Genomic deletion and promoter methylation status of Hypermethylated in Cancer 1 (HIC1) in mantle cell lymphoma

Mantle cell lymphomas (MCL), characterized by the t(11;14)(q13;q32), frequently carry secondary genetic alterations such as deletions in chromosome 17p involving the TP53 locus. Given that the association between TP53-deletions and concurrent mutations of the remaining allele is weak and based on our recent report that the Hypermethylated in Cancer 1 (HIC1) gene, that is located telomeric to the TP53 gene, may be targeted by deletions in 17p in diffuse large B-cell lymphoma (DLBCL), we investigated whether HIC1 inactivations might also occur in MCL. Monoallelic deletions of the TP53 locus were detected in 18 out of 59 MCL (31%), while overexpression of p53 protein occurred in only 8 out of 18 of these MCL (44%). In TP53-deleted MCL, the HIC1 gene locus was co-deleted in 11 out of 18 cases (61%). However, neither TP53 nor HIC1 deletions did affect survival of MCL patients. In most analyzed cases, no hypermethylation of the HIC1 exon 1A promoter was observed (17 out of 20, 85%). However, in MCL cell lines without HIC1-hypermethylation, the mRNA expression levels of HIC1 were nevertheless significantly reduced, when compared to reactive lymph node specimens, pointing to the occurrence of mechanisms other than epigenetic or genetic events for the inactivation of HIC1 in this entity

Rituximab But Not GA101 Is Partially Antagonistic With Inhibitors Of The B-Cell Receptor Pathway In Diffuse Large B-Cell Lymphoma

Introduction Anthracyclin-containing immuno-chemotherapy represents the current standard approach in diffuse large cell B cell lymphoma (DLBCL). However, especially in ABC lymphoma new therapeutic approaches are warranted. Small molecule inhibitors of the B- cell receptor pathway have recently achieved high response rates in several lymphoma subtypes. Since rituximab has been previously described to influence the PI3K- AKT pathway, we investigated the impact of rituximab as well as the novel glycoengineered type II anti-CD20 antibody GA101 (obinutuzumab) in combination with the PI3K- delta inhibitor idelalisib and BTK inhibitor ibrutinib. Methods Established DLBCL ABC (U2932, OCI-Ly10, OCI-Ly3, HBL-1) and GCB (HT, WILL-2, SU-DHL-5, SU-DHL-4, ULA) cell lines were cultivated under standard conditions and exposed to previously determined doses of compounds (rituximab [R]: 1 µg/ml, GA101 [G]: 1 µg/ml, idelalisib [ID]: 5 µM, ibrutinib [I]: 5 nM). Viable cells were determined after 24, 48 and 72 hours based on trypane blue exclusion test. Western blot analysis was performed after 1, 6, 12 and 24 h. All experiments were performed at least in triplicates. Results Rituximab in combination with idelalisib showed differential effects in ABC and GCB cell lines. In ABC cell lines the combination was not superior to single substances after 48 h (OCI-LY10: R: 70%, ID: 83%, R+ID: 76%; U2932: R: 63%, ID: 88%, R+ID: 58%) whereas after 72 h additive effects were observed (OCI-LY10: R: 78%, ID: 77%, R+ID: 57%; U2932: R: 58%, ID: 87%, R+ID: 47%). In GCB cell lines, rituximab and idelalisib again were partially antagonistic and did not increase the effect of single drugs (48 h: ULA: R: 79%, ID: 76%, R+ID: 76%; 72 h: SU-DHL-5: R: 87%, ID: 69%, R+ID: 64%). Combination treatment with GA101 and idelalisib was more effective in both subtypes. In ABC cell lines cell counts were additively reduced after 72 h (U2932: G: 40%, ID: 47%, G+ID: 33%; HBL-1: G: 83%, ID: 86%, G+ID: 63%). Similar additive effects were detected in GCB cell lines (SU-DHL-5: G: 91%, ID: 69%, G+ID: 52%; ULA: G: 59%, ID: 68%, G+ID: 50%). As expected, ibrutinib was not effective in GCB cell lines. In ABC cell lines effects of the combination with rituximab or GA101 were comparable to ibrutinib only (OCI-LY10: R: 84%, I: 51%, R+I: 49%; HBL-1: G: 76%, I: 76%, G+I: 77%). In contrast to published data downregulation of p-AKT was detected after antibody treatment in neither ABC nor GCB cell lines. Idelalisib significantly reduced expression of p-AKT already after 1 h in GCB cell lines (ULA). The combination of idelalisib and GA101 also downregulated potently p-AKT whereas the rituximab combination did not reduce p-AKT expression as pronounced. Similar differences were observed In the ABC cell line U2932. Conclusion The combination of rituximab and idelalisib induced a partially antagonistic effect in GCB cell lines. In contrast, in ABC an additive effect of the combination was observed at all time points. Combination of GA101 and idelalisib was more effective in ABC and GCB lymphoma cell lines potentially due to the more pronounced down regulation of p-AKT. These in vitro data suggest that GA101 may overcome the previously reported antagonism of anti CD20 antibodies and inhibitors of the B-cell receptor pathway. However, the relevance of these data has to be validated in clinical trials. Disclosures: Hiddemann: Hoffmann-La Roche: Support of IITs, Scientiffic advisory board, Speakers honoraria Other. Dreyling:Hoffmann-La Roche: Support of IITs, Speakers honoraria, Support of IITs, Speakers honoraria Other; Janssen: Support of IITs, Scientiffic advisory board, Speakers honoraria Other. </jats:sec

