scholarly journals TAK-243, a Small Molecule Inhibitor of Ubiquitin-Activating Enzyme (UAE), Induces ER Stress and Apoptosis in CLL Cells

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1867-1867
Author(s):  
Scott R Best ◽  
Taylor Rowland ◽  
Cody Paiva ◽  
Nur Bruss ◽  
Stephen E Spurgeon ◽  
...  
Keyword(s):  
B Cells ◽  
Er Stress ◽  
B Cell ◽  
Cell Lines ◽  
Small Molecule ◽  
Research Funding ◽  
Cell Receptor ◽  
B Cell Receptor ◽  
Ubiquitin Conjugation

Abstract Introduction: Despite the promise of B-cell receptor-associated kinase inhibitors (BCRi) in CLL, resistance to these agents is inevitable. Ubiquitin-proteasome systems are altered in cancer, leading to destabilization of tumor suppressors, overexpression of proto-oncogenes (e.g., MYC), and impaired DNA repair. Neoplastic B cells exhibit a state of heightened cellular stress and are thereby susceptible to endoplasmic reticulum (ER) stress and the unfolded protein response (UPR). The UPR is activated in CLL cells upon sIgM signaling and inhibited by ibrutinib. Proteasome inhibitors demonstrate clinical activity in certain types of B-cell neoplasia but are inactive in CLL. Here, we investigated an alternative approach to harness the pro-apoptotic UPR in CLL by using TAK-243, a first-in-class small molecule UAE inhibitor. Methods: Peripheral blood cells were obtained from patients with CLL (N=20) and isolated using Ficoll-Hypaque techniques. TAK-243 was obtained from Millennium Pharmaceuticals, Inc., a wholly owned subsidiary of Takeda Pharmaceutical Company Limited (Cambridge, MA). TAK-243 efficacy was assessed in CLL cells and 10 DLBCL (diffuse large B-cell lymphoma) cell lines. Results: TAK-243 induced ER stress and the UPR in CLL cells, followed by rapid apoptosis within 2 hours. Following 24-hour incubation, we established an IC50 of ~50 nM (Annexin V+ cells) in CLL cells. By contrast, primary B cells and T cells were less sensitive to TAK-243. Given the importance of tumor microenvironment in CLL cell survival, we evaluated the effect of TAK-243 in a CD40L-expressing stromal co-culture model. Whereas CD40L-stimulated CLL cells were resistant to BCRi, they were fully sensitive to UAE inhibition. TAK-243 had a similar IC50 (~50 nM) across DLBCL cell lines, independent of cell of origin. Treatment with TAK-243 rapidly disrupted ubiquitin conjugation and degradation of proteins controlled by the UPS in CLL cells and DLBCL cell lines. UPR induction occurred within 2 hours, as shown by activation of eIF2α (in both CLL and DLBCL cells) and oligomerization and autophosphorylation of PERK (in DLBCL cells). After 4 hours, neoplastic B cells exhibited late apoptotic phase of the UPR: transcriptional induction of CHOP, GADD34, and NOXA. These events were accompanied by upregulation of pro-apoptotic BH3-only proteins, stabilization of Mcl-1 and, ultimately, cleavage of caspase-3 and PARP. TAK-243 inhibited NFκB pathway, as shown by accumulation of IκBα, a negative pathway regulator. The extent of the UPR in CLL cells varies depending on the initiating signal. For example, B-cell receptor crosslinking induced expression of CHOP and GRP78 in CLL cells, but only weak activation of PERK and no IRE1-dependent processing of XBP1 (Krysov S, et al. Blood. 2014). Targeting UAE in CLL cells induced robust activation of eIF2α, upregulation of CHOP, GADD34 as well as NOXA mRNA, indicating high sensitivity to this pathway. TAK-243 induced a more rapid UPR and exhibited lower IC50 compared with the proteasome inhibitor bortezomib in CLL and DLBCL cells. While both drugs induced autophagy as shown by LC3 processing, only bortezomib treatment led to p62 degradation, suggesting that autophagy was inefficient in response to TAK-243 due to lack of ubiquitin conjugation. Our findings were confirmed in a mouse lymphoma xenograft model. OCI-LY3 cells were inoculated subcutaneously in the right flank of NSG mice and treatment with TAK-243 (10 or 20 mg/kg IV twice weekly) or vehicle control began when tumors reached 10 mm in size. Treatment led to reduced tumor progression, induction of ER stress, and decreased cell proliferation and survival. Conclusions: The UAE inhibitor TAK-243 induces ER stress and promotes apoptosis in CLL cells in vitro and restricts lymphoma growth in vivo. TAK-243 exhibited greater in-vitro cytotoxicity in lymphoma cells compared to bortezomib. Targeting UAE is a novel approach to disrupt the UPS which may hold promise in therapy of CLL and other B-cell malignancies. Disclosures Spurgeon: Bristol Myers Squibb: Research Funding; Gilead Sciences, Inc.: Consultancy, Research Funding; Oncternal: Research Funding; Acerta: Research Funding; Genentech: Research Funding; Janssen: Research Funding; Pharmacyclics: Consultancy, Research Funding; MEI Pharma: Consultancy. Berger:Takeda Pharmaceuticals International Co.: Employment. Danilov:TG Therapeutics: Consultancy; Aptose Biosciences: Research Funding; Astra Zeneca: Consultancy; Genentech: Consultancy, Research Funding; Bayer Oncology: Consultancy, Research Funding; Verastem: Consultancy, Research Funding; Gilead Sciences: Consultancy, Research Funding; Takeda Oncology: Research Funding.

