Combination Bortezomib (PS341, Velcade) and Rituximab Treatment Affects Multiple Survival and Death Pathways To Promote Apoptosis in Mantle Cell Lymphoma.

Abstract Mantle cell lymphoma (MCL) is a distinct histologic subtype of B cell non-Hodgkin’s lymphoma that is associated with an aggressive clinical course and a particularly poor prognosis. The mechanisms that contribute to resistance of MCL to chemotherapy are not clear, however, recent work examining the consequences of ubiquitin-proteasome pathway inhibition on cell cycle (p21, p27) and key survival/death networks (NFkB, p53, Bcl2) has provided rationale for exploring combination regimens that include tumor-specific reagents (rituximab) and the 26S proteasome inhibitor bortezomib. In this study, we examined the effects of combination treatment with bortezomib and rituximab on MCL patient samples and three patient-derived cell lines (Jeko, Mino, SP53). Cells treated with bortezomib (10 – 100nM) for 4 hours demonstrated proteasome inhibition that persisted for 24 hours but returned to baseline activity at 48 hours after treatment. Despite transient proteasome inhibition, combination therapy with bortezomib (10–100nM for 4hrs) and rituximab (1 mg/ml immobilized with 20 mg/ml goat anti-human IgG) resulted in synergistic induction of apoptosis that persisted for as long as 72 hours after treatment. While bortezomib (100 nM) induced apoptosis in 18.3 ± 6.5% and rituximab induced apoptosis in 24.5 ± 4.5% of MCL cells, combination treatment resulted in 57.4 ± 5.1% apoptosis at 48 hours (p ≤ 0.02). Pretreatment of MCL cells with the broad spectrum caspase inhibitor zVAD-FMK (10 mM) showed that bortezomib-induced cell death occurred by caspase-dependent mechanisms, however, when immobilized rituximab was added, cell death occurred via caspase dependent and independent pathways. Single agent bortezomib (10 nM) or rituximab treatment of Mino and Jeko lines resulted in decreased levels of nuclear NFkB complex(s) capable of binding p65 consensus oligonucleotides (28% and 21% reduction, respectively), while combination treatment resulted in enhanced reduction of detectable nuclear NFkB (36% reduction, p ≤ 0.0007). Similar trends were observed with primary MCL cells. Experiments with an IKK inhibitor (PS1145, Millenium Pharmaceuticals) resulted in nuclear NFkB reduction without equivalent induction of apoptosis which led us to hypothesize that other pro-death pathways might be operable with combination treatment. Western blot analysis of BCL2-family members revealed that combination treatment of MCL lines resulted in near complete elimination of Bcl-xL protein while Bcl-2 protein levels remained unchanged. The pro-death gene product Bax was induced in a synergistic fashion with combined bortezomib and rituximab treatment. Finally, we have developed a reliable preclinical animal model utilizing the severe combined immune deficient (SCID) mouse engrafted with three patient-derived MCL cell lines. Each cell line results in a characteristic pattern of tumor burden and highly reproducible time to develop advanced disease. We are currently evaluating combination therapy with bortezomib and rituximab in this preclinical animal model. Our preclinical evaluation provides clear rationale for pursuing combination strategies that inhibit the proteasome in combination with tumor-specific immunotherapy in patients with 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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Dual Targeting of NOXA/MCL-1 Synergistically Induces Cell Death in Mantle Cell Lymphoma (MCL) Cells

Blood ◽

10.1182/blood.v126.23.2757.2757 ◽

2015 ◽

Vol 126 (23) ◽

pp. 2757-2757 ◽

Cited By ~ 1

Author(s):

Elisabeth Höring ◽

German Ott ◽

Christine Bayha ◽

Kleih Markus ◽

Matthias C. Voehringer ◽

...

