Combined Targeting of Chromatin Modifying Enzymes LSD1, EZH2 and Histone Deacetylases (HDACs) Has Superior Efficacy Against Human Mantle Cell Lymphoma Cells

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2429-2429
Author(s):  
Warren Fiskus ◽  
Sunil Sharma ◽  
Rekha Rao ◽  
Ramesh Balusu ◽  
Sreedhar Venkannagari ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
Cyclin D1 ◽  
Mantle Cell Lymphoma ◽  
Core Protein ◽  
Cell Lymphoma ◽  
Amine Oxidase ◽  
Combined Treatment ◽  
Reversible Inhibitor ◽  
Mantle Cell

Abstract Abstract 2429 PRC (polycomb repressive complex) 2 contains three core protein components, i.e., EZH2, SUZ12 and EED, of which EZH2 has the SET domain with its intrinsic histone methyltransferase activity that mediates the trimethylation (Me3) of lysine (K) 27 on histone (H) 3-a repressive chromatin mark for gene expression. We have previously reported that treatment with the S-adenosylhomocysteine hydrolase and EZH2 inhibitor, DZNep as well as treatment with the pan-histone deacetylase inhibitor panobinostat (PS, Novartis Pharma) deplete PRC2 complex proteins. LSD1 (KDM1A) is a demethylase of H3K4Me2, and inhibiton of LSD1 leads to increase in H3K4Me3-a permissive mark for gene expression. In the present studies, we determined the chromatin-modifying and cytotoxic effects of LSD1 inhibition alone and in combination with PS or DZNep in cultured (JeKo-1 and Z138C) and primary human Mantle Cell Lymphoma (MCL) cells. Treatment with the non-amine oxidase reversible inhibitor of LSD1 CIT0665 (250 to 1000 nM), or the more potent analogue HCI2509 (20 to 250 nM), dose-dependently increased the levels of H3K4Me2 & Me3, p21 and p27, while decreasing the levels of cyclin D1, which was associated with inhibition of cell proliferation and accumulation of the MCL cells in the G1 phase of the cell cycle. Abrogation of LSD1 by a specific shRNA treatment also induced similar chromatin, cell cycle and growth inhibitory effects. Exposure to CIT0665 or HCI2509 disrupted the binding of LSD1 with the co-repressor CoREST and HDAC1, without affecting the levels of these proteins. As noted above, treatment with PS (10 to 50 nM) dose-dependently depleted the levels of not only EZH2, SUZ12 and the PRC1 complex protein BMI1, but also of LSD1 in MCL cells. PS treatment alone also depleted the levels of AKT, cRAF, CDK4 and cyclin D1, as well as induced cell cycle growth inhibition and apoptosis of MCL cells. Co-treatment with PS enhanced the chromatin modifying effects of CIT0665 or HCI2509. The combination synergistically induced apoptosis of the cultured MCL cells (combination indices, CI <1.0). This was associated with greater induction of p27 and depletion of cyclin D1. Treatment with PS and HCI2509 also synergistically induced loss of viability of primary MCL cells (CI <1.0). We have previously reported that DZNep dose-dependently depleted EZH2, SUZ12 and BMI1 expression, inhibited H3K27Me3 levels, induced p21, p27 and FBXO32 (muscle atrophy F-box protein, also called atrogin-1) levels in cultured and primary MCL cells. Here, we determined the effects of co-treatment with HCI2509 and DZNep in MCL cells. Combined treatment with HCI2509, although not synergistic, enhanced the apoptosis of MCL cells induced by DZNep. Taken together these findings indicate that combined targeted depletion of the level and activity of LSD1 by PS and CIT0665 or HCI2509 along with PS-mediated depletion of PRC2 proteins, BMI and HDACs exerts superior activity against MCL cells. These studies also support the in vivo testing of combined epigenetic therapies in the treatment of MCL. Disclosures: Sharma: Novartis: Research Funding.