Allosteric Akt Inhibitor MK2206 Synergizes with Bendamustine in Promoting the Apoptosis of Chronic Lymphocytic Leukemia Cells and Selectively Targets B-Cell Receptor Mediated Cytokine Production

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3928-3928
Author(s):  
Wei Ding ◽  
Tait D. Shanafelt ◽  
Connie Lesnick ◽  
Traci Sassoon ◽  
Charla Secreto ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Research Funding ◽  
Cytokine Production ◽  
Cell Receptor ◽  
B Cell Receptor ◽  
Leukemic Cells ◽  
Akt Inhibitor ◽  
Akt Activation

Abstract Abstract 3928 Background: Accumulating data support the critical role of PI3K/Akt in CLL B cell receptor (BCR) mediated signal transduction, cell proliferation and survival. In addition recent preclinical and clinical studies indicate that specific PI3K blockade results in robust preclinical and clinical efficacy in CLL. In our model system of CLL B cell-stromal cultures which feature their interaction, platelet derived growth factor (PDGF) present in CLL culture medium drives VEGF production through PI3K/Akt activation in stromal cells (Blood. 2010. 116:2984). Indeed Akt was found to be activated in leukemic cells during the CLL-stroma interaction (Leuk Res. 2008. 32:1565). Therefore, we hypothesized that Akt inhibition should promote CLL B cell apoptosis and abrogate BCR mediated cytokine production. MK2206 is an orally bioavailable highly specific allosteric Akt inhibitor. It has been tested in patients with refractory solid tumors and was demonstrated to be safely administered in a phase I trial. Therefore the goal of this study was to test the preclinical efficacy of MK2206 on both the survival and the BCR mediated cytokine production of CLL leukemic B cells. Methods: Peripheral blood mononuclear cells isolated from CLL patients (n=37) were treated with escalating concentrations of MK2206 (1–16 μM) for 24 hours, 48 hours or 72 hours. The levels of leukemic B cell viability were tested using an (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (MTT) assay. The potential impact (antagonistic/additive/synergistic) of Bendamustine in combination with MK2206 was also tested by using the MTT assay. We used the Calcusyn system to calculate the effect of drug interactions. The combination index (CI) as calculated by the program usually indicates synergy when ≤ 0.8 and indicates additive outcomes when between 0.8–1.2. A CI >1.2 indicates antagonism. Downstream signals of Akt activation in CLL B cells were evaluated by testing their expression of Mcl-1, 4EBP1 and p70S6K using immunoblot. The impact of Akt inhibition by MK2206 on cytokine production in response to B-cell receptor ligation with anti-IgM was also tested using a multiplex cytokine analysis (Invitrogen) in a time-course experiment. Results: MK2206 treatment induced concentration- and time-dependent apoptosis in CLL leukemic cells. At 72 hours, the IC50 of MK2206 in the experiments using CLL leukemic cells in vitro is ∼8 mM. MK2206 incubation at 1 or 5 mM cultured with CLL B cells over a 48-hour period abolished of Akt and p70S6K phosphorylation while native PARP was cleaved into the 85 kD polypeptide fragment. However, the expression level of the upstream signal molecule, PI3K, was not changed. Among the CLL patients tested (n = 37), we did not find any difference in sensitivity to MK2206 induced apoptosis based on critical prognostic factors of CD38, ZAP-70, IGHV and del(17p) status. Importantly, we detected synergistic or additive activity between MK2206 and Bendamustine in 11 tested CLL samples when these combinations were used to treat CLL cells in vitro for 72hrs. Thus the median CI value for this group of patients was 0.8 (0.1 – 1.1). Six were found to have CI ≤ 0.8 and five fell within the additive CI values (0.8 – 1.2). Production of immune or chemotactic cytokines (e.g. CCL3, CCL4, MCP-1, IL-1Ra, IL-8 and IL-2R) at 24 hour incubation increased significantly above baseline when CLL cells were stimulated anti-IgM. Akt inhibition with MK2206 selectively abrogated upregulation of CCL3, CCL4, MCP-1 and IL-2R production, but not for IL-8 or IL-1Ra secretion. MK2206 also abolished BCR mediated Akt activation and decreased Erk activation. Conclusion: MK2206, a robust and selective Akt inhibitor, induced significant in vitro apoptosis of CLL B-cells in vitro. Preclinical evidence of a synergistic effect between MK2206 and Bendamustine was also observed independent of prognostic risk. MK2206 abolished BCR mediated Akt activation and selectively abrogates BCR mediated production of cytokines that may promote apoptotic resistance. These findings support the use of MK2206 in treating CLL and indeed we have initiated a phase I/II trial of MK2206 in combination with Bendamustine and Rituximab for relapsed CLL patients(N1087, October 2011). Acknowledgments: This study was funded by the NCI-K23, NCCTG and CLL Global Foundation. Disclosures: Shanafelt: Cephalon: Research Funding; Genentech: Research Funding. Kumar:Genzyme: Research Funding; Novartis: Research Funding; Celgene: Consultancy, Research Funding; Millennium: Research Funding; Merck: Consultancy, Honoraria. Kay:Celgene: Research Funding.

