Pre-Clinical Evaluation of a Novel DNA Crosslinking Agent, Bo-1055 in B-Cell Lymphoma

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 5483-5483 ◽  
Author(s):  
Eloisi Caldas Lopes ◽  
Fabian Correa ◽  
Elizabeth Peguero ◽  
Srikanth R. Ambati ◽  
Jae Hung Shieh ◽  
...  
Keyword(s):  
B Cell ◽  
Cell Lines ◽  
Conflicts Of Interest ◽  
Cell Lymphoma ◽  
Crosslinking Agent ◽  
B Cell Lymphoma ◽  
Water Soluble ◽  
Therapeutic Window ◽  
Hematopoietic Stem ◽  
Normal Human

Abstract Some B-cell lymphoma including mantle cell lymphoma (MCL) and diffuse large B-cell lymphoma (DLBCL) remain incurable using conventional chemotherapeutic approaches. Therefore it is important that new treatment strategies be developed. We have evaluated the efficacy of a number of novel, third generation, DNA-directed alkylating agents that have DNA specific binding domain by linking DNA-affinic molecules to N-mustard pharmacophore via a urea, carbamate or hydrazinecarboxamide linker (Kapuriya et al. Bioorganic & Medicinal Chem. 19:471–485; 2011). One of these, the water-soluble ureidomustine BO-1055, was screened for toxicity against a panel of human lymphoma cell lines including MCL (JEKO-1, Z-138, HBL-2), DLBCL (OCILy10, OCILy19) and a spontaneous murine B-cell lymphoma. We also screened BO1055 against a panel of normal human cells including mesenchymal stromal cells (IC50 >10uM), bone marrow-derived endothelium (IC50 >10uM) lung basal epithelium, bronchial epithelium and myofibroblasts (IC50s >10uM) and purified cord blood CD34+ cells in suspension culture or colony-forming assay (for hematopoietic progenitor cells) (IC50 >10uM) and in cobblestone area-forming assay (for hematopoietic stem cells) (IC50 9.1uM). The mean IC50±SD (uM) in MCL cell lines was JEKO-1 (0.266±0.27), Z-138 (0.182±0.15), HBL2 (0.161±0.34), In DLBCL lines OCILy10 (0.117±0.21), OCILy19 (0.287±0.17) and murine B-cell lymphoma (0.463±0.39). Our results indicated that BO-1055 has a significant therapeutic window (50-100-fold) between its toxicity against human B-cell lymphomas compared to various normal human cell types. We evaluated BO-1055 cardiotoxicity in the HL-1 cardiomyocyte line and observed a 227-fold less cytotoxicity compared to Doxorubicin. Treatment with BO-1055 resulted in accumulation of cells in S-phase and up-regulation of proteins involved in DNA repair [MRE11, p-P95/NBS1 (ser343), RAD50, p-ATR (ser428)] while Bcl-6, an important B-cell lymphoma biomarker, was down-regulated. Xenograft experiments in NSG mice bearing JEKO-1 GFP/luciferase+ tumors treated with BO-1055 (30mg/kg) 3x/week showed complete tumor remission after 2 weeks of treatment as monitored by luciferase image. Our results suggest that BO-1055 has potent activity against B-cell lymphoma and present a strong rationale for its further therapeutic development as an alkylating agent due to his low toxicity against normal tissue and high toxicity against hematopoietic tumors. Disclosures No relevant conflicts of interest to declare.

Novel Alkylating Agent, Ureidomustine Exhibit Pre-Clinical Efficacy in B-Cell Lymphoma with Minimal Dose-Limiting Myelotoxicity

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1556-1556 ◽  
Author(s):  
Eloisi Caldas Lopes ◽  
Shieh Jae-Hung ◽  
Srikanth Ambati ◽  
Su Tsann-Long ◽  
Fabian Correa ◽  
...  
Keyword(s):  
B Cell ◽  
Cell Lines ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
S Phase ◽  
Water Soluble ◽  
Lymphoma Cell ◽  
Therapeutic Window ◽  
B Cell Lymphomas ◽  
Normal Human

