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 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.

Molecular Mechanisms of the mTOR Inhibitor Temsirolimus (CCI-779) Antiproliferative Effects in Mantle Cell Lymphoma: Induction of Cell Cycle Arrest, Autophagy, and Synergy with Vorinostat (SAHA).

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2493-2493 ◽  
Author(s):  
Victor Y. Yazbeck ◽  
Georgios V. Georgakis ◽  
Yang Li ◽  
Eiji Iwado ◽  
Seiji Kondo ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Mantle Cell Lymphoma ◽  
Cell Cycle Arrest ◽  
Cell Lines ◽  
Mtor Inhibitor ◽  
Cell Lymphoma ◽  
Time Dependent ◽  
Dependent Manner ◽  
Cycle Arrest ◽  
Mantle Cell

Abstract Aberrant activation of the PI3-Kinase/Akt/mTOR survival pathway has been implicated in promoting the growth and survival of a variety of cancers, including lymphoma, and is currently being explored for cancer therapy. Importantly, the small molecule mTOR inhibitor temsirolimus (CCI-779) recently demonstrated significant clinical activity in patients with relapsed mantle cell lymphoma (MCL). However, the mechanism of action of temsirolimus in MCL cells is unknown. In this study, we demonstrated that temsirolimus induced cell growth inhibition in three MCL cell lines in a time-dependent and dose-dependent manner. The activity of temsirolimus was determined in 3 mantle cell lymphoma cell lines (Jeko-1, Mino, SP53). Temsirolimus upregulated p27 without altering cyclin D1 levels, resulting in cell cycle arrest in the G0/G1 phase. The Akt/mTOR pathway has been implicated in regulating cellular autophagy in yeasts and in mammalian cells. Thus, we examined whether temsirolimus may also induce autophagy in MCL cells, which is identified by the sequestering of cytoplasmic proteins into the lytic autophagosomes and autolysosome, and the formation of acidic vesicular organelles (AVOs). Temsirolimus induced AVOs formation indicative of autophagy in all MCL cell lines at doses ranging between 1 and 1000 nM in a time-dependent manner, with the highest activity observed between 72 and 96 hours of incubation. LC3 is essential for amino acid starvation-induced autophagy in yeasts. LC3-I is the cytoplasmic form, which is processed into the LC3-II form that is associated with the autophagosome membrane. Incubation of the SP53 cells with temsirolimus (1,000 nM) for 96 hours, resulted in processing LC3-I into LC3-II, indicative of autophagy induction. To further confirm induction of autophagy, SP53 cells expressing LC3-fused green fluorescent protein (GFP-LC3) were treated with temsirolimus and the pattern of LC3 distribution was compared with untreated cells using fluorescence microscopy. Untreated control cells showed a diffuse cytoplasmic distribution of LC3, whereas temsirolimus -treated cells showed a punctate pattern of green fluorescence, indicative of its association with autophagosomes. Furthermore, temsirolimus increased acidic vesicular organelles and microtubule-associated protein 1 light chain 3 (LC3) processing as determined by Western blot, which are characteristic of autophagy. In contrast, temsirolimus had minimal induction of apoptosis. Moreover, temsirolimus inhibited ribosomal S6 phosphorylation, an mTOR downstream target. The histone deacetylase inhibitor vorinostat (suberoylanilide hydroxamic acid, SAHA) demonstrated antiproliferative activity in a dose and time dependent manner in all three MCL cell lines. SAHA enhanced the activity of temsirolimus, which was associated with ERK dephosphorylation and caspase 3 activation. In contrast, temsirolimus did not potentiate the antitumor effects of bortezomib, doxorubicin, or gemcitabine. Our results demonstrate that in short-term culture, temsirolimus is primarily a cytostatic drug, and suggest that SAHA may potentiate the clinical efficacy of temsirolimus patients with MCL.