Differential Regulation Patterns of Anti-CD20 Antibodies GA101 and Rituximab in Mantle Cell Lymphoma

Abstract Abstract 1839 Background: Mantle cell lymphoma (MCL) is a distinct lymphoma subtype characterized by a poor long-term prognosis. Rituximab, a chimeric type I anti-CD20 antibody has shown an anti-proliferative effect in MCL cell lines and is meanwhile widely clinically applied in combination with chemotherapy. GA101, a type II, glycoengineered CD20 IgG1 antibody has been shown to result in higher direct cell death induction and increased ADCC in comparison to rituximab. In previous experiments GA101 displayed a significant higher cytotoxicity in comparison to rituximab. Aim of this study was the elucidation of the involved downstream signal pathways of the two antibodies. Methods: In two sensitive MCL cell lines (Rec-1, Granta-519) we determined the effect of GA101, rituximab and the combination of both antibodies on cell viability and proliferation. Granta 519 and Rec-1 were treated at a cell density of 5×105 cells/ml with GA101 or rituximab at a previously defined dose of 10 μg/ml. After 4h of exposure samples of 3×106 cells were harvested and processed for 2D-PAGE (polyacrylamide gel electrophoresis) analysis. Protein spots with altered expression after antibody treatment from untreated controls were identified and analyzed by mass spectrometry (MALDI-TOF). In parallel, Affymetrix micro-array analysis of MCL cell lines (Granta-519, HBL-2, Jeko-1, Rec-1 and Z-138) was performed after 4h exposure with either rituximab or GA101. To determine downstream pathways, Ingenuity Pathway Analysis of the identified genes was performed. Results: After mono-exposure with GA101 70% and 40 % cell reduction was achieved in Granta-519 and Rec-1, respectively. In contrast, rituximab led to 25% and 5% in Granta-519 and Rec-1. Interestingly, combination of both antibodies resulted in a cytotoxicity comparable to rituximab monotherapy. Computer-based analysis of the respective 2D-PAGE protein maps revealed 40 and 39 distinct differently expressed protein spots after GA101 and rituximab treatment, respectively. 23 of these protein spots were commonly altered after both antibodies (e.g. CCDC158, MACF1, RAB39, RAD23B) whereas after GA101 treatment 17 proteins (e.g. ENO1, MKI67, NPM1, HSPA5) and after rituximab 16 proteins (e.g. DST, G3BP2, LMO7, PSMD13) were uniquely altered. Micro-array analysis resulted in 2–3 (Granta 519) to 14–78 (HBL; GA101 and rituximab respectively) modulated genes after antibody exposure in all five distinct MCL cell lines. Again, applying a fold-change cut-off of 2, unique candidate genes after GA101 (EGR2, EGR3, NFATC1, SPRY2, ZBTB24 (includes EG: 9841)) and rituximab exposure (BCL2A1, CHL1, LILRA4, LPL, LY9, RHEBL1, SOX11, WNT3) were affected in multiple cell lines. Interestingly, transcriptome and proteome-based analysis characterized different sets of candidate molecules, which were however were mapped to common cellular functions including e.g. “cellular growth and proliferation”, “cell death” and “cell cycle”. Combination of both antibodies resulted in a rituximab-like expression pattern, both on RNA and protein level. Conclusions: Our analyses identified different and antibody-specific downstream expression patterns of GA101 and rituximab, which may represent the molecular basis of the superior effect of GA101 in comparison to rituximab. The simultaneous application of both antibodies resulted in a rituximab-like expression pattern of affected cellular functions and canonical pathways. These data will help to identify a molecular-based rationale for future combined therapeutic approaches and avoid potential antagonist effects. Disclosures: Dreyling: Roche: Support of in vitro studies in MCL. </jats:sec