Abstract Many patients with B-cell lymphomas, including mantle cell lymphoma (MCL) and diffuse large B-cell lymphoma (DLBCL), are not cured by conventional chemo-immunotherapy. One reason for this is because these drugs, while effective, are limited by their narrow therapeutic window and significant toxicities. B-cell lymphomas are highly dependent on DNA damage checkpoints, and hence are biologically responsive to drugs that trigger these checkpoints. Hence, In order to identify superior DNA damaging anti-lymphoma drugs we evaluated a series of novel, third generation, DNA-directed alkylating agents that have DNA specific binding domains chemically linked via urea, carbamate or hydrazinecarboxamide to N-mustard pharmacophores. The chemically favorable water-soluble ureidomustine (BO-1055) was evaluated for activity against 23 human lymphoma cell lines including MCL, DLBCL (GCB and ABC subtype) and a spontaneous murine B-cell lymphoma. Fifty % of these MCL and DLBCL cell lines exhibited BO-1055 IC50 values in the nanomolar range including even markedly chemo-resistant cell lines as OCI-Ly10 (IC50 0.117μM ±0.21). In marked contrast, against normal human tissues (lung fibroblast IMR90, kidney fibroblast CV1, mesenchymal stromal, bronchial epithelium, myofibroblast, bone marrow-derived endothelium) and hematopoietic cells (cord blood CD34+ cells, colony-forming assay for hematopoietic progenitors) and in cobblestone area-forming assay for hematopoietic stem cells, BO-1055 IC50s were > 10μM. Hence BO-1055 has a significant therapeutic window (50-100-fold) between its toxicity against B-cell lymphomas compared to normal human cells. We evaluated BO-1055 cardiotoxicity in the HL-1 cardiomyocyte line and observed a 227-fold less cytotoxicity compared to Doxorubicin. Drug combination studies with BO-1055 and an Hsp90 inhibitor (PU-H71), an Hsp70 inhibitor (TT46), doxorubicin and bortezomib (Velcade) demonstrated synergistic effects based on Compusyn analysis with very low combination indices in 50% of lymphoma cell lines. The synergistic effect was not observed in normal cells. Notably, BO-1055 caused downregulation of the critical lymphoma oncoproteins MYC and BCL6, but not Bcl2. BCL6 normally suppresses the ATR-driven S-phase checkpoint. Accordingly treatment with BO-1055 resulted in accumulation of cells in S-phase and up-regulation of proteins involved in DNA repair and intra-S-phase checkpoints [MRE11, p-P95/NBS1 (ser343), RAD50, p-ATR (ser428)]. Finally, xenograft experiments in NSG mice bearing MCL JEKO1 GFP/luciferase+ tumors treated with BO-1055 (30mg/kg) 3x/week showed complete tumor remission after 2 weeks of treatment as monitored by luminescent imaging. In summary, BO-1055 is emerging as a potent therapeutic agent for B-cell lymphomas, with little toxicity against normal tissues and hence potentially wider therapeutic window than current lymphoma drugs. Disclosures Caldas Lopes: BOtique Biopharm: Employment.

NIK Is Involved In the Activation of the Classical and Alternative NF-κB Pathways In Diffuse Large B Cell Lymphoma

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3099-3099
Author(s):  
Lina Odqvist ◽  
Margarita Sánchez-Beato ◽  
Santiago Montes-Moreno ◽  
Ken H Young ◽  
Francesco Acquadro ◽  
...  
Keyword(s):  
B Cell ◽  
Cell Lines ◽  
Conflicts Of Interest ◽  
Cell Lymphoma ◽  
Alternative Pathway ◽  
B Cell Lymphoma ◽  
Classical Pathway ◽  
Strong Positive Correlation ◽  
Large B Cell Lymphoma ◽  
Large B Cell

