽  
Cell Lines ◽  
Cell Lymphoma ◽  
Side Population ◽  
B Cell Lymphoma ◽  
Canonical Wnt Signaling ◽  
Canonical Wnt

Abstract Introduction Patients with aggressive B-cell lymphoma are treated in curative intention. However, some patients experience fatal relapse, originating from refractory lymphoma cells with the capacity for clonogenic regrowth. We here addressed repopulation capacity of lymphoma cell subpopulations and the mechanisms regulating the populational composition in the growing tumor. Material & Methods We identified side population (SP) cells in diffuse large B-cell lymphoma cell lines and patient samples with the DNA-binding dye Hoechst33342, analyzed clonogenicity in vitro and in vivo and screened for differentially expressed genes and DNA-methylation patterns. A GFP-containing lentiviral vector construct was used to keep track of side population cells cultured among mixed cultures of SP and nonSP cells. Manipulation of canonical wnt-signaling was performed by lentiviral sh-RNA constructs as well as pharmacological tankyrase-inhibition by XAV-939. In vitro data were supported by in vivo experiments using a chorioallantoic membrane-assay. Results Colony assays and suspension cultures of sorted SP and nonSP cells revealed restriction of clonogenic potential to the SP cell population as well as resurgence of nonSP cells from purified SP cell progenitors, while mixed culture assays using a GFP-vector construct tracing the SP vs. nonSP-population revealed homeostasis between the two populations, showing both SP and nonSP cells contributing to either cell compartment. SP cells show enhanced canonical wnt-signaling and increased exosomal secretion of wnt3a. Suppression of canonical wnt-signaling resulted in reduced clonogenicity. Exosome stimulation of DLBCL cell lines resulted in increased clonogenicity, stabilization of beta catenin and enhanced TOP/FOP activity. Conclusion Here we show that tumor cells reversibly switch between states of autonomous and non-autonomous clonogenicity, and that such transitions are regulated by exosome-mediated wnt signaling. Disclosures: No relevant conflicts of interest to declare.

The First in Class, Alkylator-Histone-Deacetylase-Inhibitor Fusion Molecule Edo-S101 in Combination with Proteasome Inhibitors Induces Highly Synergistic Pro-Apoptotic Signaling through UPR Activation and Suppression of c-Myc and BCL2 in Multiple Myeloma

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 4466-4466 ◽  
Author(s):  
Lenka Besse ◽  
Marianne Kraus ◽  
Andrej Besse ◽  
Juergen Bader ◽  
Thomas Mehrling ◽  
...  
Keyword(s):  
B Cell ◽  
Histone Deacetylase Inhibitor ◽  
Cell Lymphoma ◽  
Proteasome Inhibitors ◽  
B Cell Lymphoma ◽  
Proteasome Inhibition ◽  
Apoptotic Signaling ◽  
Ubiquitinated Proteins ◽  
Deacetylase Inhibitor

Abstract Background. EDO-S101 is a first-in-class alkylating, histone-deacetylase inhibitor (HDACi) fusion molecule with dual activity that is currently in Phase I. It structurally combines the strong DNA damaging effect of bendamustine with a fully functional pan-HDAC inhibitor, vorinostat. Bendamustine has substantial activity against B-cell malignancies; vorinostat sensitizes the same type of cancers against alkylators or proteasome inhibitors (PI). Bendamustine combined with the PI bortezomib (BTZ) is active against multiple myeloma (MM). Cytotoxicity of PI in MM relies on excess induction of proteotoxic stress and triggering of the unfolded protein response (UPR). Upon proteasome inhibition, HDACi synergize with PI by interfering with the a-tubulin-mediated transport of poly-ubiquitinated proteasome substrates to lysosomal destruction. Indeed, EDO-S101 has strong synergistic cytotoxicity with PI in vitro against hematological malignancies, including MM, mantle cell lymphoma and ABC type diffuse large B-cell lymphoma. The aim of this work is to characterize the molecular mechanism of action of the synergy of EDO-S101 with PI in comparison to its established structurally related drugs, bendamustine and vorinostat. Methods. The cytotoxic and molecular activity of EDO-S101 in combination with BTZ and other types of PI was assessed in vitro using the RPMI-8226 and several other MM cell lines. HDAC-inhibiting activity, accumulation of poly-ubiquitinated proteins and induction of ER stress, apoptotic signaling and autophagy induction were assessed by quantitative PCR and western blotting. Proteasome activity was measured with activity based probes (ABP). Apoptosis was assessed by AnnexinV/FITC staining with flow cytometry. Cell viability was evaluated by MTS assay. Results. EDO-S101 showed substantially stronger cytotoxicity in combination with PI than melphalan, bendamustine, cyclophosphamide or PI combined with equimolar vorinostat. EDO-S101 had higher HDACi-type of activity, compared to vorinostat, as demonstrated judged in particular by increased a-tubulin acetylation, providing a potential mechanistic basis for its superior synergy with PI. Consistent with this, EDO-S101 alone induced moderate cellular accumulation of poly-ubiquitinated proteins already in the absence of proteasome inhibition, which was potentiated when EDO-S101 was combined with BTZ. EDO-S101 induced activation of the UPR-regulators XBP1 and IRE1 known to control BTZ sensitivity of MM, in contrast to vorinostat or bendamustine alone. Co-treatment with BTZ and EDO-S101 or vorinostat resulted in highly synergistic triggering of the UPR (ATF4, CHOP, BIP). Interestingly, EDO-S101 in addition induced the pro-apoptotic machinery via upregulation of NOXA, downregulation of BCL2 and an increase of the BAX/BCL2 ratio, and also activated autophagy, as evidenced by upregulation of LC3A and LC3B. While this pro-apoptotic signaling of EDO-S101 was highly synergistic with BTZ-induced apoptotic signals, co-treatment with BTZ and vorinostat reduced apoptotic signaling compared to BTZ alone. EDO-S101 reduced c-Myc expression by 60%, while vorinostat had no effect on c-Myc levels. The combination BTZ+EDO-S101 decreased c-Myc levels by approx. 90%, while these levels remained unchanged during treatment with BTZ+vorinostat. Conclusion. EDO-S101 is a first-in-class, dual-mechanism, alkylator-HDAC-inhibitor fusion molecule that combines key structural features of bendamustine and vorinostat. The molecular mode of action of EDO-S101 differs from that of its structurally related drugs by a more effective interaction with a-tubulin, which may in part explain superior synergy with PI. Most importantly, EDO-S101 has a direct pro-apoptotic activity via downregulation of c-Myc and BCL2 while upregulating NOXA, features not observed with vorinostat. This results in highly synergistic signaling with the PI-induced pro-apoptotic effects. EDO-S101 is a promising advancement of bendamustine with molecular features clearly different from and superior to a combination of bendamustine with vorinostat. EDO-S101 should be explored in combination with proteasome inhibitors in particular in poor risk B cell neoplasms with c-Myc overexpression such as aggressive MM, Burkitt lymphoma or "double hit" aggressive B cell lymphoma. Disclosures Besse: Mundipharma-EDO: Other: travel support. Mehrling:Mundipharma-EDO: Employment. Driessen:Mundipharma-EDO: Honoraria, Membership on an entity's Board of Directors or advisory committees; celgene: Consultancy; janssen: Consultancy.

Sign in / Sign up

Export Citation Format

Share Document