All Trans Retinoic Acid Nanodisks Enhance Retinoic Acid Receptor-Mediated Apoptosis and Cell Cycle Arrest in Mantle Cell Lymphoma.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3722-3722
Author(s):  
Amareshwar Singh ◽  
Andrew M Evens ◽  
Reilly Anderson ◽  
Jennifer Beckstead ◽  
Natesan Sankar ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Retinoic Acid ◽  
Mantle Cell Lymphoma ◽  
Cell Cycle Arrest ◽  
Cell Lines ◽  
Cell Lymphoma ◽  
Retinoic Acid Receptor ◽  
Ros Generation ◽  
Cycle Arrest ◽  
Mantle Cell

Abstract Abstract 3722 Poster Board III-658 Background Mantle cell lymphoma (MCL) is characterized by the translocation t (11; 14)(q13; q32), aggressive clinical behavior, and poor patient outcomes following conventional chemotherapy. New treatment approaches are needed that target novel pathways. All-trans retinoic acid (ATRA) is a key retinoid that acts through nuclear receptors that function as ligand-inducible transcription factors. We hypothesized that since MCL cells express retinoid receptors, ATRA will exert anti-proliferative effects and thus may have a role in treatment. In the present study, we pursued a novel approach to deliver an ATRA payload to MCL cells in culture. This water insoluble bioactive lipid was stably incorporated into nanoscale lipid particles, termed nanodisks (ND). ND are comprised of a disk-shaped phospholipid bilayer whose periphery is stabilized by amphipathic apolipoproteins. It was hypothesized that following cellular uptake and/or liberation of ATRA from ND, this retinoid will interact with intracellular binding proteins and, ultimately, nuclear hormone receptors, leading to target gene transactivation, cell growth arrest and/or apoptosis. Methods We studied the mantle cell lymphoma cell lines Granta, NCEB and JEKO. ATRA-ND were prepared using recombinant human apolipoprotein A-I as the scaffold protein. Empty ND, lacking ATRA, were prepared in the same manner except that ATRA was omitted from the formulation contents. In various experiments ATRA was presented to cells using dimethylsulfoxide (DMSO) as vehicle (naked ATRA). Reactive oxygen species (ROS) were measured by oxidation of 2'7'dichlorofluorescein diacetate (H2DCFDA) to dichlorofluorescein (DCF) and quantified by fluorescence intensity using FACS. Apoptosis was quantified by Annexin V-FITC and PI using flow cytometry and FACS. Cell cycle analysis was measured by flow cytometry using FACS. PARP cleavage, caspase activation and analysis of cell cycle regulator proteins was measured by Western blotting, and retinoic acid receptor activity was measured by RT-PCR. Results We found that ATRA-ND induced significantly more cell death than naked ATRA (in dimethylsulfoxide) or empty ND. In all three cell lines, ATRA-ND induced reactive oxygen species (ROS) generation to a greater extent than naked ATRA. Incubation of cells with the antioxidant, N-acetylcysteine, inhibited ATRA-ND-induced apoptosis. Compared to naked ATRA, ATRA-ND enhanced G1 cell cycle arrest. Cyclin dependent kinases (CDK) regulate checkpoints that integrate mitogenic and growth inhibitory signals during cell cycle transitions. CDK inhibitors bind to CDK and inhibit kinase activity, leading to cell cycle arrest. P21 and p27 negatively regulate CDKs, and since p21 is induced by the tumor suppressor p53 in response to DNA damage, the expression level of these proteins was examined in Granta cells as a function of ATRA exposure. Compared to untreated control cells, no changes in the level of these proteins were seen following incubation with empty-ND. Whereas naked ATRA induced a modest increase in p21, p27 and p53, much larger increases were induced by ATRA-ND. Further ATRA-ND resulted in striking decrease in cyclin-D1. At ATRA concentrations that induce apoptosis, expression levels of the retinoic acid receptor- α (RAR α) and retinoid X receptor- γ (RXR γ) were increased. Furthermore, the RAR antagonist, Ro41-5253, inhibited ATRA-ND-induced ROS generation and abrogated ATRA-ND-induced cell growth arrest and apoptosis. Taken together, we found that ATRA-ND-induced apoptosis is dependent on ROS generation and RAR activation. We do not know if ATRA in ND is more stable or resists degradation during the course of cell incubations or whether ATRA-ND are taken up by the cells via receptor mediated endocytosis. Further work is required to elucidate the molecular basis of the enhanced biological activity of ATRA when presented to cells as a component of ND. Conclusion ATRA-ND greatly enhanced apoptosis and cell cycle arrest in MCL cell lines, and resulted in increase in p21, p27 and p53 with a decrease in cyclin D1. It is conceivable that solubilization of ATRA in the ND hydrophobic milieu effectively concentrates this bioactive lipid and provides a means for more efficient delivery to target cells. Nonetheless, results obtained in this study suggest ATRA-ND represent a potentially effective approach to the treatment of MCL and should be explored further. Disclosures: No relevant conflicts of interest to declare.

