Malignant hematopoietic cell lines: in vitro models for the study of mantle cell lymphoma

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.

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Malignant hematopoietic cell lines: In vitro models for the study of Waldenström's macroglobulinemia

Leukemia Research ◽

10.1016/j.leukres.2008.04.016 ◽

2008 ◽

Vol 32 (11) ◽

pp. 1669-1673 ◽

Cited By ~ 10

Author(s):

Hans G. Drexler ◽

Roderick A.F. MacLeod

Keyword(s):

Cell Lines ◽

In Vitro Models ◽

Hematopoietic Cell ◽

Waldenström’S Macroglobulinemia ◽

Waldenstrom's Macroglobulinemia ◽

Waldenstrom’S Macroglobulinemia ◽

Waldenström's Macroglobulinemia ◽

Hematopoietic Cell Lines

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GST-n and Nitric Oxide: A Novel Approach to Mantle Cell Lymphoma Therapy.

Blood ◽

10.1182/blood.v114.22.3729.3729 ◽

2009 ◽

Vol 114 (22) ◽

pp. 3729-3729

Author(s):

Heather Gilbert ◽

John Cumming ◽

Josef T. Prchal ◽

Michelle Kinsey ◽

Paul Shami

Keyword(s):

Nitric Oxide ◽

Mantle Cell Lymphoma ◽

Cell Lines ◽

Cell Lymphoma ◽

Xenograft Model ◽

Malignant Cells ◽

Mantle Cell ◽

Tumor Viability

Abstract Abstract 3729 Poster Board III-665 Mantle cell lymphoma (MCL) is a well defined B-cell non-Hodgkin lymphoma characterized by a translocation that juxtaposes the BCL1 gene on chromosome 11q13, which encodes cyclin D1 (CD1), next to the immunoglobulin heavy chain gene promoter on chromosome 14. The resulting constitutive overexpression of CD1 leads to a deregulated cell cycle and activation of cell survival mechanisms. In addition, the gene which encodes GST-n, an enzyme that has been implicated in the development of cancer resistance to chemotherapy, is also located on chromosome 11q13 and is often coamplified along with the BCL1 gene in MCL (1). These two unique biological features of MCL - the overproduction of cyclin D1 and GST-n – may be involved in the carcinogenesis, tumor growth and poor response of this disease to treatment, and they offer potential mechanisms for targeted anti-cancer therapy. Nitric oxide (NO) is a biologic effector molecule that contributes to a host's immune defense against microbial and tumor cell growth. Indeed, NO is potently cytotoxic to tumor cells in vitro (2–4). However, NO is also a potent vasodilator and induces hypotension, making the in vivo administration of NO very difficult. To use NO in vivo requires agents that selectively deliver NO to the targeted malignant cells. A new compound has recently been developed that releases NO upon interaction with glutathione in a reaction catalyzed by GST-n. JS-K seeks to exploit known GST-n upregulation in malignant cells by generating NO directly in cancer cells, and it has been shown to decrease the growth and increase apoptosis in vitro in AML cell lines, AML cells freshly isolated from patients, multiple myeloma cell lines, hepatoma cells and prostate cancer cell lines (3, 5–7). JS-K also decreases tumor burden in NOD/SCID mice xenografted with AML and multiple myeloma cells (5, 7). Importantly, JS-K has been used in cytotoxic doses in the mouse model without significant hypotension. To evaluate whether JS-K treatment has anti-tumor activity in MCL, the human MCL cell lines Jeko1, Mino, Granta and Hb-12 were grown with media only, with JS-K at varying concentrations and with DMSO as an appropriate vehicle control. For detection of apoptotic cells, cell-surface staining was performed with FITC-labeled anti–Annexin V and PI. Cell growth was evaluated using the Promega MTS cytotoxicity assay. Results show that JS-K (at concentrations up to 10 μM) inhibits the growth of MCL lines compared to untreated controls, with an average IC50 of 1 μM. At 48 hours of incubation, all cell lines showed a significantly greater rate of apoptosis than untreated controls. A human MCL xenograft model was then created by subcutaneously injecting two NOD/SCID IL2Rnnull mice with luciferase-transfected Hb12 cells. Seven days post-injection, one of the mice was treated with JS-K at a dose of 4 μmol/kg (expected to give peak blood levels of around 17 mM in a 20 g mouse). Injections of JS-K were given intravenously through the lateral tail vein 3 times a week. The control mouse was injected with an equivalent volume of micellar formulation (vehicle) without active drug. The Xenogen bioluminescence imaging clearly showed a difference in tumor viability, with a significantly decreased signal in the JS-K treated mouse. Our studies demonstrate that JS-K markedly decreases cell proliferation and increases apoptosis in a concentration- and time-dependent manner in mantle cells in vitro. In a xenograft model of mantle cell lymphoma, treatment with JS-K results in decreased tumor viability. Proposed future research includes further defining the molecular basis of these treatment effects; using this therapy in combination with other cancer treatments both in vitro and in vivo; and studying JS-K treatment in MCL patients. Disclosures: Shami: JSK Therapeutics: Founder, Chief Medical Officer, Stockholder.