Abstract Abstract 3099 Deregulated NF-κB activity plays a role in the lymphoma pathogenesis, and has been proposed to constitute a cardinal feature of some subtypes of diffuse large B cell lymphoma (DLBCL). The NF-κB-Inducing Kinase (NIK) is essential for the activation of the alternative NF-κB pathway by inducing the phosphorylation of the NF-κB member p100, which leads to its processing to p52 and its subsequent nuclear translocation. A role for NIK in the classical NF-κB pathway as well has been shown, suggesting NIK as an attractive therapeutic target in lymphomas. Here, we study the frequency and extent of alternative and classical NF-κB activation in diffuse large B cell lymphoma, and the implication of NIK in both pathways. The activation of the classical and alternative NF-κB pathways was present in 28 and 34% of DLBCL cases respectively, as assessed by nuclear expression of p50 (classical pathway) and p52 (alternative pathway) by immunohistochemistry in a series of 301 samples. Activation of both NF-κB pathways was observed in germinal centre B-cell like (GC) and activated B-cell like (ABC) subtypes, with a slight predominance, although not significant, in ABC subtype. In contrast, the levels of p52 and p50 were significantly higher in ABC-DLBCL cell lines than those of GC subtype. The activation of both pathways was mostly overlapped and there was a strong positive correlation between nuclear p52 and p50 (p<0.001). Eighteen % of the cases expressed both p50 and p52 while only 8 and 16% expressed exclusively p50 or p52, respectively. Activation of the alternative NF-κB pathway was strongly associated with Epstein-Barr virus (EBV), since 93% of EBV+ cases expressed nuclear p52 (p<0.001). In our study, no TRAF3 deletions were detected in a panel of 25 DLBCL samples, although absence of TRAF3 was observed in one DLBCL cell line. Since NIK acts as a bottleneck in the activation of the alternative pathway but has also been described to play a role in the classical pathway, we wanted to analyze the effect of the knockdown of NIK on both pathways. Using small interference RNA in two lymphoma cell lines, we observed that the silencing of NIK had an effect on both pathways, decreasing the processing of p100 as well as p105. Taken together, our results show that the activation of NF-κB distinguishes a subset of DLBCL cases, comprising both ABC and GC subtypes, suggest a frequent overlap between the classical and alternative NF-κB pathway in DLBCL, and identify a possible role for NIK in the activation of both pathways. Disclosures: No relevant conflicts of interest to declare.

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.

5-Aza-2-Deoxycytidine Regulates Wnt Beta-Catenin Pathway in B Cell Lymphoma

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 4810-4810
Author(s):  
Xinyu Li ◽  
Lingyu Geng ◽  
Xiangxiang Zhou ◽  
Kang Lu ◽  
Peipei Li ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
B Cell ◽  
Cell Lines ◽  
Mononuclear Cells ◽  
Conflicts Of Interest ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Beta Catenin ◽  
Cell Functions ◽  
Protein Expressions

