p38 MAPK Controls Sensitivity Towards BH3 Mimetics By Regulating Mcl-1 Expression of Chronic Lymphocytic Leukemia in Hypoxia and Acquired Resistance

Abstract Background: CLL patients frequently suffer relapse after an initially successful chemotherapy. This distinct resistance towards chemotherapy is thought to be caused by microenvironmental stimulation. Within the tumor microenvironment (TME) cells are not only stimulated by well-known external stimuli like CD40 ligand (CD40L) or activation of the B cell receptor (BCR), but are also exposed to hypoxia, which was found in the bone marrow and lymphatic tissue. Despite the known importance of hypoxia in solid tumors, its impact on survival and treatment response in CLL is still poorly understood. Methods: We have established a novel in vitro model for the CLL microenvironment, which considers both the external stimulation by CD40L and the hypoxic oxygen levels (1% O2). Treatment efficacy of different drugs in normoxia (21% O2) and hypoxia were determined by AnnexinV/7-AAD staining and subsequent FACS analysis. The underlying molecular mechanisms were analyzed via qRT-PCR and immunoblot. Furthermore B-cell lines Raji, Ramos and Mec-1 were continuously exposed to increasing concentrations of fludarabine or the BH3 mimetic ABT-737. After establishment of resistance the molecular adaptation was assessed and correlated to the changes induced by hypoxia. Results: Hypoxia is known to protect solid cancers from chemotherapy. In our model we made similar observations for CLL, since sensitivity to the classical DNA-targeting drugs fludarabine and bendamustine was reduced under hypoxic conditions. Interestingly, the tyrosine kinase inhibitor ibrutinib did not benefit from hypoxia either. However, this resistance was overcome by the mitochondria-targeting BH3 mimetics ABT-199 and ABT-737, whose effect was pronounced under hypoxia. We reveal that this effect was caused by an uncoupling of major signaling pathways. Under hypoxic conditions the activity of Akt, ERK1/2 and NFκB was reduced, while p38 MAPK became hyperphosphorylated. Phospho-p38 (pp38) downregulated Mcl-1 levels, which are the main regulator of sensitivity towards BH3 mimetics. Despite the known heterogeneity in between CLL patients this effect was found in most samples analyzed. The functional importance was underlined by the observation that pharmacological inhibition of p38 MAPK could reconstitute Mcl-1 levels and thereby resistance in hypoxia. The relevance of the pp38-Mcl-1 axis for ABT efficacy was emphasized by findings in B-cell lines with acquired resistance. Each ABT-resistant clone of the three tested cell lines induced p38 activity and decreased Mcl-1 levels. In contrast, in the fludarabine-resistant clones the pp38-Mcl-1 axis was not altered. Conclusion: These are the first experiments providing evidence that hypoxia has a crucial impact on survival and response to chemotherapy in CLL. We show that hypoxia renders CLL cells resistant to classical DNA-targeting agents, while the small molecules ABT-199 and ABT-737, which specifically target mitochondria, efficiently eradicate CLL cells within the microenvironment. Furthermore, we identified the pp38-Mcl-1 axis to be a major determinant of sensitivity to these BH3 mimetics, which warrants further evaluation of p38 as a novel biomarker for prediction of sensitivity to BH3 mimetics. Disclosures No relevant conflicts of interest to declare.

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CD30 Promotes Proliferation Via ERK 1/2 and p38 MAPK Signal Pathways in ABC-Diffuse Large B Cell Lymphoma

Blood ◽

10.1182/blood-2021-146372 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 4337-4337

Author(s):

Weimin Huang ◽

Xiaolei Wei ◽

Yongqiang Wei ◽

Ru Feng

Keyword(s):

B Cell ◽

P38 Mapk ◽

Cell Lines ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

In Vivo Study ◽

Signal Pathways ◽

Large B Cell Lymphoma ◽

Large B Cell

Abstract Background CD30 was expressed in about 40% diffuse large B cell lymphoma (DLBCL), epically in activated B cell subtype DLBCL(ABC-DLBCL) and implied superior outcome. however, the underlying mechanisms remains unclear. Methods Three ABC-DLBCL cell lines (SuDHL2, TMD8, HBL1) were used for construction of stably CD30 expression cell lines. The proliferation of these cell lines was measured by MTT assay. Cell cycles were detected by Flow Cytometry after staining with PI. Expression of CyclinD1, p27, CDK2, ERK, phosphorylation-ERK, p38, phosphorylation p38 was detected by Western Blot. Nude mice were used in vivo study. Results With CD30 overexpression, the proliferation and division of SuDHL2, TMD8 and HBL1 cell lines were promoted. CyclilnD1, CDK2 were upregulated and p27 was downregulated after of CD30 overexpression. Furthermore, CD30 was knock down by treated CD30 shRNA in CD30 overexpression cell lines. CD30 KD could reverse the the proliferation induced by CD30 expression. CD30 overexpression increased the phosphorylation of ERK and p38 in ABC-DLBCL cells. In vivo study, CD30 overexpression was correlated with cell proliferation and division. Conclusion Taken together, our date showed CD30 promotes proliferation and division by activating ERK 1/2 and p38 MAPK signal pathways, and targeting CD30 may be a novel therapeutic approach for improving the effectiveness of chemotherapy in ABC-DLBCLs. Key words: CD30, ERK 1/2, p38 MAPK, ABC- diffuse large B cell lymphoma Disclosures No relevant conflicts of interest to declare.

