scholarly journals Rituximab and Obinutuzumab Induce Direct B-Cell Death Via B-Cell Receptor (BCR) Signaling, but Rituximab Elicits Stronger BCR-Derived Pro-Survival Signals Diminishing Apoptosis

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1579-1579
Author(s):  
Jennifer Edelmann ◽  
Arran Dokal ◽  
Karlheinz Holzmann ◽  
David James Britton ◽  
Emma Vilventhraraja ◽  
...  
Keyword(s):  
Cell Death ◽  
B Cell ◽  
Research Funding ◽  
B Cell Lymphoma ◽  
Cell Receptor ◽  
Improved Outcomes ◽  
Bcr Signaling ◽  
Signaling Events ◽  
Anti Cd20 ◽  
Survival Signals

The anti-CD20 monoclonal antibody (mAb) rituximab in combination with chemotherapy has improved outcomes for patients with CD20+ B-cell lymphoma. Obinutuzumab was developed as an anti-CD20 mAb with enhanced induction of direct B-cell death and antibody-dependent cellular cytotoxicity. In direct comparison to rituximab, improved outcomes with obinutuzumab have been observed for chronic lymphocytic leukemia (CLL) and follicular lymphoma (FL), but not for diffuse large B-cell lymphoma (DLBCL). The molecular basis behind these discrepancies remained unknown. Our aim was to define intracellular signaling events in the induction of direct B-cell death upon rituximab and obinutuzumab treatment. We performed LC-MS/MS phosphoproteomics on SU-DHL4 lymphoma cells treated with rituximab or obinutuzumab (0, 1 and 24 h time-points). Kinase activities were inferred by kinase-substrate enrichment analysis linking kinases to their phosphosite substrates. Immunoblotting was done where necessary to understand the nature of specific signaling events. The activity of 41 and 40 protein kinases was altered upon rituximab or obinutuzumab treatment, respectively, 32 of which were affected by both mAbs. Pathway enrichment analyses revealed up-regulated B-cell receptor (BCR) signaling and down-regulated cell cycle progression with both treatments. To delineate differences between the two mAbs we investigated signaling events in the BCR cascade in more detail. The proximal BCR kinase SYK was strongly phosphorylated on Tyr352 by both mAbs, whereas SYK Tyr525/526 phosphorylation, essential for normal kinase function, was detectable at a low level only with rituximab. Phospho-SYK Tyr352 in the absence of phospho-SYK Tyr525/526 has been associated with autoimmune checkpoint activation and B-cell apoptosis. Both mAbs phosphorylated the key BCR kinase BTK on Tyr551 but not on Tyr223. The latter site was essential for full kinase activity and, in line with incomplete BTK activation, downstream PCLγ2 phosphorylation was delayed. No evidence was found for PLCγ2 dependent de-phosphorylation of NFAT2/NFAT3 and PKCβ activation. Instead, we observed NFAT1 de-phosphorylation and PKCδ activation, both of which have been associated with an anergic B-cell reaction. The MAPK pathway and MYK were strongly activated by both mAbs serving as potent inducers of cell death in the absence of pro-survival signals. To determine if pro-survival signals were generated, we assessed PI3K and NF-κB activation. While we found no evidence for NF-κB activation, differences in the phosphorylation status of PI3K binding sites on CD19 and BCAP suggest stronger PI3K activation by rituximab than obinutuzumab. In keeping with this, the PI3K effector AKT was much more strongly activated by rituximab. AKT activity was likely also increased by Ca2+-flux following rituximab but not obinutuzumab treatment and modified by differential SHIP1 phosphorylation, a phosphatase that hydrolyzes PI3K-generated PIP3. A central AKT target in apoptosis regulation is BAD. We found increased BAD Ser99 and Ser118 phosphorylation only after rituximab treatment. Rituximab thereby sequestered BAD in the cytosol and impaired its inhibitory effects on anti-apoptotic BCL-2 and BCL-xL. Moreover, signaling events protecting against caspase 2 activation were exclusively found for rituximab. In summary, here we show that rituximab and obinutuzumab both induce BCR signaling capable of promoting B-cell apoptosis. However, rituximab generates signals that increase anti-apoptotic BCL-2 effects and diminish B-cell death, whereas obinutuzumab more readily overcomes resistance through BCL-2 overexpression characteristic for FL and CLL. The differences found between rituximab and obinutuzumab are likely less pronounced in cases with strong tonic BCR signaling, which is more prevalent in DLBCL. Thus, our findings provide a mechanistic rationale for the observations made in clinical trials comparing rituximab and obinutuzumab head-to-head. We believe that our results will help to identify new patient subgroups benefitting from obinutuzumab and to better assess the role of anti-CD20 treatment in combination therapies. Disclosures Vilventhraraja: Janssen Pharmaceutical Companies: Employment. Döhner:AbbVie, Agios, Amgen, Astellas, Astex, Celator, Janssen, Jazz, Seattle Genetics: Consultancy, Honoraria; AROG, Bristol Myers Squibb, Pfizer: Research Funding; Celgene, Novartis, Sunesis: Honoraria, Research Funding. Gribben:Acerta/Astra Zeneca: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Abbvie: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Research Funding.