Pathogenesis of Mantle-Cell Lymphoma: All Oncogenic Roads Lead to Dysregulation of Cell Cycle and DNA Damage Response Pathways

2005 ◽  
Vol 23 (26) ◽  
pp. 6364-6369 ◽  
Author(s):  
Veronica Fernàndez ◽  
Elena Hartmann ◽  
German Ott ◽  
Elias Campo ◽  
Andreas Rosenwald
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
Dna Damage ◽  
Cyclin D1 ◽  
Mantle Cell Lymphoma ◽  
Dna Damage Response ◽  
Cell Lymphoma ◽  
Mantle Cell ◽  
Damage Response ◽  
Cell Cycle Machinery

Mantle-cell lymphoma (MCL) is a well-defined subtype of B-cell non-Hodgkin's lymphomas (B-NHL), accounts for approximately 6% of all lymphoid neoplasms, and has a median survival of 3 to 4 years. The genetic hallmark of MCL is the chromosomal translocation t(11;14)(q13;q32) that leads to deregulation and upregulation of Cyclin D1, an important regulator of the G1 phase of the cell cycle. This genetic event is present in virtually all cases of MCL, whereas additional genetic alterations that occur in subsets of MCL have been described. Most of these alterations appear to disturb the cell cycle machinery/interfere with the cellular response to DNA damage, thus making MCL a paradigm for cell cycle and DNA damage response dysregulation in cancer in general. In particular, Cyclin D1 upregulation, genomic amplification of the cyclin-dependent kinase (CDK) -4, deletions of the CDK inhibitor p16INK4a and overexpression of BMI-1, a transcriptional repressor of the p16INK4a locus, are associated with dysregulation of the cell cycle machinery in MCL. The DNA damage response pathway is affected by frequent alterations of the ataxia-telangiectasia mutated (ATM) kinase as well as occasional inactivation of checkpoint kinase (CHK)-1 and CHK2 that are kinases that act downstream of ATM in response to detection of DNA damage. Moreover, p53 is frequently targeted by alterations in MCL. A recent gene expression profiling study defined the proliferation signature, a quantitative measure of gene expression of proliferation-associated genes as the strongest survival predictor available to date allowing the definition of prognostic MCL subgroups that differ in median survival by more than 5 years.

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.

FTY720 Demonstrates Promising in-Vitro and in-Vivo Pre-Clinical Activity by Down-Modulation of Cyclin D1 and Pakt in Mantle Cell Lymphoma.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3728-3728
Author(s):  
Lapo Alinari ◽  
Qing Liu ◽  
Ching-Shih Chen ◽  
Fengting Yan ◽  
James T Dalton ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cyclin D1 ◽  
Mantle Cell Lymphoma ◽  
Cell Line ◽  
Cell Lymphoma ◽  
Tumor Burden ◽  
Time Dependent ◽  
Control Group ◽  
Mantle Cell

Abstract Abstract 3728 Poster Board III-664 Over-expression of Cyclin D1 and constitutive phosphorylation of Akt has been implicated in the pathogenesis of mantle cell lymphoma (MCL). Here we describe FTY720 (fingolimod), an immunosuppressive agent currently being explored in phase III studies in renal transplantation and multiple sclerosis patients, to mediate time- and dose-dependent cell death in primary MCL cells (6 patients) and MCL cell lines, Jeko and Mino. FTY720-induced apoptosis was associated with reactive oxygen species (ROS) generation, Bax up-regulation but not associated with caspase 3 activation in MCL. FTY720 treatment resulted in time-dependent down-modulation of Cyclin D1 and phospho Akt (p-Akt) protein level, two critical disease-relevant molecules in the pathogenesis of MCL. Consistent with the modulation of Cyclin D1, FTY720-induced cell cycle arrest with accumulation of cells in G0/G1 and G2/M phases of the cell cycle with concomitant decrease in S phase entry. Importantly, FTY720 treatment was also associated with a time-dependent phospho Erk (p-Erk) induction in Mino and Jeko cells. To determine the in vivo efficacy of FTY720, we developed a preclinical, in vivo xenograft model of human MCL where MCL cell lines (Jeko, Mino and SP53) were engrafted into severe combined immune deficient (SCID) mice. Cell dose titration trials identified 4 × 107 Mino or Jeko cells injected intravenously via tail vein to result in consistent engraftment and fatal tumor burden in all mice. All mice engrafted with 4 × 107 Jeko cells developed a disseminated disease within 3 weeks and had a median survival of 28 days (compared to 43 days for Mino and 51 days for SP53). Because the Jeko cell line was established from the peripheral blood of a patient with blastic variant MCL and demonstrated a more resistant phenotype to several immuno-chemoterapeutic compounds, this cell line was chosen to create a more stringent in vivo preclinical model. SCID mice were treated with the monoclonal antibody TMβ1 to deplete murine NK cells, engrafted with 4 × 107 Jeko cells and observed daily for signs of tumor burden. Ten mice/group were treated starting at day 15 post-engraftment with intraperitoneal injection of 100 μl of saline or FTY720 (5 mg/kg resuspended in 100 μl of saline), every day, for two weeks. The median survival for FTY720-treated mice (N=10) was 38 days (95% CI:30-39) compared to 26.5 days (95% CI: 26-27 days) for the control group mice (N=10). The results from the log-rank test indicated an overall statistical significant difference in survival functions between the FTY720 treatment and the control group (p=0.001). These results provide the first evidence for a potential use of FTY720 in targeting key pathways that are operable in the pathogenesis of MCL and warrant the further investigation of FTY720 in combination with other agents in clinical trials treating patients with MCL. Disclosures: No relevant conflicts of interest to declare.

