British Journal of Haematology
Differential Regulatio n Patterns of Anti -CD20 Antibodies Obinutuzumab and Rituximab in Mantle Cell Lymphoma
Journal:For British Peer Journal of Haematology Review Manuscript ID: BJH-2013-01393.R2
Manuscript Type: Letters
Date Submitted by the Author: n/a
Complete List of Authors: Heinrich, Daniel; Klinikum der Universität München, Med. Klinik und Poliklinik IV Weinkauf, Marc; Klinikum der Universität München, Med. Klinik und Poliklinik III Hutter, Grit; Klinikum der Universität München, Med. Klinik und Poliklinik III Zimmermann, Yvonne; Klinikum der Universität München, Med. Klinik und Poliklinik III Jurinovic, Vindi; Ludwig-Maximilians-Universität München, Institut für medizinische Informationsverarbeitung, Biometrie und Epidemiologie Hiddeman, Wolfgang; Klinikum der Universität München, Med. Klinik und Poliklinik III Dreyling, Martin; Klinikum der Universität München, Med. Klinik und Poliklinik III
Mantle Cell Lymphoma, rituximab, obinutuzumab, anti-CD20, Key Words: MONOCLONAL ANTIBODIES, MCL
Page 1 of 18 British Journal of Haematology
1 2 3 Introduction 4 5 6 7 Mantle cell lymphoma (MCL) is an aggressive B cell Non Hodgkin lymphoma 8 9 characterized by a poor long term prognosis.(Dreyling and Hiddemann 2009) The 10 11 most important step in improving the outcome in MCL has been the introduction of 12 13 the type I anti CD20 monoclonal antibody (mAb) rituximab. In this study we focus on 14 15 16 the comparison of type I mAb rituximab and novel humanized and glycoengineered 17 18 type II obinutuzumab.For In order Peer to reveal common Review and distinct downstream pathways, 19 20 we conducted an in-vitro study in isolated MCL cell lines after rituximab or 21 22 obinutuzumab mono and combination treatment. 23 24
25 26 27 Materials and Methods 28 29 30 MCL cell lines Granta 519, Jeko 1 Rec 1 and Z 138 were provided by the DSMZ 31 32 33 (Braunschweig, Germany) or the respective primary investigator. Experiments were 34 35 performed in triplicates. MCL cells were treated with obinutuzumab and / or rituximab 36 37 at 10 g/ml. Cell viability was analysed by “ViCell Cell Viability Analyzer” tests at 0h, 38 39 24h, 48h and 72h. Fractional product was calculated: synergism > 0,1; antagonism < 40 41 42 0,1. Cell preparation for Affymetrix analysis was performed according to the 43 44 manufacturer’s protocol. The cells were treated for 4h with 10 g/ml obinutuzumab, 45 46 rituximab, human anti IgG antibody (isotype control) and PBS (buffer control, pH 7.2, 47 48 7 l). Genechip HU U133 Plus 2.0 was used for cRNA microarray analysis. All 49 50 samples fulfilled the filter criteria: fold change > 1, mean > 100, call > 0.5 and t test p 51 52 53 value < 0.05. Networks were generated through the use of IPA (Ingenuity® 54 55 Systems). 56 57 58 59 60 British Journal of Haematology Page 2 of 18
1 2 3 Results and discussion 4 5 6 In-vitro effects of obinutuzumab and rituximab 7 8 Obinutuzumab induced a higher reduction in cell viability in each MCL cell line than 9 10 11 rituximab. (Figure 1) Combination experiments suggested the competitive binding of 12 13 the two antibodies due to overlapping epitopes on the target cells and resulted in a 14 15 lower cytotoxicity than obinutuzumab alone, according to fractional product 16 17 calculations (Granta 519: 0.56; Jeko 1: 0.14; Rec 1: 0.44; Z 138: 0.18). Due to the 18 For Peer Review 19 20 high rate of cell kill in Granta 519, micro array data collection was not possible. 21 22 23 24 Commonly deregulated genes after both mono-treatments 25 26 27 19 of the 21 commonly deregulated genes were also deregulated after combination 28 29 therapy. Cytokines CCL3 and CCL4 are upregulated in both mono experiments. 