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Enzastaurin Treatment Affects Multiple Regulatory Pathways at Transcriptome and Cellular Proteome Level of Mantle Cell Lymphoma

Blood ◽

10.1182/blood.v116.21.2893.2893 ◽

2010 ◽

Vol 116 (21) ◽

pp. 2893-2893

Author(s):

Marc Weinkauf ◽

Grit Hutter ◽

Yvonne Zimmermann ◽

Malte Rieken ◽

Alessandro Pastore ◽

...

Keyword(s):

Cell Cycle ◽

Mantle Cell Lymphoma ◽

Cell Lines ◽

Cell Lymphoma ◽

Signal Pathways ◽

Rna Expression ◽

Cellular Functions ◽

Mantle Cell ◽

Canonical Pathways

Abstract Abstract 2893 Background: The protein kinase C beta inhibitor enzastaurin is one of the promising molecular targeted approaches currently investigated in mantle cell lymphoma (MCL), a disease still characterized by a dismal long term prognosis. Methods: Four well characterized MCL cell lines (Granta 519, HBL-2, Jeko-1 and Rec-1) as well as three patient samples were exposed to enzastaurin at a previously defined dose (10 μM). Cell viability as well as cell cycle activity were analyzed by tryphan blue exclusion test and flow cytometry, respectively, after 24 and 48 hours. To dissect the regulatory processes targeted by enzastaurin, the panel of MCL cell lines was screened on both protein and RNA expression levels (2D-gel electrophoresis and mass spectrometric peptide fingerprint analysis and Affymetrix microarray) after 4h enzastaurin treatment. Results: Enzastaurin in vitro resulted in a reduced viability and cell proliferation by 15–20% after 24h in cell lines and 9–20% in primary patient samples after 48h. This effect was related to a G2/M block of cell cycle and induction of apoptosis. Based on the proteome and transcriptome analysis of early alterations, only HSPD1 was affected on both regulation levels. Nonetheless, combined analysis of alterations on both, protein and RNA expression levels, resulted in identification of common signal pathways characterizing a more comprehensive network of affected molecular interactions mapping to distinct canonical pathways and defined cellular functions. Indicated canonical pathways included ‘calcium signalling', (CAMKK2, HDAC5, HDAC9, TP63) ‘calcium induced T-lymphocyte apoptosis' (MEF2D, NR4A1, PRKCG, TRA@), ‘NFkB signalling' (KRAS, MAP3K8, TNFAIP3, TNFRS17) and ‘molecular mechanisms of cancer' (APAF1, CDKN2D, FOS, PAK6), whereas the top ranking cellular functions were ‘cellular growth and proliferation' (CCNG2, EIF4E, PDIA3, TOP1, TPM1,), ‘cell death' (BCL6, EEF1D, PAK6, RAD50), ‘cell cycle'(AKAP9, BMF, CUL5, GADD45B, PDIA3), ‘cellular development' (APAF1, GAS7, ID1, PAX8) and ‘gene expression' (ABCG1, HOXB4, LMO4, PIM1). Alterations of these pathways were confirmed by Western Blot analysis of selected candidate proteins marker proteins of the regulated pathways. Conclusion: In summary, the combined approach of RNA and protein analysis revealed the targeted signal pathways after Enzastaurin exposure. These data will allow a more rationally designed combination of biologicals to finally improve the clinical outcome of MCL. Disclosures: Dreyling: Eli Lilly: Support of in vitro studies of Enzostaurin in MCL.