High Density Lipoprotein-like Nanoparticles Synergize with Akt and Btk Inhibitors to Induce Cell Death in Diffuse Large B Cell Lymphomas

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 3022-3022
Author(s):  
Jonathan Scott Rink ◽  
Sol Misener ◽  
Osman Cen ◽  
Shuo Yang ◽  
Leo I. Gordon ◽  
...  
Keyword(s):  
Cell Death ◽  
B Cell ◽  
Cell Lines ◽  
Cell Lymphoma ◽  
Density Lipoprotein ◽  
B Cell Lymphoma ◽  
Cell Receptor ◽  
Cholesterol Homeostasis ◽  
B Cell Lymphomas ◽  
Bcr Signaling

Abstract Introduction: We previously reported that our bio-inspired, synthetic high-density lipoprotein-like nanoparticles (HDL NP) induced apoptosis in B cell lymphoma cells expressing scavenger receptor type B1 (SCARB1), the high-affinity receptor for cholesterol-rich HDLs. HDL NPs consist of a 5nm gold nanoparticle core surface functionalized with the HDL-defining apolipoprotein A1 and a phospholipid bilayer, and bind specifically to SCARB1, inducing the efflux of free cholesterol and inhibiting cholesteryl ester influx. SCARB1 is overexpressed in a subset of follicular and diffuse large B cell lymphomas (DLBCL), and resides in cholesterol-rich plasma membrane microdomains called lipid rafts, similar to the B cell receptor (BCR) and its associated signaling kinases. Upon binding to natural HDL, SCARB1 activates a number of pro-survival signaling kinases, including Akt and PI3K. Both Akt and PI3K are also involved in B cell receptor-mediated signaling in germinal center-derived (GC) DLBCL, through tonic BCR signaling, and activated B cell (ABC) DLBCL, through chronic active BCR signaling. Additionally, PI3K was recently shown to play a role in recruitment and activation of Btk, a crucial survival kinase downstream of the BCR. We hypothesized that small molecule inhibitors against pro-survival kinases, specifically Akt and Btk, will synergize with HDL NPs against B cell lymphomas. Methods: Burkitt's lymphoma (Ramos), GC DLBCL (SUDHL4) and ABC DLBCL (TMD8 and HBL-1) cell lines were treated with the Akt inhibitor GDC-0068 or the Btk inhibitor Ibrutinib, in the absence or presence of HDL NPs, and synergy was calculated using the Calcusyn software. Phos-flow was used to assay for changes in the phosphorylation status of Akt and Btk. Results: The Burkitt's lymphoma and GC DLBCL cell lines were more sensitive to HDL NP induced cell death compared to the ABC DLBCL cell lines (Ramos HDL NP IC50 = 1.5nM; SUDHL4 HDL NP IC50 = 2.1nM; TMD8 HDL NP IC50 = 31.4nM; HBL-1 HDL NP IC50 = 89nM). HDL NPs synergized with GDC-0068 in the Ramos, SUDHL4 and TMD8 cell lines (all combination indexes < 1). Correspondingly, HDL NPs dose-dependently decreased phosphorylation of Akt in Ramos and TMD8 cells. Ibrutinib synergized with the HDL NPs in all cell lines tested (all combination indexes < 1). In TMD8 cells, HDL NPs decreased p-Btk levels comparable to treatment with 10nM Ibrutinib. Addition of the PI3K inhibitor Pilaralisib (XL147) demonstrated mild synergy in the Ramos cell line, but not the SUDHL4, TMD8 or HBL-1 cell lines (all combination index values >1). Treatment of Ramos and SUDHL4 cells with an inhibitor of PTEN, a phosphatase responsible for acting in opposition to PI3K leading to inactivation of Akt, rescued the cells from HDL NP-induced cell death. TMD8 cells treated with the PTEN inhibitor demonstrated a smaller