Histone 3 Methyltransferase (EZH2) Inhibition Enhances TRAIL-Induced Apoptosis In Mantle Cell Lymphoma Cells By Accelerated cFLIP Degradation

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4425-4425 ◽  
Author(s):  
Frank K Braun ◽  
Rohit Mathur ◽  
Lalit Sehgal ◽  
Zuzana Berkova ◽  
Felipe Samaniego
Keyword(s):  
Mantle Cell Lymphoma ◽  
Cell Lines ◽  
Conflicts Of Interest ◽  
Cell Lymphoma ◽  
Parp Cleavage ◽  
Caspase 8 ◽  
Apoptosis Resistance ◽  
Lymphoma Cells ◽  
Mantle Cell ◽  
Trail Sensitivity

Introduction Mantle cell lymphoma (MCL) is an aggressive form of non-Hodgkin lymphoma that is characterized by the t(11:14)(q13:p32) translocation. MCL cells have altered cyclinD1 levels, impaired cell cycle regulation, DNA damage response, and likely defects in apoptosis signaling. Furthermore, up-regulated anti-apoptotic mediators such as the target of NF-κB c-FLIP were correlated with decreased apoptosis signaling. Also many cancer cells and malignant tumors show a prevalent resistance to apoptosis induction by TRAIL. Thus, by understanding the underpinnings of apoptosis resistance, we will be in a better position to develop strategies that improve TRAIL-induced killing of lymphoma cells. Methods/Results MCL cell lines (Mino, JeKo-1, JVM-2 and Z-138) were treated with DZNep (3-Deazaneplanocin A; 0.2-5µM) for 24 h followed by incubation with TRAIL (10-20ng/ml, 6-16h). Cell death, DNA fragmentation, and mitochondrial membrane potential (Δψm) were determined by calcein staining, subG1 analysis, and TMRM staining, respectively. Neither DZnep alone nor in combination with TRAIL showed a significant induction of necrosis as determined by LDH-release levels, but DZNep alone showed strong antiproliferative properties at higher concentrations (Promega CellTiter 96 assay). Activation of the caspase signaling cascade (caspase-8, -9, -3, Bid, and PARP cleavage) was analyzed by Western blotting. TRAIL-induced signaling was significantly increased and caspase-8 processing enhanced in DZNep pretreated cells indicating a regulation at the TRAIL/DISC. Although a reduced expression of DR5 in total cell lysates of DZNep treated cell was observed, the surface receptor levels were not altered. Interestingly, downregulation of the well-known caspase inhibitor, cFLIP, correlated with the DZNep-induced increased TRAIL sensitivity in MCL cell lines. However, it appears that cFLIP levels are not reduced due to blocked NF-kB signaling but rather by an accelerated ubiquitin-mediated degradation. Conclusions This study reveals that inhibition of histone methyltransferase (EZH2) activity by DZNep has a profound positive impact on TRAIL signaling; it enhances TRAIL sensitivity by promoting processing of caspase-8 through enhanced cFLIP degradation. The capacity of DZNep to target stability of cFLIP, which represents a center piece in DISC regulation underscores its potential for enhancing efficacy of TRAIL-based cancer therapies. Disclosures: No relevant conflicts of interest to declare.

The Tak-1 Inhibitor AZ-Tak1 Inhibits XIAP, Activates Caspase-9, and Induces Apoptosis in Mantle Cell Lymphoma.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1692-1692 ◽  
Author(s):  
Daniela Buglio ◽  
Sangeetha Palakurthi ◽  
Katharine F. Byth ◽  
Anas Younes
Keyword(s):  
Mantle Cell Lymphoma ◽  
Cell Lines ◽  
Small Molecule ◽  
Cell Lymphoma ◽  
Small Molecule Inhibitor ◽  
Caspase 9 ◽  
Lymphoma Cells ◽  
Molecule Inhibitor ◽  
Mantle Cell ◽  
Induced Apoptosis