30 31 Cittera et al. identified the upregulation of cytokines CCL3 and CCL4 after rituximab 32 33 administration, presumably involved in the eradication of lymphoma cells.(Cittera , et 34 35 36 al 2007) Francke et al. were able to show that treatment with anti CD20 and B cell 37 38 receptor (BCR) mAbs resulted in a similar deregulation of the transcriptome in 39 40 multiple lymphoma cell lines.(Franke , et al 2011) 41 42 Both mAb treatments share an upregulation of cancer suppressor gene EGR-1 that 43 44 also belongs to the BCR signalling pathway, known to down regulate Survivin , an 45 46 47 anti apoptotic molecule, and to induce caspase signalling resulting in apoptosis 48 49 induction.(Chen , et al 2010) Survivin was silenced by siRNA in MCL cell line Jeko 1, 50 51 followed by a reduction in proliferation. 52 53 Both treatments upregulated DUSP2 , a gene that belongs to the ERK/MAPK 54 55 56 pathway that plays a crucial role in growth suppression. 57 58 (Table 1a) 59 60 2 Page 3 of 18 British Journal of Haematology
1 2 3 Uniquely deregulated genes after obinutuzumab mono-treatment 4 5 6 Obinutuzumab only treatment uniquely deregulated a subset of 21 genes. It induced 7 8 an upregulation of transcription factor AP-1, that is comprised of JUN and FOS 9 10 family members, forming a heterodimer. AP-1 is involved in apoptosis induction, in 11 12 survival and proliferation of cells.(Shaulian and Karin 2001) Low JUN expression in 13 14 Multiple Myeloma (MM) was associated with early disease related deaths.(Chen , et 15 16 17 al 2010) A down regulation of EGR-1 was found to be associated with disease 18 For Peer Review 19 progression in MM patients. EGR-1 is a direct downstream target gene of 20 21 JUN .(Chen , et al 2010) An up regulated EGR 1 gene induces a down regulation of 22 23 Survivin .(Wagner , et al 2008) In consequence, down regulation of Survivin is 24 25 26 followed by an activation of caspase induced apoptosis.(Chen , et al 2010) 27 28 In addition, Chen et al. were able to show that JUN and EGR-1 are key role players 29 30 in the bortezomib induced apoptosis downstream pathway in MM.(Chen , et al 2010) 31 32 Upregulated NFKBIE encodes for a protein known to bind to NF κB components 33 34 targeting the complex for degradation. As a result, the NF κB complex is prevented 35 36 37 from deregulating genes in the nucleus and promoting cell proliferation. The encoded 38 39 protein I,B is deactivated by phosphorylation mediated by IκB kinases. Apart from 40 41 NF B signalling, together with NFATC1, NFKBIE is involved in PI3K signalling. 42 43 (Table 1b) 44 45 46 47 48 Uniquely deregulated genes after rituximab mono-treatment 49 50 Rituximab mono treatment resulted in 29 uniquely altered genes. Rituximab’s 51 52 53 intermediate in-vitro effect might be in part due to further induction of the already 54 55 overexpressed WNT/ß catenin canonical pathway, a key pathway in MCL 56 57 pathogenesis.(Rizzatti , et al 2005) Upregulation of WNT and SOX11 might indicate a 58 59 cellular response leading to less pronounced results in apoptosis induction. 60 3 British Journal of Haematology Page 4 of 18