increase in survival when HDL NPs were applied, suggesting that PI3K may not play a major role in HDL NP-induced cell death in activated B cell DLBCLs. PTEN activity is influenced by the level of cholesterol and cholesteryl esters present in the cell, with increasing levels correlating with decreased PTEN activity. Cholesterol levels were higher in the ABC DLBCL cell lines compared to the other B cell lymphoma cell lines. HDL NPs significantly reduced the cholesterol content of Ramos cells, but not the TMD8 or HBL-1 cells, suggesting that the ability of the HDL NPs to alter cellular cholesterol homeostasis correlates with their ability to induce lymphoma cell death. Conclusion: HDL NPs demonstrated synergy with inhibitors to the pro-survival kinases Akt and Btk, suggesting that HDL NPs act to disrupt second messenger signaling pathways in lymphoma cells by directly altering signaling through SCARB1, modulating cellular cholesterol homeostasis, and/or through disruption of membrane raft organization. HDL NPs represent an innovative, targeted therapeutic, with great potential, to add to existing combination chemotherapy regimens. Disclosures Thaxton: Aurasense: Equity Ownership, Patents & Royalties: The patent for the HDL NPs has been licensed to Aurasense, a biotech company co-founded by C. Shad Thaxton..

scholarly journals SYK-dependent tonic B-cell receptor signaling is a rational treatment target in diffuse large B-cell lymphoma

Blood ◽  
2008 ◽  
Vol 111 (4) ◽  
pp. 2230-2237 ◽  
Author(s):  
Linfeng Chen ◽  
Stefano Monti ◽  
Przemyslaw Juszczynski ◽  
John Daley ◽  
Wen Chen ◽  
...  
Keyword(s):  
B Cell ◽  
Cell Lines ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Cell Receptor ◽  
Primary Tumors ◽  
Large B Cell Lymphoma ◽  
Bcr Signaling ◽  
Survival Signals

The role of B-cell receptor (BCR)–mediated survival signals in diffuse large B-cell lymphoma (DLBCL) remains undefined. Ligand-induced BCR signaling induces receptor oligomerization, Igα/β immunoreceptor tyrosine-based activation motif (ITAM) phosphorylation, and activation of the spleen tyrosine kinase (SYK), which initiates downstream events and amplifies the initial BCR signal. BCRs also transmit low-level tonic survival signals in the absence of receptor engagement. Herein, we assess the role of SYK-dependent tonic BCR survival signals in DLBCL cell lines and primary tumors and evaluate the efficacy of an ATP-competitive inhibitor of SYK, R406, in vitro. R406 induced apoptosis of the majority of examined DLBCL cell lines. In R406-sensitive DLBCL cell lines, R406 specifically inhibited both tonic- and ligand-induced BCR signaling (autophosphorylation of SYK525/526 and SYK-dependent phosphorylation of the B-cell linker protein [BLNK]). The majority of examined primary DLBCLs also exhibited tonic- and ligand-induced BCR signaling; in these primary tumors, BCR signaling was also inhibited by R406. Of note, BCR-dependent and R406-sensitive DLBCL cell lines were independently identified as “BCR-type” tumors by transcriptional profiling. Therefore, SYK-dependent tonic BCR signaling is an important and potentially targetable survival pathway in some, but not all, DLBCLs. In addition, R406-sensitive DLBCLs can be identified by their transcriptional profiles.

scholarly journals Ibrutinib in B-cell lymphoma: single fighter might be enough?