Abstract Abstract 1692 Poster Board I-718 Transforming growth factor-b-activated kinase 1 (TAK1) is a key regulator of NF-kB activation. TAK1 can be activated by a variety of pro-inflammatory cytokines and T and B cell receptors. Recent experiments demonstrated that deletion of TAK1 results in inactivation of both JNK and NF-kB signaling resulting in massive apoptotic death of hematopoietic cells in mice. In this study, we examined the expression pattern of TAK1 and its role as a potential therapeutic target for lymphoma. First, we examined TAK1 expression in a panel of lymphoid cell lines by western blot, and found it to be highly expressed in mantle cell lymphoma cell lines (Mino, SP53, and Jeko-1). These lines expressed relatively low levels of the tumor suppressor protein A20. Mino and SP53 expressed high level of p-p38. Subsequently, we investigated the in vitro activity of the novel TAK1 small molecule inhibitor AZ-Tak1 in these cell lines. AZ-Tak1 is a potent and a relatively selective inhibitor of TAK1 kinase activity, with an IC50 of 0.009 mM. It also inhibits Jak2 but at a much higher concentration (IC50=0.18 mM). AZ-Tak1 treatment decreased the level of p38 and ERK in mantle cell lymphoma cells, and induced apoptosis in a dose and time dependent manner, with an IC50 of 0.1-0.5 mM. Using the annexin-V and PI staining and FACS analysis, After 48 hours of incubation, AZ-Tak1 (0.1 mM) induced apoptosis in 28%, 34% and 86% of Mino, SP53, and Jeko cells, respectively, which was increased to 32%, 42%, and 86% when 0.5 mM concentration was used. Similar activity was also observed when primary mantle cell lymphoma cells were examined. Using pathway-specific protein arrays focusing on apoptosis, kinases, and transcription factors, AZ-Tak1 (0.5 mM) altered the level of several proteins that regulate cell growth and survival, especially members of the inhibitors of apoptosis (IAP) family. Specifically, AZ-Tak1 decreased the level of SMAC/DIABOLO and cytochrome –C in the mitochondria, which was associated with a decrease in the level of the anti-apoptotic protein X-linked IAP (XIAP) and activation of the intrinsic apoptotic pathway as evident by activation of caspase 9, cleavage of caspase 3, and induction of apoptosis. Furthermore, and consistant with its ability to inhibit Jak2 activity, AZ-Tak1 reduced STAT2 and STAT6 levels. AZ-Tak1 demonstrated no significant effect on bcl-2 family members. Finally, co-treatment with HDAC inhibitors demonstrated synergistic effect with low concentrations of AZ-Tak1. Collectively, our data demonstrate that targeting TAK1 by the small molecule inhibitor AZ-Tak1 induces cell death in mantle cell lymphoma by activating the intrinsic apoptosis pathway, suggesting that targeting TAK1 may have a therapeutic value for the treatment of mantle cell lymphoma. Disclosures Palakurthi: Astra Zeneca: Employment. Byth:Astra Zeneca : Employment.

Rictor Overexpression and mTORC2 Signaling in Chronic Lymphocytic Leukemia

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3884-3884 ◽  
Author(s):  
Gwen Jordaan ◽  
Wei Liao ◽  
Joe Gera ◽  
Sanjai Sharma
Keyword(s):  
Western Blot ◽  
Mantle Cell Lymphoma ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Conflicts Of Interest ◽  
Cell Lymphoma ◽  
Parp Cleavage ◽  
Mantle Cell ◽  
Bcr Signaling ◽  
Mtorc2 Complex

Abstract Abstract 3884 Signaling via the B-cell receptor (BCR) stimulates growth and survival of CLL leukemic cells and inhibits apoptosis by phosphorylating immunoreceptor tyrosine based activation motifs. This signaling subsequently activates PI3 Kinase/AKT, mTOR, ERK and other pathways. Activation of Akt in turn requires phosphorylation by mTOR kinase, which assembles in two complexes mTORC1 and mTORC2 and it is the mTORC2 complex that phosphorylates and activates Akt. This phosphorylation of Akt in CLL specimen's upregulates anti-apoptotic proteins such as Mcl-1, Bcl-xl and XIAP. We have identified that Rictor, a component of mTORC2 complex is over-expressed in CLL specimens as compared to normal peripheral mononuclear B cells. This over-expression was noted by real time PCR that showed 1.5 to 4 fold upregulation (n=12). Western blot analysis also showed Rictor overexpression in all the twelve CLL specimens tested. Rictor overexpression was also seen in Mantle cell lymphoma cell lines and to study its role in BCR signaling, stable Mantle cell lymphoma lines with SiRNA mediated Rictor knockdown were established. Rictor knockdown resulted in a significant decrease in Akt activation as phosphorylation (phospho S473) of Akt both in unstimulated cells and when the cells were stimulated with BCR crosslinking was decreased. To determine the effect of Rictor and mTORC2 inhibition on CLL specimens, we tested the activity of three compounds isolated via yeast two hybrid drug screen designed to identify molecules that inhibit Rictor/mTOR interaction. When tested on CLL specimens in the presence of BCR crosslinking, these mTORC2 inhibitor compounds inhibited the downstream phosphorylation of Akt S473. Functionally the inhibitors also induced apoptosis in CLL cells with 40–60% of CLL cells undergoing apoptosis (1.0mM, cells treated for 48 hours). In comparison, rapamycin (mTORC1 inhibitor) and ppp242 (mTORC1, 2 inhibitor) were comparatively less active in CLL specimens as they were less effective in the induction of apoptosis. Western blot analysis of mTORC2 inhibitor treated cells also showed PARP cleavage and an increase in the pro-apoptotic protein BAD. Our data indicates that Rictor overexpression in CLL specimens is required for Akt phosphorylation activation and downstream BCR signaling. Inhibition of this pathway by mTORC2 inhibitors in CLL will be an effective therapeutic strategy. Disclosures: No relevant conflicts of interest to declare.

