Bortezomib Induces an Antioxidant and ER-Stress Response Gene Expression Signature in Mantle Cell Lymphoma: Implications for Response Prediction and Optimized Chemotherapy Regimens.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 830-830
Author(s):  
Edgar G. Rizzatti ◽  
Helena Mora-Jensen ◽  
Raymond Lai ◽  
Masanori Daibata ◽  
Therese White ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stress Response ◽  
Er Stress ◽  
Mantle Cell Lymphoma ◽  
Cell Lines ◽  
Cell Lymphoma ◽  
Er Stress Response ◽  
Mantle Cell ◽  
Sensitive Group ◽  
Resistant Cells

Abstract Mantle cell lymphoma (MCL) is an aggressive and incurable B-cell lymphoma for which new treatment options are needed. Recent phase II clinical trials reported response to the proteasome inhibitor bortezomib (BZM) in up to 50% of pre-treated patients. Despite the successful use of BZM in the clinic, the precise molecular mechanisms underlying sensitivity or resistance to BZM in MCL remain largely unknown. To address this issue, we used U133A 2.0 microarrays to analyze gene expression in MCL cells from peripheral blood of 5 patients with previously untreated leukemic MCL. Samples were collected immediately before (0h) and at 3, 6, 24, and 72 hours after administration of BZM (1.5 mg/m2). After the blood collection at 72 hours, a second dose of BZM was given, and cells were collected 24 hours later. Two patients had major reductions in peripheral ALC already at 24h from dose 2 and normalized their blood counts by day 21 (sensitive), 1 patient had no change over a full course of 4 injections (resistant), and 2 patients had some decrease in ALC (intermediate). Genes differentially expressed with treatment were ranked according to the degree of correlation with time (Pearson). We used gene set enrichment analysis (GSEA) to detect distinct functional gene expression signatures; the most consistently up-regulated of which was a signature composed by proteasome and chaperone genes. To confirm and expand these findings, we exposed 10 MCL cell lines (7 sensitive, IC50<10nM; 3 resistant IC50>10nM) to 10nM of BZM and analyzed gene expression at 1, 3, 6 and 24 hours. The proteasome signature was again dominant, and the majority of the up-regulated genes in both clinical and cell line samples shared binding motifs for the NRF, MAF, ATF and HSF families of transcription factors (TF). Thus genes up-regulated by BZM in vivo and in cell lines predominantly belonged to a functional response to oxidative and/or endoplasmic reticulum (ER) stress. Under physiologic conditions, this is thought to help restore homeostasis and protect from apoptosis. This response could therefore contribute to drug resistance or be a marker of an overwhelming insult before the cells undergo apoptosis. To address this issue, we investigated differences in response to BZM between sensitive and resistant cell lines. The proteasome signature was more strongly up-regulated in sensitive cells than in resistant cells, and the ER-stress response as measured by genes controlled by the NRF and MAF family of TFs was also more highly expressed in the sensitive group. Consistently, expression of HMOX1, which encodes a key enzyme in the antioxidant response, was increased by 32× at 24h in the sensitive group, but only by 4× in the resistant group; the expression of DDIT3, a transcription factor implicated in a pro-apoptotic response to ER-stress was 5.5-fold up-regulated in the sensitive cells but only 1.4-fold in the resistant cells. We conclude that in sensitive cells BZM induces an overwhelming ER-stress response with high expression of proteasome components and chaperone proteins that could serve as a predictor of response to BZM.

Targeting Histone Deacetylases and Unfolded Protein Mediated Endoplasmic Reticulum (ER) Stress as a Strategy Against Human Mantle Cell Lymphoma.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1378-1378
Author(s):  
Rekha Rao ◽  
Warren Fiskus ◽  
Rajeshree Joshi ◽  
Jianguang Chen ◽  
Pravina Fernandez ◽  
...  
Keyword(s):  
Stress Response ◽  
Er Stress ◽  
Cyclin D1 ◽  
Mantle Cell Lymphoma ◽  
Cell Lymphoma ◽  
Dependent Manner ◽  
Unfolded Protein ◽  
Er Stress Response ◽  
Mantle Cell

