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 Activation of MYC, a bona fide client of HSP90, contributes to intrinsic ibrutinib resistance in mantle cell lymphoma

2018 ◽  
Vol 2 (16) ◽  
pp. 2039-2051 ◽  
Author(s):  
Jimmy Lee ◽  
Liang Leo Zhang ◽  
Wenjun Wu ◽  
Hui Guo ◽  
Yan Li ◽  
...  
Keyword(s):  
Mantle Cell Lymphoma ◽  
Cell Lines ◽  
Cell Lymphoma ◽  
Resistant Cell ◽  
Intrinsic Resistance ◽  
Mantle Cell ◽  
Myc Gene ◽  
Bona Fide ◽  
Ibrutinib Resistance ◽  
Induced Apoptosis

Abstract The BTK inhibitor ibrutinib has demonstrated a remarkable therapeutic effect in mantle cell lymphoma (MCL). However, approximately one-third of patients do not respond to the drug initially. To identify the mechanisms underlying primary ibrutinib resistance in MCL, we analyzed the transcriptome changes in ibrutinib-sensitive and ibrutinib-resistant cell lines on ibrutinib treatment. We found that MYC gene signature was suppressed by ibrutinib in sensitive but not resistant cell lines. We demonstrated that MYC gene was structurally abnormal and MYC protein was overexpressed in MCL cells. Further, MYC knockdown with RNA interference inhibited cell growth in ibrutinib-sensitive as well as ibrutinib-resistant cells. We explored the possibility of inhibiting MYC through HSP90 inhibition. The chaperon protein is overexpressed in both cell lines and primary MCL cells from the patients. We demonstrated that MYC is a bona fide client of HSP90 in the context of MCL by both immunoprecipitation and chemical precipitation. Furthermore, inhibition of HSP90 using PU-H71 induced apoptosis and caused cell cycle arrest. PU-H71 also demonstrates strong and relatively specific inhibition of the MYC transcriptional program compared with other oncogenic pathways. In a MCL patient-derived xenograft model, the HSP90 inhibitor retards tumor growth and prolongs survival. Last, we showed that PU-H71 induced apoptosis and downregulated MYC protein in MCL cells derived from patients who were clinically resistant to ibrutinib. In conclusion, MYC activity underlies intrinsic resistance to ibrutinib in MCL. As a client protein of HSP90, MYC can be inhibited via PU-H71 to overcome primary ibrutinib resistance.

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.

Cell Cycle Regulation by Iron: Novel Approaches to Mantle Cell Lymphoma Therapy.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2515-2515 ◽  
Author(s):  
Heather Gilbert ◽  
John Cumming ◽  
Josef T. Prchal
Keyword(s):  
Cell Cycle ◽  
Cell Growth ◽  
Cyclin D1 ◽  
Mantle Cell Lymphoma ◽  
Cell Lines ◽  
Cell Lymphoma ◽  
Iron Chelation ◽  
Lymphoma Cell ◽  
Protein Levels ◽  
Mantle Cell