Biology and Treatment of Mantle Cell Lymphoma in the Genomic Era.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. SCI-28-SCI-28
Author(s):  
Adrian Wiestner
Keyword(s):  
Gene Expression ◽  
Cyclin D1 ◽  
Mantle Cell Lymphoma ◽  
Gene Expression Profiling ◽  
Expression Profiling ◽  
Cell Lymphoma ◽  
Genetic Alterations ◽  
Diagnostic Methods ◽  
Genetic Changes ◽  
Mantle Cell

Abstract Abstract SCI-28 The t11;14 translocation, the genetic hallmark of MCL, drives cyclin D1 expression in the tumor cells and historically facilitated the separation of MCL into a distinct entity. FISH cytogenetics are part of the workup and can be particularly helpful to separate leukemic MCL from CLL. Morphology and clinical course of MCL are heterogeneous and might have suggested the presence of different entities. Gene expression profiling answered this concern and provided several important insights: 1. despite the clinical heterogeneity, MCL has a characteristic gene expression profile supporting the accuracy of current diagnostic methods; 2. cyclin D1 negative MCL has the same diagnostic pathologic and gene expression features as cyclin D1 positive MCL, and 3. a gene expression based measure of tumor proliferation is a potent predictor of outcome and identifies patients with survival probabilities ranging from less than 1 year (highly proliferative tumors) to more than 6 years (low proliferation).1 Biologically, the gene expression based proliferation score integrates several acquired genetic changes in the tumor that include deletions of the INK4a/ARF locus encoding the tumor suppressors p14 and p16, amplification of BMI1, and secondary changes in the cyclin D1 locus. Mutations and deletions that alter the structure of the 3'UTR can enhance cyclin D1 mRNA stability,2 and the loss of miR binding sites in this region can enhance protein translation.3 These changes increase cyclin D1 protein resulting in increased proliferation. Additional genetic lesions such as deletions of ATM and p53 affect DNA damage responses pathways. The high frequency of secondary genetic changes in MCL cells may indicate genomic instability and the presence of additional chromosomal aberrations and certain genetic alterations hold prognostic information.4 With the continued refinement of whole genome genetic approaches the goal of identifying crucial pathways and possible driver genes in the pathogenesis of MCL may be within reach. MCL characterized by an antiapoptotic phenotype combined with features of aggressive lymphomas remains an incurable disease and having the worst outcome among all B-cell lymphomas. Biologic markers that predict treatment response and that could give way to targeted therapy have remained elusive. Several new drugs could help overcome treatment resistance and new analytic tools when incorporated into clinical trials may help dissect mechanisms of drug action and resistance. Our approach has been to incorporate gene expression profiling into a clinical trial of bortezomib to directly monitor the effects of the treatment on tumor biology in vivo. We identified an integrated stress response to bortezomib in sensitive tumors that may yield clinically usefully predictors of sensitivity and that could guide the development of improved therapies. 1. Rosenwald A, Wright G, Wiestner A, et al. The proliferation gene expression signature is a quantitative integrator of oncogenic events that predicts survival in mantle cell lymphoma. Cancer Cell. 2003;3:185-197. 2. Wiestner A, Tehrani M, Chiorazzi M, et al. Point mutations and genomic deletions in Cyclin D1 create stable truncated mRNAs that are associated with increased proliferation rate and shorter survival in mantle cell lymphoma. Blood. 2007. 3. Chen RW, Bemis LT, Amato CM, et al. Truncation in CCND1 mRNA alters miR-16-1 regulation in mantle cell lymphoma. Blood. 2008;112:822-829. 4. Salaverria I, Espinet B, Carrio A, et al. Multiple recurrent chromosomal breakpoints in mantle cell lymphoma revealed by a combination of molecular cytogenetic techniques. Genes Chromosomes Cancer. 2008;47:1086-1097. Disclosures Off Label Use: Bortezomib in previously untreated patients with MCL.

