Synergistic Antilymphoma Effect of Protein Kinase C Beta (PKCβ) and mTOR Inhibition in Mantle Cell Lymphoma.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4780-4780
Author(s):  
Grit Hutter ◽  
Yvonne Zimmermann ◽  
Malte Rieken ◽  
Elena Hartmann ◽  
Vindi Jurinovic ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Cell Line ◽  
Cell Lines ◽  
Research Funding ◽  
Cell Lymphoma ◽  
Pi3k Inhibitor ◽  
Cell Cycle Profile ◽  
Kinase C ◽  
Deutschland Gmbh ◽  
Mantle Cell

Abstract Abstract 4780 Introduction The protein kinase C (PKC) family of enzymes are serine/threonine kinases essential to the cell signal cascades effecting cellular growth, proliferation and apoptosis. Accordingly PKCβ overexpression correlates with poor clinical prognosis in diffuse large cell lymphoma. The pivotal role of PKCb in neoplastic transformation renders it a potential therapeutic target in the therapy of hematologic malignancies. Aim To determine drugs which are efficiently inhibiting cell proliferation in combination with enzastaurin in MCL. Methods Five MCL cell lines (HBL-2, GRANTA 519, Jeko-1, Z138, Rec-1) and patient samples were cultured in the presence of LY317615 (PKCb inhibitor), rapamycin (mTOR inhibitor) and LY294002 (PI3K inhibitor). Cell proliferation and viability was assessed by cell count and WST-1 proliferation assay. Analysis of cell cycle profile and apoptosis was performed by flow cytometry (PI and Annexin V FITC staining). mRNA expression was measured before and after treatment (8h) by microarray and real time PCR in cell lines. Protein expression was analysed by Western blot. Results In a panel of mantle cell lymphoma cell lines, with IC50 values ranging from 2 to 5 microM for enzastaurin treatment, a refractory to enzastaurin cell line (Rec-1) was characterized. Treatment of the cell lines with enzastaurin induced apoptosis and lead to accumulation of cells in the G2, M phase in susceptible cell lines (Hbl-2, Jeko-1), whereas cell cycle profile remained unaltered in the refractory cell line (Rec-1). While enzastaurin induced increased phosphorylation of mTOR and MEK and decrease of p90RSK phosphorylation in all MCL cell lines, mTOR phosphorylation was twice as high in the refractory cell line (Rec-1). In line with this observation the combination of enzastaurin with rapamycin lead to a synergistic effect on the inhibition of cell proliferation in the Rec-1 cell line as well as in an additional MCLpatient sample. Protein expression levels (low CCND1, phAkt, php90RSK, phPDK) achieved in Rec-1 after treatment with enzastaurin were also characteristic for the cell lines more sensitive to rapamycin. In contrast in some cell lines combination of enzastaurin and the PI3K inhibitor (LY294002) displayed antagonism. Further mRNAand proteinexpression analysis of patient samples are ongoing to determine molecular predictors of drug sensitivity. Conclusion In our study a combination of rapamycin and enzastaurin acted synergistically in MCL cell lines and a patient samples whereas the combination with a PI3K inhibitor displayed partial antagonism. Based on this results we have identified the underlying signal pathways to develop new synergistic molecular combinations in MCL. Disclosures: Hutter: Lilly Deutschland GmbH: Research Funding. Zimmermann:Lilly Deutschland GmbH: Research Funding. Rieken:Lilly Deutschland GmbH: Research Funding. Weinkauf:Lilly Deutschland GmbH: Research Funding. Dreyling:Lilly Deutschland GmbH: Research Funding.

Whole Transcriptome Sequencing Reveals Recurrent NOTCH1 Mutations in Mantle Cell Lymphoma

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 436-436 ◽  
Author(s):  
Robert Kridel ◽  
Barbara Meissner ◽  
Sanja Rogic ◽  
Merrill Boyle ◽  
Adele Telenius ◽  
...  
Keyword(s):  
Overall Survival ◽  
Mantle Cell Lymphoma ◽  
Cell Line ◽  
Cell Lines ◽  
Research Funding ◽  
Cell Lymphoma ◽  
The Other ◽  
Mantle Cell ◽  
Whole Transcriptome ◽  
Notch1 Mutations