Abstract Introduction: The Wnt/beta-catenin pathway is aberrantly activated in B cell lymphomas, unphosphorylated beta-catenin accumulates and translocates into the nucleus, regulates the expression of c-myc, cyclinD1 and many other target genes which govern fundamental cell functions, such as proliferation, cell cycle regulation and apoptosis. Methylation is a highlight of epigenetic regulation research which also occurred in lymphoma, but the concrete mechanism of how the demethylation drug 5-aza-2-deoxycytidine affect Wnt/beta-catenin pathway is still unknown. This study was designed to illuminate the implications on Wnt/beta-catenin pathway via demethylation 5-aza in B cell lymphoma. Methods: Peripheral blood mononuclear cells (PBMCs) were obtained from samples of 30 primary CLL patients. The PBMCs contained more than 90% of CD19+ B lymphocytes, which were detected by flow cytometry and were referred to as primary CLL cells. The activation of Wnt/beta-catenin pathway and DNMT-1 of B cell lymphoma cells lines (MEC-1, LY8, Jeko-1, Grant519, mino and sp53) and the 30 patients were detected by qPCR and western blot. The expressions of beta-catenin in 20 cases of B cell lymphoma tissues were measured by IHC. The B cell lines and PBMCs from 10 primary CLL patients were given 5-aza-2-deoxycytidine in different concentrations, the effects in the pathway and apoptosis were observed by WB and flow cytometry. Results: The expressions of beta-catenin, c-myc, cyclinD1 and DNMT-1 were aberrantly higher in all cell lines we used ( MEC-1,LY8, Jeko-1, Grant519, mino and sp53 Fig.1-A,B), most primary CLL patients (Fig.1-C), and B cell lymphoma tissues (Fig.1-D). The protein expressions of beta-catenin in MEC-1 were higer than primary CLL patients. 0, 0.5, 1.0, 2.0¦ÌM 5-aza-2-deoxycytidine were given to the B cells lines and PBMCs from primary CLL patients for 48h, beta-catenin were found accumulated, but c-myc and cyclinD1 in the downstream were reduced (Fig.2-A,B,C,D). For further understanding of aberrant accumulation ofbeta-catenin, we extracted the nuclear protein of MEC-1, nuclear beta-catenin protein expressions were found decreased and cytoplasmic were increased (Fig.2-E). After 5-aza treatment, the apoptosis rate increased and caspase pathway were activated (Fig.2-A,F). Conclusions: The enhanced expressions of beta-catenin, c-myc, cyclinD1 in the B cell lines and the B cell lymphoma samples indicated the Wnt/beta-catenin was aberrantly activated. After 5-aza treatment with the cell lines (MEC-1, Jeko-1, LY8) and primary CLL cells, the abnormal accumulation of beta-catenin protein was observed which was discrepancy with previous reports, but the decrease of c-myc and cyclinD1 suggested the pathway was inhibited, apoptosis also occurred. The increase of totalbeta-catenin protein was supposed to be an stress reaction of the 5-aza treatment, however, the redundant beta-catenin protein in B cell lymphoma was speculated to be combined with demethylated genes and resulted in dormancy of this pathway. Our results indicated that 5-aza played a demethylation role through Wnt/beta-catenin pathway in B cell lymphoma. The data are of interest in the context of epigenetic-based therapy in B cell lymphoma. Figure 1. Figure 1. Figure 2. Figure 2. Disclosures No relevant conflicts of interest to declare.

scholarly journals Imbalance in Alternative Splicing of MCL-1 Inhibits Apoptosis in Diffuse Large B-Cell Lymphoma

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 5424-5424
Author(s):  
Nicolle H Rekers ◽  
Laura M Moesbergen ◽  
Nathalie J Hijmering ◽  
Wim Vos ◽  
Joost Oudejans ◽  
...  
Keyword(s):  
Alternative Splicing ◽  
B Cell ◽  
Cell Lines ◽  
Conflicts Of Interest ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Apoptosis Resistance ◽  
Expression Levels ◽  
Large B Cell Lymphoma ◽  
Large B Cell

Abstract Diffuse large B-cell lymphoma (DLBCL) remains eventually fatal in 30-40% of the patients, despite intensive chemotherapy (CHOP) in combination with rituximab. A subgroup of chemotherapy-refractory DLBCL is characterized by high expression levels of both pro- and anti-apoptotic genes, including MCL-1. Alternative splicing of the MCL-1 gene results in a Bcl-2-like anti-apoptotic MCL-1L protein and a BH3-only pro-apoptotic MCL-1S protein. In the present study, we investigated if a switch in alternative splicing of MCL-1 is involved in apoptosis-resistance in primary lymphoma cells of 20 DLBCL patients and 5 DLBCL cell lines. RT-MLPA analysis revealed that MCL-1L and MCL-1S are both expressed in all tested DLBCL samples and DLBCL cell lines, however expression levels varied strongly. An imbalance between the expression levels of MCL-1L and MCL-1S to an anti-apoptotic status was observed in DLBCL patient cells and DLBCL cell lines, especially in activated B-cell like (ABC)-DLBCL, compared to tonsillar germinal center B-cells. MCL-1 mRNA expression was confirmed at protein level using immunohistochemistry and western blot analysis. Co-immunoprecipitation demonstrated that MCL-1L inhibited apoptosis by binding of Bak in MCL-1L positive DLBCL cell lines. Knockdown of MCL-1L with siRNA analysis resulted in induction of apoptosis in both GCB- and ABC-DLBCL cell lines and also in increased sensitivity to the conventional chemotherapeutical drugs etoposide. Downregulation of MCL-1L using flavopiridol induced apoptotic cell death of MCL-1L-positive DLBCL cells with low Bcl-2 expression. In summary, a switch in alternative splicing of MCL-1 occurs in a subgroup of DLBCL leading to an increase in the level of anti-apoptotic MCL-1L that contributes to therapy-resistance. These preclinical data suggest that targeting of MCL-1L might be a therapeutic option for MCL-1L positive DLBCL. Disclosures No relevant conflicts of interest to declare.