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 Concurrent disruption of cell cycle associated genes in mantle cell lymphoma: a genotypic and phenotypic study of cyclin D1, p16, p15, p53 and pRb

Leukemia ◽  
1998 ◽  
Vol 12 (8) ◽  
pp. 1266-1271 ◽  
Author(s):  
K Grønbæk ◽  
T Nedergaard ◽  
MK Andersen ◽  
P thor Straten ◽  
P Guldberg ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cyclin D1 ◽  
Mantle Cell Lymphoma ◽  
Cell Lymphoma ◽  
Mantle Cell

Multiparameter Immunohistochemical Analysis of the Cell Cycle Proteins Cyclin D1, Ki-67, p21WAF1, p27KIP1, and p53 in Mantle Cell Lymphoma

2000 ◽  
Vol 124 (10) ◽  
pp. 1457-1462
Author(s):  
Keith F. Izban ◽  
Serhan Alkan ◽  
Timothy P. Singleton ◽  
Eric D. Hsi
Keyword(s):  
Cell Cycle ◽  
Cyclin D1 ◽  
Mantle Cell Lymphoma ◽  
Mitotic Index ◽  
Cell Lymphoma ◽  
Ki 67 ◽  
Exact Test ◽  
Mantle Cell ◽  
Oil Immersion ◽  
Fisher’S Exact Test

Abstract Background.—Mantle cell lymphoma (MCL) is characterized by overexpression of cyclin D1, a G1 cyclin that participates in the control of cell cycle progression at the G1 to S phase transition. In addition to cyclin D1, other cell cycle regulatory molecules may be involved in the proliferation and progression of MCL. Mutation of p53, deletion of p16INK4a, and loss of p21WAF1 expression have been reported in some cases of blastoid MCL. Objective.—We sought to examine levels of expression of these proteins in typical and blastoid MCL and to determine whether differences were present between these subtypes of lymphomas. Design.—A retrospective series of typical and blastoid MCLs was evaluated for expression of the cell cycle–related proteins cyclin D1, p21WAF1, p27KIP1, Ki-67, and p53, as well as mitotic index. Paraffin-embedded archival tissues from 24 MCL specimens (17 typical, 7 blastoid) were immunostained with antibodies to p21WAF1, p27KIP1, p53, Ki-67, and cyclin D1. The percentage of positive cells for each specimen was estimated by counting 1500 cells under oil immersion microscopy. Levels of antigen expression were compared for the typical and blastoid MCLs. The mitotic index was estimated using twenty 100× oil immersion fields (OIFs) for each specimen. Results.—Cyclin D1 expression was seen in 22/24 specimens (92%). Blastoid MCLs were characterized by a significantly higher mean mitotic index (&gt;20 mitoses/20 OIFs) and Ki-67 index (&gt;45%) when compared with typical MCLs (P &lt; .001 and P &lt; .008, respectively; Fisher's exact test). High expression of p27KIP1 (&gt;25% staining) was seen more frequently in typical MCLs than in the blastoid variants (P = .03; Fisher's exact test). No significant differences were found between typical and blastoid MCLs for the expression of p21WAF1 or p53. Conclusions.—A significantly higher mitotic index and Ki-67 index were found in blastoid MCLs as compared with typical MCLs. Low p27KIP1 expression was associated with the blastoid MCL variant. These findings confirm the high proliferative nature of blastoid MCL and suggest a role for p27KIP1 in the negative regulation of the cell cycle in MCL.

