Cyclin D1 Over-Expressing Mantle Cell Lymphoma Cells Are Hypersensitive to Inhibition of Fatty Acid Synthase (FASN)

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1656-1656
Author(s):  
Michael A. Dengler ◽  
Matthias Gutekunst ◽  
Stephanie Kopacz ◽  
Heike Horn ◽  
Ute Hofmann ◽  
...  
Keyword(s):  
Cell Death ◽  
Fatty Acid ◽  
Cyclin D1 ◽  
Mantle Cell Lymphoma ◽  
Cell Lines ◽  
Fatty Acid Synthase ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Proapoptotic Protein ◽  
Mantle Cell

Abstract Abstract 1656 Mantle cell lymphoma (MCL) is an aggressive type of B-cell lymphoma characterized by a t(11;14)(q13;q32) chromosomal translocation resulting in a constitutive over-expression of cyclin D1. Cyclin D1 is a multifunctional protein not only regulating cell cycle progression but also affecting other cellular functions including glucose and lipid metabolism. In this study, we investigated the effects of the fatty acid synthase (FASN) inhibitors orlistat and C75 on viability of different MCL cell lines including JEKO-1, MINO, Granta-519, JVM-2, and Rec-1. In all cell lines inhibition of FASN alone induced apoptosis. In contrast, normal peripheral blood lymphocytes were resistant to these compounds. This proapoptotic effect was dependent on cyclin D1 as silencing of cyclin D1 partially rescued cells from induction of cell death following FASN inhibition. Inhibition of FASN led to a strong induction of the proapoptotic protein NOXA (PMAIP) in all MCL cell lines investigated independent of the p53 status. Pre-treatment of cells with NOXA siRNA significantly reduced induction of cell death demonstrating the predominant role of this proapoptotic protein for the observed effects. We then analyzed the combined effects of inhibition of FASN with a panel of compounds with potential clinical relevance. Combination of FASN inhibitors with the BH3 mimetic ABT737, the proteasome inhibitor Bortezomib, and the glycolysis inhibitor 2-deoxy-D-glucose (2-DG) led to an almost complete loss of viability. These combinatory effects were selective for MCL cells as the same treatments had almost no effects on cell viability of primary PBMCs or fibroblasts from healthy donors. Bortezomib, 2-DG, and ABT737 enhanced the proapoptotic effect of FASN inhibitors by further disturbing the balance of pro- and antiapoptotic Bcl-2 family proteins: FASN inhibitor mediated induction of NOXA was enhanced by Bortezomib whereas 2-DG significantly reduced the NOXA antagonist Mcl-1. In summary, our results suggest that FASN inhibitors exert significant antitumoral activity especially in combination with Bcl-2 family modulators in different MCL cell lines and may therefore represent an attractive model for the design of new therapeutic approaches for this entity. Disclosures: No relevant conflicts of interest to declare.

Newer-Generation HSP90 Inhibitors Have Potent Activity Against Human Mantle Cell Lymphoma in Vitro and in Vivo

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1651-1651
Author(s):  
Oliver Weigert ◽  
Diederik van Bodegom ◽  
Liat Bird ◽  
Amy Saur ◽  
Trevor Tivey ◽  
...  
Keyword(s):  
Cell Death ◽  
Cyclin D1 ◽  
Mantle Cell Lymphoma ◽  
Cell Lines ◽  
Cell Lymphoma ◽  
Cell Killing ◽  
Hsp90 Inhibitors ◽  
Mantle Cell