scholarly journals Inhibition of Fatty Acid Synthase Suppresses c-Met Receptor Kinase and Induces Apoptosis in Diffuse Large B Cell Lymphoma.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4787-4787
Author(s):  
Shahab Uddin ◽  
Azhar Hussain ◽  
Prashant Bavi ◽  
Khawla Al-Kuraya
Keyword(s):  
Fatty Acid ◽  
B Cell ◽  
Cell Lines ◽  
Fatty Acid Synthase ◽  
Conflicts Of Interest ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Specific Chemical ◽  
Large B Cell Lymphoma ◽  
Large B Cell

Abstract Abstract 4787 Targeted approaches are expected to revolutionize cancer treatment in near future. Fatty acid synthase (FASN), the enzyme responsible for de novo synthesis of fatty acids has emerged as a potential therapeutic target for several cancers however its role in diffuse large B-cell lymphoma (DLBCL) has not been fully elucidated.. Therefore, we investigated the expression of FASN in tissue micro array cohort of 301 DLBCL patients. FASN was found to be expressed in 62.6% (162/259) DLBCL samples and was seen in highly proliferative tumors manifested by high Ki67 (p<0.0001). Significant association was found between tumors expressing high FASN and c-Met tyrosine kinase (p<0.0002) as well as p-AKT (p=0.0309). In vitro, pharmacological FASN inhibition and SiRNA targeted against FASN triggered caspase dependent apoptosis and suppressed expression of c-Met kinase in DLBCL cell lines which further highlighted the molecular link between FASN and c-Met kinase. Finally, simultaneous targeting of FASN and c-Met with specific chemical inhibitors induced a synergistically stimulated apoptotic response in DLBCL cell lines. These findings provide evidence of an active role of FASN in DLBCL evolution by specifically regulating tyrosine kinases related to malignant transformation strongly suggest that targeting FASN may have therapeutic value in treatment of DLBCL. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Efficacy of Yttrium-90 Iburitumomab Tiuxetan for Early Relapsed/Refractory Indolent B-Cell Lymphoma: A Single Institute Retrospective Analysis in the Rituximab Era

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 5333-5333
Author(s):  
Keiichi Nakata ◽  
Shigeo Fuji ◽  
Ryo Nakata ◽  
Kazuhito Tsutsumi ◽  
Shuhei Kida ◽  
...  
Keyword(s):  
Follicular Lymphoma ◽  
B Cell ◽  
Conflicts Of Interest ◽  
Cell Lymphoma ◽  
Univariate Analysis ◽  
B Cell Lymphoma ◽  
Significant Prognostic Factor ◽  
Hematopoietic Stem ◽  
The Impact ◽  
Yttrium 90