2020 ◽  
Vol 20 (1) ◽  
Author(s):  
Chao Xue ◽  
Xin Wang ◽  
Lingyan Zhang ◽  
Qingyuan Qu ◽  
Qian Zhang ◽  
...  
Keyword(s):  
B Cell ◽  
Signaling Pathway ◽  
Cell Lymphoma ◽  
Critical Role ◽  
B Cell Lymphoma ◽  
Cell Receptor ◽  
High Response Rate ◽  
Main Body ◽  
Combination Strategies ◽  
Bcr Signaling

Abstract Background In recent years, the B cell receptor (BCR) signaling pathway has become a “hot point” because it plays a critical role in B-cell proliferation and function. Bruton’s tyrosine kinase (BTK) is overexpressed in many subtypes of B-cell lymphoma as a downstream kinase in the BCR signaling pathway. Ibrutinib, the first generation of BTK inhibitor, has shown excellent antitumor activity in both indolent and aggressive B-cell lymphoma. Main body Ibrutinib monotherapy has been confirmed to be effective with a high response rate (RR) and well-tolerated in many B-cell lymphoma subgroups. To achieve much deeper and faster remission, combination strategies contained ibrutinib were conducted to evaluate their synergistic anti-tumor effect. Conclusions For patients with indolent B-cell lymphoma, most of them respond well with ibrutinib monotherapy. Combination strategies contained ibrutinib might be a better choice to achieve deeper and faster remission in the treatment of aggressive subtypes of B-cell lymphoma. Further investigations on the long-term efficacy and safety of the ibrutinib will provide novel strategies for individualized treatment of B-cell lymphoma.

Targeting B-cell receptor and PI3K signaling in diffuse large B-cell lymphoma

Blood ◽  
2021 ◽  
Author(s):  
Wendan Xu ◽  
Philipp Berning ◽  
Georg Lenz
Keyword(s):  
B Cell ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Cell Receptor ◽  
Genetic Alterations ◽  
B Cell Receptor ◽  
Special Focus ◽  
Large B Cell Lymphoma ◽  
Bcr Signaling ◽  
Large B Cell

Diffuse large B-cell lymphoma (DLBCL) is a heterogeneous diagnostic category comprising distinct molecular subtypes characterized by diverse genetic aberrations that dictate patient outcome. As roughly one-third of DLBCL patients are not cured by current standard chemo-immunotherapy a better understanding of the molecular pathogenesis is warranted to improve outcome. B-cell receptor (BCR) signaling is crucial for the development, growth and survival of both normal and a substantial fraction of malignant B-cells. Various analyses revealed genetic alterations of central components of the BCR or its downstream signaling effectors in some subtypes of DLBCL. Thus, BCR signaling and the downstream NF-κB and PI3K cascades have been proposed as potential targets for the treatment of DLBCL patients. As one of the main effectors of BCR activation, PI3K mediated signals play a crucial role in the pathogenesis and survival of DLBCL. In this review, we summarize our current understanding of BCR signaling with a special focus on the PI3K pathway in DLBCL and how to utilize this knowledge therapeutically.

scholarly journals Survival of human lymphoma cells requires B-cell receptor engagement by self-antigens

2015 ◽  
Vol 112 (44) ◽  
pp. 13447-13454 ◽  
Author(s):  
Ryan M. Young ◽  
Tianyi Wu ◽  
Roland Schmitz ◽  
Moez Dawood ◽  
Wenming Xiao ◽  
...  
Keyword(s):  
B Cell ◽  
B Cell Lymphoma ◽  
Cell Receptor ◽  
B Cell Receptor ◽  
Large B Cell Lymphoma ◽  
Bcr Signaling ◽  
Human Lymphoma ◽  
V Region ◽  
Pathway Inhibition ◽  
Self Antigens

