Resistance In Mantle Cell Lymphoma Research Articles

215: Activated B cell receptor signaling pathway contributes to bortezomib resistance in mantle cell lymphoma

215: Activated B cell receptor signaling pathway contributes to bortezomib resistance in mantle cell lymphoma

Synergistic antitumor activity of lenalidomide with the BET bromodomain inhibitor CPI203 in bortezomib-resistant mantle cell lymphoma.

Bortezomib therapy has shown promising clinical activity in mantle cell lymphoma (MCL), but the development of resistance to proteasome inhibition may limit its efficacy. To unravel the factors involved in the acquisition of bortezomib resistance in vivo, immunodeficient mice were engrafted with a set of MCL cell lines with different levels of sensitivity to the drug, followed by gene expression profiling of the tumors and functional validation of the identified gene signatures. We observed an increased tumorigenicity of bortezomib-resistant MCL cells in vivo, which was associated with plasmacytic differentiation features, like interferon regulatory factor 4 (IRF4) and Blimp-1 upregulation. Lenalidomide was particularly active in this subgroup of tumors, targeting IRF4 expression and plasmacytic differentiation program, thus overcoming bortezomib resistance. Moreover, repression of the IRF4 target gene MYC in bortezomib-resistant cells by gene knockdown or treatment with CPI203, a BET (bromodomain and extra terminal) bromodomain inhibitor, synergistically induced cell death when combined with lenalidomide. In mice, addition of CPI203 to lenalidomide therapy further decreased tumor burden, involving simultaneous MYC and IRF4 downregulation and apoptosis induction. Together, these results suggest that exacerbated IRF4/MYC signaling is associated to bortezomib resistance in MCL in vivo and warrant clinical evaluation of lenalidomide plus BET inhibitor combination in MCL cases refractory to proteasome inhibition.

BMP7 Expression Correlates with Secondary Drug Resistance in Mantle Cell Lymphoma

PurposeWe designed a gene profiling experiment to identify genes involved in secondary drug resistance in mantle cell lymphomas (MCL).Experimental DesignWe obtained paired tissue samples collected from the same patients before treatment and after relapse or progression. Variations in gene expression between the 2 samples were estimated for 5 patients. For each gene, the mean variation was estimated for patients with a refractory primary tumor and for responders who developed secondary drug resistance. Nine genes of interest were selected on the basis of the magnitude and statistical significance of the variation of expression in responders and non-responders.ResultsBMP7 was the only one with significantly increased expression at relapse in patients who developed secondary resistance. Validation of BMP7 as a key gene involved in secondary resistance was performed using cultures of cell line. Incubation of BMP7 with MCL cell lines increased their resistance to bortezomib and cytarabine, while inhibition of BMP7 expression by siRNA correlated with increased cell death linked to drug application.ConclusionVariations in gene expression after treatment point out BMP7 as a key gene involved in secondary resistance in mantle cell lymphoma.

Abstract 3389: Nuclear accumulation of c-Rel transcription factor is correlated with the proteasome inhibitor resistance in mantle cell lymphoma.

Abstract Purpose: Nuclear factor kB (NF-kB) plays essential roles in survival and drug resistance of multiple malignancies including mantle cell lymphoma (MCL) and multiple myeloma (MM). Proteasome inhibitors have been shown to block constitutively active NF-kB and clinical data indicates a high overall response of MCL and MM patients to this treatment. The 2nd generation proteasome inhibitor, carfilzomib (CFZ), has a therapeutic effect on relapsed or refractory disease and low toxicity to normal lymphocytes. However, there are increasing numbers of proteasome inhibitor-resistant cases. Thus, this study was designed to identify the molecular mechanism that leads to CFZ-resistance. Experimental design: In this study we have determined the cytotoxic effect of CFZ in a panel of MM and MCL cell lines, as well as primary tumor cells. The cell growth inhibition was assessed by the thymidine intake and MTT assay. NF-kB activity was examined by electrophoretic mobility shift assay (EMSA) using nuclear extracts from CFZ-treated and control cells. Nuclear pattern of NF-kB family members, such as RelA/p50, cRel/p50 and RelB/p52 complexes, was analyzed by supershift and western blot. Results: CFZ-induced NF-kB inhibition was not observed in two (out of eight) examined cell lines, even with the highest doses (100 nM). Consistently, cell growth was suppressed only in cells with a CFZ-sensitive NF-kB activity (IC50<20 nM), as shown by EMSA and MTT assay. In contrast, the treatment with proteasome inhibitor increased the nuclear accumulation and DNA binding of NF-kB in CFZ-resistant cells. Furthermore, we have found that only these cells had upregulated c-Rel/p50 member of NF-kB family. Supershift analysis revealed that all examined cell lines and primary cells had constitutively active classical (RelA/p50 dimer) and/or non-classical (RelB/p52 dimer) NF-kB, excluding CFZ-resistant cells with prominent nuclear accumulation of c-Rel. This data suggests that activated c-Rel may trigger resistance to CFZ. Enhanced nuclear accumulation of c-Rel is also an unwanted molecular consequence of exposure to CFZ. Conclusion: Our work indicates the need for new treatment strategies in c-Rel-positive cases. A strategic combination targeting different signaling mediators, especially upstream of c-Rel, would enhance the ultimate anticancer effect. Citation Format: Marzenna Blonska, Deng Pan, Zhishuo Ou, Liang Zhang, Xin Lin, Michael Wang. Nuclear accumulation of c-Rel transcription factor is correlated with the proteasome inhibitor resistance in mantle cell lymphoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3389. doi:10.1158/1538-7445.AM2013-3389

Abstract 2432: The bruton's tyrosine kinase inhibitor ibrutinib synergized with the proteasome inhibitor carfilzomib and overcame immunoproteasome-mediated carfilzomib resistance in mantle cell lymphoma .