Abstract Abstract 2515 Poster Board II-492 Mantle cell lymphoma is a well defined subtype of B-cell non-Hodgkin lymphoma characterized by a translocation that juxtaposes the BCL1 gene on chromosome 11q13 (which encodes cyclin D1) next to the immunoglobulin heavy chain gene promoter on chromosome 14q32. The result is constitutive overexpression of cyclin D1 (CD1) resulting in deregulation of the cell cycle and activation of cell survival mechanisms. There are no “standard” treatments for MCL. Despite response rates to many chemotherapy regimens of 50% to 70%, the disease typically progresses after treatment, with a median survival time of approximately 3-4 years. Mantle cell lymphoma represents a small portion of malignant lymphomas, but it accounts for a disproportionately large percentage of lymphoma-related mortality. Novel therapeutic approaches are needed. In 2007, Nurtjaha-Tjendraputra described how iron chelation causes post-translational degradation of cyclin D1 via von Hippel Lindau protein-independent ubiquitinization and subsequent proteasomal degradation (1). Nurtjaha-Tjendraputra demonstrated that iron chelation inhibits cell cycle progression and induces apoptosis via proteosomal degradation of cyclin D1 in various cell lines, including breast cancer, renal carcinoma, neuroepithelioma and melanoma. Our preliminary data show similar findings in mantle cell lymphoma. To establish whether iron chelation can selectively inhibit and promote apoptosis in mantle cell derived cell lines, the human MCL cell lines Jeko-1, Mino, Granta and Hb-12; the Diffuse Large B cell lymphoma line SUDHL-6; and the Burkitt's Lymphoma lines BL-41 and DG75 were grown with media only, with two different iron chelators (deferoxamine (DFO) and deferasirox) at various concentrations (10, 20, 40, 100 and 250 μM), and with DMSO as an appropriate vehicle control. Cells were harvested at 24, 48 and 72 hours. For detection of apoptotic cells, cell-surface staining was performed with FITC-labeled anti–Annexin V antibody and PI (BD Pharmingen, San Diego, CA). Cell growth was analyzed using the Promega MTS cytotoxicity assay. CD1 protein levels were assessed using standard Western blot techniques. At 24, 48 and 72 hours of incubation with iron chelators, the mantle cell lymphoma cell lines showed significantly increased rates of apoptosis compared to the non-mantle cell lymphoma cell lines (p<0.0001 for all time points). DFO and deferasirox inhibted cell growth with an IC50 of 18 and 12 μM respectively. All of the mantle cell lines had measurable cyclin D1 levels at baseline. None of the non-mantle cell lines expressed baseline measurable cyclin D1. In the mantle cell lines, cyclin D1 protein levels were no longer apparent on western blot after 24 hours of incubation with chelation. We then added ferrous ammonium sulfate (FAS) to DFO in a 1:1 molarity ratio and to deferasirox in a 2:1 ratio, and then treated the same lymphoma cell lines with the FAS/chelator mixture and with FAS alone for 72 hours. Adding iron to the chelators completely negated all the pro-apoptotic effects that were seen with iron chelation treatment. Treating with FAS alone had no effect on cell growth or apoptosis. Iron chelation therapy with both DFO and deferasirox results in decreased cell growth, increased cellular apoptosis, and decreased cyclin D1 protein levels in vitro in mantle cell lymphoma. The cytotoxic effects are prevented by coincubation with ferrous ammonium citrate, confirming that the effects are due to iron depletion. Proposed future research includes further defining the molecular basis of iron chelation effects; studying these therapies in combination with other cancer treatments both in vitro and in vivo; and studying iron chelation therapy in mantle cell lymphoma patients. 1. Nurtjahja-Tjendraputra, E., D. Fu, et al. (2007). “Iron chelation regulates cyclin D1 expression via the proteasome: a link to iron deficiency-mediated growth suppression.” Blood109(9): 4045–54. Disclosures: No relevant conflicts of interest to declare.

The Bioactive Polyphenol Curcumin (diferuloylmethane) In Human Apolipoprotein A-1 Nanodisks Enhances Apoptosis and G1 Cell Cycle Arrest In Mantle Cell Lymphoma Compared with Free Curcumin

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3934-3934
Author(s):  
Amareshwar T.K. Singh ◽  
Mistuni Ghosh ◽  
C. Shad Thaxton ◽  
Trudy M. Forte ◽  
Robert O. Ryan ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Drug Delivery ◽  
Cyclin D1 ◽  
Cell Cycle Arrest ◽  
Cell Lines ◽  
Cell Lymphoma ◽  
Parp Cleavage ◽  
Cycle Arrest ◽  
Mantle Cell ◽  
Induced Apoptosis