Abstract Abstract 1651 Mantle cell lymphoma (MCL) is associated with particularly poor outcome, with long-term survival achieved in less than 40% of patients. In addition to the characteristic t(11;14) that results in overexpression of cyclin D1, a variety of other molecular pathways are dysregulated in MCL. Cyclin D1 is a known client of heat shock protein 90 (HSP90), suggesting that inhibitors of HSP90 may destabilize cyclin D1 and have activity in this disease. Yet, first-generation HSP90 inhibitors such as 17-AAG generally lack potency in MCL cell lines. We assessed the pre-clinical activity of second- (NVP-AUY922, PU-H71) and third-generation (NVP-HSP990) HSP90 inhibitors, which have greater potency and superior in vivo pharmacokinetics, in the MCL cell lines Granta519, JeKo1, MAVER1, Rec1, and Z-138. To define the genetics of these lines, we utilized an exon-capture followed by next-generation sequencing approach to identify single nucleotide variants and insertions/deletions across the entire coding sequence of 197 genes known to be recurrently altered in lymphoid malignancies. Sequencing to a median depth of coverage∼400 recovered alterations previously described in MCL (e.g. in ATM, RB1, TP53, NOTCH1) as well as variants in genes that have not previously been associated with MCL (e.g. in MLL2, KDM6A, FLT3, IKZF3, JAK3, RFXAP). Dose response curves of these cell lines treated with structurally diverse HSP90 inhibitors showed 10–100-fold greater potency for NVP-AUY922 (IC50, 3–11 nM), NVP-HSP990 (IC50, 5–24 nM) and PU-H71 (IC50, 40–287 nM), compared with 17-AAG (IC50, 29–1503 nM). In vitro exposure of all lines to 50 nM AUY922 resulted in G0/G1 cell cycle arrest within 6–8 hrs followed by apoptosis within 24–72 hours. Immunoblotting after exposure to AUY922 demonstrated rapid reductions in HSP90 client proteins, including cyclin D1, CDK4 and AKT, in all lines as well as accumulation of HSP70 in all lines except REC1, which harbors an HSP70 locus deletion. Cell killing by AUY922 (based on Annexin V/PI flow cytometry, caspase 3/7 activation and PARP cleavage) varied between cell lines, with Granta519 being the most sensitive (>50% cell death after 24 hr exposure) and Rec1 being the least sensitive (<15% cell death under the same conditions). Co-culture of Granta519, JeKo1, and Z-138 cells with bone marrow stroma had no effect on killing by AUY922, suggesting that HSP90 inhibition may overcome cell non-autonomous pathways that support resistance to other antineoplastic agents. To build on these findings in vivo, we xenografted luciferized MAVER1 (harbors TP53 D281E and JAK3 V722I mutations) and Z-138 (TP53 and JAK3 wild-type) cells into SCID beige mice (10 million cells per mouse). Upon evidence of measurable engraftment, mice (10 per arm) were randomized to receive either AUY922 (50 mg/kg by tail vein injection thrice weekly) or vehicle. Tumors were analyzed from sentinel mice that were sacrificed after 5 days of treatment. Tumors from mice receiving AUY922 had complete loss of cyclin D1 and Ki67 staining by immunohistochemistry. 18F-FLT PET scanning performed on mice xenografted with Z-138 cells demonstrated ∼75% reduction in activity after 5 days of AUY922 treatment. Consistent with these findings, tumor growth was significantly slowed among AUY922-treated animals for both lines, which translated into a survival advantage (p<0.01 for MAVER1 and p=0.03 for Z-138). Finally, in an effort to enhance cell killing, we combined AUY922 with compounds in clinical use for MCL. In JeKo1, MAVER1, Rec1 and Z-138 cells, combinations with AUY922 were either antagonistic (with cytarabine or doxorubicin) or lacked synergistic effects (with bortezomib). AUY922 also failed to block the accumulation of MCL1 induced by exposure to bortezomib. Thus, appropriate drug combination partners for AUY922 in MCL remain to be determined. In conclusion, newer-generation HSP90 inhibitors such as AUY922 have significant single-agent activity across a genetically diverse spectrum of MCLs, can target cyclin D1, CDK4, AKT and other drivers of malignant phenotype, and warrant evaluation in clinical trials. Disclosures: Weinstock: Novartis: Consultancy, Research Funding.

scholarly journals PRMT5 Inhibition Drives Therapeutic Vulnerability to BCL-2 Inhibition with Venetoclax and Provides Rationale for Combination Therapy in Mantle Cell Lymphoma

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 302-302 ◽  
Author(s):  
Fiona Brown ◽  
Yang Zhang ◽  
Claire Hinterschied ◽  
Alexander Prouty ◽  
Shelby Sloan ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Cell Death ◽  
Mantle Cell Lymphoma ◽  
Cell Lines ◽  
Combination Treatment ◽  
Cell Lymphoma ◽  
Targeted Agents ◽  
Mantle Cell ◽  
Anti Tumor Activity