Novel CRM-1 Inhibitors for Therapy In Mantle Cell Lymphoma

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2734-2734
Author(s):  
Kejie Zhang ◽  
Lan V Pham ◽  
Liang Zhang ◽  
Archito T. Tamayo ◽  
Zhishuo Ou ◽  
...  
Keyword(s):  
B Cells ◽  
Western Blot ◽  
Mantle Cell Lymphoma ◽  
Cell Lines ◽  
Cell Lymphoma ◽  
Parp Cleavage ◽  
Mantle Cell ◽  
Dose Dependent

Abstract Abstract 2734 Chromosomal Region Maintenance 1 (CRM1) overexpression has been associated with cancer progression and mortality in several human cancers, suggesting that activation of nuclear export may play a role in human neoplasia and may serve as a novel target for the treatment of cancers. This overexpression of CRM1 may be related to the export of most tumor suppressor and growth regulatory proteins out of the nucleus, thereby functionally inactivating them. Mantle cell lymphoma (MCL) is an aggressive histotype of B-cell non-Hodgkin lymphoma that is not yet curable. The objective of our study was to investigate the status of CRM1 in MCL, both in MCL cell lines and primary MCL cells, in comparison to normal B cells, and to evaluate the therapeutic efficiency of CRM1 inhibition in MCL in vitro and in vivo, and to elucidate the mechanism of CRM1 inhibitor-mediated MCL cell apoptosis. We used 8 established MCL cell lines and primary cells from 4 patients with relapsed/refractory MCL. KPT185 and KPT276 are novel, highly selective, drug-like small molecular CRM1 inhibitors. Western Blot analysis showed that CRM1 was expressed in both the cytoplasm and nuclei of 8 MCL cell lines. CRM1 was mainly detected in nuclei of normal resting B cells; In contrast, CRM1 was primarily detected in the cytoplasm of freshly isolated primary MCL cells from patients with relapsed/refractory MCL. In 3H-thymidine incorporation assays, inhibition of CRM1 by KPT185 resulted in a significant dose-dependent growth inhibition of 8 MCL cells, with IC50 values range between 10 nM to 120 nM. The blastoid-variant MCL cell lines (Z-138 and Rec-1) were significantly more sensitive to KPT185 than the non-blastoid variant MCL cell lines. Flow cytometry analysis with fluorescence-labeled Annexin V and propidium iodide showed that KPT185 induced MCL cells apoptosis in both time- and dose-dependent manners, but had no effect on cell cycle arrest. MCL cells treated with KPT185 for 12 hours showed caspase 3 activation and PARP cleavage. As shown in Western blot and confocal microscopy, blocking CRM1 activity by KPT185 in MCL cells up-regulated the protein expression of p53, a known CRM1-mediated export protein, and also induced CRM1 translocation to the nucleus and decreased CRM1 expression. In severe combined immunodeficient (SCID) mice bearing palpable Z-138 tumors, treatment with KPT-276 (similar structure to KPT-185 but improved animal pharmacokinetics), 50mg/kg or 150 mg/kg PO QDx5 each week, or cyclophosphamide 100 mg/kg on days 1–3, was initiated. Tumor growth was significantly inhibited (>75%) in all of treatment groups compared with vehicle control. Neutropenia and other cytotoxic-agent specific effects have not been observed in treated animals. In conclusion, CRM1 inhibitors inhibited growth of MCL cells in vitro and in vivo, and induced apoptosis of MCL cells via inhibition of CRM1 expression and blockage of its translocation with functional nuclear proteins. Our data suggest that novel CRM1 inhibitors provide a potential therapy for patients with relapsed/refractory MCL. Disclosures: No relevant conflicts of interest to declare.