Abstract Abstract 3934 Background: Mantle cell lymphoma (MCL) is a pre–germinal center neoplasm characterized by cyclin D1 overexpression resulting from translocation of the cyclin D1 gene on 11q13 to the promoter of the immunoglobulin heavy chain locus on 14q32. Since MCL is incurable with standard lymphoma therapies, new treatment approaches are needed that target specific biologic pathways. The bioactive polyphenol curcumin (Curc), derived from the rhizome of Curcuma longa Linn, has been shown to have pleiotropic activities related to its complex chemistry and its influence on multiple signaling pathways including NF-kB, Akt, Nrf2 and pathways involved in metastasis and angiogenesis. Curc has been shown to cause growth arrest and apoptosis of BKS-2 immature B-cell lymphoma by downregulating growth and survival promoting genes (Clin Immunol 1999; 93:152). However, because of poor aqueous solubility Curc has had limited clinical utility, so investigators have explored nanoparticle drug delivery approaches (J Nanobiotech 2007, 5:3, MCT 2010; 9:2255). We reasoned that effective and targeted drug delivery by nanoparticles required appropriate receptors to facilitate binding. We therefore screened lymphoma cell lines for receptors that recognize apolipoprotein (apo) A-1. We hypothesized that a novel discoidal nanoparticle (ND) consisting of apoA-1, phospholipid and Curc (Curc ND) would bind to such receptors to facilitate drug delivery. Methods: We compared biologic activity of free Curc vs. Curc-ND in MCL cell lines expressing receptors for apoA-1. Cell lines were grown and maintained in culture, treated, and apoptosis and cell cycle progression was measured by flow cytometry. Relevant signaling intermediates and presence of apoA-1 receptors were measured by immunoblotting using specific antibodies. Results: Granta and Jeko cells (both MCL cell lines) expressed apoA-1 receptors including class B scavenger receptor (SR-B1) and the ATP-binding cassette transporter of the sub-family G1 (ABCG1). To compare the pro-apoptotic effect of free Curc and Curc-ND, Granta cells were incubated with free Curc, Curc-ND, empty ND, and medium alone (untreated). Compared to medium alone, empty ND had no effect while free Curc (20 μM) induced apoptosis. Curc-ND produced a dose-dependent increase in apoptosis, with ∼70% apoptosis at 20 μM. To investigate the mechanism of Curc-ND induced apoptosis, apoptosis-related proteins were studied in cultured Granta cells. A time-dependent decrease in caspase-9 levels was observed following incubation with Curc-ND or free Curc. The decrease in caspase-9 seen with Curc-ND, however, occurs much earlier (between 2–4 h of incubation) than for free-Curc. Caspase-3 was undetectable after 16 h with either treatment. Loss of this band implies activation of caspase-3, which was confirmed by PARP cleavage, wherein a decrease in the 116 kD band was accompanied by an increase in the 85 kD cleavage product. Unlike free Curc, Curc-ND induced PARP cleavage even at 16 h of incubation, suggesting sustained drug release. Curc-ND downregulated cyclin D1, decreased Akt phosphorylation and enhanced cleavage of caspases-9 and -3, and PARP. In addition, Curc-ND induced G1 cell cycle arrest to a greater extent than free Curc in Granta and Jeko cells (Granta: Control 34% G1, Curc 37% G1, Curc-ND 46% G1; Jeko: Control 39% G1, Curc 49% G1, Curc-ND 54% G1). Conclusion: We have shown that the MCL cell lines Granta and Jeko express apoA-1 receptors, making them likely targets for discoidal nanoscale delivery vehicles stabilized with Apo-A1. These nanodisks, when carrying the polyphenol Curc, can result in increased caspase -dependent apoptosis, cell cycle arrest, downregulation of cyclin-D1 and decreased p-Akt. These data suggest that the pleiotropic polyphenol Curc has cell killing/arrest activity in MCL and that Curc-ND may be a potential therapeutic with drug targeting ability. Disclosures: Forte: Lypro Biosciences: Employment.

The mTOR inhibitor CCI-779 (temsirolimus) downregulates p21 and induces cell cycle arrest and autophagy in mantle cell lymphoma (MCL)

2006 ◽  
Vol 24 (18_suppl) ◽  
pp. 7573-7573 ◽  
Author(s):  
V. Y. Yazbeck ◽  
G. V. Georgakis ◽  
Y. Li ◽  
A. Younes
Keyword(s):  
Cell Cycle ◽  
Cyclin D1 ◽  
Mantle Cell Lymphoma ◽  
Cell Cycle Arrest ◽  
Cell Lines ◽  
Antiproliferative Activity ◽  
Cell Lymphoma ◽  
Mtor Inhibition ◽  
Cycle Arrest ◽  
Mantle Cell