scholarly journals Pharmacologic Inhibition of B Cell-Receptor-Associated Kinases with CG-806 Induces Apoptosis and Metabolic Reprogramming in Aggressive Non-Hodgkin Lymphoma (NHL) Models

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 29-29
Author(s):  
Elana Thieme ◽  
Vi Lam ◽  
Nur Bruss ◽  
Fei Xu ◽  
Stephen E Kurtz ◽  
...  
Keyword(s):  
B Cell ◽  
Cell Lines ◽  
Research Funding ◽  
Cell Lymphoma ◽  
Cell Receptor ◽  
B Cell Receptor ◽  
Mitochondrial Mass ◽  
Bcr Signaling ◽  
Pdx Models

Introduction: Activated B cell receptor (BCR) signaling is a hallmark of NHL. BCR-associated kinases LYN, SYK, BTK and PI3K activate pro-survival signaling pathways including MEK/ERK, AKT/mTOR, and NFκB. While targeting BTK (ibrutinib, acalabrutinib) and PI3K (idelalisib, duvelisib) has shown efficacy in CLL, clinical responses fall short in aggressive NHL, necessitating the development of novel approaches to suppress BCR signaling. CG-806 is a BTK/cluster-selective kinase inhibitor currently under investigation in phase 1 clinical trials for patients with hematological malignancies. CG-806 targets both WT BTK (IC50 ~ 8 nM) and the BTKC481S (IC50 ~ 2.5 nM; www.aptose.com). Here we investigate the anti-tumor effects of CG-806 in mantle cell lymphoma (MCL) and diffuse large B cell lymphoma (DLBCL). Methods: CG-806 was provided by Aptose Biosciences, Inc. (San Diego, CA). DLBCL and MCL cell lines were assayed for apoptosis/proliferation, metabolic phenotype (Seahorse), mitochondrial mass and mitophagy. Ibrutinib (ibr) resistance was induced by exposure over 6 months. Primary peripheral blood mononuclear cells were incubated for 24 h in media conditioned by stromal cells engineered to express CD40L or BAFF prior to drug treatment. Two MCL PDX models were used (chemo-resistant and ibr-resistant). MCL cells were injected into the tail vein of NSG mice and tracked weekly by flow cytometry (CD5+ CD19+ CD45+). Upon MCL detection in the peripheral blood, mice began daily treatment with 30.8 or 308 mg/kg CG-806 or vehicle control via oral gavage until moribund. Splenocytes were harvested 1 h after the final drug treatment. Results: CG-806 potently inhibited proliferation of both parental and ibr-resistant MCL cell lines (Mino, JeKo-1) with IC50<0.01 μM at 72 h. DLBCL cell lines (U2932, OCI-LY3 OCI-LY19) demonstrated moderate sensitivity to CG-806 (IC50 0.3-1 μM), while SU-DHL10 was highly sensitive (IC50<0.01 µM). Treatment with CG-806, but not ibrutinib, induced apoptosis of primary MCL cells in CD40L- or BAFF-expressing stromal co-cultures. Following anti-IgM crosslinking of primary cells, treatment with CG-806 decreased phosphorylation of SYK, BTK, AKT and ERK, indicating disrupted BCR signaling. Treatment with CG-806 increased respiratory reserve capacity but did not impact the basal oxygen consumption rate in both parental and ibr-resistant MCL cell lines. Basal extracellular acidification rate (ECAR) was increased following CG-806 treatment, indicating heightened glycolytic activity. Furthermore, CG-806-treated cells demonstrated potent induction of mitophagy accompanied by a reduction in mitochondrial mass. CG-806 slowed expansion of circulating MCL cells and reduced proliferation of spleen-resident MCL cells in both chemo- and ibr-resistant MCL PDX models. CG-806 and ibrutinib extended survival of chemoresistant PDX mice without evidence of toxic events. Treatment with CG-806 led to decreased phosphorylation of SYK, BTK, and AKT but also upregulated expression of BCL2 and BCLX. RNA-seq analysis of spleen-resident cells revealed downregulation of NFκB targets and JAK/STAT signaling in ibr-resistant PDX mice treated with CG-806. This was accompanied by enrichment of metabolic pathways (oxidative phosphorylation, fatty acid metabolism) and MYC targets. Next, we evaluated CG-806 for synthetic lethality in a functional in vitro screening assay using a panel of 189 small molecule inhibitors that target a variety of distinct signaling pathways activated in cancer (Tyner et al, 2018). Consistent with the above observations, synergy was observed between CG-806 and inhibitors of metabolic enzymes (teleglenastat, perhexiline maleate) and BH3-mimetics targeting BCL2/X proteins (venetoclax, AZD4320). Conclusions: Our data demonstrate preliminary efficacy of CG-806 in MCL and DLBCL in vitro and in MCL DPX models. CG-806 treatment led to metabolic reprograming towards glycolysis and induced mitophagy. BCL2 family proteins may be implicated in resistance to CG-806. These results provide rationale for further investigation of CG-806 in aggressive NHL. Disclosures Tyner: Array: Research Funding; AstraZeneca: Research Funding; Constellation: Research Funding; Genentech: Research Funding; Incyte: Research Funding; Janssen: Research Funding; Petra: Research Funding; Seattle Genetics: Research Funding; Syros: Research Funding; Takeda: Research Funding; Gilead: Research Funding; Agios: Research Funding; Aptose: Research Funding. Danilov:Pharmacyclics: Consultancy; Astra Zeneca: Consultancy, Research Funding; Verastem Oncology: Consultancy, Research Funding; Takeda Oncology: Research Funding; Gilead Sciences: Research Funding; Bayer Oncology: Consultancy, Research Funding; Genentech: Consultancy, Research Funding; TG Therapeutics: Consultancy; Nurix: Consultancy; Celgene: Consultancy; Aptose Biosciences: Research Funding; Bristol-Myers Squibb: Research Funding; Rigel Pharmaceuticals: Consultancy; Karyopharm: Consultancy; BeiGene: Consultancy; Abbvie: Consultancy.