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

Blood ◽

10.1182/blood-2011-04-346601 ◽

2012 ◽

Vol 119 (2) ◽

pp. 476-487 ◽

Cited By ~ 72

Author(s):

Mamta Gupta ◽

Andrea E. Wahner Hendrickson ◽

Seong Seok Yun ◽

Jing Jing Han ◽

Paula A. Schneider ◽

...

Keyword(s):

Mantle Cell Lymphoma ◽

Cell Lines ◽

Transcriptional Activation ◽

Cell Lymphoma ◽

Lymphocytic Leukemia ◽

Clinical Samples ◽

Lymphoid Malignancies ◽

Mantle Cell ◽

Induced Apoptosis

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

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The Effects of PI3K-δ/γ Inhibitor, Duvelisib, in Mantle Cell Lymphoma in Vitro and in Patient-Derived Xenograft Studies

Blood ◽

10.1182/blood.v128.22.3016.3016 ◽

2016 ◽

Vol 128 (22) ◽

pp. 3016-3016 ◽

Cited By ~ 4

Author(s):

Jack Wang ◽

Victoria Zhang ◽

Taylor Bell ◽

Yang Liu ◽

Hui Guo ◽

...

Keyword(s):

Growth Inhibition ◽

Mantle Cell Lymphoma ◽

Cell Lines ◽

Cell Lymphoma ◽

Oral Gavage ◽

Patient Derived Xenograft ◽

Pdx Model ◽

Mantle Cell

Abstract Background: Mantle cell lymphoma (MCL) is an incurable subtype of B-cell lymphoma. Ibrutinib, a first-in-class, once-daily, oral covalent inhibitor of Bruton's tyrosine kinase (BTK) was approved by the FDA for the treatment of MCL in patients previously treated. In our prior multicenter Phase 2 clinical trial, the overall response rate in relapsed/refractory MCL was 68%, with a median progression-free survival (PFS) of 13.9 months. However, the majority of MCL patients treated with ibrutinib relapsed; in these relapsed patients, the one-year survival rate was only 22%. Therefore, there exists an urgent need for additional novel targeted therapies to improve the mortality rate in these patients. In this study, we assessed the in vitro and in vivo effects of duvelisib, a PI3K-δ,-γ inhibitor, in MCL. Methods: The PI3K/AKT/mTOR and other cell survival signaling pathways were investigated by RNASeq and reverse phase protein array (RPPA) in ibrutinib-sensitive and -resistant MCL samples. The expression of PI3K isoforms, α, β, γ, and δ was tested in 11 MCL cell lines, patient and patient-derived xenograft (PDX) MCL cells by western blot analysis. We then investigated the growth inhibition and apoptosis of duvelisib (IPI-145, Infinity Pharmaceuticals, Inc.) in MCL cells by CellTiter-Glo® Luminescent Cell Viability Assay (Promega) and Annexin V-binding assay (BD Biosciences). We established a primary MCL-bearing PDX model and passaged the primary MCL tumor to next generations. Mice were administrated with 50 mg/kg duvelisib daily by oral gavage. Tumor burden and survival time were investigated in the MCL-PDX model. Results: We found that the PI3K/AKT/mTOR signaling pathway was activated in both primary and acquired ibrutinib-resistant MCL cell lines and PDX MCL cells. We immunoblotted PI3K isoforms, α, β, γ, and δ in 11 MCL cell lines and the result demonstrated that both ibrutinib-sensitive and ibrutinib-resistant MCL cells dominantly expressed PI3K-δ and -γ. Next, we tested the effects of duvelisib on these MCL cells. Duvelisib had effects on the growth inhibition and apoptosis in both ibrutinib-sensitive and ibrutinib-resistant MCL cells as good as the PI3K-δ inhibitor, idelalisib (Cal-101, GS-1101). The PI3K-δ isoform could play a very important role in PI3K-mediated signals in MCL. We then investigated the effects of duvelisib in vivo through our established MCL-bearing PDX mouse models. These models are created by inoculating the primary tumor cells from MCL patients into a human fetal bone chip implanted into NSG mice to provide a microenvironment that reconstitutes the human environment. MCL tumor mass was then passaged to next generations for therapeutic investigation of duvelisib. Mice were treated with 50 mg/kg duvelisib daily by oral gavage. Our data demonstrated that duvelisib significantly inhibited tumor growth and prolonged survival of MCL-PDX mice. Conclusion: Duvelisib, an oral dual inhibitor of PI3K-δ,-γ, inhibits MCL growth both in vitro and in PDX mice. These preclinical results suggests duvelisib may be effective in the treatment of patients with relapsed/refractory MCL. Disclosures No relevant conflicts of interest to declare.