1 2 3 Downregulated BCL2A belongs to a group of cell death regulators, involved in the 4 5 NF κB pathway facilitating cell survival in healthy and cancer cells.(Vogler 2012). 6 7 Vogler argues that the development of small molecule inhibitors for this target gene 8 9 10 might contribute to anti tumour therapy.(Vogler 2012) (Table 1c) 11 12 13 14 Deregulated genes by combination treatment 15 16 17 The combination experiments disclosed 37 up and 6 downregulated genes. 16 18 For Peer Review 19 genes were unique to both rituximab mono and combination treatments, while 20 21 obinutuzumab and combination exposure shared only two uniquely deregulated 22 23 genes, indicating a rituximab like expression pattern in combination experiments, 24 25 26 similar to the in-vitro findings. Both mAbs have approximately the same affinity 27 28 (obinutuzumab: 4.0 nM vs. rituximab: 4.5 nM KD value) to the human CD20. The in- 29 30 vitro effect is related to the overlapping epitopes, although it may be argued that on a 31 32 particular cell CD20 molecules can be occupied by rituximab and obinutuzumab 33 34 simultaneously.(Mossner , et al 2010) Niederfellner et al . hypothesized that the 35 36 37 excess binding sites for type I mAbs are either due to an abundance of various CD20 38 39 molecules that are targeted by either mAb type or due to steric binding orientations 40 41 and elbow hinge angle conformations.(Niederfellner , et al 2011) Furthermore, the 42 43 CD20 epitope displays twice as many binding sites for type I mAbs as for type II. 44 45 46 (Mossner , et al 2010, Niederfellner , et al 2011) 47 48 49 50 51 52 53 54 55 56 57 58 59 60 4 Page 5 of 18 British Journal of Haematology
1 2 3 In conclusion, new combination experiments are needed to reveal potential additive 4 5 effects in MCL therapy. PI3K inhibitors such as idelalisib, proteasome inhibitors such 6 7 as bortezomib, anti BCR mAbs, siRNA of Survivin or IKK inhibitors are promising 8 9 10 combination partners for obinutuzumab. 11 12 These findings indicate that a combination with obinutuzumab may be more 13 14 promising than rituximab in MCL patients. However, patients recently pre treated with 15 16 rituximab should only be exposed to obinutuzumab after rituximab levels have 17 18 decreased. The understandingFor Peer of the deregulation Review patterns presented in our work 19 20 21 might guide more rational approaches for new combination experiments in MCL. 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 5 British Journal of Haematology Page 6 of 18
1 2 3 Acknowledgements and “Conflict of Interest” statement 4 5 6 7 DH designed the research study, performed the research, analysed the data and 8 9 wrote the paper. MW acted in a supervisory role in the laboratory and assisted in 10 11 analysing the data. GH helped to conceptualize the structure of the study. YZ 12 13 contributed to the execution of the research. VJ also helped to analyse the data. As 14 15 16 chief of the department WH helped conceptualize the study and provided essential 17 18 reagents or tools.For MD acted Peer in an advisory Review role, designed the research study and 19 20 contributed essential reagents or tools. The “Lymphoma Research Foundation” and 21 22 the “European Mantle Cell Lymphoma Network” supported this work. 23 24
25 26 27 DH declares to have a patent pending on obinutuzumab combination treatment. MW, 28 29 GH, YZ, VJ declare no potential conflict of interest. WH declares speaker's honoraria, 30 31 support of investigator initiated trials from Roche. He is on the scientific advisory 32 33 board of Roche. MD declares to receive speaker's honoraria, support of investigator 34 35 36 initiated trials from Roche. He declares to be on the scientific advisory board of 37 38 Roche. He declares to have a patent pending on obinutuzumab combination 39 40 treatment. 41 42 43 44 We thank the “Helmholtz Zentrum München – German Research Center for 45 46 47 Environmental Health” for providing research facilities. We thank Dr. Christian Klein, 48 49 Roche Glycart AG, Schlieren for discussion and providing GA101 and Dr, Helmut 50 51 Burtscher and Ute Baer, Roche Diagnostics GmbH, Penzberg for mRNA profiling 52 53 experiments. 54 55 56 57 58 59 60 6 Page 7 of 18 British Journal of Haematology