Vitamin D3 and Lenalidomide Synergize To Induce Apoptosis In Mantle Cell Lymphoma Via Demethylation Of BIK

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1830-1830
Author(s):  
Carole Brosseau ◽  
Christelle Dousset ◽  
Cyrille Touzeau ◽  
Sophie Maiga ◽  
Philippe Moreau ◽  
...  
Keyword(s):  
Cell Death ◽  
Mantle Cell Lymphoma ◽  
Cell Lines ◽  
Vitamin D3 ◽  
Conflicts Of Interest ◽  
Cell Lymphoma ◽  
Treatment Options ◽  
Annexin V ◽  
Dna Demethylation ◽  
Mantle Cell

Abstract Among new treatment options for mantle cell lymphoma (MCL), the targeted drug lenalidomide appears as one of the most efficient molecules. Lenalidomide has multiple modes of action targeting the tumor cell and its environment including the immune system. It is widely reported that cancer patients are deficient in vitamin D3 (1,25-dihydroxyvitamin D3, VD3) and recent studies have shown in non Hodgkin lymphomas (NHL) that VD3 levels have a prognostic value on survival (Drake, J Clin Oncol. 2010;28:4191). While the relations between VD3 and cancer incidence remain unresolved, it has been shown that VD3 displays anti-tumoral properties via its anti-proliferative, pro-differentiation, anti-inflammatory and anti-angiogenic properties. We assessed the efficacy of VD3 to potentiate cell death induced by lenalidomide in MCL cell lines and patients’ samples and explored the mechanisms of cell death in this context. Experiments were conducted on a panel of 6 MCL cell lines (JEKO-1, MINO, GRANTA-519, UPN-1, REC-1 and Z138) and 8 primary peripheral blood samples. After 6 days of treatment, MCL cells were weakly sensitive to low doses of lenalidomide (1µM and 10µM for cell lines and samples, respectively). Addition of physiological doses of VD3 (100nM) significantly and synergistically increased cell death in 67% of cell lines (Z138, JEKO-1, MINO, REC-1) and in 63% of primary samples (p<0.05). However resistance to lenalidomide alone was not reversed by VD3 since both GRANTA-519 and UPN-1 remained unsensitive. Apoptosis, characterized by Annexin V staining, appearance of a subG1 peak and caspase 9 activation, was dependent on Bax expression, since transient extinction of BAX by siRNA in JEKO-1 cells inhibited cell death (mean of inhibition 30%±5%, p=0.03). The combination of lenalidomide and VD3 dramatically increased expression of the BH3-only Bik (Bcl2-Interacting Killer) protein in sensitive (Z138, JEKO-1, MINO, REC-1) but not resistant (GRANTA-519, UPN-1) cell lines, without affecting the expression of other molecules of the Bcl2 family. By immunoprecipitation assays, we showed that induced-Bik was not bound to the anti-apoptotic molecules Bcl2, BclxL or Mcl1 in treated cells but was free to activate such pro-apoptotic molecules as Bax. Moreover, siBIK RNA significantly decreased the proportion of Annexin V+ cells observed after treatment with lenalidomide and VD3, respectively by 36%±9% (p=0.04) and 28%±4% (p=0.04) in JEKO-1 and MINO cells. This confirmed the involvement of Bik in the cell death induced by this synergistic combination. Q-RT-PCR assays disclosed that Bik accumulation was related to an increase in BIK mRNA expression. BIK expression is controlled by the transcription factor TEF and is regulated by epigenetic modifications, its expression being silenced by methylation in many cancer cells. We showed that Bik accumulation induced by lenalidomide and VD3 was not related to an increase in TEF expression. To determine whether Bik expression could be induced or increased upon demethylation in MCL, we treated cell lines for 3 days with 1µM 5-azadecytidine (5-aza). Indeed, higher expression of Bik was observed after this treatment in the four cell lines sensitive to lenalidomide. Of note, cell death induced by 5-aza correlated linearly to that induced by lenalidomide and VD3 (p<0.001, r=0.95, n=6), suggesting that BIK demethylation could be a key point in the response to this combination. To directly assess the level of BIK methylation in MCL cell lines, we then performed a DNA methylation specific PCR assay on bisulfite-treated DNA, which targets the CpG rich region located within intron 1, as previously described by Hatzimichael et al (Leuk Lymphoma. 2012;53:1709). Indeed, we showed that lenalidomide and VD3 increased the proportion of unmethylated over methylated BIKDNA CpG islands in sensitive (2 to 5-fold increase) but not in resistant cell lines. These data show that the association of lenalidomide and VD3, by increasing BIK expression through DNA demethylation, is an efficient combination to induce the apoptosis of MCL cells. They also underline the interest of measuring the level of VD3 in MCL patients especially those receiving lenalidomide, since supplementation in deprived patients might improve the effect of therapy. Disclosures: No relevant conflicts of interest to declare.