Abstract Abstract 436 Background: Mantle cell lymphoma (MCL) is an aggressive subtype of non-Hodgkin's lymphoma that is characterized by the hallmark t(11;14)(q13;q32) translocation, as well as a high number of secondary chromosomal alterations. Further, a small number of genes such as TP53, ATM and CCND1 have been reported to be recurrently mutated in MCL, but do not fully explain the biology and do not adequately account for the wide spectrum of clinical manifestations, response to treatment and prognosis. The aim of this study was to discover new somatic mutations that could contribute to our understanding of the pathogenesis of MCL. Methods: In our discovery cohort, we sequenced the transcriptomes of 18 clinical samples (11 diagnostic and 7 progression biopsies) and 2 mantle cell lymphoma-derived cell lines (Mino and Jeko-1). For this purpose, whole transcriptome shotgun sequencing was performed on RNA extracted from fresh frozen tissue. We assembled an extension cohort of 103 diagnostic patient samples and 4 additional cell lines (Rec-1, Z-138, Maver-1, JVM-2), and performed Sanger sequencing of NOTCH1 exons 26, 27 and 34 on genomic DNA. We further exposed the 6 cell lines to 1 μM of the γ-secretase inhibitor XXI (compound E) for 7 days and measured cellular proliferation with an EdU incorporation assay. Survival analysis was carried out in the 113 patients with diagnostic biopsies and available outcome data. Results: NOTCH1 mutations were found in 14 out of 121 patient samples (11.6%) and in 2 out of 6 cell lines, Mino and Rec-1 (33.3%). The majority of these mutations (12 out of 14) lie in exon 34 that encodes the PEST domain of NOTCH1 and consist of either small frameshift-causing indels (10 cases) or nonsense mutations (2 cases). These mutations are predicted to cause truncations of the C-terminal PEST domain. To gain further insight into functional relevance, we treated 6 cell lines with compound E, an inhibitor of the γ-secretase complex that plays a critical role in the release of the intracellular domain of NOTCH1 after ligand-induced activation. In Rec-1, that harbours a NOTCH1 mutation, we observed a significant decrease in proliferation (mean percentage of cells in culture incorporating EdU decreasing from 47.5% to 1.4%, p<.001). No effect of compound E was observed in Mino, the other cell line with a NOTCH1 mutation, nor in the 4 cell lines that are wild type for NOTCH1. Outcome correlation analysis showed that NOTCH1 mutations are associated with poor overall survival (1.56 versus 3.86 years respectively, p=.001), but not with significantly shortened progression-free survival (0.88 versus 1.73 years respectively, p=.07). Discussion: We have identified recurrent mutations in NOTCH1 in a subset of patients with MCL (11.6%). The frequency and the pattern of mutations are strikingly similar to what has recently been reported in chronic lymphocytic leukemia, the other major CD5 positive B-cell malignancy (Nature, 2011 Jun 5, 475:101–105 and J Exp Med, 2011 Jul 4, 208:1389–1401). NOTCH1 mutations are associated with adverse prognosis as evidenced by shortened overall survival. This latter finding, however, should ideally be validated in a larger and uniformly treated cohort. Finally, the sensitivity of the Rec-1 cell line to compound E suggests that NOTCH1 mutations could serve as the target for tailored therapy in mantle cell lymphoma. Disclosures: Sehn: Roche/Genentech: Consultancy, Honoraria, Research Funding. Connors:Roche: Research Funding.

scholarly journals Genome-Wide Analysis of BRD4-Targets Reveals the Therapeutic Relevance of Simultaneous Targeting of BCR Pathway and IKZF-MYC Axis in Mantle Cell Lymphoma

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 4100-4100
Author(s):  
Junya Kuroda ◽  
Taku Tsukamoto ◽  
Shingo Nakahata ◽  
Kazuhiro Morishita ◽  
Ryuichi Sato ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Mantle Cell Lymphoma ◽  
Cell Lines ◽  
Research Funding ◽  
Target Genes ◽  
Cell Lymphoma ◽  
Genome Wide ◽  
Mantle Cell ◽  
Bcr Signaling ◽  
Dose Dependent

Abstract Mantle cell lymphoma (MCL) has been mostly incurable, and there is an urgent need to identify targetable molecules for development of a more effective treatment strategy. Bromodomain and extraterminal domain (BET) proteins associate with acetylated histones and facilitate transcription of target genes, and bromodomain-containing 4 (BRD4), a member of BET proteins, recruits the P-TEFb complex to genomic lesions in chromatin and thereby activates RNA Pol II at specific promoter sites of target genes. In addition, super-enhancers have been recognized as regulatory regions with a high level of acetylated histones, mediator complexes and BRD4, and super-enhancers in cancer cells are enriched at oncogenes. Recent studies have shown that BRD4 promotes expression of pivotal molecules in disease development, maintenance and progression in various cancers, including lymphoma. Given, we in this study examined the effect of BRD4 inhibition on human MCL-derived cell lines, Jeko-1, JVM-2, MINO and Z138, and performed broad screening of BRD4-regulated molecules using genome-wide approaches to identify therapeutic targets for MCL. As the results, treatment with a BRD4 inhibitor I-BET151 for 72 h showed a dose-dependent inhibitory effect on cell proliferation in all four cell lines, with half maximal inhibitory concentrations (IC50s) of 15.6 nM, 3.6 nM, 2.6 nM and 3.0 nM in Jeko-1 cells, JVM2 cells, MINO cells and Z138 cells, respectively, which was accompanied by G1/S cell cycle arrest and the induction of apoptosis. Next, we performed comprehensive gene expression profile (GEP) analysis for JVM2 and Z138 cells with or without I-BET151 treatment, and BRD4 chromatin immunoprecipitation sequencing (ChIP-Seq) in JVM2 cells treated with 10 nM I-BET151 or DMSO. Accordingly, GEP analyses revealed that more than 600 genes were commonly upregulated by more than 1.5-fold and downregulated by less than 0.67-fold, respectively, in JVM2 and Z138 cells treated by I-BET151, while ChIP-Seq showed that 7988 BRD4-binding regions were dysregulated by I-BET151, with most of these sites in enhancer regions, and 547 BRD4-binding regions were characterized as super-enhancers. Integrated analysis using the Reactome Pathway Database and the results of GEP and ChIP-Seq showed that a series of genes involved in the B cell receptor (BCR) signaling pathway and IKZF-MYC axis are regulated by BRD4 in MCL cells. To confirm whether each BRD4 target contributes to survival and proliferation of MCL cells, we focused on several candidate targets: the BCR pathway, IKZF and MYB. However, ibrutinib, a Bruton kinase inhibitor, suppressed cell growth in only two of the four cell lines (MINO and JVM2) in a dose-dependent manner, while lenalidomide, an inhibitor of the IKZF family, did not affect cell survival, despite its potency in decreasing IKZF1 and IKZF3 proteins. MYB silencing using shMYB did not decrease cell proliferation in any of the four MCL cell lines. In conclusion, our study disclosed that BRD4 regulates transcription of multiple genes by binding to enhancer region, partly involving super-enhancers and multiple known pathways, such as BCR signaling and the IKZF-MYC axis, which play essential roles in survival of MCL cells. While the efficacy of single targeting of BCR-signaling, IKZF, or MYB was limited, I-BET151 concomitantly inactivated the BCR pathway and IKZF and had a high growth inhibitory efficacy in MCL cells. These results suggest that simultaneous targeting of multiple molecules involved in the BCR pathway and IKZF-MYC axis may overcome resistance to ibrutinib and/or lenalidomide in MCL, and that BRD4 inhibitors are promising candidates for MCL treatment. Disclosures Kuroda: Chugai Pharma: Honoraria, Research Funding. Taniwaki:Bristol-Myers Squibb: Research Funding; Chugai Pharmaceutical Co., Ltd.,: Research Funding; Astellas Pharma Inc,: Research Funding.