The activated B-cell–like (ABC) subtype of diffuse large B-cell lymphoma (DLBCL) relies on chronic active B-cell receptor (BCR) signaling. BCR pathway inhibitors induce remissions in a subset of ABC DLBCL patients. BCR microclusters on the surface of ABC cells resemble those generated following antigen engagement of normal B cells. We speculated that binding of lymphoma BCRs to self-antigens initiates and maintains chronic active BCR signaling in ABC DLBCL. To assess whether antigenic engagement of the BCR is required for the ongoing survival of ABC cells, we developed isogenic ABC cells that differed solely with respect to the IgH V region of their BCRs. In competitive assays with wild-type cells, substitution of a heterologous V region impaired the survival of three ABC lines. The viability of one VH4-34+ ABC line and the ability of its BCR to bind to its own cell surface depended on V region residues that mediate the intrinsic autoreactivity of VH4-34 to self-glycoproteins. The BCR of another ABC line reacted with self-antigens in apoptotic debris, and the survival of a third ABC line was sustained by reactivity of its BCR to an idiotypic epitope in its own V region. Hence, a diverse set of self-antigens is responsible for maintaining the malignant survival of ABC DLBCL cells. IgH V regions used by the BCRs of ABC DLBCL biopsy samples varied in their ability to sustain survival of these ABC lines, suggesting a screening procedure to identify patients who might benefit from BCR pathway inhibition.

Discrepancy of CD20 Protein Expression In IHC and FCM Analyses In Primary B-Cell Lymphoma: Relationship Between FCM-Negative Phenotype and Rituximab Binding with Lymphoma Cells

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 5087-5087 ◽  
Author(s):  
Takashi Tokunaga ◽  
Akihiro Tomita ◽  
Kazuyuki Shimada ◽  
Junji Hiraga ◽  
Takumi Sugimoto ◽  
...  
Keyword(s):  
B Cell ◽  
Research Funding ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Primary Lymphoma ◽  
Newly Diagnosed ◽  
Rt Pcr ◽  
Lymphoma Cells ◽  
Anti Cd20