Abstract Introduction: Indolent B-cell lymphoma is a type of non-Hodgkin's lymphoma that grows slowly and is not a curative disease. Yttrium-90 (90Y) iburitumomab tiuxetan is used in patients with relapsed/refractory indolent B-cell lymphoma, data is still limited in the rituximab era. In addition, previous studies did not assess the impact of early progression of disease within 2 years (POD24) which was reported to be associated with a poor prognosis in follicular lymphoma (Casulo et al, J Clin Oncol 2015) on the efficacy of 90Y iburitumomab tiuxetan. Thus, here we assessed the efficacy of 90Y iburitumomab tiuxetan in relapsed/refractory indolent B-cell lymphoma, and analyzed the impact of POD24 in this setting. Methods: We retrospectively analyzed the clinical outcomes of 51 patients with a relapsed/refractory indolent B-cell lymphoma who received 90Y iburitumomab tiuxetan at our institute from February 2009 to January 2018. POD24 was defined as progression within 24 months from the beginning of induction chemotherapy. Survival outcomes including overall survival (OS) and progression-free survival (PFS) were analyzed by Kaplan-Meier analysis and Cox proportional hazard models. Results: The median age was 64 years (range 37-88 years) at the time of receiving 90Y iburitumomab tiuxetan, and 29 (56.9%) were male. Disease subtypes were as follows: follicular lymphoma (n=44, 86.3%), mantle cell (n=4, 7.8%), marginal zone (n=2, 3.9%), and MALT lymphoma (n=1, 2.0%). The median number of previous regimens was 2 (range 1-9) and included rituximab-based therapy in all patients. Disease status at the time of receiving 90Y iburitumomab tiuxetan were as follows: complete remission (CR n=3, 5.9%), partial remission (PR n=13, 25.5%), stable disease (SD n=3, 5.9%), progression disease (PD n=32, 62.7%). The median follow-up time was 3.7 years (range 0.3-8.8 years) and overall response rate (ORR) was 94.1% (CR 56.9%, PR 37.3%). The ORR in patients with POD 24 was 95.0% (CR 60.0%, PR 35.0%), compared to 93.5% (CR 54.8%, PR 38.7%) with non-POD24. The 3-year OS and PFS rates for all patients receiving 90Y iburitumomab tiuxetan were 83.6% and 41.0%, respectively. POD24 was a significant prognostic factor for PFS in univariate analysis (1.2 years in patients with POD24 vs. 3.3 years in patients with non-POD24, (P=0.02) (Figure1). In multivariate analysis, POD24 was an independent prognostic marker of PFS (hazard ratio (HR) 0.43, 95% confidence interval (CI) 0.22-0.87, P=0.02). Conclusion: 90Y iburitumomab tiuxetan was highly effective in patients with relapsed/refractory indolent B-cell lymphoma patients. However, in patients with POD24, duration of response was short. Thus, another treatment strategy including hematopoietic stem cell transplantation should be considered. Disclosures No relevant conflicts of interest to declare.

scholarly journals Targeting Histone Acetyltransferase Gene Inactivation in Diffuse Large B Cell Lymphoma

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 671-671 ◽  
Author(s):  
Stefanie Meyer ◽  
Sofija Vlasevska ◽  
Laura Garcia Ibanez ◽  
Claudio Scuoppo ◽  
Riccardo Dalla-Favera ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
De Novo ◽  
Conflicts Of Interest ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Therapeutic Window ◽  
Acetyltransferase Activity ◽  
Large B Cell Lymphoma ◽  
Large B Cell