Anti-CD20 Antibody GA101 Shows Higher Cytotoxicity but Is Competitively Displaced by Rituximab in Mantle Cell Lymphoma.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2704-2704
Author(s):  
Daniel A. Heinrich ◽  
Christian Klein ◽  
Kristina Decheva ◽  
Marc Weinkauf ◽  
Grit Hutter ◽  
...  
Keyword(s):  
Western Blot ◽  
Cell Viability ◽  
Mantle Cell Lymphoma ◽  
Cell Lines ◽  
Research Funding ◽  
Cell Lymphoma ◽  
Deutschland Gmbh ◽  
Mantle Cell ◽  
Cd20 Antibody ◽  
Anti Cd20

Abstract Abstract 2704 Poster Board II-680 Background: Mantle cell lymphoma (MCL) is characterized by a poor long-term prognosis with a median survival of 3–5 years. Type I anti-CD20 antibody rituximab has demonstrated a clear anti-proliferative effect in MCL and achieves increased response rates in combination with chemotherapy. GA101, a third-generation IgG1 anti-CD20 antibody displays improved ADCC and superior direct cell death induction by virtue of glycoengineering compared to rituximab and its targeting a type II epitope on CD20, respectively. Methods: Using a panel of MCL cell lines (Rec-1, HBL-2, Jeko-1, Granta-519, JVM-2 and Z-138) we determined the effect of GA101 alone as well as in combination with rituximab on cell viability and proliferation. Karpas-422 (Diffuse Large B-Cell Lymphoma) was used as a control cell line. MCL and Karpas-422 cells were treated with GA101 or rituximab at concentrations of 1 – 20μg/ml and rituximab. Cell viability was analyzed by trypan-blue exclusion tests at 0h, 24h, 48h and 72h. The panel of MCL cell lines and Karpas-422 were then treated with GA101 and rituximab each at 1 and 10 μg/ml to determine potential synergism of antibody combinations. Accordingly, a fractional product calculation was performed: synergism > 0,1; antagonism < −0,1. In addition, Western-blot and RNA-array-analyses were performed to elucidate potential intra-cellular downstream pathway mechanisms. Results: After mono-exposure with GA101 (1 μg/ml), Granta-519 and Rec-1 showed the highest sensitivity (65–75% cell reduction in Granta-519 and 35–40% in Rec-1). Intermediate results were gained for Z-138, HBL-2, Jeko-1 and JVM-2 and Karpas-422 (15–20%). rituximab mono-exposure at 12,5 μg/ml showed a 25% reduction of cell count in Granta-519, 20% in HBL-2 and < 5% in Rec-1, Jeko-1 and Z-138. Combination experiments suggested the competitive binding of the two antibodies. Thus, GA101 plus rituximab combination experiments resulted in a lower cytotoxicity than GA101 alone, according to fractional product calculations. Conclusions: Although GA101 is competitively displaced by rituximab, GA101 demonstrates higher efficacy in MCL cell lines than rituximab, even at a more than 10-fold lower concentration. Currently RNA-array- and Western blot analysis are being performed to identify the critical pathways responsible for the superior cytotoxicity of GA101. Disclosures: Klein: Discovery Oncology, Roche Diagnostics GmbH: Employment. Weinkauf:Lilly Deutschland GmbH: Research Funding. Hutter:Lilly Deutschland GmbH: Research Funding. Zimmermann:Lilly Deutschland GmbH: Research Funding. Dreyling:Roche: Honoraria, Research Funding.