Investigational Agent MLN2238/MLN9708, a Specific, Orally Available, Small Molecule Proteasome Inhibitor, Shows Promising In Vitro Activity Against Multiple Myeloma Cell Lines

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3014-3014
Author(s):  
Giada Bianchi ◽  
Vijay G. Ramakrishnan ◽  
Teresa Kimlinger ◽  
Jessica Haug ◽  
S. Vincent Rajkumar ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Er Stress ◽  
Cell Lines ◽  
Small Molecule ◽  
Proteasome Inhibitor ◽  
Research Funding ◽  
Proteasome Inhibitors ◽  
Biologically Active ◽  
Investigational Agent

Abstract Abstract 3014 Background: Proteasome inhibitors have proven particularly effective in treatment of multiple myeloma, the second most frequent hematologic malignancy in the western world. Bortezomib, the first in class proteasome inhibitor in clinical use, was first approved in 2003 via fast FDA track, given the remarkable activity shown during phase II clinical trials. Nevertheless, more than 50% of multiple myeloma patients did not respond to single agent bortezomib when administered as second line agent. Moreover, bortezomib is only available for intravenous administration, representing a cumbersome therapy for patients, and its use is limited by significant toxicities (especially peripheral neuropathy). MLN9708 (Millennium Pharmaceuticals, Inc.), an investigational orally available, small molecule, is a potent, specific and reversible inhibitor of the 20S proteasome. It is currently under clinical investigation for the treatment of hematologic and non-hematologic malignancies. Upon exposure to aqueous solutions or plasma, MLN9708 rapidly hydrolyzes to MLN2238, the biologically active form, and MLN2238 was used for all of the preclinical studies reported here. In vitro biochemistry studies have shown that MLN2238 has a faster dissociation rate from the proteasome compared to bortezomib, and in vivo studies of MLN2238 have shown antitumor activity in a broader range of tumor xenografts when compared to bortezomib. Given these encouraging preclinical results, we set to investigate the anti-myeloma activity of MLN2238 in vitro. Results: MLN2238 proved to have anti-proliferative and pro-apoptotic activity against a broad range of MM cell lines with EC50 at 24 hours ranging between 10 and 50 nM, even in relatively resistant MM cell lines (OPM2, DOX6, RPMI, etc.). In MM.1S cells, induction of apoptosis was time and dose dependent and related to activation of both caspase 8 and 9. When compared to MM.1S treated for 24 hours with EC50 dose of bortezomib, treatment with EC50 dose of MLN2238 resulted in the same extent of caspases cleavage occurring at an earlier time point (8-12 hours), possibly suggesting more rapid onset and/or irreversibility of apoptosis in cells treated with MLN2238. Treatment with MLN2238 was associated with early, but persistent induction of endoplasmic reticulum (ER) stress with BiP being induced 2–4 hours after treatment with EC50 dose and gradually increasing over time. While bortezomib has been associated with early induction and late decrease in proteins involved in ER stress, MLN2238 appears to induce a persistent rise in these factors, suggesting either more sustained proteasome blockade with stabilization of proteasome substrates or de-novo induction of unfolded protein response (UPR) genes. MLN2238 also proved effective in reducing phosphorylation of ERK1-2 with no overall alteration in the total ERK level, thus accounting for the observed reduction in proliferation upon treatment. Preliminary data indicate potential for additive and synergistic combination with widely used drugs, including doxorubicin and dexamethasone. Conclusion: While further clinical data are needed to establish the effectiveness of MLN2238 in the treatment of multiple myeloma, these preliminary nonclinical data, together with the favorable biochemical and pharmacokinetic properties, including oral bioavailability, make the investigational agent MLN9708 an appealing candidate for treatment of multiple myeloma. Further in vitro data could help establish whether a difference in the apoptotic mechanisms exist between MLN2238 and other proteasome inhibitors, primarily bortezomib, and could also help inform combination treatment approaches aimed at increasing effectiveness, overcoming bortezomib resistance and decreasing toxicity. Disclosures: Kumar: Celgene: Consultancy, Research Funding; Millennium: Research Funding; Merck: Consultancy, Research Funding; Novartis: Research Funding; Genzyme: Consultancy, Research Funding; Cephalon: Research Funding.