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.

A Novel mTOR Kinase Inhibitor Causes Growth Inhibition, Cell Cycle Arrest, Apoptosis and Autophagic Cell Death in Mantle Cell Lymphoma Cell Lines: A Distinct Profile from Rapamycin.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1579-1579
Author(s):  
Weiming Xu ◽  
Christine Kang ◽  
Mercedes Delgado ◽  
Sophie M Perrin-Ninkovic ◽  
Patrick W Papa ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Death ◽  
Cyclin D1 ◽  
Mantle Cell Lymphoma ◽  
Kinase Inhibitor ◽  
Cell Lymphoma ◽  
Autophagic Cell Death ◽  
Mtor Kinase ◽  
Cycle Arrest ◽  
Mantle Cell

Abstract Mantle cell lymphoma (MCL) is a distinct sub-type non-Hodgkin lymphoma characterized by overexpression of cyclin D1 (CCND1) in 95% of patients due to the cytogenetic change of chromosome translocation t(11;14) (q13;q32). It remains one of the most challenging lymphomas associated with shorter response duration to conventional chemotherapy as well as continuous relapses and refractory to current drugs. However, dysregulation of cyclin D1 biology alone is insufficient to develop MCL. The emerging data suggest that the mammalian target of rapamycin (mTOR) plays a crucial role in the proper transmission of proliferative and anti-apoptotic signals through the PI3K/AKT pathway that makes it an attractive therapeutic target for hematological malignances including mantle cell lymphoma. As a single agent, rapamycin analogs such as temsirolimus (CCI-779) achieved 38% overall response rate in heavily pretreated MCL and prolonged progression free survival (PFS) in relapsed and refractory mantle cell lymphoma (4.8 months in temsirolimus vs. 1.9 months in investigator’s choice, ASCO 2008). mTOR regulates two distinct complexes TORC1 and TORC2. TORC1 complex is involved in cell cycle regulation by phosphorylating p70S6K and 4E-BP1, two molecules that are important for translational control of cyclin D1 and c-myc as well as ribosomal biogenesis whereas TORC2 complex mainly regulates phospho- AKT serine 473 leading to cell survival and proliferation. mTOR kinase also negatively regulates autophagy, a process of cellular bulk protein degradation by fusion to lysosomes upon the nutrient deprivation. We have developed mTOR kinase selective inhibitors which exhibit distinct biological profile from rapamycin in many cancer cell lines. Here we demonstrate that a selective mTOR kinase inhibitor displays potent anti-proliferative activity in JeKo-1 and Mino cells associated with decreased phosphorylation of S6, p70S6K, AKT S473, 4E-BP1 as well as decreased cyclin D1 levels leading to G1 arrest. The inhibitor also promotes autophagic cell death at 72h and 96h post-treatment. Furthermore a selective mTOR kinase inhibitor but not rapamycin induces a significant apoptosis in JeKo-1 and Mino cells. The observed apoptosis is correlated with caspases mediated PARP cleavage as well as inhibition of anti-apoptotic protein Mcl-1, suggesting TORC2/AKT S473 complex may provide survival signaling for mantle cell lymphoma. A timecourse study demonstrated that JeKo-1 and Mino cells undergo apoptosis at 24h and 48h followed by significant autophagic cell death at 72h and 96h in a dose dependent manner when exposed to our mTOR kinase inhibitor. In conclusion, mTOR kinase inhibitors are able to induce G1 cell cycle arrest, caspase-dependent apoptosis and autophagic cell death that contribute to the anti-tumor activity. Therefore it may provide a powerful alternative targeted therapy for mantle cell lymphoma.