Activity of Rituximab and Ofatumumab Against Mantle Cell Lymphoma (MCL) in Vitro in MCL Cell Lines and Ex Vivo in Tumor Cells Derived From Patient Tumor Samples

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 4972-4972
Author(s):  
Matthew J. Barth ◽  
Gopichand Pendurty ◽  
Cory Mavis ◽  
Natalie M Czuczman ◽  
John Gibbs ◽  
...  
Keyword(s):  
Cell Viability ◽  
Cell Line ◽  
Tumor Cells ◽  
Cell Lines ◽  
Research Funding ◽  
Cell Lymphoma ◽  
Clinical Investigations ◽  
Mantle Cell ◽  
Anti Cd20

Abstract Abstract 4972 Mantle cell lymphoma (MCL) is an aggressive form of non-Hodgkin lymphoma (NHL) that frequently presents with advanced stage disease. The addition of rituximab, a monoclonal anti-CD20 antibody, to high dose chemotherapy regimens often followed by stem cell transplant has improved outcomes, but survival still remains low at 3–5 years. Novel agents are needed to improve outcomes in MCL. Ofatumumab is a fully human anti-CD20 monoclonal antibody directed against a novel epitope on the CD20 antigen. Ofatumumab has been shown to be more potent than rituximab against B-NHL cells in pre-clinical investigations. Ofatumumab is FDA approved for the treatment of CLL that is fludarabine and alemtuzumab refractory or with bulky disease resistant to fludarabine and is being investigated in clinical trials in NHL. In order to characterize the activity of ofatumumab against MCL, we performed pre-clinical investigations into the activity of ofatumumab against MCL cell lines and primary MCL tumor cells derived from patient tumor samples (n=2). Antibody-dependant cellular cytotoxicity (ADCC) and complement dependent cytotoxicity (CDC) assays were performed in the MCL cell lines Mino, Jeko, Rec-1 and Z-138 to demonstrate sensitivity to rituximab and ofatumumab. Lymphoma cells were labeled with 51Cr prior to incubation with rituximab or ofatumumab at 10ug/mL plus human serum or effector cells (efector:target ratio of 20:1). 51Cr-release was measured and the percentage of lysis was calculated. Patient tumor cells were isolated from tumor biopsy samples by MACS sorting (negative selection). Patient tumor cells were incubated with ofatumumab or rituximab at 10ug/mL in the presence of human serum as a complement source. Cell viability was determined at 48 hours by CellTiterGlo assay. Means were compared using a t-test. Expression of CD20 and the complement inhibitory proteins (CIPs) CD55 and CD59 in MCL cell lines were determined by flow cytometry and compared to the rituximab-sensitive cell line Raji and the rituximab-resistant cell line Raji 4RH. Surface density of CD20, CD55 and CD59 were determined by Imagestream analysis. Western blot was performed to measure total CD20 protein expression. Ofatumumab induced significantly higher levels of cell lysis compared to rituximab in CDC assays of all MCL cell lines tested (Mino: 65.9% vs 0.5%; JeKo 43.9% vs 13.3%; REC-1 25.4% vs 4.7%; Z-138: 56.4% vs 0.65%; all p-values <0.05). The ADCC assays showed a similar degree of lysis with ofatumumab when compared to rituximab in all cell lines tested. In primary tumor cells, ofatumumab and rituximab demonstrated similar levels of decreased cell viability following 48 hours of antibody exposure. MCL cell lines demonstrated similar expression of surface and total CD20 when compared to the rituximab-sensitive B-NHL Raji cell line. CIP expression was increased in all MCL cell lines compared to Raji cells and was similar to the rituximab-resistant Raji 4RH cell line. Our data suggest ofatumumab is more potent than rituximab against MCL cells in vitro and retains CDC activity despite high expression levels of CIPs. This increased activity was not seen in patient tumor samples; however we were limited by the number of available patient samples. In vivo experiments investigating the activity of ofatumumab in a SCID mouse MCL xenograft model and investigations into the activity of ofatumumab in MCL cells in combination with cytotoxic agents and novel small molecule inhibitors are ongoing. Disclosures: Czuczman: Genmab: Consultancy, Honoraria, Research Funding; GlaxoSmithKline: Consultancy, Honoraria, Research Funding. Hernandez-Ilizaliturri:Genmab: Research Funding.

Anti-CD20 Antibody GA101 in Combination with Chemotherapy or Flavopiridol in Mantle Cell Lymphoma.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4781-4781
Author(s):  
Daniel A. Heinrich ◽  
Christian Klein ◽  
Kristina Decheva ◽  
Marc Weinkauf ◽  
Grit Hutter ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Mantle Cell Lymphoma ◽  
Cell Lines ◽  
Research Funding ◽  
Cell Lymphoma ◽  
Cdk Inhibitor ◽  
Type Ii ◽  
Deutschland Gmbh ◽  
Mantle Cell ◽  
Anti Cd20