Abstract Introduction: Mantle cell lymphoma (MCL) is not curable and novel therapeutic agents and their rational combinations are needed. The ubiquitin-proteasome pathway is an attractive target for antitumor therapy, as its persistent activity is associated with tumor formation, growth, metastasis, and drug resistance in many cancer types, including B-cell non-Hodgkin lymphoma. 20S proteasome activity is regulated by the catalytic subunits β1, β2, and β5 or in the case of the immunoproteasome (i20S) by the inducible catalytic subunits LMP2 (β1i), MECL-1(β2i), and LMP7 (β5i). Carfilzomib (CFZ), a proteasome inhibitor, inhibits the proteasome by binding specifically and irreversibly to the catalytic subunit β5/β5i. Materials and Methods: CFZ was supplied by Onyx Pharmaceuticals (South San Francisco, CA). The Btk inhibitor ibrutinib (PCI-32765) was provided by Pharmacyclics (Sunnyvale, CA). Human MCL cell lines (Mino, Jeko-1, Z138, and Rec-1) were either created by our laboratory or obtained from American Type Culture Collection. Primary cells were obtained from newly diagnosed MCL patients with informed consent. Cell viability was assessed by an MTT assay, apoptosis by Annexin V binding assays, and proteasome expression by Western blotting. Results: We found that MCL cells had different responses to CFZ. For the CFZ-sensitive MCL cell lines Mino and Z138 and freshly isolated MCL cells from 6 patients, CFZ significantly induced the growth inhibition and apoptosis at a low dose (IC50 2-6 nM). In contrast, for the CFZ-resistant MCL cell line (Rec-1) and fresh primary MCL cells from patients with clinical resistance, CFZ did not induce cell death (IC50 not reached at 100 nM). Next, Western blot analysis showed that the CFZ-resistant cells lacked expression of the i20S subunits LMP2, LMP7, and MECL-1. By contrast, the CFZ-sensitive cells showed high levels of LMP2, LMP7, and MECL-1. These results suggest that an intact immunoproteasome is necessary for CFZ-induced anti-MCL activity. In an attempt to overcome the CFZ resistance, we studied the effect of ibrutinib in CFZ-resistant MCL cells. Ibrutinib alone was toxic to both CFZ-sensitive and CFZ-resistant MCL cells with IC50 values ranging from 3 to 12.5 μM. Furthermore, we found that ibrutinib synergized with CFZ in CFZ-sensitive cell lines, and primary MCL cells. The Chou-Talay combination index was < 1, indicating synergism between the two drugs. Most importantly, the CFZ-resistant MCL cells Rec-1 were sensitive to ibrutinib, with an IC50 of 6 uM. Conclusion: Our data suggest that LMP2, LMP7, and MECL-1 may serve as potential biomarkers for patients who are likely to be sensitive to CFZ. Our data also suggest that ibrutinib may be useful in patients with CFZ-resistant MCL. Our preclinical data provide a strong rationale for testing the ibrutinib-CFZ combination clinically. Citation Format: Zhishuo Ou, Liang Zhang, Kate Newberry, Luhong Sun, Kirk J. Christopher, Alex Rollo, Archito Tamayo, John Lee, Richard J. Ford, Michael Wang, Lan V. Pham. The bruton's tyrosine kinase inhibitor ibrutinib synergized with the proteasome inhibitor carfilzomib and overcame immunoproteasome-mediated carfilzomib resistance in mantle cell lymphoma . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2432. doi:10.1158/1538-7445.AM2013-2432

Abstract 4944: Combined treatment of BTK and PI3K inhibitors synergistically disrupts BCR-signaling, overcomes microenviroment-mediated survival and drug resistance in mantle cell lymphoma.

