Resistant Mantle Cell Lymphoma Research Articles

Abstract B77: The BET bromodomain inhibitor OTX015 (MK-8628) in mantle cell lymphoma (MCL): in vivo activity and identification of novel combinations to overcome adaptive resistance

Abstract OTX015 (MK-8628) has shown anti-lymphoma activity in both the preclinical and clinical settings (Boi et al, CCR 2015; Stathis et al, EORT-NCI-AACR 2014). Now, we report in vivo and in vitro data on OTX015 in MCL, including combination regimens. Methods. NOD-Scid (NOD.CB17-Prkdcscid/NCrHsd) mice were subcutaneously inoculated with 15 x106 MCL REC1 cells. Tumor size was measured twice weekly using digital calipers. Tumor volumes were calculated using the equation V = [length x width2]/2, in which the width and the length are the shortest and the longest diameters of each tumor, respectively. Gene expression profiling (GEP) was performed with Illumina HumanHT12 Expression BeadChips. Combinations were evaluated using the Chou-Talalay combination index (CI): <0.9, synergism; 0.9-1.1 additive effect; >1.2, no benefit. Results. Since we had previously reported in vitro activity of OTX015 as single agent in MCL cell lines (Boi et al, CCR 2015), we first confirmed its antitumor activity in an in vivo model. Mice were treated with OTX015 (50 mg/kg once daily; QDx7/w x5w) started 3 days after the graft. OTX015-treated mice did not show any weight loss. OTX015-treated REC1 xenografts presented a 2-fold decrease in volume compared with the control group (p< 0.05). To better understand the activity of OTX015 in MCL and to identify rational combinations that may overcome adaptive resistance mechanisms, GEP was performed on three sensitive (Rec1, Maver1, Jeko1) and one resistant MCL cell lines (Granta519, IC50 >10000 nM) exposed to DMSO or OTX015 (500 nM) for 2, 4, 8 or 12 hr. Despite the difference in the IC50 values between the three OTX015 sensitive cell lines and Granta-519, GEP changes were similar, and support the previously reported synergisms of OTX015 with ibrutinib and lenalidomide (Bernasconi et al, ASH 2014). Downregulated transcripts were enriched for MYC targets, genes involved in interferon response, in NFKB signaling, in MYD88 signaling, in B cell activation, genes more expressed in MCL than in CLL or DLBCL. TNFRSF17, TNFRSF13B, TLR10, CD72, VPREB3, PLEK, PTPN6 were among the most down-regulated transcripts, while the most upregulated ones comprised genes coding different histones of the clusters 1-2, PPP1R13B, CDKN2D (p19), IRF7, FGFR3, HES6. The upregulation of few transcripts, including HES and FGFR3, more pronounced in the resistant cell line, were suggestive of adaptive resistance mechanisms. We thus evaluated the combination of OTX015 with inhibitors of FGFR (PD173074) or MEK (pimasertib), which might counteract FGFR3 upregulation. Both combinations increased the antiproliferative effect of OTX015 in the sensitive cell line Jeko1 (CI values of 0.83 and 0.37, respectively). The combination with pimasertib was able to increase the sensitivity to OTX015 also in the resistant cell line (CI = 0.18), although it was not able to completely reverse its primary resistance. Conclusions. OTX015 showed in vivo activity as a single agent in MCL. GEP identified new combinations with MEK and FGFR inhibitors with the potential to overcome adaptive resistance. Citation Format: Eugenio Gaudio, Elena Bernasconi, Chiara Tarantelli, Luciano Cascione, Ivo Kwee, Andrea Rinaldi, Maurilio Ponzoni, Maria E. Riveiro, Emanuele Zucca, Francesco Bertoni. The BET bromodomain inhibitor OTX015 (MK-8628) in mantle cell lymphoma (MCL): in vivo activity and identification of novel combinations to overcome adaptive resistance. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B77.

Harnessing Noxa demethylation to overcome Bortezomib resistance in mantle cell lymphoma.