Mantle cell lymphoma (MCL) is an incurable B cell malignancy, defined by the t(11;14) translocation and comprises 3-6% of non-Hodgkin lymphomas diagnosed annually. MCL is associated with a poor prognosis due to emergence of resistance to immuno-chemotherapy and targeted agents. Due to the late median age of diagnosis, aggressive chemotherapy and stem cell transplantation are often not realistic options. The average overall survival of patients with MCL is 5 years and for the majority of patients who progress on targeted agents like ibrutinib, survival remains at a dismal 3-8 months. There is a major unmet need to identify new therapeutic approaches that are well tolerated by elderly patients to improve treatment outcomes and quality of life. Our group has identified the type II protein arginine methyltransferase enzyme, PRMT5, to be dysregulated in MCL and to promote growth and survival by supporting the cell cycle, PRC2 activity, and signaling via the BCR and PI3K/AKT pathways. We have developed first-in-class selective inhibitors of PRMT5 and, in collaboration with Prelude Therapeutics, we have demonstrated that novel SAM-competitive PRMT5 inhibitors provide potent anti-tumor activity in aggressive preclinical models of human MCL. Selective inhibition of PRMT5 in these models and MCL cell lines leads to disruption of constitutive PI3K/AKT signaling, dephosphorylation and nuclear translocation of FOXO1, and enhanced recruitment of this tumor suppressor protein to chromatin. We identified 136 newly emerged FOXO1-bound genomic loci following 48 hours of PRMT5 inhibition in the CCMCL1 MCL line by performing chromatin immunoprecipitation-seq analysis. These genes were markedly upregulated in CCMCL1 cells treated with the PRMT5 inhibitor PRT382 as determined by RNA-seq analysis. Among those genes, we identified and confirmed FOXO1 recruitment to the promoter of BAX, a pro-apoptotic member of the BCL2 family of proteins. Treatment of MCL cell lines (Granta-519, CCMCL1, Z-138, and SEFA) with the selective PRMT5 inhibitor PRT382 (10, 100nM) led to upregulation of BAX protein levels and induction of programmed cell death as measured by annexin V/PI staining and flow cytometry. We hypothesized that induction of BAX would trigger a therapeutic vulnerability to the BCL2 inhibitor venetoclax, and that combination PRMT5/BCL2 inhibitor therapy would drive synergistic cell death in MCL. Single agent and combination treatment with venetoclax and PRT382 was performed in eight MCL lines including a new cell line generated from our ibrutinib-refractory PDX model (SEFA) and IC50 and synergy scores were calculated. The Z-138 line was most sensitive to venetoclax (IC50&lt;10nM) while CCMCL-1, SP53, JeKo-1, and Granta-519 demonstrated relative resistance (IC50&gt;1uM). All lines reached an IC50 &lt;1uM when co-treated with PRT382, with IC50 values ranging from 20 - 500nM. Combination treatments showed high levels of synergy (scores &gt; 20) in 4 lines and moderate synergy (scores 10-20) in 2 lines. The two lines with the highest levels of synergy, Z-138 and SEFA, express high levels of BCL-2 and are Ibrutinib resistant. Overall there was a strong positive correlation between BCL2 expression and synergy score (r=0.707), and no correlation between PRMT5 expression and synergy score (r=0.084). In vivo evaluation in two preclinical MCL models (Granta-519 NSG mouse flank and an ibrutinib-resistant MCL PDX) showed therapeutic synergy with combination venetoclax/PRT382 treatment. In both models, mice were treated with sub-therapeutic doses of venetoclax and/or PRT543 (Granta) or PRT382 (IR-MCL PDX) and tumor burden assessed weekly via flank mass measurement (Granta) or flow cytometry (IR-MCL-PDX). Combination treatment with well-tolerated doses of venetoclax and PRMT5 inhibitors in both MCL in vivo models showed synergistic anti-tumor activity without evidence of toxicity. This preclinical data provides mechanistic rationale while demonstrating therapeutic synergy and lack of toxicity in this preclinical study and justifies further consideration of this combination strategy targeting PRMT5 and BCL2 in MCL in the clinical setting. PRT543, a selective PRMT5 inhibitor, has been advanced into clinical studies for the treatment of patients with solid tumors and hematologic malignancies, including MCL (NCT03886831). Disclosures Zhang: Prelude Therapeutics: Employment. Vaddi:Prelude Therapeutics: Employment. Scherle:Prelude Therapeutics: Employment. Baiocchi:Prelude: Consultancy.

scholarly journals Blockade of Fatty Acid Synthase Triggers Significant Apoptosis in Mantle Cell Lymphoma

PLoS ONE ◽  
2012 ◽  
Vol 7 (4) ◽  
pp. e33738 ◽  
Author(s):  
Pascal Gelebart ◽  
Zoulika Zak ◽  
Mona Anand ◽  
Andrew Belch ◽  
Raymond Lai
Keyword(s):  
Fatty Acid ◽  
Mantle Cell Lymphoma ◽  
Fatty Acid Synthase ◽  
Cell Lymphoma ◽  
Mantle Cell