Cell Cycle Dysregulation Represents an Early Effect of the Proteasome Inhibitor Bortezomib in Mantle Cell Lymphoma.

Abstract Mantle cell lymphoma (MCL) is a distinct subtype of malignant lymphoma with an especially poor clinical outcome, a median survival time of 3 years and virtually no long-term survivors. On the molecular level, MCL is characterized by the chromosomal translocation t(11;14)(q13;q32) resulting in the constitutive overexpression of cyclin D1. However, additional genetic alterations of cell cycle regulators, e.g. deletions of the INK4A gene cluster, are detectable in the majority of cases. In various phase II studies the proteasome inhibitor bortezomib (Velcade) has demonstrated a high clinical efficacy with up to 60% remission rates in relapsed MCL. Additionally, in a previous in vitro study, the inhibitor induced a downregulation of cyclin D1 expression and a concomitant G(1) cell cycle arrest. However, little is known which molecules represent the critical targets of proteosome inhibition and how different regulators of cell cycle and apoptosis (inhibitors of CDK/INK4: p15INK4A, p16INK4B -and p14ARF and other kinase inhibitor proteins/KIP: 21CIP1, p27KIP1 and p57KIP2) are affected. 4 MCL cell lines (HBL2, GRANTA 519, Jeko-1, NCEB-1) and 2 hematological control cell lines (Jurkat, Karpas 422) were exposed to bortezomib at the minimal cytotoxic concentration (25 nmol) which corresponds to clinically achieved drug levels and results in a significant cytolysis after 48 – 72 hours. Real-time RT-PCR and protein expression levels of various CDK inhibitors (INK4s, KIPs) and cyclin D1 were determined a 0, 4, 8 and 12 hours after treatment with bortezomib. In addition, RNA- and protein expression data were compared to functional cell cycle phase (FACS) and cell apoptosis. Prelimenary data indicate that downregulation of cyclin D1 RNA expression after 12 and 24h of treatment represents a rather late event whereas alterations of other cell cycle regulators (like p21CIP1) were detected siginificantly earlier in all four MCL cell lines. Thus, expression of cell cycle regulators may indicate early events of proteasome inhibition. A comparative analysis of the cell cycle regulation network is currently being performed and will be presented at the conference.</jats:p

