Efficient, Non-Viral and Reproducible Protocol for Stable Knockdown of Genes in Mantle Cell Lymphoma Cell Lines

Introduction The biological role of specific genes can be investigated using gene silencing by cell transfection with small interfering RNA (siRNA), which leads to degradation of the corresponding mRNA and reduced target protein expression. The study of biological functions in modified cell cultures often requires a long-lasting knockdown (KD) of gene expression, which can be obtained by viral delivery of short hairpin RNA (shRNA) or Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9 systems. However, these methods are laborious and require high level of laboratory safety. Hematopoietic cells, typically grown in suspension, are known to be difficult to transfect. Previous efforts to transfect lymphocytes using liposomes (H Guven et al, ExpHem2005) showed limited transfection efficiencies of only 10-30%. Transfection by electroporation leads to decreased viability, and therefore poses a risk of unintentional selection of cell subsets. Transfection of leukemia cells with siRNAs using Accell siRNA Delivery Media showed KD efficiencies of 56-65% and a viability of 57-95% (H Larsen et al,ExpHem2011). However, this was only investigated 24-72h post-transfection. By whole transcriptome sequencing, we previously identified a number of candidate genes, includingLILRA4, PTPRJ and SYNE2, which were significantly upregulated in mantle cell lymphoma (MCL) cells compared to B cells from healthy donors (MH Hansen et al, ExpHem 2020). To investigate the biological function of these genes, an efficient and stable KD was required. Aim The aim of this study was to identify the most effective non-viral method for siRNA-mediated knockdown in MCL cells. Methods The MCL cell lines Mino, Jeko-1 and Granta 519, were transfected using three different methodologies; Lipofectamine 2000 (Invitrogen), Electroporation with Amaxa Nucleofector (Lonza), and Accell siRNA Delivery Media (Dharmacon). For Accell delivery a pool of 4 siRNAs targeting each gene was used (SMARTpool siRNA, Dharmacon). For the others, 2 pre-designed siRNAs (Ambion) targeting each gene were used. For all, a positive control siRNA targetingGAPDHwas included and different conditions tested e.g. siRNA concentration, incubation time and addition of serum. KD efficiency was evaluated by qPCR usingABLandGUSreference genes. Viability of cells was measured either by flow cytometry staining with a live/dead marker or by trypan blue. Optimization of the Accell protocol was performed in Granta 519 cells transfected with SMARTpool siRNAs targeting eitherSYNE2,LILRA4,orGAPDH. KD efficiency was evaluated with qPCR and viability was counted with trypan blue. Results Using Lipofectamine, at 24h a non-reproducible and non-efficient KD (<40%) was achieved forLILRA4in the Jeko-1 cell line, while only 5-22% gene silencing was achieved for Mino. Using electroporation, a KD efficiency of 45-70% was achieved in Mino at 24h forGAPDH,LILRA4andPTPRJ, while inferior efficiency (<15%) and viability were observed in Jeko-1 cells. However, in the Mino cell line,PTPRJwas almost fully re-expressed (80% expression) after 48h. Using transfection with Accell delivery medium,GAPDH,LILRA4,andPTPRJwere successfully knocked down in all tested cell lines with a median efficiency of 75% (range 50%-98%) after 72h. When performing KD in Accell delivery medium, the viability of the cells decreased after 72h and dropped to 26-39% at day 5 and 6, and a decrease was found in the total number of cells, suggesting that proliferation may also be affected. By adding 10% fetal bovine serum (FBS) to this medium after 48h, the viability was kept above 66.7% for up to 6 days, and the total number of cells was comparable to that observed in normal culture medium, while the KD efficiency was still preserved. In Ganta 519 cells with addition of FBS, an average KD efficiency of 67% was achieved forLILRA4(range 56-72%) at 72h-5 days, while the average KD efficiency ofSYNE2was 77% (range: 67-95%) at 72h-5 days. Conclusion This study propose that transfection with SMARTpool siRNAs in Accell Delivery Medium with addition of 10% FBS 48h post transfection is an applicable procedure for siRNA-mediated KD in MCL suspension cell lines. This protocol permits efficient, non-viral, and stable KD across different MCL cell lines without affecting the cells' ability to proliferate and survive, and is therefore suitable for investigating biological processes. Disclosures No relevant conflicts of interest to declare.

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Whole Transcriptome Sequencing Reveals Recurrent NOTCH1 Mutations in Mantle Cell Lymphoma

Blood ◽

10.1182/blood.v118.21.436.436 ◽

2011 ◽

Vol 118 (21) ◽

pp. 436-436 ◽

Cited By ~ 3

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.