Abstract Abstract 4781 Background Mantle cell lymphoma (MCL) responds only transiently to conventional chemotherapy resulting in a dismal long-term prognosis. At a molecular level it is characterised by the chromosomal translocation t(11;14)(q13;q32), which leads to constitutive over-expression of the cell cycle regulatory protein cyclin D1. GA101 is a third generation, glycoengineered type II IgG1 anti-CD20 monoclonal antibody with superior direct cell death induction by targeting a type II epitope and enhanced antibody dependent cellular cytotoxicity (ADCC). High efficacy in lymphoma cell lines has led to combination experiments with various chemotherapeutic compounds or the CDK-inhibitor Flavopiridol. Methods Using a MCL cell line panel (Granta-519, HBL-2, Jeko-1, Rec-1 and Z-138) and a Diffuse Large B-Cell Lymphoma cell line (Karpas-422) we determined the effect of GA101 (1 μg/ml) monotherapy as well as in combination with Fludarabine (0,25 μg/ml), Bendamustine (5 μg/ml), Mitoxantrone (0,25 and 0,5 μg/ml) and Flavopiridol (100nM) on cell proliferation and viability. Trypan-blue exclusion tests were used to analyze cell viability at 0h, 24h, 48h and 72h. The panel of MCL cell lines was treated to determine potential synergism of agent combinations. Accordingly, fractional product was calculated: synergism > 0,1; additive effect -0,1<x<0,1; antagonism < -0,1. Results After mono-exposure with GA101 (1 μg/ml), Granta-519 and Rec-1 showed the highest sensitivity (Granta: 65-75% cell reduction, Rec-1: 30-45%). Intermediate results were achieved for HBL-2 (20-30%), Z-138 and Karpas-422 (10-15%), Jeko-1 (5%). Fludarabine alone resulted in a 20-40% cell reduction. Bendamustine showed a higher efficacy in Jeko-1, Rec-1 and Z-138 (40-90%) than in Granta-519, Karpas-422 and HBL-2 (10%). Mitoxantrone treatment demonstrated a high impact on all cell lines (80-95% cell reduction). Flavopiridol induced a 65-85% cell reduction in Jeko-1, Rec-1 and Karpas-422in comparison to 30-45% in Granta-519, HBL-2 and Z-138. Additional experiments showed additive effects of all GA101 combinations resulting in 40-80% cell reduction (Fludarabine), 30-90% (Bendamustine), 85-95% (Mitoxantrone) and 60-80% (Flavopiridol). Conclusions These in vitro results demonstrate that the anti-CD20 monoclonal antibody GA101 alone or in combination with various chemotherapeutical compounds or the CDK-inhibitor (Flavopiridol) show a promising efficacy in MCL cell lines (additive in combination), supporting the clinical evaluation of such an innovative immuno-chemotherapy in mantle cell lymphoma. Disclosures: Klein: Roche (Glycart): Employment, Equity Ownership, Patents & Royalties. Weinkauf:Lilly Deutschland GmbH: Research Funding. Hutter:Lilly Deutschland GmbH: Research Funding. Zimmermann:Lilly Deutschland GmbH: Research Funding. Dreyling:Roche: Honoraria, Research Funding.

Foxo 3a Signaling Mediates Apoptosis in Lymphoma Cells By the Diterpenoid Lactone Andrographolide (Andro), the Active Component of Andrographis Paniculata

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2760-2760
Author(s):  
Shuo Yang ◽  
Bo Ding ◽  
Fei Ying ◽  
Jana Svetlichnaya ◽  
Austin Tom ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Mantle Cell Lymphoma ◽  
Signaling Pathways ◽  
Cell Line ◽  
Cell Lines ◽  
Cell Lymphoma ◽  
Wild Type ◽  
Mantle Cell ◽  
Golgi Fragmentation ◽  
Bcl2 Expression

Abstract Introduction: Andrographolide is a diterpenoid lactone isolated from Andrographis paniculata (King of Bitters), an herbal medicine used in Asia. It has been reported to have anti-inflammatory, antihypertensive, antiviral, and immune-stimulant properties. Furthermore, it has been shown to inhibit cancer cell proliferation and induce apoptosis in lymphoma, leukemia and other solid tumor cell lines. We have shown that Andro caused ROS-dependent apoptosis in lymphoma cell lines and in primary tumor samples that was mediated through mitochondrial pathways and enhanced by depletion of GSH and inhibited by NAC or the pan-caspase inhibitor Z-VAD-FMK (Yang et al Clin Cancer Res 2010; 16(19):4755). We hypothesized that the tumor suppressor, FOXO3a may be involved in signaling pathways that lead to apoptosis and to test that hypothesis we investigated the role of FOXO3A in Andro induced signaling in lymphoma. Methods: We studied the Burkitt p53-mutated Ramos cell line, the mantle cell lymphoma (MCL) line Granta, the transformed follicular lymphoma (FL) cell line HF-1, and the diffuse large B-cell lymphoma (DLBCL) cell line SUDHL4, as well as primary cells from patients with FL and MCL. We transfected shRNA FOXO3a by electroporation to build stable cells with constant knockdown of FOXO3a in Ramos and SUDHL4 cell lines. We then compared the cell viability (MTT and Golgi fragmentation), apoptosis (Annexin V by flow), c-MYC and Bcl2 expression, death receptors 4 (DR4) expression and cell cycle related proteins in wild type and FOXO3a knockdowns. Results: We found that Andro resulted in nuclear translocation of FOXO3a in Ramos at early time points. We found that shRNA stable knockdown of FOXO3a in Ramos and SUDHL4 cell lines protected cells (Ramos and SUDHL4) from Andro-induced apoptosis (Figure 1). Moreover, in multiple cell lines, we found that Andro decreased c-MYC expression, which was abrogated in part by FOXO3A knockdown compared with wild type cells. Similarly, reduction in mitochondrial membrane potential by Andro is abrogated in the FOXO 3a knockdown cells. These data suggest that FOXO3a regulates c-MYC stabilization by mitochondrial proteins (for example TFAM and MAD-1). In the Granta cell line, derived from Mantle Cell Lymphoma (MCL) and in an MCL patient sample, Andro reduced c-MYC expression. We also found that Andro induced Death Receptor 4 (DR4) at the mRNA and protein level in Granta cells in a dose-dependent manner. The cell cycle control proteins Aurora, p21, p27 (the latter 2 regulated by FOXO3a), are also increased by Andro. When cell death was measured by Golgi fragmentation and subsequent collapse, we found that Andro induced Golgi fragmentation in Granta and SUDHL4 cells Conclusion: Andro-induced lymphoma cell apoptosis is mediated through multiple signaling pathways, including FOXO3a, which appears to play a significant role, perhaps by regulating c-MYC stabilization and BCL2 expression and cell cycle proteins. These data suggest that this novel diterpenoid lactone compound deserves further pre-clinical and clinical testing in malignant lymphoma. Figure 1. Figure 1. Disclosures No relevant conflicts of interest to declare.