B-Cell Receptor Signaling Suppresses TAp63-Induced Apoptosis Via PI3-K/mTOR Pathway and Upregulation of MiR-21 in Chronic Lymphocytic Leukemia (CLL)

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 562-562
Author(s):  
Leigh Ann Humphries ◽  
Darcy Bates ◽  
Claire Godbersen ◽  
Prabhjot Kaur ◽  
Alexey V. Danilov
Keyword(s):  
B Cells ◽  
B Cell ◽  
Cell Lines ◽  
B Cell Lymphoma ◽  
Cell Receptor ◽  
Mtor Pathway ◽  
B Cell Receptor ◽  
Lymphoma Cell ◽  
Raji Cells ◽  
Transcript Levels

Abstract Abstract 562 p63, an ancestral homolog of p53, encodes two major variants that have variable expression and context-specific functions in malignant tissues. We and others have shown that N-terminally truncated ΔNp63 promotes tumor growth in carcinomas. Meanwhile, the full-length TAp63 variant, which predominates in lymphoid malignancies, is anti-oncogenic in solid tumor models, where it mediates Ras-induced cellular senescence, suppresses anchorage-independent growth, and induces apoptosis. In hepatoma cells, TAp63 activates both extrinsic and intrinsic apoptosis pathways and enhances chemosensitivity. CLL clonal B cells have a low proliferative potential and disrupted apoptotic mechanism as a result of intrinsic defects and interaction with the microenvironment. At the crossroads of those pathways, the B-cell receptor (BCR) serves as a key survival molecule in CLL. Little is known about whether p63 regulates B-cell survival in CLL. Here we sought to investigate the role of TAp63 in regulation of apoptosis in CLL B cells and lymphoma cell lines and determined whether B-cell receptor signaling modulates p63. Forty-eight patients with B-CLL were enrolled in this study. CLL B cells were isolated from peripheral blood using standard Ficoll-Hypaque technique and co-cultured with M210B4 murine stroma cell line layers in RPMI supplemented with 15% fetal bovine serum (FBS). B-cell lymphoma cell lines Daudi, DOHH, Raji, OCI-LY3, OCI-LY19, SU DHL-4 and SUDHL-10 were maintained in RPMI with 10% FBS. CLL B cells and Raji cells were transfected with TAp63α expression vector or with siRNAs targeting p63 or miR-21 using Lonza Nucleofector with B-cell nucleofection solution (CLL B cells) and Solution V (Raji cells). Apoptosis was quantified by means of Annexin V/7-AAD staining and flow cytometry. B-cell receptor was engaged with 5 mg/mL (Raji cells) or 10 mg/mL IgM (CLL B cells). Expression of p63 and miR-21 was evaluated by immunoblotting and real-time RT-PCR. Median age of patients was 63 years. Median follow up was 3 years. Most patients presented in Rai stage 0–1 (80%). TAp63α was the predominantly expressed p63 variant in CLL cells and 7 lymphoma cell lines. Compared with normal B cells, TAp63 mRNA transcript levels were low in 28 CLL patient samples (using an arbitrary cutoff of <10% normal; 58.3%) and normal/elevated in 20 samples (41.7%). Patients with high expression of p63 were more likely to exhibit unmutated IGHV (U-CLL; p=0.016). siRNA-mediated knockdown of p63 in CLL cells resulted in protection from spontaneous apoptosis of CLL cells cultured on M210B4 murine bone marrow stroma (p<0.01) and was accompanied by a reduction in Noxa, Puma and Bax transcript levels. By contrast, enforced expression of TAp63α enhanced apoptosis. p63 knockdown in the Raji lymphoma cells resulted in increased proliferation and metabolic activity (p<0.05). B-cell receptor engagement suppressed p63 expression in CLL cells and Raji lymphoma cells. Pre-incubation of Raji cells with Syk inhibitor R406 and inhibitors of the PI-3K/mTOR pathway (LY294002, rapamycin, and CAL-101) resulted in the reversal of this phenomenon. Meanwhile, chemical inhibition of MEK, Erk, JNK, and p38 and siRNA-mediated knockdown of the transcription factor FOXO (a downstream targets of PI-3K) had no effect on p63 expression. Since TAp63α is a known target of miR-21, a microRNA that has been implicated in the pathogenesis of CLL, we examined their relationship in CLL and lymphoma. We found that TAp63 transcript levels inversely correlated with the expression of miR-21 in CLL B cells, but not in lymphoma cell lines. BCR stimulation led to increased miR-21 levels in CLL B cells, whereas knockdown of miR-21 resulted in upregulation of TAp63 in 3 out of 5 tested samples. TAp63α is the predominantly expressed p63 variant in the peripheral blood CLL cells and B-cell lymphoma cell lines, where it modulates the apoptosis program. BCR signaling repressed TAp63α via PI-3K/mTOR pathway and via upregulation of miR-21. This may be particularly relevant in U-CLL, where baseline p63 levels were higher. These data provide additional insights and rationale for targeting the BCR pathway and miR-21 in CLL and lymphoma. Disclosures: No relevant conflicts of interest to declare.