Bortezomib (BZM) is the first proteasome inhibitor approved for relapsed Mantle Cell Lymphoma (MCL) with durable responses seen in 30%-50% of patients. Given that a large proportion of patients will not respond, BZM resistance is a significant barrier to use this agent in MCL. We hypothesized that a subset of aberrantly methylated genes may be modulating BZM response in MCL patients. Genome-wide DNA methylation analysis using a NimbleGen array platform revealed a striking promoter hypomethylation in MCL patient samples following BZM treatment. Pathway analysis of differentially methylated genes identified molecular mechanisms of cancer as a top canonical pathway enriched among hypomethylated genes in BZM treated samples. Noxa, a pro-apoptotic Bcl-2 family member essential for the cytotoxicity of BZM, was significantly hypomethylated and induced following BZM treatment. Therapeutically, we could demethylate Noxa and induce anti-lymphoma activity using BZM and the DNA methytransferase inhibitor Decitabine (DAC) and their combination in vitro and in vivo in BZM resistant MCL cells. These findings suggest a role for dynamic Noxa methylation for the therapeutic benefit of BZM. Potent and synergistic cytotoxicity between BZM and DAC in vitro and in vivo supports a strategy for using epigenetic priming to overcome BZM resistance in relapsed MCL patients.

Poor Overall Survival of Patients with Ibrutinib-Resistant Mantle Cell Lymphoma

IntroductionThe United States Food and Drug Administration approved ibrutinib for treatment of patients with mantle cell lymphoma (MCL) that have received at least one prior therapy based on a phase II trial demonstrating a 68% response rate with a median progression-free survival of 13.9 months (Wang et al, NEJM 2013). Despite this significant efficacy, recent data suggest that primary or secondary resistance to ibrutinib is common and the mechanisms of ibrutinib resistance are varied (Chiron et al, Cancer Discovery 2014). The outcomes of MCL patients with ibrutinib resistance have not been previously reported, and the ideal management of patients with ibrutinib-resistant MCL is not known.MethodsFollowing IRB approval, we performed a retrospective cohort study of all patients with MCL at WCMC and OSU that had experienced disease progression while receiving ibrutinib in a clinical trial or in routine clinical use. We evaluated historical medical records for clinical, pathological, and radiological data. Time-to-event statistics were estimated using the Kaplan-Meier method.ResultsWe identified 32 subjects (19 male, 13 female) with primary or acquired ibrutinib resistant MCL. The median age at start of ibrutinib was 70 years (range 50-83), and the median number of prior therapies was 3 (0-9), including 2 patients with no prior therapy. The median time from diagnosis to start of ibrutinib was 32.5 months (range 0-223). Of the 30 patients that had received prior treatment, all had received rituximab, 21 had received bendamustine, 19 received bortezomib, 10 received an IMiD, 5 had received cytarabine, 5 had received methotrexate, 4 had received a purine analog, 2 received a PI3K inhibitor, and 1 had received an mTOR inhibitor. Five had undergone prior stem cell transplantation (3 auto, 2 allo). Only 11/30 patients had responded to their prior treatment, with a median time of 2.5 months between the prior treatment and ibrutinib. Four of 18 patients with morphology evaluated within 6 months of start of ibrutinib had blastoid MCL, and 11/19 patients had a previously reported Ki67 of >30%. The MIPI scores were as follows: 18 high risk, 5 intermediate risk, and 8 low risk, 1 not evaluable. Best response to ibrutinib was reported as follows: PD 20, SD 2, PR 6, CR 2, not evaluable 2. The median time on ibrutinib was 2.5 months (range 0-11). Among 27 patients with available follow-up data, 19 received treatment following ibrutinib with a median time of 0 months between stopping ibrutinib and starting subsequent treatment. The MIPI scores at start of subsequent therapy were as follows: 11 high risk, 3 intermediate risk, 3 low risk, and 2 unknown. Subsequent treatments included the following: 11 rituximab, 6 lenalidomide, 6 alkylator, 3 bendamustine, 2 anthracycline, 1 bortezomib, 1 purine analog. Two patients underwent allogeneic stem cell transplantation. Six of 17 patients evaluated for response demonstrated an objective response to subsequent treatment, including 2 patients that received bendamustine, 2 patients that received lenalidomide (one in combination with bendamustine), one that received an anthracycline, and one that received a purine analog. The median overall survival following cessation of ibrutinib was 4 months (95% CI 2-10 months). Both patients that underwent allogeneic stem cell transplantation died, one at 4 months from toxicity and one at 5 months from disease progression. Twelve patients were still alive, including four patients with more than 6 months of follow up (17, 22, 25, and 32+ months). There was no clear association between number of prior therapies, response to ibrutinib, morphology, Ki67 prior to ibrutinib, response to ibrutinib, duration of ibrutinib, or choice of subsequent therapy and OS.ConclusionsIn a group of patients with highly treatment-resistant MCL, primary and secondary ibrutinib resistance was associated with poor clinical outcomes, with limited duration of response to subsequent therapy and short overall survival. As yet, there are no identifiable predictors of clinical outcome following development of ibrutinib resistance, nor are there any clear signs that specific therapies might be particularly effective in this setting. Additional investigation into the outcomes of these patients is warranted. Clinical trials evaluating combinations with ibrutinib should be prioritized, as should trials of novel agents in the post-ibrutinib setting. DisclosuresMartin:Janssen: Honoraria. Maddocks:Pharmacyclics: Advisory Board Other, Research Funding. Furman:Pharmacyclics: Consultancy, Speakers Bureau. Jones:Pharmacyclics: Consultancy, Research Funding. Ruan:Pharmacyclics: Speakers Bureau; Janssen: Speakers Bureau. Blum:Janssen, Pharmacyclics : Research Funding.