Keyword(s):

Cell Death ◽

Protein Expression ◽

Mantle Cell Lymphoma ◽

Cell Lymphoma ◽

Dependent Manner ◽

Stabilizing Agents ◽

Dual Targeting ◽

Promising Strategy ◽

Mantle Cell ◽

Induction Of Apoptosis

Abstract Mantle cell lymphoma (MCL) cells are characterized by a discrepancy between high mRNA and low protein levels of the pro-apoptotic BH3-only protein NOXA. Modulation of NOXA protein expression has been described as a major mechanism of cell death induction in MCL cells. However, the efficiency of induction of apoptosis at lower concentrations is limited despite effective stabilization of NOXA. We therefore investigated whether the main binding partner of NOXA, the anti-apoptotic protein MCL-1 is co-regulated by these agents and whether dual targeting of NOXA and MCL-1 could be a promising strategy to enhance effectiveness of cell death induction in MCL cell lines. Screening a panel of compounds supposed to lead to NOXA stabilization in MCL, we identified the proteasome inhibitor Bortezomib, the fatty acid synthase inhibitor Orlistat and the ROS inducing agents Helenalin, a sesquesterpenone lactone from Arnica and the naphtoquinone derivative Menadione to kill MCL cells very effectively in a NOXA -dependent manner. Investigating the NOXA-/MCL-1-protein expression upon treatment with these agents, we could observe that at lower, sublethal concentrations of Bortezomib and Orlistat, NOXA protein was increased to a certain extent but also the anti-apoptotic MCL-1 was highly induced, counteracting induction of apoptosis. From this observation it has to be concluded that MCL-1 limits the apoptotic activity of NOXA stabilizing agents and dual targeting of MCL-1 and NOXA is required for optimal killing of MCL cells. In search for MCL-1 regulating agents we could identify the cdk-inhibitor Dinaciclib to be the most effective one. This compound rapidly downregulates Mcl-1 protein in a dose- and time-dependent manner presumably due to cdk 9-mediated inhibition of phosphorylation of the RNA-Polymerase II-subunit RPB1. To study the efficiency of dual targeting of NOXA/MCL-1 in killing of MCL cells we combined sublethal doses of Dinaciclib with NOXA stabilizing agents and observed a synergistic induction of apoptosis in MCL cells. Western Blot analysis showed that combination treatment decreased Mcl-1 and increased NOXA protein expression compared to single-agents. It could be shown that induction of cell death by treatment with the combined agents depends on NOXA as transfection of cells with NOXA-siRNA rescued cells from induction of apoptosis. Cell death upon treatment with Dinaciclib and ROS-inducing agents Helenalin and Menadione could furthermore be rescued by preincubation with the antioxidant GSH. Interestingly, combined treatment of cells with Orlistat and Dinaciclib killed most effectively when Dinaciclib was added for 8 hours after 16 hours of preincubation with the NOXA stabilizing agent. This observation contributes to the hypothesis that stabilization of NOXA protein leads to priming of MCL cells to induction of apoptosis by pharmacological downregulation of Mcl-1 with Dinaciclib. In summary, the NOXA-MCL-1 balance is critical for survival of MCL cells. Dual targeting of MCL-1 and NOXA efficiently kills MCL cells and appears to be of particular importance especially at lower concentrations of these compounds. These culture conditions most likely resemble the limited exposure after in vivo treatment. Therefore the combination of NOXA stabilizing agents with Dinaciclib appears to be a promising strategy to be tested in clinical trials. Disclosures No relevant conflicts of interest to declare.

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FTY720 Increases CD74 Expression and Sensitizes Mantle Cell Lymphoma Cells to Milatuzumab-Mediated Cell Death

Blood ◽

10.1182/blood.v118.21.600.600 ◽

2011 ◽

Vol 118 (21) ◽

pp. 600-600

Author(s):

Lapo Alinari ◽

Emilia Mahoney ◽

John T. Patton ◽

Xiaoli Zhang ◽

Lenguyen Huynh ◽

...

Keyword(s):

