Developing a Novel Class of Drug to Inhibit Protein Arginine Methyltransferase 5 (PRMT5) Enzyme Dysregulation in Mantle Cell Lymphoma

Abstract Abstract 595FN2 Introduction: Mantle cell lymphoma (MCL) is an incurable B-cell non-Hodgkin lymphoma characterized by aberrant genetic (t(11;14)(q13;q32)) and epigenetic (DNA hypermethylation) dysregulation. Chromatin remodeling complexes and associated co-repressors such as histone deacetylases (HDAC), DNA methyltransferases (DNMT) and protein arginine methyltransferase 5 (PRMT5), are involved in silencing tumor suppressor and regulatory gene expression and may contribute to B-cell transformation. PRMT5 silences the transcription of key regulatory genes by symmetric di-methylation (S2Me) of arginine (R) residues on histone proteins (H4R3 and H3R8). We have previously identified PRMT5 over expression to be relevant to MCL pathogenesis and shown it to work concertedly with HDAC2, methyl-CpG binding domain protein 2 (MBD2) and DNMT3a to silence genes with anti-cancer and immune modulatory activities. siRNA-mediated knockdown of PRMT5 in MCL cell lines leads to growth arrest and apoptosis, thus, we explored methods to inhibit PRMT5 activity as a novel experimental therapeutic strategy for this disease. Methods and Results: A rational design of small molecule compounds to inhibit PRMT5 activity led us to construct an in silico model of the human PRMT5 catalytic domain based on available homologous crystal structures from Protein Data Bank (MODELLER9v1 software). We screened a library of 10,000 compounds and eight small molecules were identified for biological investigation based on binding energy in the PRMT5 catalytic site. Enzyme inhibition assays using purified PRMT1 (type I PRMT) and PRMT5 (type II PRMT) showed that two compounds (BLL1 and BLL3) were capable of selectively inhibiting PRMT5 and not PRMT1 activity (p<0.0001). PRMT methylation assays were also performed with SWI/SNF complexes containing PRMT5, PRMT7 (type II) or PRMT4 (type I) and both BLL1 and BLL3 demonstrated selective PRMT5 inhibition. Both drugs interfered with maintenance of S2Me-H4R3 and S2Me-H3R8 in MCL cell lines by western blot and confocal microscopy. Dose titration experiments with BLL1 (10uM - 100uM) showed a dose-dependent response of inhibition of cellular proliferation, induction of cell cycle arrest, and promotion of caspase-independent cell death in 7 MCL cell lines. BLL1 treatment of MCL cells resulted in down modulation of cyclin D1 and Mcl1, critical molecules involved in the pathogenesis of MCL. The loss of cyclin D1 and Mcl1 expression occurred as early as 1 hour after treatment with BLL1 (50uM). PRMT5 associates with the co-repressors HDAC2, MBD2 and DNMT3a on target gene promoters, thus we next evaluated the effect of BLL1 on transcriptional repression of known target anti cancer genes. The association with other co-repressors provided rationale for examining PRMT5 inhibition alone and in combination with agents that target epigenetic processes. Combination treatment of MCL cells with subtoxic doses of BLL1 (25uM), hypomethylating agent (5-azacitidine, 500nM) and HDAC inhibitor (TSA 75nM) showed synergistic induction of cell death and loss of S2Me-H4R3. Analysis of the ST7 tumor suppressor, a target repressed by PRMT5, showed mRNA levels to increase 5–7-fold following treatment with BLL1. Preclinical in vivo studies have shown favorable toxicity and pharmacokinetic profiles for both BLL1 and BLL3. In vivo evaluation of BLL1 in a preclinical, xenograft model of human MCL are currently in progress. Primary tumors of 46 patients with MCL (common, blastoid or pleomorphic histology) demonstrated abundant PRMT5 expression in both cytoplasmic and nuclear compartments (87% PRMT5 pos). Conclusions: We have successfully developed a new class of drug to selectively target PRMT5 enzymatic activity. PRMT5 over expression is linked with post translational modification of both histone and non histone proteins that contribute to key oncogenic pathways in MCL. Inhibition of type II PRMT enzymes reverses transcriptional repression of anti cancer genes and restores important regulatory cellular checkpoints of cell growth and survival. We are currently developing drugs with improved selectivity and potency. The anti tumor activity of this novel class of drug and PRMT5 expression profiles seen in MCL primary tumor specimens, supports further exploration of targeting this pathway in hematologic malignancies. Disclosures: No relevant conflicts of interest to declare.