Abstract Poor clinical outcome of therapy of Mantle Cell Lymphoma (MCL) has generated the need to develop and test novel treatments for human MCL. Here we have determined that treatment with hydroxamic acid analogue (HA) pan-histone deacetylase (HDAC) inhibitor (HDI), e.g., LBH589 (Novartis Pharmaceuticals Inc) and vorinostat (Merck Pharmaceuticals), induces the CDK inhibitors p21 and p27, and attenuates the levels of c-Myc, CDK4 and cyclin D1 in the cultured (Jeko-1, MO-2058 and Granta-519) and in primary patient-derived MCL cells. In a dose-dependent manner, HA-HDI also induced Bax, Bak and Bim, and attenuated Bcl-xL, XIAP, survivin, AKT and c-Raf levels, resulting in growth inhibition and apoptosis of MCL cells. We have previously demonstrated that HDAC6 deacetylates heat shock protein (hsp) 90. By inhibiting HDAC6, both LBH589 (10 to 50 nM) and vorinostat (0.5 to 2.0 uM) induced acetylation of hsp90 in MCL cells. This inhibited the ATP binding and co-chaperone association, and abrogated the chaperone function of hsp90 for the MCL- relevant, hsp90 client proteins, e.g., cyclin D1, CDK4, c-Raf and AKT in the cultured and primary MCL cells. HDAC6 has been shown to shuttle and sequester misfolded and polyubiquitylated proteins into the protective perinuclear aggresome. Present studies demonstrate that inhibition of HDAC6 abrogates formation of the aggresome and augments the ER-based unfolded protein response (UPR). Treatment of MCL cells with the proteasome inhibitor bortezomib (BZ) induced the formation of aggresome (as detected by confocal immuno-fluorescence microscopy and electron microscopy), as well as induced UPR and ER stress response. The latter was associated with BZ-mediated increased levels of the spliced form of XBP1 (XBP1s) and p-eIF2α protein. It was also associated with increased levels of the protective ER chaperone protein GRP78, and increased expression of pro-death proteins, CHOP and Noxa. Treatment with BZ or HA-HDI also increased the expression of the transcriptional repressor, PRDM1. Co-treatment of MCL cells with LBH589 abrogated BZ-induced aggresome formation, but increased the levels of BZ-induced XBP1s and p-eIF2α, indicating increased ER stress response. Concomitantly, higher CHOP and Noxa levels suggested a protracted ER-stress, associated with significantly increased apoptosis of MCL cells (p < 0.01). These findings suggest that co-treatment with LBH589 accentuates BZ-induced ER-stress and cell death of MCL cells despite up-regulation of GRP78 levels. Next, we determined the effects of knocking down GRP78 on BZ-induced ER-stress response. As compared to the control siRNA treated cells, knockdown by siRNA to GRP78 markedly increased BZ-induced CHOP and Noxa levels and significantly augmented BZ-induced apoptosis of cultured MCL cells. Collectively, these findings strongly support the in vivo testing of the efficacy of the combination of HA-HDI with BZ in inducing protracted and lethal ER stress in MCL cells. These results also create the rationale to develop targeted knockdown of GRP78 as a novel strategy to augment the lethal ER stress in human MCL cells.

Co-Treatment with Panobinostat Enhances Bortezomib-Induced Unfolded Protein Response, Endoplasmic Reticulum Stress and Apoptosis of Human Mantle Cell Lymphoma Cells

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 887-887 ◽  
Author(s):  
Rekha Rao ◽  
Warren Fiskus ◽  
Yonghua Yang ◽  
Rajeshree Joshi ◽  
Pravina Fernandez ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Stress Response ◽  
Er Stress ◽  
Mantle Cell Lymphoma ◽  
Cell Lymphoma ◽  
Unfolded Protein ◽  
Er Stress Response ◽  
Mantle Cell ◽  
Protein Response