1 2 3 References 4 5 6 Chen, L., Wang, S., Zhou, Y., Wu, X., Entin, I., Epstein, J., Yaccoby, S., Xiong, W., Barlogie, B., Shaughnessy, J.D., Jr. & Zhan, 7 F. (2010) Identification of early growth response protein 1 (EGR-1) as a novel target for JUN-induced 8 apoptosis in multiple myeloma. Blood, 115, 61-70. 9 Cittera, E., Leidi, M., Buracchi, C., Pasqualini, F., Sozzani, S., Vecchi, A., Waterfield, J.D., Introna, M. & Golay, J. (2007) The 10 CCL3 family of chemokines and innate immunity cooperate in vivo in the eradication of an established 11 lymphoma xenograft by rituximab. Journal of immunology, 178, 6616-6623. 12 Dreyling, M. & Hiddemann, W. (2009) Current treatment standards and emerging strategies in mantle cell lymphoma. Hematology / the Education Program of the American Society of Hematology. American Society of Hematology. 13 Education Program , 542-551. 14 Franke, A., Niederfellner, G.J., Klein, C. & Burtscher, H. (2011) Antibodies against CD20 or B-cell receptor induce 15 similar transcription patterns in human lymphoma cell lines. PloS one, 6, e16596. 16 Mossner, E., Brunker, P., Moser, S., Puntener, U., Schmidt, C., Herter, S., Grau, R., Gerdes, C., Nopora, A., van Puijenbroek, 17 E., Ferrara, C., Sondermann, P., Jager, C., Strein, P., Fertig, G., Friess, T., Schull, C., Bauer, S., Dal Porto, J., Del 18 Nagro, C., Dabbagh, K., Dyer, M.J., Poppema, S., Klein, C. & Umana, P. (2010) Increasing the efficacy of CD20 antibody therapyFor through the Peer engineering of a newReview type II anti-CD20 antibody with enhanced direct and 19 immune effector cell-mediated B-cell cytotoxicity. Blood, 115, 4393-4402. 20 Niederfellner, G., Lammens, A., Mundigl, O., Georges, G.J., Schaefer, W., Schwaiger, M., Franke, A., Wiechmann, K., 21 Jenewein, S., Slootstra, J.W., Timmerman, P., Brannstrom, A., Lindstrom, F., Mossner, E., Umana, P., Hopfner, 22 K.P. & Klein, C. (2011) Epitope characterization and crystal structure of GA101 provide insights into the 23 molecular basis for type I/II distinction of CD20 antibodies. Blood, 118, 358-367. Rizzatti, E.G., Falcao, R.P., Panepucci, R.A., Proto-Siqueira, R., Anselmo-Lima, W.T., Okamoto, O.K. & Zago, M.A. (2005) 24 Gene expression profiling of mantle cell lymphoma cells reveals aberrant expression of genes from the PI3K- 25 AKT, WNT and TGFbeta signalling pathways. Br J Haematol, 130, 516-526. 26 Shaulian, E. & Karin, M. (2001) AP-1 in cell proliferation and survival. Oncogene, 20, 2390-2400. 27 Vogler, M. (2012) BCL2A1: the underdog in the BCL2 family. Cell Death Differ, 19, 67-74. 28 Wagner, M., Schmelz, K., Dorken, B. & Tamm, I. (2008) Transcriptional regulation of human survivin by early growth 29 response (Egr)-1 transcription factor. Int J Cancer, 122, 1278-1287. 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 7 British Journal of Haematology Page 8 of 18
1 2 3 4 120 5 6 7 100 8 9 10 80 11 12 13 60 14 15 untreated cells 16 40 17 18 Viable cells/ml (x10^6) in % of 19 20 20 For Peer Review 21 22 23 0 Granta-519 Jeko-1 Rec-1 Z-138 24 25 26 27 28 29 Figure 1: In-vitro reduction of cell viability of MCL cell lines Granta-519, Jeko-1, Rec- 30 31 1 und Z-138 after obinutuzumab (dots), rituximab (diagonal lines) and combination 32 33 34 therapy (dark grey) at equimolar concentrations (10 µg/ml) at 72 hours after 35 36 treatment. The indicator of error displays standard deviation by percentage. 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Page 9 of 18 British Journal of Haematology