Cell-cycle reprogramming for PI3K inhibition overrides a relapse-specific C481S BTK mutation revealed by longitudinal functional genomics in mantle cell lymphoma.

Despite the unprecedented clinical activity of the Bruton tyrosine kinase (BTK) inhibitor ibrutinib in mantle cell lymphoma (MCL), acquired resistance is common. By longitudinal integrative whole-exome and whole-transcriptome sequencing and targeted sequencing, we identified the first relapse-specific C481S mutation at the ibrutinib binding site of BTK in MCL cells at progression following a durable response. This mutation enhanced BTK and AKT activation and tissue-specific proliferation of resistant MCL cells driven by CDK4 activation. It was absent, however, in patients with primary resistance or progression following transient response to ibrutinib, suggesting alternative mechanisms of resistance. Through synergistic induction of PIK3IP1 and inhibition of PI3K-AKT activation, prolonged early G1 arrest induced by PD 0332991 (palbociclib) inhibition of CDK4 sensitized resistant lymphoma cells to ibrutinib killing when BTK was unmutated, and to PI3K inhibitors independent of C481S mutation. These data identify a genomic basis for acquired ibrutinib resistance in MCL and suggest a strategy to override both primary and acquired ibrutinib resistance. We have discovered the first relapse-specific BTK mutation in patients with MCL with acquired resistance, but not primary resistance, to ibrutinib, and demonstrated a rationale for targeting the proliferative resistant MCL cells by inhibiting CDK4 and the cell cycle in combination with ibrutinib in the presence of BTK(WT) or a PI3K inhibitor independent of BTK mutation. As drug resistance remains a major challenge and CDK4 and PI3K are dysregulated at a high frequency in human cancers, targeting CDK4 in genome-based combination therapy represents a novel approach to lymphoma and cancer therapy. Cancer Discov; 4(9); 1022-35. ©2014 AACR. This article is highlighted in the In This Issue feature, p. 973.

Characterization of ibrutinib-sensitive and -resistant mantle lymphoma cells.