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Glucocorticoid Resistant Pediatric Acute Lymphoblastic Leukemia Samples Display Altered Splicing Profile and Vulnerability to Spliceosome Modulation

Cancers ◽

10.3390/cancers12030723 ◽

2020 ◽

Vol 12 (3) ◽

pp. 723

Author(s):

Rocco Sciarrillo ◽

Anna Wojtuszkiewicz ◽

Irsan E. Kooi ◽

Leticia G. Leon ◽

Edwin Sonneveld ◽

...

Keyword(s):

Acute Lymphoblastic Leukemia ◽

Cell Lines ◽

Molecular Mechanisms ◽

Therapeutic Option ◽

Lymphoblastic Leukemia ◽

Leukemia Cell ◽

Differential Splicing ◽

Pediatric Acute Lymphoblastic Leukemia ◽

Response To Chemotherapy ◽

The Impact

Glucocorticoid (GC) resistance is a crucial determinant of inferior response to chemotherapy in pediatric acute lymphoblastic leukemia (ALL); however, molecular mechanisms underlying this phenomenon are poorly understood. Deregulated splicing is a common feature of many cancers, which impacts drug response and constitutes an attractive therapeutic target. Therefore, the aim of the current study was to characterize global splicing profiles associated with GC resistance and determine whether splicing modulation could serve as a novel therapeutic option for GC-resistant patients. To this end, 38 primary ALL samples were profiled using RNA-seq-based differential splicing analysis. The impact of splicing modulators was investigated in GC-resistant leukemia cell lines and primary leukemic specimens. Our findings revealed, for the first time, markedly distinct splicing landscapes in ALL samples of B-cell precursor (BCP)-ALL and T-ALL lineages. Differential splicing events associated with GC resistance were involved in RNA processing, a direct response to GCs, survival signaling, apoptosis, cell cycle regulation and energy metabolism. Furthermore, our analyses showed that GC-resistant ALL cell lines and primary samples are sensitive to splicing modulation, alone and in combination with GC. Together, these findings suggest that aberrant splicing is associated with GC resistance and splicing modulators deserve further interest as a novel treatment option for GC-resistant patients.

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CTCF and Nucleophosmin Mediate Long Distance Cyclin D1 Deregulation in t(11;14) B Cell Malignancies.

Blood ◽

10.1182/blood.v106.11.742.742 ◽

2005 ◽

Vol 106 (11) ◽

pp. 742-742

Author(s):

Elliot M. Epner ◽

Jin Wang ◽

Hui Liu ◽

Bruce Clurman

Keyword(s):

B Cell ◽

Cyclin D1 ◽

Cell Lines ◽

Molecular Mechanisms ◽

Growth Arrest ◽

Regulatory Elements ◽

Similar Distribution ◽

Long Distance ◽

B Cell Malignancies ◽

Regulatory Regions

Abstract Translocations involving the immunoglobulin heavy chain gene locus (IgH) are common in B cell malignancies. One common target gene is cyclin D1, which is deregulated in most patients with mantle cell lymphoma (MCL) and 20–30% of patients with multiple myeloma (MM). Cyclin D1 is known not to be expressed in normal lymphocytes, yet is deregulated by IgH translocations and insertions. The molecular mechanisms of long distance cyclin D1 activation by IgH regulatory elements in B cell malignancies have been obscure. Chromatin immunoprecipitation (ChIP) assays demonstrated the presence of the CCCTC-binding factor (CTCF) at both the cyclin D1 promoter and 3′ IgH regulatory regions only in cyclin D1 expressing malignant B cell lines containing IgH translocations or insertions. The nucleolar protein nucleophosmin (B23; NPM), a newly identified CTCF interaction partner, exhibited a similar distribution in ChIP assays. Combined FISH/immunofluorescence labelling studies have shown the presence of the cyclin D1 loci colocalized with CTCF and nucleophosmin at the nucleolus in MCL cell lines and patient samples, “Knockdown” of NPM by shRNA shows a specific growth arrest in MCL and MM cell lines containing the t(11;14) and cell context dependent apoptosis. This growth arrest is not mediated by changes in cyclin D1 RNA or protein levels. Tethering of the cyclin D1 promoter to the 3′Cα IgH regulatory regions at the nucleolar membrane by CTCF/nucleophosmin allows spatial juxtaposition of the cyclin D1 promoter and 3′IgH regulatory elements and provides a mechanism for long distance cyclin D1 deregulation. Nucleophosmin is also identified as a target for novel therapies in t(11;14) B cell malignancies.