1 2 3 Table 1a 4 Commonly deregulated genes after rituximab or obinutuzumab mono-treatment up- / down- Symbol Entrez Gene Name Location Type(s) 5 regulated 6 CCL3 chemokine (C-C motif) ligand 3 7 CCL4 chemokine (C-C motif) ligand 4 Extracellular Space cytokine 8 CSF1 colony stimulating factor 1 (macrophage) 9 MAP2 microtubule-associated protein 2 Cytoplasm other NR4A3 nuclear receptor subfamily 4, group A, member 3 ligand-dependent nuclear receptor 10 HIST1H1D histone cluster 1, H1d 11 HIST1H2AJ histone cluster 1, H2aj 12 HIST1H3A (includes others) histone cluster 1, H3a other 13 RGS2 regulator of G-protein signaling 2, 24kDa MALAT1 metastasis associated lung adenocarcinoma transcript 1 (non-protein coding) 14 Nucleus DUSP2 dual specificity phosphatase 2 15 phosphatase DUSP5 dual specificity phosphatase 5 16 EGR1 early growth response 1 17 EGR2 early growth response 2 transcription regulator 18 EGR3 early growth responseFor 3 Peer Review 19 TFEC transcription factor EC 20 AMIGO2 adhesion molecule with Ig-like domain 2 RGS1 regulator of G-protein signaling 1 other 21 SPRED1 sprouty-related, EVH1 domain containing 1 Plasma Membrane 22 SPRY2 sprouty homolog 2 (Drosophila) 23 SLC34A3 solute carrier family 34 (sodium phosphate), member 3 transporter 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 British Journal of Haematology Page 10 of 18
1 2 3 Table 1b 4 5 Uniquely deregulated genes after obinutuzumab mono-treatment up- / down- 6 Symbol Entrez Gene Name Location Type(s) 7 regulated 8 IER2 immediate early response 2 other 9 PLOD2 procollagen-lysine, 2-oxoglutarate 5-dioxygenase 2 Cytoplasm enzyme 10 11 SOCS3 suppressor of cytokine signaling 3 phosphatase 12 C16orf54 chromosome 16 open reading frame 54 other 13 DKK2 dickkopf 2 homolog (XenopusFor laevis) Peer ReviewExtracellular Space 14 PDGFA platelet-derived growth factor alpha polypeptide growth factor 15 16 FOS FBJ murine osteosarcoma viral oncogene homolog 17 IKZF2 IKAROS family zinc finger 2 (Helios) 18 NAB2 NGFI-A binding protein 2 (EGR1 binding protein 2) transcription regulator 19 NFATC1 nuclear factor of activated T-cells, cytoplasmic, calcineurin-dependent 1 20 Nucleus 21 NFKBIE nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, epsilon 22 PPIL4 peptidylprolyl isomerase (cyclophilin)-like 4 enzyme 23 CCNG2 cyclin G2 24 other 25 ZBTB24 zinc finger and BTB domain containing 24 26 ANXA1 annexin A1 Plasma Membrane other 27 CD69 CD69 molecule 28 29 ICAM1 intercellular adhesion molecule 1 Plasma Membrane transmembrane receptor 30 IL21R interleukin 21 receptor 31 MIR155HG MIR155 host gene (non-protein coding) unknown other 32 33 TMEM107 transmembrane protein 107 unknown other 34 FAM65B family with sequence similarity 65, member B unknown other 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Page 11 of 18 British Journal of Haematology
1 2 3 Table 1c 4 Uniquely deregulated genes after rituximab mono-treatment 5 up- / down- Symbol Entrez Gene Name Location Type(s) 6 regulated 7 BCL2A1 BCL2-related protein A1 8 DCAF12 DDB1 and CUL4 associated factor 12 9 other 10 HSPA1A/HSPA1B heat shock 70kDa protein 1A 11 LPL lipoprotein lipase Cytoplasm RHEBL1 Ras homolog enriched in brain like 1 12 enzyme 13 TRIM2 tripartite motif containingFor 2 Peer Review 14 PPP2R4 protein phosphatase 2A activator, regulatory subunit 4 phosphatase 15 CRIM1 cysteine rich transmembrane BMP regulator 1 (chordin-like) kinase 16 SPRED2 sprouty-related, EVH1 domain containing 2 cytokine 17 Extracellular Space COL4A2 collagen, type IV, alpha 2 18 other 19 WNT3 wingless-type MMTV integration site family, member 3 20 IFIH1 interferon induced with helicase C domain 1 enzyme 21 ESRRG estrogen-related receptor gamma ligand-dependent nuclear receptor 22 NFKBID nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, delta 23 RELB v-rel reticuloendotheliosis viral oncogene homolog B Nucleus 24 RFX3 regulatory factor X, 3 (influences HLA