7573 Background: Mantle cell lymphoma (MCL) is a distinct type of B-cell lymphoma associated with transient response to conventional chemotherapy, continuous relapses and median survival of only 3–4 years. The mammalian target of rapamycin (mTOR) pathway is activated in many human malignancies where it regulates cyclin D1 translation. In a phase II trial, temsirolimus (CCI-779), an inhibitor of mTOR kinase used as single agent achieved an overall response rate of 38% in relapsed MCL patients. Our goal was to determine the activity and the mechanism of action of CCI-779 in MCL cell lines and to examine whether CCI-779 may synergizes with proteasome inhibitors. Methods: The activity of CCI-779 was determined in 3 mantle cell lymphoma cell lines (Jeko 1, Mino, Sp 53). Cell viability was determined by MTS assay, and autophagy by Acridine orange. Analysis of cell cycle was performed by flow cytometry and apoptosis by Annexin-V binding. Molecular changes were determined by western blot . Results: CCI-779 induced cell growth arrest in all cell lines in a time and dose dependent manner. The antiproliferative activity was due to cell cycle arrest in the G0/G1 phase followed by autophagy. CCI-779 decreased S6 phosphorylation in Jeko 1,Sp 53 indicative of mTOR inhibition. Furthermore, CCI-779 downregulated p21 expression in all three cell lines, without altering p 27 expression. Moreover, CCI-779 decreased the expression of the antiapoptotic protein cFLIP and ERK in both Jeko1 and Sp 53, but had no effect on cyclin D1 expression. The proteasome inhibitor bortezomib was also effective in all MCL cell lines, but failed to demonstrate significant synergy with CCI-779. Conclusions: The antiproliferative activity of CCI-779 in MCL is mediated by p21 downregulation and autophagy, without significant effect on cyclin D1 expression. The lack of synergy between bortezomib and CCI-779 should be confirmed using fresh MCL tumor cells. No significant financial relationships to disclose.

scholarly journals Dual mTORC1/mTORC2 inhibition diminishes Akt activation and induces Puma-dependent apoptosis in lymphoid malignancies

Blood ◽  
2012 ◽  
Vol 119 (2) ◽  
pp. 476-487 ◽  
Author(s):  
Mamta Gupta ◽  
Andrea E. Wahner Hendrickson ◽  
Seong Seok Yun ◽  
Jing Jing Han ◽  
Paula A. Schneider ◽  
...  
Keyword(s):  
Mantle Cell Lymphoma ◽  
Cell Lines ◽  
Transcriptional Activation ◽  
Cell Lymphoma ◽  
Lymphocytic Leukemia ◽  
Clinical Samples ◽  
Lymphoid Malignancies ◽  
Mantle Cell ◽  
Induced Apoptosis

Abstract The mammalian target of rapamycin (mTOR) plays crucial roles in proliferative and antiapoptotic signaling in lymphoid malignancies. Rapamycin analogs, which are allosteric mTOR complex 1 (mTORC1) inhibitors, are active in mantle cell lymphoma and other lymphoid neoplasms, but responses are usually partial and short-lived. In the present study we compared the effects of rapamycin with the dual mTORC1/mTORC2 inhibitor OSI-027 in cell lines and clinical samples representing divers lymphoid malignancies. In contrast to rapamycin, OSI-027 markedly diminished proliferation and induced apoptosis in a variety of lymphoid cell lines and clinical samples, including specimens of B-cell acute lymphocytic leukemia (ALL), mantle cell lymphoma, marginal zone lymphoma and Sezary syndrome. Additional analysis demonstrated that OSI-027–induced apoptosis depended on transcriptional activation of the PUMA and BIM genes. Overexpression of Bcl-2, which neutralizes Puma and Bim, or loss of procaspase 9 diminished OSI-027–induced apoptosis in vitro. Moreover, OSI-027 inhibited phosphorylation of mTORC1 and mTORC2 substrates, up-regulated Puma, and induced regressions in Jeko xenografts. Collectively, these results not only identify a pathway that is critical for the cytotoxicity of dual mTORC1/mTORC2 inhibitors, but also suggest that simultaneously targeting mTORC1 and mTORC2 might be an effective anti-lymphoma strategy in vivo.

scholarly journals Cyclin D1 Expression in Mantle Cell Lymphoma Is Accompanied by Downregulation of Cyclin D3 and Is Not Related to the Proliferative Activity

Blood ◽  
1997 ◽  
Vol 90 (8) ◽  
pp. 3154-3159 ◽  
Author(s):  
M. Michaela Ott ◽  
Jirina Bartkova ◽  
Jiri Bartek ◽  
Alexander Dürr ◽  
Lars Fischer ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Cyclin D1 ◽  
Mantle Cell Lymphoma ◽  
Cell Lines ◽  
Cell Lymphoma ◽  
Chromosomal Translocation ◽  
Germinal Centers ◽  
Cyclin D3 ◽  
Mantle Cell