SOX11 is useful in differentiating cyclin D1-positive diffuse large B-cell lymphoma from mantle cell lymphoma

2012 ◽  
Vol 61 (4) ◽  
pp. 685-693 ◽  
Author(s):  
Shih-Chuan Hsiao ◽  
Inmaculada Ribera Cortada ◽  
Luis Colomo ◽  
Hongtao Ye ◽  
Hongxiang Liu ◽  
...  
Keyword(s):  
B Cell ◽  
Cyclin D1 ◽  
Mantle Cell Lymphoma ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Large B Cell Lymphoma ◽  
Mantle Cell ◽  
Large B Cell

Iron Chelation in Mantle Cell Lymphoma.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1395-1395
Author(s):  
Heather M. Gilbert ◽  
Josef T. Prchal ◽  
Miles C. Deneris
Keyword(s):  
Cyclin D1 ◽  
Mantle Cell Lymphoma ◽  
Cell Lymphoma ◽  
Iron Chelation ◽  
B Cell Lymphoma ◽  
Iron Chelators ◽  
Large B Cell Lymphoma ◽  
Mantle Cell ◽  
Lymphoma Line ◽  
Large B Cell

Abstract Cell proliferation is dependent upon iron, and numerous studies have shown that iron limitation arrests cells in the G1 phase of the cell cycle. A recent study of the molecular basis of these observations (Richardson, et al. Blood2007;109:4045) examined the ability of iron chelators to inhibit cell proliferation and to induce apoptosis, focusing on the role of iron chelation on cyclin D1. Cyclin D1 assembles with cdk-4 or cdk-6, generating an active holo-enzyme that catalyzes a rate limiting step in G1/S progression. This complex phosphorylates substrates, including the retinoblastoma protein, which regulate S phase entrance. Richardson’s group demonstrated that the G1/S arrest after Fe depletion is mediated, in part, by a decrease in cyclin D1 via ubiquitin-independent proteasomal degradation. Studies looking specifically at mantle cell lymphoma cell lines, however, have not yet been reported. Mantle Cell lymphoma is an interesting target for potential iron chelation as it is associated with a balanced translocation (t11;14) which leads to upregulation of BCL1 and to the constitutive overproduction of cyclin D1. We studied five different cell lines - JeKo (Mantle Cell Lymphoma), BL-41 (Burkitt Cell Lymphoma), DG-75 (Burkitt Cell Lymphoma), SUDHL-6 (Diffuse Large B cell Lymphoma) and EBV-immortalized lymphocytes from normal controls - and incubated them with four different iron chelators - deferoxamine (DFO), 2-hydroxy-1-naphthylaldehyde isonicotinoyl hydrazone (311), Pyridoxal Isonicotinoyl Hydrazone (PIH), and Salicylaldehyde Isocotinoyl Hydrazone (SIH). We then measured and compared cell cycle proliferation (using the Cellometer Auto T4, an instrument that measures cell count, cell viability, and cell size) and the rate of apoptosis (via propidium iodide FACS analysis). At 24 hours incubation, the mantle cell lymphoma lines showed significantly increased rates of apoptosis compared with non-chelated mantle cell controls (5% vs. 48%, p=0.04). The diffuse large B cell lymphoma line showed a lesser increase in apoptosis that did not reach statistical significant (6.5% vs. 14%, p=0.07), while the Burkitt’s lymphoma lines and the EBV immortalized lymphocytes showed no significant difference (BL-41, 3.4% vs. 4.1%, p=0.50; DG-75, 6% vs. 5.9%, p=0.99; EBV lymphocytes, 12.5% vs. 12.7%, p=0.96). At 72 hours of incubation with chelators, the EBV lymphocytes showed increased apoptosis compared to untreated controls (2.5% vs. 44.5%, p=0.002), while the apoptotic rate increased in the diffuse large B cell lymphoma line (3.8% vs. 48%, p=0.001) and even more dramatically in the mantle cell lymphoma line (1.5% vs. 64%, p=0.0006). The two Burkitt’s lymphoma lines were affected to a lesser degree at 72 hours by the presence of iron chelators (BL-41, 0.9% vs, 3.9%, p=0.02; DG-75, 5.5% vs. 8.9%, p=0.11). Although iron chelation, especially at longer incubation times, did affect all cell lines to various degrees, the chelator-mediated effects do appear to be specific for cell type, with mantle cell lymphoma cells displaying higher rates of apoptosis compared with other lymphomas and normal lymphocytes. These initial results will now be followed by examination of cyclin D1 expression after iron chelation. If overexpression of cyclin D1 in mantle cell lymphoma releases cells from their normal controls and acts as an oncogene, then a decrease in cyclin D1 levels via iron chelation could be added to the therapeutic armamentarium of mantle cell lymphoma.