class II expression) transcription regulator 25 SOX11 SRY (sex determining region Y)-box 11 26 TP63 tumor protein p63 27 28 CHL1 cell adhesion molecule with homology to L1CAM (close homolog of L1) 29 FAIM3 Fas apoptotic inhibitory molecule 3 30 LILRA4 leukocyte immunoglobulin-like receptor, subfamily A (with TM domain), member 4 other 31 LY9 lymphocyte antigen 9 Plasma Membrane 32 NINJ1 ninjurin 1 33 OCA2 oculocutaneous albinism II 34 transporter SLC38A1 solute carrier family 38, member 1 35 36 HIST1H2AG (includes others) -- MBNL2 muscleblind-like splicing regulator 2 37 unknown other 38 OTUD1 OTU domain containing 1 39 UBALD2 UBA-like domain containing 2 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 British Journal of Haematology Page 12 of 18
1 2 3 4 5 6 7 Introduction 8 9 10 Mantle cell lymphoma (MCL) is an aggressive B cell Non Hodgkin lymphoma 11 12 characterized by a poor long term prognosis.(Dreyling and Hiddemann 2009) The 13 14 most important step in improving the outcome in MCL has been the introduction of 15 16 the type I anti CD20 monoclonal antibody (mAb) rituximab. In this study we focus on 17 18 the comparison of type I mAbFor rituximab Peerand novel humanized Review and glycoengineered 19 type II obinutuzumab. In order to reveal common and distinct downstream pathways, 20 21 we conducted an in-vitro study in isolated MCL cell lines after rituximab or 22 23 obinutuzumab mono and combination treatment. 24 25 26 27 Materials and Methods 28 29 30 MCL cell lines Granta 519, Jeko 1 Rec 1 and Z 138 were provided by the DSMZ 31 32 (Braunschweig, Germany) or the respective primary investigator. Experiments were 33 34 performed in triplicates. MCL cells were treated with obinutuzumab and / or rituximab 35 36 at 10 g/ml. Cell viability was analysed by “ViCell Cell Viability Analyzer” tests at 0h, 37 38 24h, 48h and 72h. Fractional product was calculated: synergism > 0,1; antagonism < 39 40 0,1. Cell preparation for Affymetrix analysis was performed according to the 41 42 manufacturer’s protocol. The cells were treated for 4h with 10 g/ml obinutuzumab, 43 44 rituximab, human anti IgG antibody (isotype control) and PBS (buffer control, pH 7.2, 45 46 7 l). Genechip HU U133 Plus 2.0 was used for cRNA microarray analysis. All 47 48 samples fulfilled the filter criteria: fold change > 1, mean > 100, call > 0.5 and t test p 49 50 value < 0.05. Networks were generated through the use of IPA (Ingenuity® 51 52 Systems). 53 54 55 56 57 58 59 60 Page 13 of 18 British Journal of Haematology
1 2 3 4 5 6 7 Results and discussion 8 9 In-vitro effects of obinutuzumab and rituximab 10 11 Obinutuzumab induced a higher reduction in cell viability in each MCL cell line than 12 13 rituximab. (Figure 1) Combination experiments suggested the competitive binding of 14 15 the two antibodies due to overlapping epitopes on the target cells and resulted in a 16 17 lower cytotoxicity than obinutuzumab alone, according to fractional product 18 For Peer Review 19 calculations (Granta 519: 0.56; Jeko 1: 0.14; Rec 1: 0.44; Z 138: 0.18). Due to the 20 21 high rate of cell kill in Granta 519, micro array data collection was not possible. 22 23 24 25 Commonly deregulated genes after both mono-treatments 26 27 19 of the 21 commonly deregulated genes were also deregulated after combination 28 29 therapy. Cytokines CCL3 and CCL4 are upregulated in both mono experiments. 30 31 Cittera et al. identified the upregulation of cytokines CCL3 and CCL4 after rituximab 32 33 administration, presumably involved in the eradication of lymphoma cells.(Cittera , et 34 35 al 2007) Francke et al. were able to show that treatment with anti CD20 and B cell 36 37 receptor (BCR) mAbs resulted in a similar deregulation of the transcriptome in 38 39 multiple lymphoma cell lines.