Ibrutinib inhibits Bruton tyrosine kinase (BTK), a key component of early B-cell receptor (BCR) signalling pathways. A multicentre phase 2 trial of ibrutinib in patients with relapsed/refractory mantle cell lymphoma (MCL) demonstrated a remarkable response rate. However, approximately one-third of patients have primary resistance to the drug while other patients appear to lose response and develop secondary resistance. Understanding the molecular mechanisms underlying ibrutinib sensitivity is of paramount importance. In this study, we investigated cell lines and primary MCL cells that display differential sensitivity to ibrutinib. We found that the primary cells display a higher BTK activity than normal B cells and MCL cells show differential sensitivity to BTK inhibition. Genetic knockdown of BTK inhibits the growth, survival and proliferation of ibrutinib-sensitive but not resistant MCL cell lines, suggesting that ibrutinib acts through BTK to produce its anti-tumour activities. Interestingly, inhibition of ERK1/2 and AKT, but not BTK phosphorylation per se, correlates well with cellular response to BTK inhibition in cell lines as well as in primary tumours. Our study suggests that, to prevent primary resistance or to overcome secondary resistance to BTK inhibition, a combinatory strategy that targets multiple components or multiple pathways may represent the most effective approach.

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.

Ofatumumab (OFA) Is a Novel Anti-CD20 Monoclonal Antibody (mAb) with Improved Anti-Tumor Activity in Vitro and in Vivo in Mantle Cell Lymphoma (MCL) Pre-Clinical Models.

AbstractAbstract 2757MCL is typically characterized by an aggressive clinical course and inevitable development of refractory disease despite early intervention that often includes: immunotherapy (e.g., rituximab), multi-agent induction chemotherapy and consolidation with high dose chemotherapy and autologous stem cell transplant in first remission. Residual disease at the time of stem cell collection is an important cause for treatment failure. There is a need to evaluate more potent anti-CD20 mAbs capable to kill lymphoma cells with low CD20 surface levels. In Burkitt’s lymphoma (BL) and diffuse large B-cell lymphoma (DLBCL) pre-clinical models we previously demonstrated that OFA was more potent than rituximab (RIT) in vitro and in vivo. In order to characterize the activity of OFA against MCL, we evaluated the activity of OFA against cytarabine (Ara-C)-sensitive (eg. Mino, Jeko-1, Rec-1, HBL-2, Granta and Z-138); –resistant MCL cell lines (eg. MinoAraCR, Jeko-1AraCR, Rec-1AraCR, HBL-2AraCR and GrantaAraCR); and primary tumor cells derived from MCL patients (n=2). Antibody-dependent cellular cytotoxicity (ADCC) and complement dependent cytotoxicity (CDC) were measured by standard 51Cr release assays in MCL exposed to OFA, RIT or isotype control. OFA vs. RIT direct anti-proliferative effects were measured in by alamar blue reduction assay. Apoptosis following in vitro exposure to OFA or RIT was detected by caspase 3/PARP cleavage. Patient tumor cells were isolated from biopsy specimens by negative selection using magnetic beads and incubated with OFA or RIT +/− human serum as a complement source. Cell viability was determined at 48 hours by CellTiterGlo assay. Surface CD20 and the complement inhibitory proteins (CIPs) (CD55 and CD59) density in MCL cell lines was determined by flow cytometry (Image stream) and compared to BL or DLBCL cell lines. For in vivo experiments 6–8 week-old SID mice were inoculated subcutaneously with 5×106 matrigel suspended Z-138 cells. Upon tumor engraftment, mice were assigned to RIT (10mg/kg), OFA (10mg/kg) or control groups. Tumor growth curves were calculated for each group. Mice were sacrificed if tumor size reached >2cm in any dimension. After 6 months, survival was analyzed by Kaplan-Meier analysis and compared by log-rank test. OFA induced significantly higher levels of CDC associated cell lysis compared to RIT in almost all MCL cell lines tested (10/11) (Mino: 53.2% vs 0.2%; MinoAraCR: 72.6% vs. 0.6%; Jeko-1: 33.4% vs. 9.8%; Jeko-1AraCR: 38.3% vs. 2.8%; REC-1: 17% vs 3%; Rec-1AraCR: 7.8% vs. 0.2%; HBL-2: 27.1% vs. 19.2%; HBL-2AraCR: 86.6% vs. 72.2%; GrantaAraCR: 17% vs 0.9%; Z-138: 56.4% vs. 0.65%; all p-values <0.05). No differences in RIT or OFA mediated ADCC or direct signaling was observed. As previously noted in BL and DLBCL models, OFA was capable of inducing a higher degree of CMC even at low CD20 levels in contrast to RIT. In vivo, OFA slowed tumor growth, and prolonged survival in Z-138 bearing SCID mice compared to RIT (median survival for RIT was 127 days vs. not reached for OFA treated animals; p<0.05). Our data suggest that, OFA is more potent than RIT against Ara-C-sensitive and –resistant MCL cells in vitro, delays tumor growth and prolongs survival compared to RIT in an in vivo MCL SCID mouse model, and retains CDC activity despite low CD20 and high CIP surface expression levels. OFA appears to be a promising mAb targeting CD20 in MCL and is undergoing clinical testing in the front-line setting (NCT01527149). Disclosures:Czuczman:Genmab: Unrestricted Research Grant Other.