Abstract The 26S proteasome inhibitor bortezomib (BZ), which increases intracellular unfolded protein levels and toxicity through endoplasmic reticulum (ER) stress response, was shown to have a single agent activity in relapsed mantle cell lymphoma (MCL). Here we have determined that treatment with hydroxamic acid analogue (HA) pan-histone deacetylase (HDAC) inhibitor (HDI), e.g., panobinostat (LBH589, Novartis Pharmaceuticals Inc) induces the CDK inhibitors p21 and p27, and attenuates the levels of c-Myc, CDK4 and cyclin D1 in the cultured (Jeko-1, MO-2058 and Granta-519) and in primary patient-derived MCL cells. In a dose-dependent manner, panobinostat also induced Bax and Bak, and attenuated Bcl-xL, XIAP, survivin, AKT and c-Raf levels, resulting in growth inhibition and apoptosis of MCL cells. We have previously demonstrated that HDAC6 deacetylates heat shock protein (hsp) 90, as well as shuttles and sequesters misfolded and polyubiquitylated proteins into the protective perinuclear aggresome.. By inhibiting HDAC6, panobinostat (10 to 50 nM) induced acetylation of hsp90 in MCL cells. This inhibited the ATP binding and co-chaperone association, and abrogated the chaperone function of hsp90 for the MCL- relevant, hsp90 client proteins, e.g., cyclin D1, CDK4, c-Raf and AKT in the cultured and primary MCL cells. Panobinostat mediated inhibition of HDAC6 abrogated formation of the aggresome and augmented endoplasmic reticulum (ER)-based unfolded protein response (UPR). Treatment of MCL cells with BZ induced the formation of aggresome (as detected by confocal immuno-fluorescence microscopy and electron microscopy), as well as induced UPR and ER stress response. The latter was associated with BZ-mediated increased levels of GRP78, the spliced form of XBP1 (XBP1s) and p-eIF2α protein. As compared to the control siRNA treated cells, knockdown of GRP78 by siRNA markedly increased BZ-induced CHOP and Noxa levels and significantly augmented BZ-induced apoptosis of cultured MCL cells. Co-treatment of MCL cells with panobinostat abrogated BZ-induced aggresome formation, decreased the levels of ATF4, XBP1s and p-eIF2α, as well as increased the levels of CHOP, Noxa and GADD34. Ultrastructural analysis of Jeko-1 cells also revealed that co-treatment with panobinostat and BZ showed pronounced ER dilatation compared to panobinostat treatment alone, suggestive of enhanced ER stress. Higher and persistent CHOP and Noxa levels suggested a protracted ER-stress, associated with synergistic increase in apoptosis of MCL but not normal CD34+ bone marrow progenitor cells (p < 0.01). Conversely, knockdown of CHOP levels by siRNA significantly inhibited panobinostat and BZ-induced cell death of MCL cells. Results of ongoing in vivo studies of panobinostat and/or BZ in the NOD/SCID mouse xenograft model of Jeko-1 MCL cells will be presented. These findings strongly support further in vivo evaluation of the efficacy of the combination of panobinostat with BZ against human MCL. Additionally, the findings create the rationale to develop targeted knockdown of GRP78 as a novel strategy to augment lethal ER stress due to panobinostat and BZ and resulting activity against MCL cells.

Treatment with Histone Deacetylase 6-Specific Inhibitor WT-161 Disrupts hsp90 Function, Abrogates Aggresome Formation and Sensitizes Human Mantle Cell Lymphoma Cells to Lethal ER Stress Induced by Proteasome Inhibitor Carfilzomib

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2856-2856 ◽  
Author(s):  
Rekha Rao ◽  
Warren Fiskus ◽  
Ramesh Balusu ◽  
Hongwei Ma ◽  
James Bradner ◽  
...  
Keyword(s):  
Stress Response ◽  
Er Stress ◽  
Histone Deacetylase ◽  
Proteasome Inhibitor ◽  
Cell Lymphoma ◽  
Specific Inhibitor ◽  
Er Stress Response ◽  
Mantle Cell ◽  
Aggresome Formation ◽  
Induced Apoptosis