(Franke , et al 2011) 40 41 Both mAb treatments share an upregulation of cancer suppressor gene EGR-1 that 42 43 also belongs to the BCR signalling pathway, known to down regulate Survivin , an 44 anti apoptotic molecule, and to induce caspase signalling resulting in apoptosis 45 46 induction.(Chen , et al 2010) Survivin was silenced by siRNA in MCL cell line Jeko 1, 47 48 followed by a reduction in proliferation. 49 50 Both treatments upregulated DUSP2 , a gene that belongs to the ERK/MAPK 51 52 pathway that plays a crucial role in growth suppression. 53 54 (Figure 2Table 1a ) 55 56 57 2 58 59 60 British Journal of Haematology Page 14 of 18
1 2 3 4 5 6 7 Uniquely deregulated genes after obinutuzumab mono-treatment 8 9 Obinutuzumab only treatment uniquely deregulated a subset of 21 genes. It induced 10 11 an upregulation of transcription factor AP-1, that is comprised of JUN and FOS 12 13 family members, forming a heterodimer. AP-1 is involved in apoptosis induction, in 14 15 survival and proliferation of cells.(Shaulian and Karin 2001) Low JUN expression in 16 17 Multiple Myeloma (MM) was associated with early disease related deaths.(Chen , et 18 al 2010) A down regulationFor of EGR-1 Peer was found to beReview associated with disease 19 20 progression in MM patients. EGR-1 is a direct downstream target gene of 21 22 JUN .(Chen , et al 2010) An up regulated EGR 1 gene induces a down regulation of 23 24 Survivin .(Wagner , et al 2008) In consequence, down regulation of Survivin is 25 26 followed by an activation of caspase induced apoptosis.(Chen , et al 2010) 27 28 In addition, Chen et al. were able to show that JUN and EGR-1 are key role players 29 30 in the bortezomib induced apoptosis downstream pathway in MM.(Chen , et al 2010) 31 32 Upregulated NFKBIE encodes for a protein known to bind to NF κB components 33 34 targeting the complex for degradation. As a result, the NF κB complex is prevented 35 36 from deregulating genes in the nucleus and promoting cell proliferation. The encoded 37 38 protein I,B is deactivated by phosphorylation mediated by IκB kinases. Apart from 39 40 NF B signalling, together with NFATC1, NFKBIE is involved in PI3K signalling. 41 42 (Figure 2Table 1b ) 43 44 45 46 Uniquely deregulated genes after rituximab mono-treatment 47 48 Rituximab mono treatment resulted in 29 uniquely altered genes. Rituximab’s 49 50 intermediate in-vitro effect might be in part due to further induction of the already 51 52 overexpressed WNT/ß catenin canonical pathway, a key pathway in MCL 53 pathogenesis.(Rizzatti , et al 2005) Upregulation of WNT and SOX11 might indicate a 54 55 cellular response leading to less pronounced results in apoptosis induction. 56 57 3 58 59 60 Page 15 of 18 British Journal of Haematology
1 2 3 4 5 6 7 Downregulated BCL2A belongs to a group of cell death regulators, involved in the 8 NF κB pathway facilitating cell survival in healthy and cancer cells.(Vogler 2012). 9 10 Vogler argues that the development of small molecule inhibitors for this target gene 11 12 might contribute to anti tumour therapy.(Vogler 2012) (Figure 2Table 1c ) 13 14 15 16 Deregulated genes by combination treatment 17 18 The combination experimentsFor disclosed Peer 37 up and 6 Reviewdownregulated genes. 16 19 20 genes were unique to both rituximab mono and combination treatments, while 21 22 obinutuzumab and combination exposure shared only two uniquely deregulated 23 24 genes, indicating a rituximab like expression pattern in combination experiments, 25 26 similar to the in-vitro findings. Both mAbs have approximately the same affinity 27