Integrative Gene Expression Profiling Reveals G6PD-Mediated Resistance to RNA-Directed Nucleoside Analogues in B-Cell Neoplasms

The nucleoside analogues 8-amino-adenosine and 8-chloro-adenosine have been investigated in the context of B-lineage lymphoid malignancies by our laboratories due to the selective cytotoxicity they exhibit toward multiple myeloma (MM), chronic lymphocytic leukemia (CLL), and mantle cell lymphoma (MCL) cell lines and primary cells. Encouraging pharmacokinetic and pharmacodynamic properties of 8-chloro-adenosine being documented in an ongoing Phase I trial in CLL provide additional impetus for the study of these promising drugs. In order to foster a deeper understanding of the commonalities between their mechanisms of action and gain insight into specific patient cohorts positioned to achieve maximal benefit from treatment, we devised a novel two-tiered chemoinformatic screen to identify molecular determinants of responsiveness to these compounds. This screen entailed: 1) the elucidation of gene expression patterns highly associated with the anti-tumor activity of 8-chloro-adenosine in the NCI-60 cell line panel, 2) characterization of altered transcript abundances between paired MM and MCL cell lines exhibiting differential susceptibility to 8-amino-adenosine, and 3) integration of the resulting datasets. This approach generated a signature of seven unique genes including G6PD which encodes the rate-determining enzyme of the pentose phosphate pathway (PPP), glucose-6-phosphate dehydrogenase. Bioinformatic analysis of primary cell gene expression data demonstrated that G6PD is frequently overexpressed in MM and CLL, highlighting the potential clinical implications of this finding. Utilizing the paired sensitive and resistant MM and MCL cell lines as a model system, we go on to demonstrate through loss-of-function and gain-of-function studies that elevated G6PD expression is necessary to maintain resistance to 8-amino- and 8-chloro-adenosine but insufficient to induce de novo resistance in sensitive cells. Taken together, these results indicate that G6PD activity antagonizes the cytotoxicity of 8-substituted adenosine analogues and suggests that administration of these agents to patients with B-cell malignancies exhibiting normal levels of G6PD expression may be particularly efficacious.

Novel therapy for therapy-resistant mantle cell lymphoma: Multipronged approach with targeting of hedgehog signaling