An Alternatively Spliced Form of CD79b Gene May Account for Altered B-Cell Receptor Expression in B-Chronic Lymphocytic Leukemia

Blood ◽  
1999 ◽  
Vol 93 (7) ◽  
pp. 2327-2335 ◽  
Author(s):  
A. Alfarano ◽  
S. Indraccolo ◽  
P. Circosta ◽  
S. Minuzzo ◽  
A. Vallario ◽  
...  
Keyword(s):  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Cell Lines ◽  
Cell Receptor ◽  
B Cell Receptor ◽  
Lymphocytic Leukemia ◽  
Receptor Expression ◽  
Rt Pcr ◽  
Alternatively Spliced

Several functional anomalies of B-chronic lymphocytic leukemia (B-CLL) cells may be explained by abnormalities of the B-cell receptor (BCR), a multimeric complex formed by the sIg homodimer and the noncovalently bound heterodimer Ig/Igβ (CD79a/CD79b). Because the expression of the extracellular Ig-like domain of CD79b has been reported to be absent in the cells of most CLL cases, we have investigated the molecular mechanisms that may account for this defect. Peripheral blood lymphocytes (PBL) from 50 patients and two cell lines (MEC1, MEC2) obtained from the PBL of one of them were studied. MEC1, MEC2, and 75% of CLL cases did not express detectable levels of the extracellular Ig-like domain of CD79b, which was nevertheless present in greater than 80% CD19+ cells from normal donors. In healthy subjects the expression of CD79b was equally distributed in CD5+ and CD5− B-cell subsets. Reverse transcription-polymerase chain reaction (RT-PCR) analysis of CD79b RNA from all patients and from MEC1 and MEC2 cell lines consistently yielded two fragments of different size (709 bp and 397 bp). The 709-bp band corresponds to CD79b entire transcript; the 397-bp band corresponds to an alternatively spliced form lacking exon 3 that encodes the extracellular Ig-like domain. Both fragments were also visible in normal PBL. The expression of the 397-bp fragment was increased in normal activated B cells, while no difference was seen between CD5+ and CD5− B cells. To obtain a more accurate estimate of the relative proportions of the two spliced forms, a radioactive PCR was performed in 13 normal and 22 B-CLL samples and the results analyzed using a digital imager. The mean value of the CD79b to the CD79b internally deleted ratio was 0.64 ± 0.20 SD in normal donors and 0.44 ± 0.27 SD in B-CLL (P = .01). Direct sequencing of 397-bp RT-PCR products and of genomic DNA corresponding to exon 3 from MEC1, MEC2, their parental cells, and five fresh B-CLL samples did not show any causal mutation. Single-strand conformation polymorphism analysis of exon 3 performed in 18 additional B-CLL cases showed a single abnormal shift corresponding to a TGT → TGC polymorphic change at amino acid 122. We propose a role for the alternative splicing of CD79b gene in causing the reduced expression of BCR on the surface of B-CLL cells. As normal B cells also present this variant, the mechanism of CD79b posttranscriptional regulation might reflect the activation stage of the normal B cell from which B-CLL derives.

scholarly journals B Lymphocyte Chemotaxis Regulated in Association with Microanatomic Localization, Differentiation State, and B Cell Receptor Engagement

1998 ◽  
Vol 187 (5) ◽  
pp. 753-762 ◽  
Author(s):  
Conrad C. Bleul ◽  
Joachim L. Schultze ◽  
Timothy A. Springer
Keyword(s):  
B Cells ◽  
B Cell ◽  
B Lymphocytes ◽  
Messenger Rna ◽  
B Lymphocyte ◽  
Somatic Hypermutation ◽  
Cell Receptor ◽  
B Cell Receptor ◽  
Cxc Chemokine

Migration of mature B lymphocytes within secondary lymphoid organs and recirculation between these sites are thought to allow B cells to obtain T cell help, to undergo somatic hypermutation, to differentiate into effector cells, and to home to sites of antibody production. The mechanisms that direct migration of B lymphocytes are unknown, but there is evidence that G protein–coupled receptors, and possibly chemokine receptors, may be involved. Stromal cell– derived factor (SDF)-1α is a CXC chemokine previously characterized as an efficacious chemoattractant for T lymphocytes and monocytes in peripheral blood. Here we show with purified tonsillar B cells that SDF-1α also attracts naive and memory, but not germinal center (GC) B lymphocytes. Furthermore, GC B cells could be converted to respond to SDF-1α by in vitro differentiation into memory B lymphocytes. Conversely, the migratory response in naive and memory B cells was significantly reduced after B cell receptor engagement and CD40 signaling. The receptor for SDF-1, CXC chemokine receptor 4 (CXCR4), was found to be expressed on responsive as well as unresponsive B cell subsets, but was more rapidly downregulated on responsive cells by ligand. Finally, messenger RNA for SDF-1 was detected by in situ hybridization in a layer of cells surrounding the GC. These findings show that responsiveness to the chemoattractant SDF-1α is regulated during B lymphocyte activation, and correlates with positioning of B lymphocytes within a secondary lymphoid organ.