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BET bromodomain inhibitor birabresib in mantle cell lymphoma: in vivo activity and identification of novel combinations to overcome adaptive resistance

ESMO Open ◽

10.1136/esmoopen-2018-000387 ◽

2018 ◽

Vol 3 (6) ◽

pp. e000387 ◽

Cited By ~ 10

Author(s):

Chiara Tarantelli ◽

Elena Bernasconi ◽

Eugenio Gaudio ◽

Luciano Cascione ◽

Valentina Restelli ◽

...

Keyword(s):

Mantle Cell Lymphoma ◽

Cell Lines ◽

Cell Lymphoma ◽

Antitumour Activity ◽

Single Agent ◽

Treatment Strategies ◽

Bet Inhibitor ◽

Mantle Cell

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

Blood ◽

10.1182/blood-2019-130797 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 302-302 ◽

Cited By ~ 1

Author(s):

Fiona Brown ◽

Yang Zhang ◽

Claire Hinterschied ◽

Alexander Prouty ◽

Shelby Sloan ◽

...

Keyword(s):

Flow Cytometry ◽

Cell Death ◽

Mantle Cell Lymphoma ◽

Cell Lines ◽

Combination Treatment ◽

Cell Lymphoma ◽

Targeted Agents ◽

Mantle Cell ◽

Anti Tumor Activity

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

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

Blood ◽

10.1182/blood.v112.11.886.886 ◽

2008 ◽

Vol 112 (11) ◽

pp. 886-886 ◽

Cited By ~ 1

Author(s):

Lapo Alinari ◽

Erin Hertlein ◽

David M. Goldenberg ◽

Rosa Lapalombella ◽

Fengting Yan ◽

...

Keyword(s):

Monoclonal Antibody ◽

B Cell ◽

Mantle Cell Lymphoma ◽

Cell Lines ◽

Combination Treatment ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

Preclinical Model ◽

Mantle Cell

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

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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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Differential Patterns of Gene Expression and CD20 Localisation by the Type II Anti-CD20 Antibody Obinutuzumab (GA101) Compared with the Type I Antibody Rituximab Suggests Binding to Functionally Distinct CD20 Subpopulations on B-Cell Lymphoma Cell Lines,

Blood ◽

10.1182/blood.v118.21.3721.3721 ◽

2011 ◽

Vol 118 (21) ◽

pp. 3721-3721

Author(s):

Gerhard Niederfellner ◽

Olaf Mundigl ◽

Alexander Lifke ◽

Andreas Franke ◽

Ute Baer ◽

...

Keyword(s):