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Molecular Mechanisms Regulating BAFF and APRIL Receptor Expression in B Cells: Promoter Structure and Epigenetics

Blood ◽

10.1182/blood.v112.11.4765.4765 ◽

2008 ◽

Vol 112 (11) ◽

pp. 4765-4765

Author(s):

Stephen A. Mihalcik ◽

Renee C. Tschumper ◽

Diane F. Jelinek

Keyword(s):

B Cells ◽

B Cell ◽

Cell Lines ◽

Promoter Activity ◽

Molecular Mechanisms ◽

Cpg Island ◽

Epigenetic Modification ◽

Regulatory Region ◽

Receptor Expression ◽

B Lineage

Abstract Throughout differentiation, mature B cells express distinct combinations of the BAFF and APRIL receptors, BAFF-R, TACI, and BCMA. The patterns of B lineage cell receptor expression reflect their stage of differentiation and impart the ability to respond to ligands, in some cases delivering a powerful anti-apoptotic signal. B cell malignancies arise from each stage of differentiation, typically exhibit the patterns of receptor expression that reflect their cell of origin, and have been shown to exploit the generally anti-apoptotic effects of BAFF and/or APRIL. For example, there is evidence for a role for BAFF in mature B cell cancers, including B cell chronic lymphocytic leukemia (B-CLL). As the majority of circulating CLL B cells are quiescent cells, prolonged survival is a significant hallmark, a trait that signals through BAFF-R could initiate or reinforce. Therapeutic strategies designed to interrupt this pro-survival pathway have thus far primarily focused on blocking ligand binding. Therapeutic modalities impacting receptor expression may be similarly effective. However, despite the apparent precise activation stage-dependent orchestration of B cell BAFF-R, TACI, and BCMA expression, the genetic mechanisms regulating expression of the three receptors remain undefined, and the question of whether each receptor governs expression of the other two remains unanswered. In agreement with previous studies in our own lab and others, analyses of the receptor profiles of CLL B cells continue to show BAFF-R surface expression, albeit at lower levels than seen on normal peripheral B cells, and the curious variable presence of BCMA and TACI. Similarly, multiple myeloma (MM) plasma cells (PCs), like normal PCs, uniformly lack BAFF-R expression, express BCMA, and variably express TACI. Our current study explores the mechanisms of receptor regulation in B cells, with an emphasis on BAFF-R, the receptor that is most consistently expressed on the CLL B cell population and that has the most clearly defined survival function. We began by analyzing the BAFF-R gene’s genomic context. We identified a possible regulatory element 2 kb upstream of the first exon with significant homology across seven mammalian species that overlapped a cluster of B cell lineage transcription factor binding sites, and, thus, we called the 2.5 kb directly upstream of the gene the putative BAFF-R promoter. We cloned the region and created promoter-reporter vectors in which the full-length promoter and 0.5 kb 5’ truncations thereof drive firefly luciferase production. While studies of primary B cells continue, studies with malignant B cell lines suggest that we have successfully cloned a powerful positive regulatory region. Specifically, B cell lines that express surface BAFF-R show positive inductions of firefly to control renilla luciferase activity in all of the promoter constructs over the empty construct with the greatest promoter activity in the longest constructs: 6-, 18-, and 3-fold inductions with the 2.5 kb promoter in RAJI, Loukes, and MEC-1 cells, respectively. To further test the promoter specificity, we transfected malignant PC lines, ALMC-1, ALMC-2, and KAS-6/1, which are negative by RT-PCR and surface staining for BAFF-R. These lines showed little promoter activity over baseline, with fold inductions between 0.5 and 2.5 for all of the promoter constructs. These results suggest that the MM lines no longer express the transcription factors required to drive BAFF-R expression and underscore our conclusion that we have identified the BAFF-R promoter. At the same time, investigations into epigenetic modification may reveal a crucial level of control. The transcriptional start site of the BAFF-R gene falls within a region of high CG content, and may be a possible CpG island. Upon treatment with the methyltransferase inhibitor, 5-azacytidine, primary blood B cells and MM cells showed no change in receptor expression. However, in CLL B cells, treatment of cultured cells caused a slight (9%) decrease in BAFF-R expression and prevented TACI upregulation in cells stimulated with CpG (a 76% increase fell to 36%). This evidence suggests that methylation indirectly suppresses expression of BAFF-R and TACI. It is essential to understand the regulation of survival receptors critical to normal B lineage cell survival, which may also be crucial for their malignant counterparts, in order to target those mechanisms as therapy.