Abstract Abstract 2856 The proteasome inhibitor bortezpmib has been shown to markedly increase the intracellular levels of misfolded proteins, induce aggresome formation and cause endoplasmic reticulum (ER) stress, resulting in apoptosis of human Mantle Cell Lymphoma (MCL) cells. Consistent with this, Bortezomib displays clinical efficacy in patients with relapsed and refractory MCL. We have recently reported that the pan-histone deacetylase (HDAC) inhibitor panobinostat, by also inhibiting HDAC6, abrogates aggresome formation and induces Endoplasmic Stress (ER) stress, as well as potentiates bortezomib-induced apoptosis of MCL cells. Here, we determined the anti-MCL cell activity of an HDAC6-specific inhibitor, WT-161 alone and in combination with the novel, orally bio-available, proteasome inhibitor carfilzomib (Proteolix Inc.) against human, cultured and primary, patient-derived MCL cells. Treatment with WT-161 (0.1 to 1.0 uM) resulted in a dose-dependent increase in the acetylation of alpha-tubulin and heat shock protein (hsp) 90, without any appreciable increase in the levels of acetylated histone (H) 3. Consistent with WT-161 mediated hyperacetylation and inhibition of hsp90 chaperone function, treatment with WT-161 increased the intracellular levels of polyubiuitylated proteins in the cultured MCL JeKo-1 and Z138 cells. WT-161 was also noted to dose-dependently deplete the levels of cyclin D1 in the cultured MCL cells. Treatment with WT-161 also induced ER stress response in the MCL cells, demonstrated by increase in the protein levels of Glucose regulated protein (GRP) 78, phosphorylated eIF2 (eukaryotic initation factor 2) α, and induction of the pro-apoptotic transcription factor CHOP (CAAT/Enhancer Binding Protein Homologous Protein). We next determined the effects of co-treatment with WT-161 on carfilzomib-induced aggresome formation, ER stress response and apoptosis of the cultured and primary MCL cells. Co-treatment with WT-161 (0.25 uM) abrogated carfilzomib-induced aggresome formation in MCL cells, as evidenced by confocal immunofluorescent staining of aggresomes with anti-HDAC6 and anti-ubiquitin antibodies. Compared to each agent alone, co-treatment with WT-161 and carfilzomib induced more intracellular polyubiquitylated proteins and induced higher levels of CHOP in the cultured MCL cells. Co-treatment with WT-161 and carfilzomib also synergistically induced apoptosis of the cultured MCL cells (combination indices < 1.0). Notably, co-treatment with WT-161 and carfilzomib also synergistically induced apoptosis of primary MCL cells (combination indices < 1.0). These findings strongly support the in vivo testing of the combination of an HDAC6-specific inhibitor such as WT-161 with the proteasome inhibitor carfilzomib against human MCL cells. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Bortezomib resistance in mantle cell lymphoma is associated with plasmacytic differentiation

Blood ◽  
2011 ◽  
Vol 117 (2) ◽  
pp. 542-552 ◽  
Author(s):  
Patricia Pérez-Galán ◽  
Helena Mora-Jensen ◽  
Marc A. Weniger ◽  
Arthur L. Shaffer ◽  
Edgar G. Rizzatti ◽  
...  
Keyword(s):  
Gene Expression ◽  
Mantle Cell Lymphoma ◽  
Cell Lines ◽  
Expression Profiling ◽  
Plasma Cells ◽  
Cell Lymphoma ◽  
Proteasome Inhibition ◽  
Plasmacytic Differentiation ◽  
Mantle Cell ◽  
Parental Lines

Abstract Bortezomib 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.

Integrative Analysis of MicroRNA and Gene Expression Profiling Contributes to Understand Mantle Cell Lymphoma Pathogenesis.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2936-2936
Author(s):  
Lorena Di Lisio ◽  
Gonzalo Gómez-López ◽  
Margarita Sáanchez-Beato ◽  
Maria Elena Rodríguez ◽  
Cristina Gómez Abad ◽  
...  
Keyword(s):  
Gene Expression ◽  
Mantle Cell Lymphoma ◽  
Cell Lines ◽  
Expression Profile ◽  
Nuclear Translocation ◽  
Conflicts Of Interest ◽  
Cell Lymphoma ◽  
Gene Set Enrichment Analysis ◽  
Simultaneous Increase ◽  
Mantle Cell