Gene Expression ◽

B Cell ◽

Cell Lines ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

Mechanisms Of Action ◽

Type I ◽

Type Ii ◽

Cd20 Antibody ◽

Anti Cd20

Abstract Abstract 3721 The anti-CD20 antibody rituximab has become central to the treatment of B-cell malignancies over the last decade. Recently, it has been shown that anti-CD20 antibodies can be divided into two types based on their mechanisms of action on B cells. Rituximab is a type I antibody that redistributes CD20 into lipid rafts and promotes complement-dependent cytotoxicity (CDC), while the type II, glycoengineered antibody GA101 has lower CDC activity but higher antibody-dependent cellular cytotoxicity and direct cell death activity. In preclinical studies GA101 was superior to rituximab in B-cell killing in vitro, depletion of B cells from whole blood, and inhibition of tumour cell growth in lymphoma xenograft models. GA101 is currently being evaluated in Phase II/III trials, including comparative studies with rituximab. To investigate the differences in direct effects of GA101 and rituximab on B-cell lymphoma signaling, we have analysed the effects of antibody binding on gene expression in different B-cell lines using a GeneChip Human Genome U133 Plus 2.0 Array (Affymetrix). Rituximab and GA101 rapidly induced gene expression changes in SUDHL4 and Z138 cells, including regulation of genes associated with B-cell-receptor activation such as EGR2, BCL2A1, RGS1 and NAB2. The effects on gene expression differed markedly between different cell lines and between the two antibodies. SUDHL4 cells showed pronounced changes in the gene expression pattern to rituximab treatment, while Z138 cells, which represent a different B-cell stage, showed less pronounced changes in gene expression. The reverse was true for GA101, suggesting not only that the signaling mediated by CD20 differs in different cell lines, but also that in a given cell line the two types of antibodies bind CD20 molecules with different signaling capacity. For each cell line, gene expression induced by other type I antibodies (LT20, 2H7, MEM97) was more like rituximab and that induced by other type II antibodies (H299/B1, BH20) was more like GA101 in terms of the number of genes regulated and the magnitude of changes in expression. Unbiased hierarchical clustering analysis of gene expression in SUDHL4 could discriminate type I from type II antibodies, confirming that the two classes of antibody recognised CD20 complexes with inherently different signalling capacities. By confocal and time-lapse microscopy using different fluorophores, rituximab and GA101 localised to different compartments on the membrane of lymphoma cells. GA101/CD20 complexes were relatively static and predominantly associated with sites of cell–cell contact, while rituximab/CD20 complexes were highly dynamic and predominantly outside areas of contact. These findings suggest that type II antibodies such as GA101 bind distinct subpopulations of CD20 compared with type I antibodies such as rituximab, accounting for the differences in mechanisms of action and anti-tumour activity between these antibodies. Disclosures: Niederfellner: Roche: Employment. Mundigl:Roche: Employment. Lifke:Roche: Employment. Franke:Roche: Employment. Baer:Roche: Employment. Burtscher:Roche: Employment. Maisel:Roche: Employment. Belousov:Roche: Employment. Weidner:Roche: Employment. Umana:Roche: Employment, Patents & Royalties. Klein:Roche: Employment, Equity Ownership, Patents & Royalties.

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Microrna-16 Regulates BMI1 in the Clonogenic Side Population of Refractory Mantle Cell Lymphoma

Blood ◽

10.1182/blood.v120.21.861.861 ◽

2012 ◽

Vol 120 (21) ◽

pp. 861-861

Author(s):

Kazuaki Teshima ◽

Miho Nara ◽

Atsushi Watanabe ◽

Mitsugu Ito ◽

Yoshiaki Hatana ◽

...

Keyword(s):