Abstract Abstract 5087 Background Rituximab is an anti-CD20 chimeric-monoclonal antibody, and its effectiveness for treatment of CD20-positive B-cell lymphomas has been proven over the past 10 years. Although rituximab is now a key molecular targeting drug for CD20-positive lymphomas, some patients with rituximab resistance have emerged. We previously reported that the CD20-protein-negative phenotypic change after using rituximab is one of the critical mechanisms in rituximab resistance (Hiraga J, Tomita A, et al., Blood, 2009., Sugimoto T, Tomita A, et al., Biochem Biophys Res Commun, 2009.). Recently, we have recognized that some newly-diagnosed B-cell lymphomas show CD20-protein-positive in immunohistochemistry (IHC) but -negative in flow cytometry (FCM) analyses. For these patients, so far, neither the molecular mechanisms of CD20 IHC(+)/FCM(−) phenotype, nor the relationship between this phenotype and rituximab resistance are clear. Thus, the clinical significance of introducing rituximab therapy for these patients must be elucidated. Aims Analyses of the molecular backgrounds of CD20 IHC(+)/FCM(−) phenotype in primary B-lymphoma cells, and confirmation of the effectiveness of rituximab therapy for the patients who show CD20 IHC(+)/FCM(−) phenotype. Results Primary B-cell lymphoma (diffuse large B-cell (DLBCL), follicular, MALT, mantle cell, and Burkitt) tissues and cells were analyzed by IHC and FCM. Four newly-diagnosed B-cell lymphoma patients showed IHC CD79(+)/CD20(+) and FCM CD19(+)/CD20(−) phenotype using anti-CD20 antibodies L26 for IHC and B1 for FCM, and all were diagnosed as DLBCL. Chromosomal analysis showed complex karyotypes in 3 out of 3 patients analyzed, and no shared abnormalities were confirmed. Primary lymphoma cells from 3 patients were available for further molecular analyses, and the genomic DNA, the total RNA, and the protein from whole cell lysate were obtained from these lymphoma cells. DNA sequencing analysis indicated no significant genetic mutations on the coding sequences (CDS) of MS4A1 (CD20) gene. Semi-quantitative and quantitative RT-PCR indicated that CD20 mRNA expression was almost normal in 2 patients and ≂~f10 times lower in 1 patient compared to the positive control B-lymphoma/leukemia cells. Almost the same expression tendency with RT-PCR was confirmed in immunoblot analysis using whole cell lysate and the two different anti-CD20 antibodies. The molecular weight of the CD20 protein in immunoblotting corresponded to the wild type in these patients. Rituximab binding assay in vitro was performed using primary lymphoma cells from a patient and the fluorescent-labeled rituximab (Alexa488-rituximab). Interestingly, rituximab binding on the surface of the CD19 positive lymphoma cells was confirmed in vitro. Rituximab containing combination chemotherapy was performed, resulting in complete response in all 4 cases after completing 4 to 8 courses. Conclusions and Discussion CD20 IHC(+)/FCM(−) phenotype was confirmed in newly-diagnosed DLBCL patients. Significant abnormalities in CD20 protein and mRNA expression in immunoblotting and RT-PCR were not confirmed, and genetic mutations on CDS of MS4A1 gene, resulting in the conformation change of CD20 protein, were not detected. The possibility of abnormal post-translational modification or aberrant localization of CD20 protein, leading to interference with antibody binding, can not be excluded. Rituximab binding with CD19-positive primary lymphoma cells was confirmed in a patient, suggesting that CD20 IHC(+)/FCM(-) phenotype does not directly indicate the ineffectiveness of rituximab for these cells. Further investigations, performing in vitro CDC and ADCC assay using primary lymphoma cells, are still warranted to show rituximab effectiveness and sensitivity to those cells. Disclosures: Kinoshita: Zenyaku Kogyo Co.: Research Funding; Chugai Pharmaceutical Co., Ltd.: Research Funding. Naoe:Zenyaku Kogyo Co.: Research Funding; Chugai Pharmaceutical Co., Ltd.: Research Funding.

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

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4420-4420
Author(s):  
Anna-Katharina Zoellner ◽  
Nico Peter ◽  
Grit Hutter ◽  
Yvonne Zimmermann ◽  
Wolfgang Hiddemann ◽  
...  
Keyword(s):  
B Cell ◽  
Cell Lines ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Cell Receptor ◽  
Advisory Board ◽  
B Cell Receptor ◽  
Additive Effects ◽  
Anti Cd20 ◽  
The Impact

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.

Inhibitors Of B-Cell Receptor Molecules Affect Surface CD20 and Impair Antitumor Activity Of Anti-CD20 Monoclonal Antibodies

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4217-4217
Author(s):  
Kamil Bojarczuk ◽  
Magdalena Winiarska ◽  
Malgorzata Bobrowicz ◽  
Michal Dwojak ◽  
Nina Miazek ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Monoclonal Antibodies ◽  
Antitumor Activity ◽  
B Cell ◽  
Cell Receptor ◽  
B Cell Receptor ◽  
Bcr Signaling ◽  
Anti Cd20 ◽  
Constitutively Active