Abstract Abstract 2936 Poster Board II-912 Mantle cell lymphoma (MCL) pathogenesis is still partially unexplained. Although the overexpression of CyclinD1 dependent of t(11;14) is a distinctive molecular hallmark of this neoplasm, this event alone cannot account for the increased survival signaling that characterizes this lymphoma type. Here we investigate whether microRNA (miRNA) expression profile may help to explain the changes in the expression of gene pathways that are characteristic of MCL. Twenty-three frozen MCL samples, 11 frozen control tissues (7 lymph nodes and 4 tonsils), 8 MCL cell lines and 3 samples of CD19+IgD+CD27- cells obtained from tonsils, were studied for miRNAs and gene expression. MiRNA one color microarray data for 470 human miRNA were analyzed using SAM (Significance Analysis of Microarrays) algorithm. MiRNA targets were predicted by miRanda and TargetScan methods. Pathways identification and analysis was carried out by GSEA (Gene Set Enrichment Analysis) online resource. The analysis of 23 MCL cases compared to 11 control tissues showed a miRNA signature that included 117 miRNAs with FDR<0.05, 85 of which downregulated and 32 upregulated. Combined analysis of these miRNAs and changes in the gene expression profile, paired with bioinformatic target prediction, revealed a group of genes and pathways potentially targeted by the miRNAs, including essential pathways for lymphoma survival. An interesting correspondence was found between the simultaneous increase in CD40, MAPK, NFKB and others pathway signaling with the downregulation of 15 miRNA predicted to target genes belonging to these pathways. Functional validation in MCL cell lines demonstrated NF-kB nuclear translocation to be regulated by the expression of one of these miRNAs. Most of the MCL cell lines exhibit a strong expression of the mir-17-92 polycistron (Oncomir-1). MiRNAs were used also for the identification of survival prognostic markers; using different analysis (22 frozen specimens) and validation (54 paraffin embedded cases) series. A single miRNA distinguished a group of MCL cases with a 72.2% survival at 60 m. This study identifies a set of miRNAS involved in MCL pathogenesis, which could be used in MCL recognition and clinical prognostication. Disclosures: No relevant conflicts of interest to declare.

Epigenetic Determinants of Pathogenesis and Resistance to Proteosome Inhibition in Mantle Cell Lymphoma.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3373-3373 ◽  
Author(s):  
Tobias Gellen ◽  
Pei-Yu Kuo ◽  
Rita Shaknovich ◽  
Maria E Figueroa ◽  
Ari Melnick ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Transcription Factor ◽  
Tumor Suppressor ◽  
Mantle Cell Lymphoma ◽  
Cell Lines ◽  
Tumor Suppressor Genes ◽  
Cell Lymphoma ◽  
Mantle Cell ◽  
Hypermethylated Genes

Abstract Mantle Cell Lymphoma (MCL) is an aggressive tumor accounting for 5% of non Hodgkin’s lymphomas(NHL). Given the poor clinical outcomes in this disease with current therapy, it is of great importance to better understand disease pathogenesis. Since aberrant epigenetic gene regulation is a hallmark of cancer, we hypothesized that an unbiased genome-wide DNA methylation study would identify genes involved in MCL pathogenesis. Moreover, we hypothesized that aberrantly silenced/hypermethylated genes responsible for drug sensitivity in MCL could be pharmacologically manipulated for maximizing clinical benefit. To address these questions, we examined the abundance of DNA methylation in 25,000 promoter fragments using the HELP (HPA II Enzyme Ligation mediated PCR amplification) assay in MCL cell lines. This study revealed s substantial heterogeneity in genomic methylation between cell lines, possibly reflective of underlying biologic diversity in this disease. However, MCL cell lines did display consistent aberrant methylation when compared to naïve B cells extracted from normal human tonsils. Remarkably, among the 102 genes with a methylation fold change in >3 and p<0.0001 the majority (100) were hypermethylated in MCL vs. normal B cells. Amongst these genes we identified five tumor suppressor genes implicated in the pathogenesis of other cancers: Protocadherin 8,Paternally expressed gene 3 (PEG3),Myeloid leukemia factor 1, Transcription Factor AP-2 gamma and Homeobox D8. Interestingly. PEG3 and Protocadherin 8 are both known to be silenced by hypermethylation at their gene promoters in gastric and breast cancer respectively. These genes may also possibly function as tumor suppressor genes in MCL pathogenesis and are excellent candidates for future functional studies. We then integrated methylation and gene expression data to identify loci that were both significantly methylated and suppressed in MCL. To identify the pathways and biological processes most relevant to our data set of methylated genes, we queried the Ingenuity Pathway Analysis (IPA) Knowledge base with the top 560 hypermethylated genes (>2 Standard Deviation) across all MCL cell lines. An unsupervised core analysis from the IPA knowledge base using our gene set revealed gene networks revolving around tumor suppressor TP53 and the NPM1 transcription factor in MCL cell lines. Locus specific confirmation by MassArray confirmed methylation of the distal portion of the TP53 promotor in the MCL cell lines. Recently, the proteosome inhibitor Bortezomib(BZM) has been shown to have specific activity in MCL, with single agent response rates ~40% in heavily pretreated patients. We wondered whether aberrant epigenetic programming might contribute to resistance to this agent and whether reversal of DNA methylation could overcome cellular mechanisms of resistance to Bortezomib. Therefore, to determine whether pharmacologic re-expression of genes could overcome bortezomib resistance, we treated MINO (a Bortezomib-refractory MCL cell line) with two low doses of Decitabine (0.1 uM and 1 uM) for 48 hours and sequentially with 15 nM of Bortezomib (IC50) for an additional 48 hours. Pretreatment of BZM-resistant MINO cells with Decitabine significantly (p<0.001) reduced cell viability as compared to control, suggesting that Decitabine can overcome BZM resistance in this refractory cell line. We are currently determining which genes contribute to this effect and validating these results in patient samples from a prospective trial of BZM+EPOCH. We predict that this will provide the basis for future trials individualizing BZM based therapy based on methylation and gene expression signatures in MCL.