scholarly journals Tolinapant (ASTX660), a Non-Peptidomimetic Antagonist of cIAP1/2 and XIAP, and the HDAC Inhibitor Romidepsin Are Synergistic in in Vitro Models of T Cell Lymphoma

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 4356-4356
Author(s):  
John S Manavalan ◽  
Ipsita Pal ◽  
Aidan Pursley ◽  
George A. Ward ◽  
Tomoko Smyth ◽  
...  
Keyword(s):  
T Cell ◽  
Cell Line ◽  
Board Of Directors ◽  
Cell Lines ◽  
Research Funding ◽  
Cell Lymphoma ◽  
T Cell Lymphoma ◽  
Tnf Alpha ◽  
Sensitive Cell ◽  
Advisory Committees

Abstract Background: The PTCL are a heterogeneous group of non-Hodgkin lymphomas originating from mature T-lymphocytes. They are aggressive diseases, often resistant to conventional chemotherapy. Despite the fact that a number of new agents have been approved, treatment paradigms tailored to the biology of the disease have yet to emerge. Tolinapant (ASTX660) is a potent antagonist of both cellular and X-linked inhibitors of apoptosis proteins (cIAP1/2 and XIAP), and is presently in phase I/II trials in patients with advanced solid tumors and lymphomas (NCT02503423). IAP antagonists enhance tumor necrosis factor (TNF) receptor superfamily mediated apoptosis (Ward GA, et al. Mol Cancer Ther. 2018), are potent anti-tumor immune enhancers and induce markers of immunogenic cell death such as damage associated molecular patterns (DAMPs; Ye W, et al, Oncoimmunology, 2020). Objectives: We explored the sensitivity of a range of T-cell lymphoma (TCL) cell lines to tolinapant. We establish the synergy coefficient between tolinapant and the HDAC inhibitor, romidepsin, and interrogated the molecular basis of their synergistic interaction. Methods: A panel of human T-cell lymphoma cell lines were tested in proliferation assays (CellTiterGlo) for sensitivity to tolinapant in the presence or absence of 10ng/ml of TNF alpha. For combination studies, with tolinapant and romidepsin, each drug was tested at the IC10 and IC40 concentrations in the presence or absence of TNF alpha. Synergy scores using the Excess over Bliss (EOB) model were calculated using SynergyFinder (Aleksandr Ianevski et al; Nucleic Acids Research, 2020). Additionally, the effects of tolinapant and romidepsin on the IAPs and caspases were analyzed by western blots. TNFR1 receptor expression and induction of DAMPs were also analyzed by flow cytometry. Results: TCL Lines demonstrated varying sensitivities to tolinapant in the presence or absence of TNF alpha. The most sensitive cell lines, ALK+ ALCL and SUP-M2, had IC50 concentrations ranging from 200nM ± 100nM to 20nM ± 1nM in the absence or presence of TNF alpha, respectively, at 24, 48 and 72hrs, while a resistant CTCL cell line HH had an IC50 concentration of over 20mM, even in the presence of TNF alpha. Interestingly, using western blot analysis, we found that the presence of TNF alpha increased the levels of cIAP1 in the tolinapant sensitive SUP-M2 cell line, but not in the resistant HH cell line. However, there was a concentration dependent decrease in cIAP1 but not in XIAP in both cell lines treated with tolinapant. Flow cytometry analysis demonstrated that tolinapant increases the expression of TNFR1 and DAMPs in a dose dependent manner on the sensitive SUP-M2, but not in the resistant HH cells. In combination experiments, using the EOB model, tolinapant plus romidepsin was found to be synergistic in the absence of TNF alpha, at 36hrs, in both the sensitive cell line SUP-M2 and the resistant cell line HH. In the presence of TNF alpha, synergism was seen only in the sensitive cell line SUP-M2 and antagonistic in the HH cell line (Fig. 3). In the tolinapant plus romidepsin treated samples, cIAP1 levels decreased in the SUP-M2 cell line, in the absence of TNF alpha, however, addition of TNF alpha did not alter the levels of cIAP1 in the SUP-M2 cells. The cIAP1 levels decreased in the HH cells treated with the combination, in both the presence or absence of TNF alpha (Figure). Our findings indicate that the synergy of the tolinapant plus romidepsin is not dependent on the presence of TNF alpha. Conclusion: Tolinapant has demonstrated potent cytotoxic effects against a broad range of TCL lines both as a monotherapy and in combination with the HDAC Inhibitor, romidepsin. In in vitro studies, T cell lymphoma cell lines demonstrated varying sensitivity to tolinapant with certain cell lines being more resistant, even in the presence of TNF alpha. Interestingly, the addition of romidepsin appeared to overcome the intrinsic resistance to tolinapant in the absence of TNF alpha. These data provide the rationale to continue to explore the combination of tolinapant and romidepsin in vivo and to investigate additional combinations with T-cell specific agents (e.g. pralatrexate, belinostat, azacitidine and decitabine). Figure 1 Figure 1. Disclosures Smyth: Astex Pharmaceuticals: Current Employment. Sims: Astex Pharmaceuticals: Current Employment. Loughran: Kymera Therapeutics: Membership on an entity's Board of Directors or advisory committees; Bioniz Therapeutics: Membership on an entity's Board of Directors or advisory committees; Keystone Nano: Membership on an entity's Board of Directors or advisory committees; Dren Bio: Membership on an entity's Board of Directors or advisory committees. Marchi: Kyowa Kirin: Honoraria; Myeloid Therapeutics: Honoraria; Astex: Research Funding; BMS: Research Funding; Merck: Research Funding; Kymera Therapeutics: Other: Scientific Advisor.

