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

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 63-63
Author(s):  
Nagendra K Chaturvedi ◽  
Rajkumar Rajule ◽  
Shukla Ashima ◽  
Prakash Radhakrishnan ◽  
Amarnath Natarajan ◽  
...  

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

2013 ◽  
Vol 20 (2) ◽  
pp. 393-403 ◽  
Author(s):  
Alexandra Moros ◽  
Sophie Bustany ◽  
Julie Cahu ◽  
Ifigènia Saborit-Villarroya ◽  
Antonio Martínez ◽  
...  

2010 ◽  
Vol 16 (12) ◽  
pp. 3182-3192 ◽  
Author(s):  
Qing Liu ◽  
Lapo Alinari ◽  
Ching-Shih Chen ◽  
Fengting Yan ◽  
James T. Dalton ◽  
...  

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 769-769
Author(s):  
Elena Beltran ◽  
Vicente Fresquet ◽  
Javier Martinez-Useros ◽  
Jose A. Richter-Larrea ◽  
Ainara Sagardoy ◽  
...  

Abstract Abstract 769 Despite the many and diverse therapeutic approaches used to treat patients with mantle cell lymphoma (MCL), it remains an incurable disease. Recently, attention has turned into novel therapies targeting MCL-specific oncogenic pathways important for the growth and maintenance of the transformed phenotype. The chromosomal translocation t(11;14)(q13;q32) leading to cyclin-D1 over-expression is the hallmark of MCL. Constitute cyclin-D1 activation in B-lymphocytes maintains retinoblastoma protein in a phosphorylated state and promotes cell cycling, thus initiating the tumorigenesis process. Cyclin-D1 has been postulated as a putative target for therapeutic intervention, however its evaluation has been hampered by the incomplete understanding of the mechanism underlying this cyclin oncogenic function and by the lack of valid MCL models. To investigate these issues, we developed a combined cellular-genomics screening whereby responses to known cytotoxic compounds targeting cancer-related molecular pathways were correlated with genomic, gene expression and proteomic profiles of human MCL cells. Results showed that cyclin-D1 silencing had minimal antitumoral effects but significantly increased the therapeutic efficacy of several compounds, especially the BH3 mimetics that inhibited anti-apoptotic protein BCL-2. To further evaluate this finding we generated a MCL mouse model by transducing a tetracycline-regulatable cyclin-D1-expressing vector in murine pro-B cells, which allowed modulating cyclin-D1 expression levels. These mice generated lymphomas recapitulating most of the cellular, histopathological and molecular features of human MCL. Similar to the previous in vitro findings, cyclin-D1 inhibition in this model did not induce lymphoma regression, but sensitized cells to apoptosis. Analysis of the mechanisms underlying this therapeutic synergy identified a novel role for cyclin-D1 as a pro-survival molecule. Specifically, cyclin-D1 sequestrated the pro-apoptotic effector protein BAX in MCL cells, thereby favoring BCL2 anti-apoptotic function. Accordingly, therapeutic cyclin-D1 inactivation released BAX, thus sensitizing cells to apoptosis and inducing lymphoma regression. Interestingly, pharmacological blockade in vivo of cyclin-D1 with Roscovitine synergistically cooperated with the BH3 mimetic ABT-737 to effectively inhibit MCL tumor growth. In summary, our study reveals a novel role for cyclin-D1 in deregulating apoptosis in MCL cells and highlights the potential benefit of cyclin-D1 targeting, thus providing the rationale for the clinical evaluation of drugs targeting cell proliferation and survival pathways in MCL. Disclosures: Siebert: Abbott: Honoraria.


Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1651-1651
Author(s):  
Oliver Weigert ◽  
Diederik van Bodegom ◽  
Liat Bird ◽  
Amy Saur ◽  
Trevor Tivey ◽  
...  

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.


2013 ◽  
Vol 12 (11) ◽  
pp. 2494-2504 ◽  
Author(s):  
Liang Zhang ◽  
Lan V. Pham ◽  
Kate J. Newberry ◽  
Zhishuo Ou ◽  
Rong Liang ◽  
...  