Clinical Results of Gemox-R in Mantle CELL Lymphoma: The Role of Oxaliplatin

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2722-2722
Author(s):  
Marta García-Recio ◽  
Antonio Gutierrez ◽  
Antonia Obrador-Hervia ◽  
Lucia García Mañó ◽  
Leyre Bento ◽  
...  
Keyword(s):  
High Risk ◽  
Cell Viability ◽  
Mantle Cell Lymphoma ◽  
Cell Lines ◽  
Conflicts Of Interest ◽  
Cell Lymphoma ◽  
Effective Drug ◽  
Combination Regimen ◽  
Entire Series ◽  
Mantle Cell

Abstract Introduction: Mantle cell lymphoma (MCL) is mostly incurable. The current standard therapy achieves a high rate of complete remission (CR), but the pattern of continuous relapses still marks this disease as a challenge. We previously reported the efficacy of GemOx-R, a combination regimen of gemcitabine, oxaliplatin and rituximab, in patients with refractory and relapsing MCL. Our aim is to confirm our previous results in a larger retrospective series and evaluate the efficacy of each component of GemOx-R in a panel of MCL cell lines and in patient-derived primary cells. Methods: Between 2003 and 2015, 30 patients with MCL were included in a retrospective study of treatment with GemOx-R from the University Hospital Son Espases: 10 cases frontline and 20 in the salvage setting. Frontline cohort was consolidated with radioimmunotherapy and received maintenance therapy with rituximab. The translational study was performed in established cell lines as well as primary MCL lines from patients by cell viability, cell cycle, apoptosis and western blot analysis. Drug synergy was determined by the isobologram and combination index methods. Results: This is a high risk series of patients: median age 70 years, 87% stage IV and 86% intermediate or high risk MIPI. Overall response rate and CRR was 80% and 60% in the frontline cohort as well as 85 % and 60% for salvage patients, respectively. Median progression-free survival was 28 months in the entire series: 66 and 22 months, respectively, for the two cohorts. Median overall survival was 34 months in the entire series: not reached and 20 months, respectively, for the two cohorts. Grade 3 and 4 toxicity was as follows: neutropenia (63%), anemia (34%) and thrombocytopenia (30%) as well as 24% of grade 1 and 2 neurotoxicity. Cell viability and apoptosis analysis showed that oxaliplatin is the most effective drug in this regimen in contrast to the poor responses induced by gemcitabine and rituximab. Oxaliplatin had a profound effect on cellular viability, consistent with the induction of caspase activityand the downregulation of pro-survival proteins. We further present synergistic efficacy of oxaliplatin combined with cytarabine in MCL cells. Conclusions: (1) GemOx-R shows excellent results in MCL both in the frontline and salvage settings considering the high risk patients included. (2) Oxaliplatin is the most effective drug in GemOx-R; (3) oxaliplatin has a robust in vitro activity comparable to that of cytarabine, and the combination of both oxaliplatin and cytarabine shows a significant synergism; (4) taken together, our findings suggest that oxaliplatin alone or combined with cytarabine could constitute a new or alternative backbone for promising new regimens in MCL. Disclosures No relevant conflicts of interest to declare.

Integrative Analysis of MicroRNA and Gene Expression Profiling Contributes to Understand Mantle Cell Lymphoma Pathogenesis.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2936-2936
Author(s):  
Lorena Di Lisio ◽  
Gonzalo Gómez-López ◽  
Margarita Sáanchez-Beato ◽  
Maria Elena Rodríguez ◽  
Cristina Gómez Abad ◽  
...  
Keyword(s):  
Gene Expression ◽  
Mantle Cell Lymphoma ◽  
Cell Lines ◽  
Expression Profile ◽  
Nuclear Translocation ◽  
Conflicts Of Interest ◽  
Cell Lymphoma ◽  
Gene Set Enrichment Analysis ◽  
Simultaneous Increase ◽  
Mantle Cell

Abstract Abstract 2936 Poster Board II-912 Mantle cell lymphoma (MCL) pathogenesis is still partially unexplained. Although the overexpression of CyclinD1 dependent of t(11;14) is a distinctive molecular hallmark of this neoplasm, this event alone cannot account for the increased survival signaling that characterizes this lymphoma type. Here we investigate whether microRNA (miRNA) expression profile may help to explain the changes in the expression of gene pathways that are characteristic of MCL. Twenty-three frozen MCL samples, 11 frozen control tissues (7 lymph nodes and 4 tonsils), 8 MCL cell lines and 3 samples of CD19+IgD+CD27- cells obtained from tonsils, were studied for miRNAs and gene expression. MiRNA one color microarray data for 470 human miRNA were analyzed using SAM (Significance Analysis of Microarrays) algorithm. MiRNA targets were predicted by miRanda and TargetScan methods. Pathways identification and analysis was carried out by GSEA (Gene Set Enrichment Analysis) online resource. The analysis of 23 MCL cases compared to 11 control tissues showed a miRNA signature that included 117 miRNAs with FDR<0.05, 85 of which downregulated and 32 upregulated. Combined analysis of these miRNAs and changes in the gene expression profile, paired with bioinformatic target prediction, revealed a group of genes and pathways potentially targeted by the miRNAs, including essential pathways for lymphoma survival. An interesting correspondence was found between the simultaneous increase in CD40, MAPK, NFKB and others pathway signaling with the downregulation of 15 miRNA predicted to target genes belonging to these pathways. Functional validation in MCL cell lines demonstrated NF-kB nuclear translocation to be regulated by the expression of one of these miRNAs. Most of the MCL cell lines exhibit a strong expression of the mir-17-92 polycistron (Oncomir-1). MiRNAs were used also for the identification of survival prognostic markers; using different analysis (22 frozen specimens) and validation (54 paraffin embedded cases) series. A single miRNA distinguished a group of MCL cases with a 72.2% survival at 60 m. This study identifies a set of miRNAS involved in MCL pathogenesis, which could be used in MCL recognition and clinical prognostication. Disclosures: No relevant conflicts of interest to declare.