scholarly journals Abemaciclib-Based Combinational Therapies to Overcome Therapeutic Resistance in Mantle Cell Lymphoma

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 33-34
Author(s):  
Yuxuan Che ◽  
Yang Liu ◽  
Lingzhi Li ◽  
Holly Hill ◽  
Joseph McIntosh ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cyclin D1 ◽  
Cell Lines ◽  
Research Funding ◽  
Cell Lymphoma ◽  
Single Agent ◽  
Therapeutic Resistance ◽  
Cycle Arrest ◽  
Mantle Cell ◽  
Ic50 Values

Introduction The past decades witnessed dramatic improvement of overall survival rate of mantle cell lymphoma (MCL) patients by constant efforts in developing novel therapeutic strategies that include ibrutinib and venetoclax. Nevertheless, resistance is still a major challenge in refractory/relapsed MCL patients. Chromosomal translocation t(11:14)(q13:q32) of the cyclin D1 (CCND1) gene is the hallmark of MCL, which leads to overexpression of cyclin D1. This overexpression promotes aberrant cell cycle progression by activating CDK4/6. Abemaciclib is a selective CDK4/6 inhibitor used as a clinical treatment of breast cancer and has been shown to be effective in preclinical human MCL xenograft models. It has also been used in a phase II clinical trial as a single agent among refractory/relapsed MCL patients with an objective response rate of 35.7%. In this preclinical study, we aim to evaluate the benefit of a combinational therapeutic strategy using abemaciclib with other molecular targeting agents among MCL patients with therapeutic resistance. Methods Cytotoxic efficacy of abemaciclib as a single agent and in combination with other drugs on different MCL cell lines and primary lymphoma cells from MCL patients with or without resistance was used as a key criterion for screening beneficial therapeutic strategies. Cell apoptosis and cell cycle arrest assays were conducted to further evaluate those effective combinations. Western blot was performed to investigate the mechanism of action of the combinations. Finally, the efficacy of abemaciclib alone or in combination were assessed in ibrutinib-resistant or venetoclax-resistant MCL PDX models in vivo. Results Our preliminary data showed that all MCL cell lines involved in this study were highly sensitive to abemaciclib treatment with IC50 values ranging from 50 nM to 1 µM. Further investigation of abemaciclib cytotoxicity on ibrutinib and/or venetoclax resistant MCL cell lines showed effective inhibition with a higher IC50 values ranging from 5 µM to 10 µM. More importantly, abemaciclib had potent efficacy on cells from primary MCL patients as well as from patients with acquired ibrutinib resistance. Our recent findings revealed that the addition of PI3K inhibitor TGR-1202 significantly enhanced cytotoxicity of abemaciclib in both sensitive and resistant MCL cell lines. Abemaciclib significantly inhibited phosphorylation of Rb1, the active form of the protein, in 4 different MCL cell lines. The active Rb1 maintains the cell in the G1 phase, preventing progression through the cell cycle and acting as a growth suppressor. The result suggests that CDK4/6 inhibition with abemaciclib disrupts CDK4/6 suppressive activity towards pRb-E2F and induce cell cycle arrest in the MCL cells. Interestingly, abemaciclib somehow interrupted phosphorylation of Chk1, which is continuously phosphorylated and hence activated in the MCL cell lines. Inhibiting activation of Chk1 by abemaciclib may induce cell death via unmonitored and accumulated DNA damage. The efficacy of abemaciclib in combination with Bcl-2 or BTK inhibitors in MCL cell lines and isolated cells from MCL patients are ongoing. These data suggest that abemaciclib in combination with other therapeutic drugs could be beneficial in targeting therapeutic resistant MCL cells. Conclusions Abemaciclib showed impressive therapeutic potency on both MCL cell lines and isolated primary cells from MCL patients, which is likely due to the predominant contribution of cyclin D1-CDK4/6 pathway to malignancy. Other agents, such as PI3K inhibitors, can sensitize abemaciclib in therapeutic resistant MCL cells. Thus, an abemaciclib based multi-drug combinational strategy may be a promising therapy for refractory/relapsed MCL patients in the near future. Disclosures Wang: Beijing Medical Award Foundation: Honoraria; Lu Daopei Medical Group: Honoraria; Kite Pharma: Consultancy, Other: Travel, accommodation, expenses, Research Funding; Pulse Biosciences: Consultancy; Loxo Oncology: Consultancy, Research Funding; Targeted Oncology: Honoraria; OMI: Honoraria, Other: Travel, accommodation, expenses; Nobel Insights: Consultancy; Guidepoint Global: Consultancy; Dava Oncology: Honoraria; Verastem: Research Funding; Molecular Templates: Research Funding; OncLive: Honoraria; Celgene: Consultancy, Other: Travel, accommodation, expenses, Research Funding; AstraZeneca: Consultancy, Honoraria, Other: Travel, accommodation, expenses, Research Funding; Oncternal: Consultancy, Research Funding; Juno: Consultancy, Research Funding; BioInvent: Research Funding; VelosBio: Research Funding; Acerta Pharma: Research Funding; InnoCare: Consultancy; MoreHealth: Consultancy; Pharmacyclics: Consultancy, Honoraria, Other: Travel, accommodation, expenses, Research Funding; Janssen: Consultancy, Honoraria, Other: Travel, accommodation, expenses, Research Funding.