Mantle cell lymphoma (MCL) is one of the most aggressive B-cell lymphomas with a median patient survival of only 5-7 years. The failure of existing therapies is mainly due to disease relapse when therapy-resistant tumor cells remain after chemotherapy. Therefore, development and testing of novel therapeutic strategies to target these therapy-resistant MCL are needed. Here, we developed an in vivo model of therapy-resistant MCL by transplanting a patient-derived MCL cell line (Granta 519) into NOD/SCID mice followed by treatment with combination chemotherapy. Cytomorphologic, immunophenotypic, in vitro and in vivo growth analyses of these therapy-resistant MCL cells confirm their MCL origin and resistance to chemotherapy. Moreover, quantitative real-time PCR revealed the upregulation of GLI transcription factors, which are mediators of the hedgehog signaling pathway, in these therapy-resistant MCL cells. Therefore, we developed an effective therapeutic strategy for resistant MCL by treating the NOD/SCID mice bearing Granta 519 MCL with CHOP chemotherapy to reduce tumor burden combined with GLI-antisense oligonucleotides or bortezomib, a proteosome inhibitor, to target therapy-resistant MCL cells that remained after chemotherapy. This regimen was followed by treatment with MCL-specific cytotoxic T lymphocytes to eliminate all detectable leftover minimal residual disease. Mice treated with this strategy showed a significantly increased survival and decreased tumor burden compared to the mice in all other groups. Such therapeutic strategies that combine chemotherapy with targeted therapy followed by tumor-specific immunotherapy are effective and have excellent potential for clinical application to provide long-term, disease-free survival in MCL patients.

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.

ABT-737 Induces Apoptosis in Mantle Cell Lymphoma Cells with a Bcl-2high/Mcl-1low Profile and Synergizes with Other Antineoplastic Agents

Mantle cell lymphoma (MCL) is considered to be incurable. ABT-737 is a BH3 mimetic that targets Bcl-2, which is overexpressed in MCL and implicated in drug resistance. The present work investigated the antitumor effect of ABT-737. Six MCL cell lines and primary MCL cells (n = 13) were used. Sensitivity to ABT-737 was assessed, and expression levels of Bcl-2 and Mcl-1 were analyzed. Finally, ABT-737 was combined with other cytotoxic agents to promote tailored therapy. MINO and GRANTA-519 cell lines were highly sensitive to ABT-737 [the median lethal dose (LD₅₀) = 20 and 80 nmol/L, respectively], whereas other cell lines were resistant. In primary MCL cells, 46% of patients' samples were sensitive to ABT-737. The analysis of protein expression levels revealed that both sensitive cell lines and primary MCL cells could be characterized by a Bcl-2(high)/Mcl-1(low) profile, whereas resistant MCL cells contained high levels of Mcl-1. ABT-737 induced a rapid disruption of both Bcl-2/Bax and Bcl-2/Bik complexes. In addition, silencing of Mcl-1 by siRNA sensitized MCL cell lines to ABT-737. Similarly, flavopiridol, which induces Mcl-1 downregulation, in combination with ABT-737 led to a synergistic anti-MCL effect in ABT-737-resistant cell lines. This synergy was also observed when ABT-737 was combined with either bortezomib or cytarabine. The present work shows that ABT-737 induces strong apoptosis in MCL cells expressing a Bcl-2(high)/Mcl-1(low) profile. In ABT-737-resistant MCL cells, downregulation of Mcl-1 overcomes Mcl-1-induced resistance and synergizes ABT-737 effects. Our results strongly support the use of ABT-737 according to the Bcl-2/Mcl-1 tumor cell profiles in the treatment of MCL.

Tackling mantle cell lymphoma (MCL): Potential benefit of allogeneic stem cell transplantation

Mantle cell lymphoma (MCL) is a type of non-Hodgkins lymphoma (NHL) associated with poor progression-free and overall survival. There is a high relapse rate with conventional cytotoxic chemotherapy. Intensive combination chemotherapy including rituximab, dose intense CHOP- (cyclophosphamide-doxorubicin-vincristine-prednisone) like regimens, high dose cytarabine, and/or consolidation with autologous stem cell transplant (autoSCT) have shown promise in significantly prolonging remissions. Data from phase II studies show that even in patients with chemotherapy refractory MCL, allogeneic stem cell transplant (alloSCT) can lead to long term disease control. Most patients with MCL are not candidates for myeloablative alloSCT due to their age, comorbidities, and performance status. The advent of less toxic reduced intensity conditioning (RIC) regimens, which rely more on the graft-versus-lymphoma (GVL) effect, have expanded the population of patients who would be eligible for alloSCT. RIC regimens alter the balance of toxicity and efficacy favoring its use. Treatment decisions are complicated by introduction of novel agents which are attractive options for older, frail patients. Further studies are needed to determine the role and timing of alloSCT in MCL. Currently, for selected fit patients with chemotherapy resistant MCL or those who progress after autoSCT, alloSCT may provide long term survival.