Stem Cell ◽

Cell Lines ◽

Target Gene ◽

Cell Lymphoma ◽

Side Population ◽

Upstream Regulator ◽

Bmi1 Expression ◽

Mantle Cell

Abstract Abstract 861 Background: Mantle cell lymphoma (MCL) is categorized as an indolent CD5+ B-cell lymphoma and is associated with numerous genomic copy number alterations, including 9p21 deletion (CDKN2A) and 10p12 amplification (BMI1). The target gene of the 10p12 amplification has been identified as BMI1, whose overexpression is frequently observed in the blastoid variant of MCL. CDKN2A is also well-known target of BMI1 in solid tumor. So, it has been hypothesized that BMI1 regulates CDKN2A in MCL. However there are the MCL cases with both 10p12 amplification and 9p21 homozygous deletion, suggesting that BMI1 might regulate the other target gene(s). The proto-oncogene, BMI1 is crucially involved in cancer stem cell maintenance and the upregulation has been demonstrated in aggressive or relapsed cases of solid tumors. Cancer stem cells are often identified in the side population (SP) of cancer cells, which is detected based on the cell's ability to export Hoechst 33342 dye via an ATP-binding cassette (ABC) membrane transporter, which gives the SP a distinct low-staining pattern. Aim of the study: The aim of this study is to determine the role of BMI1 in MCL initiating cells, especially in the relapsed cases. In this presentation, we show that the SP fraction has stem cell-like characteristics and high tumorigenic potential, and that BMI1 expression is upregulated in the SP in both relapsed MCL cases and MCL cell lines. Further we show that miR-16 is upstream regulator of the BMI1 in MCL. Results: To determine the role of BMI1 in the pathogenesis of MCL-initiating cells, we firstly examined BMI1 expression at primary MCL cases and found that its expression is stronger in cases of recurrent MCL than at initial diagnosis. We next characterized the MCL SP and found that the SP cells exhibit cancer stem cell-like features and upregulated BMI1 expression, which appears to enhance anti-apoptosis activity. Knocking down of BMI1 increases apoptosis and reduces tumorigenicity in CDKN2A−/− MCL cell lines (REC1 and Z138c). Subcutaneous inoculation of NOD/Shi-scid IL-2γnul (NOG) mice with CDKN2A−/− MCL cell lines, siBMI1-expressing cells were significantly smaller than those in mice receiving control siRNA in vivo. Chip assay showed that BMI1 interacts with BCL2L11/Bim and PMAIP3/Noxa, which were recently shown to be Bmi-1 target. These results suggest that BMI1/Bmi-1 may regulate Bim and/or Noxa to inhibit apoptosis in MCL cells. Furthermore, upon screening for upstream regulator of BMI1, we found that expression of a non-cording regulatory RNA, microRNA-16 (miR-16) is weaker in MCL SP cells than in non-SP cells. To investigate relationship between BMI1 and miR-16, we transfected miR-16 into MCL cell lines, and found that it directly downregulated BMI1, leading to reductions in tumor size following in vivo lymphoma xenograft (NOG mice). Finally, we find that bortezomib, which is known to be a proteasome inhibitor, led to dose-and time- dependent reductions in Bmi-1 expression with re-upregulation of miR-16 in both cell lines and a primary sample. Conclusion: We conclude that dysregulation of miR-16 and BMI1 plays a key role in lymphomagenesis by reducing MCL cell apoptosis, especially in refractory/recurrent cases via enhancement of anti apoptotic function. Disclosures: No relevant conflicts of interest to declare.

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Type I NKT cells protect (and type II NKT cells suppress) the host's innate antitumor immune response to a B-cell lymphoma

Blood ◽

10.1182/blood-2007-05-092866 ◽

2008 ◽

Vol 111 (12) ◽

pp. 5637-5645 ◽

Cited By ~ 106

Author(s):

Gourapura J. Renukaradhya ◽

Masood A. Khan ◽

Marcus Vieira ◽

Wenjun Du ◽

Jacquelyn Gervay-Hague ◽

...

Keyword(s):

T Cell ◽

B Cell ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

Nkt Cells ◽

Type I ◽

Type Ii ◽

Nkt Cell ◽

Tumor Bearing

Abstract Natural killer T (NKT) cells are a T-cell subpopulation known to possess immunoregulatory functions and recognize CD1d molecules. The majority of NKT cells express an invariant T-cell receptor (TCR) α chain rearrangement (Vα14Jα18 in mice; Vα24Jα18 in humans) and are called type I NKT cells; all other NKT cells are type II. In the current study, we have analyzed the roles for these NKT-cell subsets in the host's innate antitumor response against a murine B-cell lymphoma model in vivo. In tumor-bearing mice, we found that type I NKT cells conferred protection in a CD1d-dependent manner, whereas type II NKT cells exhibited inhibitory activity. Pro- and anti-inflammatory cytokines secreted by splenocytes from tumor-bearing mice correlated with tumor progression. Myeloid cells (CD11b+Gr1+) were present in large numbers at the tumor site and in the spleen of tumor-bearing type I NKT–deficient mice, suggesting that antitumor immunosurveillance was inhibited by CD11b+Gr1+ cells. Overall, these data suggest that there are distinct roles for NKT-cell subsets in response to a B-cell lymphoma in vivo, pointing to potential novel targets to be exploited in immunotherapeutic approaches against blood cancers.