Abstract The cell cycle regulatory protein cyclin D1 is essential for G1-S phase transition in several epithelial and mesenchymal tissues but is apparently not essential in normal mature B cells. An overexpression of cyclin D1 is induced by the chromosomal translocation t(11; 14)(q13; q32), which characterizes non-Hodgkin's lymphomas (NHLs) of mantle cell type. We studied 26 cases of mantle cell lymphoma (MCL) for the expression of cyclins D1 and D3. A total of 23 lymphomas showed a nuclear staining for cyclin D1, whereas reactive B cells of residual germinal centers were constantly negative. When compared with cyclin D3, an inverse staining pattern emerged. Whereas the B cells of residual germinal centers reacted strongly positive for cyclin D3, there was low or missing expression of cyclin D3 in MCL cells. In other B-cell lymphomas (n = 55), including chronic lymphocytic leukemia, low-grade lymphomas of mucosa-associated lymphatic tissue, follicular lymphomas, and diffuse large B-cell lymphomas, no cyclin D1 expression could be detected and 89% of these cases displayed cyclin D3 positivity. Lymphoma cell lines harboring the t(11; 14) showed cyclin D1 protein but no or very low levels of cyclin D3; three other B-cell lines, a T-cell line, and peripheral blood lymphocytes strongly expressed cyclin D3 and reacted negatively for cyclin D1. We conclude that the chromosomal translocation t(11; 14) leads to an abnormal protein expression of cyclin D1 in the tumor cells of MCL and induces a consecutive downregulation of cyclin D3. In contrast to other B-NHLs, cyclin D1 and D3 expression in MCL is not related to the growth fraction.

Arsenic Trioxide in Mantle Cell Lymphoma.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 4814-4814 ◽  
Author(s):  
Yok Lam Kwong ◽  
Chit Chow ◽  
Cyrus R. Kumana ◽  
Gopesh Srivastava ◽  
Wing Yan Au
Keyword(s):  
Cyclin D1 ◽  
Mantle Cell Lymphoma ◽  
Arsenic Trioxide ◽  
Cell Lymphoma ◽  
Down Regulation ◽  
Synergistic Interactions ◽  
Mantle Cell ◽  
Induced Apoptosis ◽  
Mtt Assays

Abstract Background. Mantle cell lymphoma (MCL) is incurable for many patients. Arsenic trioxide (As2O3) has activity in vitro against lymphoid malignancies. The effects of As2O3 on MCL in vitro and in patients with refractory disease were investigated. Materials and methods. Mantle cell lymphoma (MCL) lines (Jeko-1, Granta-519) were treated with As2O3, in combination with mitoxantrone (MTZ) and ascorbic acid (AA). Consenting patients with refractory MCL were treated with oral-As2O3 (10 mg/day), AA (1 g/day) and chlorambucil (4 mg/day) as outpatients until maximum response or the disease judged refractory. Responses were defined by standard NCI criteria. In patients showing an initial response, vincristine (2 mg intravenously) and prednisolone (30 mg/day) might also be added. After achievement of maximum response, patients were maintained with As2O3 (10 mg/day) and AA (1 g/day) for two weeks every month, for a planned two years. Results. As2O3 and MTZ but not AA induced a dose dependent apoptosis in the MCL lines, as shown by flow cytometry and MTT assays. As2O3, MTZ and AA were tested in various combinations in MTT assays. Synergistic interactions were observed only in the combinations As2O3 (1 uM) + MTZ (0.2 mg/L), and As2O3 (1 uM) + MTZ (0.2 mg/L) + AA (100 uM). Western blotting showed that As2O3-induced apoptosis was associated with a dose and time dependent down-regulation of cyclin D1. However, quantitative polymerase chain reaction showed no change in cyclin D1 gene transcription during As2O3-induced apoptosis. Eleven patients (10 men, 1 women) at a median age of 69 (51–70) years with refractory MCL were studied, at a median of 33 (8–85) months from diagnosis. At the time of As2O3 treatment, they had already had a median of 2 (1 – 4) relapses managed with a median of 2 (1 – 6) previous chemotherapeutic regimens. Eight of eleven patients were evaluable (for the other three, one died after 4 days of treatment, and the two were still receiving therapy). At a median follow up of 9 (4 – 20) months, 4 patients had reached complete remission (CR) or probable CR (CRu), two were in good partial remission, and two had died with progressive disease. Conclusion. As2O3 induced apoptosis of MCL cells by post-transcription down-regulation of cyclin D1. Synergistic interactions were observed with AA and cytotoxics. Oral-As2O3, AA and chlorambucil were an active regimen for relapsed and therapy-refractory MCL, and treatment results compared favorably with other salvage regimens. This entirely oral regimen has several attractions, including outpatient treatment, low toxicity and cost.