Abstract Mantle cell lymphoma (MCL) is recognized as an aggressive lymphoma characterized by overexpression of cyclin D1 as a result of t (11:14) translocation. It carries poor prognosis due to the emergence of drug resistance and currently no effective cure is available for this disease. Dynamic interactions between the lymphoma cell and its microenvironment play a critical role in lymphoma development and response to therapy. Recently, inhibitors of BCR signaling have become an area of substantial clinical interest. B-cell receptor (BCR) provides essential growth and survival signals to lymphomas. We therefore studied the role of BCR signal pathways in stroma mediated survival and drug resistance of mental cell lymphoma. We demonstrate that all mantle cell lymphoma cell lines and primary MCL cells express key BCR downstream signaling molecules, BTK (Bruton's tyrosine kinase), AKT and ERK, in activated (phosphorylated) form. Phosphorylation statutes of pBTK, pAKT and pERK are further up regulated upon adhesion to lymph node stroma cells. Cell adhesion between lymphoma cells and tumor stroma cells is essential for tumor cell survival and drug resistance. Inhibition of BTK by PCI 32765 or PI3Kδ by CAL101 abolishes intrinsic as well as stroma induced activation of BTK, AKT, ERK. It also significantly disrupts tumor - stromal cell adhesion and induces cell apoptosis. Moreover, combined treatment of PCI 32765 and CAL101 synergetically inhibit BCR signaling and induce cell apoptosis in absence and presence of HK stroma cells. CXCR4 has been reported to play a critical role in lymphoma cell homing, migration, and adhesion. We evaluated CXCR4 expression upon MCL adhesion to stroma cells. We demonstrate that the CXCR4 level was significantly increased in Jeko-1 and HBL2 cell lines and primary MCL cells when co-cultured with HK stroma cells. The stroma cells-induced CXCR4 increase was significantly abolished by BCR signaling inhibitors, PCI 32765 and Cal101. We further explored the effect of those inhibitors on cyclin D1. We show that cyclin D1 is constitutively expressed in MCL cell lines and primary MCL cells and that its expression was further increased when co-cultured with HK stoma cells. Both PCI 32765 and Cal101 decreased intrinsic cyclin D1 expression in the absence and presence of HK cells. Furthermore, combined treatment of PCI 32765 and Cal101 more strikingly attenuated cyclin D1 expression than either agent alone. Collectively, Our study demonstrates (1) the constitutive expression and biological function of BCR signaling in MCL, (2) the essential role of BCR signaling in microenvironment (stroma cell) mediated MCL survival, adhesion and drug resistance, and (3) the therapeutic potential of using novel BCR inhibitors, PCI 32765 and Cal101, in single or combine for MCL therapy. Citation Format: Jiangchuan Tao, Tint Lwin, Xiaohong Zhao, Bijal Shah, Ling Zhang, Lynn Moscinski, William Dalton, Eduardo M. Sotomayor, Jianguo Tao. Combined treatment of BTK and PI3K inhibitors synergistically disrupts BCR-signaling, overcomes microenviroment-mediated survival and drug resistance in mantle cell lymphoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4944. doi:10.1158/1538-7445.AM2013-4944

Evaluation of the Anti-Tumor Activity of MLN4924, A Novel NEDD8 Activating Enzyme Inhibitor, in Pre-Clinical Models of Rituximab Chemotherapy-Sensitive or -Resistant B-Cell Lymphoma.

AbstractAbstract 2761Clinical outcome of patients with relapsed/refractory diffuse large B-cell lymphoma (DLBCL) and mantle cell lymphoma (MCL) remains poor with currently available therapies. Recently, the ubiquitin-proteasome system (UPS) appears to play an important role in the development of resistance in MCL and some subtypes of DLBCL. Targeting UPS represents a rational approach in an attempt to eradicate drug-resistant lymphoma clones. MLN4924 is a novel, potent and selective inhibitor of the NEDD8-activating enzyme (NAE) that is necessary for the modification of cullin-RING ubiquitin ligases. We evaluated the anti-tumor activity of MLN4924 against a panel of rituximab-sensitive (RSCL) or rituximab/chemotherapy–resistant (RCRCL) DLBCL and Burkitt lymphoma cell lines, cytarabine-sensitive or -resistant (AraCR) MCL cell lines, and primary tumor cells freshly isolated from lymphoma patients (n=13). Lymphoma cells were exposed to escalating doses of MLN4924 alone or in combination with selected chemotherapy agents for up to 72 hrs. Changes in the cell viability or ATP content were determined by alamar Blue reduction or CellTiterGlo assays, respectively. Induction of apoptosis and changes in the levels of NFkB and UPS regulatory proteins were analyzed by Western blotting. Cell cycle alterations were determined by propidium iodide staining and NFkB activity was quantified by flow cytometry using the Imagestream technology. MLN4924 demonstrated time- and dose-dependent anti-lymphoma activity in all cell lines tested. The IC50 in RSCLs were Raji=400nM, RL=1uM and U2932=>3uM. All RCRCLs were less responsive to MLN4924 as a single agent with IC50 concentrations 4–10× those of their respective sensitive parental cell lines. The MCL cell lines Mino, MinoAraCR, Z-138, HBL-2 and HBL-2AraCR were most sensitive to MLN4924 anti-tumor effects (IC50=250nM) with no significant difference between cytarabine-sensitive and -resistant cell lines; while the MCL cell lines Rec-1, Rec-1AraCR, Jeko-1 and Jeko-1AraCR were less sensitive (IC50=500–1000nM). A variable degree of anti-tumor activity was also observed in primary lymphoma cells. In addition to single-agent activity, MLN4924 plus selected anti-lymphoma chemotherapy agents (bortezomib, bendamustine and cytarabine) demonstrated synergy in cytarabine-sensitive and (to a lesser degree) cytarabine-resistant MCL cell lines. Combinations with additional chemotherapeutic agents (doxorubicin and vincristine) resulted in additive effects. Exposure of MCL cells to MLN4924 resulted in G1 cell cycle arrest. In vitro exposure of the more sensitive MCL cell lines Mino and MinoAraCR to MLN4924 resulted in an increase in p-IkBα and down-regulation of both total and nuclear NFkB. The less sensitive cell lines Rec-1 and Rec-1AraCR demonstrated little to no change in NFkB activation following exposure to MLN4924. Additional studies are ongoing to further define the molecular mechanisms of the anti-tumor activity observed following NAE inhibition by MLN4924 in these pre-clinical models and to further evaluate the activity of MLN4924 in in vivo SCID mouse models of B-cell lymphoma. Our data suggests that MLN4924, a novel NAE inhibitor, is active against B-cell lymphomas, particularly MCL, and is a promising agent warranting further investigation in relapsed/refractory aggressive B-cell lymphomas. Disclosures:No relevant conflicts of interest to declare.