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In Vitro and In Vivo Anti Lymphoma Effect of GX15-070 in Mantle Cell Lymphoma.

Blood ◽

10.1182/blood.v108.11.4756.4756 ◽

2006 ◽

Vol 108 (11) ◽

pp. 4756-4756 ◽

Cited By ~ 1

Author(s):

Gwyn Bebb ◽

Huong Muzik ◽

Sophia Nguyen ◽

Don Morris ◽

Douglas A. Stewart

Keyword(s):

Mantle Cell Lymphoma ◽

Cell Line ◽

Cell Lines ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

Cytotoxic Agents ◽

In Vivo Experiments ◽

Mantle Cell

Abstract Introduction Mantle cell lymphoma (MCL), an incurable B cell lymphoma, consistently over expresses bcl-2 despite not carrying the t(14;18). The attenuation of apoptosis by bcl-2 is thought to contribute to the malignant process and increase resistance to some cytotoxic agents. We recently demonstrated that GX15-070, a small molecular inhibitor of the BH3 binding groove of bcl-2, has activity against MCL cell lines in vitro. We set out to assess the effect of GX15-070 alone and in combination with Vincristine on the viability of MCL cells in vitro and in vivo. Methods 3 previously characterized bcl-2 over expressing MCL cell lines (JVM-2, Hbl-2, granta) were used. Cells were grown in standard media and exposed to a range of concentrations of GX15-070 with and without Vincristine. Dose-response was assessed by measuring viability at 48 hours using the WST-1 assay. In vivo experiments were conducted on immune deficient mice in which 5×106 cells were injected in the flank then treated IV with GX15-070 (q 2days × 5 doses), Vincristine (q4 days × 3 doses) or both starting 5 days later. Tumours were measured three times weekly. Results All three MCL cell lines over-expressed bcl-2 by western blot. Each MCL cell line showed sensitivity to GX15-070 at a range of concentrations. The addition of GX15-070 to low dose Vincristine (10−6) caused significant growth inhibition of each MCL cell line (see table 1). Discussion Our results demonstrate that using GX15-070 to target bcl-2 is an effective anti neoplastic approach against MCL cell lines in vitro. In addition, our results suggest that combining Vincristine and GX15-070 is a promising strategy in treating MCL. In vivo experiments to confirm this additive activity are still ongoing and will be presented in full. Initial impressions suggest that there is a rationale for the addition of GX15-070 to current cytotoxic regimens used to treat MCL in the setting of clinical trials. Table 1: Effect of Vincristine and GX15-070 on in vitro growth of 3 MCL cell lines Growth as % age of Control Cell Line JVM-2 HBL-2 Granta Vincristine alone (10-6 mg/ml) 92% 48% 89% GX15-070 alone (0.08 uM) 75% 76% 60% Vincristine 10-6 mg/ml and GX15-070 0.08 uM 52% 24% 52%

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