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Bak and Bcl-xL Participate in Regulating Sensitivity of Solid Tumor Derived Cell Lines to Mcl-1 Inhibitors

Cancers ◽

10.3390/cancers14010181 ◽

2021 ◽

Vol 14 (1) ◽

pp. 181

Author(s):

Viacheslav V. Senichkin ◽

Nikolay V. Pervushin ◽

Alexey V. Zamaraev ◽

Elena V. Sazonova ◽

Anton P. Zuev ◽

...

Keyword(s):

Cell Lines ◽

Acquired Resistance ◽

Preclinical Models ◽

Bh3 Mimetics ◽

Promising Tool ◽

Tumor Tissues ◽

Induced Apoptosis ◽

Low Expression ◽

Dependent Mechanism

BH3 mimetics represent a promising tool in cancer treatment. Recently, the drugs targeting the Mcl-1 protein progressed into clinical trials, and numerous studies are focused on the investigation of their activity in various preclinical models. We investigated two BH3 mimetics to Mcl-1, A1210477 and S63845, and found their different efficacies in on-target doses, despite the fact that both agents interacted with the target. Thus, S63845 induced apoptosis more effectively through a Bak-dependent mechanism. There was an increase in the level of Bcl-xL protein in cells with acquired resistance to Mcl-1 inhibition. Cell lines sensitive to S63845 demonstrated low expression of Bcl-xL. Tumor tissues from patients with lung adenocarcinoma were characterized by decreased Bcl-xL and increased Bak levels of both mRNA and proteins. Concomitant inhibition of Bcl-xL and Mcl-1 demonstrated dramatic cytotoxicity in six of seven studied cell lines. We proposed that co-targeting Bcl-xL and Mcl-1 might lead to a release of Bak, which cannot be neutralized by other anti-apoptotic proteins. Surprisingly, in Bak-knockout cells, inhibition of Mcl-1 and Bcl-xL still resulted in pronounced cell death, arguing against a sole role of Bak in the studied phenomenon. We demonstrate that Bak and Bcl-xL are co-factors for, respectively, sensitivity and resistance to Mcl-1 inhibition.

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Lenalidomide Efficacy in Activated B-Cell-Like Subtype Diffuse Large B-Cell Lymphoma Is Dependent Upon IRF4 and Cereblon Expression

Blood ◽

10.1182/blood.v120.21.3287.3287 ◽

2012 ◽

Vol 120 (21) ◽

pp. 3287-3287

Author(s):

Ling-Hua Zhang ◽

Jolanta Kosek ◽

Maria Wang ◽

Carla Heise ◽

Peter H Schafer ◽

...

Keyword(s):