Acquired in Vitro Resistance to Ibrutinib Is Associated with Transcriptional Re-Programming and Sustained Survival Signaling in Waldenströms Macroglobulinemia and Mantle Cell Lymphoma, Independent of BTK Cys481 Mutation

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2250-2250 ◽  
Author(s):  
Kasyapa S. Chitta ◽  
Aneel Paulus ◽  
Maja Kuranz-Blake ◽  
Sharoon Akhtar ◽  
Anne J. Novak ◽  
...  
Keyword(s):  
Mantle Cell Lymphoma ◽  
Cell Lines ◽  
Conflicts Of Interest ◽  
Cell Lymphoma ◽  
Clinical Success ◽  
Expression Profiles ◽  
Transcriptional Networks ◽  
Dependent Manner ◽  
Mantle Cell ◽  
Mts Assay

Abstract Background: While B-cell receptor (BCR) signaling is essential for the development, of normal B cells, its aberrant hyper-activation results in neoplastic transformation of B-lymphocytes. Recent investigations using small molecule inhibitors validate the BCR pathway as a valuable target. Bruton’s tyrosine kinase (BTK) is one of the components of a signaling hub that transduces signals from the BCR into the cell for its activation and has been shown to be a therapeutic target. Ibrutinib (PCI-32765), an irreversible BTK inhibitor has shown clinical efficacy in CLL, mantle cell lymphoma (MCL) and Waldenströms macroglobulinemia (WM). Ibrutinib binds to cysteine-481 of the BTK protein and blocks its phosphorylation, resulting in termination of BCR-mediated activation of cells with a concomitant induction of death. Despite the clinical success of ibrutinib, a high percentage of patients achieve only partial response and eventually acquire resistance to the drug, resulting in aggressive relapse of the disease. A mutation of Cys481-Ser in BTK (ibrutinib-BTK binding site) has been reported to be one of the reasons for the development of ibrutinib resistance (IR). To understand the mechanisms resulting in acquisition of IR, we developed preclinical models of IR in WM and MCL. Materials: Ibrutinib was obtained from Pharmacyclics, CA. Validated human WM models (BCWM.1, RPCI-WM1 and MWCL.1 cell lines) and human MCL models (Jeko-1 and Maver cell lines) were used for the study. Results: BTK was constitutively phosphorylated at Y223 and Y551 in all the cell lines tested and this was inhibited by ibrutinib in a dose dependent manner. Phosphorylation of other kinases in the cascade such as SYK (Y323 and Y525/526) and PLCg2 (Y759 and Y1217) were also inhibited while AKT phosphorylation at both Ser473 and Thr308 was consistently increased in presence of ibrutinib. Treatment with ibrutinib induced cell cycle arrest in the G1 phase by 24h followed by apoptosis. Cell growth assays (MTS assay) showed that BCWM.1 was the most sensitive cell line followed by MWCL-1, RPCI-WM1, Maver and Jeko-1. Exposure of WM and MCL cells for prolonged periods of time with progressively increasing concentrations of ibrutinib resulted in outgrowth of clones (IR WM and MCL cell lines) that were resistant to apoptosis with a slow growth rate as compared to wild type parental cells. IR cells attained 2 – 20 fold resistance to ibrutinib as compared to the respective parental lines as determined by MTS assay. Sequence analysis of the BTK gene in all the cell lines revealed no mutation in IR cells at Cys481 suggesting that in an acquired IR state, resistance to ibrutinib can be developed independent of BTK Cys481 mutation. Interestingly, we found p-BTK levels to be markedly reduced in IR cells. Ibrutinib reversal experiments suggested that while a continuous presence of ibrutinib is needed for inhibition of BTK phosphorylation, a stable IR state could be maintained (for >1 month) in the absence of ibrutinib. This suggested the cells reliance on a parallel survival pathway, independent of BTK phosphorylation. Focused mRNA (Nanostring nCounter assay) and immunoblot analysis revealed significant changes in the expression profiles of several cellular elements. These included transcription factors such as PU.1, IRF4, BLIMP1, BCL-6 b-catenin as well as the phosphorylated ERK1/2, STAT1 and 3 suggesting a reprogramming of critical cellular networks, which IR tumor cells might be utilizing to overcome ibrutinib-induced cytotoxicity. Importantly, we observed that IR cells retained high levels of p-AKT and showed an increase in expression of BCL2 family members, as well as BCL-2 itself. Treatment of IR cells with ibrutinib +/- MK2206 (AKT inhibitor), or ABT-199 (BCL-2 inhibitor), synergistically induced cytotoxicity in IR cells, suggesting the importance of these parallel survival pathways (AKT/BCL2) in maintaining an IR state. Conclusion: Here we demonstrate that in the absence of BTK Cys481 mutation, an IR state is associated with reprogramming of transcriptional networks countering ibrutinib-induced toxicity by activation of AKT and BCL-2. Our current data exposes multiple vulnerabilities within IR cells, which can be therapeutically exploited to potentially delay onset of IR, by targeting alternative oncogenic mechanisms that are activated in presence of sustained BTK inhibition. Disclosures No relevant conflicts of interest to declare.