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

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 287-287
Author(s):  
Patricia Perez Galan ◽  
Helena Mora Jensen ◽  
Marc A Weniger ◽  
Colby M Chapman ◽  
Poching Liu ◽  
...  
Keyword(s):  
B Cell ◽  
Mantle Cell Lymphoma ◽  
Cell Lines ◽  
Plasma Cell ◽  
Cell Lymphoma ◽  
Proteasome Activity ◽  
Gene Set Enrichment Analysis ◽  
Mantle Cell ◽  
Parental Lines ◽  
Resistant Cells

Abstract Abstract 287 Mantle 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.

scholarly journals Using the Iron Chelator Deferasirox to Overcome Drug Resistance in Mantle Cell Lymphoma

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 4364-4364
Author(s):  
Aladin Samara ◽  
Saar Shapira ◽  
Ido Lubin ◽  
Pia Raanani ◽  
Galit Granot
Keyword(s):  
Drug Resistance ◽  
B Cell ◽  
Mantle Cell Lymphoma ◽  
Cell Lines ◽  
Conflicts Of Interest ◽  
Cell Lymphoma ◽  
Synergistic Effects ◽  
Iron Chelator ◽  
Mantle Cell ◽  
Resistant Cells

Abstract Mantle cell lymphoma (MCL) is a difficult-to-treat B-cell malignancy characterized by the t(11,14) translocation, resulting in cyclin D1 (CD1) overexpression. In addition to CD1 overexpression, pathways such as the B-cell receptor, PI3K/AKT/ GSK3β, NFκB and Wnt have been reported to be deregulated in MCL. Despite advancements in MCL treatment, most patients still relapse. Although the introduction of ibrutinib to relapsed/refractory MCL significantly improved the outcome of MCL patients, ibrutinib resistance has become a clinical obstacle. MCL treatments are thus pursued by studying novel agents with a broad spectrum of targets or by rationally combining existing therapies aiming for synergistic antitumor activities. Deferasirox (DFX) is a clinically approved iron chelator with only few side effects. DFX has been reported to exert anti-tumoral and synergistic effects in several types of cancers by affecting a multitude of targets. We have previously shown that DFX exerts a vigorous anti-tumoral effect via growth inhibition and induction of apoptosis in MCL cells through ROS elevation, triggering of oxidative stress, induction of DNA damage, modulation of PI3K/AKT/GSK3β signaling and most importantly by elimination of CD1 expression. The capacity of DFX to affect a multitude of targets establishes a solid basis for a possible synergistic interaction with other drugs, such that may overcome drug resistance in MCL. In this study we focused on assessing the efficiency of DFX combined with the established therapeutic-agents etoposide, cytarabine and ibrutinib in MCL cell-lines. We found that DFX synergizes with etoposide, cytarabine and ibrutinib, prompting remarkable anti-tumoral effects in MCL cells with combination index (CI) values &lt; 1. Interestingly, the DFX-drug combinations achieved synergism regardless of the innate sensitivity of the cell-lines to the treatment: ibrutinib-resistant cells restored their sensitivity to the drug when it was combined with DFX. In addition, we found that the sensitivity of MCL cells towards the drugs correlated with the drugs ability to induce CD1 degradation. In agreement, DFX co-treatment enhanced CD1 degradation, especially in resistant cells. We show here that DFX is a putative promising drug-sensitizing agent for the treatment of MCL. DFX-co-treatment not only enhances the efficacy of the tested drugs, but also restores the anti-proliferative activity of the drugs in resistant MCL cells. To the best of our knowledge, this study is the first to provide evidence on the potential of DFX to overcome drug resistance in MCL. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