B Cell ◽

Cell Lines ◽

Molecular Mechanisms ◽

Cell Lymphoma ◽

Xenograft Model ◽

B Cell Lymphoma ◽

Cell Receptor ◽

Employment Equity ◽

Large B Cell Lymphoma ◽

Equity Ownership

Abstract Abstract 3287 Background: Durable responses with lenalidomide monotherapy have been reported in patients with non-Hodgkin lymphoma. In relapsed/refractory diffuse large B-cell lymphoma (DLBCL), higher responses were observed in the activated B-cell-like (ABC) subtype than in the germinal centre B-cell (GCB)-like subtype (Czuczman, et al. British Journal of Haematology, 2011, 154, 477–481). Herein, the molecular mechanisms involved in the differential efficacy of lenalidomide in DLBCL subtypes were investigated. Methods: A panel of DLBCL cell lines, with 5 of ABC-subtype and 11 of non-ABC subtype, was collected and cell of origin subtype was confirmed based on literature, molecular and genetic analysis. The direct antiproliferative effect of lenalidomide on DLBCL cells was assessed using the 3H-thymidine incorporation assay and apoptosis analysis. The molecular mechanisms involved in the antiproliferative efficacy of lenalidomide in DLBCL subtypes were investigated by western blot, immunohistochemistry (IHC) and qRT-PCR analysis of key signaling events during B-cell receptor (BCR)-dependent NF-κB activation. The critical roles of interferon regulatory factor 4 (IRF4), and cereblon (CRBN) in lenalidomide efficacy were established by knock-in or knock-down of these proteins in sensitive ABC cells. Finally, a mouse xenograft model was used to confirm the antitumor effect of lenalidomide and the relevance of the molecular mechanism involved. Results: Using DLBCL cell lines, lenalidomide treatment was found to preferentially suppress proliferation of ABC-DLBCL cells in vitro at a concentration range of 0.01–100 μM (the median plasma concentration at Cmax for patients receiving 25 mg lenalidomide is 2.2 μM) and delay tumor growth in a human tumor xenograft model of OCI-Ly10 cells (lenalidomide 3–30 mg/kg, p.o. qdX28), with minimal effect on non-ABC-DLBCL cells. This tumoricidal effect of lenalidomide was associated with downregulation of IRF4, a survival factor in ABC-DLBCL cells. Treatment with lenalidomide for 1–3 days, similar to the inhibitors of PKCb and MALT1 (LY-333,531 and z-VRPR-fmk, respectively), was found to significantly (p<0.05) downregulate IRF4 protein levels in sensitive cell lines such as OCI-Ly10 and U2932. IRF4 inhibition by lenalidomide reduced CARD11-BCL-10-MALT1 complex activity of ABC-DLBCL cells (as measured by BCL-10 cleavage) and resulted in downregulation of B-cell receptor (BCR)-dependent NF-κB activity. An NF-κB-driven luciferase assay revealed that lenalidomide (1 μM) inhibited transcriptional activity of NF-κB up to 56% in the sensitive ABC-DLBCL cell lines OCI-Ly10 (p <0.05) and U2932 (p <0.01) after 2-day drug treatment. Lenalidomide also significantly (p <0.05) inhibited DNA binding by Rel A/p65, p50 and c-rel/p70 in 4 lines of ABC cells. While IRF4-specific siRNA mimicked the effects of lenalidomide reducing NF-κB activation, IRF4 overexpression conferred cell resistance to lenalidomide, indicating the crucial role of IRF4 inhibition in lenalidomide efficacy in ABC DLBCL. Furthermore, knockdown of CRBN in OCI-Ly10 (p <0.05) and U2932 (p <0.01) conferred resistance to lenalidomide as demonstrated by the abrogation of the inhibitory effects of lenalidomide on IRF4 expression, BCL-10 cleavage, NF-κB activity, and proliferation of these cells, whereas the activity of inhibitors to PKC β and IKKα/β (LY-333,531 and CC-415501, respectively) remained unaffected. These data indicate that antitumor effects of lenalidomide on ABC-DLBCL cells require the presence of cereblon. Conclusions: These data may provide a mechanism for the preferential efficacy of lenalidomide in ABC-DLBCL observed in clinical studies. These findings suggest that lenalidomide has direct antitumor activity against DLBCL cells, preferentially ABC-DLBCL cells, by blocking IRF4 expression and the BCR-NF-κB signaling pathway in a cereblon-dependent manner (also see Figure below). Disclosures: Zhang: Celgene Corp: Employment, Equity Ownership. Kosek:Celgene Corp: Employment, Equity Ownership. Wang:Celgene Corporation: Employment, Equity Ownership. Heise:Celgene Corporation: Employment, Equity Ownership. Schafer:Celgene: Employment, Equity Ownership. Chopra:Celgene Corporation: Employment, Equity Ownership.

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Impact of Hypoxia As Novel Microenvironmental Factor On Drug Susceptibility of Primary CLL Cells

Blood ◽

10.1182/blood.v120.21.3918.3918 ◽

2012 ◽

Vol 120 (21) ◽

pp. 3918-3918

Author(s):

Malte Huelsemann ◽

Lukas P. Frenzel ◽

Dunja Baatout ◽

Julia Claasen ◽

Sebastian Theurich ◽

...

Keyword(s):