Abstract Background Anti-CD20 monoclonal antibodies (mAbs) are widely used in the treatment of non-Hodgkin's lymphomas (NHL) and chronic lymphocytic leukemia (CLL). Combining new agents with already used anti-CD20 mAbs seems to be a reasonable approach to further improve current therapeutic options. It seems that signaling via the aberrantly activated B-cell receptor (BCR) plays a key role in the pathogenesis of certain types of B-cell tumors. Blocking BCR pro-survival pathway holds a great therapeutic potential in both NHL and CLL. Several trials are currently being conducted to investigate the effects of combination of BCR-targeting agents with anti-CD20 mAbs–based therapies. To improve these therapeutic approaches in a planned manner it will be utterly important to decipher actual mechanisms of interactions between BCR-targeted therapies and anti-CD20 mAbs in established in vitro models. Aims The aim of this study is to elucidate the role of BCR signaling pathways in the regulation of CD20 levels in B-cell-derived tumor cells and antitumor activity of anti-CD20 mAbs. Methods The project is undertaken fully in in vitro settings in the models of human lymphoma cells as well as primary cells from patients with B-cell tumors. Cells are pre-incubated for 48h with inhibitors of BCR signaling (SYK, BTK, PI3K, AKT, PLC-γ, PKC, mTOR, ERK 1/2) and subsequently tested using flow cytometry for their susceptibility to antitumor activity of anti-CD20 mAbs. Membrane level of CD20 antigen is assessed with FITC-conjugated anti-CD20 antibody staining, total level of CD20 protein is assessed in Western blotting. Transcription processes are analyzed with qPCR, ChIP and EMSA. Moreover, stably transduced lymphoma cells with silenced or constitutively active proteins of interest are employed. Results The results of our preliminary experiments show that blocking BCR network at many stages of the signaling cascade with specific chemical inhibitors or selective shRNA-mediated silencing of SYK or BTK results in considerable down-regulation of CD20 level as determined with flow cytometry. Moreover, a 48-hour incubation with BCR inhibitors leads to a substantial impairment of antitumor activity of anti-CD20 mAbs. Selected inhibitors of BCR signaling considerably decrease CD20 protein level in total cellular lysates as analyzed using Western blotting. In Raji cells incubated with selected BCR inhibitors quantitative real-time PCR shows a significant decrease in CD20 mRNA level. Noteworthy, washout experiments showed that surface CD20 reaches level of control after 96 h from the time that inhibitors were eliminated from the culture media. Studies performed on cell line expressing constitutively active AKT showed up-regulation of CD20 levels at both levels of protein and mRNA. Moreover, constitutively active AKT protects cells from BCR inhibitors-induced decrease of surface CD20. Summary/conclusions Blocking BCR complex network on nearly every step of signal initiation and propagation considerably down-regulates CD20 levels what might have extremely important consequences for the anti-cancer therapy that is based on the use of anti-CD20 mAbs. These studies should provide us with extensive knowledge on the biology of CD20 protein and pathways involved in CD20 regulation. In light of our recent experiments therapeutic combinations of BCR inhibitors and anti-CD20 mAbs-based modalities should be rationally and consciously introduced into clinic in optimized therapeutic schemes. We hypothesize that results of our experiments may lead to identification of the most beneficial therapeutic modalities and schedules that would improve the quality of life of patients suffering from B-cells originating tumors. Disclosures: No relevant conflicts of interest to declare.

Self-Enforcing Feedback Activation Between BCL6 and Tonic Pre-B Cell Receptor Signaling in Acute Lymphoblastic Leukemia

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 284-284
Author(s):  
Huimin Geng ◽  
Christian Hurtz ◽  
Dirk Baumjohann ◽  
Zhengshan Chen ◽  
Wei-Yi Chen ◽  
...  
Keyword(s):  
B Cell ◽  
Tyrosine Kinases ◽  
B Cell Lymphoma ◽  
Cell Receptor ◽  
Bcr Signaling ◽  
Btk Inhibitor ◽  
Bcl6 Expression ◽  
Targeting Strategy