Targeting Mir-548m-HDAC6 Axis Circumvents Stroma- Mediated Drug Resistance (CAM-DR) and Clonogenicity in Non-Hodgkin B-Cell Lymphomas

AbstractAbstract 5094A dynamic interaction occurs between the lymphoma cell and its microenvironment, with each profoundly influencing the behavior of the other. Here, we demonstrate that adhesion of mantle cell lymphoma (MCL) and other non-Hodgkin lymphoma cells to lymph node stromal cells, follicular dendritic cells (FDCs), confers drug resistance, enhances lymphoma cell clonogenicity, and is associated with induction of histone deacetylase 6 (HDAC6). Furthermore, stroma down-regulated miR-548m contributes to HDAC6 up-regulation, and HDAC6 is a key determinant for FDC-mediated lymphoma cell survival and colony formation. We further showed that stroma-mediated drug resistance and clonogenic growth are reversed by enforced expression of miR-548m and inhibition of HDAC6. The HDAC6-selective inhibitor tubastatin significantly enhances cell death, abolishes cell adhesion-mediated drug resistance, and suppresses clonogenicity and lymphoma growth suppression ex vivo and in vivo. Together, these data suggest that the lymphoma–stroma interaction in lymph node microenvironment directly impacts the biology of lymphoma through epigenetic regulation of miRNA and HDAC, with HDAC6 as a potential therapeutic target to overcome drug resistance in MCL and other B-cell lymphomas. Disclosures:No relevant conflicts of interest to declare.

Abstract 3364: Calcium blockers decrease the bortezomib resistance in mantle cell lymphoma (MCL) via manipulation of tissue transglutaminase activities.

Abstract Although bortezomib is clinically approved for the treatment of Mantle Cell Lymphoma (MCL), only limited effects have been demonstrated. To improve outcomes for bortezomib resistant patient groups, it is necessary to introduce the new therapeutic strategies. In this report, we utilized biochemical and molecular methodologies to demonstrate tissue transglutaminase (TG) activates downstream NF-kB signaling pathways. The signaling axis from TG to NF-kB could be a new therapeutic target to overcome bortezomib resistance in MCL. TG2 is a calcium-dependent protein crosslinking enzyme, and was reported to over-expressed in various cancer cells. MCL expressed elevated levels of TG2, and the modification of TG2 activities altered NF-κB expression and downstream signaling in MCL cells. When the TG2 signaling was inhibited via calcium blockers, the combination of a calcium blocker perillyl alcohol (POH) with bortezomib suppressed NF-κB expression and improved the cytotoxicity of bortezomib in MCL cells. Our study is the first to show the expression of TG2 and the contribution of TG2 to NF-κB signaling in MCL. TG2 inhibition can be an alternative target as anti-MCL therapy, and calcium blockers could be effective combinatory drugs with bortezomib to overcome the bortezomib resistance of MCL. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3364. doi:1538-7445.AM2012-3364

Calcium blockers decrease the bortezomib resistance in mantle cell lymphoma via manipulation of tissue transglutaminase activities

AbstractAlthough bortezomib is clinically approved for the treatment of mantle cell lymphoma (MCL), only limited effects of this treatment have been demonstrated. To improve survival for bortezomib-resistant patients, it is necessary to develop new therapeutic strategies. In the present study, we used biochemical and molecular methodologies to demonstrate that tissue transglutaminase (TG) activates downstream NF-κB signaling pathways. The signaling axis from TG to NF-κB could be a new therapeutic target to overcome bortezomib resistance in MCL. TG2 is a calcium-dependent protein cross-linking enzyme reported to be overexpressed in various cancer cells. We found that MCL cells expressed elevated levels of TG2 and that the modification of TG2 activities altered NF-κB expression and downstream signaling in MCL cells. When TG2 signaling was inhibited by calcium blockers, the combination of a calcium blocker (perillyl alcohol) with bortezomib suppressed NF-κB expression and improved the cytotoxicity of bortezomib in MCL cells. Our study is the first to show the expression of TG2 and the contribution of TG2 to NF-κB signaling in MCL. TG2 inhibition may be used as an alternative target anti-MCL therapy, and calcium blockers may be combined with bortezomib to overcome the bortezomib resistance in MCL.

High-Resolution Sequencing Identifies NOXA1 De-Methylation As a Novel Strategy to Overcome Bortezomib Resistance in Mantle Cell Lymphoma