Monoclonal Antibody ◽

Cell Death ◽

Mantle Cell Lymphoma ◽

Cell Lines ◽

Median Survival ◽

Combination Treatment ◽

Cell Lymphoma ◽

Mantle Cell

Abstract Abstract 600 Mantle cell lymphoma (MCL) is an aggressive B-cell malignancy with a short median survival despite multimodal therapy. FTY720, an immunosuppressive drug approved for the treatment of multiple sclerosis, promotes MCL cell death via down-modulation of phospho-Akt and Cyclin D1, and subsequent cell cycle arrest (1). However, the mechanism of FTY720-mediated MCL cell death remains to be fully clarified. Here we show features of autophagy blockage by FTY720 treatment, including accumulation of autolysosomes, increased LC3-II and p62 levels. FTY720 is phosphorylated in vivo by sphingosine kinase 2 and converted to p-FTY720, which binds to sphingosine-1-phosphate (S1P) receptors. A non-phosphorylatable FTY720 derivative (OSU-2S) was recently developed at the Ohio State University (2): OSU-2S treatment induces MCL cell death and shows features of autophagy blockage that led us to conclude that FTY720 phosphorylation and its interaction with SP1 receptors are not required for FTY720-mediated cell death and blockage of autophagy in MCL cells. We also demonstrate that FTY720-induced cell death is mediated by lysosomal membrane permeabilization with subsequent translocation of lysosomal hydrolases to the cytosol. FTY720-mediated disruption of the autophagic-lysosomal pathway led to increased levels of CD74, a potential therapeutic target in MCL that is degraded in the lysosomal compartment. We have recently reported CD74 to be expressed on MCL cells and that milatuzumab (Immunomedics, Morris Plains, NJ), a humanized anti-CD74 monoclonal antibody, has significant anti-MCL activity in vitro and in vivo (3). This finding provided the rationale for examining combination therapy with FTY720 and milatuzumab. The in vitro survival of 4 MCL cell lines treated with FTY720, immobilized milatuzumab, and the combination was determined at 24 hours by Annexin-V/PI staining and flow cytometry. Incubation of 4 MCL cell lines with FTY720 and milatuzumab (1 μg/ml) resulted in a statistically significant decrease in cell viability compared to either agent alone for each of the four cell lines (P< 0.01), despite using FTY720 at concentrations lower than the LC50 previously published [Jeko-1 FTY720: 10 μM (LC50: 12.5 μM), Z-138 and UPN-1: 6 μM (LC50: 7.5 μM); Mino 3.75 μM (LC50: 7.5μM)] (1). Notably, combination treatment resulted in synergistic killing in cell lines derived from patients with blastoid variant MCL (Jeko-1, Z-138, UPN-1), despite the fact that both FTY720 and milatuzumab as single agents showed only modest activity. Incubation of primary tumor cells from 6 MCL patients (3 blastoid variant and 3 classic MCL) with FTY720 (2.5 μM, LC50: 5 μM) and miltauzumab induced an average 78.5% cell death compared to 47% of FTY720 treated cells and 50% the milatuzumab-treated cells (P=0.0005 and P=0.0014, respectively). To examine the in vivo activity of FTY720 and milatuzumab, a preclinical model of human MCL using the SCID (CB17 scid/scid) mouse depleted of NK cells was used. In this model, i.v. injection of 40×106 JeKo cells results in disseminated MCL 3 weeks after engraftment. The primary end-point was survival, defined as the time to develop cachexia/wasting syndrome or hind limb paralysis. Mice (n=10/group) were treated starting at day 15 post engraftment. Twenty control mice received either placebo (saline) or trastuzumab (15 mg/kg) treatment. The third group was treated with FTY720 (5 mg/kg) every day for 2 weeks via i.p injection. The fourth group received milatuzumab (15 mg/kg) every three days, via i.p. injection. The fifth group received the combination of FTY720 and milatuzumab. The median survival for the combination-treated group was 36 days (95% CI:31,36), compared to 28 days for the saline-treated mice (95% CI:24,31), 27 days for the trastuzumab-treated mice (95% CI:23,29), 31 days for the FTY720-treated mice (95% CI:28,32), and 33.5 days for the milatuzumab-treated mice (95% CI:23,34). The combination treatment significantly prolonged survival of this group compared to control groups (P<0.0001), FTY720 (P=0.0001) and milatuzumab (P=0.0048). The most clinically relevant aspect of these findings is that we demonstrate that a potent anti-MCL agent (FTY720) has also the ability to modulate a druggable target (CD74) by preventing its degradation in the autophagic-lysosomal pathway. We believe these findings support clinical evaluation of this combination in patients with MCL. Disclosures: Off Label Use: fty720 immunosupressive drug milatuzumab fully humanized anti-CD74 monoclonal antibody. Goldenberg:Immunomedics: Equity Ownership, Immunomedics owns milatuzumab, Patents & Royalties.