High Efficiency Of the PDPK1-Inhibitor, BX912, In MCL

Abstract Background Mantle cell lymphoma (MCL) is an aggressive lymphoid malignancy with a median survival of 3-5 years. New strategies including proteasome inhibitors, immune modulatory drugs (IMiDs) and mTOR inhibitors achieve high response rate. Emerging data support the clinical efficiency of inhibitors of the B-cell receptor pathway. PDPK1 (3-phosphoinositide dependent protein kinase-1) is an important downstream target of this crucial pathway. Methods MCL cell lines (Granta 519, Jeko-1, Rec-1) were exposed to various PDK1-Inhibitors (OSU-03012, BX912; BX517; GSK2334470; 0,0625-1µM) and cell proliferation was analysed by WST assay. The effect of BX912 was tested on 6 MCL. Cell proliferation (trypanblue staining), induction of apoptosis (Annexin V PE/7-AAD staining) and cell cycle (FACS) were investigated. In MCL cell lines protein expression of the PI3K/Akt/mTOR pathway candidates (Akt, mTOR, eIF4E, PDK) was analysed after 24h BX912 exposure. Combined approaches were evaluated by cell proliferation analysis (WST-assay, trypan blue staining). In an alternative approach PDPK1 expression was downregulated by siRNA and consequently investigated in detail. Results BX912 appeared to be the most potent PDPK1-Inhibitor in the MCL cell lines tested. Sensitivity to BX912 was detected in 4 out of 6 MCL- (IC50: 0,25 -0,75µM). In addition sensitive MCL cells showed strong G2 arrest. In contrast healthy donor lymphocytes did not respond to PDPK1-inhibition. In MCL cell lines response to BX912 correlated with the PDPK1 protein expression status. Treatment with BX912 led to downregulation of PDPK1 protein expression and dephosphorylation of Rictor, Raptor, RSK and eIF4E proteins in the most sensitive to the inhibitor MCL cell line, Z138, suggesting a mode of action of BX912 mainly through the mTOR pathway. Combination of the PDPK1-inhibitors (BX912; OSU-03012, GSK2334470) with each other revealed synergism especially in combinations with GSK2334470 (BX912 and GSK2334470: CI 0,89; OSU-03012 and GSK2334470: CI 0,484; BX912 and OSU-03012: CI: 1,3), substantiating the therapeutic benefit of comprehensive PDPK1 – inhibition in MCL. Combination experiments of BX912 with inhibitors of the B-cell receptor pathway (PI3K, mTOR, PKCß) and the JAK/STAT-pathway (PIM1, JAK1/2) exhibited BX912 and the PI3K-inhibitor, CAL101, as the most potent combination (CI 0,7 -0,91) in MCL cell lines. To uncover the molecular mode of action of this combination, results of protein expression analysis will be shown. Conclusions The PDPK1 inhibitor BX912 shows high efficiency in MCL cells. Our data let suggest PDPK1 inhibition as a new targeted approach in MCL. However further exploration of the underlying molecular mechanisms is warranted to guide the future development of combined treatment approaches. Disclosures: Dreyling: Janssen: Support of IITs, Scientiffic advisory board, Speakers honoraria Other; Hoffmann-La Roche: Support of IITs, Speakers honoraria, Support of IITs, Speakers honoraria Other. </jats:sec

Proteasome Inhibition Leads to Dephosphorylation and Downregulation of Protein Expression of Members of the Akt/mTOR Pathway In MCL

Abstract Abstract 4449 Background: Mantle cell Lymphoma (MCL) is a distinct B-cell subtype characterized by the chromosomal translocation t(11;14)(q13;q32), an especially poor clinical outcome and low response to chemotherapy. The proteasome inhibitor bortezomib is approved for treatment of relapsed and refractory MCL and achieves a response rate of 30–40%. However, little is known which molecules represent the critical targets of proteasome inhibition and how different regulators of cell cycle and apoptosis are affected. Bortezomib has been shown to inactivate the NFkB pathway in MCL, but recent findings indicate Bortezomib is also active in a proteasome –independent manner suggesting Bortezomib targets multiple pathways. Method: Four MCL cell lines (HBL2, Granta 519, Jeko-1, NCEB-1), two CLL cell lines (Mec1, Mec2) and two hematological control cell lines (Jurkat, Karpas 422) were exposed to Bortezomib at a previously defined cytotoxic concentration (25nmol). Western blot and mRNA analysis were performed for various members of the PI3K/Akt/mTOR and the MEK/ERK pathway after 24h Bortezomib exposure. Results were compared to cell proliferation (WST1, trypan blue staining), induction of apoptosis (Annexin V PE/7-AAD staining) and cell cycle data (FACS). Result: Western blot analysis revealed reduced phosphorylation of Akt at Ser473 in all cell lines while autophosphorylation of mTOR at Ser2481 was completely downregulated only in the susceptible cell lines. In addition further members of the mTOR pathway were affected by bortezomib treatment. Dephosphorylation of the 4EBP1, downregulation of p70S6 protein expression was detected in all cell lines whereas EIF4E dephosphorylation was only observed in the sensitive MCL cell lines. In contrast Bortezomib did not affect members of the MEK/ERK pathway (MEK1/2, p42/44MAPK). Interestingly the mRNA and protein expression profile of CCND1 were differently altered suggesting an involvement of bortezomib in the regulation of translation initiation. This data were also confirmed by microarray analysis. Conclusion: In this study Bortezomib treatment was shown to target the Akt/mTOR pathway especially by dephosphorylation of the translation initiation factor EIF4E and other molecules of this signal pathway. This knowledge will play a crucial role in the development of future combinations of biologicals to target the molecular pathogenesis of MCL. Disclosures: Dreyling: Johnson &amp; Johnson: support of investigators initial trials. </jats:sec