Abstract 557Mantle cell lymphoma (MCL) is an aggressive B-lymphoid neoplasm with frequent relapses after initial response to standard chemotherapy. Bortezomib is the first proteasome inhibitor with promising clinical efficacy in relapsed MCL, where it achieved durable responses in 30%–50% of patients. The mechanisms contributing to Bortezomib resistance remain poorly understood. Our group and others have identified aberrantly methylated genes in MCL controlling critical cellular processes like cell cycle, transcription and regulation of gene expression. We therefore hypothesized that a subset of aberrantly methylated genes may be contributing to Bortezomib resistance.Using a massively parallel sequencing approach, HELP-tagging, we analyzed genomic methylation in MCL patients treated in a Phase II trial at the NIH. This high-resolution technique covers about 1.8 million CpGs in the human genome including gene promoters, gene bodies and CpG shores and is more sensitive and yields greater coverage of the genome as compared to array-based platforms. Unsupervised analysis of genome-wide promoter methylation of Bortezomib sensitive patients (having more than 50% reduction in absolute lymphocyte count after one cycle of Bortezomib) as compared to resistant patients revealed that methylation patterns could independently distinguish between these two groups. Similarly, three-dimensional Principal Component Analysis also clustered Bortezomib resistant patients distinctly from Bortezomib sensitive patients, confirming genomic differences in methylation between these patients. We further analyzed the differentially methylated genes using pathway analysis software, gene ontology and gene set enrichment analysis. We found that majority of differentially methylated genes were relevant to cancer biology involving metabolic pathways, regulation of transcription, cell cycle and programmed cell death. Comparing methylation profiles of patient samples taken before and 96 hours after Bortezomib treatment showed a striking genome-wide hypomethylation following Bortezomib. Western blotting of MCL cell lines showed a significant reduction in DNMT1 levels following Bortezomib treatment, which may help us understand the post-treatment hypomethylation seen in patients.NOXA1 is a pro-apoptotic Bcl-2 family member essential for the cytotoxicity of Bortezomib. The NOXA1 gene promoter was differentially methylated in Bortezomib-sensitive MCL patients. NOXA1 was also significantly hypomethylated to a greater extent 96 hours following Bortezomib treatment in Bortezomib sensitive patients. NOXA1 hypomethylation was associated with induction of NOXA1 mRNA and protein expression in sensitive patients alone. These findings suggest a possible role for dynamic NOXA1 methylation and expression as biomarkers for Bortezomib sensitivity.Treatment of MCL cell lines Z138 and MINO with demethylating agents Decitabine also induced NOXA1 hypomethylation and increased NOXA1 protein expression. Low dose (0.1-0.5μM) Decitabine pretreatment potentiated low (IC25) dose Bortezomib causing more than 80–90% cell kill in MINO and Z138 cells. The combination of Decitabine and Bortezomib led to increased NOXA1 hypomethylation and protein expression as compared to the individual drugs. Depletion of NOXA1 by specific shRNA rescued 40–50% of the cells from the cytotoxicity of this combination. Tumor progression and chemoresistance are commonly associated with the overexpression of the prosurvival Bcl-2 family members (Bcl-2, Bcl-xL, and Mcl-1). ABT-263 is a small molecular inhibitor of the Bcl-2 family of proteins, excepting Mcl-1, which can be neutralized by an increase in NOXA1. Decitabine treatment also potently enhanced the cytotoxicity of ABT-263 in MCL cell lines.Our data suggest that inducing hypomethylation of NOXA1 promoter can be a novel therapeutic strategy for overcoming Bortezomib resistance in MCL, and that genomic methylation patterns and specifically NOXA1 hypermethylation may be useful biomarkers for Bortezomib response. More broadly, these data demonstrate that high-resolution genomic methylation profiling can identify genes and signatures with important prognostic and therapeutic implications. Disclosures:No relevant conflicts of interest to declare.

Bortezomib-resistant nuclear factor κB expression in stem-like cells in mantle cell lymphoma

Mantle cell lymphoma (MCL) is a subtype of B-cell Non-Hodgkin's Lymphoma (NHL) and accounts for approximately 6% of all lymphomas. Unlike small lymphocytic lymphoma and chronic lymphocytic lymphoma, which are relatively sensitive to chemotherapy, MCL is highly refractory to most chemotherapy, and has the worst survival rate among NHL patients. Stem-like cells in MCL, which we have termed mantle cell lymphoma-initiating cells (MCL-ICs), enriched in the population that are lack of prototypic B-cell marker CD19. These cells were able to self-renew upon serial transplantation and are highly tumorigenic. Importantly, these stem-like cells confer chemotherapeutic resistance to MCL. In this report, we show that stem-like MCL-ICs are resistant to bortezomib, as well as chemotherapeutic regimens containing bortezomib, despite constitutive nuclear factor-κB (NF-κB) expression. Interestingly, bortezomib treatment induced MCL-IC differentiation in plasma-like cells with upregulated expression of CD38 and CD138. This process was accompanied by expression of plasma cell differentiation transcriptional factors, BLIMP-1 and IRF4. This article is the first to show that stem-like MCL cells utilize constitutive NF-κB expression for survival. Given that the NF-κB expression in MCL-ICs is resistant to bortezomib, it will be important to find alternative therapeutic strategies to inhibit NF-κB expression.