A multicentre phase II trial of gemcitabine for the treatment of patients with newly diagnosed, relapsed or chemotherapy resistant mantle cell lymphoma: SAKK 36/03

Mantle cell lymphoma (MCL) has a poor prognosis with often short and incomplete remissions. We aimed to test the efficacy and tolerability of gemcitabine in treating MCL. Gemcitabine was given in doses of 1000 mg/m(2) as a 30 min infusion on days 1 and 8 of each 3 week cycle for a maximum of nine cycles. Eighteen patients with a median age of 70 years were recruited. MCL was newly diagnosed in half of patients and relapsed in the remainder. Fifteen patients had Ann Arbor stage IV. The best-recorded responses were 1 CR (complete remission), 4 PRs (partial responses), 8 SDs (stable diseases) and 4 PDs (diseases progression). The response rate (RR) (CR + PR) was 5 (28%; 95% confidence interval: 7.1, 48.5). The patient achieving a CR had stage IV disease. Most haematological adverse events occurred during the first chemotherapy cycle. Three patients developed non-haematological serious adverse events: dyspnea, glomerular microangiopathy with haemolytic uremic syndrome (HUS) and hyperglycaemia. The median time-to-progression and treatment response duration (TRD) was 8.0 (95% confidence interval: 5.5, 9.3) and 10.6 (95% confidence interval: 5.5, 10.9) months, respectively. We conclude that Gemcitabine is well tolerated, moderately active and can induce disease stabilization in patients with MCL.

2‐D PAGE‐based comparison of proteasome inhibitor bortezomib in sensitive and resistant mantle cell lymphoma

Although gene expression following bortezomib treatment has been previously explored, direct effects of bortezomib-induced proteasome inhibition on protein level has not been analyzed so far. Using 2-D PAGE in five mantle cell lymphoma cell lines, we screened for cellular protein level alterations following treatment with 25 nM bortezomib for up to 4 h. Using MS, we identified 38 of the 41 most prominent reliably detected protein spots. Twenty-one were affected in all cell lines, whereas the remaining 20 protein spots were exclusively altered in sensitive cell lines. Western blot analysis was performed for 17 of the 38 identified proteins and 70.6% of the observed protein level alterations in 2-D gels was verified. All cell lines exhibited alterations of the cellular protein levels of heat shock-induced protein species (HSPA9, HSP7C, HSPA5, HSPD1), whereas sensitive cell lines also displayed altered cellular protein levels of energy metabolism (ATP5B, AK5, TPI1, ENO-1, ALDOC, GAPDH), RNA and transcriptional regulation (HNRPL, SFRS12) and cell division (NEBL, ACTB, SMC1A, C20orf23) as well as tumor suppressor genes (ENO-1, FH). These proteins clustered in a tight interaction network centered on the major cellular checkpoints TP53. The results were confirmed in primary mantle cell lymphoma, thus confirming the critical role of these candidate proteins of proteasome inhibition.

Noxa mediates bortezomib induced apoptosis in both sensitive and intrinsically resistant mantle cell lymphoma cells and this effect is independent of constitutive activity of the AKT and NF-kappaB pathways