NRF2 at the Interface of Oxidative and Endoplasmic Reticulum Stress Pathways Is a Critical Integrator of Bortezomib Response in Mantle Cell Lymphoma in Vitro and In Vivo.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 727-727 ◽  
Author(s):  
Marc A Weniger ◽  
Edgar Gil Rizzatti ◽  
Patricia Perez Galan ◽  
Delong Liu ◽  
Peter J Munson ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stress Response ◽  
Er Stress ◽  
Tumor Cells ◽  
Cell Lines ◽  
Target Genes ◽  
Resistant Cell ◽  
Er Stress Response

Abstract Abstract 727 The proteasome inhibitor bortezomib (BZM) is effective as single-agent in relapsed and refractory mantle cell lymphoma (MCL) but more than half of patients remain insensitive to BZM. Suggested mechanisms of action include activation of Noxa, p53, oxidative, and endoplasmic reticulum (ER) stress. To define mechanisms relevant for BZM-induced cytotoxicity we pursued two approaches: first, we characterized gene expression changes in 10 MCL cell lines exposed for 24h to 10nM BZM, a concentration that kills >50% of sensitive, but <20% of resistant cells. Secondly, we analyzed gene expression changes in tumor cells of patients with leukemic MCL undergoing BZM treatment in a clinical trial. RNA was extracted from MCL cell lines at early (1h and 3h), intermediate (6h) and late (24h) time points. Virtually no changes in gene expression were detectable at 1 and 3h of drug exposure and only about 100 genes changed by 6h. After 24h of treatment 524 genes were significantly changed in sensitive and 271 genes in resistant cell lines respectively. The delayed onset of gene expression changes is consistent with the reversibility of BZM toxicity for up to 8 hours. Using Ingenuity pathway analysis (IPA) and gene set enrichment analysis (GSEA) we identified two dominant responses induced by BZM: 1) an oxidative stress response mediated by NRF2 and related transcription factors, and 2) an ER stress/ubiquitin proteasome response (FDR by GSEA <0.1). Both responses were primarily apparent in sensitive cell lines. A set of 20 experimentally validated NRF2 target genes was used as a core NRF2 signature: this signature was increased 15-fold on average in sensitive cell lines but only 2-fold in resistant cell lines (P=0.006). Similarly, an XBP1 and ATF6 signature, reflecting activation of the ER stress response, was stronger induced in sensitive than in resistant cell lines (average 1.9-fold vs 1.3-fold; P=0.003). Activation of these stress pathways upon BZM treatment was confirmed by demonstrating accumulation of nuclear NRF2 in sensitive Jeko1 but not in resistant Mino cells. Also markers of ER stress such as phosphorylation of ER resident nuclease Ire1 that splices the transcription factor XBP1 and activation of ATF3, ATF4, and CHOP downstream of PERK were readily detected in Jeko1 but not in Mino cells. Finally, Noxa, the BH3-only protein primarily responsible for BZM-induced apoptosis, was only induced in sensitive Jeko1 cells. We next analyzed the effect of BZM on purified tumor cells from five patients with leukemic MCL treated with BZM (1.5mg/m2, day 1, 4, 8 and 11). Two patients showed a >50% reduction in circulating tumor cells after 2 injections of drug (day 8) and >75% reduction after 4 injections (day 2, sensitive), while in three patients there was no change or an intermediate response (resistant). Western blotting demonstrated Noxa up-regulation in circulating tumor cells of sensitive but not resistant samples. This is consistent with the demonstrated importance of Noxa for induction of apoptosis in response to BZM in cell line studies. Next we performed gene expression profiling immediately before, at 6h, and 24h after the first and 24h after the second dose of BZM. Using IPA and GSEA up-regulation of the ubiquitin/proteasome pathway and the NRF2-mediated oxidative stress response was again prominent, but an ER stress response was less apparent. XBP1 splicing was not detected in tumor cells from sensitive samples indicating that an ER stress response was not fully activated by BZM in vivo. Consistent with in vitro data the NRF2 signature was induced 2.3-fold on average in sensitive but not in resistant samples (P<0.05). Intriguingly, baseline expression of the NRF2 signature genes was significantly higher in resistant than in sensitive cells (P=0.0007). In summary, we identify NRF2 as critical integrator of different stress pathways in response to BZM in MCL. Thus, rapid induction of NRF2 target genes might be a useful biomarker of BZM-induced cellular stress and predict clinical response. Our data suggest a more complex function of NRF2 than previously appreciated. NRF2-regulated genes serve primarily homeostatic roles and enable cells to deal with oxidative and xenobiotic insults; a function that may come to play in BZM resistant cells with higher baseline expression of NRF2 target genes. On the other hand, our data suggest a possible pro-apoptotic role of acute induction of high levels of NRF2 that is currently under investigation. Disclosures: No relevant conflicts of interest to declare.