The Hsp90 inhibitor IPI-504 overcomes bortezomib resistance in mantle cell lymphoma in vitro and in vivo by down-regulation of the prosurvival ER chaperone BiP/Grp78

AbstractDespite the promising introduction of the proteasome inhibitor bortezomib in the treatment of mantle cell lymphoma (MCL), not all patients respond, and resistance often appears after initial treatment. By analyzing a set of 18 MCL samples, including cell lines with constitutive or induced resistance to bortezomib, we found a high correlation between loss of sensitivity to the proteasome inhibitor and up-regulation of the prosurvival chaperone BiP/Grp78. BiP/Grp78 stabilization was ensured at a posttranscriptional level by an increase in the chaperoning activity of heat shock protein of 90 kDa (Hsp90). In bortezomib-resistant cells, both BiP/Grp78 knockdown and cell pretreatment with the Hsp90 inhibitor of the ansamycin class, IPI-504, led to synergistic induction of apoptotic cell death when combined with bortezomib. Cell exposure to the IPI-504–bortezomib combination provoked the dissociation of Hsp90/BiP complexes, leading to BiP/Grp78 depletion, inhibition of unfolded protein response, and promotion of NOXA-mediated mitochondrial depolarization. The IPI-504–bortezomib combination also prevented BiP/Grp78 accumulation, thereby promoting apoptosis and inhibiting the growth of bortezomib-resistant tumors in a mouse model of MCL xenotransplantation. These results suggest that targeting unfolded protein response activation by the inhibition of Hsp90 may be an attractive model for the design of a new bortezomib-based combination therapy for MCL.

Bortezomib resistance in mantle cell lymphoma is associated with plasmacytic differentiation

AbstractBortezomib induces remissions in 30%-50% of patients with relapsed mantle cell lymphoma (MCL). Conversely, more than half of patients' tumors are intrinsically resistant to bortezomib. The molecular mechanism of resistance has not been defined. We generated a model of bortezomib-adapted subclones of the MCL cell lines JEKO and HBL2 that were 40- to 80-fold less sensitive to bortezomib than the parental cells. Acquisition of bortezomib resistance was gradual and reversible. Bortezomib-adapted subclones showed increased proteasome activity and tolerated lower proteasome capacity than the parental lines. Using gene expression profiling, we discovered that bortezomib resistance was associated with plasmacytic differentiation, including up-regulation of IRF4 and CD38 and expression of CD138. In contrast to plasma cells, plasmacytic MCL cells did not increase immunoglobulin secretion. Intrinsically bortezomib-resistant MCL cell lines and primary tumor cells from MCL patients with inferior clinical response to bortezomib also expressed plasmacytic features. Knockdown of IRF4 was toxic for the subset of MCL cells with plasmacytic differentiation, but only slightly sensitized cells to bortezomib. We conclude that plasmacytic differentiation in the absence of an increased secretory load can enable cells to withstand the stress of proteasome inhibition. Expression of CD38 and IRF4 could serve as markers of bortezomib resistance in MCL. This study has been registered at http://clinicaltrials.gov as NCT00131976.

Overcoming Drug Resistance in Mantle Cell Lymphoma Using a Combination of Dose-Dense and Intense Therapy

ABSTRACTWe present a study of the prevalence of genetic polymorphisms and expression of genes encoding the drug-resistance proteins glutathione S-transferases (GSTs) in order to gain insights into the pattern of failure evident in mantle cell lymphoma. We note a high preponderance of genetic alterations conferring resistance to standard chemotherapy in this illness. Concurrent with this investigation, we present a series of patients who were provided dose-dense and intense chemotherapy to circumvent these drug-resistance mechanisms. High responses were noted, though durable remissions were few, indicating non-traditional chemotherapy options are important to investigate in this illness.

Bortezomib Resistance in Mantle Cell Lymphoma Is Associated with Expression of a Plasmacytoid Differentiation Program.

Abstract 287Mantle cell lymphoma (MCL) is a lymphoproliferative disorder of mature B-cells with an aggressive course and short survival. The proteasome inhibitor bortezomib (BZM) induces clinical responses in up to 50% of patients. Conversely, in half of the cases the lymphoma cells are intrinsically resistant or rapidly develop resistance to BZM. To investigate the mechanisms of BZM resistance, we generated HBL2 and JEKO bortezomib resistant (HBL2-BR, JEKO-BR) derivative lines by continuous culture in sub-lethal concentrations of BZM. After several months, clones of HBL2-BR and JEKO-BR were obtained showing BZM IC50 at 48h of 41.6 and 44.6 nM, compared to 6 and 4.9 nM for the respective parental lines. Acquired resistance to BZM remained stable over months but gradually decreased with extended passages in the absence of BZM, suggesting adaptive changes rather than a single gene mutation as the basis of BZM resistance. BR cells exhibited higher proteasome activity, which was dose-dependently inhibited by higher concentrations of BZM. However, BR cells were able to survive with lower proteasome activity than the parental cells, indicating that BR cells had acquired additional changes. To investigate these changes, we use gene expression profiling (GEP) on Affymetrix U133A plus 2 arrays to compared HBL2-BR (in triplicate) and JEKO-BR (in duplicate) subclones to the corresponding parental lines. Unexpectedly, Gene Set Enrichment Analysis (GSEA) of microarray data revealed reduced expression of the mature B-cell gene signature (including genes for CD19, BLNK, SPIB, SYK) and increased expression of plasma cell differentiation signatures (including genes for CD38, IRF4, BLIMP, CD138) in both HBL-2 BR and JEKO-BR. BR lines also expressed higher protein levels of the master plasma cell regulators BLIMP and IRF4, but did not show enhanced expression of the secretory program controlled by XBP1. Flow cytometry analysis confirmed that BR cells had dramatically reduced expression of B-cell surface markers, including CD19, CD24 and CD52, and expressed plasma cell markers, such as CD38 and CD138. Consistent with a partial plasmacytoid phenotype, BR cells tended to be somewhat larger and more granular than parental cells. Loss of BZM resistance over months of culture in the absence of BZM was paralleled by the recovery of CD19 and CD24 expression and down-regulation of CD38, supporting a mechanistic link between the acquisition of a plasmacytoid phenotype and BZM resistance. We have previously shown that the MCL cell lines Mino and REC-1 are less sensitive to BZM than HBL-2, JEKO and most other MCL cell lines. Here we found that these constitutively resistant cells also showed plasmacytoid features including CD38 and CD138 surface expression, increased granularity and size, and an enlarged endoplasmic reticulum (ER). Combined these changes may enhance the ability of the cells to deal with an increased protein load due to bortezomib inhibition. In addition, we also observed higher expression of IRF4 and its target genes in the constitutively resistant cells, as well as higher IRF4 and CD38 expression in primary tumor cells of patients with poor response to BZM. Given the important role of IRF4 as a survival factor in multiple myeloma, we tested whether BZM treatment could decrease IRF4 expression in MCL cells. Indeed, within 24 hours BZM dose-dependently decreased IRF4 expression and the degree of downregulation of IRF4 correlated with the induction of apoptosis. Knockdown of IRF4 expression by shRNA has been shown to be toxic to myeloma cells (Shaffer et al, Nature 2008). Surprisingly, we found a similar toxic effect of IRF4 knockdown using the same inducible shRNA system in the MCL cell lines HBL2, JEKO and REC, which was more prominent in the latter BZM resistant cell line. These results identify loss of IRF4 expression as an additional mechanism by which BZM may induce cell death. However, overexpression of IRF4 in MCL cells is not sufficient to induce bortezomib resistance, indicating that several components of the plasma cell program cooperate to protect cells from BZM induced apoptosis. Furthermore, we have identified markers of BZM resistance that may be clinically relevant predictors of outcome. Disclosures:No relevant conflicts of interest to declare.