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Inhibition of Tak-1 by AZ-Tak1 Impairs NF-κB Activation, Downregulates XIAP and Activates Caspase-9 Inducing Apoptosis In Mantle Cell lymphoma

Blood ◽

10.1182/blood.v116.21.2852.2852 ◽

2010 ◽

Vol 116 (21) ◽

pp. 2852-2852 ◽

Cited By ~ 1

Author(s):

Daniela Buglio ◽

Sangeetha Palakurti ◽

Francisco Vega ◽

Sattva S. Neelapu ◽

Donald Berry ◽

...

Keyword(s):

Cell Death ◽

Mantle Cell Lymphoma ◽

Cell Lines ◽

Small Molecule ◽

Cell Lymphoma ◽

Small Molecule Inhibitor ◽

Caspase 9 ◽

Molecule Inhibitor ◽

Mantle Cell ◽

Induced Apoptosis

Abstract Abstract 2852 TGF-b-activated kinase 1 (TAK1), a member of the mitogen-activated protein kinase kinase kinase (MAPKKK) family, plays a key role in regulating inflammation, immunity, metabolism, and cell death in a variety of cell types. It is activated in response to a variety of cytokines, including tumor necrosis factor (TNF), TGF-b, and interleukin 1 (IL-1). Upon receptor binding, TAK1 binds to adaptor proteins, and subsequently phosphorylate downstream molecules leading to activation of p38MAPK, JNK, and NF-kB. In this study, we examined the expression pattern of TAK1 and its potential therapeutic role as a target for lymphoma. First, we examined TAK1 expression in a panel of lymphoid cell lines by western blot, and found it to be highly expressed in mantle cell lymphoma cell lines (Mino, SP53, and Jeko-1). In contrast, PBL from healthy donors had no expression of TAK1 protein. TAK1 was also highly expressed in primary lymph node sections of MCL compared with benign reactive lymph nodes. Subsequently, we investigated the in vitro activity of the novel TAK1 small molecule inhibitor AZ-Tak1 in these cell lines. AZ-Tak1 is a potent and a relatively selective inhibitor of TAK1 kinase activity, with an IC50 of 0.009 mM. AZ-Tak1 treatment decreased the level of p38 and ERK in mantle cell lymphoma cells, and induced apoptosis in a dose and time dependent manner, with an IC50 of 0.1–0.5 mM. Using the annexin-V and PI staining and FACS analysis, After 48 hours of incubation, AZ-Tak1 (0.1 mM) induced apoptosis in 28%, 34% and 86% of Mino, SP53, and Jeko cells, respectively, which was increased to 32%, 42%, and 86% when 0.5 mM concentration was used. Similar activity was also observed when TAK1 expression in MCL cells was downregulated by TAK1- specific SiRNA and when primary mantle cell lymphoma specimens were examined after treatment with AZ-Tak1 for 24h (300nM). Using pathway-specific protein arrays focusing on apoptosis, kinases, and transcription factors, AZ-Tak1 (0.5 mM) altered the level of several proteins that regulate cell growth and survival, especially members of the inhibitors of apoptosis (IAP) family. Specifically, nuclear NF-κB p65 levels were decreased, cytosolic levels of SMAC/DIABLO and cytochrome-C were increased in AZ-Tak1 treated cells, which were associated with a decrease in the level of the anti-apoptotic protein X-linked IAP (XIAP) and activation of the intrinsic apoptotic pathway as evident by activation of caspase 9, cleavage of caspase 3, and consequent cells apoptosis. Collectively, our data demonstrate that TAK1 is essential for MAPK and NF-κB activation. Inhibition of TAK1 by the small molecule inhibitor AZ-Tak1 or TAK1-SiRNA induces cell death in mantle cell lymphoma by activating the intrinsic apoptosis pathway, suggesting that targeting TAK1 may have a therapeutic value for the treatment of mantle cell lymphoma. Disclosures: No relevant conflicts of interest to declare.