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

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

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

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 HDACi Romidepsin Markedly Synergizes With Inhibition Of ATM By KU60019 In Mantle Cell Lymphoma

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3066-3066 ◽  
Author(s):  
Luigi Scotto ◽  
Kelly Zullo ◽  
Xavier Jirau Serrano ◽  
Laura K Fogli ◽  
Owen A. O'Connor
Keyword(s):  
Cell Cycle ◽  
Cell Death ◽  
Mantle Cell Lymphoma ◽  
Cell Cycle Arrest ◽  
Cell Lines ◽  
Cell Lymphoma ◽  
Cdk Inhibitor ◽  
Protein Levels ◽  
Cycle Arrest ◽  
Mantle Cell

Abstract Mantle cell lymphoma (MCL) is a disease characterized by gross cell cycle dysregulation driven by the constitutive overexpression of cyclin D1. The identification of a “proliferation signature” in MCL, underscores the necessity of new therapeutic approaches aimed at lowering the proliferative signature of the disease, theoretically shifting the prognostic features of the disease. Romidepsin, an HDAC inhibitor (HDACi) approved for the treatment of relapsed T-cell lymphoma, is thought to induce cell cycle arrest and apoptosis. Central to the block of cell proliferation is the up-regulation of the cdk inhibitor p21Cip1/Waf1. However up-regulation of p21Cip1/Waf1 has also been shown to reduce sensitivity to romidepsin. HDACi activates p21Cip1/Waf1 expression via ATM and KU60019, a specific ATM inhibitor, has been shown to decrease the p21Cip1/Waf1 protein levels in a concentration dependent manner. We sought to explore the effect of the combination of romidepsin and KU60019 in inducing cell death in MCL. Analysis of romidepsin treated Jeko-1 cell extracts showed a marked effect on the expression of proteins involved in cell cycle regulation. Decrease expression of Emi1, a mitotic regulator required for the accumulation of the APC/C substrates was observed. Emi1 is also responsible for the stability of the E3 ubiquitin ligase Skp2 that specifically recognizes and promotes the degradation of phosphorylated cdk inhibitor p27. However, decrease in Emi1 protein levels, upon addition of romidepsin, was not followed by an increased expression of the cdk inhibitor p27. On the other end, increased expression of the cdk inhibitor p21Cip1/Waf1, was a common feature of all romidepsin treated MCL lines analyzed. Cell cycle analysis via Fluorescent Activated Cell Sorting (FACS) of romidepsin treated Jeko-1 cells showed an accumulation of romidepsin treated cells in the G2/M phase when compared to the control suggesting a p21Cip1/Waf1 induced cell cycle arrest. For all cytotoxicity assays, luminescent cell viability was performed using CellTiter-GloTM followed by acquisition on a Biotek Synergy HT and IC50s calculated using the Calcusyn software. Drug: drug interactions were analyzed using the calculation of the relative risk ratios (RRR). Synergy analyses were performed using Jeko-1, Maver-1 and Z-138 cells treated with different concentrations of romidepsin corresponding to IC10-20 in combination with KU60019 at a concentration of 2.5, 5.0, 7.5 and 15 umol/L for 24, 48 and 72 hours. A synergistic cytotoxic effect was observed in all MCL cell lines when the HDACi was combined with KU60019 throughout the range of all concentrations. The RRR analysis showed a strong synergism at 48 and 72 hours in virtually all combinations of HDACi and KU60019 in all three cell lines. The results of drug:drug combination in two of the three cell lines are shown below. Protein expression analysis of Jeko-1 and Maver-1cells treated with single agents or combinations for 48 hours revealed changes in a host of proteins known to be involved in cell cycle control and apoptosis. The increased p21 protein expression upon addition of romidepsin, was not observed when the romidepsin treatment was combined with the KU60019. Increased activation of the programmed cell death proteins Caspase 8, induced by Fas, and Caspase 3 was observed upon combinations of the single agents in all three cell lines, resulting in an increased cleavage of Poly (ADP-ribose) polymerase (PARP-1). Finally, the abundance of the anti-apoptotic proteins Bcl-XL and BCL-2 showed a significant decrease after treatment with romidepsin plus increase concentrations of KU60019 when compared with their abundance in the presence of the single agents. Cell cycle analysis of Jeko-1 cells treated for 24 hours with single agents and combination suggests that the increased apoptosis is the result of inhibition of the p21Cip1/Waf1 induced G2/M cell cycle arrest by KU60019. Overall, these data demonstrated that the combination of romidepsin and KU60019 was synergistically effective in inhibiting the in vitro growth of the mantle cell lymphoma lines. Jeko-1 Maver-1 Disclosures: O'Connor: Celgene: Consultancy, Research Funding.