2013 ◽  
Vol 2013 ◽  
pp. 1-11 ◽  
Author(s):  
Hongyan Lv ◽  
Yan Li ◽  
Hengfei Du ◽  
Jie Fang ◽  
Xiaoning Song ◽  
...  

This study aimed to elucidate the antitumor activity of norcantharidin (NCTD) against human mantle cell lymphoma (MCL). Cell proliferation and apoptosis were examined by MTS and flow cytometry. Caspase-3, -8, and -9 activities were detected with a colorimetric caspase protease assay. Apoptotic proteins—including PARP, cyclin D1, Bcl-2 family proteins, XIAP, and cIAP I—were studied by western blot. The phosphoinositide 3 kinase (PI3K) inhibitor LY294002 was used to investigate the involvement of the PI3K/Akt signaling pathway. In vivo studies were performed using Z138 cell xenografts in nude mice. NCTD inhibited proliferation and induced apoptosis of Z138 and Mino cells, both in vitro and in vivo. PI3Kp110αand p-Akt expressions were downregulated by NCTD treatment. NCTD downregulated NF-κB activity by preventing NF-κB phosphorylation and nuclear translocation. This effect was correlated with the suppression of NF-κB-regulated gene products, such as cyclin D1, BAX, survivin, Bcl-2, XIAP, and cIAP. This phenomenon was blocked by the PI3K inhibitor LY294002. Our results demonstrated that NCTD can induce growth arrest and apoptosis in MCL cells and that the mechanism may involve the PI3K/Akt/NF-κB signaling pathway. NCTD may have therapeutic and/or adjuvant therapeutic applications in the treatment of MCL.


ESMO Open ◽  
2018 ◽  
Vol 3 (6) ◽  
pp. e000387 ◽  
Author(s):  
Chiara Tarantelli ◽  
Elena Bernasconi ◽  
Eugenio Gaudio ◽  
Luciano Cascione ◽  
Valentina Restelli ◽  
...  

BackgroundThe outcome of patients affected by mantle cell lymphoma (MCL) has improved in recent years, but there is still a need for novel treatment strategies for these patients. Human cancers, including MCL, present recurrent alterations in genes that encode transcription machinery proteins and of proteins involved in regulating chromatin structure, providing the rationale to pharmacologically target epigenetic proteins. The Bromodomain and Extra Terminal domain (BET) family proteins act as transcriptional regulators of key signalling pathways including those sustaining cell viability. Birabresib (MK-8628/OTX015) has shown antitumour activity in different preclinical models and has been the first BET inhibitor to successfully undergo early clinical trials.Materials and methodsThe activity of birabresib as a single agent and in combination, as well as its mechanism of action was studied in MCL cell lines.ResultsBirabresib showed in vitro and in vivo activities, which appeared mediated via downregulation of MYC targets, cell cycle and NFKB pathway genes and were independent of direct downregulation of CCND1. Additionally, the combination of birabresib with other targeted agents (especially pomalidomide, or inhibitors of BTK, mTOR and ATR) was beneficial in MCL cell lines.ConclusionOur data provide the rationale to evaluate birabresib in patients affected by MCL.


2015 ◽  
Vol 21 (19) ◽  
pp. 4391-4397 ◽  
Author(s):  
Matthew J. Barth ◽  
Cory Mavis ◽  
Myron S. Czuczman ◽  
Francisco J. Hernandez-Ilizaliturri

Blood ◽  
2016 ◽  
Vol 128 (21) ◽  
pp. 2517-2526 ◽  
Author(s):  
Caron Jacobson ◽  
Nadja Kopp ◽  
Jacob V. Layer ◽  
Robert A. Redd ◽  
Sebastian Tschuri ◽  
...  

Key Points Inhibition of HSP90 targets multiple dependences in mantle cell lymphoma. Clinically available HSP90 inhibitors overcome ibrutinib resistance in vitro and in vivo.


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