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.

siRNAs Against Cyclin D1/D2 Improves Cytotoxicity of Chemotherapy In Mantle Cell Lymphoma

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1816-1816
Author(s):  
Robert W Chen ◽  
Katrin Tiemann ◽  
Jessica Alluin ◽  
Stephen Forman ◽  
John Rossi
Keyword(s):  
Cell Cycle ◽  
Cyclin D1 ◽  
Mantle Cell Lymphoma ◽  
Cell Lines ◽  
Cytotoxic Effect ◽  
Cell Lymphoma ◽  
Cyclin D2 ◽  
Down Regulation ◽  
Dual Targeting ◽  
Mantle Cell

Abstract Abstract 1816 Introduction: Mantle cell lymphoma is an aggressive B cell neoplasm with a median survival of 3 years. Cyclin D1 overexpression is the genetic hallmark of MCL and regulates cell cycle progression. However, the significance of cyclin D1 in the pathogenesis and treatment of MCL still remains to be defined. The aim of this study is to determine whether down regulation of cyclin D1 with siRNA will lead to enhanced therapeutic effect of chemotherapy in MCL. We used siRNA technology in three well characterized MCL cell lines, and tested traditional chemotherapy agents (doxorubicin and etoposide) as a model system. Material and Methods: We designed three different siRNA targeting cyclin D1 (si-224, 391, 778), one siRNA against cyclin D2 (si-D2), and a dual targeting siRNA against both cyclin D1 and D2 (si-D1/D2). The siRNAs used were 27 mer asymetric duplexes with a 2nd 3′ overhang. Granta-519 cells were transfected by lipofection (Lipofectamin RNAimax, Invitrogen), Z-138 and Jeko-1 cells were transfected with electroporation (BioRad). Western Blot analysis and real time PCR were performed to examine the down regulatory efficiency of the siRNAs on cyclin D1 mRNA and protein. Chemotherapeutics doxorubicin and etoposide were tested for enhancement of cytotoxicity by siRNA. The effect on cell viability of cyclin D1 reduction in combination with chemotherapeutics was analyzed by MTS assay. Results: We achieved cyclin D1 mRNA and protein down regulation in all 3 MCL cell lines, although the efficiency of knockdown varied among the siRNAs and the cell lines of interests. (Table 1) Si-224 has the best activity in Granta-519 while si-778 has the best activity in Jeko-1. We determined the cytotoxic effect of chemotherapy alone as well as in combination with siRNAs by MTS assays. The combination of chemotherapeutic with our siRNAs decreased the IC50 of both doxorubicine and etoposide. In Granta 519, si-224 decreased the IC 50 of doxorubicin by 32% and etoposide by 28%. In Jeko-1, si-778 decreased the IC 50 of doxorubicin by 49% but no effect on etoposide was seen. The magnitude of cyclin D1 down regulation seems to correlates with the percentages changes in IC 50. Klier et al previously reported that knockdown of cyclin D1 leads to an upregulation of cyclin D2 in MCL. Hence we mixed si-224 as well as si-778 targeting cyclin D1 with a si-D2 against cyclin D2 in combination with doxorubicine and etoposide in Granta-519. We also designed a dual-targeting siRNA against CCND1 and CCND2 (si-D1/D2). Targeting both cyclin D1 and D2 decreased the IC 50 of doxorubicin further than targeting cyclin D1 alone. Si224/D2 decreased the IC 50 of doxorubicin by 57% (si-224 alone 32%) and etoposide by 39% (si-224 alone 28%), and si778/D2 decreased the IC 50 of doxorubicine by 58% (si-778 alone 49%). The dual-targeting siRNA showed a decrease in IC 50 of doxorubicin by 45% and etoposide by 48%. Conclusions: Down regulation of cyclin D1 in MCL with siRNA improves the IC 50 of chemotherapeutic agents. Dual inhibition of both cyclin D1 and D2 further enhances the cytotoxic effect of doxorubicine and etoposide. Besides being a cell cycle regulator, cyclin D1 also seems to regulate chemosensitivity in MCL. Footnotes: This work was supported by grants from the Tower Cancer Research Foundation and Tim Nesvig Lymphoma Research Fund and Fellowship, Think Cure, Keck-foundation, SPORE. Disclosures: No relevant conflicts of interest to declare.

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