Drug Resistance ◽

Oxygen Tension ◽

Drug Efficacy ◽

Protein Translation ◽

Malignant Cells ◽

Dna Targeting ◽

Hypoxic Conditions ◽

Response To Chemotherapy ◽

The Impact

Abstract Abstract 3918 Background: CLL cells circulating in the peripheral blood are sensitive to therapy while malignant cells residing in the microenvironment survive and are the source of relapse. One of the strongest microenvironmental stimuli is CD40 ligand (CD40L)-CD40 interaction, which induces proliferative/anti-apoptotic genes in CLL cells, protecting them from apoptosis and many cytotoxic drugs. Despite the evident importance of CD40 activation further stimuli have to be considered, especially hypoxia. Lymph nodes, particularly those being infiltrated by malignant cells, show a low oxygen tension (<1%). Prior CLL investigations never took this important factor into account, hence the impact of hypoxia on cell survival and drug-resistance is still unrevealed. Methods: We have established an in vitro model, which mimics hypoxic conditions and CD40L-CD40 interaction, in order to understand the molecular basis of drug resistance of CLL cells resident in the microenvironment. CLL cells were cultured on CD40L feeder cells and kept up to 96 hours in hypoxia (1% O2) or normoxia (21% O2). We determined how proliferation rates in CLL are affected by these conditions and subsequently applied several drugs to investigate differences in drug efficacy between normoxia and hypoxia. Apoptosis was determined by AnnexinV/7AAD-staining and subsequent flow cytometry. Expression of potential target molecules was determined by qRT-PCR and Western Blotting. Results: Hypoxia is known to protect malignant cells in solid cancers from chemotherapy. We made similar observations, since classical DNA-targeting drugs were inefficient to kill CLL cells under hypoxic conditions. However, we identified ABT-737, which affects mitochondrial integrity, to be even more efficient under hypoxic conditions compared to normoxia. In order to explain this discrepancy we investigated the expression of several mitochondrial localized anti-/proapoptotic genes on RNA and protein level. We show that the de-regulation of BclXL and Mcl-1 under hypoxic conditions is essential for ABT-737 sensitivity. BclXL deregulation depends on a general reduction in protein translation in hypoxic cells. Mcl-1 protein expression differs from its mRNA expression, hence we expected regulation subsequent to protein synthesis. Indeed we could identify an increased activity of the proteasome in hypoxia, as Mcl-1 is a short-lived protein with a rapid proteasomal turnover this is a feasible explanation for the observed downregulation. Interestingly, hypoxia has a great impact on proliferation of primary CLL cells under different stimuli in vitro. Conclusion: These are the first experiments investigating the impact of oxygen tension on survival and response to chemotherapy of CLL cells. We show that hypoxia renders CLL cells resistant to classical DNA-targeting agent Fludarabine and Bendamustine. Furthermore we point out that small molecules like ABT-737, which specifically target mitochondria, might be efficient in targeting CLL cells protected by hypoxia and CD40L-CD40 interaction within the microenvironment. Development of novel in vitro models like ours will help us understand the specific molecular changes induced by microenvironmental stimuli and their impact on drug efficacy. These findings will allow us to identify novel therapeutic targets. M.Hu. and L.P.F. contributed equally to this work Disclosures: No relevant conflicts of interest to declare.

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Novel BAFF-Receptor Antibody to Natively Folded and Glycosylated Recombinant Protein Eliminates Drug Resistant B Cell Malignancies In Vivo

Blood ◽

10.1182/blood.v128.22.468.468 ◽

2016 ◽

Vol 128 (22) ◽

pp. 468-468

Author(s):

Hong Qin ◽

Guowei Wei ◽

Ippei Sakamaki ◽

Zhenyuan Dong ◽

Diane Lynne Smith ◽

...

Keyword(s):