MEK1/2 Inhibition By MEK162 Is Effective Against Chronic Lymphocytic Leukaemia Cells Under Conditions That Mimic Stimulation of B-Cell Receptor-Mediated Signaling

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3330-3330 ◽  
Author(s):  
Kyle Crassini ◽  
William S Stevenson ◽  
Stephen P. Mulligan ◽  
Oliver Giles Best
Keyword(s):  
Protein Kinase ◽  
B Cell ◽  
Cell Survival ◽  
Research Funding ◽  
Cell Receptor ◽  
B Cell Receptor ◽  
Spontaneous Apoptosis ◽  
Viable Cells ◽  
Cells Cultured

Abstract BACKGROUND Chronic Lymphocytic Leukemia (CLL) is characterised by the clonal expansion of apoptosis resistant B-lymphocytes. However, in vitro and in the absence of pro-survival factors primary CLL cells undergo spontaneous apoptosis. B-cell receptor (BCR) signalling plays a major role in the survival and proliferation of CLL cells, which is highlighted by the clinical efficacy of the Btk and PI3-kinase inhibitors, ibrutinib and idelalisib. Mitogen activated protein kinase (MAPK) is an important mediator of signals downstream of both Btk and PI3-kinase but few studies have shown that inhibitors of MEK1/2, a critical component in the MAPK pathway, have any potential benefit for therapy of CLL. METHODS We sought to investigate the potential of the MEK1/2 inhibitor MEK162 against CLL cells in vitro. To mimic the tonic BCR stimulation experienced in vivo, primary CLL cells were stimulated using an immobilised antibody to IgM or were treated with PMA, a less specific B-cell activator which promotes protein kinase C-dependent MAPK-ERK1/2 signaling. Sensitivity to MEK162 and effects on MAPK-ERK1/2 pathway activity were assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and western blot analyses respectively. RESULTS MEK162 treatment of CLL cells cultured in media alone resulted in a modest but significant (P < 0.01) reduction in cell viability; 20µM MEK162 reduced the proportion of viable cells remaining after 48 h to 77.40 +/- 7.81 % relative to vehicle-treated controls. In contrast, BCR stimulation through IgM ligation promoted cell survival 3.2 +/- 1.01 fold and sensitised CLL cells to MEK162; 20µM MEK162 reduced the proportion of viable cells by 56.28 +/- 2.37 % (P < 0.001 relative to control). A similar effect was observed in response to PMA stimulation; cell viability increased 1.78 +/- 0.15 fold and was reduced by 59.55 +/- 10.33 % (P < 0.001 relative to control) following treatment with 20µM MEK162 (Figure 1). At concentrations > 0.05 µM MEK162 was significantly (P < 0.05) more effective against CLL cells stimulated with either anti-IgM or PMA than against cells cultured in media alone. By western blotting we observed low levels of MAPK-ERK1/2 activity in cells cultured in media alone, which we suggest may contribute to the spontaneous apoptosis of these cells and the low degree of sensitivity to MEK162 under these conditions. We confirmed that stimulation with either IgM or PMA results in activation of MAPK-ERK1/2 and show that this response can be effectively blocked using MEK162. The effects of anti-IgM and PMA on cell survival and response to MEK162 were independent of ZAP-70 expression or ATM/TP53 functional status. CONCLUSIONS Our data illustrate the important role of MAPK-ERK1/2 activity in BCR-mediated CLL cell survival and suggest that MEK162 may have potential for CLL therapy. These data highlight the importance of employing appropriate culture conditions in order to make accurate assessments concerning the efficacy of novel agents for the treatment of CLL. Figure 1. Stimulation with IgM or PMA sensitises B-CLL cells to MEK1/2 inhibition by MEK162. Figure 1. Stimulation with IgM or PMA sensitises B-CLL cells to MEK1/2 inhibition by MEK162. Disclosures Mulligan: Roche: Consultancy, Honoraria, Research Funding, Speakers Bureau; Sanofi Aventis: Research Funding; Janssen: Consultancy, Honoraria, Speakers Bureau; Celgene: Consultancy, Honoraria.

scholarly journals A B Cell Receptor with Two Igα Cytoplasmic Domains Supports Development of Mature But Anergic B Cells

2004 ◽  
Vol 199 (6) ◽  
pp. 855-865 ◽  
Author(s):  
Amy Reichlin ◽  
Anna Gazumyan ◽  
Hitoshi Nagaoka ◽  
Kathrin H. Kirsch ◽  
Manfred Kraus ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Cell Receptor ◽  
Cytoplasmic Domain ◽  
B Cell Receptor ◽  
B1a Cells ◽  
Bcr Signaling ◽  
Membrane Bound