Abstract Diffuse Large B-cell Lymphoma (DLBCL) is the most common form of non-Hodgkin lymphoma, accounting for ~30% of de-novo diagnoses and also arising as a frequent clinical evolution of indolent lymphomas. Although curable in a substantial fraction of cases, one third of patients do not achieve durable remissions, highlighting the need for novel, targeted therapies. Over the past decade, we and others have identified the CREBBP acetyltransferase and, less frequently, its paralogue EP300 as highly recurrent targets of inactivating somatic mutations/deletions in DLBCL and follicular lymphoma (FL) (30% and 60% of patients, respectively), indicating a prominent role in the pathogenesis of these tumors (Pasqualucci et al., Nature 2011). In most cases, mutations are heterozygous and the residual wildtype (WT) allele is expressed, suggesting a haploinsufficient tumor suppressor role. Indeed, germinal center (GC)-specific loss of Crebbp perturbs the expression of genes that are relevant to the normal biology of this structure, i.e. the lymphoma cell of origin, and cooperates with BCL2 deregulation to increase the incidence of tumors recapitulating the features of the human disease (Zhang et al., Cancer Discovery 2017). Intringuingly, while CREBBP binds to virtually all GC-specific superenhancers, no detrimental effects were observed upon its deletion in mice, suggesting the existence of compensatory mechanisms. Consistent with this hypothesis, inactivation of CREBBP and EP300 rarely coexist in human DLBCL and FL, suggesting that cells require a certain amount of acetyltransferase activity. To investigate whether EP300 compensates for CREBBP loss in the GC, we analyzed the GC responses in compound mouse models engineered to specifically delete these two genes (alone and in combination) upon SRBC immunization and induction of a Cγ1-driven Cre-recombinase. While CrebbpKOmice showed a mild increase in GC formation, as reported, loss of Ep300 led to ~40% reduction in the percentage of GC cells (mean: 1.8% vs 3.1% in WT littermates; p<0.05), documenting that these two enzymes play non-entirely overlapping roles in this population. Importantly, GC formation was completely abrogated in CrebbpKOEp300KO mice and dramatically impaired in CrebbpHETEp300KO mice, as compared to both WT and single EP300KO mice. These data suggest that GC B cells require a minimum amount of acetyltransferase activity, and reveal a potential therapeutically exploitable dependency of Crebbp-mutated GC B cells on Ep300. In order to probe if a similar dependency exists in neoplastic GC B cells, we used an inducible CRISPR/Cas9 system to delete EP300 (or a control non-genic region) in 4 DLBCL cell lines representative of the various CREBBP genotypes found in DLBCL, and monitored cell proliferation and survival in competition assays over 12 days. Compared to CREBBPWT, CREBBP heterozygous and homozygous mutant cells were significantly counter-selected from the total population following doxycycline induced EP300 deletion (~30% at day 7). Moreover, no EP300-edited clones were recovered from the CREBBP mutant lines in single cell plating assays, compared to CREBBP WT (p<0.01). Thus, DLBCL cells remain addicted to the residual EP300 aceyltransferase activity, supporting the existence of a therapeutic window for EP300 inhibitors. To explore this concept further, we generated isogenic DLBCL clones carrying WT or defective CREBBP alleles (n=4 each), and performed drug-sensitivity assays with 2 novel small molecule inhibitors that specifically target the CREBBP/EP300 HAT or BRD domain. While, at higher doses, both inhibitors interfered with cell growth in all clones, CREBBPKO cells were significantly more sensitive than their isogenic WT pairsat low nanomolar ranges (IC50: 60nM vs 300nM). Importantly, we were able to design an in vitro protocol that was toxic to CREBBPKO cells but tolerated by CREBBPWT cells, providing a proof of concept for therapeutically targeting these molecules. In conclusion, we show that CREBBP and EP300 have differential roles in normal GC B cell development and that CREBBP mutated cells are addicted to the residual EP300 activity. This dependency is maintained in DLBCL cells, providing the basis for the potential application of acetyl transferase inhibition into the clinical settings. Disclosures No relevant conflicts of interest to declare.

scholarly journals HIF-1a Regulating the Chemoresistance By Upregulating the Expression of xCT and GCLM in the Diffuse Large B Cell Lymphoma

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 5236-5236
Author(s):  
Yongqiang Wei ◽  
Hong Zeng ◽  
Xiaolei Wei ◽  
Weimin Huang ◽  
Jialin Song ◽  
...  
Keyword(s):  
Drug Resistance ◽  
B Cell ◽  
Cell Lines ◽  
Conflicts Of Interest ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Response To Treatment ◽  
Chop Regimen ◽  
Large B Cell Lymphoma ◽  
Large B Cell