B Cell ◽

Cell Lines ◽

Cell Lymphoma ◽

Acquired Resistance ◽

B Cell Lymphoma ◽

Lymphoma Cell ◽

Drug Resistant ◽

L Cells

Abstract Background: Targeted monoclonal antibodies (mAbs) such as the anti-CD20 rituximab, are proven therapies in lymphoma, yet these diseases remain incurable because of primary or acquired resistance. Using a eukaryotic expression system to produce antigen closely representing endogenous protein, we developed a new therapeutic antibody against an alternative lymphoma target. B cell activating factor receptor (BAFF-R/TNFRSF13C) is a tumor-necrosis factor receptor superfamily member specifically involved in B lymphocyte development and mature B cell survival. Although earlier attempts to target the BAFF/BAFF-R axis therapeutically for B cell tumors yielded limited success, BAFF-R remains an attractive target for B cell lymphoma therapeutic antibody development, particularly for rituximab-resistant tumors. Methods and Results: We generated 2 mAbs to human BAFF-R expressed as a natively folded, eukaryotically glycosylated cell-surface immunogen on engineered mouse fibroblast (L) cells. Both mAbs specifically bound BAFF-R-expressing L cells, but not the parental counterparts. Each of the complementarity determining regions (CDRs) of the 2 mAbs are unique, suggesting different binding epitopes. Both mAbs bound with high affinity to the human B cell lymphoma cell lines JeKo-1 (mantle cell lymphoma; MCL), SU-DHL6 (diffuse large B cell lymphoma; DLBCL), Raji (Burkitt's lymphoma) and RL (follicular lymphoma). Because our goal is to develop antibodies for clinical use, we substituted in human IgG1 Fc to generate the chimeric mAbs C55 and C90. The chimeric mAbs retained the binding specificity and affinity of the mouse antibodies to their target cells. By immunohistochemistry C55 and C90 staining was specific to the B cell-containing organs tonsil and spleen. No detectable staining was observed in heart, lung, brain, liver, and kidney. Using primary human natural killer (NK) cells as effectors, we demonstrated the chimeric antibodies induced potent antibody-dependent cellular cytotoxicity (ADCC) against BAFF-R-expressing L cells and the JeKo-1, SU-DHL6, Raji and RL human lymphoma cell lines. C55 and C90 were also able to elicit ADCC in primary human lymphomas; they efficiently killed tumor cells from patients with MCL, DLBCL, follicular lymphoma and chronic lymphocytic leukemia (CLL) (n=8). Notably, 5 of these primary lymphomas were from patients who had relapsed after rituximab treatment (2 MCL, 3 CLL). We next determined the activity of C55 and C90 in models of drug-resistant lymphoma. Both ibrutinib and rituximab are effective anti-lymphoma agents, however, primary or acquired resistance to these drugs is common. We derived a rituximab-resistant JeKo-1 variant (JeKo-1 CD20KO) using the CRISPR/HDR system to knock-out CD20 gene expression, and also used the naturally ibrutinib-resistant (Z-138) lymphoma cell line. We confirmed that the C55 and C90 anti-BAFF-R antibodies induced ADCC in both drug-resistant cell lines in vitro. Using xenogenic tumor models in NOD scid gamma (NSG) mice we observed remarkable in vivo anti-tumor effects of both the C55 and C90 chimeric antibodies. We found our antibodies significantly inhibited growth of implanted Z-138 and JeKo-1 CD20KO lymphomas (P<0.001; Figure). Conclusion: In contrast to previously reported BAFF-R antibodies, our in vitro and in vivo results strongly support the translational development of our novel BAFF-R-specific monoclonal antibodies, especially as an alternative immunotherapy against ribuximab- or ibrunitib-resisitant B cell maglinancies. Other preliminary data also suggest BAFF-R may be an effective target of CAR T cells. Figure Figure. Disclosures No relevant conflicts of interest to declare.

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Heterogeneous Pattern of Dependence on Anti-Apoptotic BCL-2 Family Proteins upon CHOP Treatment in Diffuse Large B-Cell Lymphoma

International Journal of Molecular Sciences ◽

10.3390/ijms20236036 ◽

2019 ◽

Vol 20 (23) ◽

pp. 6036 ◽

Cited By ~ 3

Author(s):

Mathilde Rikje Willemijn de Jong ◽

Myra Langendonk ◽

Bart Reitsma ◽

Marcel Nijland ◽

Anke van den Berg ◽

...

Keyword(s):

B Cell ◽

Cell Lines ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

Therapy Resistance ◽

Specific Cell ◽

Bh3 Mimetics ◽

Enhanced Sensitivity ◽

Large B Cell Lymphoma ◽

Large B Cell

Expression of the anti-apoptotic B-cell lymphoma 2 (BCL-2) protein in patients with diffuse large B-cell lymphoma (DLBCL) strongly correlates with resistance to standard therapy with cyclophosphamide, vincristine, doxorubicin, prednisolone, and rituximab (R-CHOP). Although studies focus mainly on the contribution of BCL-2, here we also investigate the contribution of other anti-apoptotic proteins to CHOP-therapy resistance in DLBCL. Functional dynamic BCL-2 homology (BH)3 profiling was applied to DLBCL cell lines upon CHOP treatment or single CHOP compounds. Cell-specific anti-apoptotic dependencies were validated with corresponding BH3-mimetics. We found high expression of anti-apoptotic BCL-2, MCL-1, and BCL-XL in DLBCL cell lines and patients. CHOP treatment resulted in both enhanced and altered anti-apoptotic dependency. Enhanced sensitivity to different BH3-mimetics after CHOP treatment was confirmed in specific cell lines, indicating heterogeneity of CHOP-induced resistance in DLBCL. Analysis of single CHOP compounds demonstrated that similar changes could also be induced by doxorubicin or vincristine, providing evidence for clinical combination therapies of doxorubicin or vincristine with BH3-mimetics in DLBCL. In conclusion, we show for the first time that CHOP treatment induces increased anti-apoptotic dependency on MCL-1 and BCL-XL, and not just BCL-2. These results provide new perspectives for the treatment of CHOP-resistant DLBCL and underline the potential of BH3 profiling in predicting therapy outcomes.