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Fibroblast Growth Factor-1 Inhibition Enhances FAS-Induced Apoptosis in Mantle Cell Lymphoma Cells By Accelerated BIRC2/3 Degradation

Blood ◽

10.1182/blood-2018-99-116691 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 2859-2859

Author(s):

Lalit Sehgal ◽

Neeraj Jain ◽

Khashab Tamer ◽

Natalie Willingham ◽

Felipe Samaniego

Keyword(s):

Cell Death ◽

Growth Factor ◽

Mantle Cell Lymphoma ◽

Cell Lymphoma ◽

Cancer Center ◽

Fibroblast Growth ◽

Lymphoma Cells ◽

Autocrine Loop ◽

Mantle Cell ◽

Induced Apoptosis

Abstract Fibroblast growth factor-1 Inhibition Enhances FAS-Induced Apoptosis in Mantle Cell Lymphoma Cells by Accelerated BIRC2/3 Degradation. Neeraj Jain1, Tamer Khashab1, Natalie Willingham2, Felipe Samaniego1 and Lalit Sehgal2 1 Department of Lymphoma/Myeloma, UT MD Anderson Cancer center, Houston, TX 77054 2 Division of Hematology, Comprehensive Cancer Center, The Ohio State University, Columbus, OH, USA Introduction Mantle cell lymphoma (MCL) is an aggressive form of non-Hodgkin lymphoma that is characterized by the t(11:14)(q13:p32) translocation. MCL cells have altered cyclinD1 levels, impaired cell cycle regulation, DNA damage response, and likely defects in apoptosis signaling. Furthermore, up-regulated anti-apoptotic mediators such as the target of NF-κB BIRC2 and BIRC3 were correlated with decreased apoptosis signaling. Also many cancer cells and malignant tumors show a prevalent resistance to apoptosis induction by FAS. Thus, by understanding the underpinnings of apoptosis resistance, we will be in a better position to develop strategies that improve Fas-induced killing of lymphoma cells. Methods More detailed insight into MCL pathogenesis has been delayed until the recent development of a tissue culture system, using human mesenchymal stromal cells (hMSC), suitable for propagating primary MCL cells. Isolates of primary MCL cells were co-cultured with human mesenchymal stem cells (hMSCs) and the content of MCL-ICs was analyzed by flow-cytometry based on marker expression profile; CD34-CD3-CD45+CD19-. Cytokine array was used to identify the soluble factors enriched in the co-cultures and the expression of these factors was confirmed by RT-PCR analysis. The signaling pathways employed by the newly-identified factors were blocked in 3 MCL cell lines (JVM2, Mino, Z138) to confirm their essential role in survival of MCL cells and, more importantly, for MCL-ICs. Results Co-cultures of primary MCL isolates with hMSCs supported the growth of MCL cells for over 4 weeks with continued presence of MCL-ICs (CD34-CD3-CD45+CD19-) representing about 1% of MCL cells. We found that IL-6 produced by hMSCs triggered an FGF/FGFR autocrine loop in MCL-ICs. The extent of FGFR expression correlated tightly with expression of SOX11, a pathology related negative prognostic marker in MCL. MCL cell survival and growth was regulated via the FGFR-1 mediated BIRC2/3 axis. Blocking of this signaling pathway with FGFR-1 inhibitors consistently induced early degradation in BIRC2/3 levels and subsequently MCL cell death. Conclusion We established that propagation of primary MCL in co-cultures with hMSCs depends on activation of FGF/FGFR-1 autocrine loop that enhances BIRC2/3 protein expression and thus, supports survival of MCL cells. We identified the factors essential for survival of MCL and MCL-ICs that present new targets for improved MCL treatment strategies. This study reveals that inhibition of FGFR-1 signaling by specific inhibitor has a profound positive impact on extrinsic cell death signaling; it enhances FAS sensitivity by promoting processing of caspases through enhanced BIRC2/3 degradation. The capacity of FGFR-1 inhibition to target stability of BIRC2/3, underscores its potential for enhancing efficacy of conventional cancer therapies. Disclosures Samaniego: ADC Therapeutics: Research Funding.

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c-FLIP Level Determines the Sensitivity of Mantle Cell Lymphoma Cells to TRAIL-Induced Apoptosis Following Inhibition of NFkB Signalling or Proteasome Activity.