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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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The Nucleoside Analogue Acadesine Exerts Antitumoral Activity and Cooperates with Conventional Agents In In Vitro and In Vivo Models of Mantle Cell Lymphoma

Blood ◽

10.1182/blood.v116.21.3918.3918 ◽

2010 ◽

Vol 116 (21) ◽

pp. 3918-3918

Author(s):

Arnau Montraveta ◽

Mercè de Frías ◽

Clara Campàs ◽

Elias Campo ◽

Gael Roue ◽

...

Keyword(s):

Mantle Cell Lymphoma ◽

Cell Lines ◽

Nucleoside Analogue ◽

Cell Lymphoma ◽

Primary Cultures ◽

Antitumoral Activity ◽

In Vivo Models ◽

Mantle Cell

Abstract Abstract 3918 Mantle cell lymphoma (MCL) is a mature B-cell neoplasm characterized by the t(11;14)(q13:q32) that involves cyclin D1 overexpression and consequent cell cycle deregulation at the G1 phase. This entity is generally characterized by an aggressive course and a bad prognosis. Recently, a specific subtype of MCL has been described, showing best outcomes and that might be managed more conservatively than conventional MCL. These cases are characterized by non-nodal presentation, predominantly hypermutated IgVH, lack of genomic complexity, and absence of SOX11 expression. Acadesine is a nucleoside analogue initially developed as a cardioprotective agent, and which has shown a wide range of metabolic effects, including the activation of AMP-activated protein kinase (AMPK). Acadesine was shown to induce apoptosis in primary cells from several B lymphoid neoplasms and has been entered in a phase I/II clinical trial with relapsed/refractory chronic lymphocytic leukemia (CLL) patients. This clinical study has shown that acadesine plasmatic levels in the micro molar range are achievable and safe when CLL patients are treated with the drug. To evaluate the antitumoral properties of acadesine in MCL, we exposed a set of 11 MCL primary cultures and 9 MCL cell lines for up to 48h with increasing doses of the drug. Cytotoxicity and cytostatic effects were then assessed by flow cytometry detection of annexinV/propidium iodide labeling and MTT proliferation assay, respectively. In both MCL cell lines and MCL primary cultures, we observed a heterogeneous response to the drug, with no correlation to common genetic alterations such as deletion/mutation of P53, ATM or P16 genes. JVM2, Jeko-1, Rec-1 and UPN-1 were the more sensitive cell lines, with a mean lethal dose 50 (LD50) of 1.57 mM at 24 h and 0.95 mM at 48h, while 2 cell lines (HBL-2 and Granta-519) showed a primary resistance to the compound (LD50 > 50 mM). Among MCL primary cultures, acadesine showed selective cytotoxic activity against malignant B cells while sparing accompanying T cells. Of note, those cases corresponding to the indolent MCL group showed increased sensitivity to the drug at 24h of treatment, when compared to conventional MCL cases (p=0.03). We observed that acadesine efficiently activates the intrinsic apoptotic pathway in MCL cells by modulating Bcl-2 family protein levels, leading to conformational activation of Bax and Bak, mitochondrial depolarization, generation of reactive oxygen species and caspases processing. In drug combination assays, acadesine showed a synergistic effect when combined with Rituximab, being the Rituximab-acadesine combination more potent than other Rituximab-based polychemotherapies such as R-bendamustine and R-CHOP. Finally, a daily administration of 400mg/kg acadesine in mice previously inoculated with a MCL xenotransplant significantly reduced tumor burden when compared to control animals, as soon as 7 days of treatment. In summary, these results suggest that acadesine exerts significant antitumoral activity in both in vitro and in vivo model of MCL, and may represent an attractive model for the design of a new therapeutic approach for this entity, especially in patients presenting with the indolent form. Disclosures: de Frías: Advancell therapeutics: Employment. Campàs:Advancell therapeutics: Employment.

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