The Functional Impact of PKCβ/PI3K/AKT Signalling on Translational Initiation in MCL

Abstract Introduction: Mantle cell lymphoma (MCL) is an aggressive form of B-cell non-Hodgkin’s lymphoma (NHL). It is characterized by the t(11;14)(q13;q32) translocation, which results in the overexpression of cyclinD1, a cyclin regulated by the PI3K/AKT pathway. Activation of the PI3K/AKT pathway has been shown to be involved in the pathogenesis of MCL. In addition overexpression of the protein kinase C beta (PKCβ) has been described for most cases of MCL, inhibited by enzastaurin which in turn induces apoptosis and reduces proliferation through the PKCβ/PI3K/AKT pathways. 4EBP1 is described as one of the downstream targets of PI3K/mTOR pathway linking translation initiation with PI3K/mTOR signalling as a EIF4E binding protein and playing therefore critical role in the control of protein synthesis, survival and cell growth. Targeting 4EBP1 and/or EIF4E via the PI3K/AKt/mTOR signalling or directly will affect tumor tissue. Aim of the study: The aim of the study was to determine the functional impact of PKCβ/PI3K/AKt/mTOR signaling on the translation initiation factor EIF4E, its binding protein and regulated proteins in MCL cell lines. Methods: MCL cell lines were treated with inhibitors of the PKCβ/PI3K/AKt/mTOR pathways (enzastaurin, LY294002, rapamycin) for up to 48h.The impact of the drugs on the proliferation rate of the cells was accessed after 48h by WST-assay and/or cell count. mRNA expression levels were determined using Taqmanassays. Protein phosphorylation status and protein expression were identified by westernblot. For downregulation of EIF4E in the cells sodium arsenite was used. Specific silencing of EIF4E was achieved by transfection of cells with siRNA against EIF4E. Results: The MCL celllines (5) responded to the treatment with the inhibitors of the PI3K/AKt/mTOR pathway at a IC50 for rapamycin between 5nM-50nM and for the PI3Kinhibitor between 0,31μM-5μM. Treatment of the cells with the PI3K/AKt/mTOR inhibitors induced dephosphorylation of 4EBP1 in a time-and dosedependent manner while a potential effect of the PI3K and mTOR inhibitors on the EIF4E expression and its target genes (cyclinD1, BCL2) could not be shown consistently. 4 out of 5 MCL cell lines were susceptible to enzastaurin with an IC50 between 2μM-5μM. In the not responding to enzastaurin and most resistant to rapamycin cell line (Rec-1) no 4EBP1 proteinexpression was detectable. Dephosphorylation of 4EBP1 achieved by treatment of the cells with sodium arsenit was accompanied by downregulation of EIF4E, cyclinD1 and BCL2 proteins but also stop of proliferation. The potential involvement of eIF4E gene expression in the NaAsO2-induced cytotoxicity and cell death in MCL cell lines was shown by silencing the expression of the eIF4E gene by transfection with siRNA specifically targeting the eIF4E gene expression leading to downregulation of cyclinD1, 4EBP1 proteins and cell proliferation. Conclusion: Eventhough treatment of the cells with the PI3K/AKt/mTOR inhibitors induced dephosphorylation of 4EBP1 a potential effect of the PI3K and mTOR inhibitors on the EIF4E expression and its target genes (cyclinD1, BCL2) could not be shown consistently. Instead dephosphorylation of 4EBP1 accomponied by downregulation of EIF4E or targeted downregulation of eIF4E gene expression lead to downregulation of cyclinD1 and BCL2 proteins as well as cell death in MCL. Therefore targeting the downstream targets of the PI3K/AKt/mTOR signalling 4EBP1 and/or EIF4E directly seems to be a promising anticancer strategy.</jats:p