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A Novel Anti-a Proliferation-Inducing Ligand Hapril.01A Monoclonal Antibody Targets Multiple Myeloma Cells in the Bone Marrow Microenvironment

Blood ◽

10.1182/blood.v124.21.2098.2098 ◽

2014 ◽

Vol 124 (21) ◽

pp. 2098-2098

Author(s):

Yu-Tzu Tai ◽

Chirag Acharya ◽

Gang An ◽

Mike Y Zhong ◽

Michele Cea ◽

...

Keyword(s):

Multiple Myeloma ◽

Monoclonal Antibody ◽

Bone Marrow ◽

Dna Binding ◽

B Cell ◽

Cell Lines ◽

Plasma Cell ◽

Molecular Mechanisms ◽

Myeloma Cells ◽

Micromolar Range

Abstract A proliferation-inducing ligand (APRIL), a close member of B-cell-activating factor (BAFF) belonging to the TNF superfamily, is mainly produced by bone marrow (BM) accessory cells to stimulates growth and survival of multiple myeloma (MM) cells. Unlike BAFF, APRIL is dispensable in B cell homeostasis but more critical in plasma cell differentiation and survival. It has higher affinity than BAFF (nanomolar vs micromolar range) to B cell maturation antigen (BCMA) (nanomolar vs micromolar range) which expresses at high levels in all patient MM cells. APRIL also binds to a common plasma cell (PC) marker syndecan-1 (CD138) to induce signaling cascade via TACI, the other APRIL receptor in PC. We here characterize molecular mechanisms regulating APRIL activation in the BM microenvironment and further determine whether a novel anti-APRIL monoclonal antibody hAPRIL.01A inhibits its functional sequelae in MM. First, in vitro osteoclast and macrophage cultures were performed by stimulating CD14+ monocytes from MM patient samples with M-CSF/RANKL and M-CSF, respectively. Osteoclasts and macrophages secret significantly higher levels of APRIL than unstimulated CD14+ monocytes and BM stromal cells (BMSC), as confirmed by ELISA and qRT-PCR. All MM cell lines express cell surface BCMA in significantly higher level than TACI (p < 9.06e-15). H929 MM cells (expressing only BCMA, but not TACI), and other MM cell lines were next stimulated with APRIL, in the presence or absence of hAPRIL.01 Ab followed by immunoblotting and TaqMan® Array assays on harvested protein lysates and mRNA. APRIL stimulation consistently activated NF-kB, PI3K/AKT, and ERK1/2 signaling in MM cells. Importantly, NF-kB-DNA binding activities of p65 and p50 (p52, to a less extent), were significantly upregulated as early as 15 minute and sustain to 4h in all MM cell lines after stimulation. Conversely, hAPRIL.01 Ab completely blocked these signaling cascades, consistent with significantly decreased NF-kB-DNA binding activities in BCMA-knock-downed MM cells by shBCMA lentivirus transfection. APRIL further induced pro-survival proteins (Mcl1, Bcl2, BIRC3, XIAP) and MM cell growth-stimulating regulators (CCDN2, CDK4, CDK6, c-myc), which were completely inhibited by hAPRIL.01 Ab. These results correlated with blockade of hAPRIL.01 Ab in APRIL-promoted viability and colony formation of MM cells, in the presence of osteoclasts or macrophages. APRIL also induces adhesion of MM cells to BMSC, which was blocked by hAPRIL.01 Ab. This concurred with hAPRIL.01 Ab-reduced adhesion molecules (CD44, ICAM-1) induced by APRIL. APRIL-increased VEGF-A and PECAM-1 in MM cells was also significantly reduced by this mAb. APRIL-upregulated chemotactic/osteoclast-activating factors (MIP-1α, MIP1β, SDF-1) were also inhibited by this Ab. Other angiogenesis and adhesion/chemoattractant factors, i.e., IL-8, CXCL10, RANTES and MDC (ccl22), were changed in a similar fashion, indicating specific blocking of hAPRIL.01 Ab to APRIL-induced downstream target genes. This mAb further inhibited APRIL-increased viability of plasmacytoid dendritic cells (pDC) and diminished MM cell viability protected by pDC in 3-d cocultures. Finally, hAPRIL.01 specifically overcame APRIL-, but not IL-6, induced protection in lenalidomide- or dexamethasone-treated MM1S and H929 MM cells. These studies confirm a constitutive APRIL activation via BCMA and TACI in promoting malignancies of myeloma cells, supporting a novel therapeutics of hAPRIL.01 Ab to target MM in the BM microenvironment. Disclosures van Eenennaam: BioNovion: Employment. Elsas:BioNovion: Employment. Anderson:Celgene: Consultancy; Onyx: Consultancy; Gilead Sciences: Consultancy; Sanofi-Aventis US: Consultancy; Acetylon: Scientific Founder Other; Oncoprep: Scientific Founder Other.

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