Epstein-Barr Virus and Kaposi's Sarcoma Herpesvirus Lytic Cycle Induction with Bortezomib Is a Response to ER Stress

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1736-1736 ◽  
Author(s):  
Courtney O'Farrell ◽  
Meir Shamay ◽  
Nene Kalu ◽  
Andrew DuFresne ◽  
Sunetra Biswas ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stress Response ◽  
Er Stress ◽  
Cell Lines ◽  
Family Members ◽  
Lytic Cycle ◽  
Early Gene ◽  
Immediate Early ◽  
Antitumor Effects ◽  
Er Stress Response

Abstract Abstract 1736 EBV and KSHV are associated with a variety of lymphoid malignancies. Bortezomib, a proteasome inhibitor, is among the most potent activators of EBV and KSHV lytic infection and is active at nanomolar concentrations. Studying EBV Burkitt's cell lines, KSHV primary effusion lymphoma cell lines and EBV myeloma cell lines, we found that lytic gene expression is a class effect shared by other proteasome inhibitors, such as MG-132 and epoxomicin. Proteasome inhibition results in IKB stabilization and inhibition of NFKB activity and this effect has been implicated in many of the effects of bortezomib. We studied the promoters of EBV and KSHV immediate early viral protein reporters (EBV Zta and KSHV Rta) and found that IKB super-repressor did not activate promoter reporters suggesting that other pathways must be important in activation. Therefore, we sought to better understand the drug's impact on viral gene expression in virus-associated tumor cells. ER stress has been implicated in bortezomib's antitumor effects. Thapsigargin and tunicamycin are classic activators of ER stress that do not act through proteasomal inhibition. These agents were found to be potent activators of the EBV and KSHV lytic cycle. Previous studies have identified C/EBP family members (C/EBP alpha and beta) as activators of EBV and KSHV immediate early gene expression. In other studies, C/EBP family members (C/EBP beta, CHOP10) have been implicated in regulating the ER stress response. We found that bortezomib, thapsigargin and tunicamycin increased levels of the activating C/EBP beta LAP isoform as assessed by immunoblot and by real-time RT-PCR. Treatment also led to increase in ATF4, XBP1(s), CHOP10, and ATF6 cleavage, all consistent with induction of the ER stress response. Additionally, we showed that treatment with bortezomib increased C/EBP beta binding to previously characterized binding sites in the Zta promoter and expression of C/EBP beta LAP isoform was sufficient to activate EBV immediate early lytic promoters. Finally, we demonstrated that in tumor cell lines with C/EBP beta silenced by doxycycline regulated siRNA, induction of EBV lytic induction by bortezomib and thapsigargin was blocked. These results demonstrate that both human lymphoma associated herpesviruses (EBV and KSHV) are activated into lytic cycle by bortezomib and that these effects are mediated through ER stress pathways and specifically involve C/EBP beta. Disclosures: No relevant conflicts of interest to declare.

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