Blood ◽

10.1182/blood.v106.11.4820.4820 ◽

2005 ◽

Vol 106 (11) ◽

pp. 4820-4820

Author(s):

Gael Roue ◽

Patricia Perez Galan ◽

Neus Villamor ◽

Elies Campo ◽

Dolors Colomer

Keyword(s):

Cell Death ◽

Mantle Cell Lymphoma ◽

Cell Lines ◽

Cell Lymphoma ◽

Proteasome Inhibition ◽

Caspase 8 ◽

Synergistic Activity ◽

Mantle Cell ◽

Nfkb Pathway ◽

In Cells

Abstract Mantle cell lymphoma (MCL) is a lymphoproliferative disorder derived from a subset of naive pregerminal center cells with a mature B-cell phenotype and an aggressive course. MCL cells are characterized by the chromosomal translocation t(11;14)(q13;q32) which results in cyclin D1 overexpression, and also present a constitutive activation of the NFkB pathway which leads to the overexpression of several anti-apoptotic regulators. As a consequence, these cells poorly respond to common chemotherapeutic agents acting via the intrinsic mitochondrial pathway. However, recent results indicate that proteasome inhibition represents a promising way to initiate or to potentiate apoptotic cell death in MCL cells, mainly by regulating the levels of several members of the Bcl-2 family implicated in the mitochondrial apoptotic pathway. On the other hand, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a potent activator of the extrinsic cell death pathway and has been shown to exert in vivo a tumoricidal activity via its receptors TRAIL-R1 and -R2, with minimal toxicity on normal cells mainly expressing the inhibitory decoy receptors TRAIL-R3 and -R4. Moreover, proteasome inhibition has been show to increase cancer cells sensitivity to TRAIL, mainly by regulating TRAIL-R1 and TRAIL-R2 membrane level. In this context, our purpose was 1) to assess the sensitivity of MCL cells to recombinant human TRAIL on primary and established MCL cell lines and 2) to potentiate its effects in the less sensitive cells by co-treating cells either with inhibitors of the NFkB pathway, or with the proteasome inhibitor bortezomib. On the 6 MCL cell lines tested, three (Jeko, HBL-2, UPN-1) presented a high sensitivity to TRAIL, two (Rec-1 and Granta-519) were less sensible and one cell line (JVM-2) remained resistant, without apparent correlation to the TRAIL-R1 and -R2 receptors or to the Bcl-2 family protein levels. TRAIL-induced apopotosis was characterised by a time- and dose-dependent loss of membrane potential, Bax and Bak activation, caspase activation and phophatidylserine exposure. In cells with reduced response to TRAIL, we detected a higher transcriptional activity of the TRAIL-R3 vs TRAIL-R1 gene and higher level of the DISC inhibitor c-FLIP, both phenomenons presumably impeding caspase-8 cleavage upon TRAIL treatment. The same observations were done in 3 out of 6 (50%) primary cultures from MCL patients which also harboured a lack of sensitivity to TRAIL. Co-treatment of primary and established MCL cell lines with sub-toxic doses of bortezomib let to the upregulation of the TRAIL-R1 and -R2 agonistic receptors, but also to intracellular accumulation of c-FLIP, presumably impeding synergistic activity of bortezomib and TRAIL in cells with highest c-FLIP basal level. In contrast, the IKK inhibitor BMS-354451 allowed to consistent reduction of NFkB activity, decrease in total and DISC-associated c-FLIP, and sensitization of all MCL cells to TRAIL cytotoxic effects. Indeed, although NFkB inhibition was also associated with slight reduction of TRAIL receptors, BMS-354451 effects were associated with an increased formation of TRAIL-dependent DISC, caspase 8 activation and increase of XIAP-unbound and actived caspase 3. These results indicate that pharmacological enhancement of MCL cells sensitivity to TRAIL does not depend on TRAIL-R1 and -R2 levels but is rather regulated by NFkB-induced anti-apoptotic factors that act at both DISC activation and caspase regulation.

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PRMT5 INHIBITION RESTARTS A PRO‐APOPTOTIC PROGRAM AND CREATES VULNERABILITY TO COMBINATION TREATMENT WITH BCL‐2 INHIBITOR VENETOCLAX IN MANTLE CELL LYMPHOMA

Hematological Oncology ◽

10.1002/hon.143_2880 ◽

2021 ◽

Vol 39 (S2) ◽

Author(s):

F Brown ◽

I Hwang ◽

S Sloan ◽

C Hinterschied ◽

J Helmig‐Mason ◽

...

Keyword(s):

Mantle Cell Lymphoma ◽

Combination Treatment ◽

Cell Lymphoma ◽

Mantle Cell

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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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