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

Blood ◽

10.1182/blood.v126.23.2760.2760 ◽

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.

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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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Treatment with Intermediate Subviral Particles Overcomes Resistance to Reovirus Induced Oncolysis in Mantle Cell Lymphoma Cell Lines.

Blood ◽

10.1182/blood.v106.11.2417.2417 ◽

2005 ◽

Vol 106 (11) ◽

pp. 2417-2417

Author(s):

Gwyn Bebb ◽

Huong Muzik ◽

Tom Alain ◽

Karen Dong ◽

Chandini Thirukkamaran ◽

...

Keyword(s):

Mantle Cell Lymphoma ◽

Cell Line ◽

Cell Lines ◽

Cell Lymphoma ◽

Lymphoma Cell ◽

Complete Medium ◽

Mantle Cell ◽

Time Point ◽

Subviral Particles

Abstract Introduction: Mantle Cell Lymphoma (MCL), a B-cell, non-Hodgkin lymphoma (NHL) characterized by cyclin D1 and Bcl-2 over-expression, is considered incurable with standard therapies. Reovirus, a double stranded RNA oncolytic virus has shown a consistent anti-neoplastic effect in a variety of cell lines both in vitro, in vivo and ex vivo. We recently demonstrated an anti-lymphoma effect of reovirus in two MCL cell lines and reovirus resistance in three. Here we report the effect of trypsinization of the reovirus envelope to generate intermediate subviral particles (ISVP) on the resistance of these cell lines in vitro. Methods Five MCL cell lines (Z138C, NCEB-1, Hb-12, JVM2 and Granta 519) and one Follicular Lymphoma cell line (DoHH2) were counted and resuspended in serum-free medium. 104 cells in 20ul were dispensed per well in a 96-well plate. 10ul of reovirus at appropriate MOI in serum-medium or 40MOI of ISVP was added to the cells. ISVP was prepared as follows; 1ul of reovirus at 40MOI was treated with 9ul of chymotrypsin at 37°C for 30 minutes before being added to the cells. Cells and viruses were allowed to be in contact at 4°C for 45 min -1 hour. 170 ul of warm complete medium RPMI 1640 containing 10% FCS and L-glutamine was then added and the plate was incubated at 37 °C. Each time point was performed in quadruplicate. WST-1 reagent was used to quantify cell proliferation and cell viability was assessed by absorbance at 450nm using a plate reader. Results MCL cell lines NCEB-1 and JVM-2 showed exquisite sensitivity to reovirus induced oncolysis after 4 days’ incubation at 40 and 80 MOI. Granta exhibited intermediate sensitivity to reovirus while Hb12 and Z138C demonstrated an innate resistance to reovirus induced oncolyis at this time point. Incubation of the resistant cell lines with ISVP overcame this resistance in the Granta and Z138C cell lines (as well as DoHH2). Viability of Granta and Z138C after 4 days’ incubation with ISVP approached that of the sensitive cell lines NCEB-1 and JVM-2 at the same time point. However, the Hb12 cell line retained a significant degree of resistance to reovirus when incubated with ISVP. Percent Viability of 5 MCL Cell Lines at 4 Days’ Incubation with Reovirus or ISVP Treatment NCEB-1 JVM-2 Hb-12 Z138C Granta DoHH2 Control 100 100 100 100 100 100 Reovirus 40 MOI 26 29 113 89 58 54 Reovirus 80 MOI 20 1 114 94 50 45 ISVP 40 MOI 22 5 45 9 12 8 Conclusion Although each of the five cell lines investigated has the karyotypic and molecular hallmarks of MCL, they exhibit heterogeneity of sensitivity to reovirus induced oncolysis. The resistance to reovirus oncolysis exhibited by the two cell lines Z138C and Granta can be overcome by 40 MOI ISVP. In contrast, incubation with the ISVP only partly overcomes reovirus resistance in the HB12 cell line. The molecular basis of this variation and the mechanism underlying reovirus resistance warrants further investigation at the proteome, transcriptome and genome level.

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The Tumor Microenvironment in Mantle Cell Lymphoma (MCL): Novel Targets to Overcome Chemo-Resistance in MCL

Blood ◽

10.1182/blood.v124.21.494.494 ◽

2014 ◽

Vol 124 (21) ◽

pp. 494-494 ◽

Cited By ~ 1

Author(s):

Lan V Pham ◽

Archito T. Tamayo ◽

Elizabeth Pogue ◽

Gary Lu ◽

Pramoda Challagundla ◽

...