Novel Treatment for Therapy-Resistant Mantle Cell Lymphoma Targeting NF-κB and mTOR Signaling Pathways in Vitro and in Vivo

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 63-63
Author(s):  
Nagendra K Chaturvedi ◽  
Rajkumar Rajule ◽  
Shukla Ashima ◽  
Prakash Radhakrishnan ◽  
Amarnath Natarajan ◽  
...  
Keyword(s):  
Cyclin D1 ◽  
Mantle Cell Lymphoma ◽  
Therapeutic Efficacy ◽  
Cellular Proliferation ◽  
Conflicts Of Interest ◽  
Cell Lymphoma ◽  
Thymidine Uptake ◽  
Mantle Cell

Abstract Abstract 63 Background: Mantle cell lymphoma (MCL) is one of the most aggressive B-cell non-Hodgkin lymphomas (NHL) with a median survival of less than five years. Currently, there is no curative therapy available for refractory MCL because of relapse from therapy-resistant tumor cells. It has been well documented that the NF-κB and mTOR pathways are constitutively active in MCL leading to increased survival, proliferation and decreased apoptosis. Therefore, in an effort to improve therapy for refractory MCL, we investigated the antilymphoma activity in vitro and in vivo and associated molecular mechanism of action of 13–197, a quinoxaline analog that specifically perturbs IκB kinase (IKK) β, an upstream kinase of the NF-κB and mTOR pathways. Methods: Established therapy-resistant from Granta 519 (Ahrens and Chaturvedi et al, Leukemia and Lymphoma doi:10.3109/10428194.2012.691481), other MCL cell lines Mino and Rec-1 and primary MCL cells from patients were used in this study. These MCL cells were treated in vitro with varying concentrations of 13–197 for the different time points. Cellular proliferation/viability, cytomorphology, frequency of cells undergoing apoptosis in treated and control cells were evaluated using 3[H]-thymidine uptake, MTT assay, cytomorphology and Annexin-V staining methods respectively. The status of key molecules in the NF-κB and mTOR pathways were examined in therapy-resistant and parental MCL cells following treatment with 13–197 using western blot analyses. The results of these analyses were compared to untreated control cells as appropriate and statistical significance of the results were determined using student‘t’ test. In addition, in vivo therapeutic efficacy of 13–197 was investigated using NOD-SCID mouse bearing therapy-resistant MCL. Results: Our results showed that 13–197 significantly decreased the proliferation and induced a ∼four-fold (P<0.005) increase in apoptosis in parental and therapy-resistant MCL cells compared to control cells. At the molecular level, we observed down-regulation of IκBα phosphorylation and inhibition of NF-κB nuclear translocation by the 13–197 in MCL cells. In addition, NF-κB regulated genes such as cyclin D1, Bcl-XL and Mcl-1 were down-regulated in 13–197-treated cells. 13–197 also inhibited the phosphorylation of S6K and 4E-BP1, the downstream molecules of mTOR pathway that are also activated in refractory MCL. Further, to investigate the therapeutic efficacy of 13–197 against therapy-resistant MCL in vivo, we treated NOD-SCID mice bearing therapy-resistant MCL with 13–197; there was significantly reduced tumor burden in the kidney (p>0.05), liver (p>0.01), and lungs (p>0.03) of 13–197 treated mice compared to vehicle treated mice. Indeed, 13–197 treatment significantly increased the survival (p>0.001) of MCL transplanted mice. Taken together, our results suggest that 13–197 targets IKKβ which leads to both the transcriptional (NF-κB) and translational (mTOR) downregulation of gene products (cyclin D1, Bcl-XL and Mcl-1) misregulated in therapy-resistant MCL. Summary/Conclusions: Overall, results suggest that 13–197 perturbs the NF-κB and mTOR pathways leading significant antilymphoma effects in vitro and in vivo thus demonstrates its potentials to be a therapeutic agent for refractory MCL. (This work was supported by the Lymphoma Research Foundation New York, NY) 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.

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