scholarly journals Differential B-Cell Receptor Signaling Requirement for Adhesion of Mantle Cell Lymphoma Cells to Stromal Cells

Cancers ◽  
2020 ◽  
Vol 12 (5) ◽  
pp. 1143 ◽  
Author(s):  
Laia Sadeghi ◽  
Gustav Arvidsson ◽  
Magali Merrien ◽  
Agata M. Wasik ◽  
André Görgens ◽  
...  
Keyword(s):  
B Cell ◽  
Mantle Cell Lymphoma ◽  
Cell Lines ◽  
Stromal Cells ◽  
Cell Lymphoma ◽  
Cell Receptor ◽  
B Cell Receptor ◽  
Model Framework ◽  
Lymphoma Cells ◽  
Mantle Cell

Interactions between lymphoma cells and stromal cells play a key role in promoting tumor survival and development of drug resistance. We identified differences in key signaling pathways between the JeKo-1 and REC-1 mantle cell lymphoma (MCL) cell lines, displaying different patterns of stromal cell adhesion and chemotaxis towards stroma-conditioned medium. The identified adhesion-regulated genes reciprocated important aspects of microenvironment-mediated gene modulation in MCL patients. Five-hundred and ninety genes were differently regulated between the cell lines upon adhesion to stromal cells, while 32 genes were similarly regulated in both cell lines. Regulation of B-cell Receptor (BCR) signature genes in adherent cells was specific for JeKo-1. Inhibition of BCR using siRNA or clinically approved inhibitors, Ibrutinib and Acalabrutinib, decreased adhesion of JeKo-1, but not REC-1 cells. Cell surface levels of chemokine receptor CXCR4 were higher in JeKo-1, facilitating migration and adhesion of JeKo-1 but not REC-1 cells. Surface levels of ICAM1 adhesion protein differ for REC-1 and JeKo-1. While ICAM1 played a positive role in adherence of both cell lines to stromal cells, S1PR1 had an inhibitory effect. Our results provide a model framework for further investigation of mechanistic differences in patient-response to new pathway-specific drugs.

The Multi-Kinase Inhibitor Sorafenib Blocks Migration, BCR Survival Signals, Protein Translation and Stroma-Mediated Bortezomib Resistance in Mantle Cell Lymphoma

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1647-1647
Author(s):  
Silvia Xargay-Torrent ◽  
Monica López-Guerra ◽  
Ifigènia Saborit-Villarroya ◽  
Laia Rosich ◽  
Alba Navarro ◽  
...  
Keyword(s):  
Cell Migration ◽  
Cyclin D1 ◽  
Mantle Cell Lymphoma ◽  
Cell Lines ◽  
Conflicts Of Interest ◽  
Cell Lymphoma ◽  
Protein Translation ◽  
Multikinase Inhibitor ◽  
Mantle Cell ◽  
Induced Apoptosis