B cell receptor (BCR) signaling is mediated through immunoglobulin (Ig)α and Igβ a membrane-bound heterodimer. Igα and Igβ are redundant in their ability to support early B cell development, but their roles in mature B cells have not been defined. To examine the function of Igα–Igβ in mature B cells in vivo we exchanged the cytoplasmic domain of Igα for the cytoplasmic domain of Igβ by gene targeting (Igβc→αc mice). Igβc→αc B cells had lower levels of surface IgM and higher levels of BCR internalization than wild-type B cells. The mutant B cells were able to complete all stages of development and were long lived, but failed to differentiate into B1a cells. In addition, Igβc→αc B cells showed decreased proliferative and Ca2+ responses to BCR stimulation in vitro, and were anergic to T-independent and -dependent antigens in vivo.

scholarly journals BAR-Bodies: B-Cell Receptor Antigens As the Targeting Moiety of Antibodies in Substitution for the Variable Region of Heavy and Light Chains

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 2940-2940
Author(s):  
Moritz Bewarder ◽  
Lorenz Thurner ◽  
Frank Neumann ◽  
Natalie Fadle ◽  
Evi Regitz ◽  
...  
Keyword(s):  
B Cell ◽  
Cell Line ◽  
Board Of Directors ◽  
Cell Lines ◽  
Research Funding ◽  
Cell Receptor ◽  
B Cell Receptor ◽  
Lymphoma Cell ◽  
Lymphoma Cells ◽  
Advisory Committees

Abstract Background Chronic antigenic stimulation of the B-cell receptor (BCR) seems to play a critical role in the pathogenesis of B-cell lymphomas. We recently identified ARS2 and LRPAP1 as the autoantigenic targets of the B-cell receptors of approximately 25% of diffuse large B cell lymphomas (DLBCLs) of the ABC type and 45% of mantle cell lymphomas (MCLs), respectively. These BCR antigens can be used to target lymphoma cells in an approach we designated as BAR (B-cell receptor antigens for reverse targeting). The optimal therapeutic format BARs can be integrated in has yet to be found. Since the most established approach to deliver therapeutic payloads to specific targets are antibodies which have well-defined pharmacokinetics, we constructed and tested an antibody like construct (BAR-body) incorporating the DLBCL-BAR ARS2 in substitution for the variable domains of the heavy and light chains. Material and methods To create the ARS2 BAR-body, we exchanged the heavy and light chain variable region sequences of an IgG1 antibody with a sequence of similar length (approximately 120 amino acids) of the ARS2 protein (aa 343 - 466) containing the DLBCL reactive epitope (aa 343 - 375). The construct was assembled in a pCR2.1 vector, then transferred to a pSfi FLAG Tag vector for fusion with the FLAG tag and transfected into HEK293 cells for production. Purification of the BAR-body was performed via anti-FLAG antibody affinity chromatography. The BAR-body was detected by western blot analysis and binding capacity to the ARS2-reactive lymphoma cell lines U2932 and OCI-Ly3 and the not ARS2-reactive control DLBCL cell line TMD8 was assessed by flow cytometry. ARS2 BAR-body induced cytotoxicity of lymphoma cells with an ARS2 reactive BCR was measured by LDH release assays with human PBMCs as effector cells at an E:T ratio of 10:1. Results We cloned, expressed and characterized an ARS2 containing BAR-body incorporating 4 molecules of the lymphoma-reactive epitope of ARS2 resulting in an antibody like construct using a BAR (ARS2) as binding moiety instead of normal variable regions. The ARS2 BAR-body could successfully be cloned and expressed as confirmed by western blot analysis, which showed the construct at approximately 150 kD as was to be expected. The BAR-body bound specifically to the ARS2-reactive lymphoma cell lines U2932 and OCI-Ly3 and did not bind to the DLBCL cell line TMD8, which has a B-cell receptor of different specificity or to lymphoma cell lines of different entities. In LDH release assays with 5 x 104 PBMCs and 5 x 103 lymphoma cells (E:T ratio of 10:1) the ARS2 BAR-body induced PBMC mediated specific lysis of the ARS2 reactive lymphoma cell lines U2932 and OCI-Ly3 but not the control DLBCL cell line TMD8 starting at a concentration of 0,1µg/ml. Cytotoxic effects were dose dependent, reached a maximum of 50% specific lysis at a concentration of 1µg/ml and did not increase at concentrations of 10µg/ml. Conclusion Here, we show that BARs can substitute for the variable domains as binding moiety in antibody like constructs to target the BCR of B-cell lymphomas. Because approaches using their specific cognate antigen for targeting the malignant B cells have an exclusive specificity for the BCR of the malignant clone, they can be expected to be less toxic than the currently available antibody derived therapies targeting B-cells, because they leave normal B-lymphocytes unaffected. By incorporating BARs into the well-known format of an antibody we hope to capitalize on years of experience with this therapeutic format from conducting and interpreting in vivo experiments to the translation of the BAR approach into the clinic. Disclosures Stilgenbauer: Genentech: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Hoffmann La-Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Genzyme: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; GSK: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Mundipharma: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Boehringer-Ingelheim: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; AbbVie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharmcyclics: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Sanofi: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

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