scholarly journals Inhibition of Cyclin Dependent Kinase 9 Synergistically Enhanced Venetoclax Activity in Mantle Cell Lymphoma Cells

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1593-1593
Author(s):  
Xiaoxian Zhao ◽  
Juraj Bodo ◽  
Ruoying Chen ◽  
Lisa Durkin ◽  
Andrew J. Souers ◽  
...  
Keyword(s):  
Rna Polymerase Ii ◽  
Cell Lines ◽  
Research Funding ◽  
Cell Lymphoma ◽  
Single Agent ◽  
Cyclin Dependent Kinase ◽  
Bcl2 Family ◽  
Mantle Cell ◽  
Induced Apoptosis ◽  
Cdk9 Inhibitor

Abstract Better therapeutic strategies are needed for patients with mantle cell lymphoma (MCL), an aggressive and largely incurable subtype of Non-Hodgkin Lymphoma. Concurrent expression of anti-apoptotic BCL2 family proteins in lymphoma cells contribute to their evasion of apoptosis. Therefore, targeting only one anti-apoptotic protein may lead to or uncover resistance associated with activity of other anti-apoptotic BCL2 family members. A variety of cyclin-dependent kinase (CDK) inhibitors are undergoing clinical trials either as a single agent or in combination with other approved drugs. CDK9, a portion of the elongation factor P-TEFb, phosphorylates Ser-2 in the C-terminal domain of RNA Polymerase II, which is required for transcript elongation. The effect of CDK9 inhibition is observed most immediately on those proteins with rapid turnover rates such as the BCL2 family protein MCL1, which is associated with both intrinsic and acquired resistance to venetoclax in B-cell malignancies. Here we report the responses of 4 MCL cell lines (Mino, Jeko-1, CCMCL1 and JVM2) and 5 primary MCL samples (representing de novo and relapsed cases, including two relapsed cases after ibrutinib failure and a relapsed case harbor Myc rearrangement) to venetoclax and a novelCDK9 inhibitor A-1467729. Exposure of Mino and Jeko-1 cells to venetoclax rapidly induced apoptosis (IC50 at 5 hours were 235 and 955 nM, respectively). In contrast, CCMCL1 and JVM2 cells were not sensitive to venetoclax with IC50s > 3000 nM. However, CCMCL1 cells were more responsive to A-1467729 alone than the other 3 lines, while JVM2 cells were much less sensitive to A-1467729. All primary samples were sensitive to venetoclax, ex vivo, at the doses between 1 - 100 nM, although their IC50s were variable (range: 2-90 nM). A-1467729 at doses of 1-20 nM had modest single agent effects on the primary samples; however, its combination with venetoclax synergistically induced apoptosis and decreased the IC50 of venetoclax by 2-10 times in all cell lines and primary samples. The strongest synergy was observed in Jeko-1 cells with all combined indexes < 0.1. Studies on mechanisms through immunoblotting and immunohistochemical staining demonstrated that A-1467729 quickly down-regulated phospho-RNA Polymerase II (Ser2) and MCL1 protein levels. CCMCL1 cells lack BCL2 expression, while JVM2 displayed higher expression of MCL1 than other cells. The expression levels of BCL2 and MCL1 in primary samples were case-dependent as well. The expression pattern and level of anti-apoptotic BCL2 family proteins in cell lines and primary cases may be responsible for their variable reactions to these two agents. To further confirm that CDK9 inhibition was affecting cell viability at least partially through its function on MCL1, A-1210477, a MCL1 inhibitor, was applied to the same study. Strong synergistic apoptotic induction was also observed when A-1210477 was combined with venetoclax, especially in MCL1-"dependent" CCMCL1 cells as evidenced by flow cytometry based apoptotic assay and PARP cleavage. Further mechanism studies aiming the effects of CDK9 inhibitor/venetoclax on MCL1/BIM association is being under investigation. MCL mouse xenograft study for such a combined effect has been planned. In summary, the combination of a CDK9 inhibitor and venetoclax showed synergistic induction of apoptosis in both MCL cell lines and primary patient samples. These findings support further evaluation of the efficacy of such a combination in MCL, including ibrutinib-resistant MCL. Disclosures Souers: AbbVie: Employment. Phillips:AbbVie Inc.: Employment. Hsi:HTG Molecular Diagnostics: Consultancy; Abbvie: Honoraria, Research Funding; Seattle Genetics: Honoraria; Cellerant: Honoraria, Research Funding; Eli Lilly: Research Funding; Onyx Pharmaceuticals: Honoraria.

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

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 4756-4756 ◽  
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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