Bortezomib is more active against mantle cell lymphoma (MCL) than against most other lymphoma subtypes. Nevertheless, up to half of patients with MCL have bortezomib resistant disease. Factors contributing to intrinsic resistance to bortezomib have not been determined. Here we used a panel of eight bortezomib sensitive (median IC50 5.9 nM) and three relatively bortezomib resistant cell lines (median IC50 12.9 nM) to investigate differences in tumor biology that could determine sensitivity to bortezomib. Bortezomib effectively inhibited high baseline proteasome activity and induced a comparable degree of proteasome inhibition in both sensitive and resistant cells. At 10 nM, bortezomib induced the proapoptotic BH3-only protein Noxa in sensitive but not resistant cells. At higher concentrations of bortezomib, however, Noxa was also upregulated in resistant cells and this effect was sufficient to induce apoptosis. Silencing of Noxa with siRNA rescued these cells from apoptosis, arguing against a defect in Noxa regulation or function as the basis of bortezomib resistance. Bortezomib was equally effective against cells with high and low constitutive NF-κB signaling. Also, sensitive and resistant MCL cell lines showed comparable activation of the AKT pathway. We conclude that bortezomib can overcome classic mechanisms of resistance to apoptosis and that determinants of bortezomib sensitivity in MCL are due to differences in signaling or stress pathways upstream of Noxa.

A Multicentre Phase II Trial Testing Gemcitabine in the Treatment of Patients with Newly Diagnosed, Relapsed or Chemotherapy Resistant Mantle Cell Lymphoma: SAKK 36/03.

Mantle cell lymphoma (MCL) has a very poor prognosis. Remissions are often incomplete and short with a median survival of less than 3 years and a 5 year survival of < 30%. Young patients are usually treated with intensive chemotherapy regimens or a stem cell transplant. In elderly, unfit patients aggressive treatments are not a realistic approach. This trial set out to assess Gemcitabine in the treatment of patients with newly diagnosed, relapsed or chemotherapy resistant MCL not fit enough for intensive chemotherapy regimens. The primary endpoint was the objective response. Eligibility criteria were a WHO performance status < 2, age ≥ 18 years, a histologically confirmed diagnosis of MCL along with at least one measurable lesion of diameter ≥ 11 mm, ≤ 2 previous lines of chemotherapy. Gemcitabine was given in doses of 1000mg/m2 as a 30 minute infusion on days 1 and 8 of each 3 week cycle for a total of 9 cycles. To prevent flu-like symptoms, one dose of steroids per Gemcitabine infusion was administered. Response duration was read as the time from trial registration to a relapse for patients reaching a remission on the trial therapy. Progression-free survival was defined as the time from trial registration to death or disease progression, treatment failure as early termination of the trial treatment. Patient enrollment began in Aug 2004. Stage I analysis concluded the trial treatment was not promising for further investigation, due to only 1 (10%) patient reaching a remission, and patient accrual stopped after the inclusion of 18 patients in March 2006. Median age at enrollment was 70 years. 66% of the patients were male. MCL was newly diagnosed in 50% of the patients and relapsed in the remainder. Ann Arbor stage IV rating was present in 15 patients, stage II in 1 patient and stage I in 2 patients. Bone marrow involvement was reported in 10 patients. Gastrointestinal tract involvement was reported in 4 patients. Involvement of > 1 extranodal organ was seen in 3 patients. One CR, 4 PRs, 8 SDs and 4 PDs were recorded as a best response. The patient achieving a CR was one of the patients presenting with a stage IV disease. Most myelotoxicities occurred during the first chemotherapy cycle. Neutropenia CTC grade 1 in 24 (19.8%), grade 2 in 35 (28.9%) and grade 3/4 was seen in 24 (19.8%) of the cycles. Thrombocytopenia CTC grade 1 was recorded in 55 (45.5%) cycles and grade 2/3 in 9 (7.7%) cycles. Three patients developed non-hematological serious adverse events, defined as an inpatient or prolonged hospitalisation or a life threatening illness related to the trial medication; dyspnea, glomerular microangiopathy with hemolytic uremic syndrome, and hyperglycemia. The median time-to-progression and response duration was 8.0 (95% confidence interval: 5.5 – 9.3) and 10.6 (95% confidence interval: 5.5 – 10.9) months respectively. The median time-to-treatment failure was not reached. We conclude that Gemcitabine is well tolerated and that it can stabilise MCL in elderly patients. However, Gemcitabine as a monotherapy has only limited activity in MCL patients in terms of treatment response. Further trials should therefore assess gemcitabine in frail patients with MCL.