Keyword(s):

Drug Resistance ◽

Tumor Microenvironment ◽

Mantle Cell Lymphoma ◽

Cell Line ◽

Tumor Cells ◽

Cell Lines ◽

Stromal Cells ◽

Cell Lymphoma ◽

Mantle Cell ◽

Leukemic Phase

Abstract Mantle Cell Lymphoma (MCL) is a relatively uncommon, aggressive form of B-cell non-Hodgkin’s lymphoma (NHL), defining approximately 5-8% of adult NHL in the United States. Although individual survival times have increased in recent years, prognosis remains among the poorest of NHL, with a median survival of 5 to 7 years. Despite an initial response to chemotherapy, MCL cells remain residually in the marrow and digestive tract, making MCL prone to recur leading to short survival prognosis. The characteristic re-emergence of MCL is likely related to drug resistance generated from the tumor microenvironment (TME). In particular, stromal cells found in bone marrow and secondary lymphoid sites promote the movement and proliferation and possibly drug resistance from cross-talk between stromal and lymphoma cells. While studies show a correlation between stromal cells and tumor survival in various forms of NHL, the mechanisms are poorly understood and its role in MCL has not extensively explored. Based on our experimental studies on an extensive spectrum of representative patient samples, we have shown that in vitro culturing of primary leukemic phase MCL cells (15 cases) usually leads to the spontaneous formation of activated CD68+ monocytic macrophages and stromal cells. The MCL tumor cells tended to form rosettes, clustering around and adhering to these cultured lymphoma-associated macrophages (LAM). The LAM formed in culture coexisted with rosetted MCL tumor cells for up to 4 months. During this period, a small subset of MCL cells showed limited mitotic figures, suggesting that these cells were proliferating and possibly becoming incipient immortalized MCL cells. When these cells were weaned from the adherent LAM, they began to expand autonomously and proliferate as an autonomous cell line. Using this methodology, we have developed 3 MCL cell lines (PF-1, PF-2, and PF-3) from 15 leukemic phase MCL cases. PF-1 MCL cell line represents classic typical MCL cells, while PF-2 and PF-3 MCL cell lines represent the in situ indolent MCL cells that are CD200 positive. When these cells were xeno-transplanted into SCID mice, lymphoma cell uptakes were disseminated throughout the lymphatic system, including the spleen, lymph nodes, and GI tract, representing excellent in vivo models for MCL. Next, we delineate the biologic functions of LAM in the MCL microenvironment milieu. Our data showed that when we initially removed the accessory cells (monocytes, etc.) from the MCL primary culture, no LAM were formed and the purified MCL cells died out in <2-3 weeks in culture, suggesting that the LAM developed in culture are biologically and pathologically functional in providing growth/survival signals to the tumor cells. We then examined several therapeutic agents that have shown efficacy against relapsed-MCL in the clinic, including the proteasome inhibitor carfilzomib (CFZ) and the BTK inhibitor ibrutintib (IB), on targeting LAM. Our results showed that both CFZ and IB, at very low drug concentrations could affectively eliminate LAM, followed by spontaneous apoptosis of the MCL cells. We have also developed a unique mesenchymal stromal cell line (PF-MSC), derived from a leukemic phase MCL patient. These cells grow in culture as spindle-shaped morphologies and can enhance the growth of MCL cells as well as protect MCL cells from chemotherapy. However, PF-MSC cells did not protect MCL cells from CFZ or IB therapies. Our findings clearly indicate that MCL TME is a critical mediator for growth/survival and chemoresistance mechanisms in the pathophysiology of MCL. Therapeutically targeting these MCL-associated macrophages/stromal cells with CFZ and/or IB should lead to better therapeutic strategies for refractory MCL patients. Disclosures Pham: Onyx/Amgen: Research Funding.

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Constitutive activation of the Wnt canonical pathway in mantle cell lymphoma

Blood ◽

10.1182/blood-2008-02-139212 ◽

2008 ◽

Vol 112 (13) ◽

pp. 5171-5179 ◽

Cited By ~ 58

Author(s):

Pascal Gelebart ◽

Mona Anand ◽

Hanan Armanious ◽

Anthea C. Peters ◽

Jennifer Dien Bard ◽

...