Abstract Background and hypothesis: Like mature B cell lymphoma, pre-B ALL originates from B cell precursors that critically depend on survival signals emanating from a functional (pre-) B cell receptor (BCR). While recent work successfully introduced BCR signaling inhibitors into patient care for various subtypes of mature B cell lymphoma, it is not known whether pre-BCR signaling represents a therapeutic target in pre-B ALL and in which cytogenetic subsets targeting of pre-BCR signaling will be effective. In this study we demonstrated that ALL can be subdivided into two groups that fundamentally differ with respect to pre-BCR signalling. We identified a novel mechanism of self-enforcing feedback activation between the transcription factor BCL6 and tonic pre-BCR signaling in pre-BCR+ ALL and proposed a dual targeting strategy of both BCL6 and pre-BCR related tyrosine kinases for the treatment of patients with pre-BCR+ ALL. Results: Flow cytometry analysis of surface pre-BCR expression (λ5, VpreB), cytoplasmic μ heavy chain (μHC) expression and intracellular Ca2+ signal in 29 patient-derived pre-B ALL xenograft samples and cell lines showed pre-BCR expression and activity in a subset of pre-B ALL, including all TCF3-PBX1 cases studied (n=4) and two cases with deletions at 6q21. Studying 830 pre-B ALL cases from four clinical trials (MDACC, St. Jude, COG P9906 and ECOG E2993), tonic pre-BCR signaling and constitutive PI3K-AKT activation was found in 112 cases (13.5%), including 93% TCF3-PBX1 (53 of 57), del (6)(q21) (7 of 7), PBX1 (1q23) duplication (4 of 4), MLL-rearrangement (3 of 86), hyperdiploid (2 of 43) and other (43 of 406) pre-B ALL cases. In other major ALL subtypes, we found no evidence for pre-BCR expression and activity, including BCR-ABL1 (0 of 196) and ETV6-RUNX1 (0 of 31). We found frequent 1q23 (PBX1) duplication, TCF3-PBX1 or other PBX1-rearrangement, 6q21 (PRDM1) deletion in ALL cells with tonic pre-BCR signaling. Development of a genetic mouse model for inducible ablation of Bcl6. Pre-BCR-induced activation of BCL6 relieves PRDM1-mediated repression of pre-BCR signaling components and positively regulates pre-BCR signaling output at the transcriptional level. The clinical data (COG P9906, ECOG E2993) revealed that high mRNA levels of BCL6 at the time of diagnosis is predictive of poor clinical outcome specifically in patients with pre-BCR+ ALL but not ALL cells lacking pre-BCR expression. These findings suggest an important role of BCL6 as a cofactor of pre-BCR signaling in a large subset of ALL. To directly test the role of Bcl6- and pre-BCR interactions, we generated a novel mouse model for inducible Cre-mediated deletion of Bcl6 exons 5-10, flanked by loxP sites. For lineage-specific deletion in vivo, we crossed these mice with an Mb1-Cre deleter strain, in which Bcl6 was deleted in pro-B cells, resulting in a differentiation block at the pre-B cell stage. Deletion of Bcl6 in mouse pre-BCR+ ALL and expression of a dominate-negative form of BCL6 in human primary pre-BCR+ALL cells, both rapidly induced cell death, indicating BCL6 cooperates with the pre-BCR in leukemic transformation. Cooperation between pre-BCR and BCL6 signaling. Inhibition of BCL6 via the specific BCL6 inhibitor RI-BPI showed compromised colony formation and induced cell cycle arrest. Interestingly, constitutive BCL6 expression was sensitive to inhibition of SYK and SRC tyrosine kinases downstream of the pre-BCR. Treating 6 pre-BCR+ and 8 pre-BCR- patient-derived ALL samples with the SYK inhibitor (PRT06207), BTK inhibitor (Ibrutinib) or a broader SRC and BTK inhibitor Dasatinib, we observed remarkably decreased BCL6 expression and increased apoptosis in pre-BCR+ but not pre-BCR- ALL cells. In vivo treatments with Dasatinib prevented leukemia initiation and significantly prolonged survival of the recipient mice that were injected with primary pre-BCR+ ALL cells, compared to non-treatment or Nilotinib-treatment. These data demonstrate that both inhibition of BCL6 and pre-BCR signaling selectively killed patient-derived pre-BCR+ ALL cells. Conclusions: Our study identified two distinct subtypes of pre-B ALL that fundamentally differ with respect to pre-BCR signaling. Tonic pre-BCR signaling engages a BCL6-dependent, self-enforcing amplification loop. Based on these findings, we propose a dual targeting strategy of BCL6 and pre-BCR tyrosine kinases for the treatment of patients with pre-BCR+ALL. Disclosures No relevant conflicts of interest to declare.

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