Abstract Abstract 1647 Mantle cell lymphoma (MCL) is an incurable B-lymphoid neoplasm harboring the t(11;14)(q13;q32) translocation which leads to the overexpression of cyclin D1, with the consequent cell cycle deregulation. Typically, MCL is characterized by bad prognosis and an aggressive course of the disease. Unfortunately, current therapies have shown limited efficacy and relapses occur early, thus our purpose was to evaluate the antitumoral properties of the multikinase inhibitor sorafenib in MCL. Sorafenib is an oral multikinase inhibitor that targets several cancer-specific pathways and directly affects tumor cell proliferation, cell survival and neovascularization. We analyzed the sensitivity to sorafenib in 9 MCL cell lines and 17 primary MCL cells by flow cytometry after annexin V staining. Sorafenib induced apoptosis in MCL cell lines with a mean LD50 of 11.5 ± 5.0 μM at 24 hours, while at 48 hours decreased to 7.1 ± 2.7 μM. In primary MCL cells, the mean LD50 was 13.0 ± 3.6 μM at 24 hours, while at 48 hours it notably decreased to 9.4 ± 3.4 μM. These data indicated that sorafenib exerted a time- and dose-dependent cytotoxic effect in MCL cells. Both in cell lines and primary MCL cells, sorafenib induces rapid dephosphorylation of the BCR (B-Cell Receptor)-associated tyrosine kinases, SYK and LYN, as well as of FAK, a downstream SRC target involved in focal adhesion. In line with this, we demonstrate a strong synergy when combining sorafenib with the SYK inhibitor, R406. In parallel, we show that sorafenib also blocks Mcl-1 and cyclin D1 translation, which promotes an unbalance between pro- and anti-apoptotic proteins and facilitates the release of Bax from cyclin D1. This process leads to the induction of the mitochondrial apoptotic pathway and caspase-dependent and independent mechanisms. Moreover, sorafenib inhibits MCL cell migration as well as actin polymerization in response to CXCL12. FAK siRNA-mediated knockdown partially prevents this inhibitory effect, indicating that FAK is a relevant target for the action of sorafenib in MCL cells. Importantly, this compound resensitizes MCL cells cocultured with bone marrow-derived stromal and follicular dendritic-like cells to bortezomib-induced apoptosis indicating that sorafenib was able to antagonize stroma-mediated resistance in MCL. In conclusion, we provide first evidence on the molecular mechanism of action of the multikinase inhibitor sorafenib in MCL. We propose that this compound inhibits cell migration and stroma-mediated bortezomib resistance by interfering BCR signaling and protein translation. All these results suggest that sorafenib, alone or in combination with bortezomib-based therapies, may represent a promising approach for the treatment of MCL patients. Research funding This work was supported by grants from Ministerio de Ciencia e Innovación (SAF 09/9503) and Redes Temáticas de Investigación Cooperativa de Cáncer from the Instituto de Salud Carlos III RED 2006-20-014 (D.C.). S.X-T. is a recipient of predoctoral fellowship from Ministerio de Ciencia e Innovación (FPU) and M.L-G. holds a contract from Fundación Científica de la Asociación Española contra el Cáncer. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Differential B-cell receptor signaling requirement for adhesion of mantle cell lymphoma cells to stromal cells

2020 ◽  
Author(s):  
Laia Sadeghi ◽  
Gustav Arvidsson ◽  
Magali Merrien ◽  
Agata M. Wasik ◽  
André Görgens ◽  
...  
Keyword(s):  
B Cell ◽  
Mantle Cell Lymphoma ◽  
Cell Lines ◽  
Stromal Cells ◽  
Cell Lymphoma ◽  
Cell Receptor ◽  
B Cell Receptor ◽  
Model Framework ◽  
Lymphoma Cells ◽  
Mantle Cell

AbstractInteractions between lymphoma cells and stromal cells play a key role in promoting tumor survival and development of drug resistance. We identified differences in key signaling pathways between the JeKo-1 and REC-1 mantle cell lymphoma (MCL) cell lines, which exhibited different patterns of stromal cell adhesion and chemotactic migration towards stroma-conditioned medium. The identified adhesion-regulated genes reciprocated important aspects of microenvironment-mediated gene modulation in MCL patients. 590 genes were differently regulated between the cell lines upon adhesion to stromal cells, while 32 genes were similarly regulated in both cell lines. Regulation of B-cell Receptor (BCR) signature genes in adherent cells was specific for JeKo-1. Inhibition of BCR signaling by siRNA or clinically approved inhibitors, Ibrutinib and Acalabrutinib, decreased adhesion of JeKo-1 but not REC-1 cells to stromal cells. Cell surface levels of CXCR4 were higher in JeKo-1 cells and CXCR4 was important for migration and adhesion of JeKo-1 but not REC-1 cells. Surface levels of ICAM1 adhesion protein differ for REC-1 and JeKo-1. While ICAM1 plays a positive role in adherence of both cell lines to stromal cells, S1PR1 had an inhibitory effect. The results presented here provide a model framework for further investigation of mechanistic differences in patient-response to new pathway-specific drugs.

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