Selective Inhibition of IκB Kinase Sensitizes Mantle Cell Lymphoma B Cells to TRAIL by Decreasing Cellular FLIP Level

In an attempt to circumvent the intrinsic resistance of mantle cell lymphoma (MCL) cells to apoptosis, we have analyzed their sensitivity to the extrinsic apoptotic signal triggered by TRAIL. We show here that TRAIL can trigger apoptosis in a majority of MCL cell lines and primary cultures, irrespective of receptor levels, Bcl-2 family members, or caspase regulator expression. MCL sensitivity to TRAIL was closely linked to the activity of the NF-kappaB p50 factor and to the consequent expression of cellular FLIP (c-FLIP), which accumulated into the TRAIL-dependent complex in resistant cells. c-FLIP transient knockdown overcame MCL resistance to TRAIL, while NF-kappaB inhibitors differentially modulated TRAIL cytotoxicity. Indeed, bortezomib increased TRAIL cytotoxic effects in sensitive cells, but led to the intracellular accumulation of c-FLIP, impeding full synergistic interaction. In contrast, the IkappaB kinase inhibitor BMS-345541 led to decreased c-FLIP expression and allowed all MCL samples to undergo TRAIL-mediated apoptosis. These results present the combination of TRAIL stimulation and IkappaB kinase inhibition as a new approach to MCL therapy.

Resistance to Bortezomib Develops Slowly in MCL Cells, Extends to the Class of Proteasome Inhibitors, and Is Associated with Decreased Proliferation of the Resistant Cells.

Mantle Cell Lymphoma (MCL) is a malignancy of mature B-cells. MCL has a poor prognosis and a limited response to traditional chemotherapy. Bortezomib (BZM), a new powerful inhibitor of the proteasome, can induce responses in up to 50% of relapsed MCL patients, suggesting that in at least half of the patients the lymphoma cells are intrinsically resistant to BZM or rapidly develop resistance during single agent therapy. To investigate possible mechanisms of BZM resistance, we cultured MCL cell lines continuously in sub-lethal concentrations of BZM that were then gradually increased. Resistance was slow to develop taking several months for truly resistant clones to grow out. We generated a bortezomib resistant (BR) clone of HBL-2 with an IC50 of 30nM compared to 5nM in the parental clone and several BR subclones of Jeko-1, the most resistant of which had an IC50 of 200nM compared to 3nM for the parental clone. All BR subclones also showed decreased sensitivity to three other proteasome inhibitors: MG-132, Lactacystin, and NLVS. The increase in IC50 to these drugs was between 3 and 8-fold, consistent with more off-target effects of these drugs compared to BZM. BAY11-7082, an inhibitor of NF-kB signaling, maintained its activity against the resistant cells. Resistance to BZM, once acquired, has remained stable over several months. This is remarkable because the resistant subclones grow significantly slower than the parental lines, even after having been removed from selection for extended periods of time. Consistent with slower cell proliferation, we found reduced Cyclin D1 protein expression in the BZM resistant Jeko clones; however, mRNA levels were comparable to the parental line, indicating that changes in Cyclin D1 protein translation and/or stability may be responsible for the decreased proliferation. BZM resistance has been associated with up-regulation of proteasome components and heat-shock proteins. Indeed, in the resistant HBL-2 subclone we found marked upregulation of two proteasome components (PSMA5 and PSMC1) and of Hsp70 by RT-PCR, but there was only a small change in Hsp70 protein expression. Nevertheless, upregulation of these genes could be part a more global gene expression response as seen with ER-stress and could thus reflect an adaptive change to BZM in the HBL-2 BR subclone. All three Jeko BR clones in contradistinction showed only minor changes in PSMA5, PSMC1 and Hsp70 mRNA expression and surprisingly had markedly reduced Hsp70 protein levels. Thus, in these subclones, BR resistance appears to correlate primarily with changes affecting cell cycle control. We conclude that resistance to BZM may be determined by several mechanisms that affect cell cycle control as well as expression of proteasome components and heat-shock proteins. While the slow development of resistance suggests adaptive changes, its persistence over time is more consistent with mutations or other genomic alterations that are not readily reversible. Ongoing studies aim to more precisely define the basis for BZM resistance in MCL.