The Phenotype High Noxa mRNA/Low NOXA Protein Levels Constitutes a Critical Achilles Heel Of Mantle Cell Lymphoma (MCL) Cells

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 644-644
Author(s):  
Michael A. Dengler ◽  
Andrea Weilbacher ◽  
Matthias Gutekunst ◽  
Annette M. Staiger ◽  
Heike Horn ◽  
...  
Keyword(s):  
Cell Death ◽  
Cyclin D1 ◽  
Mantle Cell Lymphoma ◽  
Cell Lymphoma ◽  
Mtor Signaling ◽  
Mrna Levels ◽  
Transcript Levels ◽  
Protein Levels ◽  
Apoptotic Protein ◽  
Mantle Cell

Abstract Mantle cell lymphoma (MCL) is an aggressive type of non-Hodgkin lymphoma with comparatively short response to chemotherapy and frequent relapses. New treatment strategies for this malignancy are urgently needed. The genetic hallmark of MCL is the t(11;14)(q13;q32) translocation. This alteration leads to deregulated expression of the oncogene Cyclin D1 and is considered the primary event in the pathogenesis of MCL. Additionally, deregulations of different oncogenic signaling and cell death pathways have been described in MCL. In this study, we investigated the role of the BH3-only protein NOXA for life/death decision in MCL. We found a stunning discrepancy between constitutive Noxa (PMAIP1) mRNA and NOXA protein levels in MCL cell lines and primary cells. Noxa mRNA was found to be highly expressed whereas NOXA protein levels were low. Remarkably, constitutive high gene expression of this pro-apoptotic Bcl2 family member was dependent on Cyclin D1 overexpression and chronic active B cell receptor (BCR) signaling, two of the major oncogenic alterations in MCL. We identified the PI3K/AKT/mTOR pathway to be crucial in the maintenance of Noxa transcript levels downstream of BCR. Cyclin D1 overexpression contributed to the high Noxa mRNA levels by exerting a positive feedback loop on PI3K/AKT/mTOR signaling. Intriguingly, the high constitutive Noxa transcript levels do not impair cell viability. MCL cells adapt to this constitutive pro-apoptotic signal by keeping NOXA protein low due to extensive ubiquitination and rapid proteasomal degradation of NOXA protein (T½ ∼ 15-30 min). As expected, treatment of the cells with the proteasome inhibitor Bortezomib accumulated NOXA protein and efficiently induced cell death. Additionally, we identified the neddylation inhibitor MNL4924 and the fatty acid synthase (FASN) inhibitor Orlistat as potent inducers of NOXA protein, and thereby apoptosis, in MCL. Cell death upon Bortezomib as well as MLN4924 and Orlistat treatment was dependent on NOXA since RNAi mediated silencing of the pro-apoptotic protein significantly reduced induction of apoptosis. We found that all three inhibitors targeted the rapid NOXA protein turnover by stabilizing the preexisting pool of NOXA. In contrast to Bortezomib, however, MLN4924 and Orlistat inhibited the ubiqutination process of NOXA protein and stabilized the pro-apoptotic protein by a proteasome independent manner. These findings could be of great clinical relevance as Bortezomib resistance is a frequently observed phenomenon. Indeed, both inhibitors were still able to induce NOXA and cell death in Bortezomib resistant clones through targeting ubiquitine-proteasome system-mediated NOXA turnover upstream of the proteasome. Interestingly, active PI3K/AKT/mTOR signaling was needed for effective accumulation of NOXA protein and induction of cell death by all these compounds indicating that the high constitutive Noxa mRNA levels are essential for sensitivity of MCL cells. Together, our data highlight that NOXA regulation may represent an important Achilles heel of MCL cells. Stabilization of NOXA protein by inhibition of the ubiquitin-proteasome system on different levels might be an effective strategy to kill MCL cells and offer novel treatment options. Disclosures: No relevant conflicts of interest to declare.

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.

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