scholarly journals The ATM Inhibitor KU60019 Synergizes the Antineoplastic Effect of Romidepsin in Mantle Cell Lymphoma (MCL)

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3703-3703
Author(s):  
Luigi Scotto ◽  
Xavier Jirau-Serrano ◽  
Kelly Zullo ◽  
Michael Mangone ◽  
Jennifer E Amengual ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Western Blot ◽  
Cell Cycle Arrest ◽  
Cell Lines ◽  
Research Funding ◽  
Cell Lymphoma ◽  
Cdk Inhibitor ◽  
Genetics Research ◽  
Cycle Arrest ◽  
Subcutaneous Xenograft

Abstract Introduction: Romidepsin (R), 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. Interestingly up-regulation of p21Cip1/Waf1 has also been shown to reduce sensitivity to romidepsin. HDAC inhibitors as a class appear to activate p21Cip1/Waf1 expression via ATM. 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 potential synergistic interaction of an ATM inhibitor with R, given the potential complementary effects around p21Cip1/Waf1. Methods: For all cytotoxicity assays, luminescent cell viability was performed using CellTiter-GloTM. Gene expression analysis was performed via Western blot and semi-quantitative PCR assay. Apoptosis and cell cycle analysis were analyzed via Fluorescence-activated cell sorting (FACS) and Western blot. The efficacy of the compounds as single agent and combination is evaluated using a subcutaneous xenograft MCL mouse model. Results: Synergy analyses were performed using Jeko-1, Maver-1 and Z-138 cells. A synergistic cytotoxic effect was observed in all MCL cell lines when the HDACi was combined with KU60019 throughout the range of all tested concentrations. Flow cytometry analysis of all three cell lines treated with single agents and combination indicated a substantial increase in the apoptotic cell fraction when R was combined with KU60019. Furthermore protein expression analysis revealed changes in a host of proteins known to be involved in cell cycle control and apoptosis. Increased activation of the programmed cell death proteins Caspase 8 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) and accumulation of the DNA damage marker gammaH2AX. Finally, the abundance of the anti-apoptotic proteins Bcl-XL and BCL-2 showed a significant decrease after treatment with R plus increased concentrations of KU60019 when compared with their abundance in the presence of the single agents. Romidepsin kill tumor cells by driving premature exit from aberrant mitosis and inducing the rapid onset of apoptosis. The increased p21 protein expression upon addition of R, responsible for cell cycle arrest in the G2/M phase, was not observed when the R treatment was combined with the KU60019 suggesting that the increased apoptosis observed in the combination treatment was due to the ability of the KU60019 to affect the up-regulation of the cdk inhibitor p21Cip1/Waf1 by the HDACi. Indeed cell cycle and protein expession analysis of Jeko-1 cells treated with single agents and the combination confirmed that addition of KU60019 results in the inhibition of the p21Cip1/Waf1 induced G2/M cell cycle arrest. Western blot and transfection analysis of p21 promoter constructs indicated that activation of p21(WAF1/Cip1) transcription by HDACi in Jeko-1 cells occurs through Sp1 sites and KU60019 affected R ability to induce p21 expression at transcriptional level. A decreased in the expression level of SP1 protein upon addition of KU60019 would suggest that KU60019 affect the ability of HDACi to induce p21(WAF1/Cip1) by modulating the expression levels of SP1. A survey of the effect of R in combination with KU60019 in other tumor types derived cell lines (CTCL, DLBCL, TALL and ATLL) also shown an overall synergistic effect similar to the one observed in MCL. The potential therapeutic effect of the HDACi in combination with KU60019 is currently evaluated in a MCL subcutaneous xenograft mouse model using the Z-138 lymphoma derived cell line. Conclusions: These data support the novel concept that dual targeting of HDAC and ATM inhibition may be a synergistic and effective strategy in MCL. Disclosures Amengual: Acetylon Pharmaceuticals, INC: Consultancy, Research Funding. Deng:TG Therapeutics, Inc.: Honoraria, Research Funding; Seattle Genetics: Research Funding. O'Connor:Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Acetylon: Consultancy, Other: Consultancy fee; Spectrum Pharmaceuticals: Consultancy, Honoraria, Research Funding; Bristol-Myers Squibb Company: Consultancy, Other: Consultancy fee; Novartis: Consultancy, Honoraria, Other: Consultancy fee; Takeda Millenium: Consultancy, Honoraria, Other: Consultancy fee, Research Funding; Seattle Genetics: Research Funding; Bayer: Consultancy, Honoraria; Mundipharma: Consultancy, Research Funding.