28 (obinutuzumab: 4.0 nM vs. rituximab: 4.5 nM KD value) to the human CD20. The in- 29 30 vitro effect is related to the overlapping epitopes, although it may be argued that on a 31 32 particular cell CD20 molecules can be occupied by rituximab and obinutuzumab 33 34 simultaneously.(Mossner , et al 2010) Niederfellner et al . hypothesized that the 35 36 excess binding sites for type I mAbs are either due to an abundance of various CD20 37 38 molecules that are targeted by either mAb type or due to steric binding orientations 39 40 and elbow hinge angle conformations.(Niederfellner , et al 2011) Furthermore, the 41 42 CD20 epitope displays twice as many binding sites for type I mAbs as for type II. 43 44 (Mossner , et al 2010, Niederfellner , et al 2011) 45 46 47 48 49 50 51 52 53 54 55 56 57 4 58 59 60 British Journal of Haematology Page 16 of 18
1 2 3 4 5 6 7 In conclusion, new combination experiments are needed to reveal potential additive 8 effects in MCL therapy. PI3K inhibitors such as idelalisib, proteasome inhibitors such 9 10 as bortezomib, anti BCR mAbs, siRNA of Survivin or IKK inhibitors are promising 11 12 combination partners for obinutuzumab. 13 14 These findings indicate that a combination with obinutuzumab may be more 15 16 promising than rituximab in MCL patients. However, patients recently pre treated with 17 18 rituximab should only be For exposed to Peer obinutuzumab afReviewter rituximab levels have 19 20 decreased. These results mayThe understanding of the deregulation patterns 21 22 presented in our work might guide a more rationale approach of Formatted: English (U.S.) 23 Formatted: English (U.S.) 24 obinutuzumabrational approaches for combined treatment optionsnew combination Formatted: English (U.S.) 25 26 experiments in MCL. Formatted: English (U.S.) 27 Formatted: Font: Bold 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 5 58 59 60 Page 17 of 18 British Journal of Haematology
1 2 3 4 5 6 7 8 9 10 11 12 Acknowledgements and “Conflict of Interest” statement 13 14 15 DH designed the research study, performed the research, analysed the data and 16 17 wrote the paper. MW acted in a supervisory role in the laboratory and assisted in 18 For Peer Review 19 analysing the data. GH helped to conceptualize the structure of the study. YZ 20 21 contributed to the execution of the research. VJ also helped to analyse the data. As 22 23 chief of the department WH helped conceptualize the study and provided essential 24 25 reagents or tools. MD acted in an advisory role, designed the research study and 26 27 contributed essential reagents or tools. The “Lymphoma Research Foundation” and 28 the “European Mantle Cell Lymphoma Network” supported this work. 29 30
31 32 DH declares to have a patent pending on obinutuzumab combination treatment. MW, 33 34 GH, YZ, VJ declare no potential conflict of interest. WH declares speaker's honoraria, 35 36 support of investigator initiated trials from Roche. He is on the scientific advisory 37 38 board of Roche. MD declares to receive speaker's honoraria, support of investigator 39 40 initiated trials from Roche. He declares to be on the scientific advisory board of 41 42 Roche. He declares to have a patent pending on obinutuzumab combination 43 44 treatment. 45 46 47 48 We thank the “Helmholtz Zentrum München – German Research Center for 49 50 Environmental Health” for providing research facilities. We thank Dr. Christian Klein, 51 52 Roche Glycart AG, Schlieren for discussion and providing GA101 and Dr, Helmut 53 54 Burtscher and Ute Baer, Roche Diagnostics GmbH, Penzberg for mRNA profiling 55 experiments. 56 57 6 58 59 60 British Journal of Haematology Page 18 of 18
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