Background Diffuse large B-cell lymphoma (DLBCL) is the most common subtype of non-hodgkin lymphoma with great heterogeneity in clinical behavior and response to treatment. Although the addition of rituximab to CHOP regimen has significantly improved the survival of DLBCL, 1/3 will be eventually relapse and progression. HIF1a has been reported to be related with the drug resistance in DLBCL, but the mechanism remains unknown. Methods Two DLBCL cell lines (Riva and SuDHL2) were treated with doxorubicin and HIF-1a inhibitors digoxin, YC-1. The proliferation of these cell lines (Riva and SuDHL2) were measured by MTT and apoptosis were detected by FCM after staining with Annexin V/SYTOX Green. Expression of IF-1α, PHD, xCT, GCLM and apoptosis-related proteins was detected by Western Blot. Results With the increase concentration of doxorubicin treatment, the proliferation of Riva and SuDHL2 cell lines could be gradually inhibited. xCT and GCLM expression were upregulated after treated with doxorubicin and can be reversed by N-acetylcysteine. HIF-1a inhibitors digoxin and YC-1 could inhibited the expression of xCT and GCLM, proliferation and apoptosis induced by doxorubicin. Furthermore, we treated shHIF-1a RNA to significantly reduce the expression of HIF-1a, and the decrease of HIF-1a expression enhanced the proliferation inhibition and apoptosis of lymphoma cells by Dox. Downregulation the expression of HIF-1a by shRNA enhanced the doxorubicin induced apoptosis and inhibited the proliferation in DLBCL cell lines. Conclusions Taken together, our results showed that HIF-1a could regulate the expression of xCT and GCLM and further mediate drug resistance in the diffuse large B cell lymphoma. Disclosures No relevant conflicts of interest to declare.

Curcumin Enhances the Sensitivity of Bortezomib By Inhibiting NF-Κb in ABC Diffuse Large B-Cell Lymphoma Cells

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 5237-5237
Author(s):  
Xiaolei Wei ◽  
Jialin Song ◽  
Yongqiang Wei ◽  
Hong Zeng ◽  
Weimin Huang ◽  
...  
Keyword(s):  
B Cell ◽  
Cell Lines ◽  
Conflicts Of Interest ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Response To Treatment ◽  
Proliferation Inhibition ◽  
Cell Type ◽  
Large B Cell Lymphoma ◽  
Large B Cell

Background Diffuse large B-cell lymphoma (DLBCL) is the most common subtype of non-hodgkin lymphoma with great heterogeneity in clinical behavior and response to treatment. According to gene expression profile, DLBCL can be divided into at least two subtypes: germinal center B cell type (GCB) and activated B cell type (ABC). ABC-type DLBCL is commonly activated by the NF-κB pathway, but the proteasome inhibitor bortezomib does not significantly improve the prognosis of ABC-DLBCL. Curcumin inhibit the proliferation of various tumor cells by inhibiting the activation of NF-κB. Our purpose was to evaluate whether curcumin could enhance the ensitivity of Bortezomib in ABC-DLBCL. Methods MTT assay was used to evaluate the proliferation of 2 ABC-DLBCL cell lines by treated with curcumin and bortezomib. Apoptosis were detected by FCM after staining with Annexin V/SYTOX Green.Western Blot was used to evaluated the expression of PARP, NF-κB, I κBα/p-IκBα and caspase-3 in ABC-DLBCL cells treated with curcumin and bortezomib. Results Both curcumin and bortezomib could inhibit the proliferation and induce apoptosis in ABC-DLBCL cell lines. Curcumin decreased the expression of p-IκBα, NF-κB/p65 and increased the expression of Cleaved PARP in ABC-DLBCL cell lines. Caspase Inhibitor Z-VAD could reverse the curcumin induced proliferation inhibition and apoptosis by decreasing the cleaved PARP expression. The combination of curcumin and bortezomib could further enhance the proliferation inhibition and apoptosis in ABC-DLBCL cell lines by inhibiting NF-κB. Conclusions Curcumin induced proliferation inhibition and apoptosis by inhibiting NF-κB in ABC-DLBCL. It may sensitize ABC-DLBCL cell lines to the cytotoxic effects of bortezomib by inhibiting NF-κB. Disclosures No relevant conflicts of interest to declare.

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