Selective Inhibition of I kappaB Kinase Sensitizes Mantle Cell Lymphoma B Cells to TRAIL by Decreasing c-FLIP Level.

Mantle cell lymphoma (MCL) is an aggressive B lymphoid neoplasm with a mature B-cell phenotype and genetically characterized by the t(11;14)(q13;q32) leading to cyclin D1 overexpression with the consequent deregulation of cell cycle at the G1-S checkpoint. MCL cells also present a constitutive activation of the NF-kappaB pathway which leads to the overexpression of several anti-apoptotic regulators. We have analyzed sensitivity to the extrinsic apoptotic signal triggered by tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) on six human MCL cell lines and primary cells from 10 MCL patients, which differ in their p53-dependent pathway status, growth characteristics and sensitivity to cytotoxic drugs. TRAIL has been shown to exert in vivo a selective anti-tumor activity with minimal toxicity on normal cells. We observed that TRAIL was able to trigger apoptosis in a majority of MCL cell lines and primary MCL tumor cells, while sparing normal peripheral B cells. TRAIL-induced cell death was characterized by a time- and dose-dependent loss of membrane potential, Bax and Bak activation, caspase activation and phophatidylserine exposure. MCL sensitivity to TRAIL was irrespective of TRAIL-R1 and TRAIL-R2 receptor levels, Bcl-2 family members or caspase regulators expression, but was closely linked to the activity of the NF-kappaB p50 factor and to the expression of c-FLIP, a NF-kappaB-regulated factor. C-FLIP accumulated into the TRAIL-dependent complex in resistant cells and its transient knockdown overcame MCL resistance to TRAIL. In parallel, NF-kappaB inhibitors differentially modulated TRAIL cytotoxicity. Indeed, sub-toxic doses of bortezomib increased TRAIL cytotoxic effects by up-regulating TRAIL-R2 receptor expression, but also led to the intracellular accumulation of c-FLIP, impeding full synergistic interaction in cells with highest c-FLIP basal level. In contrast, the IkappaB kinase (IKK) inhibitor BMS-354451 allowed to consistent reduction of NF-kappaB activity, decreased total and DISC-associated c-FLIP levels, and sensitized all MCL cells to TRAIL cytotoxic effects. These results indicate that pharmacological enhancement of MCL cells sensitivity to TRAIL does not depend on TRAIL receptors level but is rather regulated by NF-kappaB-regulated c-FLIP expression. Considering that both TRAIL and BMS-345541 have already demonstrated selective cytotoxicity against malignant cells, combining TRAIL, with pharmacological inhibitors of IkappaB kinase signaling may represent an attractive model for the design of a new and rational combination therapy for MCL.

Curcumin (diferuloylmethane) inhibits constitutive NF-κB activation, induces G1/S arrest, suppresses proliferation, and induces apoptosis in mantle cell lymphoma

Human mantle cell lymphoma (MCL), an aggressive B cell non-Hodgkin's lymphoma, is characterized by the overexpression of cyclin D1 which plays an essential role in the survival and proliferation of MCL. Because of MCL's resistance to current chemotherapy, novel approaches are needed. Since MCL cells are known to overexpress NF-κB regulated gene products (including cyclin D1), we used curcumin, a pharmacologically safe agent, to target NF-κB in a variety of MCL cell lines. All four MCL cell lines examined had overexpression of cyclin D1, constitutive active NF-κB and IκB kinase and phosphorylated forms of IκBα and p65. This correlated with expression of TNF, IκBα, Bcl-2, Bcl-xl, COX-2 and IL-6, all regulated by NF-κB. On treatment of cells with curcumin, however, downregulated constitutive active NF-κB and inhibited the consitutively active IκBα kinase (IKK), and phosphorylation of IκBα and p65. Curcumin also inhibited constitutive activation of Akt, needed for IKK activation. Consequently, the expression of all NF-κB-regulated gene products, were downregulated by the polyphenol leading to the suppression of proliferation, cell cycle arrest at the G1/S phase of the cell cycle and induction of apoptosis as indicated by caspase activation, PARP cleavage, and annexin V staining. That NF-κB activation is directly linked to the proliferation of cells, is also indicated by the observation that peptide derived from the IKK/NEMO-binding domain and p65 suppressed the constitutive active NF-κB complex and inhibited the proliferation of MCL cells. Constitutive NF-κB activation was found to be due to TNF, as anti-TNF antibodies inhibited both NF-κB activation and proliferation of cells. Overall, our results indicate that curcumin inhibits the constitutive NF-κB and IKK leading to suppression of expression of NF-κB-regulated gene products that results in the suppression of proliferation, cell cycle arrest, and induction of apoptosis in MCL.