Keyword(s):

Mantle Cell Lymphoma ◽

Cell Lines ◽

Cell Lymphoma ◽

Selective Inhibition ◽

Canonical Pathway ◽

Oligonucleotide Arrays ◽

Downstream Target ◽

Naturally Occurring ◽

Inactive Form ◽

Mantle Cell

Abstract Aberrations of the Wnt canonical pathway (WCP) are known to contribute to the pathogenesis of various types of cancer. We hypothesize that these defects may exist in mantle cell lymphoma (MCL). Both the upstream and downstream aspects of WCP were examined in MCL cell lines and tumors. Using WCP-specific oligonucleotide arrays, we found that MCL highly and consistently expressed Wnt3 and Wnt10. β-catenin, a transcriptional factor that is a downstream target of WCP, is localized to the nucleus and transcriptionally active in all 3 MCL cell lines examined. By immunohistochemistry, 33 (52%) of 64 MCL tumors showed nuclear localization of β-catenin, which significantly correlated with the expression of the phosphorylated/inactive form of GSK3β (p-GSK3β; P = .011, Fisher). GSK3β inactivation is directly linked to WCP stimulation, since addition of recombinant sFRP proteins (a naturally occurring decoy for the Wnt receptors) resulted in a significant decrease in p-GSK3β. Down-regulation of DvL-2 (an upstream signaling protein in WCP) by siRNA or selective inhibition of β-catenin using quercetin significantly decreased cell growth in MCL cell lines. To conclude, WCP is constitutively activated in a subset of MCL and it appears to promote tumorigenesis in MCL.

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Expression of STAT3 and its phosphorylated forms in mantle cell lymphoma cell lines and tumours

The Journal of Pathology ◽

10.1002/path.1253 ◽

2002 ◽

Vol 199 (1) ◽

pp. 84-89 ◽

Cited By ~ 31

Author(s):

Raymond Lai ◽

George Z Rassidakis ◽

L Jeffrey Medeiros ◽

Vasiliki Leventaki ◽

Micheal Keating ◽

...

Keyword(s):

Mantle Cell Lymphoma ◽

Cell Lines ◽

Cell Lymphoma ◽

Lymphoma Cell ◽

Mantle Cell Lymphoma Cell ◽

Mantle Cell

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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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Activation of MYC, a bona fide client of HSP90, contributes to intrinsic ibrutinib resistance in mantle cell lymphoma

Blood Advances ◽

10.1182/bloodadvances.2018016048 ◽

2018 ◽

Vol 2 (16) ◽

pp. 2039-2051 ◽

Cited By ~ 16

Author(s):

Jimmy Lee ◽

Liang Leo Zhang ◽

Wenjun Wu ◽

Hui Guo ◽

Yan Li ◽

...

Keyword(s):

Mantle Cell Lymphoma ◽

Cell Lines ◽

Cell Lymphoma ◽

Resistant Cell ◽

Intrinsic Resistance ◽

Mantle Cell ◽

Myc Gene ◽

Bona Fide ◽

Ibrutinib Resistance ◽

Induced Apoptosis

Abstract The BTK inhibitor ibrutinib has demonstrated a remarkable therapeutic effect in mantle cell lymphoma (MCL). However, approximately one-third of patients do not respond to the drug initially. To identify the mechanisms underlying primary ibrutinib resistance in MCL, we analyzed the transcriptome changes in ibrutinib-sensitive and ibrutinib-resistant cell lines on ibrutinib treatment. We found that MYC gene signature was suppressed by ibrutinib in sensitive but not resistant cell lines. We demonstrated that MYC gene was structurally abnormal and MYC protein was overexpressed in MCL cells. Further, MYC knockdown with RNA interference inhibited cell growth in ibrutinib-sensitive as well as ibrutinib-resistant cells. We explored the possibility of inhibiting MYC through HSP90 inhibition. The chaperon protein is overexpressed in both cell lines and primary MCL cells from the patients. We demonstrated that MYC is a bona fide client of HSP90 in the context of MCL by both immunoprecipitation and chemical precipitation. Furthermore, inhibition of HSP90 using PU-H71 induced apoptosis and caused cell cycle arrest. PU-H71 also demonstrates strong and relatively specific inhibition of the MYC transcriptional program compared with other oncogenic pathways. In a MCL patient-derived xenograft model, the HSP90 inhibitor retards tumor growth and prolongs survival. Last, we showed that PU-H71 induced apoptosis and downregulated MYC protein in MCL cells derived from patients who were clinically resistant to ibrutinib. In conclusion, MYC activity underlies intrinsic resistance to ibrutinib in MCL. As a client protein of HSP90, MYC can be inhibited via PU-H71 to overcome primary ibrutinib resistance.

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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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