Inhibition of AKT/mTOR Signaling Induces Cell Cycle Arrest and Apoptosis in Mantle Cell Lymphoma.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2415-2415 ◽  
Author(s):  
Evangelia Peponi ◽  
Elias Drakos ◽  
Guadalupe Reyes ◽  
Vassiliki Leventaki ◽  
L. Jeffrey Medeiros ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Tumor Cell ◽  
Mantle Cell Lymphoma ◽  
Cell Cycle Arrest ◽  
Cell Lymphoma ◽  
Mtor Signaling ◽  
Cycle Arrest ◽  
Mantle Cell ◽  
Tumor Cell Survival ◽  
Biologic Effects

Abstract Mantle cell lymphoma (MCL) is a distinct type of B-cell lymphoma associated with aggressive clinical course. The PI3K/AKT pathway is activated in a variety of human malignancies including hematopoeitic tumors. Activated (serine 473-phosphorylated) AKT (pAKT) is believed to mediate its effects, at least in part, via phosphorylation/activation of the mammalian target of rapamycin (mTOR). However, the biologic significance of AKT/mTOR signaling in MCL is unknown. We hypothesized that the PI3K/AKT signaling pathway is activated in MCL and contributes to tumor cell survival through activation of mTOR and its downstream effectors 4EBP1, p70S6K, ribosomal protein (rp) S6 (rpS6) and eIF-4E. We used 3 mantle cell lymphoma cell lines (Mino, Jeko and Z-138), a PI3K inhibitor (LY294002), and small interfering RNA (siRNA) specific for mTOR gene to investigate the biologic effects after inhibition of AKT/mTOR signaling in MCL. Treatment of MCL cells with LY294002 resulted in apoptotic cell death in a dose-dependent manner that was associated with downregultion of the anti-apoptotic proteins cFLIP and Mcl-1. Inhibition of PI3K also resulted in decreased cyclin D1 levels and cell cycle arrest as shown by decreased S-phase fraction assessed by BrdU incorporation. Changes in cell cycle and apoptosis were associated with decreased phosphorylation (activation) of mTOR and its downstream targets 4EBP1, p70S6K, and rpS6. Silencing of mTOR expression, using mTOR-specific siRNA also decreased phosphorylation of mTOR signaling proteins and induced cell cycle arrest and apoptosis in MCL cell lines. These biologic effects were associated with downregulation of cyclin D1 and modulation of apoptosis regulators. In addition, expression of activated AKT, and effectors downstream of AKT/mTOR pathway (p-p70S6K, p-rpS6, 4EBP1, eIF-4E) was assessed in MCL tumors. Using a 10% cutoff to define positivity, 10/20 (33%) expressed Ser473pAKT, 22/22 (100%) p-p70S6K, 5/20 (25%)p- rpS6, 13/14 (93%) 4E-BP1, and 16/29 (55%) eIF-4E. p-p70S6K phosphoprotein was detected predominantly in the nucleus of tumor cells. All other phosphoproteins were expressed with a cytoplasmic pattern. We conclude that activation of PI3K/AKT may contribute to cell cycle progression and tumor cell survival in MCL cells through activation of the mTOR signaling pathway. Our data suggest that inhibition of mTOR with rapamycin analogues currently used in clinical trials may be a novel therapeutic strategy for patients with MCL.

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